Note: Descriptions are shown in the official language in which they were submitted.
X344?~'~
This is a divisional application of Canadian serial no.
475,940, filed March 7, 1985.
H,ACKGROUND OF THE INVENTION
The present invention concerns pre-S gene coded hepatitis
B immunogens, vaccines and diagnostics. More especially, this
invention concerns novel pre-S gene coded peptides and novel
carriers, particularly carriers for pre-S gene coded peptides.
Even more especially, the present invention relates to synthetic
pre-S gene coded peptides covalently linked to lipid vesicle
carriers.
There are approximately 600,000 persistent carriers of
hepatitis B virus (HBV) in the United States; the estimated
total number of carriers in the world is 200 million. A
considerable portion of HBY carriers have chronic liver disease.
The involvement of HBY in liver cancer has been demonstrated
(W. Szmuness, P_rog., Med. Yirol. 24, 40 (1978) and R.P. Beasley.
L.-Y. Hwang, C.-C. Ling, C.-S. Chien, Lancet Nov., 21, 1129
( 1981 ) ).
HBY infections thus represent a major public health
problem worldwide. Already available vaccines (S. Krugman,
in Yiral Hepatitis;; Laboratory and Clinical Science,
F. Deinhardt, J. Deinhardt, Eds., Marcel Dekker, Inc., New
York-Basel, 1983, pp. 257-263) produced from the serum of HBV
carriers, because of limited resources and production costs
involved, do not provide the appropriate
-1-
~~~fl~~~'
means to control and eradicate the disease worldwide. There
is hope, however, that this may be accomplished by vaccines
based on recombinant DNA technology and/or synthetic
peptides.
The biology, structure and immunochemistry of HBV
and the genetic organization of its DNA genome have been
reviewed (B.S. Blumberg, Science, 197 17, (1977)). The
clotting and sequencing of the genome of several hepatitis
virus (HBV) isolates led to the elucidation of the genetic
structure of the viral DNA (P. Tiollais, P. Charnay, G.N.
Vyas, Science, 213, 406, (1981)).
The i.mmunologic markers of HBV infection include
the surface antigen (HRsAg), the core antigen (HBcAg), the
"e" antigen (HBeAgI and their respective antibodies.
Antibodies against HBsAg are protective against HBV
infection.
Several antigenic subtypes of HBV and of subviral
approximately :?2 nm diameter particles (hepatitis B surface
antigen; HBsAg) have been recognized (G. Le Bouvier, A.
Plilliams, Am. ~1. Med. Sci., 270, 165 (1975) ) . All of these
subtypes (for Example, ayw, adyw, adw2, adw and adr) share
common (group-;specificl envelope epitopes, the immune
response against which appears sufficient for protection
against infection by any of the virus subtypes (W. Szmuness,
C.E. Stevens, '.E.J. Harley, E.A. Zang, H.J. Alter, P.E.
Taylor, A. DeVera, G.T.S. Chen, A. Kellner, et al., N. Engl.
J. Med., 307, 1481, (1982)).
The physical structure and proposed genetic
organization of the HBV genome are described by Tiollais et
-2-
al, 1981, supra at pp. 408-409. There are two DPdA strands,
namely the lone (Ll strand and the short (S1 strand. The L
strand transcript has four open reading frame regions which
are termed ( S -~- pre-S ) , C , P and X .
The open reading frame region (S + pre-S)
corresponds to the envelope (env) gene of HBV DNA and codes
for a family oi: proteins found in the HBV envelope and in
virus related particles.
A schematic representation of the potential
translation products of the env genes) of HBV DNA is as
follows:
Pre-S Region S Region 400
1 12 120 174 175 400
I I I ~ i
pre-S(1~ pre-S(1:?) pre- (120) pre-S(174) S(11 S(2~6)
175 400
S region only:
S(2 6)
400
I i
pre-S(120) S(2~6)
400
pre-S ( 1:? ) S ( 2 6 )
400
l
pre-S ( 1 ) S ( 2 _ 6 )
The .numbers in the above schematic refers to amino
acids (AA). A translation initiation site at Met 1 exists
-3-
for the adw2 and adr substypes only. The first amino acid
for the other subtypes correspond to position pre-S 12.
Hereinafter, amino acid sequences corresponding to
the pre-S region (env 1 to 174) are designated with the
prefix "pre-S" and amino acid sequences corresponding to the
S region (env 1'75 to 400) are designated by the prefix "S".
In the env gene product representation, the S region spans
amino acids 175 to 400 as compared to amino acids 1 to 226
in the "S region only" representation.
In th~~ above schematic, the pre-S region is
defined by amino acid sequence positions pre-S 1 to amino
acid sequence position pre-S 174. The S region is defined by
sequence positions S 1 (amino acid 175 of the open reading
frame and adjacent to pre-S 174) to sequence position S 226
(amino acid 400 of the open reading frame). The s-gene
product (S-protein) consists of this 226 amino acid
sequence.
The epitope(s) essential for eliciting
virus-neutralizing antibodies have not yet been
unambiguously defined. It has been reported that the
group-specificity is represented by a complex of
determinants located on each of the two major approximately
22 and approximately 26 kilodalton constituent proteins (P22
and P26) of the virus envelope and of the hepatitis B
surface antigen (HBsAg). See J.W.-K. Shih, J.L. Gerin, J.
Immunol., 115, 634, (1975); J.W.-K. Shih, P.L. Tan, J.L.
Gerin, J. Immun.ol., 120, 520, (1978); S. Mishiro, M. Imai,
K. Takahashi. ~~. M=chida, T. Gotanda, Y. rliyakawa, M.
-4-
Mayumi, J. Immunol., 124, 1589, (1980); and G.R. Dreesman,
R. Chairez, M. ~~uarez., F.B. Hollinger, R.J. Courtney, J.L.
rselnick, J. Virol., 16, 508, (1975).
These proteins have identical amino acid sequences
coded for by they S-gene of HBV DNA (Tiollais et al, supra),
but the larger protein also carries carbohydrate chains.
Peptides corresponding to selected segments of the S-gene
product were synthesized and shown to elicit antibodies to
HBsAg (anti-HBs). However, immunization of chimpanzees with
these peptides resulted in only partial protection against
HBV infection (1\f. Williams, Nature, 306, 427, (1983)).
It has. been reported recently that the minor
glycoprotein components of HBsAg with rIr of approximately 33
and approximately 36 kilodaltons (P33, P36) are coded for
HBV DNA and contain the sequence of P22 (226 amino acids
corresponding to the S region) and have 55 additional amino
acids at the amino-terminal part which are coded by the
pre-S region of the env genes) of HBV DNA. See W. Stibbe,
W.H. Gerlich, Virology, 123, 436, (1982); M.A. Feitelson,
P.L. Marion, W.~~. Robinson, Virology, 130, 76, (1983); W.
Stibbe, W.H. Gerlich, J. Virol., 46, 626, (1983); and A.
Machida, S. Kishimoto, H. Ohnuma, H. Miyamoto, K. Baba, K.
Oda, T. Nakamura, Y. Miyakawa, M. Mayumi, Gastroenterol_ogy,
85, 268, (1983). Machida et al describe an amino acid
sequence composition as a receptor for polymerized serum
albumin.
Heretofore, amino acid sequences coded for by the
pre-S region of the hepatitus B virus DNA were virtually
-5-
completely ignored for purposes of producing synthetic
vaccines. The hepatitis B vaccine currently in use in the
United States :Lacks the pre-S gene coded sequences (and
therefore does not elicit antibodies to such sequences) and
thus elicits an immune response to the HBV envelope which is
incomplete as compared with that occurring during recovery
from natural infection.
The generation of antibodies to proteins by
immunization with short peptides having the amino acid
sequence corresponding to the sequence of preselected
protein fragments appears to be a frequent event (Nima,
N.L., Houghten, R.A., Walker, L.E., Reisfeld, R.A., Wilson,
I.A., Hogle, J.M. and Lerner, R.A., "Generation Of
Protein-Reactive Antibodies By Short Peptides Is An Event Of
High Frequency: Implications For The Structural Basis Of
Immune Recognition", Proceedings of the National Academy of
Sciences USA, 80, 4949-4953, (1983)). Nevertheless, the
generation of antibodies which recognize the native protein
may depend on the appropriate conformation of the synthetic
peptide immunogen and on other factors not yet understood.
See Pfaff, E., Mussgay, M., BBhm, H.O., Schulz, G.E. and
Schaller, H., "Antibodies Against A Preselected Peptide
Recognize And Neutralize Foot And Mouth Disease Virus", The
EMBO Journal, 7, 869-874, (1982); Neurath, A.R., Kent,
S.B.h. and Strick, N., "Specificity Of Antibodies Elicited
By A Synthetic Peptide having A Sequence In Common ~~'ith A
-6-
Fragment. Of A Virus Protein, The Hepatitis B Surface
Antigen," Proceedings O.f The~tJational Academy Of Sciences
USA, 79, 7871-7875, (1982); Ionescu-Matiu, I., Kennedy,
R.C., Sparrow, J.T., Culwell, A.R., Sanchez, Y., Melnick,
J.L. and Dreesman, G.R., "Epitopes Associated With A
Synthetic Hepatitis B Surface Antigen Peptide", The Journal
Of_ Immunology, 13U, 1947-1952,(1983); and Kennedy, R.C.,
Dreesman, G.R., Sparrow, J.T., Culwell, A.R., Sanchez, Y.,
Ionescu-Matiu, I., Hollinger, F.B. and Melnick, J.L. (1983);
"Inhibition Of A Common Human Anti-Hepatitis B Surface
Antigen Idiotype By A Cyclic Synthetic Peptide," Journal of
Virology, 46, 653-655, (1983). For this reason, immunization
with synthetic peptide analogues of various virus proteins
has only rarely resulted in production of virus-neutralizing
antisera comparable to those elicited by the viruses (virus
proteins) themselves (Pfaff et al., 1982, supra). Thus, the
preparation of synthetic immunogens optimally mimicking
antigenic determinants on intact viruses remains a
challenge.
Replacement of commonly used protein carriers,
namely keyhole limpet hemocyanin (KLH), albumin, etc., by
synthetic carriers, represents part of such challenge.
Although recent reports indicate that free synthetic
peptides can be immunogenic, (Dreesman, G.R., Sanchez, Y.,
Ionescu-Matiu, I., Sparrow, J.T., Six, H.R., Peterson, D.L.,
Hollinger, F.B. .and Melnick, J.L., "Antibody To Iiepatitis B
_7_
~~~~'t
Surface AntigE:n After A Single Inoculation Of Uncoupled
Synthetic HBsAg Peptides" Nature, 295, 158-160, (1982), and
Schmitz, H.E.,, Atassi, Fi., and Atassi, M.Z., "Production Of
Monoclonal Antibodies To Surface Regions That Are
Non-Immunogenic In A Protein Using Free Synthetic Peptide As
Immunogens: Demonstration With Sperm-whale Myoglobin'',
Immunological Communications, 12, 161-175, (198311, even in
these cases the antibody response was enhanced by linking of
the peptides t:o a protein carrier (Sanchez, Y.,
Ionescu-Matiu,, I., Sparrow, J.T., Melnick, J.L., Dreesman,
G.R., "Immunoctenicity Of Conjugates And Micelles Of
Synthetic Hepatitis B Surface Antigen Peptides",
Intervirology" 18, 209-213, (.1982)).
For commonly used protein carriers there is a
strong immune response to the carrier, as well as the
synthetic pepi~ide. Thus, it would be advantageous to evoke
an anti-HBs response with peptides by use of non-protein
carriers, which themselves do not evoke an antibody
response.
The possible use of several distinct vaccines in
prophylaxis would be facilitated by the availability of
fully synthetic immunogens.
-8-
DEFINITIONS
AminoAcid Code Words (as appearing in Fig.
?.)
D Asp aspartic acid
N Asn asparagine
T Thr threonine
S Ser serine
E Glu glutamic acid
Q Gln glutamine
P Pro proline
G Gly glycine
A Ala alanine
C Cys cysteine
V Val valine
M Met methionine
I Ile isoleucine
L Leu leucine
Y Tyr tyrosine
F Phe phenylalanine
W Trp tryptophane
K Lys lysine
H His histidine
R Arg arginine
HBV hepatitis B virus
fIBsllg hepatitis B surface antigen.
DNA ~ deoxyribonucleic acid
_g_
~.~ 4~~ ~~
SUMf9ARY Or TIIIJ INVENTION
The applicants have found that antibodies to the
pre-S protein appear early in the course of hepatitis B
infection and probably play the role of antibodies
eliminating HBV from the circulation and thus interrupting
further spread of the infection. Antibodies to the pre-S
protein are likely to represent virus-neutralizing
antibodies. The failure of some hepatitis B vaccines to
elicit such antibodies may be of considerable biological
significance, as indicated by poor immunoprophylactic
effects elicited by such vaccines in some populations,
despite a detectable immune response to the S-protein.
Applicants have discovered that amino acid
sequences coded for by the pre-S region of the env gene of
hepatitis B virus (HBV) DNA carry dominant antigenic
determinants common to intact and denatured HBsAg.
Applicants have found that immuno-dominant disulfide bond-
independent epitopes recognized by human antibodies to
hepatitis B virus (HBV) exist within proteins containing
amino acid sequences coded by the pre-S region of HBV DNA,
and more particularly within proteins containing an
N-terminal portion (coded for the pre-S region of HBV DNA)
having an N-terminal methionine at amino acid sequence
position pre-S 120. Applicants further discovered that
peptides corrE~sponding to amino acid sequences in the pre-S
region, and more particularly in the aforementioned region
-10-
starting at amino acid 120 of the env gene open reading
frame, inhibit the reaction between human anti-IIHs and P33
fP36), are highly immunogeni_c, and elicit high levels of
group-specific antibodies against HBsAg and HBV. The
immunogenicity of such peptides is enhanced by covalent
linking to nov~'1 lipid vesicle (liposome) carriers also
discovered by applicants.
Glutaraldehyde-fixed liposomes were found by
applicants to lbe preferred carriers for the peptides of the
invention for .inducing anti-HBs.
The present invention thus concerns a hepatitis B
peptide immunogen including a peptide containing an amino
acid chain corresponding to at least six consecutive amino
acids within the pre-S gene coded region of the envelope of
HBV. The hepatitis B peptide immunogen is free of an
amino acid chain corresponding to the naturally occurring
envelope proteins of hepatitis B virus.
