Language selection

Search

Patent 2320958 Summary

Third-party information liability

Some of the information on this Web page has been provided by external sources. The Government of Canada is not responsible for the accuracy, reliability or currency of the information supplied by external sources. Users wishing to rely upon this information should consult directly with the source of the information. Content provided by external sources is not subject to official languages, privacy and accessibility requirements.

Claims and Abstract availability

Any discrepancies in the text and image of the Claims and Abstract are due to differing posting times. Text of the Claims and Abstract are posted:

  • At the time the application is open to public inspection;
  • At the time of issue of the patent (grant).
(12) Patent Application: (11) CA 2320958
(54) English Title: ANTIGEN LIBRARY IMMUNIZATION
(54) French Title: IMMUNISATION PAR BIBLIOTHEQUE D'ANTIGENES
Status: Dead
Bibliographic Data
(51) International Patent Classification (IPC):
  • C12N 15/31 (2006.01)
  • C07K 14/02 (2006.01)
  • C07K 14/035 (2006.01)
  • C07K 14/16 (2006.01)
  • C07K 14/18 (2006.01)
  • C07K 14/24 (2006.01)
  • C07K 14/245 (2006.01)
  • C07K 14/28 (2006.01)
  • C07K 14/31 (2006.01)
  • C07K 14/315 (2006.01)
  • C07K 14/35 (2006.01)
  • C07K 14/435 (2006.01)
  • C07K 14/445 (2006.01)
  • C07K 19/00 (2006.01)
  • C12N 15/10 (2006.01)
  • C12N 15/36 (2006.01)
  • C12N 15/48 (2006.01)
  • C12N 15/51 (2006.01)
  • C12N 15/86 (2006.01)
  • A61K 39/00 (2006.01)
(72) Inventors :
  • PUNNONEN, JUHA (United States of America)
  • BASS, STEVEN H. (United States of America)
  • WHALEN, ROBERT GERALD (France)
  • HOWARD, RUSSELL (United States of America)
  • STEMMER, WILLEM P.C. (United States of America)
(73) Owners :
  • MAXYGEN, INC. (United States of America)
(71) Applicants :
  • MAXYGEN, INC. (United States of America)
(74) Agent: FETHERSTONHAUGH & CO.
(74) Associate agent:
(45) Issued:
(86) PCT Filing Date: 1999-02-10
(87) Open to Public Inspection: 1999-08-19
Availability of licence: N/A
(25) Language of filing: English

Patent Cooperation Treaty (PCT): Yes
(86) PCT Filing Number: PCT/US1999/002944
(87) International Publication Number: WO1999/041383
(85) National Entry: 2000-08-10

(30) Application Priority Data:
Application No. Country/Territory Date
09/021,769 United States of America 1998-02-11
60/074,294 United States of America 1998-02-11
60/105,509 United States of America 1998-10-23

Abstracts

English Abstract




This invention is directed to antigen library immunization, which provides
methods for obtaining antigens having improved properties for therapeutic and
other uses. The methods are useful for obtaining improved antigens that can
induce an immune response against pathogens, cancer, and other conditions, as
well as antigens that are effective in modulating allergy, inflammatory and
autoimmune diseases.


French Abstract

L'invention concerne une immunisation par bibliothèque d'antigènes mettant en oeuvre des méthodes qui produisent des antigènes présentant des propriétés améliorées et destinés à un usage thérapeutiques ou autres. Ces méthodes permettent d'obtenir des antigènes améliorés pouvant produire une réponse immunitaire contre des agents pathogènes, le cancer ou d'autres affections, ainsi que des antigènes pouvant moduler efficacement des maladies allergiques, inflammatoires ou auto-immunes.

Claims

Note: Claims are shown in the official language in which they were submitted.




118

WHAT IS CLAIMED IS
1. An recombinant multivalent antigenic polypeptide that comprises a first
antigenic determinant of a first polypeptide and at least a second antigenic
determinant from
a second polypeptide.
2. The multivalent antigenic polypeptide of claim 1, wherein the
polypeptide comprises at least a third antigenic determinant from a third
polypeptide.
3. The multivalent antigenic polypeptide of claim 1, wherein the first and
second polypeptides are selected from the group consisting of cancer antigens,
antigens
associated with autoimmunity disorders, antigens associated with inflammatory
conditions,
antigens associated with allergic reactions, and antigens from infectious
agents.
4. The multivalent antigenic polypeptide of claim 3, wherein the antigens
are from a virus, a parasite, or a bacteria.
5. The multivalent antigenic polypeptide of claim 4, wherein the antigens
are from a virus selected from the group consisting of a Venezuelan equine
encephalitis virus
or a related alphavirus, a virus of the Japanese encephalitis virus complex, a
virus of the
tick-borne encephalitis virus complex, a Dengue virus, a Hanta virus, an HIV,
a hepatitis B virus,
a hepatitis C virus, and a Herpes simplex virus.
6. The multivalent antigenic polypeptide of claim 5, wherein the antigens
are envelope proteins.
7. The multivalent antigenic polypeptide of claim 4, wherein the antigens
are from a bacteria and are selected from the group consisting of a Yersinia V
antigen, a
Staphylococcus aureus enterotoxin, a Streptococcus pyogenes enterotoxin, a
Vibrio cholera
toxin, an enterotoxigenic Escherichia coli heat labile enterotoxin, a OspA and
a OspC
polypeptide from a Borrelia species, an Antigen 85 polypeptide from a
Mycobacterium
species, a VacA and a CagA polypeptide from Helicobacter pylori, and an MSP
antigen
from Plasmodium falciparum.



119
8. The multivalent antigenic polypeptide of claim 1, wherein the
multivalent antigenic polypeptide exhibits reduced affinity to IgE from a
mammal compared
to the first or second polypeptides.
9. The multivalent antigenic polypeptide of claim 1, wherein the first
antigenic determinant and the second antigenic determinant are from different
serotypes of a
pathogenic organism.
10. The multivalent antigenic polypeptide of claim 1, wherein the first
antigenic determinant and the second antigenic determinant are from different
species of
pathogenic organism.
11. The multivalent antigenic polypeptide of claim 1, wherein the first
polypeptide and the second polypeptide are allergens.
12. The multivalent antigenic polypeptide of claim 11, wherein the
allergens are dust mite allergens, grass pollen allergens, birch pollen
allergens, ragweed
pollen allergens, hazel pollen allergens, cockroach allergens, rice allergens,
olive tree pollen
allergens, fungal allergens, mustard allergens, and bee venom.
13. The multivalent antigenic polypeptide of claim 1, wherein the first
polypeptide and the second polypeptide are associated with an inflammatory or
autoimmune
disease.
14. The multivalent antigenic polypeptide of claim 13, wherein the first
polypeptide and the second polypeptide are autoantigens associated with a
disease selected
from the group consisting of multiple sclerosis, scleroderma, systemic
sclerosis, systemic
lupus erythematosus, hepatic autoimmune disorder, skin autoimmune disorder,
insulin-dependent
diabetes mellitus, thyroid autoimmune disorder, and rheumatoid arthritis.
15. The multivalent antigenic polypeptide of claim 1, wherein the first
polypeptide and the second polypeptide are cancer antigens or sperm antigens.



120
16. A recombinant antigen library comprising recombinant nucleic acids
that encode antigenic polypeptides, wherein the library is obtained by
recombining at least
first and second forms of a nucleic acid which comprises a polynucleotide
sequence that
encodes a disease-associated antigenic polypeptide, wherein the first and
second forms differ
from each other in two or more nucleotides, to produce a library of
recombinant nucleic
acids.
17. The recombinant antigen library of claim 16, wherein the first and
second polypeptides are toxins.
18. A method of obtaining a polynucleotide that encodes a recombinant
antigen having improved ability to induce an immune response to a disease
condition, the
method comprising:
(1) recombining at least first and second forms of a nucleic acid which
comprises a polynucleotide sequence that encodes an antigenic polypeptide that
is associated
with the disease condition, wherein the first and second forms differ from
each other in two
or more nucleotides, to produce a library of recombinant nucleic acids; and
(2) screening the library to identify at least one optimized recombinant
nucleic acid that encodes an optimized recombinant antigenic polypeptide that
has improved
ability to induce an immune response to the disease condition.
19. The method of claim 18, wherein the method further comprises:
(3) recombining at least one optimized recombinant nucleic acid with a
further form of the nucleic acid, which is the same or different from the
first and second
forms, to produce a further library of recombinant nucleic acids;
(4) screening the further library to identify at least one further
optimized recombinant nucleic acid that encodes a polypeptide that has
improved ability to
induce an immune response to the disease condition; and
(5) repeating (3) and (4), as necessary, until the further optimized
recombinant nucleic acid encodes a polypeptide that has improved ability to
induce an
immune response to the disease condition.



121
20. The method of claim 18, wherein the disease-associated polypeptides
are selected from the group consisting of cancer antigens, antigens associated
with
autoimmunity disorders, antigens associated with inflammatory conditions,
antigens
associated with allergic reactions, and antigens associated with infectious
agents.
21. The method of claim 18, wherein the disease condition is an infectious
disease and the first and second forms of the nucleic acid each encode an
antigen of a
different serotype of a pathogenic agent.
22. The method of claim 21, wherein the first and second forms of the
nucleic acid are each from a different species of pathogen.
23. The method of claim 21, wherein the screening is accomplished by:
introducing into a test animal either:
a) the library of recombinant nucleic acids, or
b) recombinant polypeptides encoded by the library of recombinant
nucleic acids;
introducing the pathogenic agent into the test animal; and
determining whether the test animal is resistant to challenge by the
pathogenic agent.
24. The method of claim 23, wherein the pathogenic agent introduced into
the test animal is of a different serotype than that used as a source of the
first and second
forms of the nucleic acid.
25. The method of claim 23, wherein the library is subdivided into a
plurality of pools, each of which pools is introduced into a test animal to
identify those pools
that include an optimized recombinant nucleic acid that encodes a polypeptide
which has
improved ability to induce an immune response to the pathogenic agent.
26. The method of claim 25, wherein the pools that include an optimized
recombinant nucleic acid are further subdivided into a plurality of subpools,
each of which



122
subpools is introduced into a test animal to identify those pools that include
an optimized
recombinant nucleic acid that encodes a polypeptide which has improved ability
to induce an
immune response to the pathogenic agent.
27. The method of claim 18, wherein the optimized recombinant nucleic
acid encodes a multivalent antigenic polypeptide and the screening is
accomplished by:
expressing the library of recombinant nucleic acids in a phage display
expression vector such that the recombinant antigen is expressed as a fusion
protein with a
phage polypeptide that is displayed on a phage particle surface;
contacting the phage with a first antibody that is specific for a first
serotype of the pathogenic agent and selecting those phage that bind to the
first antibody;
contacting those phage that bind to the first antibody with a second
antibody that is specific for a second serotype of the pathogenic agent and
selecting those
phage that bind to the second antibody;
wherein those phage that bind to the first antibody and the second
antibody express a multivalent antigenic polypeptide.
28. The method of claim 27, wherein the screening further comprises
contacting those phage that bind to the first and second antibodies with one
or more
additional antibodies, each of which is specific for an additional serotype of
the pathogenic
agent, and selecting those phage that bind to the respective additional
antibodies.
29. The method of claim 27, wherein the phage display expression vector
comprises a suppressible stop codon between the recombinant nucleic acid and
the phage
polypeptide, whereby expression in a host cell which comprises a corresponding
suppressor
tRNA results in production of the fusion protein and expression in a host cell
which lacks a
corresponding suppressor tRNA results in production of the recombinant antigen
not as a
fusion protein.
30. The method of claim 18, wherein the optimized recombinant antigen
exhibits an enhanced expression level in a host cell and the screening is
accomplished by
expression of each recombinant nucleic acid in the host cell and subjecting
the host cells to



123
flow cytometry-based cell sorting to obtain those host cells that display the
recombinant
antigen on the host cell surface.
31. The method of claim 18, wherein the improved property is selected
from the group consisting of
improved immunogenicity;
enhanced cross-reactivity against different forms of the
disease-associated antigenic polypeptide;
reduced toxicity;
improved adjuvant activity in vivo; and
improved production of the immunogenic polypeptide.
32. The method of claim 31, wherein the improved property is enhanced
cross-reactivity against different forms of the disease-associated polypeptide
and the first
and second forms of the nucleic acid are from a first and a second form of the

disease-associated polypeptide.
33. The method of claim 32, wherein the first and second forms of the
disease-associated polypeptide are obtained from at least a first and second
species of a
pathogenic agent and the optimized recombinant nucleic acid encodes a
recombinant
polypeptide that induces a protective response against both species of the
pathogenic agent.
34. The method of claim 33, wherein the recombinant polypeptide induces a
protective response against at least one additional species of the pathogenic
agent.
35. The method of claim 33, wherein the pathogenic agent is a toxin.
36. The method of claim 33, wherein the pathogenic agent is a virus or a
cell.
37. The method of claim 33, wherein the disease-associated polypeptide is a
Yersinia V-antigen.



124
38. The method of claim 37, wherein the at least first and second forms of a
nucleic acid are obtained from at least a first and second species of
Yersinia.
39. The method of claim 38, wherein the Yersinia species are selected from
the group consisting of Y. pestis, Y. enterocolitica, and Y.
pseudotuberculosis.
40. The method of claim 33, wherein the pathogenic agent is a bacterial
toxin.
41. The method of claim 18, wherein the disease condition is cancer and the
screening step involves introducing the optimized recombinant nucleic acids
into a genetic
vaccine vector and testing library members for ability to inhibit
proliferation of cancer cells
or inducing death of cancer cells.
42. The method of claim 41, wherein the optimized recombinant nucleic
acid comprises a nucleotide sequence that encodes a tumor specific antigen.
43. The method of claim 41, wherein the optimized recombinant nucleic
acid comprises a nucleotide sequence that encodes a molecule which is capable
of inhibiting
proliferation of cancer cells.
44. The method of claim 18, wherein the disease condition is an
inflammatory response which has an unknown or no antigen specificity and the
screening
step involves one or more of the following:
a) determining the ability of the genetic vaccine vector to induce
cytokine production by PBMC, synovial fluid cells, purified T cells,
monocytes/macrophages, dendritic cells, or T cell clones;
b) determining the ability of the genetic vaccine vector to induce T cell
activation or proliferation; and
c) determining the ability of the genetic vaccine vector to induce T cell
differentiation to TH1 or TH2 cells.



125


45. The method of claim 18, wherein the disease condition is an
autoimmune response.
46. The method of claim 45, wherein the optimized recombinant antigenic
polypeptide shifts the immune response from a TH1-mediated response to a TH2-
mediated
response.
47. The method of claim 18, wherein the disease condition is an allergic
immune response.
48. The method of claim 47, wherein the optimized recombinant antigenic
polypeptide shifts the immune response from a TH2-mediated response to a TH1-
mediated
response.
49. The method of claim 47, wherein the optimized recombinant antigenic
polypeptide induces an immune response characterized by predominant IgG and
IgM
expression and reduced IgE expression.
50. The method of claim 47, wherein the optimized recombinant antigenic
polypeptide is not recognized by pre-existing IgE molecules present in sera of
atopic
mammals.
51. The method of claim 50, wherein the optimized recombinant antigenic
polypeptide retains T cell epitopes that are involved in modulating a T cell
response.
52. A method of obtaining a recombinant viral vector which has an
enhanced ability to induce an antiviral response in a cell, the method
comprising the steps of:
(1) recombining at least first and second forms of a nucleic acid which
comprise a viral vector, wherein the first and second forms differ from each
other in two or
more nucleotides, to produce a library of recombinant viral vectors;
(2) transfecting the library of recombinant viral vectors into a
population of mammalian cells;
(3) staining the cells for the presence of Mx protein; and




126
(4) isolating recombinant viral vectors from cells which stain positive
for Mx protein, wherein recombinant viral vectors from positive staining cells
exhibit
enhanced ability to induce an antiviral response.
53. The method of claim 52, wherein the viral vector comprises an
influenza viral genomic nucleic acid.

Description

Note: Descriptions are shown in the official language in which they were submitted.



CA 02320958 2000-08-10
WO 99/41383 PCTNS99/02944
ANTIGEN LIBRARY IMMUNIZATION
BACKGROUND OF THE INVENTION
Field of the Invention
This invention pertains to the field of methods for developing immunogens
that can induce efficient immune responses against a broad range of antigens.
Background
The interactions between pathogens and hosts are results of millions of years
of evolution, during which the mammalian immune system has evolved
sophisticated means
to counterattack pathogen invasions. However, bacterial and viral pathogens
have
1 S simultaneously gained a number of mechanisms to improve their virulence
and survival in
hosts, providing a major challenge for vaccine research and development
despite the powers
of modern techniques of molecular and cellular biology. Similar to the
evolution of pathogen
antigens, several cancer antigens are likely to have gained means to
downregulate their
immunogenicity as a mechanism to escape the host immune system.
Efficient vaccine development is also hampered by the antigenic
heterogeneity of different strains of pathogens, driven in part by
evolutionary forces as
means for the pathogens to escape immune defenses. Pathogens also reduce their
immunogenicity by selecting antigens that are difficult to express, process
and/or transport in
host cells, thereby reducing the availability of immunogenic peptides to the
molecules
initiating and modulating immune responses. The mechanisms associated with
these
challenges are complex, multivariate and rather poorly characterized.
Accordingly, a need
exists for vaccines that can induce a protective immune response against
bacterial and viral
pathogens. The present invention fulfiils this and other needs.
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/02944
2
SUIIiMARY OF THE INVENTION
The present invention provides recombinant multivalent antigenic
polypeptides that include a first antigenic determinant from a first disease-
associated
polypeptide and at least a second antigenic determinant from a second disease-
associated
polypeptide. The disease-associated polypeptides can be selected from the
group consisting
of cancer antigens, antigens associated with autoimmunity disorders, antigens
associated
with inflammatory conditions, antigens associated with allergic reactions,
antigens
associated with infectious agents, and other antigens that are associated with
a disease
condition.
In another embodiment, the invention provides a recombinant antigen library
that contains recombinant nucleic acids that encode antigenic polypeptides.
The libraries are
typically obtained by recombining at least first and second forms of a nucleic
acid which
includes a polynucleotide sequence that encodes a disease-associated antigenic
polypeptide,
wherein the first and second forms differ from each other in two or more
nucleotides, to
produce a library of recombinant nucleic acids.
Another embodiment of the invention provides methods of obtaining a
polynucleotide that encodes a recombinant antigen having improved ability to
induce an
immune response to a disease condition. These methods involve: ( 1 )
recombining at least
first and second forms of a nucleic acid which comprises a polynucleotide
sequence that
encodes an antigenic polypeptide that is associated with the disease
condition, wherein the
first and second forms differ from each other in two or more nucleotides, to
produce a library
of recombinant nucleic acids; and (2) screening the library to identify at
least one optimized
recombinant nucleic acid that encodes an optimized recombinant antigenic
polypeptide that
has improved ability to induce an immune response to the disease condition.
These methods optionally further involve: (3) recombining at least one
optimized recombinant nucleic acid with a further form of the nucleic acid,
which is the
same or different from the first and second forms, to produce a further
library of recombinant
nucleic acids; (4) screening the further library to identify at least one
further optimized
recombinant nucleic acid that encodes a polypeptide that has improved ability
to induce an
immune response to the disease condition; and (5) repeating (3) and (4), as
necessary, until
the further optimized recombinant nucleic acid encodes a polypeptide that has
improved
ability to induce an immune response to the disease condition.
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/01944
In some embodiments, the optimized recombinant nucleic acid encodes a
multivalent antigenic polypeptide and the screening is accomplished by
expressing the
library of recombinant nucleic acids in a phage display expression vector such
that the
recombinant antigen is expressed as a fusion protein with a phage polypeptide
that is
displayed on a phage particle surface; contacting the phage with a first
antibody that is
specific for a first serotype of the pathogenic agent and selecting those
phage that bind to the
first antibody; and contacting those phage that bind to the first antibody
with a second
antibody that is specific for a second serotype of the pathogenic agent and
selecting those
phage that bind to the second antibody; wherein those phage that bind to the
first antibody
and the second antibody express a multivalent antigenic polypeptide.
The invention also provides methods of obtaining a recombinant viral vector
which has an enhanced ability to induce an antiviral response in a cell. These
methods can
include the steps of ( 1 ) recombining at least first and second fonms of a
nucleic acid which
comprise a viral vector, wherein the first and second forms differ from each
other in two or
more nucleotides, to produce a library of recombinant viral vectors; (2)
transfecting the
library of recombinant viral vectors into a population of mammalian cells; (3)
staining the
cells for the presence of Mx protein; and (4) isolating recombinant viral
vectors from cells
which stain positive for Mx protein, wherein recombinant viral vectors from
positive
staining cells exhibit enhanced ability to induce an antiviral response.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows a schematic representation of a method for generating a
chimeric, multivalent antigen that has immunogenic regions from multiple
antigens.
Antibodies to each of the non-chimeric parental immunogenic polypeptides are
specific for
the respective organisms (A, B, C). After carrying out the recombination and
selection
methods of the invention, however, a chimeric immunogenic polypeptide is
obtained that is
recognized by antibodies raised against each of the three parental immunogenic
polypeptides.
Figure 2 shows the principle of family DNA shuffling. A family of antigen
genes from related pathogens are subjected to shuffling, which results in a
library of
chimeric and/or mutated antigens. Screening methods are employed to identify
those
SUBSTITUTE SHEET (RULE 26)


:CA 02320958 2000-08-10
W
y
. .:..:.. .. ..:'. ... . ..:. . . .....: y 1 y 1 1::::::_:._.; ::: :::::_.. .:
~~ 1 ~ all a1 _...........!
~1 ~ 1
~ y 1 ~ v 1
1 1 1 ~ ~ ~ ~ ~ ~ 1 1 1 ~ 1 1
1 1 1 ~ ~ ~ ~ 1
1 1 111 ~~ ~1 11 11 1~
4
recombinant antigens that are the most immunogenic and/or cross-protective.
These can, if
desired, be subjected to additional rounds of shuffling and screening.
Figure 3A-Figure 3B shows a schematic for a method by which one can
obtain recombinant polypeptides that can induce a broad-spectrum immune
response. In
Figure 3A, wild-type immunogenic polypeptides from the pathogens A, B, and C
provide
protection against the corresponding pathogen from which the polypeptide is
derived, but
little or no cross-protection against the other pathogens (left panel). After
shuffling, an
A!B/C chimeric polypeptide is obtained that can induce a protective immune
response
against all three pathogen types (right panel). In Figure 3B, shuffling is
used with substrate
nucleic acids from two pathogen strains (A, B), which encode polypeptides that
are
protective only against the corresponding pathogen. After shuffling, the
resulting chimeric
polypeptide can induce an immune response that is effective against not only
the two
parental pathogen strains, but also against a third strain of pathogen (C}.
Figure 4 diagrams some of the possible factors that can determine whether a
I S particular polynucleotide encodes an immunogenic polypeptide having a
desired property,
such as enhanced immunogenicity and/or cross-reactivity. Those sequence
regions that
positively affect a particular property are indicated as plus signs along the
antigen gene,
while those sequence regions that have a negative effect are shown as minus
signs. A pool of
related antigen genes are shuffled and screened to obtain those that
recombinant nucleic
acids that have gained positive sequence regions and lost negative regions. No
pre-existing
knowledge as to which regions are positive or negative for a particular trait
is required.
Figure 5 shows a schematic representation of the screening strategy for
antigen library screening.
Figure 6 shows a schematic representation of a strategy for pooling and
deconvolution as used in antigen library screening.
Figure 7 is an alignment of the nucleotide sequences of glycoprotein D (gD)
from HSV-1 (SEQ ID N0:1) and HSV-2 (gD-1 (SEQ ID N0:2) and gD-2 (SEQ ID
N0:3)).
Figure 8A shows a diagram of a method for expressing HN gp120 using
genetic vaccine vectors and generation of a library of shuffled gp 120 genes.
Figure 8B
shows PCR primers that are useful for obtaining gp120 nucleic acid substrates
for DNA
AMENDED SHEET
~.: .;:, ~...~:. t ~..: :: J ~:: .. ~~ y:.. .: ; ~; %v''':
:::::::~~.v.~.:..:: . ;:.~ .:


CA 02320958 2000-08-10
a ~ . ~ ~ ~ ~ ~ ~ ~
...... . . ..... . .. ~ ~...::~ ~... .. ..~~ ~ ~ ~ ~ ~ ~ ~ ~..... '..: ~
.~:::........::.
1 ~ ~ ~ y ~ ~ ~ y~ ~ ~ ~
~ ~ ~ ~~~ ~ ~ ~ ~ ~ ~ ~~~ ~~~
~ ~ ~ ~ ~ ~ ~ ~ ~
' . ~ ~ ~~~ ~~ ~~ ~~ ~~ ~~
shuffling reactions. Primers suitable for generating substrates include 6025F
(SEQ ID
N0:4), 77738 (SEQ ID NO:S), and primers suitable for amplifying the shuffled
nucleic
acids include 6196F (SEQ ID N0:6) and 77468 (SEQ ID N0:7). The primer BssH2-
6205F
(SEQ ID N0:8) can be used to clone the resulting fragment into a genetic
vaccine vector.
5 Figure 9 shows the domain structure of hepadnavirus envelope genes.
Figure I O shows a schematic representation of the use of shuffling to obtain
hepadnavirus proteins in which the immunogenicity of one antigenic domain is
improved.
Figure 11 shows a strategy in which genes that encode the hepadnavirus
proteins having one antigenic domain that has improved immunogenicity are
shuffled to
obtain recombinant proteins in which all three domains have improved
immunogenicity.
Figure 12 shows the transmembrane organization of the HBsAg polypeptide.
Figure 13 shows a method for using phage display to obtain recombinant
allergens that are not bound by pre-existing IgE.
Figure 14 shows a strategy for screening of recombinant allergens to identify
those that are effective in activating TH cells. PBMC or T cell clones from
atopic individuals
are exposed to antigen-presenting cells that display the antigen variants
obtained using the
methods of the invention. To identify those allergen variants that are
effective in activating T
cells, the cultures are tested for induction of T cell proliferation or for a
pattern of cytokine
synthesis that is indicative of the particular type of T cell activation that
is desired. If
desired, the allergen variants that test positive in the in vitro assay can be
subjected to in vivo
testing.
Figure 15 shows a strategy for screening of recombinant cancer antigens to
identify those that are effective in activating T cells of cancer patients.
Figure 16A and Figure 16B show two different strategies for generating
vectors that contain multiple T cell epitopes obtained, for example, by DNA
shuffling. In
Figure 16A, each individual shuffled epitope-encoding nucleic acid is linked
to a single
promoter, and multiple promoter-epitope gene constructs can be placed in a
single vector.
The scheme shown in Figure 16B involves linking multiple epitope-encoding
nucleic acids
to a single promoter.
Figure 17 shows the sequences of PreS2-S coding regions (SEQ ID NOS:9
and I 1 ) and corresponding amino acid sequences (SEQ )D NOS:10 and 12) of
different
.:.::~~~~~,~~~., MENDED SHEET .::.;.;


CA 02320958 2000-08-10:
.. .. .. :. . .
~ i: :.. .... , i . . . . . . _..:~.~.. :. ::.;;: :.::
. . . . 1 ~~ . . . .
.~. . . ~ ~ . . ... ...
. . . . . . . .
... .. .. .. .. .
6
hepatitis B surface antigen (HBsAg) or woodchuck hepatitis B (WHV) proteins
(SEQ ID
NOS:15 and 16). Primers suitable for amplification of this region are also
shown (HBV,
SEQ ID NOS:13 and 14; WHV, SEQ ID NOS:l7 and 18).
Figure 18 shows primers (SEQ ID NOS:19-22) that are suitable for
amplification of large fragments that contain the S2S coding sequences. The
primers
hybridize to regions that are approximately 200 by outside the desired
sequences.
Figure 19 shows an alignment of the amino acid sequences of surface
antigens from different HVB subtypes (SEQ ID NOS:10 and 12).
Figure 20 shows a diagram of multimeric particles that assemble when an
appropriate number of chimeric polypeptides and native HBsAg S monomers are
mixed.
DETAILED DESCRIPTION
Definitions
The term "screening" describes, in general, a process that identifies optimal
antigens. Several properties of the antigen can be used in selection and
screening including
antigen expression, folding, stability, immunogenicity and presence of
epitopes from several
related antigens. Selection is a form of screening in which identification and
physical
separation are achieved simultaneously by expression of a selection marker,
which, in some
genetic circumstances, allows cells expressing the marker to survive while
other cells die (or
vice versa). Screening markers include, for example, luciferase, beta-
galactosidase and
green fluorescent protein. Selection markers include drug and toxin resistance
genes, and
the like. Because of limitations in studying primary immune responses in
vitro; in vivo
studies are particularly useful screening methods. In these studies, the
antigens are first
introduced to test animals, and the immune responses are subsequently studied
by analyzing
protective immune responses or by studying the quality or strength of the
induced immune
response using lymphoid cells derived from the immunized animal. Although
spontaneous
selection can and does occur in the course of natural evolution, in the
present methods
selection is performed by man.
A "exogenous DNA segment", "heterologous sequence" or a "heterologous
nucleic acid", as used herein, is one that originates from a source foreign to
the particular
host cell, or, if from the same source, is modified from its original form.
Thus, a
heterologous gene in a host cell includes a gene that is endogenous to the
particular host cell,
:::~:~~~x~y:c::: ::
:::.::::.::::.:::::».:.:::"::.:::::::::..AMEND
.. ::.:: ::.::._ ::.:::::: :.:. ::::::._:::::::::.: :::: ED SHEET ..:::;:::