The naturally occurring envelope proteins of
hepatitis B virus include the following:
(11 a full length translational product of the
env gene of HBV, i.e., for adw2 and adr pre-S(1-174) +
S(175-400)=400 amino acids, for ayw, adyw and adw
pre-S(12-174) + S(1-226) - 389 amino acids (env 12-400);
(2) pre-S(120-174) + S(175-400) - 281 amino acids
(env 120-400) - terminal 55 amino acids in the pre-S region
-11-
,.--,
13~~~'~~
+ 226 amino acids comprising the entire S region (the
corresponding proteins approximately 33 and 36 kD in size
(P33 and P36), and differing from each other in the extent
of glycosylationl; and
(3) S(1-226 - 226 amino acids, i.e., the entire
S region (env 175-400); representing the approximately 22
and 26 kD major constituents of the HBV envelope (P22 and
P26) in their non-glycosylated and glycosylated forms (the
"S-protein").
In an embodiment of the hepatitis B peptide
immunogen of the present invention, the corresponding chain
of amino acids lies between the sequence positions pre-S 120
and pre-S 174. In another embodiment of the invention, the
chain of amino acids is between sequence positions pre-S 1
and pre-S 120. In a further embodiment of the invention,
the corresponding chain of amino acids includes the
methionine amino acid at sequence position pre-S 120. In
still another embodiment, the chain of amino acids is an
amino acid chain containing at least 26 amino acids in the
pre-S region. Still further, the chain of amino acids
containing at least 26 amino acids can correspond to a chain
of at least 26. consecutive amino acids disposed between
Sequence position pre-S 120 and sequence position pre-S 174.
Generally the peptide has no more than 280 amino acids,
preferably no more than 225 amino acids, more preferably no
-12-
more than 174 amino acids, even more preferably no more than
100 amino acids, and still more preferably, no more than 50
amino acids. The vaccine of the present invention can be
composed solely of a peptide, or preferably of a peptide
joined to a carrier. Such carrier can be a conventional
carrier, or a novel carrier according to the present
invention as described hereinbelow.
The hepatitis B peptide immunogen of. the present
invention is free of any serum proteins, e.g., blood serum
proteins.
The present invention also concerns a hepatitis B
vaccine including a peptide containing an amino acid chain
corresponding to at least six consecutive amino acids within
the pre-S gene coded region of the envelope of HBV, and a
physiologically acceptable diluent, e.g., phosphate buffered
saline. The hepatitis B peptide vaccine being free of an
amino acid chain corresponding to the naturally occurring
envelope proteins of hepatitis B virus.
The present invention is also directed to a novel
carrier for peptides. In a particularly preferred embodiment
of the present: invention, the hepatitis B vaccine containing
an amino acid chain corresponding to a chain of amino acids
in the pre-S region is linked to a carrier via active sites
on the carrier. Still more preferred, the carrier is a lipid
vesicle carrier. Even more preferred, the lipid vesicle
carrier is stabilized by cross-linking.
-13-
13~~'~ j~
The carrier of the present invention includes a
lipid vesicle stabilized by cross-linking and having
covalently bonded active sites on the outer surface thereof.
The synthetic peptide is bonded via such active sites on the
carrier to the outer surface of the lipid vesicle. Such
active sites include -COOH, -CHO, -NIi2 and -SH. Such
stabilization by cross-linking is accomplished by a
stabilizing agE~nt such as an aldehyde having at least two
functional groups, such as a bifunctional aldehyde, for
example, glutaraldehyde. The carrier of the present
invention is chemically cross-linked with pendant functional
groups. -
The present application also concerns diagnostic
methods. The present invention relates to processes for
detecting the presence of either pre-S gene coded -
hepatitis B antigens or antibodies in a serum.
Antibodies to the synthetic peptides disclosed
herein can be detected in samples by a process which
comprises:
a) contacting the sample with a solid substrate
coated with a non-labelled peptide containing an amino acid
chain corresponding to at least six consecutive amino acids
within the pre--S gene coded region of the envelope of HBV,
the peptide free of an amino acid sequence corresponding to
the naturally occurring envelope proteins of hepatitis B
virus, incubating and washing said contacted sample;
b) contacting the incubated washed product
obtained from .step a above with a labelled peptide
-14-
containing an amino acid chain corresponding to at least six
consecutive amino acids within the pre-S gene coded region
of the envelope of IiBV, said peptide free of an amino acid
sequence corresponding to the naturally occurring envelope
protein of hepatitis B virus, incubating and washing the
resultant mass; and
c) determining the extent of labelled peptide
present in the resultant mass obtained by step b above.
Such a process is normally performed using a solid
substrate which is substantially free of available protein
binding sites. Such as by binding sites unbound by
unlabelled peps=ide with a protein binding site occupier,
e.g., albumin.
Another process for detecting such antibodies
comprises:
a) contacting the sample with a solid substrate
coated with a non-labelled peptide containing an amino acid
chain corresponding to at least six consecutive amino acids
within the pre-~S gene coded region of the envelope of HBV,
the peptide free of an amino acid sequence corresponding to
the naturally occurring envelope proteins of hepatitis B
virus, incubating and washing said contacted sample;
b) contacting the incubated washed product
obtained from step a above with labelled antibody to human
or animal immunoglobulin product by contact with an
immunogen comprising a peptide corresponding to at least six
consecutive amino acids within the pre-S gene coded region
of the envelope of HBY. the peptide immunogen free of an
amino acid sequence corresponding to the naturally occurring
-15-
f
1
j
envelope proteins of hepatitis B virus, incubating and
washing the contacted sample, and
c) determining the extent of labe).l_ed antibody
present in the resultant mass of step b.
fiBV or ffl3sAg can be detected in ~ sample by a
process which comprises:
a) contacting a first portion of a composition
containing an antibody produced by introducing into an
animal or human an immunogen comprising a peptide
corresponding i.o at least six consecutive amino acids within
the pre-S gene coded region of the envelope of HBV, the
peptide immunoden free of an amino acid sequence
corresponding t:o the naturally occurring envelope proteins
of hepatitis B virus, with a mixture of said sample and the
immunogen which has been labelled, incubating and washing
the first portion;
b) contacting a second portion of the
composition containing antibody with the same amount of the
labelled immunogen in an antigen free control, incubating
and washing the second portion;
c) adding the same amount of Staphylococci
bearing protein A to each of the compositions of steps a and
b above, incubating both of_ the compositions, centrifuging
each of the compositions and separating liquid from the
solids therein;
d) determining the.extent of labelled immunogen
in each of the :resultant compositions from step c above, and
e) comparing the relative amount of labelled
immunogen in each such that if the activity of the resultant
-16-
composition containing the first portion is less than the
activity for the resultant composition of the second
portion, then t:he sample contains HBV or HBsAg.
The synthetic immunogens can be used in general in
both sandwich t:ype immunoassays and competition type
immunoassays, such as those immunoassays in which antigen in
the sample competes with labelled immunogen for antibody.
ThesE: and other suitable immunoassay schemes for
use in connection with the synthetic immunogens of this
invention and antibodies thereto are disclosed in
United States patent 4,591,552.
The present invention also concerns a diagnostic
test kit for dE~tecting hepatitis B virus in sera comprising
a) antibodies to a peptide containing an amino
acid chain corresponding to at least six consecutive amino
acids within the pre-S gene coded region of the envelope of
HBV, the peptide being free of an amino acid chain
corresponding t:o the naturally occurring envelope proteins
of hepatitis B virus, attached to a solid support,
c) labelled antibodies to the peptide or to
hepatitis B virus.
The ~;it can comprise a set of instructions for
effecting an immunoassay wherein the effect of formation of
an immune comp7lex is revealed by said labelled antibody.
The present invention also concerns a diagnostic
kit for detect:ung the presence of antibodies to pre-S gene
-17-
r.
coded antigens of hepatitis B virus in a test sample
comprising
al a given amount of a peptide containing
an amino acid chain corresponding to at least six
consecutive amino acids within the pre-S gene coded region
of the envelope of HBV, the peptide being free of an amino
acid chain corresponding to the naturally occurring envelope
proteins of hepatitis B virus. The petide is attached to a
solid support, e.g., a water insoluble solid support.
b) labelled antibodies, e.g., radiolabeled
or enzyme labelled, to human IgG and/or Igh2.
The kit can comprise a set of instructions for
effecting an immunoassay, wherein the extent of formation of
an immune complex is revealed by said labelled antibodies.
In a particular aspect, the present invention
concerns a process for the detection of antigens coded for
the pre-S gene in sera of HBV infected humans and certain
animals, for example, chimpanzees, comprising the following
steps:
(a) coating a solid substrate with
antibodies to a peptide having an amino acid chain
corresponding to at least six consecutive amino acids within
the pre-S genes of HBV DNA, the peptide being free of an
amino acid sequence corresponding to the naturally occurring
envelope proteins of HBV,
(b) washing the coated substrate;
(c) contacting the washed coated substrate,
e.g., polystyrene beads; wit:: a protein-contai:.i::g solution;
(,d) washing the substrate from step c;
-18-
(e) incubating the substrate from step d
with a sample suspected to contain HBV or HBsAg;
(f) washing the substrate from step e;
(g) adding radiolabeled or enzyme-labeled
antibody, the antibody being an antibody to the peptide or
HBsAg;
(h) incubating the substrate from step g;
(i) washing the substrate from step h; and
(j) subjecting the substrate of step i to
counting in a gamma counter, or measuring its enzymatic
activity.
The above process can be conducted using ELISA
techniques rather than RIA detection techniques.
In a particular embodiment, the present invention
also relates to a process for the detection of antibodies to
proteins coded for by the pre-S region of hepatitis B virus
DNA, comprising the following steps:
(a) adsorbing on a solid substrate
containing binding sites thereon, e.g., polystyrene beads, a
peptide having an amino acid sequence corresponding to at
least six consecutive amino acids within the pre-S gene
coded region of the HBV envelope, the peptide being free of
an amino acid ~~equence corresponding to the naturally
occurring envelope proteins of hepatitis B virus,
(b) contacting the substrate from step a
with a material. to saturate the binding sites thereon,
(c) washing the substrate from step b,
(d) contacting the substrate from step c
with a specimen comprising human sera,
-19-
(e) incubating the resultant mass of step d,
(f) washing the resultant mass of step e,
(g) adding radiolabeled antibodies to human
IgG or IgM to the resultant mass of step f to form a second
resultant mass,
(h) subjecting the second resultant mass of
step g to counting in a gamma counter,
(i) subjecting normal sera utilized as a
control to steps (a) to (h) and
(j) comparing the counts of steps h and i.
In the above process for the detection of
antibodies, ELISA techniques can be substituted for RIA
techniques.
The present invention also relates to a process
for predicting the outcome of hepatitis B infection which
comprises carrying out an immunoassay on serum of a human to
detect the presence of an antibody to an antigen coded for
by the pre-S gene coded region of the envelope of hepatitis
B virus employing the above-described hepatitis B peptide
immunogen at regular intervals and evaluating the data.
The present invention further relates to a process
for determining if a human who has been vaccinated with a
vaccine against hepatitis B has become immune to hepatitis B
virus. Such process involves effecting a plurality of
immunoassays of serum from such human to determine if there
are antibodies in the serum to an antigen coded by the pre-S
gene coded region of the envelope of hepatitis B virus
employing the above-des~:~ibed hepatitis. 0 pcrti3e imm~~nogAn,
the immunoassay; being performed on serum taken from the
-20-
human at different times.
The present invention further concerns a method
for detecting thE: presence of hepatitis B virus infection
comprising effeci:ing quantitative immunoassays on a serum
sample taken from a human to determine the amount of
antibodies present therein which are antibodies to an
antigen coded by the pre-S gene coded region of the envelope
of the hepatitis B virus employing the above-described
hepatitis B peptide immunogen and comparing the value with a
known standard.
The present invention further concerns a method
for detecting the presence of hepatitis B virus infection
comprising effecting quantitative immunoassays on a serum
sample taken from a human to determine the amount of
antigens coded by the pre-S gene coded region of the
envelope of the hepatitis B virus employing the above-
described antibodies to the hepatitis B peptide immunogen
and comparing th.e value with a known standard.
The present invention also related to a process
for raising antibodies which involves introducing into an
animal the above-described hepatitis B peptide immunogen.
Still further, the present invention concerns a
process for synthesizing His and Trp containing peptides
which includes t:he steps of
a. 7Linking a first amino acid containing an
alpha-amino proi:ecting group to a resin;
b. removal of the alpha-amino protecting group;
c:oupli~ig a sacond amino acid containing an
alpha-amino proitecting group to the first amino acid;
-21-
y ~ x~'~
d. repeating steps b and c by coupling further
alpha-protected amino acids to produce a desired peptide,
wherein at least one of the amino acids is His and wherein
at least one of said amino acids is Trp,
e. cleaving the peptide from the resin and
removing remaining protective groups to said first amino
acids;
f. substituting a His(ImDNP) for the His;
g. substituting a Trp(InFormyl) for the Trp;
h. removing the DNP prior to the cleavage and
the removing of protective groups, and
i. removing the Formyl during the cleavage and
the removing of protective groups.
The present invention further concerns a
prophylatic method of protecting a patient against becoming
infected with hepatitis B comprising administering to such
patient, e.g., a human, an effective dosage of a vaccine as
described hereinabove
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 shows the results of submitting reduced
HBsAg disassociated into its constituent polypeptides to
SDS-polyacryla.mide gel electrophoresis ("SDS-PAGE") in urea.
Panel a shows the separated proteins detected by a silver
stain and panel b is a Western blot with human antiserum to
hepatitis B.
. Fig. 2 shows amino acid sequences of the
translational products of the pre-S gene region deduced from
sequences of ~BV DNA. The sequences are presented in
one-letter amino acid code words (such code words are
-22-
-.--.-.~-._~__ ~. -_
r
defined in the Definitions herein). Sequences for five
distinct HBV subtypes are presented. The 6th bottom line
shows amino acid residues common to all five subtypes.
Fig. 3 shows a profile of. relative hydrophilicity
corresponding ~to the amino acid sequence of the pre-S gene
product. Profiles for subtypes ether than ayw are similar.
The portion of the profile to the right from methionine 175
represents the S-gene translation product.
Fig. 4 shows two sets of bar graphs for mean
antibody responses of rabbits immunized with free pre-S
120-145 (Fig. CIA) and with the same peptide linked to
cross-linked liposomes containing L-tyrosine-azobenzene
-p-arsonate (RAT) groups (Fig. 4B). Anti-HBs lantibodies to
HBsAg), cross-hatched columns; anti-pre-S 120-145,
diagonally hatched columns. Similar results to Fig. 4B were
obtained with 7!iposomes lacking RAT groups, except that
responses after- six weeks were lower. Columns corresponding
to time = 0 represent sera before immunization.