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/02944
7
but has been modifed. Modification of a heterologous sequence in the
applications
described herein typically occurs through the use of DNA shuffling. Thus, the
terms refer to
a DNA segment which is foreign or heterologous to the cell, or homologous to
the cell but in
a position within the host cell nucleic acid in which the element is not
ordinarily found.
Exogenous DNA segments are expressed to yield exogenous polypeptides.
The term "gene" is used broadly to refer to any segment of DNA associated
with a biological function. Thus, genes include coding sequences and/or the
regulatory
sequences required for their expression. Genes also include nonexpressed DNA
segments
that, for example, form recognition sequences for other proteins. Genes can be
obtained from
a variety of sources, including cloning from a source of interest or
synthesizing from known
or predicted sequence information, and may include sequences designed to have
desired
parameters.
The term "isolated", when applied to a nucleic acid or protein, denotes that
the nucleic acid or protein is essentially free of other cellular components
with which it is
associated in the natural state. It is preferably in a homogeneous state
although it can be in
either a dry or aqueous solution. Purity and homogeneity are typically
determined using
analytical chemistry techniques such as polyacrylamide gel electrophoresis or
high
performance liquid chromatography. A protein which is the predominant species
present in a
preparation is substantially purified. In particular, an isolated gene is
separated from open
reading frames which flank the gene and encode a protein other than the gene
of interest.
The term "purified" denotes that a nucleic acid or protein gives rise to
essentially one band
in an electrophoretic gel. Particularly, it means that the nucleic acid or
protein is at least
about SO% pure, more preferably at least about 85% pure, and most preferably
at least about
99% pure.
The term "naturally-occurring" is used to describe an object that can be found
in nature as distinct from being artificially produced by man. For example, a
polypeptide or
polynucleotide sequence that is present in an organism (including viruses,
bacteria, protozoa,
insects, plants or mammalian tissue) that can be isolated from a source in
nature and which
has not been intentionally modified by man in the laboratory is naturally-
occurring.
The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and
polymers thereof in either single- or double-stranded form. Unless
specifically limited, the
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99Id 1383 PCT/US99/02944
8
term encompasses nucleic acids containing known analogues of natural
nucleotides which
have similar binding properties as the reference nucleic acid and are
metabolized in a manner
similar to naturally occurring nucleotides. Unless otherwise indicated, a
particular nucleic
acid sequence also implicitly encompasses conservatively modified variants
thereof (e.g.
S degenerate codon substitutions) and complementary sequences and as well as
the sequence
explicitly indicated. Specifically, degenerate codon substitutions may be
achieved by
generating sequences in which the third position of one or more selected (or
all) codons is
substituted with mixed-base and/or deoxyinosine residues (Batzer et al. (1991)
Nucleic Acid
Res. 19: 5081; Ohtsuka et al. (1985) J. Biol. Chem. 260: 2605-2608; Cassol et
al. (1992} ;
Rossolini et al. (1994) Mol. Cell. Probes 8: 91-98). The term nucleic acid is
used
interchangeably with gene, cDNA, and mRNA encoded by a gene.
"Nucleic acid derived from a gene" refers to a nucleic acid for whose
synthesis the gene, or a subsequence thereof, has ultimately served as a
template. Thus, an
mRNA, a cDNA reverse transcribed from an mRNA, an RNA transcribed from that
cDNA, a
DNA amplified from the cDNA, an RNA transcribed from the amplified DNA, etc.,
are all
derived from the gene and detection of such derived products is indicative of
the presence
and/or abundance of the original gene and/or gene transcript in a sample.
A nucleic acid is "operably linked" when it is placed into a functional
relationship with another nucleic acid sequence. For instance, a promoter or
enhancer is
operably linked to a coding sequence if it increases the transcription of the
coding sequence.
Operably linked means that the DNA sequences being linked are typically
contiguous and,
where necessary to join two protein coding regions, contiguous and in reading
frame.
However, since enhancers generally function when separated from the promoter
by several
kilobases and intronic sequences may be of variable lengths, some
polynucleotide elements
may be operably linked but not contiguous.
A specifc binding affinity between two molecules, for example, a ligand and
a receptor, means a preferential binding of one molecule for another in a
mixture of
molecules. The binding of the molecules can be considered specific if the
binding affinity is
about 1 x 10° M -~ to about 1 x 106 M '~ or greater.
The term "recombinant" when used with reference to a cell indicates that the
cell replicates a heterologous nucleic acid, or expresses a peptide or protein
encoded by a
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/~i1383 PCT/US99/QZ944
9
heterologous nucleic acid. Recombinant cells can contain genes that are not
found within
the native (non-recombinant) form of the cell. Recombinant ceils can also
contain genes
found in the native form of the cell wherein the genes are modified and re-
introduced into
the cell by artificial means. The term also encompasses cells that contain a
nucleic acid
endogenous to the cell that has been modified without removing the nucleic
acid from the
cell; such modifications include those obtained by gene replacement, site-
specific mutation,
and related techniques.
A "recombinant expression cassette" or simply an "expression cassette" is a
nucleic acid construct, generated recombinantly or synthetically, with nucleic
acid elements
that are capable of effecting expression of a structural gene in hosts
compatible with such
sequences. Expression cassettes include at least promoters and optionally,
transcription
termination signals. Typically, the recombinant expression cassette includes a
nucleic acid
to be transcribed (e.g., a nucleic acid encoding a desired polypeptide), and a
promoter.
Additional factors necessary or helpful in effecting expression may also be
used as described
herein. For example, an expression cassette can also include nucleotide
sequences that
encode a signal sequence that directs secretion of an expressed protein from
the host cell.
Transcription termination signals, enhancers, and other nucleic acid sequences
that influence
gene expression, can also be included in an expression cassette.
A "multivalent antigenic polypeptide" or a "recombinant multivalent
antigenic polypeptide" is a non-naturally occurring polypeptide that includes
amino acid
sequences from more than one source polypeptide, which source polypeptide is
typically a
naturally occurring polypeptide. At least some of the regions of different
amino acid
sequences constitute epitopes that are recognized by antibodies found in a
mammal that has
been injected with the source polypeptide. The source polypeptides from which
the different
epitopes are derived are usually homologous (i.e., have the same or a similar
structure and/or
function), and are often from different isolates, serotypes, strains, species,
of organism or
from different disease states, for example.
The terms "identical" or percent "identity," in the context of two or more
nucleic acid or polypeptide sequences, refer to two or more sequences or
subsequences that
are the same or have a specified percentage of amino acid residues or
nucleotides that are the
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/02944
same, when compared and aligned for maximum correspondence, as measured using
one of
the following sequence comparison algorithms or by visual inspection.
The phrase "substantiaily identical," in the context of two nucleic acids or
polypeptides, refers to two or more sequences or subsequences that have at
least 60%,
S preferably 80%, most preferably 90-95% nucleotide or amino acid residue
identity, when
compared and aligned for maximum correspondence, as measured using one of the
following
sequence comparison algorithms or by visual inspection. Preferably, the
substantial identity
exists over a region of the sequences that is at least about SO residues in
length, more
preferably over a region of at least about 100 residues, and most preferably
the sequences are
10 substantially identical over at least about 150 residues. In some
embodiments, the sequences
are substantially identical over the entire length of the coding regions.
For sequence comparison, typically one sequence acts as a reference sequence
to which test sequences are compared. When using a sequence comparison
algorithm, test
and reference sequences are input into a computer, subsequence coordinates are
designated,
if necessary, and sequence algorithm program parameters are designated. The
sequence
comparison algorithm then calculates the percent sequence identity for the
test sequences)
relative to the reference sequence, based on the designated program
parameters.
Optimal alignment of sequences for comparison can be conducted, e.g., by
the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981
), by the
homology alignment algorithm ofNeedleman & Wunsch, J. Mol. Biol. 48:443
(1970), by the
search for similarity method of Pearson & Lipman, Proc. Nat 'l. Acad. Sci. USA
85:2444
(1988), by computerized implementations of these algorithms (GAP, BESTFIT,
FASTA,
and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer
Group, 575
Science Dr., Madison, WI), or by visual inspection (see generally Ausubel et
al., infra).
One example of an algorithm that is suitable for determining percent
sequence identity and sequence similarity is the BLAST algorithm, which is
described in
Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing
BLAST analyses
is publicly available through the National Center for Biotechnology
Information
(http://www.ncbi.nlm.nih.gov~. This algorithm involves first identifying high
scoring
sequence pairs (HSPs) by identifying short words of length W in the query
sequence, which
either match or satisfy some positive-valued threshold score T when aligned
with a word of
SU6STITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/02944
11
the same length in a database sequence. T is referred to as the neighborhood
word score
threshold (Altschul et al., supra). These initial neighborhood word hits act
as seeds for
initiating searches to find longer HSPs containing them. The word hits are
then extended in
both directions along each sequence for as far as the cumulative alignment
score can be
increased. Cumulative scores are calculated using, for nucleotide sequences,
the parameters
M (reward score for a pair of matching residues; always > 0) and N (penalty
score for
mismatching residues; always < 0). For amino acid sequences, a scoring matrix
is used to
calculate the cumulative score. Extension of the word hits in each direction
are halted when:
the cumulative alignment score falls off by the quantity X from its maximum
achieved
value; the cumulative score goes to zero or below, due to the accumulation of
one or more
negative-scoring residue alignments; or the end of either sequence is reached.
The BLAST
algorithm parameters W, T, and X detenmine the sensitivity and speed of the
alignment. The
BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of
11, an
expectation (E) of 10, a cutoff of I00, M=5, N=-4, and a comparison of both
strands. For
amino acid sequences, the BLASTP program uses as defaults a wordiength (W) of
3, an
expectation (E) of 10, and the BLOSLrM62 scoring matrix (see Henikoff &
Henikoff (1989)
Proc. Natl. Acad Sci. USA 89:10915).
In addition to calculating percent sequence identity, the BLAST algorithm
also performs a statistical analysis of the similarity between two sequences
(see, e.g., Karlin
& Altschul (1993) Proc. Nat'l. Acad. Sci. USA 90:5873-5787). One measure of
similarity
provided by the BLAST algorithm is the smallest sum probability (P(N}), which
provides an
indication of the probability by which a match between two nucleotide or amino
acid
sequences would occur by chance. For example, a nucleic acid is considered
similar to a
reference sequence if the smallest sum probability in a comparison of the test
nucleic acid to
the reference nucleic acid is less than about 0.1, more preferably less than
about 0.01, and
most preferably less than about 0.001.
Another indication that two nucleic acid sequences are substantially identical
is that the two molecules hybridize to each other under stringent conditions.
The phrase
"hybridizing specifically to", refers to the binding, duplexing, or
hybridizing of a molecule
only to a particular nucleotide sequence under stringent conditions when that
sequence is
present in a complex mixture (e.g., total cellular) DNA or RNA. "Bind(s)
substantially"
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PC1'/US99/02944
12
refers to complementary hybridization between a probe nucleic acid and a
target nucleic acid
and embraces minor mismatches that can be accommodated by reducing the
stringency of
the hybridization media to achieve the desired detection of the target
polynucleotide
sequence.
S "Stringent hybridization conditions" and "stringent hybridization wash
conditions" in the context of nucleic acid hybridization experiments such as
Southern and
northern hybridizations are sequence dependent, and are different under
different
environmental parameters. Longer sequences hybridize specifically at higher
temperatures.
An extensive guide to the hybridization of nucleic acids is found in Tijssen
(I993)
Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization
with Nucleic
Acid Probes part I chapter 2 "Overview of principles of hybridization and the
strategy of
nucleic acid probe assays", Elsevier, New York. Generally, highly stringent
hybridization
and wash conditions are selected to be about 5° C lower than the
thermal melting point (Tm)
for the specific sequence at a defined ionic strength and pH. Typically, under
"stringent
conditions" a probe will hybridize to its target subsequence, but to no other
sequences.
The Tm is the temperature (under defined ionic strength and pH) at which
50% of the target sequence hybridizes to a perfectly matched probe. Very
stringent
conditions are selected to be equal to the Tm for a particular probe. An
example of stringent
hybridization conditions for hybridization of complementary nucleic acids
which have more
than 100 complementary residues on a filter in a Southern or northern blot is
50%
formamide with 1 mg of heparin at 42°C, with the hybridization being
carried out overnight.
An example of highly stringent wash conditions is O.15M NaCI at 72°C
for about 15
minutes. An example of stringent wash conditions is a 0.2x SSC wash at
65°C for 15
minutes (see, Sambrook, infra., for a description of SSC buffer). Often, a
high stringency
wash is preceded by a low stringency wash to remove background probe signal.
An example
medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is lx
SSC at 45°C
for 15 minutes. An example low stringency wash for a duplex of, e.g., more
than 100
nucleotides, is 4-6x SSC at 40°C for 15 minutes. For short probes
(e.g., about 10 to 50
nucleotides), stringent conditions typically involve salt concentrations of
less than about 1.0
M Na+ ion, typically about 0.01 to 1.0 M Na+ ion concentration (or other
salts) at pH 7.0 to
8.3, and the temperature is typically at least about 30°C. Stringent
conditions can also be
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PGTNS99/02944
13
achieved with the addition of destabilizing agents such as fotmamide. In
general, a signal to
noise ratio of 2x (or higher) than that observed for an unrelated pmbe in the
particular
hybridization assay indicates detection of a specific hybridization. Nucleic
acids which do
not hybridize to each other under stringent conditions are still substantially
identical if the
polypeptides which they encode are substantially identical. This occurs, e.g.,
when a copy of
a nucleic acid is created using the maximum codon degeneracy permitted by the
genetic
code.
A further indication that two nucleic acid sequences or polypeptides are
substantially identical is that the polypeptide encoded by the first nucleic
acid is
immunologicaIly cross reactive with, or specifically binds to, the polypeptide
encoded by the
second nucleic acid. Thus, a polypeptide is typically substantially identical
to a second
polypeptide, for example, where the two peptides differ only by conservative
substitutions.
The phrase "specifically (or selectively) binds to an antibody" or
"specifically
(or selectively) immunoreactive with", when referring to a protein or peptide,
refers to a
binding reaction which is determinative of the presence of the protein, or an
epitope from the
protein, in the presence of a heterogeneous population of proteins and other
biologics. Thus,
under designated immunoassay conditions, the specified antibodies bind to a
particular
protein and do not bind in a significant amount to other proteins present in
the sample. The
antibodies raised against a multivalent antigenic polypeptide will generally
bind to the
proteins from which one or more of the epitopes were obtained. Specific
binding to an
antibody under such conditions may require an antibody that is selected for
its specificity for
a particular protein. A variety of immunoassay formats may be used to select
antibodies
specifically immunoreactive with a particular protein. For example, solid-
phase ELISA
immunoassays, Western blots, or immunohistochemistry are routinely used to
select
monoclonal antibodies specifically immunoreactive with a protein. See Harlow
and Lane
(1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New
York
"Harlow and Lane"), for a description of immunoassay formats and conditions
that can be
used to determine specific immunoreactivity. Typically a specific or selective
reaction will
be at least twice background signal or noise and more typically more than 10
to 100 times
background.
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/02944
14
"Conservatively modified variations" of a particular polynucleotide sequence
refers to those polynucleotides that encode identical or essentially identical
amino acid
sequences, or where the polynucleotide does not encode an amino acid sequence,
to
essentially identical sequences. Because of the degeneracy of the genetic
code, a large
number of functionally identical nucleic acids encode any given polypeptide.
For instance,
the codons CGU, CGC, CGA, CGG, AGA, and AGG all encode the amino acid
arginine.
Thus, at every position where an arginine is specified by a codon, the codon
can be altered to
any of the corresponding codons described without altering the encoded
polypeptide. Such
nucleic acid variations are "silent variations," which are one species of
"conservatively
modified variations." Every polynucleotide sequence described herein which
encodes a
polypeptide also describes every possible silent variation, except where
otherwise noted.
One of skill will recognize that each codon in a nucleic acid (except AUG,
which is
ordinarily the only codon for methionine) can be modified to yield a
functionally identical
molecule by standard techniques. Accordingly, each "silent variation" of a
nucleic acid
which encodes a polypeptide is implicit in each described sequence.
Furthermore, one of skill will recognize that individual substitutions,
deletions or additions which alter, add or delete a single amino acid or a
small percentage of
amino acids (typically less than 5%, more typically less than 1%) in an
encoded sequence are
"conservatively modified variations" where the alterations result in the
substitution of an
amino acid with a chemically similar amino acid. Conservative substitution
tables providing
functionally similar amino acids are well known in the art. The following five
groups each
contain amino acids that are conservative substitutions for one another:
Aliphatic: Glycine (G), Alanine (A), Valine (V), Leucine (L), Isoleucine (I);
Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan (W);
Sulfur-containing: Methionine (M), Cysteine (C);
Basic: Arginine (R), Lysine (K), Histidine (H);
Acidic: Aspartic acid (D), Glutamic acid (E), Asparagine (N), Glutamine (Q}.
See also, Creighton (1984) Proteins, W.H. Freeman and Company, for additional
groupings
of amino acids. In addition, individual substitutions, deletions or additions
which alter, add
or delete a single amino acid or a small percentage of amino acids in an
encoded sequence
are also "conservatively modified variations".
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/02944
A "subsequence" refers to a sequence of nucleic acids or amino acids that
comprise a part of a longer sequence of nucleic acids or amino acids (e.g.,
polypeptide)
respectively.
Description of the Preferred Embodiments
The invention provides a new approach to vaccine development, which is
termed "antigen library immunization." No other technologies are available for
generating
libraries of related antigens or optimizing known protective antigens. The
most powerful
previously existing methods for identification of vaccine antigens, such as
high throughput
sequencing or expression library immunization, only explore the sequence space
provided by
10 the pathogen genome. These approaches are likely to be insufficient,
because they generally
only target single pathogen strains, and because natural evolution has
directed pathogens to
downregulate their own immunogenicity. In contrast, the immunization protocols
of the
invention, which use shuffled antigen libraries, provide a means to identify
novel antigen
sequences. Those antigens that are most protective can be selected from these
pools by in
15 vivo challenge models. Antigen library immunization dramatically expands
the diversity of
available immunogen sequences, and therefore, these antigen chimera libraries
can also
provide means to defend against newly emerging pathogen variants of the
future. The
methods of the invention enable the identification of individual chimeric
antigens that
provide efficient protection against a variety of existing pathogens,
providing improved
vaccines for troops and civilian populations.
The methods of the invention provide an evolution-based approach, such as
DNA shuffling in particular, that is an optimal approach to improve the
immunogenicity of
many types of antigens. For example, the methods provide means of obtaining
optimized
cancer antigens useful for preventing and treating malignant diseases.
Furthermore, an
increasing number of self antigens, causing autoimmune diseases, and
allergens, causing
atopy, allergy and asthma, have been characterized. The immunogenicity and
manufacturing
of these antigens can likewise be improved with the methods of this invention.
The antigen library immunization methods of the invention provide a means
by which one can obtain a recombinant antigen that has improved ability to
induce an
immune response to a pathogenic agent. A "pathogenic agent" refers to an
organism or virus
that is capable of infecting a host cell. Pathogenic agents typically include
and/or encode a
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/02944
16
molecule, usually a polypeptide, that is immunogenic in that an immune
response is raised
against the immunogenic polypeptide. Often, the immune response raised against
an
immunogenic polypeptide from one serotype of the pathogenic agent is not
capable of
recognizing, and thus protecting against, a different serotype of the
pathogenic agent, or
other related pathogenic agents. In other situations, the polypeptide produced
by a
pathogenic agent is not produced in sufficient amounts, or is not sufficiently
immunogenic,
for the infected host to raise an effective immune response against the
pathogenic agent.
These problems are overcome by the methods of the invention, which
typically involve recombining two or more forms of a nucleic acid that encode
a polypeptide
of the pathogenic agent, or antigen involved in another disease or condition.
These
recombination methods, referred to herein as "DNA shuffling", use as
substrates fonms of
the nucleic acid that differ from each other in two or more nucleotides, so a
library of
recombinant nucleic acids results. The library is then screened to identify at
least one
optimized recombinant nucleic acid that encodes an optimized recombinant
antigen that has
improved ability to induce an immune response to the pathogenic agent or other
condition.
The resulting recombinant antigens often are chimeric in that they are
recognized by
antibodies (Abs) reacting against multiple pathogen strains, and generally can
also elicit
broad spectrum immune responses. Specific neutralizing antibodies are known to
mediate
protection against several pathogens of interest, although additional
mechanisms, such as
cytotoxic T lymphocytes, are likely to be involved. The concept of chimeric,
multivalent
antigens inducing broadly reacting antibody responses is further illustrated
in Figure 1.
In preferred embodiments, the different forms of the nucleic acids that encode
antigenic polypeptides are obtained from members of a family of related
pathogenic agents.
This scheme of performing DNA shuffling using nucleic acids from related
organisms,
known as "family shuffling," is described in Crameri et al. ((1998) Nature
391: 288-291)
and is shown schematically in Figure 2. Polypeptides of different strains and
serotypes of
pathogens generally vary between 60-98%, which will allow for efficient family
DNA
shuffling. Therefore, family DNA shuffling provides an effective approach to
generate
multivalent, crossprotective antigens. The methods are useful for obtaining
individual
chimeras that effectively protect against most or all pathogen variants
(Figure 3A).
Moreover, immunizations using entire libraries or pools of shuffled antigen
chimeras can
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/02944
17
also result in identification of chimeric antigens that protect against
pathogen variants that
were not included in the starting population of antigens (for example,
protection against
strain C by shuffled library of chimeras/mutants of strains A and B in Figure
3B).
Accordingly, the antigen library immunization approach enables the development
of
S immunogenic polypeptides that can induce immune responses against poorly
characterized,
newly emerging pathogen variants.
Sequence recombination can be achieved in many different formats and
permutations of formats, as described in further detail below. These formats
share some
common principles. For example, the targets for modification vary in different
applications,
as does the property sought to be acquired or improved. Examples of candidate
targets for
acquisition of a property or improvement in a property include genes that
encode proteins
which have immunogenic and/or toxigenic activity when introduced into a host
organism.
The methods use at least two variant forms of a starting target. The variant
forms of candidate substrates can show substantial sequence or secondary
structural
similarity with each other, but they should also differ in at least one and
preferably at least
two positions. The initial diversity between forms can be the result of
natural variation, e.g.,
the different variant forms (homologs) are obtained from different individuals
or strains of
an organism, or constitute related sequences from the same organism (e.g.,
allelic
variations), or constitute homologs from different organisms (interspecific
variants).
Alternatively, initial diversity can be induced, e.g., the variant forms can
be generated by
error-prone transcription, such as an error-prone PCR or use of a polymerase
which lacks
proof reading activity (see, Liao ( 1990) Gene 88:107-111 ), of the f rst
variant form, or, by
replication of the first form in a mutator strain (mutator host cells are
discussed in further
detail below, and are generally well known). A mutator strain can include any
mutants in any
organism impaired in the functions of mismatch repair. These include mutant
gene products
of mutS, mutT, mutes, mutt, ovrD, dcm, vsr, umuC, umuD, sbcB, recJ, etc. The
impairment
is achieved by genetic mutation, allelic replacement, selective inhibition by
an added reagent
such as a small compound or an expressed antisense RNA, or other techniques.
Impairment
can be of the genes noted, or of homologous genes in any organism. Other
methods of
generating initial diversity include methods well known to those of skill in
the art, including,
for example, treatment of a nucleic acid with a chemical or other mutagen,
through
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PGT/US99/02944
18
spontaneous mutation, and by inducing an error-prone repair system (e.g., SOS)
in a cell that
contains the nucleic acid. The initial diversity between substrates is greatly
augmented in
subsequent steps of recombination for library generation.
Properties involved in immunogenicity
The effectiveness of an antigen in inducing an immune response against a
pathogen can depend upon several factors, many of which are not well
understood. Most
previously available methods for increasing the effectiveness of antigens are
dependent upon
understanding the molecular basis for these factors. However, DNA shuffling
and antigen
library immunization according to the methods of the invention are effective
even where the
molecular bases are unknown. The methods of the invention do not rely upon a
priori
assumptions.
Polynucleotide sequences that can positively or negatively affect the
immunogenicity of an antigen encoded by the polynucleotide are often scattered
throughout
the entire antigen gene. Several of these factors are shown diagrammatically
in Figure 4. By
recombining different forms of polynucleotide that encode the antigen using
DNA shuffling,
followed by selection for those chimeric poIynucleotides that encode an
antigen that can
induce an improved immune response, one can obtain primarily sequences that
have a
positive influence on antigen immunogenicity. Those sequences that negatively
affect
antigen immunogenicity are eliminated (Figure 4). One need not know the
particular
sequences involved.
DNA Shuffling Methods
Generally, the methods of the invention entail performing DNA
recombination ("shuffling") and screening or selection to "evolve" individual
genes, whole
plasmids or viruses, multigene clusters, or even whole genomes (Stemmer (I995)
BiolTechnology 13:549-553). Reiterative cycles of recombination and
screening/selection
can be performed to further evolve the nucleic acids of interest. Such
techniques do not
require the extensive analysis and computation required by conventional
methods for
polypeptide engineering. Shuffling allows the recombination of large numbers
of mutations
in a minimum number of selection cycles, in contrast to natural pair-wise
recombination
events (e.g., as occur during sexual replication). Thus, the sequence
recombination
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PC'T/US99/02944
19
techniques described herein provide particular advantages in that they provide
recombination
between mutations in any or all of these, thereby providing a very fast way of
exploring the
manner in which different combinations of mutations can affect a desired
result. In some
instances, however, structural and/or functional information is available
which, although not
required for sequence recombination, provides opportunities for modification
of the
technique.
The DNA shuffling methods of the invention can involve at least one of at
least four different approaches to improve immunogenic activity as well as to
broaden
specificity. First, DNA shuffling can be performed on a single gene. Secondly,
several
highly homologous genes can be identified by sequence comparison with known
homologous genes. These genes can be synthesized and shuffled as a family of
homologs, to
select recombinants with the desired activity. The shuffled genes can be
cloned into
appropriate host cells, such as E. toll, yeast, plants, fungi, animal cells,
and the like, and
those that encode antigens having the desired properties can be identified by
the methods
described below. Third, whole genome shuffling can be performed to shuffle
genes that
encode antigenic polypeptides (along with other genomic nucleic acids). For
whole genome
shuffling approaches, it is not even necessary to identify which genes are
being shuffled.
Instead, e.g., bacterial cell or viral genomes are combined and shuffled to
acquire
recombinant polypeptides that have enhanced ability to induce an immune
response, as
measured in any of the assays described below. Fourth, antigenic polypeptide-
encoding
genes can be codon modified to access mutational diversity not present in any
naturally
occurring gene. Details on each of these procedures can be found below.
Exemplary formats and examples for sequence recombination, sometimes
refereed to as DNA shuffling, evolution, or molecular breeding, have been
described by the
present inventors and co-workers in co-pending applications U.S. Patent
Application Serial
No. 08/198,431, filed February 17, 1994, Serial No. PCT/LTS95/02126, filed,
February 17,
1995, Serial No. 08/425,684, filed April 18, 1995, Serial No. 081537,874,
filed October 30,
1995, Serial No. 08/564,955, filed November 30, 1995, Serial No. 08/621,859,
filed March
25, 1996, Serial No. 08/621,430, filed March 25, 1996, Serial No.
PCT/LJS96/05480, filed
April 18, 1996, Serial No. 08/650,400, filed May 20, 1996, Serial No.
08/675,502, filed 3uly
3, 1996, Serial No. 08/721, 824, filed September 27, 1996, Serial No.
PCT/US97/17300,
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PC'T/US99/OZ944
filed September 26, 1997, and Serial No. PCT/US97/24239, filed December 17,
1997;
Stemmer, Science 270:1510 (1995); Stemmer et al., Gene 164:49-53 (1995);
Stemmer,
BiolTechnology 13:549-553 (1995); Stemmer, Proc. Natl. Acad. Sci. U.S.A.
91:10747-10751
( 1994); Stemmer, Nature 370:389-391 ( 1994); Crameri et al., Nature Medicine
2( 1 ): l -3
(1996); Crameri et al., Nature Biotechnology 14:315-319 (1996), each of which
is
incorporated by reference in its entirety for all purposes.
Other methods for obtaining recombinant polynucleotides and/or for
obtaining diversity in nucleic acids used as the substrates for shuffling
include, for example,
homologous recombination (PCT/US98/05223; Publ. No. W098/42727);
oligonucleotide-
10 directed mutagenesis (for review see, Smith, Ann. Rev. Genet. 19: 423-462
(1985); Botstein
and Shortle, Science 229: 1193-1201 (1985); Carter, Biochem. J. 237: 1-7
(1986); Kunkel,
"The efficiency of oligonucleotide directed mutagenesis" in Nucleic acids &
Molecular
Biology, Eckstein and Lilley, eds., Springer Verlag, Berlin (1987)). Included
among these
methods are oligonucleotide-directed mutagenesis (Zoller and Smith, Nucl.
Acids Res. 10:
15 6487-6500 (1982), Methods in Enrymol. 100: 468-500 (1983), and Methods in
Enrymol.
154: 329-350 (1987)) phosphothioate-modified DNA mutagenesis (Taylor et al.,
Nucl. Acids
Res. 13: 8749-8764 (1985); Taylor et al., Nucl. Acids Res. 13: 8765-8787
(1985); Nakamaye
and Eckstein, Nucl. Acids Res. 14: 9679-9698 (1986); Sayers et al., Nucl.
Acids Res. 16:
791-802 (1988); Sayers et al., Nucl. Acids Res. 16: 803-814 (1988)),
mutagenesis using
20 uracil-containing templates (Kunkel, Proc. Nat'l. Acad. Sci. USA 82: 488-
492 (1985) and
Kunkel et al., Methods in Enrymol. 154: 367-382)); mutagenesis using gapped
duplex DNA
(Kramer et al., Nucl. Acids Res. 12: 9441-9456 (1984); Kramer and Fritz,
Methods in
Enrymol. 154: 350-367 (1987}; Kramer et al., Nucl. Acids Res. 16: 7207
(1988)); and Fritz et
al., Nucl. Acids Res. 16: 6987-6999 (1988)). Additional suitable methods
include point
mismatch repair (Kramer et al., Cell 38: 879-887 (1984)), mutagenesis using
repair-deficient
host strains (Carter et al., Nucl. Acids Res. 13: 4431-4443 (1985); Carter,
Methods in
Enrymol. 154: 382-403 (1987)}, deletion mutagenesis (Eghtedarzadeh and
Henikoff, Nucl.
Acids Res. 14: 5115 (1986)), restriction-selection and restriction-
purification (Wells et al.,
Phil. Traps. R. Soc. Lond. A 317: 415-423 (1986)), mutagenesis by total gene
synthesis
(Nambiar et al., Science 223: 1299-1301 (1984); Sakamar and Khorana, Nucl.
Acids Res. 14:
6361-6372 (1988); Wells et al., Gene 34: 315-323 (1985); and Grundstrtim et
al., Nucl.
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99141383 PCT/US99/02944
21
Acids Res. 13: 3305-3316 (1985). Kits for mutagenesis are commercially
available (e.g.,
Bio-Rad, Amersham International, Anglian Biotechnology).
The breeding procedure starts with at least two substrates that generally show
some degree of sequence identity to each other (i.e., at least about 30%, SO%,
70%, 80% or
90% sequence identity), but differ from each other at certain positions. The
difference can be
any type of mutation, for example, substitutions, insertions and deletions.
Often, different
segments differ from each other in about 5-20 positions. For recombination to
generate
increased diversity relative to the starting materials, the starting materials
must differ from
each other in at Least two nucleotide positions. That is, if there are only
two substrates, there
should be at least two divergent positions. If there are three substrates, for
example, one
substrate can differ from the second at a single position, and the second can
differ from the
third at a different single position. The starting DNA segments can be natural
variants of
each other, for example, allelic or species variants. The segments can also be
from
nonallelic genes showing some degree of structural and usually functional
relatedness (e.g.,
different genes within a superfamily, such as the family of Yersinia V-
antigens, for
example). The starting DNA segments can also be induced variants of each
other. For
example, one DNA segment can be produced by error-prone PCR replication of the
other,
the nucleic acid can be treated with a chemical or other mutagen, or by
substitution of a
mutagenic cassette. Induced mutants can also be prepared by propagating one
(or both) of
the segments in a mutagenic strain, or by inducing an error-prone repair
system in the cells.
In these situations, strictly speaking, the second DNA segment is not a single
segment but a
large. family of related segments. The different segments forming the starting
materials are
often the same length or substantially the same length. However, this need not
be the case;
for example; one segment can be a subsequence of another. The segments can be
present as
part of larger molecules, such as vectors, or can be in isolated form.
The starting DNA segments are recombined by any of the sequence
recombination formats provided herein to generate a diverse library of
recombinant DNA
segments. Such a library can vary widely in size from having fewer than 10 to
more than
105, 109, 10'2 or more members. In some embodiments, the starting segments and
the
recombinant libraries generated will include full-length coding sequences and
any essential
regulatory sequences, such as a promoter and polyadenylation sequence,
required for
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99I02944
22
expression. In other embodiments, the recombinant DNA segments in the library
can be
inserted into a common vector providing sequences necessary for expression
before
performing screening/selection.
Substrates for Evolution of Optimized Recombinant Antigens
The invention provides methods of obtaining recombinant polynucleotides
that encode antigens that exhibit improved ability to induce an immune
response to a
pathogenic agent. The methods are applicable to a wide range of pathogenic
agents,
including potential biological warfare agents and other organisms and
polypeptides that can
cause disease and toxicity in humans and other animals. The following examples
are merely
illustrative, and not limiting.
1. Bacterial Pathogens and Toxins
In some embodiments, the methods of the invention are applied to bacterial
pathogens, as well as to toxins produced by bacteria and other organisms. One
can use the
methods to obtain recombinant polypeptides that can induce an immune response
against the
pathogen, as well as recombinant toxins that are less toxic than native toxin
polypeptides.
Often, the polynucleotides of interest encode polypeptides that are present on
the surface of
the pathogenic organism.
Among the pathogens for which the methods of the invention are useful for
producing protective immunogenic recombinant polypeptides are the Yersinia
species.
Yersinia pestis, the causative agent of plague, is one of the most virulent
bacteria known
with LDSO values in mouse of less than I O bacteria. The pneumonic form of the
disease is
readily spread between humans by aerosol or infectious droplets and can be
lethal within
days. A particularly preferred target for obtaining a recombinant polypeptide
that can protect
against Yersinia infection is the V antigen, which is a 37 kDa virulence
factor, induces
protective immune responses and is currently being evaluated as a subunit
vaccine (Brubaker
(1991) Current Investigations of the Microbiology of Yersinae, 12: 127). The V-
antigen
alone is not toxic, but Y. pestis isolates that lack the V-antigen are
avirulent. The Yersinia V-
antigen has been successfully produced in E. coli by several groups (Leary et
al. (1995)
Infect. Immun. 3: 2854). Antibodies that recognize the V-antigen can provide
passive
protection against homologous strains, but not against heterologous strains.
Similarly,
immunization with purified V antigen protects against only homologous strains.
To obtain
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/02944
23
cross-protective recombinant V antigen, in a preferred embodiment, V antigen
genes from
various Yersinia species are subjected to family shuffling. The genes encoding
the V antigen
from Y. pestis, Y. enterocolitica, and Y. pseudotuberculosis, for example, are
92-99%
identical at the DNA level, making them ideal for optimization using family
shuffling
according to the methods of the invention. After shuffling, the library of
recombinant nucleic
acids is screened and/or selected for those that encode recombinant V antigen
polypeptides
that can induce an improved immune response and/or have greater cross-
protectivity.
Bacillus anrhracis, the causative agent of anthrax, is another example of a
bacterial target against which the methods of the invention are useful. The
anthrax protective
antigen (PA) provides protective immune responses in test animals, and
antibodies against
PA also provide some protection. However, the immunogenicity of PA is
relatively poor, so
multiple injections are typically required when wild-type PA is used. Co-
vaccination with
lethal factor (LF) can improve the efficacy of wild-type PA vaccines, but
toxicity is a
limiting factor. Accordingly the DNA shuffling and antigen library
immunization methods
of the invention can be used to obtain nontoxic LF. Polynucieotides that
encode LF from
various B. anthracis strains are subjected to family shuffling. The resulting
library of
recombinant LF nucleic acids can then be screened to identify those that
encode recombinant
LF polypeptides that exhibit reduced toxicity. For example, one can inoculate
tissue culture
cells with the recombinant LF polypeptides in the presence of PA and select
those clones for
which the cells survive. If desired, one can then backcross the nontoxic LF
polypeptides to
retain the immunogenic epitopes of LF. Those that are selected through the
first screen can
then be subjected to a secondary screen. For example, one can test for the
ability of the
recombinant nontoxic LF polypeptides to induce an immune response (e.g., CTL
or antibody
response) in a test animal such as mice. In preferred embodiments, the
recombinant nontoxic
LF polypeptides are then tested for ability to induce protective immunity in
test animals
against challenge by different strains of B. anthracis.
The protective antigen (PA) of B. anthracis is also a suitable target for the
methods of the invention. PA-encoding nucleic acids from various strains of B.
anthracis are
subjected to DNA shuffling. One can then screen for proper folding in, for
example, E. coli,
using polyclonal antibodies. Screening for ability to induce broad-spectrum
antibodies in a
test animal is also typically used, either alone or in addition to a
preliminary screening
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99141383 PCT/US99/02944
24
method. In presently preferred embodiments, those recombinant polynucleotides
that exhibit
the desired properties can be backcrossed so that the immunogenic epitopes are
maintained.
Finally, the selected recombinants are tested for ability to induce protective
immunity
against different strains of B. anthracis in a test animal.
S The Staphylococcus aureus and Streptococcus toxins are another example of
a target polypeptide that can be altered using the methods of the invention.
Strains of
Staphylococcus aureus and group A Streptococci are involved in a range of
diseases,
including food poisoning, toxic shock syndrome, scarlet fever and various
autoimmune
disorders. They secrete a variety of toxins, which include at least five
cytolytic toxins, a
coagulase, and a variety of enterotoxins. The enterotoxins are classified as
superantigens in
that they crosslink MHC class II molecules with T cell receptors to cause a
constitutive T
cell activation (Fields et al. (1996) Nature 384: 188). This results in the
accumulation of
pathogenic levels of cytokines that can lead to multiple organ failure and
death. At least
thirty related, yet distinct enterotoxins have been sequenced and can be
phylogenetically
grouped into families. Crystal structures have been obtained for several
members alone and
in complex with MHC class II molecules. Certain mutations in the MHC class II-
binding site
of the toxins strongly reduce their toxicity and can form the basis of
attenuated vaccines
(Woody et al. (1997) Vaccine 15: 133). However, a successful immune response
to one type
of toxin may provide protection against closely related family members,
whereas little
protection against toxins from the other families is observed. Family
shuffling of enterotoxin
genes from various family members can be used to obtain recombinant toxin
molecules that
have reduced toxicity and can induce a cross-protective immune response.
Shuffled antigens
can also be screened to identify antigens that elicit neutralizing antibodies
in an appropriate
animal model such as mouse or monkey. Examples of such assays can include
ELISA
formats in which the elicited antibodies prevent binding of the enterotoxin to
the MHC
complex and/or T cell receptors on cells or purified forms. These assays can
also include
formats where the added antibodies would prevent T cells from being cross-
linked to
appropriate antigen presenting cells.
Cholera is an ancient, potentially lethal disease caused by the bacterium
Vibrio cholerae and an effective vaccine for its prevention is still
unavailable. Much of the
pathogenesis of this disease is caused by the cholera enterotoxin. Ingestion
of microgram
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/01944
quantities of cholera toxin can induce severe diarrhea causing loss of tens of
liters of fluid.
Cholera toxin is a complex of a single catalytic A subunit with a pentameric
ring of identical
B subunits. Each subunit is inactive on its own. The B subunits bind to
specific ganglioside
receptors on the surface of intestinal epithelial cells and trigger the entry
of the A subunit
5 into the cell. The A subunit ADP-ribosylates a regulatory G protein
initiating a cascade of
events causing a massive, sustained flow of electrolytes and water into the
intestinal lumen
resulting in extreme diarrhea.
The B subunit of cholera toxin is an attractive vaccine target for a number of
reasons. It is a major target of protective antibodies generated during
cholera infection and
10 contains the epitopes for antitoxin neutralizing antibodies. It is nontoxic
without the A
subunit, is orally effective, and stimulates production of a strong IgA-
dominated gut mucosal
immune response, which is essential in protection against cholera and cholera
toxin. The B
subunit is also being investigated for use as an adjuvant in other vaccine
preparations, and
therefore, evolved toxins may provide general improvements for a variety of
different
15 vaccines. The heat-labile enterotoxins (LT) from enterotoxigenic E. coli
strains are
structurally related to cholera toxin and are 75% identical at the DNA
sequence level. To
obtain optimized recombinant toxin molecules that exhibit reduced toxicity and
increased
ability to induce an immune response that is protective against V. cholerae
and E. toll, the
genes that encode the related toxins are subjected to DNA shuffling.
20 The recombinant toxins are then tested for one or more of a several
desirable
traits. For example, one can screen for improved cross-reactivity of
antibodies raised against
the recombinant toxin polypeptides, for lack of toxicity in a cell culture
assay, and for ability
to induce a protective immune response against the pathogens and/or against
the toxins
themselves. The shuffled clones can be selected by phage display and/or
screened by phage
25 ELISA and ELISA assays for the presence of epitopes from the different
serotypes. Variant
proteins with multiple epitopes can then be purified and used to immunize mice
or other test
animal. The animal serum is then assayed for antibodies to the different B
chain subtypes
and variants that elicit a broad cross-reactive response will be evaluated
further in a virulent
challenge model. The E. toll and V. cholerae toxins can also act as adjuvants
that are
capable of enhancing mucosal immunity and oral delivery of vaccines and
proteins.
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCTNS99/02944
26
Accordingly, one can test the library of recombinant toxins for enhancement of
the adjuvant
activity.
Shuffled antigens can also be screened for improved expression levels and
stability of the B chain pentamer, which may be less stable than when in the
presence of the
A chain in the hexameric complex. Addition of a heat treatment step or
denaturing agents
such as salts, urea, and/or guanidine hydrochloride can be included prior to
ELISA assays to
measure yields of correctly folded molecules by appropriate antibodies. It is
sometimes
desirable to screen for stable monomeric B chain molecules, in an ELISA
format, for
example, using antibodies that bind monomeric, but not pentameric B chains.
Additionally,
the ability of shuffled antigens to elicit neutralizing antibodies in an
appropriate animal
model such as mouse or monkey can be screened. For example, antibodies that
bind to the B
chain and prevent its binding to its specific ganglioside receptors on the
surface of intestinal
epithelial cells may prevent disease. Similarly antibodies that bind to the B
chain and prevent
its pentamerization or block A chain binding may be useful in preventing
disease.
The bacterial antigens that can be improved by DNA shuffling for use as
vaccines also include, but are not limited to, Helicobacter pylori antigens
CagA and VacA
(Blaser (1996) Aliment. Pharmacol. Ther. 1: 73-7; Blaser and Crabtree (1996)
Am. J. Clin.
Pathol. 106: 565-7; Censini et al. (1996} Proc. Nat'l. Acad. Sci. USA 93:
14648-14643).
Other suitable H. pylori antigens include, for example, four immunoreactive
proteins of 45-
65 kDa as reported by Chatha et al. (1997) Indian J. Med. Res. 105: 170-175
and the H.
pylori GroES homologue (HspA) (Kansau et al. (1996) Mol. Microbiol. 22: 1013-
1023.
Other suitable bacterial antigens include, but are not limited to, the 43-kDa
and the fimbrilin
(41 kDa) proteins of P. gingivalis (Boutsl et al. ( 1996) Oral Microbiol.
Immunol. 11: 236-
241}; pneumococcal surface protein A (Briles et al. (1996) Ann. NYAcad. Sci.
797: 118-
126); Chlamydia psittaci antigens, 80-90 kDa protein and 110 kDa protein
(Buendia et al.
{1997) FEMSMicrobiol. Lett. 150: 1 I3-9); the chiamydial exoglycolipid antigen
(GLXA)
(Whittum-Hudson et al. (1996) Nature Med. 2: 11 I6-1121); Chlamydia pneumoniae
species-
specific antigens in the molecular weight ranges 92-98, 51-55, 43-46 and 31.5-
33 kDa and
genus-specific antigens in the ranges 12, 26 and 65-70 kDa (Halme et al.
(1997) Scand J.
Immunol. 45: 378-84}; Neisseria gonorrhoeae (GC} or Escherichia coli phase-
variable
opacity (Opa) proteins (Chen and Gotschlich ( 1996) Proc. Nat'l. Acad. Sci.
USA 93: 14851-
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCTNS99/02944
77
14856), any of the twelve immunodominant proteins of Schistosoma mansoni
(ranging in
molecular weight from 14 to 208 kDa) as described by Cutts and Wilson ( 1997)
Parasitologv 114: 245-55; the 17-kDa protein antigen of Brucella abortus (De
Mot et al.
( 1996) Czzrr. Microbiol. 33: 26-30); a gene homolog of the 17-kDa protein
antigen of the
Gram-negative pathogen Brucella abortus identified in the nocardioform
actinomycete
Rhodococcus sp. NI86/21 (De Mot et al. ( 1996) Curr. Microbiol. 33: 26-30);
the
staphylococcal enterotoxins (SEs) (Wood et al. ( 1997) FEMS Immunol. Med.
Microbiol. 17:
1-10), a 42-kDa M. hyopneunzoniae NrdF ribonucleotide reductase R2 protein or
15-kDa
subunit protein of M. hyopneumoniae (Fagan et al. ( 1997) Infect. Immun. 65:
2502-2507),
the meningococcal antigen PorA protein (Feavers et al. (1997) Clin. Diagn.
Lab. Immunol.
3: 444-SO); pneumococcal surface protein A (PspA) (McDaniel et al. (1997) Gene
Ther. 4:
375-377); F. tularensis outer membrane protein FopA (Fulop et al. (1996)
FEMSImmunol.
Med. Microbiol. 13: 245-247); the major outer membrane protein within strains
of the genus
Actinobacillus (Hartmann et al. ( 1996) Zentralbl. Bakteriol. 284: 255-262);
p60 or
listeriolysin (Hly) antigen ofListeria monocytogenes (Hess et al. (1996) Proc.
Nat'l. Acad.
Sci. USA 93: 1458-1463); flagellar (G) antigens observed on Salmonella
enteritidis and S.
pullorum (Holt and Chaubal (1997) J. Clin. Microbial. 35: 1016-1020); Bacillus
anthracis
protective antigen (PA) (Ivins et al. (1995) Vaccine 13: 1779-1784);
Echinococcus
granulosus antigen 5 (Jones et al. (1996) Parasitology 113: 213-222); the rol
genes of
Shigella dvsenteriae 1 and Escherichia coli K-12 (Klee et al. (1997) J.
Bacteriol. 179: 2421-
2425); cell surface proteins Rib and alpha of group B streptococcus (Larsson
et al. (1996)
Infect. Immun. 64: 3518-3523); the 37 kDa secreted polypeptide encoded on the
70 kb
virulence plasmid of pathogenic Yersinia spp. (Leary et al. (1995) Contrib.
Microbiol.
Immunol. 13: 216-217 and Roggenkamp et al. ( 1997) Infect. Immun. 65: 446-51
); the OspA
(outer surface protein A) of the Lyme disease spirochete Borrelia burgdorferi
(Li et al.
(1997) Proc. Nat'1. Acad. Sci. USA 94: 3584-3589, Padilla et al. (1996) J.
Infect. Dis. 174:
739-746, and Wallich et al. (1996) Infection 24: 396-397); the Brucella
melitensis group 3
antigen gene encoding Omp28 (Lindler et al. (1996) Infect. Immun. 64: 2490-
2499); the PAc
antigen of Streptococcus mutans (Murakami et al. ( 1997} Infect. Immun. 65:
794-797);
pneumolysin, Pneumococcal neuraminidases, autolysin, hyaluronidase, and the 37
kDa
pneumococcal surface adhesin A (Paton et al. (1997) Microb. Drug Resist. 3: 1-
10); 29-32,
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PC'f/US99/OZ944
28
41-45, 63-71 x 10(3) MW antigens of Salmonella typhi (Perez et al. (1996)
Immunology 89:
262-267); K-antigen as a marker of Klebsiella pneumoniae (Priamukhina and
Morozova
(1996) Klin. Lab. Diagn. 47-9); nocardial antigens of molecular mass
approximately 60, 40,
20 and 15-10 kDa (Prokesova et al. (1996) Int. J. Immunopharmacol. 18: 661-
668);
S Staphylococcus aureus antigen ORF-2 (Rieneck et al. (1997) Biochim Biophys
Acta 1350:
128-132); GIpQ antigen of Borrelia hermsii (Schwan et al. ( 1996) J. Clin.
Microbiol. 34:
2483-2492); cholera protective antigen (CPA) (Sciortino ( 1996) J. Diarrhoea)
Dis. Res. 14:
16-26}; a 190-kDa protein antigen of Streptococcus mutans (Senpuku et al.
(1996) Oral
Microbiol. Immunol. 11: 121-128); Anthrax toxin protective antigen (PA)
(Shanna et al.
(1996) Protein Expr. Purif. 7: 33-38); Clostridium perfringens antigens and
toxoid (Strom et
al. (1995) Br. J. Rheumatol. 34: 1095-1096); the SEF14 fimbrial antigen of
Salmonella
enteritidis (Thorns et al. ( 1996) Microb. Pathog. 20: 235-246); the Yersinia
pestis capsular
antigen (F1 antigen) (Titball et al. (1997) Infect. Immun. 65: 1926-1930); a
35-kilodalton
protein of Mycobacterium leprae (Triccas et al. (1996) Infect. Immun. 64: S
I71-5177); the
major outer membrane protein, CD, extracted from Moraxella (Branhamella)
catarrhalis
(Yang et al. (1997) FEMS Immunol. Med Microbiol. 17: 187-199); pH6 antigen
(PsaA
protein) of Yersinia pestis (Zav'yalov et al. (1996) FEMS Immunol. Med.
Microbiol. 14: 53-
57); a major surface glycoprotein, gp63, of Leishmania major (Xu and Liew
(1994) Vaccine
12: 1534-1536; Xu and Liew (1995) Immunology 84: 173-176); mycobacterial heat
shock
protein 65, mycobacterial antigen (Mycobacterium leprae hsp65) (Lowrie et al.
(1994)
Vaccine 12: 1537-1540; Ragno et al. (1997) Arthritis Rheum. 40: 277-283; Silva
(1995}
Braz. J. Med. Biol. Res. 28: 843-851 ); Mycobacterium tuberculosis antigen 85
(Ag85)
(Huygen et al. (1996) Nat. Med. 2: 893-898}; the 45/47 kDa antigen complex
(APA) of
Mycobacterium tuberculosis, M. bovis and BCG (Horn et al. (1996) J. Immunol.
Methods
197: 151-159); the mycobacterial antigen, 65-kDa heat shock protein, hsp65
(Tascon et al.
(1996) Nat. Med. 2: 888-892); the mycobacterial antigens MPB64, MPB70, MPB57
and
alpha antigen (Yamada et al. (1995) Kekkaku 70: 639-644); the M. tuberculosis
38 kDa
protein (Vordermeier et al. (1995) Vaccine 13: 1576-1582); the MPT63, MPT64
and MPT-
59 antigens from Mycobacterium tuberculosis (Manca et al. ( 1997) Infect.
Immun. 65: 16-
23; Oettinger et al. (1997) Scand J. Immunol. 45: 499-503; Wilcke et al.
(1996) Tuber. Lung
Dis. 77: 250-256); the 35-kilodalton protein of Mycobacterium leprae (Triccas
et al. (1996)
SUBSTITUTE SHEET (RULE 2fi)