Fig. 5 depicts radioimmunoassays with serial
dilutions of a serum from a rabbit immunized with pre-S
120-145 linked to liposomes. Anti-HBs (antibodies to HBsAg),
anti-pre-S 120- 145, ~. Counts per minute (cpm)
corresponding t:o distinct dilutions of the pre-immune serum
were subtracted from cpm corresponding to dilutions of
anti-pre-S 120-145; the difference was plotted. The endpoint
titer of. the serum (1/163,840) corresponds to its highest
dilution at which the cpm were L 2.1 higher than those
corresponding t:o the same dilution of the pre-immune set~um.
-23-
t
Fig. 6 shows the reaction of anti-pre-S 120-145
with P33 and P36 in a Western blot (similar to Fig. 1).
Fig. 7 shows a graph depicting a diagnostic test
for hepatitis B antigens based on polystyrene beads coated
with anti-pre-S 120-145.
Fig. 8 depicts a plot representing the compilation
of antibody responses of individual rabbits to conjugates of
S135-155 (amino acids 309 to 329 of the open reading frame
of the HBV env gene). The type of conjugates is indicated by
numbers defined in Table 1. Antibodies in sera obtained two
weeks after the third immunization were assayed using a
S135-155- beta-galactosidase conjugate and Pansorbin
(Neurath et al., 1982, supra.). Their relative titer is given
in comparison with antibody levels induced by a S135-155-KLH
conjugate. Results of anti-HBs assays by RIA (AUSRIA test,
Abbott Laboratories, North Chicago, Illinois) are given in
international milliunits (mIU/ml; Neurath et al., 1982
supra). The line corresponds to the calculated linear
regression that best fits the set of all data concerning
rabbits with an anti-HBs response. The calculated
correlation coefficient (= 0.55) indicates a poor
correlation between anti-Figs and anti-S135-155 responses.
Fig. 9 shows four sets of bar graphs (Fig. 9A,
Fig. 9B. Fig. 9C and Fig. 9D) depicting examples of time
courses of antibody responses in rabbits immunized with
distinct.S135-155-conjugates (indicated by numbers in each
panel and defined in Table 1). Fig. 9A corresponds to
conjugate No. 5; Fig. 9B corresponds to conjugage No. 11;
Fig. 9C corresponds to conjugage No. 12 and Fig. 9D
-24-
1340'~~~
corresponds to conjugate No. 19. Anti-HBs (dashed columns)
and anti-S-135-155 (black columns) were assayed as described
for Fig. 8.
Fig. 10 shows four plots (A, B, C and D) which
depict the kinetics of antibody responses to peptide
pre-S(120-145) ( Q ) and to pre-S protein within
approximately 22nm spherical HBsAG particles ( ~ ) elicited
by unconjugated peptide pre-S(120-145) (plot A) and by the
same peptide linked to cross-linked, cysteine-activated
liposomes with attached RAT (L-tyrosine
azobenzene-p-arsenate) groups (plot B); and the effect of
carrier on anti-peptide antibody titers in sera of rabbits
immunized with 4 doses of peptides pre-S(120-145) (plot C)
and pre-S(12-3:2) (plot D) given 2 weeks apart. The carriers
for plots C and D were: lO none; 2O keyhold lympet
hemocyanin (KL1H) ; 3. alum; 4O. and 5O. cross-linked,
cysteine-activated liposomes with or without attached RAT
groups. Compl~ste and incomplete Freund's adjuvant was used
in all cases e:KCept~
Fig. 11 shows two plots for radioimmunoassays of
IgG antibodies in serial dilutions of rabbit antisera: to
pre-S(120-145) (~]; to HBV particles and tubular forms of
HBsAg (~], de,~oid of antibodies to S-protein detectable by
RIA and to a fusion protein of chloramphenicol
acetyltransferase with the sequences of pre-S protein
lacking the 41 C-terminal amino acid residues ( D ): and of
IgG (L1) and IgM (~,) antibodies in serum of a patient
recovered from hepatitis B. Tt~e latter serum wu~ ulaw:~
before antibodies to the S-protein were detectable. Immulon
ZS - I
2 Removable strips (Dynatech Laboratories) were coated with
20 y~ g/ml of either free peptide pre-S ( 120-145 ) or
pre-S(12-32) and post-coated with gelatin (2.5 mg/ml in 0.1
M Tris, pH 8.8). The conditions for coating and the double
antibody RIA are described in A.R. Neurath, S.B.H. Kent, N.
Strick, Science, 224, 392 (1984) and A.R. Neurath, S.B.H.
Kent, N. Strick, Proc. Natl. Acad. Sci USA, 79, 7871 (1982).
Fig. 12 shows a plot depicting the inhibition of
the reaction of anti-pre-S(120-145) IgG (antiserum diluted
1:100) with a pre-S(120-145)-~-galactosidase conjugate by;
free peptide p.re-S(120-145) t~ ]; by 20 nm spherical HBsAg
particles (j ] and by HBV particles f.D ]. The latter two
preparations contained the same concentration of HBsAg
S-protein as determined by radioimmunoassay (AUSRIA, Abbott
Laboratories).
Fig. 13 depicts a plot of titers of anti-pre-
S(120-145) antibodies versus days of surveillance and
indicates the development of IgM f 1 ] and IgG f ~ ]
antibodies to the pre-S gene coded protein of HBV during
acute hepatitis B.
Fig. 14 shows a plot for radioimmunoassays of
various preparations containing HBV-specific proteins on
polystyrene beads coated either with anti-pre-S(120-145) IgG
(o, o, Q ) or with IgG from a rabbit antiserum against HBV
particles and -tubular forms of HBsAg (e" p ). The tested
antigens were: HBV particles and tubular forms (s,.~);
approximately :20 nm spherical particles of HBsAg isolated
(roar plasma (u, p ) ; and the latter particles treated with
-26-
~3~fl~'~r
pepsin (1 mg/ml HBsAg, 50 pg/ml pepsin in 0.1 M glycine-HCL,
pH 2.2, 2 hours at 37°C) (Q ) .
Fig. 15 depicts a plot for radioimmunoassays of
polymerized albumin-binding sites associated with HBsAg
isolated from human plasma and containing pre-S gene coded
sequences (v) or with HBsAg produced in yeast transfected
with recombinant DNA containing the HBV DNA S-gene and thus
lacking pre-S gene coded sequences (o).
DETAILED DESCRIPTION OF THE INVENTION
Amino acid sequences deduced from sequences of the
pre-S portion of the env genes corresponding to several HBV
subtypes (see IE'ig. 2) have the following properties distinct
from those of the S-protein: (i) high hydrophilicity and
high percentage of charged residues (E. Schaeffer, J.J.
Sninsky, Pro. lVatl. Acad. Sci. USA, 81, 2902 (1984)); (ii)
absence of cysteine residues; (iii) the highest
subtype-dependent variability among HBV DNA gene products;
and (iv) little homology with analogous sequences
corresponding 'to nonhuman hepadnaviruses (F. Galibert, T.N.
Chan, E. Manda:rt, J. Virol., 41, 51, (1982)). These
properties sug~~est that the pre-S gene coded portion of the
HBV envelope i;s exposed on the surface of the virion, is a
target for the host's immune response and is responsible for
the host range of HBV (limited to humans and some primates).
Synthetic peptides and antibodies against them, having
predetermined specificity offer the opportunity to explore
the biological role of the pre-S protein moiety of the HBV
envelope.
-27-
,,.,..,.
Cleavage of disulfide bonds within HBsAg results
in:
(a) ~~ substantial decrease of binding of
polyclonal antibodies (G. N. Vyas, K.R. Rao, A.B. Ibrahim,
Science, 178, :1300, (1972); N. Sukeno, R. Shirachi, J.
Yamaguchi, N. :Ishida, J. Virol., 9, 182, (1972); G.R.
Dreesman, F.B. Hollinger, R.M. McCombs, J.L. Melnick, J.
Gen. Virol. 19, 129 (1973); and A.R. Neurath, N. Strick, J.
Med. Virol., 6, 309, (1980)) and of some monoclonal
antibodies (J. Pillot, M.M. Riottot, C. Geneste, L.
Phalente, R. M~ingalo, Develop. Biol. Stand., in press
(1984)) elicited by intact HBsAg, and
(b) reduction of immunogenicity (Y. Sanchez, I.
Ionescu-Matiu, J.L. Melnick, G.R. Dreesman, J. Med. Virol.
11, 115, (1983)). However, some epitopes are resistant to
reduction of disulfide bonds (M. Imai, A. Gotoh, K.
Nishioka, S. Ktxrashina, Y. Miyakawa, M. Diayumi, J. Immunol. ,
112, 416, (1974)). These epitopes are common to all
antigenic subtypes of HBV, but their localization on
envelope components of HBV has not been determined. The
present invent~.on takes advantage of the localization of
disulfide-bond independent antigenic determinants on the
N-terminal portion (coded for by the pre-S gene of HBV DNA)
of the minor HBsAg proteins P33 and P36, and on other
regions of prot:eins coded for by the pre-S gene.
. These' determinants represent the dominant epitopes
on reduced and dissociated HBsAg reacting with human
anti-HBs. They aie trv.imicked with high fidelity by pre-S
120-145 which elicits antibodies to HBsAg about 400 times
-28-
~~~p'?~
more efficiently than a synthetic peptide analogue
corresponding to the S-gene tA.R. Neurath, S.B.H. Kent, and
N. Strick, Proc. Natl. Acad. Sci. USA, 79, 7871 (1982)). No
precedent exist~~ for such high levels of virus-recognizing
antibodies to a synthetic peptide analogue of an HBV
protein. These antiboc3ies could be used in a diagnostic test
permitting the dLirect detection of the pre-S gene coded
antigenic determinants in serum of HBV carriers.
The pre-S gene is the most divergent among all
regions of hepadnavirus genomes (F. Galibert, T.N. Chen, E.
Mandart, J. Virol., 41, 51 (19821) (HBV is a member of. the
hepadnavirus family).
The hepatitis B vaccine of the present invention
contains a peptide, either a synthetic peptide (peptide
produced by assembling individual amino acids by chemical
means or by expression vectors (DNA route)) or a peptide
derived from natural sources, such peptide having an amino
acid chain corrE:sponding to at least six consecutive amino
acids within the' pre-S gene coded region of the surface
antigen of hepatitis B virus. Such chain can be, for
example, at lea:~t 10, 15, 20, or 26 amino acids long. A
preferred peptide according to one embodiment of the present
invention is an amino acid chain disposed between sequence
position pre-S J120 and pre-S 174, and more preferably such
chain includes t=he N-terminal methionine at sequence
position pre-S 7120. A preferred peptide is an amino acid
chain corresponding to the chain between sequence position
pre-S 120 and pre-S 145, i.e., pre-S (12C-1.45).
_29-
Preferred positions of the chain include the
following: (1) The amino acid chain entirely between and
including sequence position pre-S 1 and pre-S 11 for
subtypes adw2 and adr, (2) between and including sequence
positions pre-S 10 and pre-S 40, (3) between and including
sequence positions pre-S 15 and pre-S 320, i41 between and
including sequence position pre-S 15 and pre-S 55, and (5)
between and in~~luding sequence position pre-S 90 and pre-S
120. A particvularly preferred chain according to the
present invention has 26 amino acids, includes the
N-terminal methionine at sequence position pre-S 120 and is
disposed between sequence position pre-S 120 and pre-S 174.
Preferred peptides according to the present
invention include the following:
(1) pre-S(12-32), wherein the sequence is fsee
Fig. 2) t9GTNLSVPNPLGFFPDHQLDP for subtype adk~2;
(2) pre-S(120-145), wherein the sequence is (see
Fig. 2) MQWNST.AFHQTLQDPRVRGLYLPAGG for subtype adw2;
(3) pre-S(32-53), wherein the sequence is (see
Fig. 2) PAFGANSNNPDWDFNPVKDDWP for subtype adw2;
(4) pre-S(117-134), wherein the sequence is (see
Fig. 2) PQAMQWNSTAFHQTLQDP for subtype adw2;
(5) pre-S(94-117), wherein the sequence is (see
Fig. 2) PASTNRQSGRQPTPISPPLRDSHP for subtype adw2;
(6) pre-S(153-171), wherein the sequence is (see
Fig. 2) PAPNIASHISSISARTGDP for subtype adw2;
(7) pre-S(1-21), wherein the sequence is fsee
Fig. 2) MGGWSSKPRKGMGTNLSVPNP for subtype adw2;
-30-
(8) pre-S(57-73), wherein the sequence is (see
Fig. 2) QVGVGAFGPRLTPPHGG for subtype adw2;
(9) pre-SI1-11),
a. for adw2, wherein the sequence is (see
Fig. 2) MGGWSSKPRKG
b. for adr, wherein the sequence is (see
Fig. 2) MGGWSSKPRQG.
Any analogs of the pre-S gene coded sequences of
the present invention involving amino acid deletions, amino
acid replacements, such as replacements by other amino
acids, or by isosteres (modified amino acids that bear close
structural and spatial similarity to protein amino acids),
amino acid additions, or isosteres additions can be
utilized, so long as the sequences elicit antibodies
recognizing the pre-S protein of fiBV or hepatitis B surface
antigen.
In the formation of a peptide derived from natural
sources, a protean containing the required amino acid
sequence is subjected to selective proteolysis such as by
splitting the protein with chemical reagents or using
enzymes. Synthetic formation of the peptide requires
chemically synthesizing the required chain of amino acids.
In forming a synthetic vaccine according to the
present invention, it is preferred to insure that the amino
acid chain (pept:ide residue) corresponding to at least six
consecutive amino acids within the pre-S gene coded region
of hepatitis B virus has the steric configuration to be
recognized by antibody to hepatitis B virus. To this end,
the given chain of amino acids may have bonded thereto as
-31-
~~~~'~c~
part of the amino acid chain, one or more additional amino
acids on either, or both sides thereof. These additional
amino acids can serve as auxiliary amino acids to enhance
the stabilization of the amino acid chain so that it is
readily recognized by antibody to hepatitis D virus. The
additional amino acids can be the same amino acids in the
same sequence as they occur in the natural protein, or other
amino acids ma;y be employed.
In one form of the invention, the peptide having a
chain length of minimally six amino acids can be bounded on
either side thereof with additional amino acids, e.g., three
amino acids on either side of the residue, to form a longer
chain of amino acids. The chain of amino acids may contain
more than one amino acid sequence corresponding to at least
six consecutive amino acids within the pre-S region of the
surface antigen of hepatitis B virus.
The length of the individual amino acid sequence
would depend on the method of producing the sequence. If
the sequence i.s made by assembling individual amino acids by
chemical mean:., then the sequence length would generally not
exceed 50 amino acids, and preferably would not exceed 40
amino acids. If the synthetic peptide is obtained from a
DNA route, thE~ chain length could be longer, for example,
100 or more arnino acids. It is, however, normally shorter,
and optimally considerably shorter than the natural pre-S
protein. Thua, in the embodiment wherein the peptide has
units of both the S region and pre-S region, its peptide
portions corresponding to the S region is shorter than the
natural S protein, e.g., no more than 100 amino acids,
-32-
preferably no more than 40 amino acids and usually less than
30 amino acids. In such cases, the peptide portion
corresponding to the pre-S region can be of a length
corresponding to the entire pre-S region, but generally is
less than the entire pre S region.
When the peptide contains no components
corresponding to the amino acid sequence of the S region, it
can contain amino acid sequences corresponding to the entire
pre-S region, or shorter than the entire pre-S region.
Where, however, the amino acid sequence is part of
a long chain, such as when there are more than one sequence
of amino acids, the chain can contain residues of various
moieties, for example, segments of polyamino acids or
polysaccharides.
In addition to containing one or more different or
the same sequences of amino acids corresponding to at least
six consecutive amino acids within the pre-S region of
hepatitis B virus, e.g., containing more than one sequence
of amino acids corresponding to different epitopes
(antigenic determinants) in the pre-S region of hepatitis B
virus, the vaccine of the present invention can contain
amino acid chains cantaining epitopes of different antigens
or allergens so as to form a vaccine directed to hepatitis B
virus and to one or more additional diseases, e.g.,
measles, influenza, smallpox, polio, diptheria, just to name
a few. Such additional amino acid sequences can be of
varying amino acid chain lengths.
A hepatitis B vaccine according ~o the present
invention can include in addition to one or more amino acid
-33-
sequences corresponding to at least six consecutive amino
acids within the pre-S region of the surface antigen of
hepatitis B virus, one or more amino acid sequences
corresponding to consecutive amino acids within the S region
of the surface antigen of hepatitis B virus, for example,
141 142 143 144 145 146
Lys Pro Thr Asp Gly Asn,
or
138 139 140 141 142 143 144 145 146 147 148 149
Cys Cys Thr Lys Pro Thr Asp Gly Asn Cys Thr Cys
Other peptides corresponding to antigenic
determinants of HBsAg (S region) and thus combinable in the
same chain with one or more amino acids sequences
corresponding to at least six amino acids in the pre-S
region of HBsAg include the following:
(1)
Ser Thr Gly - Pro - Ser
117 121
Lys
122
Thr
Slr - Cys - Cys - Met - Thr
137 124
Pro Thr
Tyr Ala
I I
Met Ser Thr- Gly - Gln
(2) Position Amino Acid Series
48-81 Cys-Leu-Gly-Gln-Asn-Ser-Gln-Ser-Pro-Thr-
Ser-Asn-His-Ser-Pro-Thr-Ser-Cys-Pro-Pro-
Thr-Cys-Pro-Gly-Thr-Arg-Trp-Met-Cys-Leu-
Arg-Arg-Phe-Ile
-34-
(3) 2-16 Glu-Asn-Ile-Thr-Ser-Gly-Phe-Leu-Gly-Pro-
Leu-Leu-Val-Leu-Gln-Cys
(4) 22-35 Leu-Thr-Arg-Ile-Leu-Thr-Ile-Pro-Gln-Ser-
Leu-Asp-Ser-Trp-Cys
f5) 38-52 Ser-Leu-Asn-Phe-Leu-Gly-Gly-Thr-Thr-Val-
Cys-Leu-Gly-Gln-Asn-
(6) 47-52 Val-Cys-Leu-Gly-Gln-Asn
(7) 95-109 Leu-Val-Leu-Leu-Asp-Tyr-Gln-Gly-Met-Leu-
Pro-Val-Cys-Pro-Leu
(8) 104-109 Leu-Pro-Val-Cys-Pro-Leu
The ~;equences of amino acids can be interconnected
with one another such as by cross-linking or by being bonded
directly thereto in the form of a branched chain, or the
respective sequences can be bonded to a central "carrier".
There is realized by the present invention a
synthetic vaccine which is characterized by the absence of
naturally occuring envelope proteins of hepatitis B virus,
i.e., the vaccine of the present invention is composed of
one or more peptide sequences corresponding to a limited
portion of the :hepatitis B virus envelope protein. The
vaccine of the ;present invention is also free of other
proteins found .in the virion. Vaccines can be synthesized
which are free of biologically produced components, free of
viral components whether they be active or inactive, free of
antibodies, freE~ of deoxyribonucleic acid (DNA), and are
-35-
~4~~55
therefore likely to be substantially free from undesirable
side effects commonly found with other vaccines (e. g.,
unintentional infection with virus, allergic reactions,
fevers, etc.).
It should be understood that the vaccine of the
present invention can be in admixture with other proteins
and these proteins include the proteins of known antigens or
allergens. Thus when it is stated herein that the vaccine
is characterized by the absence of an amino acid sequence
corresponding to the naturally occurring envelope proteins
of the hepatitis B virus it is meant that notwithstanding
the absence of ouch proteins, the composition functions as a
vaccine, i.e., provides protective immunization by formation
of antibodies.
The peptide of the present invention is such that
it is capable of forming "neutralizing antibodies", i.e.,
antibodies that will protect patients against hepatitis B
virus. Accordingly, the present invention is also directed
to methods for protecting a patient against contracting
hepatitis B.
The peptides and vaccines of the present invention
can be used to improve immune response and to overcome
non-responsiveness to certain known hepatitis B virus
vaccines (e.g., containing no peptides corresponding to
amino acid sequences in the pre-S region).
The peptides of the present invention can be
utilized in conjunction with peptides containing amino acid
chains corresponding to consecutive amino acids within the S
gene coded region of FiBsAg. Also, embodied by the present
-36-
,....
invention is a peptide containing amino acids corresponding
to consecutive amino acids spanning both the pre-S and S
region, e.g., pre-S 160 to S 20.
A carrier may be provided for the synthetic
peptide of the invention. It should be understood, however,
that a carrier may not be required to practice the present
invention, i.e., a carrier may not be required to produce
antibodies according to the present invention.
The "carrier" is simply a physiologically
acceptable mass to which the synthetic peptide is attached
and which is expected to enhance the immune response. A
carrier can comprise simply a chain of amino acids or other
moieties and to that end it is specifically contemplated to
use as a carrier a dimer, oligomer, or higher molecular
weight polymer of a sequence of amino acids defining a
synthetic peptide of the invention. In other words, having
determined the desired sequence of amino acids to form the
synthetic peptide, these amino acids can be formed from
naturally available materials or synthetically and can be
polymerized to build up a chain of two or more repeating
units so that repeating sequences serve both as "carrier"
and synthetic peptide. Stated differently, an independent
carrier may not be required. Alternatively, additional amino
acids can be added to one or both ends of the amino acid
chain that defines the synthetic peptide. It is preferred
that alternative carriers comprise some substance, animal,
vegetable or mineral, which is physiologically acceptable
and functions to present the synthetic peptide so that it is
recognized by the immune system of a host and stimulates a
-37-
A,
satisfactory immunological response. Thus, a wide variety of
carriers are contemplated, and these include materials which
are inert, whi~~h have biological activity and/or promote an
immunological response. For instance, proteins can be used
as carriers. hxamples of protein carriers include tetanus
toxoid, keyhole lympet hemocyanin, etc.
Polysaccharides are also contemplated as carriers,
and these include especially those of molecular weight
10,000 to 1,000,000, including, in particular, starches,
dextran, agarose, ficoll or its carboxy methyl derivative
and carboxy methyl cellulose.
Poly,amino acids are also contemplated for use as
carriers, and these polyamino acids include, among others,
polylysine, polyalanyl polylysine, polyglutamic acid,
polyaspartic acid and poly (C2-C10) amino acids.
Organic polymers can be used as carriers, and
these polymers include, for example, polymers and copolymers
of amines, amides, alefins, vinyls, esters, acetals,
polyamides, carbonates and ethers and the like. Generally
speaking, the molecular weight of these polymers will vary
dramatically. The palymers can have from two repeating units
up to several thousand, e.g., two thousand repeating units.
Of course, the number of repeating units will be consistent
with the use of the vaccine in a host animal. Generally
speaking, such polymers will have a lower molecular weight,
say between 10,000 and 100,000 (the molecular weight being
determined by ultrac:entrifugation) .
Inorganic polymers can also be employed. These
inorganic polymers c:an be inorganic polymers containing
-38-
organic moieties. In particular, silicates and aluminum
hydroxide can he used as carriers. It is preferred that the
carrier be one which is an immunological adjuvant. In such
cases, it is particularly contemplated that the adjuvant be
muramyl dipept:ide or its analogs.
The carrier can also be the residue of a
crosslinking agent employed to interconnect a plurality of
synthetic peptide containing chains. Crosslinking agents
which have as their functional group an aldehyde (such as
glutaraldehyde;l, carboxyl, amine, amido, imido or
azidophenyl group. In particular, there is contemplated
the use of but:~raldehyde as a crosslinking agent, a divalent
imido ester or a carbodiimide. Particularly contemplated
divalent imido esters are those of the formula
R - 0 - C = NH2+
(CH2)m
R - 0 - C - NH2+
wherein m is 1 to 13 and R is an alkyl group of 1 to 4
carbon atoms. :Particularly contemplated carbodiimides for
use as crosslinking agents include cyclohexylcarboxiimide,
ethyldimethylaminopropyl carbodiimide, N-ethylmorpholino
cyclohexyl carbodiimide and diisopropyl carbodiimide.
Chemical synthesis of peptides is described in the
following publications: S.B.H. Kent, Biomedical Polymers,
eds. Goldberg, E.P. and Nakajima, A. (Academic Press, New
York), 213-242,(1980); A.R. Mitchell, S.B.H. Kent, M.
Engelhard, and R.B. Merrifield, J. Org. Chem., 43,
2845-2852, (1978); J.P. Tam, T.-W. along, tai. Riemeti, F.-G.
Tjoeng, and R.:B. Merrifield, Tet. Letters, 4033-4036,
-39-
1 (1979); S. Mojsov, A.R. Mitchell, and R.B. Merrifield, J.
2 Org. Chem., 45, 555-560, (,1980); J.P. Tacu, R.D. DiMarchi and
3 R.B. Merrifield, Tet.. Letters, 2851-2854', (1981); and S.B.H.
4 Kent, M. Rieme.n, M. Le Doux and R.B. Merrifield, Proceedings
of the IV International Symposium on Methods of Protein
6 Sequence Analysis, (Hrookhaven Press, Brookhaven, N.Y.), in
7
press, 1981.
8
Chemical Synthesis: In the so-called "Merrifield
9
solid phase procedure" the appropriate sequence of L-amino
acids is built up from the carboxyl terminal amino acid to
11
the amino terminal amino acid. Starting with the appropriate
12
carboxyl terminal amino acid attached to a polystyrene (or
13
other appropriate) resin via chemical linkage to a
14
chloromethyl group, benzhydrylamine group, or other reactive
group of the resin, amino acids are added one by one using
16
the following procedure. The peptide-resin is:
17
18 (a) washed with methylene
chloride;
19
fib) neutralized by mixing for 10 minutes at room
temperature with 5~ (v/v) diisopropyl-
21
ethylamine (or other hindered base) in
22
methylene chloride;
23
24 (c) washed with methylene chloride;
(dl an amount of amino acid equal to six times the
26 molar amount of the growing peptide chain is
27 , activated by combining it with one-half as
28 many moles of a carbodiimide (e. g.,
29 dicyclohexylcarbodiimide, or diisopropyl~~
carbodiimide) for ten minutes at 0°C, to
-40-
1 form the symmetric anhydride of the amino
2 acid. The amino acid used should be
3 provided originally as the N-alpha-tert.butyl-
4 oxycarbonyl derivative, with side chains
protected with benzyl esters (e.g. aspartic or
6 glutamic acids), benzyl ethers (e.g.,serine,
threonine, cysteine or tyrosine),
8 benzyloxycarbonyl groups (e. g., lysine) or other
9
protecting groups commonly used in peptide
synthesis
11
(e) the activated amino acid is reacted with
12
the peptide-resin for two hours at
13
room temperature, resulting in addition
14
of t:he new amino acid to the end of the
growing peptide chain.
16
(f) the peptide-resin is washed with methylene
17
chloride;
18
(g) the N-alpha-(tert. butyloxycarbonyl) group is
19
removed from the most recently added
amino acid by reacting with 30 to 65$, preferably
21
50$ (v/v) trifluoroacetic acid in methylene
22
chloride for 10 to 30 minutes at room
23
temperature;
24
(h1 the peptide-resin is washed with methylene
chloride;
26
(i) steps (a) through (h) are repeated until the
2~ ,
required peptide sequence has been
28
constructed.
29
The pept ide is then removed from the resin and
-41-
1'~~~'~
simultaneously t:he side-chain protecting groups are removed,
by reaction with anhydrous hydrofluoric acid containing 10%
v/v of anisole o:r other suitable (aromatic) scavenger.
Subsequently, the peptide can be purified by gel filtration,
ion exchange, hi~3h pressure liquid chromatography, or other
suitable means.
In soma cases, chemical synthesis can be carried
out without the aolid phase resin, in which case the
synthetic reactions are performed entirely in solution. The
reactions are sirnilar and well known in the art, and the
final product is essentially identical.
Isolation from natural sources: If sufficient
quantities of ths~ whole protein antigen are available, a
limited portion of the molecule, bearing the desired
sequence of amino acids may be excised by any of the
following procedures:
(a) Digestion of the protein by proteolytic
enzymes., especially those enzymes whose
substrate specificity results in cleavage
of the protein at sites immediately
adjacent to the desired sequence of amino
acids;
(b) Cleavage of the protein by chemical means.
Particular bonds between amino acids can be
cleaved by reaction with specific reagents.
Examples include: bonds involving
methionine are cleaved by cyanogen bromide;
asparaginyl-glycine bonds are cleaved by
-42-
1 hydroxylamine;
2 (c) A combination of proteolytic and chemical
3 cleavages.
It should also be possible to clone a small
portion of the DNA, either from natural sources or prepared
6 by synthetic procedures, or by methods involving a
combination thereof, that codes for the desired sequence of
amino acids, resulting in the production of the peptide by
bacteria, or other cells.
Analogously, one can form chains containing a
11
plurality of amino acid sequences by the following
12
technique: An aqueous solution of a peptide or peptides is
13
mixed with a water-soluble carbodiimide (e. g., ethyl-
14
dimethyl-aminopropylcarbodiimide). This results in
polymerization of the peptide(s); depending on the use of
16
the side chain blocking groups mentioned above, either
17
straight chain. or branched polymers of the peptide can be
18
19
made.