CA 02320958 2000-08-10
WO 99/41383 PCTNS99/02944
?9
Infect. In:mun. 64: 5171-5177); the ESAT-6 antigen of virulent mycobacteria
(Brandt et al.
(1996) J. Immunol. 157: 3527-3533; Pollock and Andersen (1997) J. Infect. Dis.
175: 1251
1254); Mycobacterium tuberculosis 16-kDa antigen (Hsp16.3) (Chang et al.
(1996) J. Biol.
Chem. 271: 7218-7223); and the 18-kilodalton protein of Mycobacterium leprae
(Baumgart
et al. ( 1996) Infect. Immun. 64: 2274-2281 ).
2. Viral Pathogens
The methods of the invention are also useful for obtaining recombinant
nucleic acids and polypeptides that have enhanced ability to induce an immune
response
against viral pathogens. While the bacterial recombinants described above are
typically
administered in polypeptide form, recombinants that confer viral protection
are preferably
administered in nucleic acid form, as genetic vaccines.
One illustrative example is the Hantaan virus. Glycoproteins of this virus
typically accumulate at the membranes of the Golgi apparatus of infected
cells. This poor
expression of the glycoprotein prevents the development of efficient genetic
vaccines against
these viruses. The methods of the invention solve this problem by performing
DNA
shuffling on nucleic acids that encode the glycoproteins and identifying those
recombinants
that exhibit enhanced expression in a host cell, and/or for improved
immunogenicity when
administered as a genetic vaccine. A convenient screening method for these
methods is to
express the recombinant polynucleotides as fusion proteins to PIG, which
results in display
of the polypeptides on the surface of the host cell (Whitehorn et al. (1995)
Biotechnology (N
Y) 13:1215-9). Fluorescence-activated cell sorting is then used to sort and
recover those
cells that express an increased amount of the antigenic polypeptide on the
cell surface. This
preliminary screen can be followed by immunogenicity tests in mammals, such as
mice.
Finally, in preferred embodiments, those recombinant nucleic acids are tested
as genetic
vaccines for their ability to protect a test animal against challenge by the
virus.
The flaviviruses are another example of a viral pathogen for which the
methods of the invention are useful for obtaining a recombinant polypeptide or
genetic
vaccine that is effective against a viral pathogen. The flaviviruses consist
of three clusters of
antigenically related viruses: Dengue 1-4 (62-77% identity), Japanese, St.
Louis and Murray
Valley encephalitis viruses (75-82% identity), and the tick-borne encephalitis
viruses (77-
96% identity). Dengue virus can induce protective antibodies against SLE and
Yellow fever
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/02944
(40-50% identity), but few efficient vaccines are available. To obtain genetic
vaccines and
recombinant polypeptides that exhibit enhanced cross-reactivity and
immunogenicity, the
polynucleotides that encode envelope proteins of related viruses are subjected
to DNA
shuffling. The resulting recombinant polynucleotides can be tested, either as
genetic
5 vaccines or by using the expressed polypeptides, for ability to induce a
broadly reacting
neutralizing antibody response. Finally, those clones that are favorable in
the preliminary
screens can be tested for ability to protect a test animal against viral
challenge.
Viral antigens that can be evolved by DNA shuffling for improved activity as
vaccines include, but are not limited to, influenza A virus N2 neuraminidase
(Kilbourne et
10 al. (1995) Vaccine 13: 1799-1803); Dengue virus envelope (E) and
premembrane (prM)
antigens (Feighny et al. ( I 994) Am. J. Trop. Med Hyg. 50: 322-328; Putnak et
al. ( I996)
Am. J. Trop. Med Hyg. 55: 504-10}; HIV antigens Gag, Pol, Vif and Nef (Vogt et
al. (1995)
Vaccine 13: 202-208); HIV antigens gp120 and gp160 (Achour et al. (I995) Cell.
Mol. Biol.
41: 395-400; Hone et al. (1994) Dev. Biol. Stand. 82: 159-162); gp41 epitope
of human
15 immunodeficiency virus (Eckhart et al. (1996) J. Gen. Yirol. 77: 2001-
2008); rotavirus
antigen VP4 (Mattion et al. (1995) J. Virol. 69: 5132-5137); the rotavirus
protein VP7 or
VP7sc (Emslie et al. (1995) J. Virol. 69: 1747-1754; Xu et al. (1995) J. Gen.
Virol. 76:
1971-1980); herpes simplex virus (HSV) glycoproteins gB, gC, gD, gE, gG, gH,
and gI
(Fleck et al. (1994) Med. Microbiol Immunol. (Berl) 183: 87-94 [Mattion,
1995]; Ghiasi et
20 al. (1995) Invest. Ophthalmol. Vis. Sci. 36: 1352-1360; McLean et al.
(1994) J. Infect. Dis.
170: 1100-1109); immediate-early protein ICP47 of herpes simplex virus-type 1
(HSV-1 )
(Banks et al. (1994) Virology 200: 236-245); immediate-early (IE) proteins
ICP27, ICPO,
and ICP4 of herpes simplex virus (Manickan et al. (1995) J. Virol. 69: 4711-
4716); influenza
virus nucleoprotein and hemagglutinin (Deck et al. (1997) Vaccine 15: 71-78;
Fu et al.
25 ( 1997) J. Virol. 71: 2715-2721 ); B 19 parvovirus capsid proteins VP 1
(Kawase et al. ( 1995)
Virology 211: 359-366) or VP2 (Brown et al. (1994) Virology 198: 477-488);
Hepatitis B
virus core and a antigen (Schodel et al. (1996) Intervirology 39: 104-106);
hepatitis B
surface antigen (Shiau and Murray (1997) J. Med Yirol. S 1: 159-166);
hepatitis B surface
antigen fused to the core antigen of the virus (Id.); Hepatitis B virus core-
preS2 particles
30 (Nemeckova et al. (1996) Acta Virol. 40: 273-279); HBV preS2-S protein
(Kutinova et al.
(1996) Vaccine 14: 1045-1052); VZV glycoprotein I (Kutinova et al. (1996)
Vaccine 14:
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/02944
31
1045-1052); rabies virus glycoproteins (Xiang et al. (1994) Virology 199: I32-
I40; Xuan et
al. (1995) Virus Res. 36: 151-161) or ribonucleocapsid (Hooper et al. (1994)
Proc. Nat'l.
Acad Sci. USA 91: 10908-10912); human cytomegalovirus (HCMV) glycoprotein B
(LTL55)
(Britt et al. ( 1995) J. Infect. Dis. 171: 18-25); the hepatitis C virus (HCV)
nucleocapsid
protein in a secreted or a nonsecreted form, or as a fusion protein with the
middle (pre-S2
and S) or major (S) surface antigens of hepatitis B virus (HBV) (Inchauspe et
al. (1997)
DNA Cell Biol. 16: I85-I95; Major et al. (1995) J. Virol. 69: 5798-5805); the
hepatitis C
virus antigens: the core protein (pC}; E 1 (pE 1 ) and E2 (pE2) alone or as
fusion proteins
(Saito et al. (I997) Gastroenterology 112: 1321-1330); the gene encoding
respiratory
syncytial virus fusion protein (PFP-2) (Falsey and Walsh (1996) Vaccine 14:
1214-1218;
Piedra et al. ( 1996) Pediatr. Infect. Dis. J. I 5: 23-31 ); the VP6 and VP7
genes of rotaviruses
(Choi et al. (1997) Virology 232: 129-138; Jin et al. (1996) Arch. Virol. 141:
2057-2076);
the E1, E2, E3, E4, E5, E6 and E7 proteins of human papillomavirus (Brown et
al. (1994)
Virology 201: 46-54; Dillner et al. (1995) Cancer Detect. Prev. 19: 381-393;
Krul et al.
(1996) Cancer Immunol. Immunother. 43: 44-48; Nakagawa et al. (1997) J.
Infect. Dis. 175:
927-931); a human T-lymphotropic virus type I gag protein (Porter et al.
(1995) J. Med.
Virol. 45: 469-474); Epstein-Barr virus (EBV) gp340 (Mackett et al. (1996) J.
Med Virol.
50: 263-271); the Epstein-Barr virus (EBV) latent membrane protein LMP2 (Lee
et al.
(1996) Eur. J. Immunol. 26: 1875-1883); Epstein-Barr virus nuclear antigens 1
and 2 (Chen
and Cooper (I996) J. Yirol. 70: 4849-4853; Khanna et al. (1995) Virology 214:
633-637);
the measles virus nucleoprotein (I~ (Fooks et al. (1995) Virology 210: 456-
465); and
cytomegalovirus glycoprotein gB (Marshall et al. (1994) J. Med. Yirol. 43: 77-
83) or
glycoprotein gH (Rasmussen et al. ( I 994) J. Infect. Dis. 170: 673-677).
3. Parasites
Antigens from parasites can also be optimized by the methods of the
invention. These include, but are not limited to, the schistosome gut-
associated antigens
CAA (circulating anodic antigen) and CCA (circulating cathodic antigen) in
Schistosoma
mansoni, S. haematobium or S. japonicum (Deelder et al. (1996) Parasitology
112: 2I-35); a
multiple antigen peptide (MAP) composed of two distinct protective antigens
derived from
the parasite Schistosoma mansoni (Ferro et al. (1997) Parasite Immunol. 19: 1-
11);
Leishmania parasite surface molecules (Lezama-Davila (1997) Arch. Med Res. 28:
47-53);
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10.
i ~ ~ ~ 1 ~ ~ ~
...... ......... .............. .....~.~..:.~.i....... ~y ~ ~ ~ -~ y'-~ ....
.:__ _:
~ ~ ~ ~ ~ ~ ~~ ~ ~ ~ ~
~ ~ ~ ~y ~ ~ ~ ~ ~ y w -~~v
~ ~ ~ ~ ~ ~ ~ i ~ ~
- ~ ~ ~~~ ~~ ~~ ~~ ~~ ~~
32
third-stage larval (L3) antigens of L. loa (Akue et al. (1997) J. Infect. Dis.
175: 158-63); the
genes, Tamsl-1 and Tamsl-2, encoding the 30-and 32-kDa major merozoite surface
antigens
of Theileria annulata (Ta) (d'Oliveira et al. (1996) Gene 172: 33-39);
Plasmodium
falciparum merozoite surface antigen 1 or 2 (al-Yaman et al. (1995) Trans. R.
Soc. Trop.
Ted. Hyg. 89: 555-559; Beck et al. (1997) J. Ir~ect. Dis. 175: 921-926;
Rzepczyk et al.
( 1997) Infect. Immun. 65: 1098-1100); circumsporozoite (CS) protein-based B-
epitopes from
Plasmodium berghei, (PPPPNPND)~ (SEQ ID N0:23) and Plasmodiun: yoelii,
(QGPGAP)3QG (SEQ ID N0:24), along with a P. berghei T-helper epitope
KQIRDSITEEWS (SEQ ID N0:25)(Reed et al. (1997) Iraccine 15: 482-488); NYVAC-
Pf7
encoded Plasmodium falciparum antigens derived from the sporozoite
(circumsporozoite
protein and sporozoite surface protein 2), liver (liver stage antigen 1),
blood (merozoite
surface protein 1, serine repeat antigen, and apical membrane antigen 1), and
sexual (25-kDa
sexual-stage antigen) stages of the parasite life cycle were inserted into a
single NYVAC
genome to generate NYVAC-Pf7 (Tine et al. (1996) Infect. Immun. 64: 3833-
3844);
Plasmodium falciparum antigen Pfs230 (Williamson et al. (1996) Mol. Biochem.
Parasitol.
78: 161-169); Plasmodium falciparum apical membrane antigen (AMA-1) (Lal et
al. (1996)
Infect. Immun. 64: 1054-1059); Plasrnodium falciparum proteins Pfs28 and Pfs25
(Duffy and
Kaslow (1997) Ir fect. Immun. 65: 1109-1113); Plasmodium falciparum merozoite
surface
protein, MSP1 (Hui et al. (1996) Infect. Irnrnun. 64: 1502-1509); the malaria
antigen Pf332
(Ahlborg et al. (1996) Immunology 88630-635); Plasmodium falciparum
erythrocyte
membrane protein 1 (Baruch et al. (1995) Proc. Nat'l. Acad. Sci. USA 93: 3497-
3502;
Baruch et al. (1995) Cell 82: 77-87); Plasrnodium falciparum merozoite surface
antigen,
PfMSP-1 (Egan et al. (1996) J. Infect. Dis. 173: 765-769); Plasmodium
falciparum antigens
SERA, EBA-175, RAPT and RAP2 (Riley (1997) J. Pharm. Pharmacol. 49: 21-27);
Schistosoma japonicum paramyosin (Sj97) or fragments thereof (Yang et al.
(1995)
Biochem. Biophys. Res. Commun. 212: 1029-1039); and Hsp70 in parasites
(Maresca and
Kobayashi (1994) Experientia 50: 1067-1074).
4. Aller
The invention also provides methods of obtaining reagents that are useful for
treating allergy. In one embodiment, the methods involve making a library of
recombinant
polynucleotides that encode an allergen, and screening the library to identify
those
recombinant polynucleotides that exhibit improved properties when used as
~~t~ritr~~i»»'AMENDED SH
............ ......................................... EET .....


CA 02320958 2000-08-10
WO 99/41383 PCTNS99/02944
33
immunotherapeutic reagents for treating allergy. For example, specific
immunotherapy of
allergy using natural antigens carries a risk of inducing anaphylaxis, which
can be initiated
by cross-linking of high-affinity IgE receptors on mast cells. Therefore,
allergens that are not
recognized by pre-existing IgE are desirable. The methods of the invention
provide methods
by which one can obtain such allergen variants. Another improved property of
interest is
induction of broader immune responses, increased safety and efficacy.
Synthesis of polyclonal and allergen-specific IgE requires multiple
interactions between B cells, T cells and professional antigen-presenting
cells (APC).
Activation of naive, unprimed B cells is initiated when specific B cells
recognize the
allergen by cell surface immunoglobulin (sFg). However, costimulatory
molecules expressed
by activated T cells in both soluble and membrane-bound forms are necessary
for
differentiation of B cells into IgE-secreting plasma cells. Activation of T
helper cells
requires recognition of an antigenic peptide in the context of MHC class II
molecules on the
plasma membrane of APC, such as monocytes, dendritic cells, Langerhans cells
or primed B
cells. Professional APC can efficiently capture the antigen and the peptide-
MHC class II
complexes are formed in a post-Golgi, proteolytic intracellular compartment
and
subsequently exported to the plasma membrane, where they are recognized by T
cell
receptor (TCR) (Whitton (1998) Curr. Top. Microbiol. Immunol. 232: 1-13). In
addition,
activated B cells express CD80 (B7-1) and CD86 (B7-2, B70), which are the
counter
receptors for CD28 and which provide a costimulatory signal for T cell
activation resulting
in T cell proliferation and cytokine synthesis. Since allergen-specific T
cells from atopic
individuals generally belong to the TH2 cell subset, activation of these cells
also leads to
production of IL-4 and IL-13, which, together with membrane-bound
costimulatory
molecules expressed by activated T helper cells, direct B cell differentiation
into IgE-
secreting plasma cells.
Mast cells and eosinophils are key cells in inducing allergic symptoms in
target organs. Recognition of specific antigen by IgE bound to high-affinity
IgE receptors on
mast cells, basophils or eosinophils results in crosslinking of the receptors
leading to
degranulation of the cells and rapid release of mediator molecules, such as
histamine,
prostaglandins and leukotrienes, causing allergic symptoms.
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PC'T/US99/OZ944
34
Immunotherapy of allergic diseases currently includes hyposensibilization
treatments using increasing doses of allergen injected to the patient. These
treatments result
skewing of immune responses towards TH1 phenotype and increase the ratio of
IgG/IgE
antibodies specific for allergens. Because these patients have circulating IgE
antibodies
specific for the allergens, these treatments include significant risk of
anaphylactic reactions.
In these reactions, free circulating allergen is recognized by IgE molecules
bound to high-
affinity IgE receptors on mast cells and eosinophils. Recognition of the
allergen results in
crosslinking of the receptors leading to release of mediators, such as
histamine,
prostaglandins, and leukotrienes, which cause the allergic symptoms, and
occasionally
anaphylactic reactions. Other problems associated with hyposensibilization
include low
efficacy and difficulties in producing allergen extracts reproducibly.
The methods of the invention provide a means to obtain allergens that, when
used in genetic vaccines, provide a means of circumventing the problems that
have limited
the usefulness of previously known hyposensibilization treatments. For
example, by
expressing antigens on the surface of cells, such as muscle cells, the risk of
anaphylactic
reactions is significantly reduced. This can be conveniently achieved by using
genetic
vaccine vectors that encode transmembrane forms of allergens. The allergens
can also be
modified in such a way that they are efficiently expressed in transmembrane
forms, further
reducing the risk of anaphylactic reactions. Another advantage provided by the
use of
genetic vaccines for hyposensibilization is that the genetic vaccines can
include cytokines
and accessory molecules which further direct the immune responses towards the
TH1
phenotype, thus reducing the amount of IgE antibodies produced and increasing
the efficacy
of the treatments. To further reduce IgE production, one can administer the
shuffled
allergens using vectors that have been evolved to induce primarily IgG and IgM
responses,
with little or no IgE response (see, e.g., US Patent Application Ser. No.
09/021,769, filed
February 11, 1998).
In these methods, polynucleotides encoding known allergens, or homologs or
fragments thereof (e.g., immunogenic peptides) are inserted into DNA vaccine
vectors and
used to immuni2e allergic and asthmatic individuals. Alternatively, the
shuffled allergens are
expressed in manufacturing cells, such as E. coli or yeast cells, and
subsequently purified
and used to treat the patients or prevent allergic disease. DNA shuffling or
other
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/01944
recombination method can be used to obtain allergens that activate T cells but
cannot induce
anaphylactic reactions. For example, a library of recombinant polynucleotides
that encode
allergen variants can be expressed in cells, such as antigen presenting cells,
which are than
contacted with PBMC or T cell clones from atopic patients. Those library
members that
5 efficiently activate T~ cells from the atopic patients can be identified by
assaying for T cell
proliferation, or by cytokine synthesis (e.g., synthesis of IL-2, IL-4, IFN-y.
Those
recombinant allergen variants that are positive in the in vitro tests can then
be subjected to in
vivo testing.
Examples of allergies that can be treated include, but are not limited to,
10 allergies against house dust mite, grass pollen, birch pollen, ragweed
pollen, hazel pollen,
cockroach, rice, olive tree pollen, fungi, mustard, bee venom. Antigens of
interest include
those of animals, including the mite (e.g., Dermatophagoides pteronyssinus,
Dermatophagoides jarinae, Blomia tropicalis), such as the allergens der p 1
(Scobie et al.
(1994) Biochem. Soc. Trans. 22: 4485; Yssel et al. (1992) J. Immunol. 148: 738-
745), der p2
15 (Chug et al. (1996) Clin. Exp. Allergy 26: 829-837), der p3 (Smith and
Thomas ( 1996) Clin.
Exp. Allergy 26: 571-579), der p5, der p V (Lin et al. (1994) J. Allergy Clin.
Immunol. 94:
989-996), der p6 (Bennett and Thomas ( 1996) Clin. Exp. Allergy 26: 1150-
1154), der p 7
(Shen et al. (1995) Clin. Exp. Allergy 25: 416-422), der fZ (Yuuki et al.
(1997) Int. Arch.
Allergy Immunol. 112: 44-48), der f3 (Nishiyama et al. (1995) FEBS Lett. 377:
62-66), der
20 f7 (Shen et al. (1995} Clin. Exp. Allergy 25: 1000-1006); Mag 3 (Fujikawa
et al. (1996) Mol.
Immunol. 33: 311-319}. Also of interest as antigens are the house dust mite
allergens Tyr p2
(Eriksson et al. (1998) Eur. J. Biochem. 251: 443-447), Lep dl (Schmidt et al.
(1995) FEBS
Lett. 370: 11-14), and glutathione S-transferase (O'Neill et al. (1995)
Immunol Lett. 48: 103-
107); the 25,589 Da, 219 amino acid polypeptide with homology with glutathione
S-
25 transferases (O'Neill et al. (1994) Biochim. Biophys. Acta. 1219: 521-528);
Blo t 5 (Arruda
et al. (1995) Int. Arch. Allergy Immunol. 107: 456-457); bee venom
phospholipase A2
(Carballido et al. (1994) J. Allergy Clin. Immunol. 93: 758-767; Jutel et al.
(1995) J.
Immunol. 154: 4187-4194); bovine dermal/dander antigens BDA 11 (Rautiainen et
al. (1995)
J. Invest. Dermatol. 105: 660-663) and BDA20 (Mantyjarvi et al. (1996) J.
Allergy Clin.
30 Immunol. 97: 1297-1303); the major horse allergen Equ cl (Gregoire et al.
(1996) J. Biol.
Chem. 271: 32951-32959}; Jumper ant M. pilosula allergen Myr p I and its
homologous
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/02944
36
allergenic polypeptides Myr p2 (Donovan et al. ( 1996) Biochem. Mo1 Biol.
Irrt. 39: 877-
885); 1-13, 14, 16 kD allergens of the mite Blonria tropicalis (Caraballo et
al. (1996) J.
Allergy Clirr. Immunol. 98: 573-579}; the cockroach allergens Bla g Bd90K
(Helm et al.
(1996) J. Allergy Clin. Immunol. 98: 172-80) and Bla g 2 (Arruda et al. (1995}
J. Biol.
Chem. 270: 19563-19568); the cockroach Cr-PI allergens (Wu et al. (1996) J.
Biol. Chem.
271: 17937-17943}; fire ant venom allergen, Sol i 2 (Schmidt et al. (1996) J.
Allergy Clin.
Immunol. 98: 82-88); the insect Chironomus thunrmi major allergen Chi t 1-9
(Kipp et al.
(1996) Int. Arch. Allergy Immunol. 110: 348-353); dog allergen Can f 1 or cat
allergen Fel d
1 (Ingrain et al. ( 1995) J. Allergy Clin. Immunol. 96: 449-456); albumin,
derived, for
example, from horse, dog or cat (Goubran Botros et al. ( 1996) Immunology 88:
340-347);
deer allergens with the molecular mass of 22 kD, 25 kD or 60 kD (Spitzauer et
al. ( 1997)
Clin. Exp. Allergy 27: 196-200); and the 20 kd major allergen of cow (Ylonen
et al. ( I 994) J.
Allergy Clin. Imnrunol. 93: 851-858).
Pollen and grass allergens are also useful in vaccines, particularly after
optimization of the antigen by the methods of the invention. Such allergens
include, for
example, Hor v9 (Astwood and Hill (1996) Gene 182: 53-62, Lig vl (Batanero et
al. (1996)
Clin. Exp. Allergy 26: 1401-1410); Lol p 1 (Mullet et al. (1996) Int. Arch.
Allergy Immunol.
109: 352-355), Lol p II (Tamborini et al. (1995) Mol. Immunol. 32: 505-513),
Lol pVA, Lol
pVB (Ong et al. (1995) Mol. Immunol. 32: 295-302), Lol p 9 (Blaher et al.
(1996) J. Allergy
Clin. Immunol. 98: 124-132); Par J I (Costa et al. (1994) FEBSLett. 341: 182-
186; Sallusto
et al. (1996) J. Allergy Clin. Immunol. 97: 627-637), Par j 2.0101 (Duro et
al. (1996) FEBS
Lett. 399: 295-298); Bet vl (Faber et al. (1996) J. Biol. Chem. 271: 19243-
19250), Bet v2
(Rihs et al. (1994) Int. Arch. Allergy Immunol. 105: 190-194); Dac g3 (Guerin-
Marchand et
al. (1996) Mol. Immunol. 33: 797-806); Phl p 1 (Petersen et al. (1995) J.
Allergy Clin.
Immunol. 95: 987-994), Phl p 5 (Mullet et al. (1996) Int. Arch. Allergy
Immunol. 109: 352-
355), Phl p 6 (Petersen et al. (1995) Int. Arch. Allergy Immunol. 108: 55-59);
Cry j I (Sone et
al. (1994) Biochem. Biophys. Res. Commun. 199: 619-625), Cry j II (Mamba et
al. (1994)
FEBSLett. 353: 124-128); Cor a 1 {Schenk et al. (1994) Eur. J. Biochem. 224:
717-722);
cyn dl (Smith et al. (1996) J. Allergy Clin. Immunol. 98: 331-343), cyn d7
(Suphioglu et al.
(1997) FEBS Lett. 402: 167-172); Pha a 1 and isoforms of Pha a 5 (Suphioglu
and Singh
(1995) Clin. Exp. Aller~v 25: 853-865); Cha o 1 (Suzuki et al. (1996) Mol.
Immunol. 33:
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/02944
37
451-460); profilin derived, e.g, from timothy grass or birch pollen (Valenta
et al. (1994)
Bioche»r. Biophys. Res. Commun. 199: 106-118); P0149 (Wu et al. ( I 996) Plant
Mol. Biol.
32: 1037-1042); Ory sl (Xu et al. (1995) Gene 164: 255-259); and Amb a V and
Amb t 5
(Kim et al. (1996) Mol. Immunol. 33: 873-880; Zhu et al. (1995) J. Immunol.
155: 5064-
5073).
Vaccines against food allergens can also be developed using the methods of
the invention. Suitable antigens for shuffling include, for example, profilin
(Rihs et al.
( 1994) Int. Arch. Allergy Immunol. 105: 190-194); rice allergenic cDNAs
belonging to the
alpha-amylase/trypsin inhibitor gene family (Alvarez et al. (1995) Biochim
Biophys Acta
1251: 201-204); the main olive allergen, Ole a I (Lombardero et al. (1994)
Clin Exp Allergy
24: 765-770); Sin a 1, the major allergen from mustard (Gonzalez De La Pena et
al. (1996)
Eur J Biochem. 237: 827-832); parvalbumin, the major allergen of salmon
(Lindstrom et al.
(1996) Scand. J. Immunol. 44: 335-344); apple allergens, such as the major
allergen Mal d 1
(Vanek-Krebitz et al. (1995) Biochem. Biophys. Res. Commun. 214: 538-551); and
peanut
allergens, such as Ara h I (Barks et al. (1995) J. Clin. Invest. 96: 1715-
1721).
The methods of the invention can also be used to develop recombinant
antigens that are effective against allergies to fungi. Fungal allergens
useful in these
vaccines include, but are not limited to, the allergen, Cla h III, of
Cladosporium herbarum
(Zhang et al. (1995) J. Immunol. 154: 710-717); the allergen Psi c 2, a fungal
cyclophilin,
from the basidiomycete Psilocybe cubensis (Homer et al. (1995) Int. Arch.
Allergy Immunol.
107: 298-300); hsp 70 cloned from a cDNA library of Cladosporium herbarum
(Zhang et al.
( 1996) Clin Exp Allergy 26: 88-95}; the 68 kD allergen of Penicillium notatum
(Shen et al.
(1995) Clin. Exp. Allergy 26: 350-356); aldehyde dehydrogenase (ALDH) (Achatz
et al.
(1995) Mollmmunol. 32: 213-227); enolase (Achatz et al. (1995) Mol. Immunol.
32: 213-
227); YCP4 {Id.); acidic ribosomal protein P2 {Id.).
Other allergens that can be used in the methods of the invention include latex
allergens, such as a major allergen (Hev b 5) from natural rubber latex
(Akasawa et al.
(1996) J. Biol. Chem. 271: 25389-25393; Slater et al. (1996) J. Biol. Chem.
271: 25394-
25399).
The invention also provides a solution to another shortcoming of vaccination
as a treatment for allergy and asthma. While genetic vaccination primarily
induces CD8+ T
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99141383 PCTNS99/8Z944
38
cell responses, induction of allergen-specific IgE responses is dependent on
CD4' T cells
and their help to B cells. TH2-type cells are particularly eff cient in
inducing IgE synthesis
because they secrete high levels of IL-4, IL-5 and IL-13, which direct Ig
isotype switching to
IgE synthesis. IL-5 also induces eosinophilia. The methods of the invention
can be used to
develop recombinant antigens that efficiently induce CD4+ T cell responses,
and direct
differentiation of these cells towards the TH1 phenotype.
5. Inflammatory and Autoimmune Diseases
Autoimmune diseases are characterized by immune response that attacks
tissues or cells of ones own body, or pathogen-specif c immune responses that
also are
harmful for ones own tissues or cells, or non-specific immune activation which
is harmful
for ones own tissues or cells. Examples of autoimmune diseases include, but
are not limited
to, rheumatoid arthritis, SLE, diabetes mellitus, myasthenia gravis, reactive
arthritis,
ankylosing spondylitis, and multiple sclerosis. These and other inflammatory
conditions,
including IBD, psoriasis, pancreatitis, and various immunodeficiencies, can be
treated using
antigens that are optimized using the methods of the invention.
These conditions are often characterized by an accumulation of inflammatory
cells, such as Lymphocytes, macrophages, and neutrophils, at the sites of
inflammation.
Altered cytokine production levels are often observed, with increased levels
of cytokine
production. Several autoimmune diseases, including diabetes and rheumatoid
arthritis, are
linked to certain MHC haplotypes. Other autoimmune-type disorders, such as
reactive
arthritis, have been shown to be triggered by bacteria such as Yersinia and
Shigella, and
evidence suggests that several other autoimmune diseases, such as diabetes,
multiple
sclerosis, rheumatoid arthritis, may also be initiated by viral or bacterial
infections in
genetically susceptible individuals.
Current strategies of treatment generally include anti-inflammatory drugs,
such as NSAID or cyclosporin, and antiproliferative drugs, such as
methotrexate. These
therapies are non-specific, so a need exists for therapies having greater
specificity, and for
means to direct the immune responses towards the direction that inhibits the
autoimmune
process.
The present invention provides several strategies by which these needs can be
fulfilled. First, the invention provides methods of obtaining antigens having
greater
SUBSTITUTE SHEET (RULE 2fi)