If desired the synthetic peptide of the present
invention can have bonded thereto a chain of any of the
21
following moieties: polypeptide, polyamino acid, poly-
22
23 saccharide, polyamide or polyacrylamide which can serve as a
24 stabilizing chain or as a bridge between amino acids of the
individual chains. Such chains are available commercially
26 or, in the case of polyamino acids, are formed by a process
27 which comprises: mixing a solution of the desired amino acid
28 sequence with a solution of the N-carboxylanhydride of the
29 ~ amino acid and allowing a base-catalyzed polymerization tv
-43-
1 occur, which is initiated by the amine groups of the
2
peptide.
3 Although a carrier may not be required, if a
4 carrier is employed the deposition of a chain or chains on a
"carrier" can be effected as follows:
6 1. Protein Carrier: The protein and the
7 synthetic peptide are dissolved together in water or other
8
suitable solvent, and covalently linked via amide bonds
9
formed through the action of a carbodiimide. The resulting
product may contain one or more copies of the peptide per
11
protein monomer. Alternatively, the reduced peptide may be
12
added to a carrier containing sulfhydryl groups to form
13
disulfide bonds. Yet another method involves the addition of
14
reduced peptide to protein carriers containing maleimidyl
groups to form a covalent linkage by a Michael addition, or
16
any other covalent attachment means.
17
2. Polysaccharide Carriers: Oligosaccharide
18
carriers should. have molecular weights in the range 1,000 to
19
1,000,000. In order 1.o covalently link these to synthetic
21 peptides, suitable functional groups must first be attached
22 to them. Carboxyl groups may be introduced by reacting with
23 iodoacetic acidl to yield carboxymethylated polysaccharides,
24 or by reacting with carbonyldiimidazole to yield activated
carbonyl esters. Carboxymethyl polysaccharides are coupled
26 to the peptide by a carbodimide reaction, while the
27 activated carbonyl esters react spontaneously with peptides.
28 Multiple copies; of the synthetic peptide should be attached
29 to each oligosacchar:ide unit.
-44-
~~~~~5~
3. Polyamino Acid Carriers: These carriers
should have molecular weights in the range 1,000 to
1,000,000. Po:lylysine and polyornithine have primary amino
groups on their side chains; polyaspartic acid and
polyglutamic acid have carboxyl groups. Peptides may be
coupled to these via amide bonds using' the carbodiimide
reaction. Anoi:her carrier that provides amino groups for
coupling is polylysine to which polyalanine can be attached
to the side chains of the lysine residues. The synthetic
peptide may bf~ attached to the ends of polyalanine chains,
also by a carbodiimide reaction. Multiple copies of the
synthetic peptide should be attached to each oligopep-
tide unit.
The novel carrier of the present invention
includes a lipid vesicle having active sites on the outer
surface thereof. Such active sites include -COOH, -CHO,
-NH2 and -SH. The lipid carrier can be stabilized by
cross-linking by a stabilizing agent such as an aldehyde
having at least two functional groups, such as a
bifunctional aldehyde, e.g., glutaraldehyde.
The bonding of the peptide to the lipid vesicle
carrier occurs at the active sites on the lipid vesicle on
the exterior surface of the carrier. Without wishing to be
bound by any theory of operability, it is believed that such
bonding is at least covalent bonding.
It is possible to bind a peptide to two active
sites on the outer surface of the lipid vesicle. For
example, a -NH,~ group at one end of a peptide can bind with
,_
a -COON active site on the outer surface of the lipid
-45-
1340~~~
1 vesicle. The .other end of the peptide can then bind with
another active site on the lipid vesicle, for example, a
3 -COOH group on the other end of the peptide can bind with a
4 -NH2 active site on the lipid vesicle.
The preferred carrier to support the synthetic
6 peptides of th~e present invention is a lipid vesicle. Lipid
vesicles can b~e formed by sonicating a lipid in an aqueous
8 medium, by resuspension of dried lipid layers in a buffer or
9
by dialysis of lipids dissolved in an organic solvent
against a buffer of choice. The latter procedure is
11
preferred. Lipid vesicles consist of spheres of lipid
12
bilayers that .enclose part of the aqueous medium.
13
Lipid vesicle (non-protein) carriers according to
14
the present invention can be produced in a variety of ways.
The preferred method to produce such carriers would be to
16
treat a lipid 'vesicle containing aminoalkanes and
17
diaminoalkanes having 10 to 18 carbon atoms, for example
18
stearylamine, cetylamine and myrististylamine with a
19
polyaldehyde, such as a dialdehyde, for example, butanedial
21 (succinaldehyd~e), pentanedial (glutaraldehyde), hexanedial
22 (adipoldehyde), heptanedial (pimelicaldehyde) and octanedial
23 (suberaldehyde). Alternatively, a liposome containing
24 aminoalkenes a:nd diaminoalkenes having 10 to 18 carbon
atoms, for example, oleylamine, can be treated with the
26 aforementioned polyaldehydes. The lipid vesicle carrier
27 thus formed has active aldehyde groups on the surface
28 thereof allowing the direct linking of peptides via their
29 N-terminal or lysine groups.
-46-
134~~~~
Peptides linked to lipid vesicle carriers
2 according to the present invention can also be prepared by
treating an amino captaining lipid vesicle as described
4 above with a peptide activated by carbodiimide, for example,
N-ethyl-N' (dimethylaminopropyl) carbodiimide.
6
Alternatively a carbodiimide activated peptide is
7 linked to polyaldehyde, e.g., dialdehyde, treated lipid
8
vesicles which have been further derivatized by reaction
9
with a water-soluble diaminoalkane, e.g., ethylene diamine
and propylene diamine.
11
12
13
Still further, lipid vesicles containing fatty
acids (saturated and unsaturated) having 12 to 18 carbon
atoms, e.g., stearic acid, oleic acid, palmitic acid and
14
myristic acid, are activated with carbodiimide. Thereafter,
the activated lipid vesicle is reacted with a peptide.
16
Another approach to form a carrier according to
17
the present invention involves using a fatty acid aldehyde
18
as a component of the lipid vesicle and treating such lipid
19
vesicle as described for glutaraldehyde treated lipid
21 vesicles. Such lipid vesicle reacts directly with amino
22 groups of peptides.
23 In a preferred embodiment of a carrier according
24 to the present invention, the aforementioned lipid vesicle
carrier formed by treating a amino or diaminoalkane (or
26 amino or diaminoalkene) having 10 to 18 carbon atoms with a
27 polyaldehyde is further reacted with cysteine (L-or D- or
28 LD- cysteine). These lipid vesicles are then reacted with a
2g pcr~ide having -SH groups, i.e., cysteine containing
_
1~4~'~5~
peptides. The link between the lipid vesicle and the
2 peptide is mediated by a disulfide bond.
3 Alternatively, a fatty acid mercaptan is used as a
4 component of the lipid vesicle, for example,
octadecanethio:l. A c steine containin
y g peptide is directly
6 linked to such lipid vesicle.
7
Another approach to form carriers according to the
g
present invention involves the preparation of the above
9
described fatter acid mercaptan containing lipid vesicles
which are further reacted with a dimaleimide, for example,
11
para or ortho 1J-N'-phenylenedimaleimide. Such lipid vesicle
12
is then reacted with a cysteine containing peptide.
13
Alternatively, the link between the appropriate
14
lipid vesicle and the appropriate peptide can be
accomplished by commercially available cross-linking
16
reagents such as dimethyl adipimidate; dimethyl
17
3,3'-dithiobis--propionimidate; 2-iminothiolane;
18
di-succinimidyl suberate; bist2-(succinimidooxy
19
carbonyloxy)-ei~hyl] sulfone; disuccinimidyl tartarate;
21 dithiobis (succ:inimidyl propionate); ethylene glycol
22 bis(succinimidyl succinate); N-5-azido-2-nitrobenzoyloxy-
23 succinimide; p--azidophenacyl bromide; p-azido-phenylglyoxal;
24 4-fluoro-3-nitrophenyl azide; N-hydroxysuccinimidyl-4-azide-
benzoate; N-hydroxysuccinimidyl-4-azidosalicylic acid; m-
26 maleimidobenzoyl N-hydroxy succinimide ester; methyl-4-
27 azidobenzoimidate; p-nitrophenyl 2-diazo-3,3,3-trifluoro-
28 prvprionate; N--succinimidyl-6 (4'-azido-2'-nitrophenylamino)
2g hexanoate; succ:inimidyl 4-(N-maleimidomethyl) cyclohexane-
1-carboxylate; succinimidyl 4-(p-maleimidomethyl) butyrate;
-48-
13 4~'~ ~~
1 N-(4-azidophenylthio)phthalimide; ethyl 4-aziodophenyl 1,
2 4-dithiobutyrimidate; N-succinimidyl (4-azidophenyldithio)
3 propionate; 1-5-difluoro-2, 4-dinitrobenzene;
4
4,4'-difluoro-3,3'-dinitrodiphenyl-sulfone;
4,4'-diisothiocyano-2,2'-disulfonic acid stilbene;
6 p-phenylenediisothiocyanate; 4,4'-dithiobisphenylazide;
7 er thritolbiscarbonate; N-succinimid 1 3-(2
y y -pyridyldithiol)
8
propionate; dimethyl pimelimidate and dimethyl suberimidate.
9
The lipid vesicles according to the present
11
12
13
14
invention act not only as carriers, but also as adjuvants.
The lipid vesicle synthetic carriers of the
present invention can be utilized to bind synthetic peptide
analogues (eliciting protective antibodies) of various
viral, bacterial, allergen and parasitic proteins of man and
animals, besides synthetic peptide analogues of hepatitis B
16
surface antigen, and especially the novel synthetic peptide
17
analogue of hepatitis B surface antigen containing amino
18
acid sequences corresponding to amino acid sequences in
19
pre-S gene coded region of the HBV.
~21 Accordingly, the lipid vesicle synthetic carriers
22 of the present invention can be used to bind with synthetic
23 peptide analogues of the following viruses: influenza
24 hemagglutinin (A/memphis/102/72 strain, A/Eng 1878/69
strain, A/NT/60/68/29c strain, and A/Qu/7/70 strain), fowl
26 plague virus hemagglutinin, vaccinia, polio, rubella,
27 cytomegalovirus, small pox, herpes simplex types I and II,
28 yellow fever, Infectious ectromelia virus, Cowpox virus,
29 Infectious bovine rhinotracheitis virus, Equine rhino-
pneumonitis (equine abortion) virus, Malignant catarrh virus
-49-
1 of cattle, Fel:i.ne rhinotracheitis virus, Canine herpes
2 virus, Epstein~-Barr virus (associated with infectious
3 mononucleosis ~~nd Burkitt lymphoma), Marek's disease virus,
4 Sheep pulmonary adenomatosis (Jaagziekte) virus,
Cytomegaloviruaes, Adenovirus group, Human papilloma virus,
6 Feline panleucopaenia virus, Mink enteritis virus, African
horse sickness virus (9 serotypes), Blue tongue virus (12
8 serotypes), Infectious pancreatic necrosis virus of trout,
9
Fowl sarcoma virus (various strains), Avian leukosis virus
(visceral, erythroblastic and myeloblastic), Osteopetrosis
11
virus, Newcastle disease virus, Parainfluenza virus 1,
12
Parainfluenza 'virus 2, Parainfluenza virus 3, Parainfluenza
13
4, Mumps virus, Turkey virus, CANADA/58, Canine distemper
14
virus, Measles virus, Respiratory syncytial virus,
Myxovirus, Type A viruses such as Human influenza viruses,
16
e.g., Ao/PR8/34, A1/CAM/46, and A2/Singapore/1/57; Fowl
17
plaque virus; 'Type B influenza viruses, e.g., B/Lee/40;
18
Rabies virus; :Eastern equinine encephalitis virus;
19
Venezuelan equine encephalitis virus; Western equine
21 encephalitis virus; Yellow fever virus, Dengue type 1 virus
22 (=tYPe 6), Oen~gue type 2 virus (=type 5); Dengue type 3
23 virus; Dengue type 4 virus; Japanese encephalitis virus,
24 KYasanur Forest virus; Louping ill virus; Murray Valley
encephalitis virus; Omsk haemorrhagic fever virus (types I
26 and II); St. Louis encephalitis virus; Human rhinoviruses,
2~ Foot-and-mouth disease virus; Poliovirus type 1; Enterovirus
28 Polio 2; Enterovirus Polio 3; Avian infectious bronchitis
29 virus; Human respiratory virus; Transmissible
gastro-enteritis virus of swine; Lymphocytic
-50-
~.
1 choriomeningit.is virus; Lassa virus; Machupo virus; Pichinde
2 virus; Tacaribe virus; Papillomavirus; Simian virus; Sindbis
3 virus, and the like.
4 The lipid vesicle synthetic carriers of the
present invention can be used to bind synthetic peptide
6 analogues of bacteria, for example, leprosy, tuberculosis,
7 syphilis and gonorrhea.
8 ~ The. lipid vesicle synthetic carriers of the
present invention can also be used to bind synthetic peptide
analogues of the following parasites: organisms carrying
11
malaria (P. Falciparum, P. Ovace, etc.), Schistosomiasis,
12
Onchocerca Volvulus and other filiarial parasites,
13
Trypanosomes, Leishmania, Chagas disease, amoebiasis,
14
hookworm, and the like.
The lipid vesicle carriers of the present
16
invention can be used to bind the novel peptides of the
17
present invention corresponding to amino acid sequences in
18
the pre-S region of HBsAg. The lipid vesicle carriers of
19
the present invention can also be used-to bind amino acid
sequences in the S region, as well as other amino acid
21
sequences for other virus, etc.
22
Amino acid sequences (corresponding to amino acids
23
24 in the S region) which contains an antigenic determinant for
hepatitis B surface antigen can be linked to the lipid
26 vesicle carrier of the present invention. T.P. Hopp, "A
27 Synthetic Peptide with Hepatitis B Surface Antigen
28 -Reactivity", Mol. Imm., 18, 9, 869-872, 1981, propose the
29 following sequence corresponding to the S region of HF3sAg:
138 139 140 141 142 143 144 145 146 147 148 149
-51-
1 Cys Cys Thr Lys
Pro Thr
Asp Gly
Asn Cys
Thr Cys
2 Other peptides
mimicking
the antigenic
determinant
3 of HBsAg (S region)
include
the following:
4 (1)
Pept ide 1
6
X
~Lys
7
122
8
Thr
9
Ser - Cys - ys - Met hr
G - T
~ 137 124
Pro Thr
11
y l
T A
r a
12
Met Ser Thr-
Gly--Gln
13
14 Peptide 2 contains
5 additional
amino acid
residues:
Ser - Thr -
Gly - Pro
- Ser -
X,
117 121
16
G.R. Dree sman, Y.
Sanchez,
I. Ionescu-Matiu,
J. T. Sparrow,
17
H. R. Six , D.L. Peterson,
F.B. Hollinger
and J.L.
Melnick,
18
"Antibody to Hepatitis
B Surface
Antigen
After A
Single
19
Inoculation
of Uncoupled
Synthetic
HBsAg Peptides",
Nature,
295, 158- 160, 1982;
and (2)
the following
peptides:
21
22
23 POSITION SEQUENCE
24 48-81 Cys-Leu-Gly-Gln-Asn-Ser-Gln-Ser-Pro-Thr-Ser-
Asn-F~is-Ser-Pro-Thr-Ser-Cys-Pro-Pro-Thr-Cys-
26 Pro-Gly-Tyr-Arg-Trp-Met-Cys-Leu-Arg-Arg-Phe-
27 Ile
28 2-16 Glu-Asn-Ile-Thr-Ser-Gly-Phe-Leu-Gly-Pro-Leu-
2g Leu-Val-Leu-Glr~-Cys
-52-
' ~.~~~''~ ~~a
1 22-35 Leu-Thr-Arg-Ile-Leu-Thr-Ile-Pro-Gln-Ser-Leu-
Asp-Ser-Trp-Cys
3
4 38-52 Ser-Leu-Asn-Phe-Leu-Gly-Gly-Thr-Thr-Val-Cys-
Leu-Gly-Gln-Asn
6
7 47-52 Val-Cys-Leu-Gly-Gln-Asn
8
9
95-109 Leu-Val-Leu-Leu-Asp-Tyr-Gln-Gly-Met-Leu-Pro-
Val-Cys-Pro-Leu
11
12
104-109 Leu-Pro-Val-Cys-Pro-Leu
13
R. Arnon, "Anti-influenza Response Achieved by Immunization
14
With A Synthetic Conjugate", Proc. Natl. Acad. Sci. USA, 79,
-
569-573, 1982. The peptide corresponds to the sequence
16
serine-91 to lE~ucine-108 of the amino acid chain of the
17
virus.
18
A peptide containing an amino acid sequence
19
mimicking the antigenic determinant of polyoma virus medium
21 size tumor antigen is Lys-Arg-Ser-Ars-tlis-Phe, G. Walter,
22 M.A. Hutchinson, T. Hunter and W. Eckhart, "Purification of
23 Polyoma Virus Medium-Size Tumor Antigen by Immunoaffinity
24 Chromatography", Proc. Natl. Acad. Sci USA, 79, 4025-4029,
1982.
26 A peptide containing an amino acid sequence
27 mimicking the antigenic determinant of poliovirus replicase
28 antigen is as follows:
2g Tyr-Ser-Thr-Leu-Tyr-Arg-Arg-Trp-Leu-Asp-Ser-Phe -
450 461,
-53-
1 M. H. Baron and D. Baltimore, "Antibodies Against a
2 . Synthetic Peptide of the Poliovirus Replicase Protein:
Reaction with Native, Virus-Encoded Proteins and Inhibition
4 of Virus-Specific Palymerase Activities In Vitro". Jour.
Virology, 43, 3969-3978, 1982.
6 Peptides containing an amino acid sequence
7
mimicking the antigenic determinant of simian virus 40 large
8
tumor antigen are as follows:
9
Met-Asp-Lys-Val-Leu-Asn-Arg and
Lys-Pro-Pro-Thr-Pro-Pro-Pro-Glu-Pro-Glu-Thr,
11
G. Walter, K.H. Scheidtmann, A. Carbone, A.P. Laudano and
12
R.A. Lerner, N. Green, H. Alexander, F.-T. Liu, J.G.
13
Sutcliffe and T.M. Shinnick, "Chemically Synthesized
14
Peptides Predicted From the Nucleotide Sequence of the
Hepatitis B Virus Genome Elicit Antibodies Reactive With the
16
Native Envelope Protein of Dane Particles", Proc. Natl.
17
Acad. Sci. USF~, 78, 6, 3403-3407, 1981.
18 '
A peptide containing an amino acid sequence
19
mimicking the antigenic determinant of retrovirus R antigen
21 is as follows:
22 Leu-Thr-Gln-Gln-Phe-Eiis-Gln-Leu-Lys-Pro
23 Ile-Glu-Cys-Glu-Pro,
24 J.G. SutcliffE~, T.M. Shinnick, N. Green, F.-T. Liu, H.L.
Niman and R.A" Lerner, "Chemical Synthesis of A Polypeptide
26 Predicted Frorn Nucleotide Sequence Allows Detection Of A New
27 Retroviral Gene Product", Nature, 287, 1980.
28 ~ A pE~ptide containing an amino acid sequence
29 mimicking the antigenic determinant of avian sarcoma virus
antigen is as follows:
-54-
1 Glu-Asp-Asn-Glu-Tyr-Thr-Ala-Arg-Gln-Gly,
2 T.W. Wong and Alan R. Goldberg, "Synthetic Peptide Fragment
3 Of src Gene Product Inhibits.the src Protein Kinase and
4 Cross reacts Immunologically With Avian one Kinases and
Cellular Phosphoproteins", Proc. Natl. Acad. USA, 78, 12,
6 7412-7416, 1981.
7 Peptides containing an amino acid sequence
8
mimicking the antigenic determinant of foot-and-mouth
9
disease virus antigen are as follows:
141
11 Val Pro Asn Leu Arg Gly Asp Leu Gly Val
12 160
Leu Ala Gly Lys Val Ala Arg Thr Leu Pro
13
and
14
201
His Lys Gln Lys Ile Val Ala Pro Val Lys Gln
16 Thr Leu,
17 J.L. Bittle, R..A. Houghten, H. Alexander, T.M. Shinnick,
18 J.G. Sutcliffe, R.A. Lerner, D.J. Rowlands and F. Brown,
19 "Protection Against Foot-And-Mouth Disease By Immunization
With A Chemically Synthesized Peptide Predicted From the
21 Viral Nucleotide Sequence", Nature, 298, 30-33, 1982.
22 A peptide containing an amino acid sequence
23 mimicking the antigenic determinant of hemagglutinin X-31
24 (F~3N2) influenza virus antigen is as follows:
123 125
26 Glu-Gly-Phe-Thr-Trp-Thr-Gly-
27 130 135
Val-Thr-Gln-Asn-Gly-Gly-Ser-
28 140
29 Asp-Ala-Cys-Lys-Arg-Gly-Pro-
145 150
Gly-Ser-Gly-Phe-Phe-Ser-Arg-
-55-
1 151
Leu,
2
3 D.C. Jackson, ~J.M. Murray, D.O. White, C.N. Fagan and G.W.
4 Tregear, "Antigenic Activity of a Synthetic Peptide
Comprising the 'Loop' Region of Influenza Virus
Hemagglutinin", Virology, 120, 273-276, 1982.
A peptide containing an amino acid sequence
mimicking the antigenic determinant of hemagglutinin of type
A H3N2 influenza virus antigen was synthesized by G.M.
Muller, M. Shapira and R.F. Doolittle, "Antibodies Specific
11 for the Carboxy- And Amino- Terminal Regions of Simian Virus
12 -40 Large Tumor Antigen", Proc. Natl. Acad. Sci USA, 77, 9,
13 5179-5200, 1980.
14 A peptide containing an amino acid sequence
mimicking the antigenic determinant of influenza virus
16 strain 3QB antigen is Ilel Vall Asx2 Thrl Ser2 Glx2 Prol
17 Gly3 Alal Leul Lysl, A. Aitken and C. Hannoun, "Purification
18 of Haemagglutinin and Neuraminidase from Influenza Virus
19 Strain 3QB and Isolation of a Peptide From an Antigenic
Region of Haemagluttinin", Eur. J. Biochem, 107, 51-56,
21 1980.
22 Peptides containing an amino acid sequence
23 mimicking the antigenic determinant of diptheria antigen are
24 given as follows:
Natural DT Loci
26 -Cys-Ala-Gly-Asn-Arg-Val-Arg-Arg-Ser-Val-
27 l8Ei 190 195
28 Gly--Ser-Ser-Leu-Lys-Cys-
201
'29
Synthetic Peptide
Tet~_adecapeptide Gly(188)---Cys-(201)
-56-
1 Hexadecapeptide Cys(186)---Cys-(201)
2 Octadecapeptide Ala-Ala-Cys(186)---Cys-(201)
3 F. Audibert, M. Jolivet, L. Chedid, R. Arnon and M. Sela,
4 "Successful Immunization With a Totally Synthetic Diphtheria
Vaccine", Proc. Natl. Acad. Sci. USA, _79, 5042-5046, 1982.
6
A peptide containing an amino acid sequence
7 mimicking the ~~ntigenic determinant of Streptococcus
8
pyogenes M antigen is as follows:
9
10 Asn-Phe-Ser-Thr-Ala-Asp-Ser-Ala-Lys
11 10 15
Ile-Lys-Thr-Leu-Glu-Ala-Glu-Lys-Ala-Ala-
12
20 25
13 Leu-Ala-Ala-Arg-Lys-Ala-Asp-Leu-Glu-Lys-
14 30 35
Ala-Leu-Glu-Gly-Ala-Met
E.Ii. Beachey, ,:f.M. Seyer, D.B. Dale, W.A. Simpson and A.H.
16
Kang, "Type-Specific Protective Immunity Evoked by Synthetic
17
Peptide of Streptococcus Pyogenes M Protein", Nature, 292,
18
457-459, 1981.
19
The lipid vesicle carrier of the present invention
can thus be utilized to bind with any amino acid sequence
21
which includes the antigenic determinant for a specific
22
23 antigen.
24 The lipid vesicle carriers of the present
invention can also be used to bind with enzymes.
26 The present: invention is also directed to
27 diagnostic tests for direct detection of HBV antigens and
28 HBV antibodies.
29 In order to detect HBV antigens containing
proteins coded for by the pre-S gene in sera of HBV-infected
-57-
~~~fl~~~
1 animals such ass humans, radioimmunoassay (RIA) or
2 enzyme-linked immunosorbent assay (RLISA) can be employed.
One test for detecting HBV antigens according to
4 the present invention is as follows:
(1) a solid substrate containing binding sites
6 thereon e.
g., polystyrene beads, is coated with antibodies
7
to a peptide having an amino acid chain corresponding to at
8
least six amino acids within the pre-S gene coded region of
9
the envelope of HBV, the peptide free of an amino acid
sequence corresponding to the naturally occuring proteins of
11
HBV;
12
(2) the coated beads are then washed with, for
13
example, tris buffered saline, to remove excess antibody;
14
(3) the beads are then contacted with a protein-
containing solution, such as bovine serum albumin (BSA) or
16
gelatin to saturate protein binding sites on the beads (to
17
prevent or reduce non-specific binding) - a convenient
18
concentration of such protein-containing solution can be
19
employed such as 1 mg/ml to 50 mg/ml;
21 (4) beads are then washed to remove excess BSA or
22 gelatin;
23 (5) the beads are then incubated with samples
24 suspected to contain HBV or HBsAg (normal sera is utilized
as a control);
26 (6) the beads are then washed with a solution,
27 e.g., tris buffered saline solution, and mixed with a
28 radiolabeled antibody, e.g., I125 labeled antibody (antibody
29 to either the peptide or to HHsAg);
(7) the beads are then incubated;
-58-
1 (8) the beads are then washed and counted in a
2 gamma counter.
3 If t:he specimens have counts at least 2.1 times
4 higher than counts of the control, then the specimens are
positive.
6 The pre-S gene coded peptides according to the
7
present invention can be employed as a diagnostic tool to
8 detect antibodies to the pre-S region of HBV in a given
9
sample. The pre-S gene coded peptide, for example, pre-S
re-S (12-32), pre-S (32-53), or re-S (117-134),
(120-145), p P
11 re-S(94-117), pre-S(153-171), pre-S(32-53) and
pre-S(1-21), p
12
pre-S(57-73), is adsorbed on a solid substrate, containing
13
binding sites thereon for example, polystyrene beads. The
14
substrate is thereafter contacted with a substance (protein
containing solution), for example, gelatin BSA or powdered
16
milk, to saturate the binding sites thereon. Thereafter,
17
the substrate is washed with a buffered solution and
18
thereafter the; buffer is removed. A specimen, e.g., human
19
sera diluted with animal sera is added to the substrate.
The resultant mass is then incubated and washed.
21
Thereafter, radiolabeled, e.g., iodinated, e.g., I125,
22
23 antibodies to human IgG or IgM is added to the mass. The
24 resultant mas:~ is then washed and counted, e.g., in a
gamma-counter. If the count is higher than a count
26 Performed on a normal sera control, the specimen contains
27 antibodies to the pre-S region of HBV.
28 It is believed that the above procedure for
29 detection of antibodies to the pre-S region of I-1BY can be
-59-
134~1'~J~
1 applied as a diagnostic tool in detecting hepatitis B virus
2 infection.
3 - The pre-S protein moiety appears to be directly
4 involved in ati:achment of HBV to liver cells of the host.
Similar proteins are likely to be involved in the attachment
6 of other virusE~s, the target of which is the liver. For
this reason, synthetic peptides corresponding to the pre-S
protein, as we:Ll as antibodies to them, could serve as the
basis for diagnostic assays of and vaccines against other
hepatitis viruaes reacting with the same liver receptors as
11 does hepatitis B virus.
12
In the above described procedures involving
13
radioimmunoass;~y (RIA), an enzyme linked antibody can
14
replace the radiolabeled antibody and ELISA techniques can
be performed. Furthermore, fluorescence techniques can be
16
employed in place of RIA or ELISA.
17
The ;labelling ("marking") of one of the reaction
la.
components can be brought about by use of a "marker" or
19
"marker substance" such as by incorporation of a radioactive
atom or group, or by coupling this component to an enzyme, a
21
dyestuff, e.g., chromophoric moiety or a fluorescent group.
22
The components concerned are preferably labelled
23
by coupling to an enzyme, since the estimation of this is
29
much simpler than far example, the estimation of
radioactivity, for which special apparatus is necessary.
26
The enzymes used are preferably those which can be
27
colorimetrically, spectrophotometrically, or
28
fluorimetrical.ly determined. Non-limiting examples of
29
enzymes for use in the present invention include enzymes
-60-
1340~~5
1 from the group of oxidoreductases, such as catalase,
2 peroxidase, glucose oxidise, beta-glucuronidase,
beta-D-glucosidase, beta-D-galactosidase, urease and
4 galactose oxidise.