CA 02320958 2000-08-10
WO 99/41383 PC'f/US99/02944
39
tolerogenicity and/or have improved antigenicity. In a preferred embodiment,
the antigens
prepared according to the invention exhibit improved induction of tolerance by
oral delivery.
Oral tolerance is characterized by induction of immunological tolerance after
oral
administration of large quantities of antigen. In animal models, this approach
has proven to
be a very promising approach to treat autoimmune diseases, and clinical trials
are in progress
to address the efficacy of this approach in the treatment of human autoimmune
diseases,
such as rheumatoid arthritis and multiple sclerosis. It has also been
suggested that induction
of oral tolerance against viruses used in gene therapy might reduce the
immunogenicity of
gene therapy vectors. However, the amounts of antigen required for induction
of oral
tolerance are very high and the methods of the invention provide a means for
obtaining
antigens that exhibit a significant improvement in induction of oral
tolerance.
Expression library immunization (Barry et al. (1995) Nature 377: 632) is a
particularly useful method of screening for optimal antigens for use in
genetic vaccines. For
example, to identify autoantigens present in Yersinia, Shigella, and the like,
one can screen
for induction of T cell responses in HLA-B27 positive individuals. Complexes
that include
epitopes of bacterial antigens and MHC molecules associated with autoimmune
diseases,
e.g., HLA-B27 in association with Yersinia antigens can be used in the
prevention of
reactive arthritis and ankyIosing spondylitis in HLA-B27 positive individuals.
Screening of optimized antigens can be done in animal models which are
known to those of skill in the art. Examples of suitable models for various
conditions
include collagen induced arthritis, the NFS/sld mouse model of human Sjogren's
syndrome;
a 120 kD organ-specific autoantigen recently identif ed as an analog of human
cytoskeletal
protein (a-fodrin (Haneji et al. (1997) Science 276: 604), the New Zealand
Black/White F1
hybrid mouse model of human SLE, NOD mice, a mouse model of human diabetes
mellitus,
fas/fas ligand mutant mice, which spontaneously develop autoimmune and
lymphoproliferative disorders (Watanabe-Fukunaga et al. (1992) Na~ure 356:
314), and
experimental autoimmune encephalomyelitis (EAE), in which myelin basic protein
induces a
disease that resembles human multiple sclerosis.
Autoantigens that can be shuffled according to the methods of the invention
and used in vaccines for treating multiple sclerosis include, but are not
limited to, myelin
basic protein (Stinissen et al. ( 1996) J. Neurosci. Res. 45: 500-511 ) or a
fusion protein of
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/02944
myelin basic protein and proteolipid protein (Elliott et al. (1996) J. Clin.
Invest. 98: 1602-
1612), proteolipid protein (PLP) (Rosener et al. ( 1997) J. Neuroimmunol. 75:
28-34), 2',3'-
cyclic nucleotide 3'-phosphodiesterase (CNPase) (Rosener et al. (1997) J.
Neuroimmunol.
75: 28-34), the Epstein Ban virus nuclear antigen-1 (EBNA- I ) (Vaughan et al.
( 1996) J.
5 Neuroimmunol. 69: 95-102), HSP70 (Salvetti et al. (1996) J. Neuroimmunol.
65: 143-53;
Feldmann et al. (1996) Cell 85: 307).
Target antigens that, after shuffling according to the methods of the
invention,
can be used to treat scleroderma, systemic sclerosis, and systemic lupus
erythematosus
include, for example, (-2-GPI, 50 kDa glycoprotein (Blank et al. (1994) J.
Autoimmun. 7:
10 441-455), Ku (p70/p80) autoantigen, or its 80-kd subunit protein (Hong et
al. (1994) Invest.
Ophthalmol. Yis. Sci. 35: 4023-4030; Wang et al. (1994) J. Cell Sci. 107: 3223-
3233), the
nuclear autoantigens La (SS-B) and Ro (SS-A) (Huang et al. (1997) J. Clin.
Immunol. 17:
212-219; Igarashi et al. (1995) Autoimmunity 22: 33-42; Keech et al. (1996)
Clin. Exp.
Immunol. 104: 255-263; Manoussakis et al. (1995) J. Autoimmun. 8: 959-969;
Topfer et al.
15 (1995) Proc. Nat'l. Acad. Sci. USA 92: 875-879), proteasome (-type subunit
C9 (Feist et al.
(1996) J. Exp. Med. 184: 1313-1318), Scleroderma antigens Rpp 30, Rpp 38 or
Scl-70 (Eder
et al. ( 1997) Proc. Nat 'l. Acad. Sci. USA 94: 1101-1106; Hietarinta et al. (
1994) Br. J.
Rheumatol. 33: 323-326), the centrosome autoantigen PCM-1 (Bao et al. (1995)
Autoimmunity 22: 219-228), polymyositis-scleroderma autoantigen (PM-Scl) (Kho
et al.
20 (1997) J. Biol. Chem. 272: 13426-13431), scleroderma (and other systemic
autoimmune
disease) autoantigen CENP-A (Muro et al. (1996) Clin. Immunol. Immunopathol.
78: 86-89),
U5, a small nuclear ribonucleoprotein (snRNP) (Okano et al. (1996) Clin.
Immunol.
Immunopathol. 81: 41-47), the 100-kd protein of PM-Scl autoantigen (Ge et al.
(1996)
Arthritis Rheum. 39: 1588-1595), the nucleolar U3- and Th(7-2)
ribonucleoproteins
25 (Verheijen et al. (1994) J. Immunol. Methods I69: 173-182), the ribosomal
protein L7 (Neu
et al. (1995) Clin. Exp. Immunol. 100: 198-204), hPopl (Lygerou et al. (1996)
EMBO J. 15:
5936-5948), and a 36-kd protein from nuclear matrix antigen (Deng et al.
(1996) Arthritis
Rheum. 39: 1300-1307).
Hepatic autoimmune disorders can also be treated using improved
30 recombinant antigens that are prepared according to the methods described
herein. Among
the antigens that are useful in such treatments are the cvtochromes P450 and
UDP-
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/02944
41
glucuronosyl-transferases (Obermayer-Straub and Manns ( 1996) Baillieres Clin.
Gastroenterol. 10: 501-532), the cytochromes P450 2C9 and P450 lA2 (Bourdi et
al. (1996)
Chem. Res. Toxicol. 9: 1159-1166; Clemente et al. (1997) J. Clin. Endocrinol.
Metab. 82:
1353-1361), LC-1 antigen (Klein et al. (1996) J. Pediatr. Gastroenterol. Nutr.
23: 461-465),
and a 230-kDa Golgi-associated protein (Funaki et al. (1996) Cell Struct.
Funct. 21: 63-72}.
For treatment of autoimmune disorders of the skin, useful antigens include,
but are not limited to, the 450 kD human epidermal autoantigen (Fujiwara et
al. (1996) J.
Invest. Dermatol. 106: 1125-1130), the 230 kD and 180 kD bullous pemphigoid
antigens
(Hashimoto (1995) Keio J. Med. 44: I 15-123; Murakami et al. (1996) J.
Dermatol. Sci. 13:
112-117), pemphigus foliaceus antigen (desmoglein 1 ), pemphigus vulgaris
antigen
(desmoglein 3), BPAg2, BPAgl, and type VII collagen (Batteux et al. (1997) J.
Clin.
Immunol. 17: 228-233; Hashimoto et al. (1996) J. Dermatol. Sci. 12: 10-17), a
168-kDa
mucosal antigen in a subset of patients with cicatricial pemphigoid
(Ghohestani et al. (1996)
J. Invest. Dermatol. 107: 136-139), and a 218-kd nuclear protein (218-kd Mi-2)
(Seelig et al.
(1995) Arthritis Rheum. 38: 1389-1399).
The methods of the invention are also useful for obtaining improved antigens
for treating insulin dependent diabetes mellitus, using one or more of
antigens which
include, but are not limited to, insulin, proinsulin, GAD65 and GAD67, heat-
shock protein
65 (hsp65), and islet-cell antigen 69 (ICA69) (French et al. (1997) Diabetes
46: 34-39; Roep
(1996) Diabetes 45: 1147-1156; Schloot et al. (1997) Diabetologia 40: 332-
338), viral
proteins homologous to GAD65 (Jones and Crosby ( 1996) Diabetologia 39: 1318-
1324),
islet cell antigen-related protein-tyrosine phosphatase (PTP) (Cui et al.
(1996) J. Biol. Chem.
271: 24817-24823), GM2-1 ganglioside (Cavallo et al. (1996) J. Endocrinol.
150: 113-120;
Dotta et al. (1996) Diabetes 45: 1193-1196), glutamic acid decarboxyiase (GAD)
(Nepom
(1995) Curr. Opin. Immunol. 7: 825-830; Panina-Bordignon et al. (1995) J. Exp.
Med. 181:
1923-1927), an islet cell antigen (ICA69) (Karges et al. (1997) Biochim.
Biophys. Acta 1360:
97-101; Roep et al. (1996) Eur. J. Immunol. 26: 1285-1289), Tep69, the single
T cell epitope
recognized by T cells from diabetes patients (Karges et al. (1997) Biochim.
Biophys. Acta
1360: 97-101), ICA 512, an autoantigen of type 1 diabetes (Solimena et al.
(1996) EMBO J.
15: 2102-2114), an islet-cell protein tyrosine phosphatase and the 37-kDa
autoantigen
derived from it in type 1 diabetes (including IA-2, IA-2) (La Gasse et al.
(1997) Mol. Med.
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/01944
42
3: 163-173), the 64 kDa protein from In-111 cells or human thyroid follicular
cells that is
immunoprecipitated with sera from patients with islet cell surface antibodies
(ICSA) (Igawa
et al. (1996) Endocr. J. 43: 299-306), phogrin, a homologue of the human
transmembrane
protein tyrosine phosphatase, an autoantigen of type 1 diabetes (Kawasaki et
al. (1996)
Biochem. Biophys. Res. Commun. 227: 440-447), the 40 kDa and 37 kDa tryptic
fragments
and their precursors IA-2 and IA-2 in IDDM (Lampasona et al. (1996) J.
Immunol. 157:
2707-2711; Notkins et al. (1996) J. Autoimmun. 9: 677-682), insulin or a
cholera toxoid-
insulin conjugate (Bergerot et al. (1997) Proc. Nat'l. Acad. Sci. USA 94: 4610-
4614),
carboxypeptidase H, the human homologue of gp330, which is a renal epithelial
glycoprotein involved in inducing Heymann nephritis in rats, and the 38-kD
islet
mitochondria) autoantigen (Arden et al. (1996) J. Clin. Invest. 97: 551-561.
Rheumatoid arthritis is another condition that is treatable using optimized
antigens prepared according to the present invention. Useful antigens for
rheumatoid arthritis
treatment include, but are not limited to, the 45 kDa DEK nuclear antigen, in
particular onset
juvenile rheumatoid arthritis and iridocyclitis (Murray et al. (1997) J.
Rheumatol. 24: 560-
567), human cartilage glycoprotein-39, an autoantigen in rheumatoid arthritis
(Verheijden et
al. (1997) Arthritis Rheum. 40: 1115-1125), a 68k autoantigen in rheumatoid
arthritis (Blass
et al. (1997) Ann. Rheum. Dis. 56: 317-322), collagen (Rosloniec et al. (1995)
J. Immunol.
155: 4504-4511), collagen type II (Cook et al. (1996) Arthritis Rheum. 39:
1720-1727;
Trentham (1996) Ann. N. Y. Acad. Sci. 778: 306-314), cartilage link protein
(Guerassimov et
al. (1997) J. Rheumatol. 24: 959-964), ezrin, radixin and moesin, which are
auto-immune
antigens in rheumatoid arthritis (Wagatsuma et al. (1996) Mol. Immunol. 33:
1171-1176),
and mycobacterial heat shock protein 65 (Ragno et al. (1997) Arthritis Rheum.
40: 277-283).
Also among the conditions for which one can obtain an improved antigen
suitable for treatment are autoimmune thyroid disorders. Antigens that are
useful for these
applications include, for example, thyroid peroxidase and the thyroid
stimulating hormone
receptor (Tandon and Weetman ( 1994} J. R. Coll. Physicians Lond. 28: 10-18),
thyroid
peroxidase from human Graves' thyroid tissue (Gardas et al. (1997) Biochem.
Biophys. Res.
Commun. 234: 366-370; Zimmer et al. (1997) Histochem. Cell. Biol. 107: 115-
120), a 64-
kDa antigen associated with thyroid-associated ophthalmopathy (Zhang et al.
(1996) Clin.
Immunol. Immunopathol. 80: 236-244), the human TSH receptor (Nicholson et al.
(1996) J.
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCTNS99/02944
43
Mol. Endocrinol. 16: 159-170), and the 64 kDa protein from In-111 cells or
human thyroid
follicular cells that is immunoprecipitated with sera from patients with islet
cell surface
antibodies (ICSA} (Igawa et al. (1996) Endocr. J. 43: 299-306).
Other conditions and associated antigens include, but are not limited to,
Sjogren's syndrome (-fodrin; Haneji et al. (1997) Science 276: 604-607),
myastenia gravis
(the human M2 acetylcholine receptor or fragments thereof, specifically the
second
extracellular loop of the human M2 acetylcholine receptor; Fu et al. (1996)
Clin. Immunol.
Immunopathol. 78: 203-207), vitiligo (tyrosinase; Fishman et al. (1997) Cancer
79: 1461-
1464), a 450 kD human epidermal autoantigen recognized by serum from
individual with
blistering skin disease, and ulcerative colitis (chromosomal proteins HMG1 and
HMG2;
Sobajima et al. (1997) Clin. Exp. Immunol. 107: 135-140).
6. Cancer
Immunotherapy has great promise for the treatment of cancer and prevention
of metastasis. By inducing an immune response against cancerous cells, the
body's immune
system can be enlisted to reduce or eliminate cancer. Improved antigens
obtained using the
methods of the invention provide cancer immunotherapies of increased
effectiveness
compared to those that are presently available.
One approach to cancer immunotherapy is vaccination using vaccines that
include or encode antigens that are specific for tumor cells or by injecting
the patients with
purified recombinant cancer antigens. The methods of the invention can be used
for
obtaining antigens that exhibit an enhancement of immune responses against
known tumor-
specific antigens, and also to search for novel protective antigenic
sequences. Antigens
having optimized expression, processing, and presentation can be obtained as
described
herein. The approach used for each particular cancer can vary. For treatment
of hormone-
sensitive cancers (for example, breast cancer and prostate cancer), methods of
the invention
can be used to obtain optimized hormone antagonists. For highly immunogenic
tumors,
including melanoma, one can screen for recombinant antigens that optimally
boost the
immune response against the tumor. Breast cancer, in contrast, is of
relatively low
immunogenicity and exhibits slow progression, so individual treatments can be
designed for
each patient. Prevention of metastasis is also a goal in design of cancer
vaccines.
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCTNS99/02944
44
Among the tumor-specific antigens that can be used in the antigen shuffling
methods of the invention are: bullous pemphigoid antigen 2, prostate mucin
antigen (PMA)
(Beckett and Wright (1995) Int. J. Cancer 62: 703-710), tumor associated
Thomsen-
Friedenreich antigen (Dahlenborg et al. ( 1997) Int. J. Cancer 70: 63-71 ),
prostate-specific
antigen (PSA) (Dannull and Belldegrun (1997) Br. J. Urol. 1: 97-103), luminal
epithelial
antigen (LEA.135) of breast carcinoma and bladder transitional cell carcinoma
(TCC) (Jones
et al. ( 1997) Anticancer Res. 17: 685-687), cancer-associated serum antigen
(CASA) and
cancer antigen 125 (CA 125) (Kierkegaard et al. (1995) Gynecol. Oncol. 59: 251-
254), the
epithelial glycoprotein 40 (EGP40) (Kievit et al. (1997) Int. J. Cancer 71:
237-245),
squamous cell carcinoma antigen {SCC) (Lozza et al. (1997) Anticancer Res. 17:
525-529),
cathepsin E (Mota et al. (1997) Am. J. Pathol. 150: 1223-1229), tyrosinase in
melanoma
(Fishman et al. (1997) Cancer 79: 1461-1464), cell nuclear antigen (PCNA) of
cerebral
cavernomas (Notelet et al. (1997) Surg. Neurol. 47: 364-370), DF3/MLJC1 breast
cancer
antigen (Apostolopoulos et al. (1996) Immunol. Cell. Biol. 74: 457-464; Pandey
et al. (1995)
Cancer Res. 55: 4000-4003), carcinoembryonic antigen (Paone et al. (1996) J.
Cancer Res.
Clin. Oncol. 122: 499-503; Schlom et al. (1996) Breast Cancer Res. Treat. 38:
27-39),
tumor-associated antigen CA 19-9 (Tolliver and O'Brien ( 1997) South Med. J.
90: 89-90;
Tsuruta et al. (1997) Urol. Int. 58: 20-24), human melanoma antigens MART-
1/Melan-A27-
35 and gp100 (Kawakami and Rosenberg (1997) Int. Rev. Immunol. 14: 173-192;
Zajac et al.
( 1997) Int. J. Cancer 71: 491-496), the T and Tn pancarcinoma (CA)
glycopeptide epitopes
(Springer (1995) Crit. Rev. Oncog. 6: 57-85), a 35 kD tumor-associated
autoantigen in
papillary thyroid carcinoma (Lucas et al. (1996) Anticancer Res. 16: 2493-
2496), KH-1
adenocarcinoma antigen (Deshpande and Danishefsky (I997) Nature 387: 164-166),
the A60
mycobacterial antigen (Maes et al. (1996) J. Cancer Res. Clin. Oncol. 122: 296-
300), heat
shock proteins (HSPs) (Blachere and Srivastava (1995) Semin. Cancer Biol. 6:
349-355), and
MAGE, tyrosinase, melan-A and gp75 and mutant oncogene products (e.g., p53,
ras, and
HER-2/neu (Bueler and Mulligan (1996) Mol. Med. 2: 545-555; Lewis and Houghton
(1995)
Semin. Cancer Biol. 6: 321-327; Theobald et al. (1995) Proc. Nat 'l. Acad.
Sci. USA 92:
11993-11997).
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/02944
7. Contraception
Genetic vaccines that contain optimized antigens obtained by the methods of
the invention are also useful for contraception. For example, genetic vaccines
can be
obtained that encode sperm cell specific antigens, and thus induce anti-sperm
immune
responses. Vaccination can be achieved by, for example, administration of
recombinant
bacterial strains, e.g. Salmonella and the like, which express spenm antigen,
as well as by
induction of neutralizing anti-hCG antibodies by vaccination by DNA vaccines
encoding
human chorionic gonadotropin (hCG), or a fragment thereof.
Sperm antigens which can be used in the genetic vaccines include, far
10 example, lactate dehydrogenase (LDH-C4), galactosyltransferase (GT), SP-10,
rabbit sperm
autoantigen (RSA), guinea pig (g)PH-20, cleavage signal protein (CS-1), HSA-
63, human
(h)PH-20, and AgX-1 (Zhu and Naz (1994) Arch. Androl. 33: 141-144), the
synthetic sperm
peptide, P10G (O'Rand et al. (1993) J. Reprod. Immunol. 25: 89-102), the
135kD, 95kD,
65kD, 47kD, 4lkD and 23kD proteins of sperm, and the FA-I antigen (Naz et al.
(1995)
15 Arch. Androl. 35: 225-231), and the 35 kD fragment of cytokeratin 1 (Lucas
et al. (1996)
Anticancer Res. 16: 2493-2496).
The methods of the invention can also be used to obtain genetic vaccines that
are expressed specifically in testis. For example, polynucleotide sequences
that direct
expression of genes that are specific to testis can be used (e.g.,
fertilization antigen-1 and the
20 like). In addition to sperm antigens, antigens expressed on oocytes or
hormones regulating
reproduction may be useful targets of contraceptive vaccines. For example,
genetic vaccines
can be used to generate antibodies against gonadotropin releasing hormone
(GnRH) or zona
pellucida proteins (Miller et al. (1997} Vaccine 15:1858-1862). Vaccinations
using these
molecules have been shown to be efficacious in animal models (Miller et al.
(1997) Vaccine
25 15:1858-1862). Another example of a useful component of a genetic
contraceptive vaccine
is the ovarian zona pellucida glycoprotein ZP3 (Tong et al. ( 1994} Reprod.
Fertil. Dev.
6:349-355).
Methods of Selecting and Identifying Optimized Recombinant Antigens
Once one has performed DNA shuffling to obtain a library of polynucleotides
30 that encode recombinant antigens, the library is subjected to selection
and/or screening to
identify those library members that encode antigenic peptides that have
improved ability to
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/OI9d4
46
induce an immune response to the pathogenic agent. Selection and screening of
recombinant
polynucleotides that encode polypeptides having an improved ability to induce
an immune
response can involve either in vivo and in vitro methods, but most often
involves a
combination of these methods. For example, in a typical embodiment the members
of a
library of recombinant nucleic acids are picked, either individually or as
pools. The clones
can be subjected to analysis directly, or can be expressed to produce the
corresponding
polypeptides. In a presently preferred embodiment, an in vitro screen is
performed to
identify the best candidate sequences for the in vivo studies. Alternatively,
the library can be
subjected to in vivo challenge studies directly. The analyses can employ
either the nucleic
acids themselves (e.g., as genetic vaccines), or the polypeptides encoded by
the nucleic
acids. A schematic diagram of a typical strategy is shown in Figure 5. Both in
vitro and in
vivo methods are described in more detail below.
If a recombination cycle is performed in vitro, the products of recombination,
i.e., recombinant segments, are sometimes introduced into cells before the
screening step.
Recombinant segments can also be linked to an appropriate vector or other
regulatory
sequences before screening. Alternatively, products of recombination generated
in vitro are
sometimes packaged in viruses (e.g., bacteriophage) before screening: If
recombination is
performed in vivo, recombination products can sometimes be screened in the
cells in which
recombination occurred. In other applications, recombinant segments are
extracted from the
cells, and optionally packaged as viruses, before screening.
Often, improvements are achieved after one round of recombination and
selection. However, recursive sequence recombination can also be employed to
achieve still
further improvements in a desired property, or to bring about new (or
"distinct") properties.
Recursive sequence recombination entails successive cycles of recombination to
generate
molecular diversity. That is, one creates a family of nucleic acid molecules
showing some
sequence identity to each other but differing in the presence of mutations. In
any given cycle,
recombination can occur in vivo or in vitro, intracellularly or
extracelluiarly. Furthermore,
diversity resulting from recombination can be augmented in any cycle by
applying prior
methods of mutagenesis (e.g., error-prone PCR or cassette mutagenesis) to
either the
substrates or products for recombination.
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99101944
47
In a presently preferred embodiment, polynucleotides that encode optimized
recombinant antigens are subjected to molecular backcrossing, which provides a
means to
breed the shuffled chimeras/mutants back to a parental or wild-type sequence,
while
retaining the mutations that are critical to the phenotype that provides the
optimized immune
responses. In addition to removing the neutral mutations, molecular
backcrossing can also
be used to characterize which of the many mutations in an improved variant
contribute most
to the improved phenotype. This cannot be accomplished in an efficient library
fashion by
any other method. Backcrossing is performed by shuffling the improved sequence
with a
large molar excess of the parental sequences.
The nature of screening or selection depends on what property or
characteristic is to be acquired or the property or characteristic for which
improvement is
sought, and many examples are discussed below. It is not usually necessary to
understand
the molecular basis by which particular products of recombination (recombinant
segments)
have acquired new or improved properties or characteristics relative to the
starting
substrates. For example, a gene that encodes an antigenic polypeptide can have
many
component sequences each having a different intended role (see, e.g., Figure
4). Each of
these component sequences can be varied and recombined simultaneously.
Screening/
selection can then be performed, for example, for recombinant segments that
have increased
ability to induce an immune response to a pathogenic agent without the need to
attribute
such improvement to any of the individual component sequences of the
recombinant
polynucleotide.
Depending on the particular screening protocol used for a desired property,
initial rounds) of screening can sometimes be performed using bacterial cells
due to high
transfection efficiencies and ease of culture. However, especially for testing
of immunogenic
activity, test animals are used for library expression and screening.
Similarly other types of
screening which are not amenable to screening in bacterial or simple
eukaryotic library cells,
are performed in cells selected for use in an environment close to that of
their intended use.
Final rounds of screening can be performed in cells or organisms that are as
close as possible
to the precise cell type or organism of intended use.
If further improvement in a property is desired, at least one, and usually a
collection, of recombinant segments surviving a first round of
screening/selection are subject
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99141383 PCTNS99/02944
48
to a further round of recombination. These recombinant segments can be
recombined with
each other or with exogenous segments representing the original substrates or
further
variants thereof. Again, recombination can proceed in vitro or in vivo. If the
previous
screening step identifies desired recombinant segments as components of cells,
the
components can be subjected to further recombination in vivo, or can be
subjected to further
recombination in vitro, or can be isolated before performing a round of in
vitro
recombination. Conversely, if the previous screening step identifies desired
recombinant
segments in naked form or as components of viruses, these segments can be
introduced into
cells to perform a round of in vivo recombination. The second round of
recombination,
irrespective how performed, generates further recombinant segments which
encompass
additional diversity than is present in recombinant segments resulting from
previous rounds.
The second round of recombination can be followed by a further round of
screening/selection according to the principles discussed above for the first
round. The
stringency of screening/selection can be increased between rounds. Also, the
nature of the
1 S screen and the property being screened for can vary between rounds if
improvement in more
than one property is desired or if acquiring more than one new property is
desired.
Additional rounds of recombination and screening can then be performed until
the
recombinant segments have sufficiently evolved to acquire the desired new or
improved
property or function.
The practice of this invention involves the construction of recombinant
nucleic acids and the expression of genes in transfected host cells. Molecular
cloning
techniques to achieve these ends are known in the art. A wide variety of
cloning and in vitro
amplification methods suitable for the construction of recombinant nucleic
acids such as
expression vectors are well-known to persons of skill. General texts which
describe
molecular biological techniques useful herein, including mutagenesis, include
Berger and
Kimmel, Guide to Molecular Cloning Techniques, Methods in Enrymology volume
152
Academic Press, Inc., San Diego, CA (Berger); Sambrook et al., Molecular
Cloning - A
Laboratory Manual (2nd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold
Spring
Harbor, New York, 1989 ("Sambrook") and Current Protocols in Molecular
Biology, F.M.
Ausubel et al., eds., Current Protocols, a joint venture between Greene
Publishing
Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 1998)
{"Ausubel")).
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PC'TIUS99/02944
49
Examples of techniques sufficient to direct persons of skill through in vitro
amplification
methods, including the polymerase chain reaction (PCR) the ligase chain
reaction (LCR), Q -
replicase amplification and other RNA polymerase mediated techniques (e.g.,
NASBA) are
found in Berger, Sambrook, and Ausubel, as well as Mullis et al. (1987) U.S.
Patent No.
4,683,202; PCR Protocols A Guide to Methods and Applications (Innis et al.
eds) Academic
Press Inc. San Diego, CA (1990) (Innis); Arnheim & Levinson (October 1, 1990)
CdcEN
36-47; The Journal Of NIH Research ( 1991 ) 3, 81-94; (Kwoh et al. ( 1989)
Proc. Natl. Acad.
Sci. USA 86, 1173; Guatelli et al. ( 1990) Proc. Natl. Acad Sci. USA 87, 1874;
Lomell et al.
(1989) J. Clin. Chem 35, 1826; Landegren et al. (1988) Science 241, 1077-1080;
Van Brunt
(1990) Biotechnology 8, 291-294; Wu and Wallace (1989) Gene 4, 560; Barringer
et al.
( 1990) Gene 89, 117, and Sooknanan and Malek ( 1995) Biotechnology 13: 563-
564.
Improved methods of cloning in vitro amplified nucleic acids are described in
Wallace et al.,
U.S. Pat. No. 5,426,039. Improved methods of amplifying large nucleic acids by
PCR are
summarized in Cheng et al. (1994) Nature 369: 684-685 and the references
therein, in which
PCR amplicons of up to 40kb are generated. One of skill will appreciate that
essentially any
RNA can be converted into a double stranded DNA suitable for restriction
digestion, PCR
expansion and sequencing using reverse transcriptase and a polymerase. See,
Ausubel,
Sambrook and Berger, all supra.
Oligonucleotides for use as probes, e.g., in in vitro amplification methods,
for
use as gene probes, or as shuffling targets (e.g., synthetic genes or gene
segments) are
typically synthesized chemically according to the solid phase phosphoramidite
triester
method described by Beaucage and Caruthers (1981) Tetrahedron Letts.,
22(20):1859-1862,
e.g., using an automated synthesizer, as described in Needham-VanDevanter et
al. (1984)
Nucleic Acids Res., 12:6159-6168. Oligonucleotides can also be custom made and
ordered
from a variety of commercial sources known to persons of skill.
Indeed, essentially any nucleic acid with a known sequence can be custom
ordered from any of a variety of commercial sources, such as The Midland
Certified Reagent
Company (mcrc@oligos.com), The Great American Gene Company
(http://www.genco.com), ExpressGen Inc. (www.expressgen.com), Operon
Technoloigies
Inc. (Alameda, CA) and many others. Similarly, peptides and antibodies can be
custom
ordered from any of a variety of sources, such as PeptidoGenic
(pkim@ccnet.com), HTI
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/02944
Bio-products, Inc. (http://www.htibio.com), BMA Biomedicals Ltd (U.K.),
Bio~Synthesis,
Inc., and many others.
1. Purification and in vitro analysis of recombinant nucleic acids and
..n., i.... er.~; ii or
Once DNA shuffling has been performed, the resulting library of recombinant
polynucleotides can be subjected to purification and preliminary analysis in
vitro, in order to
identify the most promising candidate recombinant nucleic acids.
Advantageously, the
assays can be practiced in a high-throughput format. For example, to purify
individual
shuffled recombinant antigens, clones can robotically picked into 96-well
formats, grown,
10 and, if desired, frozen for storage.
Whole cell lysates (V-antigen), periplasmic extracts, or culture supernatants
(toxins) can be assayed directly by ELISA as described below, but high
throughput
purification is sometimes also needed. Affinity chromatography using
immobilized
antibodies or incorporation of a small nonimmunogenic affinity tag such as a
hexahistidine
15 peptide with immobilized metal affinity chromatography will allow rapid
protein
purification. High binding-capacity reagents with 96-well filter bottom plates
provide a high
throughput purification process. The scale of culture and purification will
depend on protein
yield, but initial studies will require less than 50 micrograms of protein.
Antigens showing
improved properties can be purified in larger scale by FPLC for re-assay and
animal
20 challenge studies.
In some embodiments, the shuffled antigen-encoding polynucleotides are
assayed as genetic vaccines. Genetic vaccine vectors containing the shuffled
antigen
sequences can be prepared using robotic colony picking and subsequent robotic
plasmid
purification. Robotic plasmid purification protocols are available that allow
purification of
25 600-800 plasmids per day. The quantity and purity of the DNA can also be
analyzed in 96-
well plates, for example. In a presently preferred embodiment, the amount of
DNA in each
sample is robotically normalized, which can significantly reduce the variation
between
different batches of vectors.
Once the proteins and/or nucleic acids are picked and purified as desired,
they
30 can be subjected to any of a number of in vitro analysis methods. Such
screenings include,
for example, phage display, flow cytometry, and ELISA assays to identify
antigens that are
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCTNS99/02944
51
efficiently expressed and have multiple epitopes and a proper folding pattern.
In the case of
bacterial toxins, the libraries may also be screened for reduced toxicity in
mammalian cells.
As one example, to identify recombinant antigens that are cross-reactive, one
can use a panel of monoclonal antibodies for screening. A humoral immune
response
generally targets multiple regions of antigenic proteins. Accordingly,
monoclonal antibodies
can be raised against various regions of immunogenic proteins (Alving et al.
(1995)
Immunol. Rev. 145: 5). In addition, there are several examples of monoclonal
antibodies that
only recognize one strain of a given pathogen, and by definition, different
serotypes of
pathogens are recognized by different sets of antibodies. For example, a panel
of
monoclonal antibodies have been raised against VEE envelope proteins, thus
providing a
means to recognize different subtypes of the virus (Roehrig and Bolin (1997)
J. Clin.
Microbiol. 35: 1887). Such antibodies, combined with phage display and ELISA
screening,
can be used to enrich recombinant antigens that have epitopes from multiple
pathogen
strains. Flow cytometry based cell sorting will further allow for the
selection of variants that
are most efficiently expressed.
Phage display provides a powerful method for selecting proteins of interest
from large libraries (Bass et al. (1990) Proteins: Struct. Funct. Genet. 8:
309; Lowman and
Wells (1991) Methods: A Companion to Methods Enz. 3(3);205-216. Lowman and
Wells
(1993) J. Mol. Biol. 234;564-578). Some recent reviews on the phage display
technique
include, for example, McGregor ( 1996) Mol Biotechnol. 6(2):155-62; Dunn (
1996) Curr.
Opin. Biotechnol. 7(5}:547-53; Hill et al. (1996) Mol Microbiol 20(4):685-92;
Phage
Display of Peptides and Proteins: A Laboratory Manual. BK. Kay, J. Winter, J,
McCafferty
eds., Academic Press 1996; O'Neil et al. (1995) Curr. Opin. Struct. Biol.
5(4):443-9;
Phizicky et al. ( 1995) Microbiol Rev. 59( 1 ):94-123; Clackson et al. ( 1994)
Trends
Biotechnol. 12(5):173-84; Felici et al. (1995) Biotechnol. Annu. Rev. 1:149-
83; Burton
(1995) Immunotechnology 1(2):87-94.) See,also, Cwirla et al., Proc. Natl.
Acad. Sci. USA
87: 6378-6382 (1990); Devlin et al., Science 249: 404-406 (1990), Scott &
Smith, Science
249: 386-388 (1990); Ladner et al., US 5,571,698. Each phage particle displays
a unique
variant protein on its surface and packages the gene encoding that particular
variant. The
shuffled genes for the antigens are fused to a protein that is expressed on
the phage surface,
e.g., gene III of phage M13, and cloned into phagemid vectors. In a presently
preferred
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/OZ944
52
embodiment, a suppressible stop codon (e.g., an amber stop codon) separates
the genes so
that in a suppressing strain of E. coli, the antigen-gIIIp fusion is produced
and becomes
incorporated into phage particles upon infection with M13 helper phage. The
same vector
can direct production of the unfused antigen alone in a nonsuppressing E. coli
for protein
purification.
The genetic packages most frequently used for display libraries are
bacteriophage, particularly filamentous phage, and especially phage M 13, Fd
and F 1. Most
work has involved inserting libraries encoding polypeptides to be displayed
into either gIII
or gVIII of these phage forming a fusion protein. See, e.g., Dower, WO
91/19818; Devlin,
WO 91/18989; MacCafferty, WO 92/01047 (gene III); Huse, WO 92/06204; Kang, WO
92/18619 (gene VIII). Such a fusion protein comprises a signal sequence,
usually but not
necessarily, from the phage coat protein, a polypeptide to be displayed and
either the gene III
or gene VIII protein or a fragment thereof. Exogenous coding sequences are
often inserted
at or near the N-terminus of gene III or gene VIII although other insertion
sites are possible.
Eukaryotic viruses can be used to display polypeptides in an analogous
manner. For example, display of human heregulin fused to gp70 of Moloney
murine
leukemia virus has been reported by Han et al., Proc. Natl. Acad. Sci. USA 92:
9747-9751
(1995). Spores can also be used as replicable genetic packages. In this case,
polypeptides
are displayed from the outer surface of the spore. For example, spores from B.
subtilis have
been reported to be suitable. Sequences of coat proteins of these spores are
provided by
Donovan et al., J. Mol. Biol. 196, 1-10 (1987). Cells can also be used as
replicable genetic
packages. Polypeptides to be displayed are inserted into a gene encoding a
cell protein that
is expressed on the cells surface. Bacterial cells including Salmonella
typhimurium, Bacillus
subtilis, Pseudomonas aeruginosa, Vibrio cholerae, Klebsiella pneumonia,
Neisseria
gonorrhoeae, Neisseria meningitides, Bacteroides nodosus, Moraxella bovis, and
especially
Escherichia coli are preferred. Details of outer surface proteins are
discussed by Ladner et
al., US 5,571,698 and references cited therein. For example, the lama protein
of E. coli is
suitable.
A basic concept of display methods that use phage or other replicable genetic
package is the establishment of a physical association between DNA encoding a
polypeptide
to be screened and the polypeptide. This physical association is provided by
the replicable
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99141383 PC1'/US99/OZ944
53
genetic package, which displays a polypeptide as part of a capsid enclosing
the genome of
the phage or other package, wherein the polypeptide is encoded by the genome.
The
establishment of a physical association between polypeptides and their genetic
material
allows simultaneous mass screening of very large numbers of phage bearing
different
S polypeptides. Phage displaying a polypeptide with affinity to a target,
e.g., a receptor, bind
to the target and these phage are enriched by affinity screening to the
target. The identity of
polypeptides displayed from these phage can be determined from their
respective genomes.
Using these methods a polypeptide identified as having a binding affinity for
a desired target
can then be synthesized in bulk by conventional means, or the polynucleotide
that encodes
the peptide or polypeptide can be used as part of a genetic vaccine.
Variants with specific binding properties, in this case binding to family-
specific antibodies, are easily enriched by panning with immobilized
antibodies. Antibodies
specific for a single family are used in each round of panning to rapidly
select variants that
have multiple epitopes from the antigen families. For example, A-family
specific antibodies
can be used to select those shuffled clones that display A-specific epitopes
in the first round
of panning. A second round of panning with B-specific antibodies will select
from the "A"
clones those that display both A- and B-specific epitopes. A third round of
panning with C-
specific antibodies will select for variants with A, B, and C epitopes. A
continual selection
exists during this process for clones that express well in E. coli and that
are stable throughout
the selection. Improvements in factors such as transcription, translation,
secretion, folding
and stability are often observed and will enhance the utility of selected
clones for use in
vaccine production.
Phage ELISA methods can be used to rapidly characterize individual variants.
These assays provide a rapid method for quantitation of variants without
requiring
purification of each protein. Individual clones are anrayed into 96-well
plates, grown, and
frozen for storage. Cells in duplicate plates are infected with helper phage,
grown overnight
and pelleted by centrifugation. The supernatants containing phage displaying
particular
variants are incubated with immobilized antibodies and bound clones are
detected by anti-
M13 antibody conjugates. Titration series of phage particles, immobilized
antigen, andlor
soluble antigen competition binding studies are al! highly effective means to
quantitate
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/02944
54
protein binding. Variant antigens displaying multiple epitopes will be further
studied in
appropriate animal challenge models.
Several groups have reported an in vitro ribosome display system for the
screening and selection of mutant proteins with desired properties from large
libraries. This
S technique can be used similarly to phage display to select or enrich for
variant antigens with
improved properties such as broad cross reactivity to antibodies and improved
folding (see,
e.g., Hanes et al. (1997) Proc. Nat'l. Acad. Sci. USA 94(10):4937-42;
Mattheakis et al.
( 1994) Proc. Nat '1. Acad. Sci. USA 91 ( 19):9022-6; He et al. ( 1997) Nucl.
Acids Res.
25(24):5132-4; Nemoto et al. (1997) FEBSLett. 414(2}:405-8).
Other display methods exist to screen antigens for improved properties such
as increased expression levels, broad cross reactivity, enhanced folding and
stability. These
include, but are not limited to display of proteins on intact E. coli or other
cells (e.g.,
Francisco et al. (1993) Proc. Nat'l. Acad. Sci. USA 90: 1044-10448; Lu et al.
(1995)
BiolTechnology 13: 366-372). Fusions of shuffled antigens to DNA-binding
proteins can
link the antigen protein to its gene in an expression vector (Schatz et al. (
1996) Methods
Enrymol. 267: 171-91; Gates et al. (1996) J. Mol. Biol. 255: 373-86.}
The various display methods and ELISA assays can be used to screen for
shuffled antigens with improved properties such as presentation of multiple
epitopes,
improved immunogenicity, increased expression levels, increased folding rates
and
efficiency, increased stability to factors such as temperature, buffers,
solvents, improved
purification properties, etc. Selection of shuffled antigens with improved
expression, folding,
stability and purification profile under a variety of chromatographic
conditions can be very
important improvements to incorporate for the vaccine manufacturing process.
To identify recombinant antigenic polypeptides that exhibit improved
expression in a host cell, flow cytometry is a useful technique. Flow
cytometry provides a
method to efficiently analyze the functional properties of millions of
individual cells. One
can analyze the expression levels of several genes simultaneously, and flow
cytometry-based
cell sorting allows for the selection of cells that display properly expressed
antigen variants
on the cell surface or in the cytoplasm. Very large numbers (>10~) of cells
can be evaluated
in a single vial experiment, and the pool of the best individual sequences can
be recovered
from the sorted cells. These methods are particularly useful in the case of,
for example,
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCTNS99/OZ944
Hantaan virus glycoproteins, which are generally very poorly expressed in
mammalian cells.
This approach provides a general solution to improve expression levels of
pathogen antigens
in mammalian cells, a phenomenon that is critical for the function of genetic
vaccines.
To use flow cytometry to analyze polypeptides that are not expressed on the
5 cell surface, one can engineer the recombinant polynucleotides in the
library such that the
polynucieotide is expressed as a fusion protein that has a region of amino
acids which is
targeted to the cell membrane. For example, the region can encode a
hydrophobic stretch of
C-terminal amino acids which signals the attachment of a phosphoinositol-
glycan (PIG)
terminus on the expressed protein and directs the protein to be expressed on
the surface of
10 the transfected cell (Whitehorn et al. (1995) Biotechnology (N Y) 13:1215-
9). With an
antigen that is naturally a soluble protein, this method will likely not
affect the three
dimensional folding of the protein in this engineered fusion with a new C-
terminus. With an
antigen that is naturally a transmembrane protein (e.g., a surface membrane
protein on
pathogenic viruses, bacteria, protozoa or tumor cells) there are at least two
possibilities.
15 First, the extracellular domain can be engineered to be in fusion with the
C-terminal
sequence for signaling PIG-linkage. Second, the protein can be expressed in
toto relying on
the signalling of the host cell to direct it efficiently to the cell surface.
In a minority of cases,
the antigen for expression will have an endogenous PIG terminal linkage (e.g.,
some
antigens of pathogenic protozoa).
20 Those cells expressing the antigen can be identified with a fluorescent
monoclonal antibody specific for the C-terminal sequence on PIG-linked forms
of the
surface antigen. FACS analysis allows quantitative assessment of the level of
expression of
the correct form of the antigen on the cell population. Cells expressing the
maximal level of
antigen are sorted and standard molecular biology methods are used to recover
the plasmid
25 DNA vaccine vector that conferred this reactivity. An alternative procedure
that allows
purification of all those cells expressing the antigen (and that may be useful
prior to loading
onto a cell sorter since antigen expressing cells may be a very small minority
population), is
to rosette or pan-purify the cells expressing surface antigen. Rosettes can be
formed
between antigen expressing cells and erythrocytes bearing covalently coupled
antibody to
30 the relevant antigen. These are readily purified by unit gravity
sedimentation. Panning of the
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCTNS99/02944
56
cell population over petri dishes bearing immobilized monoclonal antibody
specific for the
relevant antigen can also be used to remove unwanted cells.
In the high throughput assays of the invention, it is possible to screen up to
several thousand different shuffled variants in a single day. For example,
each well of a
microtiter plate can be used to run a separate assay, or, if concentration or
incubation time
effects are to be observed, every 5-10 wells can test a single variant. Thus,
a single standard
microtiter plate can assay about 100 (e.g., 96) reactions. If 1536 well plates
are used, then a
single plate can easily assay from about 100 to about 1 S00 different
reactions. It is possible
to assay several different plates per day; assay screens for up to about 6,000-
20,000 different
assays {i.e., involving different nucleic acids, encoded proteins,
concentrations, etc.) is
possible using the integrated systems of the invention. More recently,
microfluidic
approaches to reagent manipulation have been developed, e.g., by Caliper
Technologies
(Palo Alto, CA).
In one aspect, library members, e.g., cells, viral plaques, or the like, are
separated on solid media to produce individual colonies (or plaques). Using an
automated
colony picker (e.g., the Q-bot, Genetix, U.K.), colonies or plaques are
identified, picked, and
up to 10,000 different mutants inoculated into 96 well microtiter dishes,
optionally
containing glass balls in the wells to prevent aggregation. The Q-bot does not
pick an entire
colony but rather inserts a pin through the center of the colony and exits
with a small
sampling of cells (or viruses in plaque applications). The time the pin is in
the colony, the
number of dips to inoculate the culture medium, and the time the pin is in
that medium each
effect inoculum size, and each can be controlled and optimized. The uniform
process of the
Q-bot decreases human handling error and increases the rate of establishing
cultures
(roughly 10,000/4 hours}. These cultures are then shaken in a temperature and
humidity
controlled incubator. The glass balls in the microtiter plates act to promote
uniform aeration
of cells dispersal of cells, or the like, similar to the blades of a
fermentor. Clones from
cultures of interest can be cloned by limiting dilution. Plaques or cells
constituting Libraries
can also be screened directly for production of proteins, either by detecting
hybridization,
protein activity, protein binding to antibodies, or the like.
The ability to detect a subtle increase in the performance of a shuffled
library
member over that of a parent strain relies on the sensitivity of the assay.
The chance of
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/02944
57
finding the organisms having an improvement in ability to induce an immune
response is
increased by the number of individual mutants that can be screened by the
assay. To increase
the chances of identifying a pool of sufficient size, a prescreen that
increases the number of
mutants processed by 10-fold can be used. The goal of the prescreen will be to
quickly
identify mutants having equal or better product titers than the parent
strains) and to move
only these mutants forward to liquid cell culture for subsequent analysis.
A number of well known robotic systems have also been developed for
solution phase chemistries useful in assay systems. These systems include
automated
workstations like the automated synthesis apparatus developed by Takeda
Chemical
Industries, LTD. (Osaka, Japan) and many robotic systems utilizing robotic
arms (Zymate II,
Zymark Corporation, Hopkinton, Mass.; Orca, Hewlett-Packard, Palo Alto,
Calif.) which
mimic the manual synthetic operations performed by a scientist. Any of the
above devices
are suitable for use with the present invention, e.g., for high-throughput
screening of
molecules encoded by codon-altered nucleic acids. The nature and
implementation of
modifications to these devices (if any) so that they can operate as discussed
herein with
reference to the integrated system will be apparent to persons skilled in the
relevant art.
High throughput screening systems are commercially available (see, e.g.,
Zymark Corp., Hopkinton, MA; Air Technical Industries, Mentor, OH; Beckman
Instruments, Inc. Fullerton, CA; Precision Systems, Inc., Natick, MA, etc.).
These systems
typically automate entire procedures including all sample and reagent
pipetting, liquid
dispensing, timed incubations, and final readings of the microplate in
detectors} appropriate
for the assay. These configurable systems provide high throughput and rapid
start up as well
as a high degree of flexibility and customization.
The manufacturers of such systems provide detailed protocols the various
high throughput. Thus, for example, Zymark Corp. provides technical bulletins
describing
screening systems for detecting the modulation of gene transcription, ligand
binding, and the
like. Microfluidic approaches to reagent manipulation have also been
developed, e.g., by
Caliper Technologies (Palo Alto, CA).
Optical images viewed (and, optionally, recorded) by a camera or other
recording device (e.g., a photodiode and data storage device} are optionally
further processed
in any of the embodiments herein, e.g., by digitizing the image and/or storing
and analyzing
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCTNS99I02944
58
the image on a computer. As noted above, in some applications, the signals
resulting from
assays are florescent, making optical detection approaches appropriate in
these instances. A
variety of commercially available peripheral equipment and software is
available for
digitizing, storing and analyzing a digitized video or digitized optical
image, e.g., using PC
(Intel x86 or Pentium chip- compatible DOS , OS2 WINDOWS , WINDOWS NT or
WINDOWS95 based machines), MACINTOSH , or UNIX based (e.g., SUN work station)
computers.
One conventional system carries light from the assay device to a cooled
charge-coupled device (CCD) camera, in common use in the art. A CCD camera
includes an
array of picture elements (pixels). The light from the specimen is imaged on
the CCD.
Particular pixels corresponding to regions of the specimen (e.g., individual
hybridization
sites on an anray of biological polymers) are sampled to obtain light
intensity readings for
each position. Multiple pixels are processed in parallel to increase speed.
The apparatus and
methods of the invention are easily used for viewing any sample, e.g., by
fluorescent or dark
field microscopic techniques.
Integrated systems for analysis in the present invention typically include a
digital computer with high-throughput liquid control software, image analysis
software, data
interpretation software, a robotic liquid control armature for transferring
solutions from a
source to a destination operably linked to the digital computer, an input
device (e.g., a
computer keyboard) for entering data to the digital computer to control high
throughput
liquid transfer by the robotic liquid control armature and, optionally, an
image scanner for
digitizing label signals from labeled assay component. The image scanner
interfaces with
the image analysis software to provide a measurement of optical intensity.
Typically, the
intensity measurement is interpreted by the data interpretation software to
show whether the
optimized recombinant antigenic polypeptide products are produced.
2. Antigen Library Immunization
In a presently preferred embodiment, antigen library immunization (ALI) is
used to identify optimized recombinant antigens that have improved
immunogenicity. ALI
involves introduction of the library of recombinant antigen-encoding nucleic
acids, or the
recombinant antigens encoded by the shuffled nucleic acids, into a test
animal. The animals
are then subjected to in vivo challenge using live pathogens. Neutralizing
antibodies and
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCTNS99/OZ944
59
cross-protective immune responses are studied after immunization with the
entire libraries,
pools and/or individual antigen variants.
Methods of immunizing test animals are well known to those of skill in the
art. In presently preferred embodiments, test animals are immunized twice or
three times at
two week intervals. One week after the last immunization, the animals are
challenged with
live pathogens (or mixtures of pathogens), and the survival and symptoms of
the animals is
followed. Immunizations using test animal challenge are described in, for
example,
Roggenkamp et al. ( 1997) Inject. Immun. 65: 446; Woody et al. ( 1997) Vaccine
2: 133;
Agren et al. (1997) J. Immunol. 158: 3936; Konishi et al. (1992) Virology 190:
454; Kinney
et al. (1988) J. Virol. 62: 4697; Iacono-Connors et al. (1996) Virus Res. 43:
125; Kochel et
al. (1997) Vaccine 15: 547; and Chu et al. (1995) J. Yirol. 69: 6417.
The immunizations can be performed by injecting either the recombinant
polynucleotides themselves, i.e., as a genetic vaccine, or by immunizing the
animals with
polypeptides encoded by the recombinant polynucleotides. Bacterial antigens
are typically
screened primarily as recombinant proteins, whereas viral antigens are
preferably analyzed
using genetic vaccinations.
To dramatically reduce the number of experiments required to identify
individual antigens having improved immunogenic properties, one can use
pooling and
deconvolution, as diagrammed in Figure 6. Pools of recombinant nucleic acids,
or
polypeptides encoded by the recombinant nucleic acids, are used to immunize
test animals.
Those pools that result in protection against pathogen challenge are then
subdivided and
subjected to additional analysis. The high throughput in vitro approaches
described above
can be used to identify the best candidate sequences for the in vivo studies.
The challenge models that can be used to screen for protective antigens
include pathogen and toxin models, such as Yersinia bacteria, bacterial toxins
(such as
Staphylococcal and Streptococcal enterotoxins, E.colilV. cholerae
enterotoxins), Venezuelan
equine encephalitis virus (VEE), Flaviviruses (Japanese encephalitis virus,
Tick-borne
encephalitis virus, Dengue virus), Hantaan virus, Herpes simplex, influenza
virus (e.g.,
Influenza A virus), Vesicular Stomatitis Virus, Pseudomonas aeruginosa,
Salmonella
typhimurium, Escherichia coli, Klebsiella pneumoniae, Toxoplasma gondii,
Plasmodium
yoelii, Herpes simplex, influenza virus (e.g., Influenza A virus), and
Vesicular Stomatitis
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PC'f/US99/02944
Virus. However, the test animals can also be challenged with tumor cells to
enable
screening of antigens that efficiently protect against malignancies.
Individual shuffled
antigens or pools of antigens are introduced into the animals intradermally,
intramuscularly,
intravenously, intratracheaily, anally, vaginally, orally, or
intraperitoneally and antigens that
can prevent the disease are chosen, when desired, for further rounds of
shuffling and
selection. Eventually, the most potent antigens, based on in vivo data in test
animals and
comparative in vitro studies in animals and man, are chosen for human trials,
and their
capacity to prevent and treat human diseases is investigated.
In some embodiments, antigen library immunization and pooling of
10 individual clones is used to immunize against a pathogen strain that was
not included in the
sequences that were used to generate the library. The level of crossprotection
provided by
different strains of a given pathogen can significantly. However, homologous
titer is always
higher than heterologous titer. Pooling and deconvolution is especially
efficient in models
where minimal protection is provided by the wild-type antigens used as
starting material for
15 shuffling (for example minimal protection by antigens A and B against
strain C in Figure
3B). This approach can be taken, for example, when evolving the V-antigen of
YerSinae or
Hantaan virus glycoproteins.
In some embodiments, the desired screening involves analysis of the immune
response based on immunological assays known to those skilled in the art.
Typically, the
20 test animals are first immunized and blood or tissue samples are collected
for example one to
two weeks after the last immunization. These studies enable one to one can
measure
immune parameters that correlate to protective immunity, such as induction of
specific
antibodies (particularly IgG) and induction of specific T lymphocyte
responses, in addition
to determining whether an antigen or pools of antigens provides protective
immunity.
25 Spleen cells or peripheral blood mononuclear cells can be isolated from
immunized test
animals and measured for the presence of antigen-specific T cells and
induction of cytokine
synthesis. ELISA, ELISPOT and cytoplasmic cytokine staining, combined with
flow
cytometry, can provide such information on a single-cell level.
Common immunological tests that can be used to identify the efficacy of
30 immunization include antibody measurements, neutralization assays and
analysis of
activation levels or frequencies of antigen presenting cells or lymphocytes
that are specific
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/02944
61
for the antigen or pathogen. The test animals that can be used in such studies
include, but
are not limited to, mice, rats, guinea pigs, hamsters, rabbits, cats, dogs,
pigs and monkeys.
Monkey is a particularly useful test animal because the MHC molecules of
monkeys and
humans are very similar.
Virus neutralization assays are useful for detection of antibodies that not
only
specifically bind to the pathogen, but also neutralize the function of the
virus. These assays
are typically based on detection of antibodies in the sera of immunized animal
and analysis
of these antibodies for their capacity to inhibit viral growth in tissue
culture cells. Such
assays are known to those skilled in the art. One example of a virus
neutralization assay is
described by Dolin R (J. Infect. Dis. 1995, 172:1175-83). Virus neutralization
assays
provide means to screen for antigens that also provide protective immunity.
In some embodiments, shuffled antigens are screened for their capacity to
induce T cell activation in vivo. More specifically, peripheral blood
mononuclear cells or
spleen cells from injected mice can be isolated and the capacity of cytotoxic
T lymphocytes
to lyse infected, autologous target cells is studied. The spleen cells can be
reactivated with
the specific antigen in vitro. in addition, T helper cell activation and
differentiation is
analyzed by measuring cell proliferation or production of TH1 (IL-2 and IFN-y)
and TH2 (IL-
4 and IL-5) cytokines by ELISA and directly in CD4+ T cells by cytoplasmic
cytokine
staining and flow cytometry. Based on the cytokine production profile, one can
also screen
for alterations in the capacity of the antigens to direct TH1/TH2
differentiation (as evidenced,
for example, by changes in ratios of IL-4/IFN-y, IL-4/IL-2, IL-S/IFN-y, IL-
5/IL-2, IL-
13/IFN-y, IL-13/IL-2). The analysis of the T cell activation induced by the
antigen variants
is a very useful screening method, because potent activation of specific T
cells in vivo
correlates to induction of protective immunity.
The frequency of antigen-specific CD8+ T cells in vivo can also be directly
analyzed using tetramers of MHC class I molecules expressing specific peptides
derived
from the corresponding pathogen antigens (Ogg and McMichael, Curr. Opin.
Immunol.
1998, 10:393-6; Altman et al., Science 1996, 274:94-6). The binding of the
tetramers can
be detected using flow cytometry, and will provide information about the
efficacy of the
shuffled antigens to induce activation of specific T cells. For example, flow
cytometry and
tetramer stainings provide an efficient method of identifying T cells that are
specific to a
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99141383 PCT/EJS99/02944
62
given antigen or peptide. Another method involves panning using plates coated
with
tetramers with the specific peptides. This method allows large numbers of
cells to be
handled in a short time, but the method only selects for highest expression
levels. The
higher the frequency of antigen-specific T cells in vivo is, the more
efficient the
immunization has been, enabling identification of the antigen variants that
have the most
potent capacity to induce protective immune responses. These studies are
particularly useful
when conducted in monkeys, or other primates, because the MHC class I
molecules of
humans mimic those of other primates more closely than those of mice.
Measurement of the activation of antigen presenting cells (APC) in response
to immunization by antigen variants is another useful screening method.
Induction of APC
activation can be detected based on changes in surface expression levels of
activation
antigens, such as B7-1 (CD80), B7-2 (CD86), MHC class I and II, CD14, CD23,
and Fc
receptors, and the like.
Shuffled cancer antigens that induce cytotoxic T cells that have the capacity
to kill cancer cells can be identified by measuring the capacity of T cells
derived from
immunized animals to kill cancer cells in vitro. Typically the cancer cells
are first labeled
with radioactive isotopes and the release of radioactivity is an indication of
tumor cell killing
after incubation in the presence of T cells from immunized animals. Such
cytotoxicity
assays are known in the art.
An indication of the efficacy of an antigen to activate T cells specific for,
for
example, cancer antigens, allergens or autoantigens, is also the degree of
skin inflammation
when the antigen is injected into the skin of a patient or test animal. Strong
inflammation is
correlated with stmng activation of antigen-specific T cells. Improved
activation of tumor-
specific T cells may lead to enhanced killing of the tumors. In case of
autoantigens, one can
add immunomodulators that skew the responses towards TH2, whereas in the case
of
allergens a TH1 response is desired. Skin biopsies can be taken, enabling
detailed studies of
the type of immune response that occurs at the sites of each injection {in
mice and monkeys
large numbers of injections/antigens can be analyzed). Such studies include
detection of
changes in expression of cytokines, chemokines, accessory molecules, and the
like, by cells
upon injection of the antigen into the skin.
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCTNS99/02944
63
To screen for antigens that have optimal capacity to activate antigen-specific
T cells, peripheral blood mononuclear cells from previously infected or
immunized humans
individuals can be used. This is a particularly useful method, because the MHC
molecules
that will present the antigenic peptides are human MHC molecules. Peripheral
blood
mononuclear cells or purified professional antigen-presenting cells (APCs) can
be isolated
from previously vaccinated or infected individuals or from patients with acute
infection with
the pathogen of interest. Because these individuals have increased frequencies
of pathogen-
specific T cells in circulation, antigens expressed in PBMCs or purified APCs
of these
individuals will induce proliferation and cytokine production by antigen-
specific CD4+ and
CD8+ T cells. Thus, antigens that simultaneously harbor epitopes from several
antigens can
be recognized by their capacity to stimulate T cells from various patients
infected or
immunized with different pathogen antigens, cancer antigens, autoantigens or
allergens. One
buffy coat derived from a blood donor contains lymphocytes from 0.5 liters of
blood, and up
to 104 PBMC can be obtained, enabling very large screening experiments using T
cells from
one donor.
When healthy vaccinated individuals (lab volunteers) are studied, one can
make EBV-transformed B cell lines from these individuals. These cell lines can
be used as
antigen presenting cells in subsequent experiments using blood from the same
donor; this
reduces interassay and donor-to-donor variation. In addition, one can make
antigen-specific
T cell clones, after which antigen variants are introduced to EBV transformed
B cells. The
efficiency with which the transformed B cells induce proliferation of the
specific T cell
clones is then studied. When working with specific T cell clones, the
proliferation and
cytokine synthesis responses are significantly higher than when using total
PBMCs, because
the frequency of antigen-specific T cells among PBMC is very low.
CTL epitopes can be presented by most cells types since the class I major
histocompatibility complex (MHC) surface glycoproteins are widely expressed.
Therefore,
transfection of cells in culture by libraries of shuffled antigen sequences in
appropriate
expression vectors can lead to class I epitope presentation. If specific CTLs
directed to a
given epitope have been isolated from an individual, then the co-culture of
the transfected
presenting cells and the CTLs can lead to release by the CTLs of cytokines,
such as IL-2,
IFN-y, or TNF, if the epitope is presented. Higher amounts of released TNF
will correspond
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/02944
64
to more efficient processing and presentation of the class I epitope from the
shuffled,
evolved sequence. Shuffled antigens that induce cytotoxic T cells that have
the capacity to
kill infected cells can also be identified by measuring the capacity of T
cells derived from
immunized animals to kill infected cells in vitro. Typically the target cells
are first labeled
with radioactive isotopes and the release of radioactivity is an indication of
target cell killing
after incubation in the presence of T cells from immunized animals. Such
cytotoxicity
assays are known in the art.
A second method for identifying optimized CTL epitopes does not require the
isolation of CTLs reacting with the epitope. In this approach, cells
expressing class I MHC
surface glycoproteins are transfected with the library of evolved sequences as
above. ARer
suitable incubation to allow for processing and presentation, a detergent
soluble extract is
prepared from each cell culture and after a partial purification of the MHC-
epitope complex
(perhaps optional) the products are submitted to mass spectrometry (Henderson
et al. (1993)
Proc. Nat'l. Acad. Sci. USA 90: 10275-10279). Since the sequence is known of
the epitope
1 S whose presentation to be increased, one can calibrate the mass spectrogram
to identify this
peptide. In addition, a cellular protein can be used for internal calibration
to obtain a
quantitative result; the cellular protein used for internal calibration could
be the MHC
molecule itself. Thus one can measure the amount of peptide epitope bound as a
proportion
of the MHC molecules.
Use of Recombinant Multivalent Antigens
The multivalent antigens of the invention are useful for treating and/or
preventing the various diseases and conditions with which the respective
antigens are
associated. For example, the multivalent antigens can be expressed in a
suitable host cell and
are administered in polypeptide form. Suitable formulations and dosage regimes
for vaccine
delivery are well known to those of skill in the art.
In presently preferred embodiments, the optimized recombinant
polynucleotides that encode improved allergens are used in conjunction with a
genetic
vaccine vector. The choice of vector and components can also be optimized for
the particular
purpose of treating allergy. For example, the polynucleotide that encodes the
recombinant
antigenic polypeptide can be placed under the control of a promoter, e.g., a
high activity or
tissue-specific promoter. The promoter used to express the antigenic
polypeptide can itself
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/OZ944
be optimized using recombination and selection methods analogous to those
described
herein. The vector can contain immunostimulatory sequences such as are
described in
copending, commonly assigned US Patent Application Serial No. , entitled
"Optimization of Immunomodulatory Molecules," filed as TTC Attorney Docket No.
18097-
5 030300US on February 10, 1999. A vector engineered to direct a TH 1 response
is preferred
for many of the immune responses mediated by the antigens described herein
(see, e.g.,
copending, commonly assigned US Patent Application Serial No. , entitled
"Genetic Vaccine Vector Engineering," filed on February 10, 1999 as TTC
Attorney Docket
No. 18097-030100US). It is sometimes advantageous to employ a genetic vaccine
that is
10 targeted for a particular target cell type (e.g., an antigen presenting
cell or an antigen
processing cell); suitable targeting methods are described in copending,
commonly assigned
US patent application Serial No. , entitled "Targeting of Genetic Vaccine
Vectors," filed on February 10, 1999 as TTC Attorney Docket No. 18097-
030200US.
Genetic vaccines that encode the multivalent antigens described herein can be
15 delivered to a mammal (including humans) to induce a therapeutic or
prophylactic immune
response. Vaccine delivery vehicles can be delivered in vivo by administration
to an
individual patient, typically by systemic administration (e.g., intravenous,
intraperitoneal,
intramuscular, subdermaI, intracranial, anal, vaginal, oral, buccal route or
they can be
inhaled) or they can be administered by topical application. Alternatively,
vectors can be
20 delivered to cells ex vivo, such as cells expIanted from an individual
patient (e.g.,
lymphocytes, bone marrow aspirates, tissue biopsy) or universal donor
hematopoietic stem
cells, followed by reimplantation of the cells into a patient, usually after
selection for cells
which have incorporated the vector.
A large number of delivery methods are well known to those of skill in the
25 art. Such methods include, for example liposome-based gene delivery (Debs
and Zhu (1993)
WO 93/24640; Mannino and Gould-Fogerite (1988) BioTechniques 6(7): 682-691;
Rose
U.S. Pat No. 5,279,833; Brigham (1991) WO 91/06309; and Felgner et al. (1987)
Proc. Natl.
Acad Sci. USA 84: 7413-7414), as well as use of viral vectors (e.g.,
adenoviral (see, e.g.,
Bems et al. (1995) Ann. NYAcad Sci. 772: 95-104; Ali et al. (1994) Gene Ther.
1: 367-384;
30 and Haddada et al. (1995) Curr. Top. Microbiol. Immunol. 199 ( Pt 3): 297-
306 for review),
papillomaviral, retroviral (see, e.g., Buchscher et al. (1992) J. Yirol. 66(5)
2731-2739;
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/413$3 PCTNS99/OZ944
66
Johann et al. (1992) J. Virol. 66 (5):1635-1640 (1992); Sommerfelt et al.,
(1990) Virol.
176:58-59; Wilson et al. (1989) J. Virol. 63:2374-2378; Miller et al., J.
Virol. 65:2220-2224
(1991); Wong-Staal et al., PCT/US94/05700, and Rosenburg and Fauci (1993) in
Fundamentallmmunology, Third Edition Paul (ed) Raven Press, Ltd., New York and
the
references therein, and Yu et al., Gene Therapy ( 1994) supra. ), and adeno-
associated viral
vectors (see, West et al. (1987) Virology 160:38-47; Carter et al. (1989) U.S.
Patent No.
4,797,368; Carter et al. WO 93/24641 (1993); Kotin (1994) Human Gene Therapy
5:793-
801; Muzyczka {1994) J. Clin. Invst. 94:1351 and Samulski (supra) for an
overview of AAV
vectors; see also, Lebkowski, U.S. Pat. No. 5,/73,414; Tratschin et al. (1985)
Mol. Cell.
Biol. 5(11):3251-3260; Tratschin, et al. (1984) Mol. Cell. Biol., 4:2072-2081;
Hermonat and
Muzyczka (1984) Proc. Natl. Acad Sci. USA, 81:6466-6470; McLaughlin et al.
(1988) and
Samulski et al. (1989) J. Virol., 63:03822-3828), and the like.
"Naked" DNA and/or RNA that comprises a genetic vaccine can be
introduced directly into a tissue, such as muscle. See, e.g., USPN 5,580,859.
Other methods
such as "biolistic" or particle-mediated transformation (see, e.g., Sanford et
al., USPN
4,945,050; USPN 5,036,006) are also suitable for introduction of genetic
vaccines into cells
of a mammal according to the invention. These methods are useful not only for
in vivo
introduction of DNA into a mammal, but also for ex vivo modification of cells
for
reintroduction into a mammal. As for other methods of delivering genetic
vaccines, if
necessary, vaccine administration is repeated in order to maintain the desired
level of
immunomodulation.
Genetic vaccine vectors (e.g., adenoviruses, liposomes, papillomaviruses,
retroviruses, etc.) can be administered directly to the mammal for
transduction of cells in
vivo. The genetic vaccines obtained using the methods of the invention can be
formulated as
pharmaceutical compositions for administration in any suitable manner,
including parenteral
(e.g., subcutaneous, intramuscular, intradermal, or intravenous), topical,
oral, rectal,
intrathecal, buccal (e.g., sublingual), or local administration, such as by
aerosol or
transdermally, for prophylactic and/or therapeutic treatment. Pretreatment of
skin, for
example, by use of hair-removing agents, may be useful in transdermal
delivery. Suitable
methods of administering such packaged nucleic acids are available and well
known to those
of skill in the art, and, although more than one route can be used to
administer a particular
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/02944
67
composition, a particular route can often provide a more immediate and more
effective
reaction than another route.
Pharmaceutically acceptable carriers are determined in part by the particular
composition being administered, as well as by the particular method used to
administer the
composition. Accordingly, there is a wide variety of suitable formulations of
pharmaceutical
compositions of the present invention. A variety of aqueous carriers can be
used, e.g.,
buffered saline and the like. These solutions are sterile and generally free
of undesirable
matter. These compositions may be sterilized by conventional, well known
sterilization
techniques. The compositions may contain pharmaceutically acceptable auxiliary
substances
as required to approximate physiological conditions such as pH adjusting and
buffering
agents, toxicity adjusting agents and the like, for example, sodium acetate,
sodium chloride,
potassium chloride, calcium chloride, sodium lactate and the like. The
concentration of
genetic vaccine vector in these formulations can vary widely, and will be
selected primarily
based on fluid volumes, viscosities, body weight and the like in accordance
with the
particular mode of administration selected and the patient's needs.
Formulations suitable for oral administration can consist of (a) liquid
solutions, such as an effective amount of the packaged nucleic acid suspended
in diluents,
such as water, saline or PEG 400; (b) capsules, sachets or tablets, each
containing a
predetermined amount of the active ingredient, as liquids, solids, granules or
gelatin; (c}
suspensions in an appropriate liquid; and (d) suitable emulsions. Tablet forms
can include
one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn
starch, potato
starch, tragacanth, microcrystalline cellulose, acacia, gelatin, colloidal
silicon dioxide,
croscarmellose sodium, talc, magnesium stearate, stearic acid, and other
excipients,
colorants, fillers, binders, diluents, buffering agents, moistening agents,
preservatives,
flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible
carriers.
Lozenge forms can comprise the active ingredient in a flavor, usually sucrose
and acacia or
tragacanth, as well as pastilles comprising the active ingredient in an inert
base, such as
gelatin and glycerin or sucrose and acacia emulsions, gels, and the like
containing, in
addition to the active ingredient, carriers known in the art. It is recognized
that the genetic
vaccines, when administered orally, must be protected from digestion. This is
typically
accomplished either by complexing the vaccine vector with a composition to
render it
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/I1S99/02944
68
resistant to acidic and enzymatic hydrolysis or by packaging the vector in an
appropriately
resistant carrier such as a liposome. Means of protecting vectors from
digestion are well
known in the art. The pharmaceutical compositions can be encapsulated, e.g.,
in liposomes,
or in a formulation that provides for slow release of the active ingredient.
The packaged nucleic acids, alone or in combination with other suitable
components, can be made into aerosol formulations (e.g., they can be
"nebulized") to be
administered via inhalation. Aerosol formulations can be placed into
pressurized acceptable
propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.
Suitable formulations for rectal administration include, for example,
suppositories, which consist of the packaged nucleic acid with a suppository
base. Suitable
suppository bases include natural or synthetic triglycerides or paraffin
hydrocarbons. In
addition, it is also possible to use gelatin rectal capsules which consist of
a combination of
the packaged nucleic acid with a base, including, for example, liquid
triglycerides,
polyethylene glycols, and paraffin hydrocarbons.
I 5 Formulations suitable for parenteral administration, such as, for example,
by
intraarticular (in the joints), intravenous, intramuscular, intradenmal,
intraperitoneal, and
subcutaneous routes, include aqueous and non-aqueous, isotonic sterile
injection solutions,
which can contain antioxidants, buffers, bacteriostats, and solutes that
render the formulation
isotonic with the blood of the intended recipient, and aqueous and non-aqueous
sterile
suspensions that can include suspending agents, solubilizers, thickening
agents, stabilizers,
and preservatives. In the practice of this invention, compositions can be
administered, for
example, by intravenous infusion, orally, topically, intraperitoneally,
intravesically or
intrathecally. Parenteral administration and intravenous administration are
the preferred
methods of administration. The formulations of packaged nucleic acid can be
presented in
unit-dose or mufti-dose sealed containers, such as ampoules and vials.
Injection solutions and suspensions can be prepared from sterile powders,
granules, and tablets of the kind previously described. Cells transduced by
the packaged
nucleic acid can also be administered intravenously or parenterally.
The dose administered to a patient, in the context of the present invention
should be sufficient to effect a beneficial therapeutic response in the
patient over time. The
dose will be detenmined by the efficacy of the particular vector employed and
the condition
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/02944
69
of the patient, as well as the body weight or vascular surface area of the
patient to be treated.
The size of the dose also will be determined by the existence, nature, and
extent of any
adverse side-effects that accompany the administration of a particular vector,
or transduced
cell type in a particular patient.
In determining the effective amount of the vector to be administered in the
treatment or prophylaxis of an infection or other condition, the physician
evaluates vector
toxicities, progression of the disease, and the production of anti-vector
antibodies, if any. In
general, the dose equivalent of a naked nucleic acid from a vector is from
about 1 p.g to 1 mg
for a typical 70 kilogram patient, and doses of vectors used to deliver the
nucleic acid are
calculated to yield an equivalent amount of therapeutic nucleic acid.
Administration can be
accomplished via single or divided doses.
In therapeutic applications, compositions are administered to a patient
suffering from a disease (e.g., an infectious disease or autoimmune disorder)
in an amount
sufficient to cure or at least partially arrest the disease and its
complications. An amount
adequate to accomplish this is defined as a "therapeutically effective dose."
Amounts
effective for this use will depend upon the severity of the disease and the
general state of the
patient's health. Single or multiple administrations of the compositions may
be administered
depending on the dosage and frequency as required and tolerated by the
patient. In any
event, the composition should provide a sufficient quantity of the proteins of
this invention
to effectively treat the patient.
In prophylactic applications, compositions are administered to a human or
other mammal to induce an immune response that can help protect against the
establishment
of an infectious disease or other condition.
The toxicity and therapeutic efficacy of the genetic vaccine vectors provided
by the invention are determined using standard pharmaceutical procedures in
cell cultures or
experimental animals. One can determine the LDso (the dose lethal to SO% of
the
population) and the EDSa (the dose therapeutically effective in 50% of the
population) using
procedures presented herein and those otherwise known to those of skill in the
art.
A typical pharmaceutical composition for intravenous administration would
be about 0.1 to 10 mg per patient per day. Dosages fram 0.1 up to about 100 mg
per patient
per day may be used, particularly when the drug is administered to a secluded
site and not
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/02944
into the blood stream, such as into a body cavity or into a lumen of an organ.
Substantially
higher dosages are possible in topical administration. Actual methods for
preparing
parenterally administrable compositions will be known or apparent to those
skilled in the art
and are described in more detail in such publications as Remington's
Pharmaceutical
S Science, 15th ed., Mack Publishing Company, Easton, Pennsylvania (1980).
The multivalent antigenic polypeptides of the invention, and genetic vaccines
that express the polypeptides, can be packaged in packs, dispenser devices,
and kits for
administering genetic vaccines to a mammal. For example, packs or dispenser
devices that
contain one or more unit dosage forms are provided. Typically, instructions
for
10 administration of the compounds will be provided with the packaging, along
with a suitable
indication on the label that the compound is suitable for treatment of an
indicated condition.
For example, the label may state that the active compound within the packaging
is useful for
treating a particular infectious disease, autoimmune disorder, tumor, or for
preventing or
treating other diseases or conditions that are mediated by, or potentially
susceptible to, a
15 mammalian immune response.
EXAMPLES
The following examples are offered to illustrate, but not to limit the present
invention.
Example 1
20 Development Of Broad-Spectrum Vaccines Against Bacterial Pathogens And
Toxins
A. Evolution of Yersinia V-antigens
This Example describes the use of DNA shuffling to develop immunogens
that produce strong cross-protective immune responses against a variety of
Yersinia strains.
Passive immunization with anti-V-antigen antibodies or active immunization
with purified
25 V-antigen can provide protection from challenge with a virulent autologous
Yersinia species.
However, protection against heterologous species is limited (Motin et al.
(1994) Infect.
Immun. 62: 4192).
V-antigen genes from a variety of Yersinia strains, including serotypes of Y.
pestis, Y. enterocolitica, and Y. pseudotuberculosis are subjected to DNA
shuffling as
30 described herein. The Yersinia pesos V antigen coding sequence, for
example, is used as a
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/02944
71
query in a database search to identify homologous genes that can be used in a
family
shuffling format to obtain improved antigens. Results for a BLAST search of
GenBank and
EMBL databases are shown in Table I, in which each line represents a unique
sequence
entry listing the database, accession number, locus name, bit score and E
value. See, Altschul
et al. (1997) Nucleic Acids Res. 25:3389-3402, for a description of the search
algorithm).
Homologous antigens have been cloned and sequenced from a number of related
yet distinct
Yersinia strains and additional natural diversity is obtained by cloning
antigen genes from
other strains. These genes and others or fragments thereof are cloned by
methods such as
PCR, shuffled and screened for improved antigens.
Table 1
Sequences producing
sigaiiicant alignments