The coupling of the enzyme and the immunological
6 component can be brought about in a known way, for example,
7 by the formation of an amide linkage by methods known from
8 peptide chemistry.
9
The :labelling with a radioactive isotope can also
be performed i:n a known way. Isotopes useful for labelling
11
are predominantly I125~ I131~ C14~ and H3.
12
The .incubation steps utilized in carrying out the
13
above procedures can be effected in a known manner, such as
14
by incubating ,at temperatures of between about 20°C and
about 50°C for between about 1 hour and about 48 hours.
16
17
Washings as described above are typically effected
using an aqueous solution such as one buffered at a pH of
18
6-8, preferably at a pH of about 7, employing an isotonic
19
saline solution.
The present invention also concerns diagnostic
21
test kits for conducting the above-described methods for
22
23 detecting antigens and antibodies.
24 A diagnostic test kit according to the present
invention for .detecting antigens coded for the pre-S gene of
26 HBV in a test sample, would include the following:
27 , a. a solid substrate coated with antibodies to a
28 peptide having an amino acid chain corresponding to at least
29 six consecutive amino acids within the pre-S gene coded
region of the envelope of HBV, the peptide free of an amino
-61-
~~4~~~~
1 acid sequence corresponding to the naturally occurring
2 proteins of HB'V,
3 b. a protein-containing solution to saturate
protein binding sites on the solid subtrate, and
c. a given amount of radiolabeled antibody, such
6 antibody to either the peptide or HBsAg.
A diagnostic test kit according to the present
8 invention for detecting antibodies to the pre-S region of
hepatitis B virus in a test sample, would include the
following:
11
a. a solid substrate having adsorbed thereon a
12
peptide having an amino acid chain corresponding to at least
13
six consecutive amino acids within the pre-S gene coded
14
region of the envelope of HBV, the peptide free of an amino
acid sequence corresponding to the naturally occurring
16
proteins of HBV, the substrate being exposed to a
17
protein-containing solution to saturate protein binding
18
sites on the solid substrate, and
19
b. a given amount of radiolabeled antibodies to
human IgG or Igl4.
21
Radiolabeled antibodies used in the
22
23 above-described test. kits can be packaged in either solution
24 form, or in lyophilized forms suitable for reconstitution.
In the above test kits, enzyme or fluorescent
26 labelled antibodies can be substituted for the described
2~ radiolabeled antibodies.
28 The .above described process and test kit for
29 detection of antibodies to the pre-S region of hepatitis B
virus can be utilized in many applications, such as
-62-
134U'~ ~r
(1) ~:letecting HBV infection in a patient by
taking serum from the patient and applying the above
described test or using the above described test kit; and
(2) predicting recovery from HBV infection by
taking serum from an infected patient and applying the above
described antibody detection procedures.
The above described test procedure and test kit
for antibody detection can be used for making qualitative
comparisons between different HBV vaccines by taking serum
from vaccinated patients and then utilize the
above-described test procedure or kit for antibody
detection. In general. all known immunoassays using this
antigen as reagent can be performed using the synthetic
peptide of this invention. Generally all known immunoassays
using antibody containing serum or reagents can be performed
using antibody serum produced through the use of a synthetic
peptide of this :invention. These immunoassays included all
those disclosed by Langone and Van Vunakis, riethods of
Enzymoloqy, Academic Press, Volumes 70, 73 and 74. See
assays disclosed in the disclosures of the following U.S.
Patents: 4,459,3~~9; 4,343,896; 4,331,761; 4,292,403;
4,228,240; 4,157,280; 4,152,911; 4,169,012; 4,016,043;
3,839,153; 3,654,090 and Re 31,006 and volumes 70, 73 and 74
of Methods of Enz molo ,
A hepatitis B vaccine can be prepared by directly
using a conjugate of a lipid vesicle and a peptide
containing an amino acid chain corresponding to at least six
consecutive amino acids within the pre-S gene coded region
-63-
of the surface antigen of hepatitis B virus in an
appropriate buffer. The conjugate having peptide in the
appropriate concentration can be used as a vaccine with or
without an adjuvant, such as, e.g., aluminum hydroxide or
others.
The .active component of the vaccine can be
employed with a physiologically acceptable diluent (medium),
e.g., phosphate buffered saline. Generally speaking, the
synthetic peptide concentration in a physiologically
acceptable medium will be between approximately less than 1
miligram and more than 10 micrograms per dose.
The 'vaccine can be prepared and used in the same
general manner as disclosed in U.S.P. 4,118,479.
The 'vaccine can be administered by subcutaneous,
intradermal or intramuscular injection. While the preferred
route would depend upon the particular vaccine, it is
believed that intramuscular injection will be generally
suitable. Frequency of administration will vary depending
upon the vaccine. Generally speaking, the vaccine will be
administered i:n two doses about one month apart followed by
a booster at six months to one year after primary
immunization. The subsequent doses or the booster will
depend on the level of antibody in the blood as a result of
the initial immunization, and in certain instances may be
unnecessary.
The lhepatitis vaccine of the present invention is
recommended fo;r all persons at risk of developing hepatitis
B infection and particularly those at especially high risk
-64-
~ ~~~~~5
1 such as patients and staff on hemodialysis unit, medical
2 personnel, persons of tropical. populations and those
3 visiting the tropics. In the case of tropical populations,
4 particularly in Africa, Asia, the Mediterranean region and
South America, where high incidence of hepatitis B
6
infections has been consistently observed, the vaccine
7
should be administered sufficiently early in life to prevent
8
acquisition of chronic carrier state infection which tend to
9
occur in these regions within the first five years of life.
In fact, the vaccine is expected to be useful for all
11
persons not already protected against hepatitis B infections
12
as a result of prior immunity.
13
In order to more fully illustrate the nature of
14
the invention and the manner of practicing the same, the
following non-limiting examples are presented:
16
EXAMPLES
17
Example 1
18
19 SDS-Polyacrylamide Gel Electrophoresis Of HBsAg.
bout 20 and 200Jug, respectively, of HBsAg were
21 separately electrophoresed for silver staining and transfer
22 to nitrocellulose, respectively. Before electrophoresis,
23 HBsAg was treated far 30 minutes at 37°C with
24 2-mercaptoethanol and NaDodS04 (10 mg/ml each in 8 M urea,
0.0625 M Tris, pH 7.2). Similar results were obtained with
26 HBsAg alkylated with iodoacetate after reduction. HBsAg was
27 purified and radiolabeled as described (A.R. Neurath, N.
28 Strick, C.Y. H:uang, Intervirology, 10, 265 (1978)).
2~ SDS-Polyacrylamide gel electrophoresis
("SDS-PAGE") was carried out following published procedures.
-65-
~. 3 4 0'~ ~ ~
See V.K. Laemmli, Nature (London), 227, 680 (1970).
However, in order to maintain proteins in fully denaturated
foam, 8M urea was utilized in the running buffers in
electrophoresis"
PolypE~ptides separated by SDS-PAGE were
transferred to nitrocellulose using the TE 42 Transphor unit
9 (Hoefer Scientific Instruments, San Francisco, California)
following the procedure recommended by the manufacturer. The
transferred proteins were tested for determinants reacting
with antibodies to intact HBsAg (anti-HBs) using
125I_labeled hurnan anti-HBs supplied as part of a commercial
test kit (Abbott: Laboratories, North Chicago, Illinois) as
described (J.C. McMichael, L.M. Greisiger, L. riillman, J.
Immunol. Meth., 45, 79, (1981)).
From i:he 20~ug sample gel, separated IiBsAg
polypeptides (their Mr given in kilodaltons) were stained by
silver in situ ~(J.H. Morrissey, Anal. Biochem, 117, 307,
(1981)), (see Fig. 1, Panel a) to yield two major and
several minor polypeptides as expected. The separated
polypeptides from the other 200 dug sample gel was then
electrophoretically transferred to nitrocellulose, reacted
(probed) with 1'~5I-labeled antibodies to intact HBsAg (anti
HBs) and submitted to autoradiography fFig. lb).
Surprisingly, the 33 and 36 kilodalton (P33 and
P36), rather then the two most abundant polypeptides reacted
preferentially with anti-FIBS (Fig. 1, Panel b). This
suggested the presence of disulfide bond independent
antigenic determinants reacting With anti-HBs on amino acid
sequences which are not coded for by the S-gene of HBV DNA.
-66-
134~'~~~
1 P33 and P36 contain the sequence corresponding to the
product of the S-gene and additional 55 residues at the
3 amino-terminal art startin with Met at
p g position 120 in the
4
pre-S gene region (See Fig. 2).
Example 2
6
Syntr~esis Of A Peptide Mimicking Antigenic
7 ,
Determinants Corresponding To Residues 120-145 Of The Pre-S
8
Gene Product
9
The location of antigenic determinants on proteins
may be predicted from computing the relative hydrophilicity
11
along the amino acid sequence. See T.P. Hopp, K.R. Woods,
12
Proc. Natl. Acad. Sci. USA, 78, 3824 (1981) and J. Kyte,
13 -"
R.F. Doolittle, J. Mol. Biol., 157., 105 (1982).
14
Results of such. computation (J. Kyte et al supra) for the
translation product of the pre-S region are shown in Fig. 3
16
and suggest the location of antigenic determinants in the
17
18 sequence to the right from Met 120 within residues 120-140.
19 The segment corresponding to residues 120-145 (Fig. 2)
(pre-S 120-145, subtype adw2) was selected for synthesis.
21 A C-terminal Cys(-SH containing) residue was added
22 to allow unambiguous conjugation to carrier molecules and
23 affinity matrices, while leaving the N-terminal unblocked
as
24 it may be in the intact protein. The molecule contains one
Tyr and c an therefore be radiolabeled. The Tyr could also
be
26 used for conjugation, although might be a part of the
it
27 antigenic determinant:.
2g The peptide was synthesized by an accelerated
2g version of stepwise solid phase peptide synthesis on the
benzhydrylamine-type resin of Gaehde and Matsueda (Int. J.
-67-
1 Peptide Protein Res., 18, 451, (1981)) using
2 Boc-NH-CH(phen;yl)-phenyl-OCEI2COOH to derivatize NH2CH2-Resin
(A. R. Mitchell, S.B.H. Kent, M. Engelhard and R.B.
4 Merrifield, J. Org. Chem., 43, 2845-2852, 11978)). After the
Cys was coupled, the protected peptide chain was assembled
according to t:he following protocol:
7
1. Deprotection: 65$ v/v trifluoroacetic acid in
8
dichloromethane, 1x10 minutes;
9
2. Wash: a flowing stream of dichloromethane was
run over the resin under suction from an aspirator for 20
11
12
13
seconds;
3. Neutralization: 10$ v/v diisopropylethylamine
in dichloromethane, 2x1 minutes;
14
4. Wash: a flowing stream of dichloromethane was
run over the resin under suction from as aspirator for 20
16
seconds;
17
5. Coupling: 2 mmol tert.Hoc-L-amino acid in 2m1
18
dichloromethan.e was added to the neutralized resin followed
19
immediately by lmmol dicyclohexylcarbodiimide in 2m1
21 dichloromethane; after 10 minutes a sample of resin
22 (aPProximately 5mg) was taken for determination of coupling
23 Yield by quantitative ninhydrin, and lOml dimethylformamide
24 was added and the coupling continued. (Asn and Gln were
coupled in they presence of hydroxybenzotriazole).
26 6. After the ninhydrin determination of a
27 satisfactory coupling, the resin was washed as in step 4,
28 above. For they addition of subsequent residues, the cycle
29 was repeated. If recoupling was necessary, steps 3-5 were
repeated. The synthesis was performed on a 0.5mmo1 scale
-68-
o~s~
1 (0.5gram aminomethyl-resin of lmmol/g loading). All volumes
2 were lOml except where noted.
Protected amino acid derivatives used were
4 N-alpha-tert.butyloxycarbonyl protected and side chain
protected as fellows: Arg(NGTosyl); Cys(4MeBz1); Tyr(BrZ);
6 Asp(OBzl); Thr(Bzl); His(ImTosyl). Met and Trp were
7
unprotected on the side chains. In another synthesis,
8
otherwise identical, use of His(ImDNPI and Trp(InFormyl)
9
gave purer product.
Assembly of the peptide chain was monitored by the
11
quantitative ninhydrin reaction (V. K. Sarin, S.B.H. Kent,
12
,T.P.Tam, R.B. ;Merrifield, Anal. Biochem, 117, 147-157,
13
(1981)) and was without difficulty except for the addition
14
of the histidine residue which was 10% incomplete despite
repeated couplings, presumably due to an impure amino acid
16
derivative. After assembly of the protected peptide chain,
17
the N-terminal Boc group was removed by trifluoroacetic acid
18
treatment and the resin neutralized as in steps 1-4 above.
19
Then the peptide was cleaved and deprotected by a 1 hour
21 treatment at 0°C with HF containing 5$ v/v p-cresol and 5$
22 v/v p-thiocresol to give the desired peptide as the
23 C-terminal cysteinamide. Where His(ImDNP) was used, the DNP
24 was removed by treatment with phenylphenol prior to HF
cleavage. Where TrP (InFormyl) was used, HF conditions were
26 adjusted to remove the Formyl group; either HF containing
27 10~ anisole and 5$ 1,4-butanedithiol, or HF containing
28 p-cresol and 5$ 1,4-butanedithiol. The product was
2g precipitated a.nd washed by the addition of ether, then
dissolved in 5~$ v/v acetic acid in water and lyophilized to
g- ,
._ ____.' __
~.3~0'~~5
give a fluffy white solid.
Quantitative Edman degradation (H. D. Niall, G.W.
Tregear, J. Jac:obs, Chemistry and Biology of Peptides, J.
Meienhofer, Ed (Ann Arbor Press, Ann Arbor, MI, 1972), pp.
659-699) of thEa assembled peptide-resin revealed a high
efficiency of chain assembly (S.B.H. Kent, M. Riemen, M.
LeDoux, R.B. Me~rrifield, Proceedings of the Fourth
International ~~ymposium on Methods in Protein Sequence
Analysis, M. EJLzinga, Ed. (Humana, Clifton, New Jersey,
1982), pp. 626--628) which proceeded at a ~ 9./9.7 percent
efficiency at Each step, except for histidine at sequence
position pre-S 128. HPLC of the peptide cleaved off the
resin revealed a single major peak corresponding to
approximately E15 percent of peptide material absorbing light
at 225 nm.
Examples 3-6
Immunologic Properties Of A Peptide Mimicking
Antigenic Determinants Corresponding To Residues 120-145 of
the Pre-S Gene Product (pre-S 120-145)
Example 3
Immunization
Immunization of rabbits with either free or
carrier-bound pre-S 120-145 (subtype adw2) were conducted
and resulted in an antibody response in all animals against
both the homologous peptide and HBsAg (Fig. 4).