Database/Accession Gene ScoreE
No. (bits)Value


gbJM26405JYEPLCR Yersinia pestis IcrG, lcrV, and 1945 0.0
lcrH genes,
co


gb~AF053946~AF053946 Yersinia pestis plasmid pCDI, 1945 0.0
complete pla


emb~X96802~YPTPIVANT Y. pseudotuberculosis V antigen 1834 0.0
gene


gbJM57893JYEPLCRGVHP Yersinia pseudotuberculosis V-antigen1818 0.0


gbJAF080155JAF080155 Yersinia enterocolitica pYV LcrV1723 0.0
(lcrV)
antigen


embJX96801 JYE96PVANTY. enterocolitica V antigen gene,1667 0.0
strain Y-...


emb~X96799JYE108VANT Y. enterocolitica V antigen gene,1659 0.0
strain Y-...


embJX96800JYE527VANT Y, enterocolitica V antigen gene,1651 0.0
serotype ...


embJX96798JYE808VANT Y. enterocolitica V antigen gene,1643 0.0
strain
8081


embJX96796JYE314VANT Y. enterocolitica V antigen gene,1237 0.0
strain WA.


emb~X96797~YENCTVANT Y. enterocolirica V antigen gene,1221 0.0
strain
NCTC


SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/02944
72
Sequences producing
significant alignments


Database/Accession Gene Score E
No.


(bits)Value


gb~S38727~S38727 IcrGVH operon: lerV=V-antigen 365 9e-99
[Yersirria


pseudo.


Shuffled clones are selected by phage display and/or screened by ELISA to
identify those recombinant nucleic acids that encode polypeptides that have
multiple
epitopes corresponding to the different serotypes. The shuffled antigen genes
are cloned into
a filamentous phage genome for polyvalent phage display or a suitable phagemid
vector for
monovalent phage display. A typical protocol for panning antigens by phage
display is as
follows.
~ Coat an appropriate surface (e.g., Nunc Maxisoip tube or multiwell plate)
at 4°C overnight with the target antibody, usually at a concentration
of 1-
pg/ml in PBS or other suitable buffer
10 ~ Rinse and Block with PBSM (PBS + 3% nonfat dry milk) at 37°C for 1-
2
hr
~ Pre-block phage if needed (PBSM, RT 1 hr)
~ Rinse tube and allow phage to bind (usually 1 hr @ 37°C)
~ Can vary time, temp, buffer, add a competitive inhibitor, etc.
~ Wash extensively (15x) with PBST (PBS + 0.1% TWEEN20), then PBS
~ Elute bound phage with low pH (e.g., 10 mM glycine), 100 mM
triethylamine, competitive Iigand, protease, etc. and then neutralize pH if
needed.
Infect E. coli with eluted phage to transduce expression phagemid into
new host. Titer and plate for colonies on drug plates
~ Pool colonies into media, grow cells and infect with helper phage to
produce phage for next round
Phage ELISA assays are a useful method to rapidly evaluate single clones
after panning of libraries. Single colonies are picked in individual wells of
a multiwell plate
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 , PCT/US99/02944
73
containing 2YT media and grown as a master plate. A replicate plate is
infected with helper
phage and grown so that phage from a single well will display a single antigen
variant. A
suitable protocol for phage ELISA assays is as follows.
~ Coat microtiter plate with 50 pl of 1 ~g/ml target antibody 4 °C
overnight
~ Rinse and block with PBSM for 2 hrs @ 37 °C
~ Rinse, add preblocked phage and allow to bind 1 hr @ 37 °C
~ Wash plates with PBST 3x, then PBS 3x with 2 min soaks
~ Add HRP(or AP)-conjugated anti-M13 antibodies for 1 hr @ 37 °C
~ Add substrate and measure absorbance
~ Identify positive clones for further evaluation
ELISA assays can also be used to screen for individual antigens with multiple
epitopes or increased expression levels. Single colonies are picked in
individual wells of a
multiwell plate containing appropriate media and grown as a master plate so
that antigens
1 S produced from a single well are a single antigen variant. A replicate
plate is grown and
induced for protein production, e.g., by addition of 0.5 mM IPTG for Lac
repressor-based
systems and grown for an appropriate time for the antigen to be produced. At
this point a
crude antigen preparation is made which depends on the antigen and where it is
produced.
Secreted proteins can be evaluated by assaying the cell supernatants after
centrifugation.
Periplasmic proteins are often readily released from cells by simple
extraction into hyper- or
hypo-tonic buffers. Intracellularly produced proteins will require some form
of cell lysis
such as detergent treatment to release them. A suitable protocol for ELISA
assays is as
follows.
~ Coat microtiter plate with 50 pl of 1 ~g/ml target antibody 4 °C
overnight
~ Rinse and block with PBSM for 2 hrs @ 37 °C
~ Rinse, add antigen prep and allow to bind 1 hr @ 37 °C
~ Wash plates with PBST 3x
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/02944
74
~ Add HRP(or AP)-conjugated secondary antibody and incubate for 1 hr @
37 °C
~ Add appropriate substrate and measure absorbance
~ Identify positive clones for further evaluation
Antibodies specific for many of the various antigens are commercially
available (e.g., Toxin Technology, Inc, Sarasota, FL) or can be generated by
immunizing
suitable animals with purified antigens. Protein A or Protein G Sepharose
(Pharmacia) can
be used to purify immunoglobulins from the serum. Various affinity
purification schemes
can be used to further purify family-specific antibodies if needed such as
immobilization of
specific antigens to NHS-, CNBr-, or epoxy-activated sepharose beads. Other
related
antigens may be included soluble form to prevent binding and immobilization of
cross-
reactive antibodies.
The multivalent polypeptides that are identified by the initial screening
protocol are purified and subjected to in vivo screening. For example, the
shuffled antigens
1 S selected by a combination of any or none of these methods are purified and
used to
immunize animals, initially mice, which are then evaluated for improved immune
responses.
Typically I 0 micrograms of protein is injected to a suitable location with or
without
appropriate adjuvant, e.g., Alhydrogel (EM Seargent Pulp and Chemical, Inc.)
and the
animals are boosted with an additional dose after 2-4 weeks. At this point
serum samples is
drawn and evaluated by ELISA assay for the presence of antibodies that cross-
react against
multiple parental antigens. In this ELISA assay format the antigens are coated
onto
multiwell plates, then serial dilutions of each sera is allowed to bind. After
washing unbound
antibodies, a secondary HRP- or AP- conjugated antibody directed against the
appropriate
test antibody constant region, e.g., goat anti-mouse IgG Fc (Sigma) is bound.
After another
washing, the appropriate substrate is added, e.g., O-phenylenediamine (Sigma).
The
absorbance of each well is read by a plate reader at the appropriate
wavelength (e.g., 490 nm
for OPD) and those producing high antibody titers to multiple antigens are
selected for
further evaluation.
Additionally, the ability of antigens to generate neutralizing antibodies can
be
evaluated in an appropriate system. Antigen variants that elicit a broad cross-
reactive
response are evaluated further in a virulent challenge model with the
appropriate pathogenic
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/02944
organism. For example, the multivalent polypeptides are used to immunize mice,
which are
then challenged with live Yersinia bacteria. Those multivalent polypeptides
that protect
against the challenge are identified and purified.
B. Evolution of broad-spectrum vaccines against bacterial toxins
This Example describes the use of DNA shuffling to obtain multivalent
polypeptides that are ef~'ective in inducing an immune response against a
broad spectrum of
bacterial toxins.
1. Staphylococcus
The Group A Streptococci, which can cause diseases such as food poisoning,
10 toxic shock syndrome, and autoimmune disorders, are highly toxic by
inhalation. The family
of Group A Streptococcus toxins numbers about 30 related members, making this
group a
suitable target for family shuffling. Accordingly, this Example describes the
use of family
DNA shuffling to create chimeric proteins that are capable of eliciting broad
spectrum r
protection.
15 Nucleic acids that encode many diverse attenuated toxins are subjected to
DNA shuffling as described herein. Table 2 shows the output of a BLAST search
of
GenBank, PDL, EMBL, and Swissprot using the S. aureus enterotoxin B protein to
identify
homologous genes that may be used in a family shuffling format to obtain
improved
antigens.
Table 2
Sequences
producing
significant
alignments


Database/Accession Gene ScoreE
No. (bits)Value


sP > e_


P ap y ococcan ero oxen omp ex e-
Tri...


Pa y 1sE$p ap y ococcusaureus>gi p e-
Staphyl...


sP ~' a _


sP e-


sP e_


91 en oxen ap yococcus e-
ero


gl en oxen ap yococcus e-
ero


91 en oxen ap yococcus e-
ero


91 y y en oxen ap yococcus e-
ero


P K am , ecep or a am e-
a a
With
S...


91 y en oxen ap yococcus e-
ero


SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99141383 PCTlUS99/OZ944
7b
Sequences producing
significant
alignments


-
-


Database/Accession Gene Score E
No.


(bits)Value


panps~;y 5 ap y ococca n ero oxen , onoc e-


Ente...


gy (u9T52~T- ype en ero oxen ap e-
lyU6USl y


intermed...


gi (L en ero oxen ap y ococcus e-


s entero-toxin=pyrogem c oxm ap e-
v y


4446, P...


g1 4ib-i6Q superan igen rep ococcus e-


gi (u4s~)--superan igen rep oco e-


pyogenes] >...


gi)11951/4 (u48'793) -superan igen rep e-
oco


pyogenes]


sp YUS09
nrwav~aav a acu ra rnrr~.vic~7viC e-
~..IUCLL' 1~ r-


91 ype exo oxen rep ococ e-


PYogenesl >gi~...


piry - exo oxen ype precursor a a a e-
518793
-


Streptococcu...


pir exo oxen ype precursor a a a e-
8


Streptococcu...


pir exo oxen precursor a a a - e-
s
a


PYo...


91 ype exo oxen rep ococ e-
~


pyogenes]


pir)~wablsa s rep ococca pyrogenic exo ox e-
- n yp


precursor -...


sp Y Zx > e-


sP > e-


pr en ero oxen ap y ococcus aureus e-


P~ [I ap y ococcus aureus >gi p e-


Staphyl...


pir en ero oxen - ap y ococcus aureu e-


sP Y > e_


91 en ero oxen ap y ococc e-


>gi~io...


gl y en ero oxen asmi p e-


91 8y en ero oxen ap y ococcus e-


91 pyrogenic exo oxen rep e-


pyogenes...


sP ~' a _


gl spe rep ococcus pyogene e-


pir~~w3o5c exo oxen precursor - rep ococcus e-


>gi~l...


91 spe rep ococcus pyogene e-


pir en ero oxen - ap y ococcus


aureus (fragments)


Shuffled recombinant clones are initially selected by phage display and/or
screened by ELISA for the presence of multiple epitopes from the different
families. Variant
proteins with multiple epitopes are purified and used to for in vivo screening
as described
above. The mouse sera are analyzed for antibodies specific for different toxin
subtypes and
variants that elicit broadly cross-reactive responses will be evaluated
further in challenge
models.
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/02944
77
2. Esclrerichia coli and Vibrio cholerae
This Example describes the use of DNA shuffling to obtain cross-reactive
multivalent polypeptides that induce an immune response against the E. coli
heat-labile toxin
(LT), cholera toxin (CT), and verotoxin (VT). Nucleic acids that encode
cholera and LT
toxin B-chains are subjected to DNA shuffling. Table 3 shows the results of a
BLAST search
using the V. cholerae toxin B-chain to identify homologous genes that can be
used in a
family shuffling format to obtain improved antigens. Homologous antigens have
been cloned
and sequenced from a number of related yet distinct Vibrio and E. coli
strains, and additional
natural diversity can be obtained by cloning antigen genes from other strains.
These genes
and others or fragments thereof can be cloned by methods such as PCR, shuffled
and
screened for improved antigens.
Table 3
Sequences producing
significant
alignments



Database/Accession Gene ScoreE
No.


(bits)Vaiue


sp CFITS VI , e-


gi c o era oxen pro em e-


cholera...


gl c x i rio c o erae e-
d


pr o oxin,c o era i rio erae e-
c o


9n Y U c o era oxen i rio c e-
o a


pir c o era en ero oxen precurso e-
c am


cholerae


gyoy5~6 c o era oxen su um pre e-
-
-


(Artificia...


Dps416SOp5 c o era- i a en ero su um e-
oxen


cholerae,...


sp e_
Y


P i rio c o erae >gi p e-


choler...


pap~lFGB~D i rio c o erae >gi p e-
-


choler...


p~lz~1 am , o era oxen en amer e-
o


With Gml...


paDlicrty i rio c o erae >gi p e-
D


choler...


panpcx~j~ i rio c o erae >gi p e-


choler...


paDycTy am , o era oxen en amer e-
D a


, Bound ...


sp e_
Y


pr oxen is ron, ea a i a sc e-
a


coli]


pirpLEC ea - a i a en ero oxa.nam prec e-
c


Escheric...


ans~131495 ea - a i a en ero oxen e-
s


~ B su...


pr oxen i rio c o erae e-


pao~lLTAp sc eric is co i >gi p , e-


Escherichia c...


SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/02944
78
Sequences producing
significant alignments



DatabaselAccession Gene ScoreE
No.


(bits)Value


p Y rio c o erae .


Those chimeric toxins that elicit high levels of neutralizing antibodies
against
both toxins and have improved adjuvant properties are identified. For example,
shuffled
clones are selected by phage display and/or screened by ELISA assays for the
presence of
epitopes from the different parental B-chains. Variants with multiple epitopes
are purified
and further studied for their capacity to act as adjuvants and to elicit cross-
protective
immune responses in challenge models.
Example 2
Evolution of Broad-spectrum Vaccines against Borrelia burgdorferi
Lyme disease is currently one of the fastest-growing infectious diseases in
the
United States. It is caused by infection of the spirochete bacterium Borrelia
burgdorjeri,
which is carned and spread by the bite of infected ticks. Early signs of
infection include skin
rash and flu-like symptoms. If left untreated Lyme disease can cause
arthritis, heart
abnormalities, and facial paralysis. Treatment of early Lyme disease with
antibiotics can stop
the infection, but a lasting immunity may not develop making reinfection
possible. A current
1 S vaccine requires three immunizations over a 1-year period to acquire
immunity.
Both passive and active immunization with the purified B. burgdorferi outer
surface protein A (OspA) protein has been successful in protecting against
infection with B.
burgdorferi, but has no effect against ongoing infections, since this antigen
is not expressed
in vertebrate hosts. OspA is normally anchored on the outside of the cell by a
covalently
attached lipid moiety through an amino terminal cysteine residue. In contrast,
the outer
surface protein C (OspC) is highly expressed by the spirochete in vertebrate
hosts and
vaccination of infected individuals with OspC may be an effective therapeutic
in curing the
infection (Zhong et al. (1997) Proc. Nat'1. Acad. Sci. USA 94 12533-12538.
A recent BLAST search (Altschul, et al., (1997) Nucleic Acids Res. 25:3389-
3402) of the non-redundant GenBank, PDB, SwissProt, Spupdate, and PIR
databases was
used to identify homologues of the OspA outer surface protein gene. This
resulted in the
identification of over 200 entries related to OspA. One hundred entries are
shown in Table 4
below from different strains of B. burgdorferi. B. garinii. B. afzelii, B.
tanukii, and B. turdi
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/02944
79
that share at least 83% DNA sequence identity to the Borrelia burgdorferi OspA
protein.
The ospA genes from these and other strains provide a source of diversity for
family
shuffling to obtain improved antigens for the prevention of Lyme disease.
These genes are
cloned by methods such as PCR, shuffled and screened for improved antigens.
Table 4
Sequences producing
significant alignments


Database/Accession Gene Score E
No.


(bits)Value


u71~~1~~ orre is sp. gene r ou er sur ,
ace


prote...


~7I~oi59~a~ orreIia s
1y~ ~ p mM'r gene or ou- .r '


surface ...


~~i~~~o y nnvidyi5 aorretia turni ge a or ou er ,
sur ace


pro...


7 orre is sp. gene or ou~'surface ,


prote...


9 ~ o p =ou er sur ace pro em orre
is '


burgdor...


9 orre is urg or eri c ne osp
'


gene frag...


a ~ . urg or eri ro osp gene ,


. urg or ri s g a


9 o re i ur r ou er
'


surface pr...


gD~ssa69a ou er su ace pr i o a is
'


burgdorferi,...


a urg or ri s gene or ou er
'


- surface p...


emDlxes~ .garinii sp gene s su ram


a .a ze ii osp gene su s rain


>gi~9... '


a . urg or eri o os gene


. urg or eri sp gene or ,


outer su...


a .a ze ii osp gene


a . urg or eri p semi osp gene ,
or


outer su...


orre is a z m major ou er me
r ne '


surfac...


a x ur


.
g o eri sp ge


a . urg r er p en ,


.a z ii o p gene a su s ra n


a . urg o eri p a mi
p gene ,


rre is a ze ii a er sur ace ,


protein A...


.a ze ii sp gene o s ram ,


. urg eri a o p a a ,


urg or eri s gene or ou er


surf . . .


9~ILi9 orre is urg or eri ou er sur ,
ace


protein...


emnlx6 u ,
_____ ' a v
rg or eri osp gene or a er


surface prot . . .


a urg or eri gene or p ou er ,


surface pro...


a x . urg or eri osp g ne


9 orre is urg o ri operon major
a er


mem...


e~ I x . urg or eri p semi osp gene '>?"',
or


outer su...


9 orre is urg or eri ou a sur ace ,


pzotein A...


SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/02944
Sequences
producing
significant
alignments



Database/Accession Gene ScoreE
No.


(bits)Value


borreiia garinia Lv4 outer sur
a a


protein A...


. urg or eri osp gene su s a


a urg or eri major ou er


surface pro...


a v sorreim burg r eri o p an osp
s


genes foz...


orre i urg r eri p asmi , ,


complet...


re a urg or eri a er
n-i-iu a c


surface pr...


e mo ur or eri p gene
y
~,


~gi~1819262~gb~I284...


a a~psuumuu~wnuumuu tsorrelia garinia gene or ou
er sur ce


p...


. a g or ezei ou er sur ace pr .
i


(ospA)...


a a . a g or eri osp gene .
a


orr is gar nii gene or ou er
sur ace


p...


a x a r eri osp gene ,
_______..


s~strain)


e mo(xibqby orre i urg or en sp gene or
ssvs~


outer surf...


9 ~ orre is sp. ou er sur ace pro
em


A pre...


orre a ur or eri a p asm


lipopr...


a x . a eri o p gene .


.gar sp gene ou su s ram


g u5 re is ga inia ou er sur ce pr
em


A (ospA...


J re a r ii gen or ou er a ce


p...


a . urg or eri ei osp gene


o a a garin i ma or ou er me
r ne


surfac...


ecn~ . urg o er sp gene .
x


rre is urg or eri gene or


outersurface pr...


orre is va ais ana gene or ou
a


surfac...


orr g rinii gene or ou er su
a a


p...


is ga inii ou er sur ace p o
in


A (ospA...


rre is ar ii gene r ou er sur
a a


p...


a garinii o ou er su ace e-


pr...


orre i garinia gene or ou er e-
sur ace


p...


re is g rinii gene or ou er su e-
ace


p...


is ga inii ou er sur ace pro e-
in


A (oBpA..


orre is ga inii gene or ou er e-
sur ce


p...


re arinii gene or ou er su ace e-


p...


o a is arinii ou er su ace p e-
em


A (OSpA...


.ga nii r osp gene e-


re is garinii gene or ou er sur e-
ace


p...


is urg or er= iso a a e-


297) ou...


SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/02944
81
Sequences
producing
significant
alignments


DatabaselAccession Gene Score E
No.


(bits)Value
-


g aps3~m o a i gariniiou er e-
[U937I1 sur ace
pro em


A (ospA...


o re is gariniigen or e-
ou er
sur ace


p...


rsorreiia terW-W a e_
ourgaor so a 6-lY7
t L


Son 188...


orre urg or eri p e_


gene, 3...


g y orre is urg eri sp e-
s or
~


gene, 3...


g orre i urg eri sp gene, e-
or


3'end ...


g xA orre is ur eri s e_
o


gene, 3'e...


g y rre is ga ou er e-
o inii sur ace
v pro em


A (ospA...


rsorreiia ferl . sp e-
burgoor


gene, 3'...


J orre is gariniigene or er sur e-
~ ou ace
~


p" I


9 orre is urg eri sp gene, e-
or


3'end ...


orre is gariniigene r er sur e-
ou ace


p...


a .garni osp gene
.,., a _


g o is garinii ou a sur e-
ace pro
em


A (ospA...


J rre is g iniigene or er su ace e-
~ov ou
v


p...


orr is a or eri e_


gene, 3'.
.


rre is gariniagene or r sur ce e-
ou


p...


o re is gariniigene or er su ace e-
ou


p...


orre is urg eri sp gene, e-
r


3'end ...


9 rre is a ze major me rane e-
ii ou er


surfac...


J orre is gar gene or er sur e-
n i ou ace


p...


. urg er osp e_
gene or ou
er


surface p...


o a i gar gen r er ur ace e-
nii ou


p..


. urg eri in e_
osp gene
s


p asmi
.....,.."~.,-,~,~,~~n~.y . p ene
e-
urg or eri


for ou...


a ur or ri a p asmi sp gene e-
,___..___


for ...


.garinii osp e_
gene s rain


orre is ga gene or er sur e-
ini ou a a


p...


orre is gariniigene or er sur 5 e_
ou ace


p...


o re is gar gene or er sur e-
nii ou ce


p...


A BLAST search with the B. burgdorferi OspC protein gene revealed over
200 related entries. Entries for one hundred sequences sharing at least 82%
DNA sequence
identity are shown in Table 5 below that provide a source of diversity for
family shuffling to
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PC'T/US99/02944
82
obtain improved therapeutics in the treatment of Lyme disease. These genes are
cloned by
methods such as PCR, shuffled and screened for improved antigens.
Table 5
Sequences
producing
significant
alignments



Database/Accession Gene Score E
No.


(bits)Value


borrelia aorreri syn a ase
aurg


(guaA) g...


g eye orre is o a i s ra n ,
urg


outer s...


orre is o eri ou er sur ce e-
a g


prote...


J y orreTia or eri gene or ou e-
y ca urg er


surface...


g v y orrelia or eri p asmi cp , e_
urg


complet...


a y . urg or osp gene or ou er e-
eri


surface
...


g o re is urg or eri ou er sur ace e-


pro...


g yt~ o re is or eri s ram ou er e-
a urg


surface...


9 ~s rre is urg r eri s ra n p ou e-
er


sur...


9 ~ orre is or en ; su s ram sensu e-
urg


strict...


9 re is urg r eri s rain ou er e-


surfac...


8 rre is urg eri s am ou er e-


surfac...


9 rre a urg a i s ra n ou er e-


surfac...


o is Japonica e-
s a n or


Out...


orre is e-
Japonica
s rain
v or


Ou...


a . urg or e-
____ eri ..,
_ _ _ osp gene s ram
'
V


rre is anu ii or e-
a er sur
ace


pr...


9 y rre is urg o eri s ram ou er e-


surfac...


rre is Japon e-
ca s ain
i o or


Ou...


a x . urg or sp gene e-
ri


a x . urg r ge a or p pro ein e-
ri


a x . urg eri p g ne, en e-


a x .a ze ii ain o o p gene e-
s


rre is a m or a er sur ace e-
z


pr...


J orre is ii gene or ou er sur e-
ze a a


p...


re is a m ge a or ou er sur e-
z ace


p...


emd x .a ze ii e-
r i osp
gene


9 o re is or en ou er sur ace e-
urg


I Pr...


rre is a m gene or ou er su
_____ z ace
J


p...


~ ~1 orre is ii or a r sur ace e-
anu


pr...


9 rre is urg or n me rane pro in e-


(ospC) ...


J ~ orre is ii gene or ou er sur e-
a ze ace


I Pro . .
.


a . . urg or . p i icus s rain e-
eri


ospC gene


SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/02944
83
Sequences producing
significant
alignments



Database/Accession Gene Score E
No.


(bits)Value


g DIL4ZH741SOR10pSPC-- o re is urg eri s ram e-
r


outer su...


soxo Y ~ $orre is a gene or ou er sur e-
ze ii ce


pro...


orr is a ze gene or ou er sur e-
ii ce


p...


g o re is a eri s ra a er e-
g or


sur...


g ey orre is a eri s ram aca e-
g o


~ outer su...


re is a ze gene or ou er sur e-
ii ace


pro...


orre i urgnoreri syn a se e_


(gusA) a...


9 a rre is a or eri syn a a a e-


(guaA) g...


~ooy or a is a gene or ou er sur e-
ze ii ce


i P...


orre is a gene or ou er ur e-
ze a.i ace


p...


9 ~ w orre is urg eri s ram ou er e-
or


sur...


orre i urg eri ou er sur ace e-
or


prot...


rre is a ze gene or ou er sur e-
ii ace


p...


a x a g or ri rain re osg gene e-
-__,__ ____ s


Eus so re is ze or a a sur a a e-
i


pr...


a x . urg o eri sp ge a e_


orr is gariniigene or ou er ur e-
ace


pro...


a is a ze or a er sur ce e-
ii


pz...


orre is urg eri ou er sur ce e-
r


pr...


9 orre is urg eri s rain e-
or


outer s...


a x . urg r eri ram osp gene e-
s


J ~a~ orr i a ze circu ar p asmi e-
ii


DNA f o .
. .


a x .gar nii s e-
rain ou osp
gene


>gi~8720...


.garinii osp e-
gene s ram


orre is a gene or ou er ur e-
ze i ce


p...


orre is gar gene or ou er su e-
nii ace


pro...


rre is ur eri ou er sur a e-
aor a


prot...


.a z i Y a ram osp gene e-
s


o a i arinii s ram o p e_


gene f...


orre is a s rain os e-
ze ii


gene ...


or a is urg eri ou er sur ace e-
or


pro...


orre is a or a er sur ace e-
ze ii


pr...


or is urg eri ou er sur ce e-
r


prote...


s orre i a z or a er sur ace e-
m


pr...


orre is anu or a r su ace e-
m


pr...
I


. ur o eri gene s r e-
osp t
I


a .. s.a ze ii e_
osp gene a
s rai


SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCTIUS99/02944
84
Sequences producing
significant
alignments


~


DatabaselAccession Gene Score E
No. ~~ ~~


(bits)Value


aorretia garinii z5r~-cir a e
ar p asmi


DNA fo...


v s x orre is garin i gene r ou er e-
sur ace


pro...


a Ycrr .garinii s ram os g ne e-


g se xF orrelia-bur or eri ou er sur
ace


pro...


orre is garin cs ~u ar p asmi e-


DNA f o . . .


g ads o~rrema burgaorteri s r in ou e-
er


surf . . .


g o y ~=re~ia~urgc~ r er a er sur e-
ace


pro...


7 uyay orre is a ze m gene or ou er e-
sur ace


p...


w~soo dys -sorreiia~zeiii gene or ou er e-
sur ace


p...


9 y~ Sorreiia burg o eri ou er sur e-
ace


pro... ~


e.y ~r . urg or er sp gene, en e-


7 a y orre is garinia s ram osp e-


gene f...


a y oo ~rre is garin i gene or ou er e-
sur ace


pro...


g too y ~rre.ria burg o eri s ram e-


outer s...


orre is ga inii s ra n osp e-


gene ...


g a orre is urg eri s ram e-


outer su...


7 a y a re is g rinii ra n osp e-


gene f...


a y o re is ga m i g ne or ou er e-
sur ace


pro...


a y . urg r eri sp g e, en e-


a x . urg or eri p gene, en e-


7 y orre is garinii ge a or ou er e-
sur ce


pro...


. urg or eri os gene or ou er e-


surface ...


a s .garinii ro s ra op gene e-


a x . urg r er g ne


rre is ur o a i a n


outer sur...


gD~L4ZS94~ orre is a g o a i s a n


outer s...


orre is gar nii gene or ou er
sur ace


pro...


9 orre is urg ri s ra ou er e-


surf . . .


J orre is upon ca o a er sur ace e-


p...


7 orre is ~apo ica o a er sur e-
ace


p...


Example 3
Evolution of Broad-spectrum Vaccines against Mycobacterium
Tuberculosis is an ancient bacterial disease caused by Mvcobacterium
tuberculosis that continues to be an important public health problem worldwide
and calls are
being made for an improved effort in eradication (Morb. Mortal Wklv Rep ( 1998
Aug 21;
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/02944
47(RR-13): 1-6). It infects over 50 million people and over 3 million people
will die from
tuberculosis this year. The currently available vaccine, Bacille Calmette-
Guerin (BCG) is
found to be less effective in developing countries and an increasing number of
multidrug-
resistant (MDR) strains are being isolated.
The major immunodominant antigen of M. tuberculosis is the 30-35 kDa
(a.k.a. antigen 85, alpha-antigen) which is normally a lipoglycoprotein on the
cell surface.
Other protective antigens include a 65-kDa heat shock protein, and a 36-kDa
proline-rich
antigen (Tascon et al. ( 1996) Nat. Med. 2: 888-92).
Table 6 shows the output of a BLAST search using the 30-35 kDa major M.
10 Tuberculosis antigen (a.k.a. antigen 85, alpha-antigen) coding sequence to
identify
homologous genes that may be used in a family shuffling format to obtain
improved
antigens. Many homologous antigens have been cloned and sequenced from a large
number
of related yet distinct mycobacterial strains. These genes are cloned by
methods such as
PCR, shuffled and screened for improved antigens.
Table 6
Sequences
producing
significant
alignments


Database/Accession Gene Score E
No.


(bits}Value


Mycoaacterium tubercu osis a


extracellu...


a . cu o is s rain r man gene or


85-H a...


y o er um a ercu osis v .


complete ge...


vis s in gene or


antigen


9 . v s gene enco ing a p a- .


antigen, comp...


yc c er um nsasii gene o p .


antigen


7 yco r um scro uraceum r .


alpha-antig...


yc a ium in race a are gen or e-


alpha-a...


. vium ne r a a-an en


yco erium in race a are r e-


alph...


rae genomic sequence, c s e-


B38 ...


a x . epra ere or an igen e-


ru~ . se o c m- i mg p em e-


~ antige...


9D r um a ercu osis e_


antigen gene.


eme y o ac um ovis gene or
x e_
___.,......,_.~


protein


yco a erium ovis genes or e-


protei...


a yc c rium a ercu osis v e_
,


complete g...


SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/02944
86
Sequences producing
significant alignments


Database/Accession Gene ScoreE
No.


l Value
- (bits)


gapa7DdS~M'1'U4'1335 MyCObaCterlum tuberCU O81& e-


extracellular 32 ...


yco ac erium ovis gene or a p e-
a


antige...


r ona gene enco ng ~ - an igen e-


u r~ycobactermm avium gene or , e-


antigen e...


g a ~c y o ac erium eprae an en gene, e-


compl...


y ac erium epra or an igen


com...


a . rinum gene or a pro in e-


(partial)


.avium yo gene or rcDa e-


protein...


yco ac erium avium gene or an
igen


85C and ...
-


e .avium gene or a e-


protein...


. n race a are gene or e-


32k... I


a y .asia scum ene or a pro em e-


(partial)


a v .asia scum gene segmen o a e-
vy


protein


emopsv~cy ru~az .avium comp ex gene or a pro e-
em


(pa...


a .avium comp ex gene o a pro ein e-


(p...


mum comp ex gene r rcDa ro em e-
r


(pa...


C~povme~rw.s~nyob M.avium complex gene ro= 3~ a e-
pro em


(pa...


.avium gene or a e-


protein...


.in race a are gene or e-


32kD...


.avium comp ex gene or a pro e-
ein


(pa...


.avium comp ex gene or a pro e-
ein


(pa...


a .avium comp ex gene or a pr a e-
n


(pa...


.aviu mp ex gen o a pro ein e-


(pa...


a .ma mo nse gene r a pr a n e-


(partial)


.avium omp ex gene o a pro em e-
(
a


...
_.__,.____.._._ p e-
a vium comp ex gen or a pro ein
(pa


...
emo(zsa . mum comp ex gene or a pro em e-
(pa...


yco ac erium a ercu osis gene e-


antigen 8...


. vium comp ex gen or a pro ein e-


(pa...


a y c erium a ercu osis v e-


complete geno...


.szu gai gene or a pro in e-


(partial)


M.avium complex genet a pro em e-


(p...


.avium comp ex gen or pro em e-


(pa... I


. ovis gene or a pro in e-


~
lparti...


SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99102944
87
Sequences producing
significant alignments


Database/Accession Gene Score E
No.


(bits)Value
-
-


a , M. triviale gene =or 3z e-
7c~a
pro em


(partial)
-
-


a M.ceiatum gene =or 3z e-
~a
protein


(partial)


a . eprae o p in gene e-


>gi~287923~emb...


gb~L7S~i16~MSGS16C5 yco ac erium eprae cosmi e-


~
" sequence.
' -


a 19 . eprae of e-
SrTFfL~SL 8~ pro in gene
F


g e-
.. .. .. ~ . ~ - w
yco ac erium eprae an igen gene,


compl...


a y a .ce a um gene or x~a pro em e-


lpartial)


a .p ei gen or a p a n e-


(partial)


a y s ru~ttw . ran eri gene or x~a pro a e-
n


(partial)
-


emn(rowma~Mr-3zxYxo4 M.ttavescens gene segmen e-
o a


~ protein


a x M.zortuitum gene for 3z xDa e-
proem


(partial)


a .avium-in ra a a are comp ex e-
gene or


32...


a y .peregrinum gene or a pro em e-


(partial)


a M.avium comp ex gene o a pro e-
em


(pa...
-
-


emn x ra~rr . e-
xenopi gene
~o pro in


(partial)


a o x .smegm i gene segmen o a
L .7G-llL


protein


emb(zsoTS~[~rA3 .amum comp ex gen r pro em e-


(pa...


a M~ M.avium compreX gene or a pro e-
em


(pa...


a ~ ~ M.vaccae gene se me o a pro e-
em


em~jx~z~o .vaccae gene or a pro em e-


(partial)


a y ruJ N 1K ~ no~romogemcum gene or a


protein (...
-


a y s rc .simiae
gent or a pro a n


(partial)
-


a v~ o A.7z lKyCObact~rmm
t;u ercu osi v,


gene


a y Hrrc3r yco ac erium a ercu osis v


complete g...


g a TTr . ume aciens c ramp enico .


acetyltransferas...


a y MsM~ .smegma s gene or a pro ein


(partial)


. errae gene or xo pro in


(partial)


.s ro a aceum gene o a pro ein


(par...


a M .gor onae gene or a .


pro...


9 n uenza i orn nu eopro ein


RNA (...


Example 4
Evolution of Broad-spectrum Vaccines against Helicobacter pylori
Chronic infection of the gastroduodenal mucosae by Helicobacter pylori
bacteria is responsible for chronic active gastritis, peptic ulcers, and
gastric cancers such as
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/02944
88
adenocarcinoma and low-grade B-cell lymphoma. An increasing occurrence of
antibiotic-
resistant strains is limiting this therapy. The use of vaccines to both
prevent and treat
ongoing infections is being actively pursued (Crabtree JE (1998) Gut 43: 7-8;
Axon AT
(1998) Gut 43 Suppl 1: S70-3; Dubois et al. (1998) Infect. Immun. 66: 4340-6;
Tytgat GN
(1998) Aliment. Pharmacol. Ther. 12 Suppl 1: 123-8; Blaser MJ (1998) BMJ 316:
1507-10;
Marchetti et al. ( I998) Vaccine 16: 33-7; Kleanthous et al. ( 1998) Br. Med.
Bull. 54: 229-4I ;
Wenneille et al. (1998) Pharm. World Sci. 20: I-17.
Identification of appropriate Helicobacter antigens for use in preventive and
therapeutic vaccines can include two-dimensional gel electrophoresis, sequence
analysis, and
serum profiling (McAtee et al. (1998) Clin. Diagn. Lab. Immunol. 5:537-42;
McAtee et al.
(1998) Helicobacter 3: 163-9). Antigenic differences between related
Helicobacter species
and strains can limit the use of vaccines for prevention and treatment of
infections (Keenan
et al. (1998) FEMS Microbiol Lett. 161: 21-7).
In this Example, DNA family shuffling of related yet immunologically
distinct antigens allows for the isolation of complex chimeric antigens that
can provide a
broad cross-reactive protection against many related strains and species of
Helicobacter.
Mouse models of persistent infection by mouse-adapted H. pylori strains that
have been used
to evaluate therapeutic use of vaccines against infection are used to evaluate
shuffled
antigens (Crabtree JE (1998) Gut 43: 7-8; Axon AT (1998) Gut 43 Suppi 1:S70-
3).
The vacuolating cytotoxin (VacA) and cytotoxin associated gene products
(CagA) have been evaluated as a vaccine against H. pylori infection in animal
models which
supports the application of this approach in htunans.
Table 7 shows the results of a BLAST search using the H. pylori VacA gene
to identify homologous genes that can be used in a family shuffling format to
obtain
improved antigens. Homologous antigens have been cloned and sequenced from a
number of
related yet distinct H. pylori strains and additional natural diversity can be
obtained by
cloning antigen genes from other strains. These genes and others or fragments
thereof are
cloned by methods such as PCR, shuffled and screened for improved antigens.
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PC"f/US99/02944
89
Table 7
Sequences producing
I significant alignments



i Database/Accession Gene Score E
No.


(bits)Value


g Dluysy~11H1'u95971 Aeii~ac er on ,
py


inactive cy...


g D ~ ~ouu5yd I rtPwEVVV59eAmco~ac er on sec o
py ion


134 of...


g DIAr'uUl.iSSIHPAF001358HellCObaC on vacuo ing ,
er py a


cytotoxi...


a nd x H.pyloW gene or ,
cy
o
oxen.


g a ~co ac er on ,
py


vacuolating .
cytot..


g Dluu56i6lHPUV56~6 a ico ac er on ,
py


cysteinyl-tRNAsyn...


g DluzyavilxPUZ94o~ a ico ac er on vacuo ing .
- py a


cytotoxin
ho...


e mDIAJU06969)Hpy696s-a 1CO ac er Orl vac ,
py gene,


strain Mz28...


g DIS/G4y415/1494 I4D k-a cy a ico ,
o oxen ac
er


pylori, Genomi...


g Dpow.~slxruo~l~ a ico ac er on ,
py


cysteinyl .
tRN..


e mDIAJUU6969~xPYSzlsea ico ac er ora vac ,
py gene,


strain Mz26...


e mDIAJUU6970~xpy a ico ac er on vac ,
py gene,


strain Mz29...


g DIAr'U779391AF07793~a ico ac er on s ram ,
py


vacuolating...


g Diwr~or-ry4vp'v~ a ico ac er on s ram ,
py


vacuolating...


g DIAFa779~1~ a ico ac er on s ram ,
py


vacuolating...


g Dlwrw-rn93a~AF a ico ac er on s ram ,
py


vacuolating
...


g DIu63zssIHPU53 a ico ac er on vacuo ing ,
py a


~ cytotoxin
ge...


g Dp63z~v[xPU a ico ac er on vacuo ing .
py a


cytotoxin
ge...


g DIu63z~z~riP a ico ac er on vacuo ing ,
py a


cytotoxin
ge...


g Dlu63za3p a ico ac er on vacuo ing
py a


cytotoxin
ge...


9 a ico ac er on vacuo ing
py a


cytotoxin
ge...


g Dlu63z6z1 a ico ac er on vacuo ing
py a


cytotoxin
ge...


g Dlu63z a ico ac er on vacuo ing ,
py a


cytotoxin
ge...


g Dlu63z~3~ a ico ac er ,
nru~sa~s n 1 ~ t pyl ori vacuolaing


I cytotoxin
ge...


gDIU63ZHZ a ico ac er on vacuo ing
py a


cytotoxin
ge...


gDlu63z59 a ico ac er on vacuo ing
py a


cytotoxin
ge...


D U63z7
9 I a ico ac er on vacuo ing
py a


cytotoxin
ge...


gDIu63Z67 a ico ac er on vacuo ing ,
py a


~ cytotoxin
ge...


gDlu53~ a ico ac er on vacuo ing
py a


~ cytotoxin
ge...


gDp63z53[ a ico ac er on vacuo ing
py a


cytotoxin
ge...


SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCTNS99/02944
Sequences producing j
significant alignments



Database/Accession Gene Score E
No.


(bits)Value


g y U63z56~HPU63Z66- Helicobacterpy on vacuo a ing


cytotoxinge...


g nEu63z75~HPU63z~~- a ico erpy on vacuo a ing .
ac


cytotoxinge...


g b~U63279~HPUS3z a ico erpy on vacuo a mg
~ ac


cytotoxinge...


g y uus~ii~riYU63lmi tteliconacterpy lorivacuo a ing ,


cytotoxinge...


g y u63zeo~HPU a ico erpy on vacuo a mg .
ac


cytotoxinge...


g Dp63z65~HPU63Z65 Hel:ico erpy on vacuo a 1ng
ac


cytotoxinge...


g D~U63z67~HPU63Zb~ a lcO~ac erpy or1vacuo a lng ,


cytotoxinge...


g a~U63261 HPU63ze~ a ico~ac erpy on vacuo a ing .


cytotoxinge...


g D~U63z61yHPU6 a 1co erpy on vacuo a a.ng ,
ac


cytotoxinge...


g b~U63Z74~ a ico erpy on vacuo a ing
ac


cytotoxinge...


g y u63ze5~ a ico erpy on vacuo a mng ,
ac


cytotoxinge...


e mo~AJOO9430~HP a ico erpy on vac gene ,
ac


(partial),. ..


a ico erpy on vac gene ,
ac


(partial),. ..


e mbpoo9~ a ico erpy on vac gene
ac


(partial),. ..


g y u63z~~ a ico erpy on vacuo a ing .
ac


cytotoxinge...


ecnywJOOS~'rH a ico erpy on vac gene
ac


(partial),. ..


ertu~~AJ00942ZJ a ico erpy on vac gene
ac


(partial),. ..


ga~U63z56 a ico erpy on vacuo a ing
ac


cytotoxinge...


gDp63z6 a ico erpy on vacuo a ing
ac


cytotoxinge...


emya.TOOg~ a ico erpy on vac gene
ac


(partial),...


emyaioo9 a ico erpy on vac gene
ac


(partial),...


emnpo a ico erpy on vac gene ,
ac


(partial),...


ga~U6326~j a ico erpy on vacuo a ing .
ac


cytotoxinge...


gD~U63Z7 a ico erpy on vacuo a mg
ac


cytotoxinge...


emn~A,lo a ico erpy on vac gene
ac


(partial),. .


a a ico erpy on vac gene
ac


(partial),...


emn~A.TO~ a ico erpy on vac gene
ac


(partial),...


e ~ ~ ~~f3 a ico erpy on vac gene
ac


(partial),...


gb~U63Z a ico erpy on vacuo a ing
ac


cytotoxinge...


a , ~ a ico erpylori vac gene ,
ac


(partial),... ~


SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/02944
91
Sequences producing significant
alignments


~


Database/Accession No. Gene Score E


~ ~ (bits)Value


e mD~AJUUJ4.il~tlPAJy43l tiellCODdcLerpylorivacA gene


(partial), .. .


e mD~AJUUy4I~~riYAJ941-/ HeIICODdcterpylorivacA gene


(partial), .. .


a ~AJ0094z1 HPAJ94z1- Heiicobacterpyon vac gene


(partial), .. .


a a ico ac er pyon vac gene


(partial), .. .


e ftID~AJUUy4.id~HPAJ9438 HellCObaCterpylon vaCA gene


(partial), .. .


g D/Ub.ilb4~riYU6dlb4 HeIICODdcLerpylorivacuolating 676


cytotoxin ge. ..


eIiIDIA~JUUy433~riPAJ9433 HellCObacterpyloriVaCR gene


(partial), .. .


e mD~AJUUy4l5~HPAJ94z5 HellcObacterpy~m vac gene


(partial), .. .


g D~U6315tfIHPUb3z59 pylorivacuo a ing
Heticobacter


~ ..
cytotoxin ge.


e mD~AJUUy441lHYAJ944Z HellCObacterpylori-vacA gene e-


(partial), .. .


eiriDI,AJUUy444~HPAJ9444 HellCObacterpylOrlVaCA gene e-


(partial), .. .


g Dpdvv6apPUavv6e Helicobacter pyloristrata z1 , e-


vacuolating . ..


eIIID~AJUUy434iHPAJ9434 HellcobacterpSIlOrlVaC~Jene e-


(partial), .. .


e mb~AJ009441~HPAJ9441- a l.cobdcter-pyorl vac gene e-


(partial), .. .


g D~Arv3s616~HPVCPZ- eiicobac pyon s ram e-
er


vacuolating . ..


e mD~AJUUy44y HPAJ944'7 xe~.icobacterpyon vac gene e-


(partial), .. .


e mD~AJ0v944U~HPAJ9440 Helicobacterp~ori vacs gene e-


(partial), .. .


e mD~AJUUJ436~HPAJ9436 HellCObacterpylori-vacA gene e-


(partial), .. .


a , vv9 H a i~ac er pyon vac gene e-


(partial), .. .


e mD~AaJU0y446~HPAJ944s -H~cobacterpyon vac gene e-


(partial), .. .


g D~UtfUUbipPU~vv67 Helicobacterpyon s ram , e-


vacuolating . ..


g D~wr~o41-i3s~AF04Z73 a ico pyon e-
ac er


vacuolating cytot...


e mD~A.~ov9445~HPA39~~5-- a icobacpyon vac gene e-
er


(partial) , . . .


g D~AF035609~AFV3~ a 1CO ac er pyOrl s rata e-


vacuolatin...


g D~Ab'U41734~AF04Z73~ a ico pyon vacuo a ing e-
ac er


~ cytoto...


g D~AF03Sb11~AF035~ a ico ac pyon s ram e-
er


~vacuolatin...


g D~AF03S613~AFU36 a ico ac er pyon s ram e-


vacuolatin...


e mDiw.~oo696y HPY a ico ac er pyon vac gene, e-


strain Mzl9.. .


g . a ico ac er pyloris rain e-


vacuolating . ..


g D~AF~'U3S614 ~AFV35614- y on strain 1~ e-
Heiicobacter-


~
vacuolatin..


SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99141383 PCT/(1S99/02944
92
Sequences producing
significant alignments



Database/Accession Gene Score E
No.


~ (bits)Value


g p~UylSnrs~HYUyIS-!tit1e11CODaCterpylOrl strataF'37 - e-
vacs.


~ gene, pa...


g a ico ac pyon s ram vac e-
er


gene, pa...


g a ico ac pyon s ram vac e-
er


gene, pa...


a Y a ico ac pyon par vac e-
er is


gene, stra...


g y xeticonacterpylori strainF94 --I~ e-
vacs-


gene, pa...


g y Y y HellcODBCLerpylori strainF~1 e-
vac


gene, pa...


g v v a ico ac pyon s ram e-
er


vacuolatin...


g n~U91560~HPU91580 HellCObaCterpylori scram vac e-


gene, pa... I I


g v a ico ac pyon s ram e-
er


vacuolatin...


e ma~Y14744~HPVACA49 HellCObaCterpylori par vac e-
is


gene, stra...


Table 8 shows the results of a BLAST search using the H. pylori CagA gene
to identify homologous genes that can be used in a family shuffling format to
obtain
improved antigens. Homologous antigens have been cloned and sequenced from a
number of
related yet distinct H. pylori strains and additional natural diversity can be
obtained by
cloning antigen genes from other strains. These genes and others or fragments
thereof are be
cloned by methods such as PCR, shuffled and screened for improved antigens.
Table 8
Sequences producing
significant alignments


Database/Accession Gene ScoreE
No. (bits)Value


gb~AF083352~AF083352 Helicobacter pylori cytotoxin 7041 0.0
associated p...


gb~AE000569~HPAE000569Helicobacter pylori section 47 5501 0.0
of 134 of...


gb~Ll 1714~HECMAJANT HeIicobacter pylori major antigen4976 0.0
gene
sequ...


emb~X70039~HPCAI H.pylori cai gene for cytotoxicity4294 0.0
associated
.. i


gb~U60176~HPU60176 Helicobacter pylori cap pathogenicity4294 0.0
island...


dbj~AB003397~AB003397Helicobacter pylori DNA for CagA,4274 0.0
complet...


gb~U80066~HPU80066 Helicobacter pylori swain 213, 349 2e-93
~ cytotoxin- ~ ~
as...


SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/02944
93
Sequences producing
significant alignments


DatabaselAccession Gene Score E
No.


(bits)Value


gb~U80065~HPU80065 Helicobacter pylori strain 184,343 le-91
cytotoxin-


as... ~


gb~AF043488~AF043488 Helicobacter pylori JK252 cytotoxicity178 4e-42
~


ass...


gb~AF043487~AF043487 Helicobacter pylori JK25 cytotoxicity170 1 e-39


asso...


gb~AF043489~AF043489 Helicobacter pylori JK269 cytotoxicity163 2e-37


ass...


emb~X70038~HPCAIDUP H.pylori DNA duplication sequence159 4e-36
within


th... ~


gb~AF043490~AF043490 Helicobacter pylori JK22 cytotoxicity153 2e-34
~ ~


asso...


Example 5
Development Of Broad-Spectrum Vaccines Against Malaria
This Example describes the use of DNA shuffling to generate improved
vaccines against malaria infection. An excellent target for evolution by DNA
shuffling is the
Plasmodium falciparum merozoite surface protein, MSP 1 (Hui et al. (1996)
Infect. Immun.
64: 1502-1509). MSP 1 is expressed on the surface of merozoites as an integral
membrane
protein. It is cleaved by parasite proteases just before and concomitant with
rupture and
release from infected cells. The cleavage appears to be obligatory for full
function in MSP 1
binding to RBC receptors. The cleaved fragments remain attached to the
membrane of the
merozoite. Other membrane proteins on merozoites also participate in the
attachment and
specific invasion events. MSP1 is a proven candidate for inclusion in a
vaccine against the
asexual blood stage of malaria.
The genes encoding MSP 1 can be isolated from various isolates of
Plasmodium falcipanun merozoites by PCR technology. Related naturally existing
genes
can be additionally used to increase the diversity of the starting genes. A
library of shuffled
MSP1 genes is generated by DNA shuffling, and this library is screened for
induction of
efficient immune responses.
The screening can be done by injecting individual variants into test animals,
such as mice or monkeys. Either purified recombinant proteins, or DNA vaccines
or viral
vectors encoding the relevant genes are injected. Typically, a booster
injection is given 2-3
weeks after the f rst injection. Thereafter, the sera of the test animals are
collected and these
SUBSTITUTE Sf~EET (RULE 28)


CA 02320958 2000-08-10
WO 99/41383 PCTNS99/02944
94
sera are analyzed for the presence of antibodies that reduce invasion of
merozoites into
uninfected erythrocytes (RBC). RBC are infected by the merozoite, immediately
inside the
12BC, the merazoite differentiates into a ring and this matures to a schizont
that contains
several nascent daughter merozoites, which then burst out of the infected
cell, destroying it,
and go on to attach and invade another RBC. In vivo, the merozoite is likely
only
extracellular for seconds. In vitro, any blockade of this event can
dramatically reduce the
level of reinfection. Antibodies against MSP 1 bind to the surface of
merozoites that are
released from schizont infected RBC when they rupture and thereby reduce the
ability of
. these merozoites to attach and engage cognate )ZBC receptors on the
uninfected RBC
surface. Merozoite attachment is reduced, merozoite entry into new RBC is
reduced, and the
numbers of newly invaded cells detected at the early ring stage is therefore
reduced if the
culture is examined several hours after the blockade of invasion test. In some
assay formats
a surrogate of merozoite invasion inhibition is to note the appearance of
agglutinated
merozoites, although this is an indirect measure of antibodies that cause
reduced invasion.
1 S The shuffled antigens that induce the most potent antibody responses
reducing invasion of merozoites into uninfected erythrocytes are selected for
further testing
and can be subjected to new rounds of shuffling and selection. In subsequent
studies, the
capacity of these antigens to induce antibodies in man is investigated. Again,
either purified
recombinant antigens, or DNA vaccines or viral vectors encoding the relevant
genes are
injected and the protective immune responses are analyzed.
Example 6
Development Of Broad-Spectrum Vaccines Against Viral Pathogens
This Example describes the use of DNA shuffling to obtain vaccines that can
induce an immune response against multiple isolates of viral pathogens.
A. Venezuelan equine encephalitis virus (VEE)
VEE belongs to the alphavirus genus, which are generally transmitted by
mosquitoes. However, VEE is an unusual alphavirus in that it is also highly
infectious by
aerosol inhalation for both humans and rodents. The disease manifestations in
humans range
from subclinical or mild febrile disease to serious infection and inflammation
of the central
nervous system. Virus clearance coincides that of production of specific anti-
VEE
antibodies, which are believed to be the primary mediators of protective
immune responses
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/02944
(Schmaljohn et al. ( 1982) Nature 297: 70). VEE is an unusual virus also
because its primary
target outside the central nervous system is the lymphoid tissue, and
therefore, replication
defective variants may provide means to target vaccines or pharmaceutically
useful proteins
to the immune system.
At least seven subtypes of VEE are known that can be identified genetically
and serologically. Based on epidemiological data, the virus isolates fall into
two main
categories: I-AB and I-C strains, which are associated with VEE
epizootics/epidemics, and
the remaining serotypes; which are associated primarily with enzootic
vertebrate-mosquito
cycles and circulate in specific ecological zones (Johnston and Peters, In
Fields Virology,
10 Third Edition, eds. B.N. Fields et al., Lippincott-Raven Publishers,
Philadelphia, 1996).
The envelope protein (E) appears to be the major antigen in inducing
neutralizing Abs. Accordingly, DNA shuffling is used to obtain a library of
recombinant E
proteins by shuffling the corresponding genes derived from various strains of
VEE. These
libraries and individuals chimeras/mutants thereof are subsequently screened
for their
15 capacity to induce widely cross-reacting and protective Ab responses.
B. Flaviviruses
Japanese encephalitis virus (JE), Tick-borne encephalitis virus (TBE) and
Dengue virus are arthropod-borne viruses belonging to the Flavivirus family,
which
comprises 69 related viruses. The heterogeneity of the viruses within the
family is a major
20 challenge for vaccine development. For example, there are four major
serotypes of Dengue
virus, and a tetravalent vaccine that induces neutralizing Abs against all
four serotypes is
necessary. Moreover, non-neutralizing antibodies induced by infection or
vaccination by
one Dengue virus may cause enhancement of the disease during a subsequent
infection by
another serotype. Therefore, cross-protective, broad spectrum vaccines for TBE
and JE
25 would provide significant improvements to the existing vaccines. In this
Example, the
ability of DNA shuffling to efficiently generate chimeric and mutated genes is
used to
generate cross-protective vaccines.
1. Japanese encephalitis virus
Japanese encephalitis virus (JE) is a prototype of the JE antigenic complex,
30 which comprises St. Louis encephalitis virus, Murrav Valley encephalitis
virus, Kunjin virus
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/02944
96
and West Nile virus (Monath and Heinz, In Fields Virology, Third Edition, eds.
B.N. Fields
et al., Lippincott-Raven Publishers, Philadelphia, pp 961-1034, 1996).
Infections caused by
JE are relatively rare, but the case-fatality is 5-40% because no specific
treatment is
available. JE is widely distributed in China, Japan, Philippines, far-eastern
Russia and India
providing a significant threat to those traveling in these areas. Currently
available JE vaccine
is produced from brain tissues of mice infected with single virus isolate.
Side effects are
observed in 10% to 30% of the vaccinees.
To obtain chimeric and/or mutated antigens that provide a protective immune
response against all or most of the viruses within the JE complex, DNA
shuffling is
performed on viral envelope genes. The amino acid identity within the JE
complex varies
between 72% and 93%. In addition, significant antigenic variation has been
observed among
JE strains by neutralization assays, agar gel diffusion, antibody absorption
and monoclonal
antibody analysis (Oda ( 1976) Kobe J. Med Sci. 22: 123; Kobyashi et al. (
1984) Infect.
Immun. 44: 1 I7). Moreover, the amino acid divergence of the envelope protein
gene among
13 strains from different Asian countries is as much as 4.2% (Ni and Barrett
(1995) J. Gen.
Virol. 76: 401 ). The resulting library of recombinant polypeptides encoded by
the shuffled
genes is screened to identify those that provide a cross-protective immune
response.
2. Tick-borne encepl:alitis virus
The tick-borne encephalitis virus complex comprises 14 antigenically related
viruses, eight of which cause human disease, including Powassan, Louping ill
and Tick-
borne encephalitis virus (TBE) (Monath and Heinz, In Fields Virology, Third
Edition, eds.
B.N. Fields et al., Lippincott-Raven Publishers, Philadelphia, pp. 961-1034,
1996). TBE has
been recognized in all Central and Eastern European countries, Scandinavia and
Russia,
whereas Powassan occurs in Russia, Canada and the United States. The symptoms
vary
from flu-like illness to severe meningitis, meningoencephalitis and
meningoencephalitis with
a fatality rate of 1 % to 2% (Gresikova and Calisher, In Monath ed., The
arboviruses:
ecology and epidemiology, vol. IV, Boca Raton, FL, CRC Press, pp. 177-203,
1988).
Family DNA shuffling is used to generate chimeric envelope proteins derived
from the TBE complex to generate crossprotective antigens. The envelope
proteins within
the family are 77-96% homologous, and viruses can be distinguished by specific
mAbs
(Holzmann et al., Vaccine, I0, 345, 1992). The envelope protein of Powassan is
78%
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/02944
97
identical at the amino acid level with that of TBE, and cross-protection is
unlikely, although
epidemiological data is limited.
Langat virus is used as a model system to analyze protective immune
responses in vivo (Iacono-Connors et al. (1996) Virus Res. 43: 125). Langat
virus belongs to
the TBE complex, and can be used in challenge studies in BSL3 facilities.
Serological
studies based on recombinant envelope proteins are performed to identify
immunogen
variants that induce high levels of antibodies against envelope proteins
derived from most or
all viruses of the TBE complex.
3. Dengue viruses
Dengue viruses are transmitted though mosquito bites, posing a significant
threat to troops and civilian populations particularly in tropical areas.
There are four major
serotypes of Dengue virus, namely Dengue 1, 2, 3 and 4. A tetravalent vaccine
that induces
neutralizing antibodies against all four strains of Dengue is required to
avoid antibody-
mediated enhancement of the disease when the individual encounters the virus
of the other
strain.
The envelope protein of Dengue virus has been shown to provide an immune
response that protects from a future challenge with the same strain of virus.
However, the
levels of neutralizing antibodies produced are relatively low and protection
from live virus
challenge is not always observed. For example, mice injected with genetic
vaccines
encoding envelope protein of Dengue-2 virus developed neutralizing antibodies
when
analyzed by in vitro neutralization assays, but the mice did not survive the
challenge with
live Dengue-2 virus (Kochel et al. (1997) Vaccine 15: 547-552). However,
protective
immune responses were observed in mice immunized with recombinant vaccinia
virus
expressing Dengue 4 virus structural proteins (Bray et al. (1989) J. Virol.
63: 2853). These
studies indicate that vaccinations with E proteins work, but significant
improvements in the
immunogenicity of the protective antigens are required.
In this Example, DNA shuffling is performed on the genes encoding the
envelope (E) protein from all four Dengue viruses and their antigenic
variants. Family DNA
shuffling is used to generate chimeric E protein variants that induce high
titer neutralizing
antibodies against all serotypes of Dengue. The E proteins of the different
dengue viruses
share 62% to 77% of their amino acids. Dengue 1 and Dengue 3 are most closely
related
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCTNS99/02944
98
(77% homologous), followed by Dengue 2 (69%) and Dengue 4 (62%). These
homologies
are well in the range that allows efficient family shuffling (Crameri et al.
(1998) Nature 391:
288-291).
The shuffled antigen sequences are incorporated into genetic vaccine vectors,
the plasmids purified, and subsequently injected into mice. The sera are
collected from the
mice and analyzed for the presence of high levels of cross-reactive
antibodies. The best
antigens are selected for further studies using in vivo challenge models to
screen for
chimeras/mutants that induce cross-protection against all strains of Dengue.
C. Improved Expression and Immunogenicity of Hantaan virus Glycoproteins
One of the advantages of genetic vaccines is that vectors expressing pathogen
antigens can be generated even when the given pathogen cannot be isolated in
culture. An
example of such potential situation was an outbreak of severe respiratory
disease among
rural residents of the Southwestern United States which was caused by a
previously
unknown hantavirus, Sin Nombre virus (Hjelle et al. (1994) J. Virol. 68: 592).
Much RNA
sequence information of the virus was obtained well before the virus could be
isolated and
characterized in vitro. In these situations, genetic vaccines can provide
means to generate
efficient vaccines in a short period of time by creating vectors encoding
antigens encoded by
the pathogen. However, genetic vaccines can only work if these antigens can be
properly
expressed in the host.
Hantaan virus belongs to the Bunyavirus family. A characteristic feature of
this family is that their glycoproteins typically accumulate at the membranes
of the Golgi
apparatus when expressed by cloned cDNAs, thereby reducing the efficacy of
corresponding
genetic vaccines (Matsuoka et al. ( 1991 ) Curr. Top. Microb. Immunol. 169:
161-179). Poor
expression of Hantaan virus glycoproteins on the cell surface is also one
explanation for
poor immune responses following injections of Hantaan virus genetic vaccines.
In this Example, family DNA shuffling is used to generate recombinant
Hantaan virus derived glycoproteins that are efficiently expressed in human
cells and that
can induce protective immune responses against the wild-type pathogen. Nucleic
acids that
encode the Hantaan virus glycoprotein are shuffled with genes that encode
other
homologous Bunyavirus glycoproteins. The resulting library is screened to
identify proteins
that are readily expressed in human cells. The screening is performed using a
dual marker
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/02944
99
expression vector that enables simultaneous analysis of transfection
efficiency and
expression of fusion proteins that are PIG-linked to the cell surface
(Whitehorn et al. (1995)
Biotechnology (N Y) 13:1215-9).
Flow cytometry based cell sorting is used to select Hantaan virus glycoprotein
variants that are efficiently expressed in mammalian cells. The corresponding
sequences are
then obtained by PCR or plasmid recovery. These chimeras/mutants are further
analyzed for
their capacity to protect wild mice against Hantaan virus infections.
Example 7
DNA Shuffling Of HSV-1 And HSV-2 Glycoproteins B And/Or D As Means To Induce
Enhanced Protective Immune Responses
This Example describes the use of DNA shuffling to obtain HSV glycoprotein
B (gB) and glycoprotein D (gD) polypeptides that exhibit improved ability to
induce
protective immune responses upon administration to a mammal. Epidemiological
studies
have shown that prior infections with HSV-1 give partial protection against
infections with
HSV-2, indicating existence of cross-reactive immune responses. Based on
previous
vaccination studies, the main immunogenic glycoproteins in HSV appear to be gB
and gD,
which are encoded by 2.7 kb and 1.2 kb genes, respectively. The gB and gD
genes of HSV-
1 are about 85% identical to the corresponding gene of HSV-2, and the gB genes
of each
share little sequence identity with the gD genes. Baboon HSV-2 gB is appr. 75%
identical to
human HSV-1 or -2 gB, with rather long stretches of almost 90% identity. In
addition, 60-
75% identity is found in portions of the genes of equine and bovine
herpesviruses.
Family shuffling is employed using as substrates nucleic acids that encode gB
and/or gD from HSV-1 and HSV-2. Preferably, homologous genes are obtained from
HSVs
of various strains. An alignment of gD nucleotide sequences from HSV-1 and two
strains of
HSV-2 is shown in Figure 7. Antigens encoded by the shuffled nucleic acids are
expressed
and analyzed in vivo. For example, one can screen for improved induction of
neutralizing
antibodies and/or CTL responses against HSV-1/HSV-2. One can also detect
protective
immunity by challenging mice or guinea pigs with the viruses. Screening can be
done using
pools or individuals clones.
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCTIUS99/02944
100
Example 8
Evolution Of HIV Gp120 Proteins For Induction Of Broad Spectrum Neutralizing
Ab
Responses
This Example describes the use of DNA shuffling to generate immunogens
that crossreact among different strains of viruses, unlike the wild-type
immunogens.
Shuffling two kinds of envelope sequences can generate immunogens that induce
neutralizing antibodies against a third strain.
Antibody-mediated neutralization of HIV-1 is strictly type-specific.
Although neutralizing activity broadens in infected individuals over time,
induction of such
antibodies by vaccination has been shown to be extremely difficult. Antibody-
mediated
protection from HIV-1 infection in vivo correlates with antibody-mediated
neutralization of
virus in vitro.
Figure 8 illustrates the generation of libraries of shuffled gp120 genes.
gp120
genes derived from HIV-1DH12 and HIV-IIIIB(NL43) are shuffled. The
chimeric/mutant
gp 120 genes are then analyzed for their capacity to induce antibodies that
have broad
spectrum capacity to neutralize different strains of HIV. Individual shuffled
gp 120 genes are
incorporated into genetic vaccine vectors, which are then introduced to mice
by injection or
topical application onto the skin. These antigens can also be delivered as
purified
recombinant proteins. The immune responses are measured by analyzing the
capacity of the
mouse sera to neutralize HIV growth in vitro. Neutralization assays are
performed against
HIV-1DH12, HIV-IIIIB and HIV-189.6. The chimeras/mutants that demonstrate
broad
spectrum neutralization are chosen for further rounds of shuffling and
selection. Additional
studies are performed in monkeys to illustrate the capacity of the shuffled
gp120 genes to
provide protection for subsequent infection with immunodeficiency virus.
Example 9
Antigen Shuffling of the Hepadnavirus Envelope Protein
The Hepatitis B virus (HBV) is one of a member of a family of viruses called
hepadnaviruses. This Example describes the use of genomes and individual genes
from this
family are used for DNA shuffling, which results in antigens having improved
properties.
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PGTNS99/02944
101
A. Shuffling of Hepadnavirus envelope protein genes
The envelope protein of the HBV assembles to form particles that carry the
antigenic structures collectively known as the Hepatitis B surface antigen
(HBsAg; this term
is also used to designate the protein itself). Antibodies to the major
antigenic site, designated
S the "a" epitope (which is found in the envelope domain called S), are
capable of neutralizing
the virus. Immunization with the HBsAg-bearing protein thus serves as a
vaccine against
viral infection. The HBV envelope also contains other antigenic sites that can
protect against
viral infection and are potentially vital components of an improved vaccine.
The epitopes are
part of the envelope protein domains known as preS 1 and preS2 (Figure 9).
DNA shuffling of the envelope gene from several members of the
hepadnavirus family is used to obtain more immunogenic proteins. Specifically,
the genes
from the following hepatitis viruses are shuffled:
~ the human HBV viruses, subtypes ayw and adw2
~ a hepatitis virus isolated from chimpanzee
~ a hepatitis virus isolated from gibbon
~ a hepatitis virus isolated from woodchuck
If desired, genes from other genotypes of the human virus are available for
inclusion in the DNA shuffling reactions. Likewise, other animal
hepadnaviruses are
available.
To promote the efficiency of the formation of chimeras resulting from DNA
shuffling, some artif cial genes are made:
~ In one case, a synthetic gene is made that contains the HBV envelope
sequences, except for those codons which specify amino acids found in
the chimpanzee and gibbon genes. For those codons, the chimpanzee or
gibbon sequence is used.
~ In a second case, a synthetic gene is synthesized in which the preS2 gene
sequence from the human HBV adw2 strain is fused with the woodchuck
S region.
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99141383 PCT/US99/02944
102
~ In a third case, all the oligonucleotides required to chemically synthesize
each of the hepadnavirus envelope genes are mixed in approximately
equal quantities and allowed to anneal to form a library of sequences.
After DNA shuffling of the hepadnavirus envelope genes, either or both of
two strategies are used to obtain improved HBsAg antigens.
Strategy A: Antigens are screened by immunizing mice using two possible
methods. The genes are injected in the form of DNA vaccines, i.e., shuffled
envelope genes
carried by a plasmid that comprises the genetic regulatory elements required
for expression
of the envelope proteins. Alternatively, the protein is prepared from the
shuffled genes and
used as the immunogen.
The sequences that give rise to greater immunogenicity for either the preS 1-,
preS2- or S-borne HBV antigens are selected for a second round of shuffling
(Figure 10).
For the second round, the best candidates are chosen based on their improved
antigenicity
and their other properties such as higher expression level or more efficient
secretion.
Screening and further rounds of shuffling are continued until a maximum
optimization for
one of the antigenic regions is obtained.
The individually optimized genes are then used as a combination vaccine for
the induction of optimal responses to preS 1-, preS2- and S-born epitopes.
Strategy B: After isolation of the individually optimized genes as in Strategy
A, the preS 1, preS2 and S candidates are shuffled together, or in a pairwise
fashion, in
further rounds to obtain genes which encode proteins that demonstrate improved
immunogenicity for at least two regions containing HBsAg epitopes (Figure 11
).
B. Use of HBsAg to carry epitopes from unrelated antigens
Several of the characteristics of the HBsAg make it a useful protein to carry
epitopes drawn from other, unrelated antigens. The epitopes can be either B
epitopes (which
induce antibodies) or T epitopes drawn from the class I type (which stimulate
CD8+ T
lymphocytes and induced cytotoxic cells) or class II type (which induce helper
T
lymphocytes and are important in providing immunological memory responses.
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/02944
103
1. B cell epitopes
Amino acid sequences of potential B epitopes are chosen from any pathogen.
Such sequences are often known to induce antibodies, but the immunogenicity is
weak or
otherwise unsatisfactory for preparation of a vaccine. These sequences can
also be
mimotopes, which have been selected based on their ability to have a certain
antigenicity or
immunogenicity.
The amino acid coding sequences are added to a hepadnavirus envelope gene.
The heterologous sequences can either replace certain envelope sequences, or
be added in
addition to all the envelope sequences. The heterologous epitope sequences can
be placed at
any position in the envelope gene. A preferred position is the region of the
envelope gene
that encodes the major "a" epitope of the HBsAg (Figure 12). This region is
likely to be
exposed on the external side of the particles formed by the envelope protein,
and thus will
expose the heterologous epitopes.
DNA shuffling is carried out on the envelope gene sequences, keeping the
1 S sequence of the heterologous epitopes constant. Screening is carried out
to choose candidates
that are secreted into the culture medium aRer transfection of plasmids from
the shuffled
library into cells in tissue culture.
Clones that encode a secreted protein are then tested for immunogenicity in
mice either as a DNA vaccine or as a protein antigen, as described above.
Clones that give
an improved induction of antibodies to the heterologous epitopes are chosen
for further
rounds of DNA shuffling. The process is continued until the immunogenicity of
the
heterologous epitope is sufficient for use as a vaccine against the pathogen
from which the
heterologous epitopes were derived.
2. Class I epitopes
MHC Class I epitopes are relatively short, linear peptide sequences that are
generally between 6 and 12 amino acids amino acids in length, most often 9
amino acids in
length. These epitopes are processed by antigen-presenting cells either after
synthesis of the
epitope within the cell (usually as part of a larger protein) or after uptake
of soluble protein
by the cells.
Polynucleotide sequences that encode one or more class I epitopes are
inserted into the sequence of a hepadnavirus envelope gene either by replacing
certain
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCTNS99/02944
104
envelope sequences, or by inserting the epitope sequences into the envelope
gene. This is
typically done by modifying the gene before DNA shuffling or by including in
the shuffling
reaction certain oligonucleotide fragments that encode the heterologous
epitopes as well as
sufficient flanking hepadnavirus sequences to be incorporated into the
shuffled products.
Preferably, the heterologous ciass I epitopes are placed into different
positions in the several hepadnavirus genes used for the DNA shuffling
reaction. This will
optimize the chances for finding chimeric gene carrying the epitopes in an
optimal position
for efficient presentation.
3. Class II epitopes
MHC Class II epitopes are generally required to be part of a protein which is
taken up by antigen presenting cells, rather than synthesized within the cell.
Preferably, such
epitopes are incorporated into a carrier protein such as the HBV envelope that
can be
produced in a soluble form or which can be secreted if the gene is delivered
in the form of a
DNA vaccine.
Polynucleotides that encode heterologous class II epitopes are inserted into
regions of the hepadnavirus envelope genes that are not involved in the
transmembrane
structure of the protein. DNA shuffling is performed to obtain a secreted
protein that also
carries the class II epitopes. When injected as a protein, or when the gene is
delivered as a
DNA vaccine, the protein can be taken up by antigen presenting cells for
processing of the
class II epitopes.
Example 10
Evolution of broad spectrum vaccines against Hepatitis C Virus.
Antigenic heterogeneity of different strains of Hepatitis C Virus (HCV) is a
major problem in development of efficient vaccines against HCV. Antibodies or
CTLs
specific for one strain of HCV typically do not protects against other
strains. Multivalent
vaccine antigens that simultaneously protect against several strains of HCV
would be of
major importance when developing efficient vaccines against HCV.
The HCV envelope genes, which encode envelope proteins E1 and E2, have
been shown to induce both antibody and lymphoproliferative responses against
these
antigens (Lee er al. (1998) J. Virol. 72: 8430-6), and these responses can be
optimized by
DNA shuffling. The hypervariable region 1 (HVR1 ) of the envelope protein E2
of HCV is
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/02944
105
the most variable antigenic fragment in the whole viral genome and is
primarily responsible
for the large inter- and intra-individual heterogeneity of the infecting virus
(Puntoriero et al.
( 1998) EMBO J. 17: 3521-33). Therefore, the gene encoding E2 is a
particularly useful
target for evolution by DNA shuffling.
DNA shuffling of HCV antigens, such as nucleocapsid or envelope proteins
E1, E2, provides a means to generate multivalent HCV vaccines that
simultaneously protect
against several strains of HCV. These antigens are shuffled using the family
DNA shuffling
approach. The starting genes will be obtained from various natural isolates of
HCV. In
addition, related genes from other viruses can be used to increase the number
of different
recombinants that are generated. A library of related, chimeric variants of
HCV antigens are
then generated and this library will be screened for induction of widely
crossreactive
immune responses. The screening can be done directly in vivo by injecting
individual
variants into test animals, such as mice or monkeys. Either purified
recombinant proteins or
DNA vaccines encoding the relevant genes are injected. Typically, a booster
injection is
given 2-3 weeks after the frst injection. Thereafter, the sera of the test
animals are collected
and these sera are tested for the presence of antibodies that react against
multiple HCV virus
isolates.
Before the in vivo testing is initiated, the antigens can be pre-enriched in
vitro
for antigens that are recognized by polyclonal antisera derived from
previously infected
patients or test animals. Alternatively, monoclonal antibodies that are
specific for various
strains of HCV are used. The screening is performed using phage display or
ELISA assays.
For example, the antigen variants are expressed on bacteriophage M13 and the
phage are
then incubated on plates coated with antisera derived from patients or test
animals infected
with various HCV isolates. The phage that bind to the antibodies are then
eluted and further
analyzed in test animals for induction of crossreactive antibodies.
Example 11
Evolution of chimeric allergens that induce broad immune responses and have
reduced
risk of inducing anaphylactic reactions
Specific immunotherapy of allergy is performed by injecting increasing
amounts of the given allergens into the patients. The therapy typically alters
the types of
' allergen-specific immune responses from a dominating T helper 2 (TH2) type
response to a
dominating T helper 1 (TH1) type response. However, because allergic patients
have
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/OZ944
106
increased levels of IgE antibodies specific for the allergens, the
immunotherapy of allergy
involves a risk of IgE receptor mediated anaphylactic reactions.
T helper (TH) cells are capable of producing a large number of different
cytokines, and based on their cvtokine synthesis pattern TH cells are divided
into two subsets
(Paul and Seder (1994) Cell 76: 241-251). TH1 cells produce high levels of IL-
2 and IFN-
gamma and no or minimal levels of IL-4, IL-5 and IL-13. In contrast, TH2 cells
produce
high levels of IL-4, IL-5 and IL-13, whereas IL-2 and IFN-gamma production is
minimal or
absent. TH1 cells activate macrophages, dendritic cells and augment the
cytolytic activity of
CD8+ cytotoxic T lymphocytes and NK cells (Id.), whereas TH2 cells provide
efficient help
for B cells and they also mediate allergic responses due to the capacity of
TH2 cells to induce
IgE isotype switching and differentiation of B cells into IgE secreting cell
(De Vries and
Punnonen (1996) In Cytokine regulation of humoral immunity: basic and clinical
aspects.
Eds. Snapper, C.M., John Wiley & Sons, Ltd., West Sussex, UK, pp. 195-215
This Example describes methods to generate chimeric allergens that can
broadly modulate allergic immune responses. This can be achieved by DNA
shuffling of
related allergen genes to generate chimeric genes. In addition,
chimeric/mutated allergens
are less likely to be recognized by preexisting IgE antibodies of the
patients. Importantly,
allergen variants that are not recognized by IgE antibodies can be selected
using patient sera
and negative selection (Figure 13).
As one example, chimeric allergen variants of Der p2, Der f2, Tyr p2 Lep d2
and Gly d2 allergens are generated. These house dust mite allergens are very
common in
exacerbating allergic and asthmatic symptoms, and improved means to
downregulate such
allergic immune responses are desired. House dust mites can be used as sources
of the genes.
The corresponding genes are shuffled using family DNA shuffling and a shuffled
library is
generated. Phage display is used to exclude allergens that are recognized by
antibodies from
allergic individuals. It is particularly important is to exclude variants that
are recognized by
IgE antibodies. Phage expressing the allergen variants are incubated with
pools of sera
derived from allergic individuals. The phage that are recognized by IgE
antibodies are
removed, and the remaining allergens are further tested in vitro and in vivo
for their capacity
to activate allergen-specific human T cells (Figure 14). Because immunotherapy
of allergy is
beiieved to function through induction of a dominating TH 1 response as
compared to allergic
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCTNS99/02944
107
TH2 response, efficient T cell activation and induction of a TH1 type response
by allergen
variants is used as a measure of the efficacy of the allergens to modulate
allergic T cell
responses.
The optimal allergen variants are then further tested in vivo by studying skin
S responses after injections to the skin. A strong inflammatory response
around the injection
site is an indication of efficient T cell activation, and the allergen
variants that induce the
most efficient delayed type T cell response (typically observed 24 hours after
the injection)
are the best candidates for further studies in vivo to identify allergens that
effectively
downregulate allergic immune responses. Accordingly, these allergen variants
re analyzed
for their capacity to inhibit allergic responses in allergic, atopic and
asthmatic individuals.
The screening of allergen variants is further illustrated in Figure 13 and
Figure 14.
Example 12
Evolution of cancer antigens that induce efficient anti-tumor immune responses
Several cancer cells express antigens that are present at significantly higher
levels on the malignant cells than on other cells in the body. Such antigens
provide excellent
targets for preventive cancer vaccines and immunotherapy of cancer. The
immunogenicity
of such antigens can be improved by DNA shuffling. In addition, DNA shuffling
provides
means to improve expression levels of cancer antigens.
This Example describes methods to generate cancer antigens that can
efficiently induce anti-tumor immune responses by DNA shuffling of related
cancer antigen
genes. Libraries of shuffled melanoma-associated glycoprotein (gp 100/pmel l
7) genes
(Huang et al. ( 1998) J. Invest. Dermatol. 111: 662-7) are generated. The
genes can be
isolated from melanoma cells obtained from various patients, who may have
mutations of
the gene, increasing the diversity in the starting genes. In addition, a gp
100 gene can be
isolated from other mammalian species to further increase the diversity of
starting genes. A
typical method for the isolation of the genes is RT-PCR. The corresponding
genes are
shuffled using single gene DNA shuffling or family DNA shuffling and a
shuffled library is
generated.
The shuffled gp100 variants, either pools or individual clones, are
subsequently injected into test animals, and the immune responses are studied
(Figure 15).
The shuffled antigens are either expressed in E. coli and recombinant,
purified proteins are
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/02944
108
injected, or the antigen genes are used as components of DNA vaccines. The
immune
response can be analyzed for example by measuring anti-gp100 antibodies, as
previous
studies indicate that the antigen can induce specif c antibody responses
(Huang et al.,
supra.). Alternatively, the test animals that can be challenged by malignant
cells expressing
gp100. Animals that have been efficiently immunized will generate cytotoxic T
cells specific
for gp 100 and will survive the challenge, whereas in non-immunized or poorly
immunized
animals the malignant cells will efficiently grow eventually resulting in
lethal expansion of
the cells. Furthermore, antigens that induce cytotoxic T cells that have the
capacity to kill
cancer cells can be identified by measuring the capacity of T cells derived
from immunized
animals to kill cancer cells in vitro. Typically the cancer cells are first
labeled with
radioactive isotopes and the release of radioactivity is an indication of
tumor cell killing after
incubation in the presence of T cells from immunized animals. Such
cytotoxicity assays are
known in the art.
The antigens that induce highest levels of specific antibodies and/or can
protect against the highest number of malignant cells can be chosen for
additional rounds of
shuffling and screening. Mice are useful test animals because large numbers of
antigens can
be studied. However, monkeys are a preferred test animal, because the MHC
molecules of
monkeys are very similar to those of humans.
To screen for antigens that have optimal capacity to activate antigen-specific
T cells, peripheral blood mononuclear cells from previously infected or
immunized humans
individuals can be used. This is a particularly useful method, because the MHC
molecules
that will present the antigenic peptides are human MHC molecules. Shuffled
cancer antigens
that induce cytotoxic T cells that have the capacity to kill cancer cells can
be identified by
measuring the capacity of T cells derived from immunized animals to kill
cancer cells in
vitro. Typically the cancer cells are first labeled with radioactive isotopes
and the release of
radioactivity is an indication of tumor cell killing after incubation in the
presence of T cells
from immunized animals. Such cytotoxicity assays are known in the art.
Example 13
Evolution of autoantigens that induce efficient immune responses
Autoimmune diseases are characterized by an immune response directed
against self antigens expressed by the host. Autoimmune responses are
generally mediated
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCTNS99/02944
109
by TH 1 cells that produce high levels of IL-2 and IFN-gamma. Vaccines that
can direct
autoantigen specific T cells towards TH2 phenotype producing increased levels
of IL-4 and
IL-5 would be beneficial. For such vaccines to work, the vaccine antigens have
to be able to
efficiently activate specific T cells. DNA shuffling can be used to generate
antigens that
have such properties. To optimally induce TH2 cell differentiation it may be
beneficial to
coadminister cytokines that have been shown to enhance TH2 cell activation and
differentiation, such as IL-4 (Racke et al. (1994) J. Exp. Med. 180: 1961-66).
This Example describes methods for generating autoantigens that can
efficiently induce immune responses. DNA shuffling is performed on related
autoantigen
genes. For example, libraries of shuffled myelin basic proteins, or fragments
thereof (Zamvil
and Steinman (1990) Ann. Rev. Immunol. 8: 579-621); Brocke et al. (1996)
Nature 379: 343-
46) are generated. MBP is considered to be an important autoantigen in
patients with
multiple sclerosis (MS). The genes encoding MBP from at least bovine, mouse,
rat, guinea
pig and human have been isolated providing an excellent starting point for
family shuffling.
A typical method for the isolation of the genes is RT-PCR. The shuffled MBP
variants,
either pools or individual clones, are subsequently injected into test
animals, and the immune
responses are studied. The shuffled antigens are either expressed in E. coli
and recombinant,
purified proteins are injected, or the antigen genes are used as components of
DNA vaccines
or viral vectors. The immune response can be analyzed for example by measuring
anti-MBP
antibodies by ELISA. Alternatively, the lymphocytes derived from immunized
test animals
are activated with MBP, and the T cell proliferation or cytokine synthesis is
studies. A
sensitive assays for cytokine synthesis is ELISPOT (McCutcheon et al. (1997)
J. Immunol.
Methods 210: 149-66). Mice are useful test animals because large numbers of
antigens can
be studied. However, monkeys are a preferred test animal, because the MHC
molecules of
monkeys are very similar to those of humans.
To screen for antigens that have optimal capacity to activate MBP specific T
cells peripheral blood mononuclear cells from patients with MS can also be
used. This is a
particularly useful method, because the MHC molecules that will present the
antigenic
peptides are human MHC molecules. Shuffled antigens that activate MBP specific
T cells
can be identified by measuring the capacity of T cells derived from MS
patients to
proliferate or produce cytokines upon culture in the presence of the antigen
variants. Such
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/02944
110
assays are known in the art. One such assay is ELISPOT (McCutcheon et al.,
supra.). An
indication of the efficacy of an MBP variant to activate specific T cells is
also the degree of
skin inflammation when the antigen is injected into the skin of a patient with
MS. Strong
inflammation is correlated with strong activation of antigen-specific T cells.
Improved
activation of MBP specific T cells, particularly in the presence of IL-4, is
likely to result in
enhanced TH2 cell responses, which are beneficial in the treatment of MS
patients.
Example 14
Method of Optimizing the Immunogenicity of Hepatitis B Surface Antigen
This Example describes methods by which the envelope protein sequence of
the hepatitis B virus can be evolved to provide a more immunogenic surface
antigen. Such a
protein is important for vaccination of low responders and for immunotherapy
of chronic
hepatitis B.
Background
Current HBV vaccines (Merck, SKB) are based on the immunogenicity of the
viral envelope protein and contain the Major (or Small) form of the envelope
protein
produced as particles in yeast. These particles induce antibodies to the major
surface antigen
(HBsAg) which can protect against infection when antibody levels are at least
10 milli-
International Units per milliliter (mU/ml). These recombinant protein
preparations are not
capable of inducing humoral immunity in chronic carriers (some 300 million
cases
worldwide) the induction of which would be important to control virus spread.
Moreover,
certain individuals respond poorly to the vaccine (up to 30-50% of vaccinees
in some
groups) and do not develop protective levels of antibody. The inclusion of the
natural
epitope sequences contained in the Middle or Large forms of the viral envelope
protein has
been used as a method to increase the immunogenicity of vaccine preparations.
An
alternative method is to introduce new (i. e., not present in the natural
virus sequence) helper
T-cell epitopes into the HBsAg sequence using DNA shuffling technology.
Method
DNA sequences of HBsAg from different subtypes of HBV (e.g., ayw and
adr) and the related woodchuck hepatitis virus are prepared for shuffling.
Comparison of the
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCTNS99/02944
111
genes encoding these proteins suggests that recombination would occur at least
ten times
within 850 base pairs when shuffling the ayw and woodchuck hepatitis virus
(WHV) DNA
sequences. Nucleotide and amino acid sequences of portions of different
subtypes of HBV
are shown in Figure 17.
The sequence of the main HBsAg B-cell antigenic site (the "a" epitope) can
be retained in the protein sequence by including the coding sequences of the
external "a"
loop in the final protein preparation. Peptide analogues) for the "a" epitope
of HBsAg have
been described (Neurath et al. (1984) J. Tirol. Methods 9:341-346), and the
immunogenicity
of the "a" epitope has been demonstrated (Bhatnagar et al. ( 1982) Proc.
Nat'l. Acad. Sci.
USA 79: 4400-4404). HBsAg and WHsAg share the major "a" determinant, and
chimps can
be protected by both antigens (Cote et al. ( 1986) J. virol. 60: 895-901 ).
Likewise, important
CTL epitopes can be included in the protein in a defined way.
One can also easily introduce B or T (helper or CTL) epitopes from other
antigens into the shuffled HBsAg sequence. This may focus the immune response
to certain
epitopes, independent of other potentially dominant epitopes from the same
protein.
Furthermore, the availability of the "a" loop on the HBsAg may provide a
region of the
envelope protein into which other artificial antigens or mimotopes could be
included.
In all cases where a novel HBV envelope sequence is prepared to include a
specific epitope (from HBV, another pathogen or a tumor cell), shuffling of
the surrounding
sequences in the HBV envelope will serve to optimize expression of the protein
and help to
ensure that the immune response is directed to the desired epitope.
Several methods of analyzing and utilizing shuffled HBsAg sequences are
described below.
A. Modulating expression levels of HBsAg
Shuffled HBsAg sequences are introduced into cells in culture and the ability
to direct expression of secreted HBsAg (measured with clinical kits for HBsAg
expression)
is evaluated. This can be used to identify shuffled HBsAg sequences which
exhibit
optimized HBsAg expression levels. Such coding sequences are particularly
interesting for
DNA vaccination.
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/02944
112
B. Circunrventing low responsiveness to tl:e HBsAg
Shuffled HBsAg sequences are evaluated for their ability to induce an
immune response to the clinically relevant HBsAg epitopes. This can be done
using mice of
the H-2s and H-2f haplotypes, which respond poorly or not at all to HBsAg
protein
immunization. In these experiments, one can verify that antibodies are
generated to the main
"a" epitope in the S protein, and a second protective epitope in the PreS2
region (a linear
sequence).
The PreS2 and S coding sequences for the envelope protein (HBsAg) from
the HBV ayw subtype (plasmid pCAG-M-Kan; Whalen) and the WHV (plasmid pWHV8
from ATCC) are amplified from the two plasmids by PCR and shuffled. Examples
of
suitable primers for PCR amplification are shown in Figure 18. The shuffled
library of
sequences is cloned into an HBsAg-expression vector and individual colonies
are chosen for
preparation of plasmid DNA. The DNA is administered to the test animals and
vectors
which induce the desired immune response are identified and recovered.
C. Presentation ojnatural HBsAg CTL epitopes by evolved HBsAg proteins
This example describes methods of using the evolved HBsAg protein to
present natural HBsAg CTL epitopes. Shuffling is used to increase overall
immunogenicity
of the HBsAg protein, as discussed above. However, some of the evolved HBsAg
sequences
are replaced with class I or class II epitope sequences from the natural HBsAg
protein in
order to stimulate immunoreactivity specifically to these natural viral
epitopes.
Alternatively, the natural viral epitopes can be added to the evolved protein
without loss of
immunogenicity of the evolved HBsAg.
D. Expression of tumor-derived CTL epitopes by evolved HBsAg proteins
This example describes methods of using the evolved HBsAg protein is used
to express tumor-derived CTL epitopes. The overall immunogenicity of the HBsAg
protein
is increased by shuffling. However, some of the evolved HBsAg sequences are
replaced with
class I or class II epitope sequences from tumor cells in order to stimulate
immunoreactivity
specifically to these natural viral epitopes. Alternatively, the tumor cells
epitopes can be
added to the evolved protein without loss of immunogenicity of the evolved
HBsAg.
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/02944
113
E. Expression of mimotope sequences by tJ:e HBsAg
This example describes the use of an evolved HBsAg protein for expression
of mimotope sequences. Again, the evolved HBsAg protein is used to increase
overall
immunogenicity of the protein. However, some of the evolved HBsAg sequences
are
replaced with mimotope sequences to stimulate immunoreactivity specifically to
the natural
sequence which cross reacts with the mimotope. Alternatively, the mimotope
sequences can
be added to the evolved protein without loss of immunogenicity of the evolved
HBsAg.
Example 15
Fusion Proteins Of The HBsAg Polypeptide and HIV gp120 Protein
This Example describes the preparation of fusion proteins ("chimeras")
formed from the HBsAg polypeptide and the extracellular fragment gp 120 of the
HIV
envelope protein, and their use as vaccines.
Background
When used as a vaccine, recombinant monomeric gp120 has failed to induce
antibodies that have strong neutralizing activity with primary isolates of the
HIV virus. It has
been suggested that oligomeric forms of the HIV envelope protein which expose
certain
regions of the tertiary structure would be better able to elicit virus-
neutralizing antibodies
(Parrin et al. ( 1997) Immunol. Lett. 57: 1 OS-112; VanCott et al. ( 1997) J.
virol. 71: 4319-
4330;
In this Example, DNA shuffling is applied to this problem, in order to obtain
gp120 polypeptides which adopt conformations slightly different from those of
previous
preparations of recombinant gp120. To allow the individual gp120 molecules to
interact as
oligomers, a fusion is prepared between gp120 sequences (on the N-terminus of
the fusion)
and HBsAg sequences (on the C-terminal of the fusion).
The N-terminal peptide sequence of the S region of the HBsAg polypeptide is
a transmembrane structure which is locked into the membrane of the endoplasmic
reticulum.
The actual N-terminus of the S region as well as the preS2 sequences are
located in the
lumenal part of the ER. They are found on the outside of the final HBsAg
particles. By
placing the gp120 sequences on the N-terminus of the HBsAg preS2 or S
sequences, the
SUBSTITUTE SHEET RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCT/US99/02944
114
gp120 sequences are also located on the outside of the particles. The gp120
molecules can
thus be brought together in three-dimensional space to interact as in the
virus.
Since the exact conformation of the final chimera which will have the most
appropriate immunogenicity cannot be predicted, DNA shuffling is employed. The
sequences of the HBsAg polypeptide, which functions as a scaffold, and of
gp120 are both
shuffled. Screening of the shuffled products can be performed by ELISA assay
using
antibodies (polyclonal or monoclonal) which have previously been determined to
have virus
neutralizing activity.
Method
The sequences encoding the gp120 fragment of the HIV envelope protein are
preferably prepared as a synthetic gene to include codons which are optimal
for gene
expression in mammals. The gp 120 sequence will typically include a signal
sequence on its
N-tenminal end.
The gp120 sequences are inserted into the preS2 region of an HBsAg-
expressing plasmid. In the preS2 region of the plasmid pMKan and its
derivatives, an EcoRI
site and an XhoI site are available for cloning. The gp 120 sequences can be
inserted between
these two sites, which brings the gp120 closer to the start of the S coding
sequences, or into
the EcoRI site alone, which leaves a spacer sequence of about SO amino acids
between the
gp120 sequence and the start of the S region of the HBsAg. These two different
cloning
strategies will give rise to chimeric molecules in which the gp120 sequences
are located at
different distances from the transmembrane region of the HBsAg sequence. This
may be
advantageous in allowing the gp 120 sequences to adopt conformations which are
more
suitable immunogens than monomeric gp 120.
DNA shuffling of the entire chimeric sequence is carned out. Family
shuffling is preferred; this involves the preparation of several gp120-HBsAg
fusion proteins
in which different gp120 and HBsAg (or WHV) sequences are used. An alignment
of
HBsAg nucleotide sequences is shown in Figure 19. After shuffling of the
different
sequences, the products are cloned into an expression vector such as pMKan.
Pools of clones
from the library of shuffled products are transfected into cultured cells and
the secretion of
chimeric proteins is assayed with broadly reactive antibodies to gp 120.
Positive clones can
be further evaluated with particular antibodies that have demonstrated HIV
neutralizing
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCTNS99/02944
115
activity, for example the anti-CD4 binding domain recombinant human monoclonal
antibody, IgGlbl2 (Kessler et al. (1997) AIDS Res. Hum. Retrovir-uses. 1: 13:
575-582;
Roben et al. ( 1994) J. Virol. 68: 4821-4828). Candidate clones can then be
used to immunize
mice and the antiserum obtained is evaluated for HIV virus-neutralizing
activity in in vitro
assays.
Because the gp120 molecule (approx. 1100 amino acids) is larger in size than
the monomeric HBsAg preS2+S protein (282 amino acids), it is likely that not
every HBsAg
monomer in an aggregated particle will contain a gp120 sequence. Internal
initiation of
protein synthesis can take place on the HBsAg coding sequences at the
initiator methionine
that marks the beginning of the S region. Thus, the chimeric molecule (which
contains the
gp120 sequences) will be mixed in the cell with the S region and the
multimeric particles
should assemble with an appropriate number of chimeric polypeptides and native
HBsAg S
monomers. Alternatively, an S-expressing plasmid can be mixed with the plasmid
expressing
the chimera, or a single plasmid which expresses the chimera and the S form
can be
constructed. A diagram of the resulting particles is shown in Figure 20.
Example 16
DNA Shuffling Of HSV-1 And HSV-2 Glycoproteins B And/Or D As Means To Induce
Enhanced Protective Immune Responses
This Example describes the use of DNA shuffling to obtain HSV glycoprotein
B (gB) and glycoprotein D (gD) polypeptides that exhibit improved ability to
induce
protective immune responses upon administration to a mammal. Epidemiological
studies
have shown that prior infections with HSV-1 give partial protection against
infections with
HSV-2, indicating existence of cross-reactive immune responses. Based on
previous
vaccination studies, the main immunogenic glycoproteins in HSV appear to be gB
and gD,
which are encoded by 2.7 kb and 1.2 kb genes, respectively. The gB and gD
genes of HSV-
1 are about 85% identical to the corresponding gene of HSV-2, and the gB genes
of each
share little sequence identity with the gD genes. Baboon HSV-2 gB is appr. 75%
identical to
human HSV-1 or -2 gB, with rather long stretches of almost 90% identity. In
addition, 60-
75% identity is found in portions of the genes of equine and bovine
herpesviruses.
Family shuffling is employed using as substrates nucleic acids that encode gB
and/or gD from HSV-1 and HSV-2. Preferably, homologous genes are obtained from
HSVs
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCTIUS99/02944
116
of various strains. An alignment of gD nucleotide sequences from HSV-1 and two
strains of
HSV-2 is shown in Figure 7. Antigens encoded by the shuffled nucleic acids are
expressed
and analyzed in vivo. For example, one can screen for improved induction of
neutralizing
antibodies and/or CTL responses against HSV-1/HSV-2. One can also detect
protective
immunity by challenging mice or guinea pigs with the viruses. Screening can be
done using
pools or individuals clones.
Example 17
Evolution Of HIV Gp120 Proteins For Induction Of Broad Spectrum Neutralizing
Ab
Responses
This Example describes the use of DNA shuffling to generate immunogens
that crossreact among different strains of viruses, unlike the wild-type
immunogens.
Shuffling two kinds of envelope sequences can generate immunogens that induce
neutralizing antibodies against a third strain.
Antibody-mediated neutralization of HIV-1 is strictly type-specific.
Although neutralizing activity broadens in infected individuals over time,
induction of such
antibodies by vaccination has been shown to be extremely difficult. Antibody-
mediated
protection from HIV-1 infection in vivo correlates with antibody-mediated
neutralization of
virus in vitro.
Figure 8 illustrates the generation of libraries of shuffled gp120 genes.
gp120
genes derived from HIV-1DH12 and HIV-IIIIB(NL43) are shuffled. The
chimeric/mutant
gp 120 genes are then analyzed for their capacity to induce antibodies that
have broad
spectrum capacity to neutralize different strains of HIV. Individual shuffled
gp 120 genes are
incorporated into genetic vaccine vectors, which are then introduced to mice
by injection or
topical application onto the skin. These antigens can also be delivered as
purified
recombinant proteins. The immune responses are measured by analyzing the
capacity of the
mouse sera to neutralize HIV growth in vitro. Neutralization assays are
performed against
HIV-1DH12, HIV-lIIIB and HIV-189.6. The chimeras/mutants that demonstrate
broad
spectrum neutralization are chosen for further rounds of shuffling and
selection. Additional
studies are performed in monkeys to illustrate the capacity of the shuffled
gp120 genes to
provide protection for subsequent infection with immunodeficiency virus.
SUBSTITUTE SHEET (RULE 26)


CA 02320958 2000-08-10
WO 99/41383 PCTNS99/OZ944
117
It is understood that the examples and embodiments described herein are for
illustrative purposes only and that various modifications or changes in light
thereof will be
suggested to persons skilled in the art and are to be included within the
spirit and purview of
this application and scope of the appended claims. All publications, patents,
and patent
applications cited herein are hereby incorporated by reference for all
purposes.
SUBSTITUTE SHEET (RULE 26)

Representative Drawing
A single figure which represents the drawing illustrating the invention.
Administrative Status

For a clearer understanding of the status of the application/patent presented on this page, the site Disclaimer , as well as the definitions for Patent , Administrative Status , Maintenance Fee  and Payment History  should be consulted.

Administrative Status

Title Date
Forecasted Issue Date Unavailable
(86) PCT Filing Date 1999-02-10
(87) PCT Publication Date 1999-08-19
(85) National Entry 2000-08-10
Dead Application 2004-02-10

Abandonment History

Abandonment Date Reason Reinstatement Date
2003-02-10 FAILURE TO PAY APPLICATION MAINTENANCE FEE

Payment History

Fee Type Anniversary Year Due Date Amount Paid Paid Date
Application Fee $300.00 2000-08-10
Maintenance Fee - Application - New Act 2 2001-02-12 $100.00 2001-01-29
Registration of a document - section 124 $100.00 2001-08-07
Maintenance Fee - Application - New Act 3 2002-02-11 $100.00 2002-01-22
Owners on Record

Note: Records showing the ownership history in alphabetical order.

Current Owners on Record
MAXYGEN, INC.
Past Owners on Record
BASS, STEVEN H.
HOWARD, RUSSELL
PUNNONEN, JUHA
STEMMER, WILLEM P.C.
WHALEN, ROBERT GERALD
Past Owners that do not appear in the "Owners on Record" listing will appear in other documentation within the application.
Documents

To view selected files, please enter reCAPTCHA code :



To view images, click a link in the Document Description column. To download the documents, select one or more checkboxes in the first column and then click the "Download Selected in PDF format (Zip Archive)" or the "Download Selected as Single PDF" button.

List of published and non-published patent-specific documents on the CPD .

If you have any difficulty accessing content, you can call the Client Service Centre at 1-866-997-1936 or send them an e-mail at CIPO Client Service Centre.


Document
Description 
Date
(yyyy-mm-dd) 
Number of pages   Size of Image (KB) 
Cover Page 2000-12-15 1 40
Representative Drawing 2000-12-15 1 4
Description 2001-02-09 132 7,439
Description 2000-08-10 117 7,023
Abstract 2000-08-10 1 56
Claims 2000-08-10 9 359
Drawings 2000-08-10 21 609
Correspondence 2000-11-23 2 3
Assignment 2000-08-10 4 128
PCT 2000-08-10 17 698
Prosecution-Amendment 2000-11-17 1 46
Correspondence 2001-02-09 16 472
Correspondence 2001-04-10 1 10
Assignment 2001-08-07 3 144
PCT 2000-08-11 11 589

Biological Sequence Listings

Choose a BSL submission then click the "Download BSL" button to download the file.

If you have any difficulty accessing content, you can call the Client Service Centre at 1-866-997-1936 or send them an e-mail at CIPO Client Service Centre.

Please note that files with extensions .pep and .seq that were created by CIPO as working files might be incomplete and are not to be considered official communication.

BSL Files

To view selected files, please enter reCAPTCHA code :