The peptide corresponding to the amino acid
sequence 120-145 (pre-S 120-145) of the pre-S region of HBV
DNA (subtype a~iw2; P. Valenzuela, P. Gray, M. Quiroga, J.
-70-
i344r1~~
1 Zaldivar, H.M. Goodman, W.J. Rutter, Nature (London), 280,
2 815, (1979)) containing an additional Cys residue at the
3 C-terminal, added for convenience of coupling to carriers,
was synthesized by an improved solid phase technique (S.B.H.
Kent, Hiomedic.al Polymers, E.P. Goldberg, A. Nakajima,Eds.
6 (Academic, New York, 1980), pp. 213-242; A.R. Mitchell,
7 S.B.H. Kent, M. Engelhard, R.B. Merrifield, J. Org. Chem.
8 43, 2845, (1978); and S. Mojsov, A.R. Mitchell, R.B.
Merrifield, _J. Org. Chem., 45, 555 (1980).
For :immunoassays and linking to carriers the
11
peptide was treated with 2-mercaptoethanol and separated
12
from low Mr components by chromatography on Sephadex G-10
13
(A. R. Neurath, S.B.H. Kent, N. Strick, Proc. Natl. Acad.
14
16
Sci. USA, 79, '7871 (1982)).
Groups of two to three rabbits were immunized with
either free pre -S 120-145 or with the peptide linked to
17
cysteine-activ<~ted liposomes containing stearylamine,
18
dilauroyl lecii~hin and cholesterol which had been fixed with
19
glutaraldehyde" and either did or did not have attached RAT
groups for enhancing antibody responses to haptens (A. R.
21
Neurath, S.B.H" Kent, N. Strick, J. Gen. Virol., in press
22
23 (1984)). The irnmunization schedule involving the use of
24 complete and incomplete Freund's adjuvant was the same as
described (Neurath, Kent, Strick, et al (1984) supra).
26 Antibodies to FiBsAg in sera of rabbits immunized with pre-S
27 120-145 were tf~sted by a double-antibody radioimmunoassay
28 (RIA) using HBsAg-coated polystyrene beads and 1251-labeled
29 art~i-rabbit IgC~ (Neurath, Kent, Strick, et al (1984) supra).
-71-
134~~1~~
1 Antibodies to the homologous peptide were tested
by a similar test except that 2.5 mg of a cellulose-peptide
3 conjugate were used instead of coated beads. This conjugate
4 was prepared in the following way: 0.5 g of sulfhydryl
cellulose, prepared as described (P. L. Feist, K.J. Danna,
6 Biochemistry, 20, 4243 (1981)), were suspended in 5 ml 0.1 M
7 sodium acetate, pH 5, and mixed with 2.5 ml of 0.25 M
8
N-N'-p-phenylenedimaleimide in dimethylformamide for one
hour at 30°C and then washed with 0.1 M phosphate-lOmM EDTA,
pH 7Ø The cellulose derivative was suspended in 10 ml of
11
the latter buffer containing 5 mg of pre-S 120-145 and mixed
12
for at least sixteen hours at 20°C. The cellulose derivative
13
was extensively washed and suspended in 0.14 P~i NaCl-10 mM
14
Tris-3 m61 Narl3 (TS) . The final preparation contained 8 mg of
pre-S 120-145 Viper g of. cellulose.
16
17
Example 4
18
Radi~~immunioassays were conducted with several
19
dilutions of a serum from one of the rabbits immunized with
pre-S 120-145 linked to liposomes (See Fig. 5).
21
22 Antibodies were still detectable when the antisera
23 were diluted up to 1.6 x 105-fold (Fig. 5).
24 Pre-.5 120-145 or anti-pre-S 120-145 inhibited the
reaction between 1251-labeled anti-figs and P33 (P36) .
26 125I-labeled HIBsAg was immunoprecipitated with anti-pre-S
27 120-145 at all dilutions positive by RIA (Fig. 5). HBV
28 particles reacted with anti-pre-S 120-145 as determined by
29 detection of HBV-DNA within the immune complexes and by
-72-
134Ur1 ~~
1 electron microscopy (A.R. Neurath, N. Strick, L. Baker, S.
2 Krugman, Proc. Nat. Acad. Sci. USA, 79, 4415 (1982)).
3
Example 5
Anti-Peptide Antibody as A Specific Frobe for
Detection of P33 and P36
7 , Anti-pre-S 120-145 was reacted with P33 and P36.
8
HBsAg polypeptides separated by SDS-PAGE run in urea were
9
transferred to nitrocellulose, reacted with anti-pre-S
120-145 diluted 1/80 in TS containing 10 mg/ml of bovine
11
serum albumin and 2.5 mg/ml of gelatine (TS-BG) for five
12
hours at 20°C. To detect bound IgG, the nitrocellulose sheet
13
was washed and exposed to 1251-labeled protein A (0.4 uC/100
14
ml TS-BG) for five hours at 20°C. For further details see
Fig. 1. In Fig. 6, arrows indicate the positions of P33 and
16
P36. The top arrow (corresponding to a molecular weight of
17
66 kilodaltons) indicates another protein reacting with
18
anti-pre-S 120-145, possibly corresponding to a dimer of
19
P33.
21
Eram~le 6
22
23 Development Of A Diagnostic Test For The
24 Detection Of Antigens Coded For By The Pre-S Gene In Sera
Of HBV-Infected Individuals
26 Fig. 7 shows the results of a diagnostic test
27 based on. polystyrene beads coated with anti-pre-S 120-145.
28 Serial dilutions of an HBsAg-positive serum in a
29 mixture of normal human and rabbit serum each diluted 1/10
in TS were tested. 1251-labeled human anti-Figs (Abbott
-73-
~ 3 ~ 4'~ ~~~
1 Laboratories) was used in the test performed essentially as
2 described for the AUSRIA II diagnostic kit (Abbott
3 Laboratories). Results are expressed as RIA ratio units,
4 determined by dividing cpm corresponding to positive samples
by cpm corresponding to positive samples by cpm
6 corresponding to normal serum controls. The endpoint titer
7
corresponds to the highest dilution at which the RIA ratio
8 was 2.1 (broken line). The endpoint titer of the serum as
9
determined by the AUSRIA test was approximately 1/106.
Negative results were obtained with control beads coated
11
with normal rabbit IgG.
12
Similar results were obtained with sera containing
13
HBsAg subtypes ad and ay, indicating that the synthetic
14
peptide with the sequence corresponding to subtype adw (Fig.
16
2) carried common group-specific antigenic determinants.
17
Example 7
18
Synthesizing and Testing
19 - S(135-155) Deriv t es
Each of the conjugates ((1) to (26)) of S(135-155)
21 listed in Table 1, except conjugate 3, was mixed 1:1 with
22 complete Freun.d's adjuvant and injected into two New Zealand
23 White rabbits (65 to 160 ~g of peptide per rabbit). The
24 rabbits were further injected at biweekly intervals with
equal doses of conjugates in incomplete Freund's adjuvant
26 (not used for conjugate 3). Blood specimens were taken two
27 weeks after each injection.
28 To prepare conjugates 1, and 4-8 (Table 1), 1 mg
29
quantities of peptide 309-329 of the env gene product
(S(135-155)) mere activated with a two times molar excess of
-74-
- 1340' ~~
1 N-ethyl-D1'(dim~sthyl-aminopropyl) carbodiimide (EDAC) and
2 N-hydroxy-benzotriazole (NHBTA) and subsequently linked to
3 equimolar quantities of poly-D-lysine and diaminoalkanes
4 (from Fluka AG, Buchs, Switzerland), respectively, as
described (Arnon, R., Sela, M., Parant, M. and Chedid., L.,
6 "Antiviral Response Elicited By A Completely Synthetic
7 Antigen With Built-In Adjuvanticity", Proceedings of The
8 National Academy of Science USA, 77,6769-6772, (19801). To
9
prepare conjug~~tes 2 and 3, 1 mg quantities of each
EDAC-activated S(135-155) and MDP (Calbiochem, San Diego,
11
California) were linked to 10 mg of poly-D-lysine. Peptide
12
309-329 of the env gene product (800pg) was oxidized with
13
ferricyanide (Dreesman et al, 1982 supra), activated with
14
EDAC as
ove ~~nd linked to 4 mg of LPH. Chromatography on
~
Sephadex G-25 :indicated complete linking of the peptide to
16
LPH (conjugate 9). The oxidized, EDAC-activated peptide (1
17
mg) was also conjugated to 1 mg of polyvaline in a
18
suspension of ;Z.S ml of 1 M NaHC03, pH 8.5, and 10 ml of
19
CHC13. The interphase and aqueous phase after centrifugation
was used for immunization (conjugate 10).
21
Lipo;~omes were prepared by the method of Oku, N.
22
Scheerer, J.F.,, and MacDonald, R.C., "Preparation of Giant
23
24 Liposomes", Biochimica et Biophysica Acta, 692, 384-388
(1982). Stear~rlamine, dilauroyl lecithin and cholesterol
26 were dissolved in glucose-saturated ethanol at final
27 concentrations of 10, 23 and 1.43 mg/ml, respectively. For
28 some liposome preparations, the concentration of dilauroyl-
29 lecithin was dE-creased to 17.5~mg/ml and sphingomyelin was
added (10 mg/m:l). Other preparations contained as an
-75-
1 additional component lipid A (420pg/ml; Calbiochem). The
2 solutions were dialyzed against 0.1 M NaCH03, pH 8.5, in
3 dialysis bags with a molecular weight cut-off of 103 daltons
4 for at least sixteen hours. The liposomes were treated for
approximately six hours with glutaraldehyde (final
6 concentration :30 mg/ml), mixed with 0.5 volumes of 33.9%
7 (w/w) sodium d:iatrizoate, floated four times into 1 M NaCH03
8
by centrifugation for ten minutes at 10,000 rpm, and reacted
9
with 0.84 to 1 mg of peptide 309-329 of the env gene product
per 10 mg stea:rylamine overnight at 20°C. The linking of
11
peptide 309-32'9 of the env gene product to liposomes under
12
these conditions was complete. Some preparations were
13
reacted additionally with 7.5 mg of RAT (Biosearch, San
14
Rafael, California) per 10 mg of stearylamine for six hours
at 20°C. The liposomes were floated three times into 0.14 M
16
NaCl, 0.01 Tris-HC1-0.02% NaN3 (TS) and dialyzed against
17 _
TS-10 4 M oxidized glutathione for at least sixteen hours.
18
In some cases (20) and (21) the stearylamine-
19
containing liposomes were not derivatized with GA but
instead directly reacted with EDAC-activated peptide 309-329
21
22 of env gene product. Alternately, (18) and (19), the
23 activated peptide 309-329 of env gene product was linked to
24 glutaraldehyde:-treated liposomes further derivatized by
reaction with 0.2 M ethylene diamine at pH 8.5 overnight at
26 20°C followed by floating two times into 0.1 M NaHC03, pH
2~7 8.5, reduction with lO~aM sodium dithionite for one hour at
28 20°C and repeated floating into the same buffer. An aliquot
29 of these liposomes was additionally reacted with
-76-
3 ~ 0'~
1 EDAC-activated RAT. The liposomes were finally dialyzed
against TS-10 ~~ M oxidized glutathione.
3 In one preparation (22), stearic acid was used
4 instead of stearylamine for the preparation of liposomes.
These were dialyzed against 0.01 M NaCl, activated with EDAC
6 (50 mg/ml for i~wo hours plus additional 25 mg/ml for one
7 hour) at pH 5.5 and 20C, floated two times into 0.01 M NaCl
8 and reacted wil~h the peptide 308-329 of the env gene product
9
in 1 M NaHC03,pH 8.5, overnight.
Polyglutaraldehyde microspheres were prepared as
11
described by Margel, S., Zisblatt, S. and Rembaum, A.
12
"Polyglutaraldcshyde: A New Reagent For Coupling Proteins To
13
Microspheres And For Labeling Cell-Surface Receptors. II.
14
Simplified Labeling Method By Means Of Non-Magnetic And
Magnetic Polyg:Lutaraldehyde Microspheres", Journal of
16
Immunological l~lethod_s, _28, 341-353 (1979), using Polysurf
17
10-36 B (Bartic~ Industries Inc., New Canaan, Conn., Margel &
18
Offarim, (1983)11. One mg of the peptide 309-329 of the env
19
gene product was linked to approximately 50 mg of
microspheres under conditions similar to those described for
21
glutaralde-hyde treated liposomes. Conjugate 25 was prepared
22
by treating the microspheres with 5 ml of 0.1 M ~ -amino
23
caproic acid a~t pH 8.5 overnight. After centrifugation, the
24
microspheres were suspended in dimethylformamide (2m1) and
reacted with 2 mg EDAC plus 670 ug NHBTA for one hour at
26
20C. After centrifugation, the microspheres were
27
2 resuspended in 2 ml of 0.1 M NaHC03, pH 8.5, containing 1 mg
8
29 of peptide 309-329 of the env gene product.
1 All reagents listed above were of analytical grade
2 and obtained from Sigma, St. Louis, Missouri, unless
indicated otherwise.
4 Free: peptide 309-329 of the env gene product (mol.
weight = 2,664 daltons) containing five cysteine residues
6 was in a predominantly monomeric form, since it was eluted
7 after moleculair exclusion chromatography in about the same
8 fractions as insulin A chain. Linking to diaminobutane and
9
to other diami.no-alkanes (data not shown) resulted in
formation of ~~(135-155) polymers which were immunogenic and
11
induced both antipeptide and anti-HBs antibodies.
12
Preparations (;4), (5) and (7) also induced anti-HBs, while
13
polymers with diaminooctane or dodecane linkers (6) and (8)
14
failed to do so (Fig. 8) for reasons not known. Oxidation of
the peptide 309-329 of the env gene product resulted in
16
polymerization (data not shown). The polymer linked to LPIi
17
(conjugate 9) induced high levels of anti-S(135-155) but no
18
anti-HBs, unlike S(135-155) linked to KLH or LPH in its
19
reduced form (Neurath et al., 1982, supra). This finding
again emphasizes the role of peptide conformation in
21
22 inducing antibodies to the native protein. Linking of the
23 oxidized peptide to highly hydrophobic poly-L-valine
24 resulted in a conjugate (10) of low immunogenicity.
S(135-155) linked to poly-D-lysine administered with
26 Freund's adju~aant (1) or having covalently linked ~1DP and
27 given without adjuvant (3) induced both anti-S(135-155) and
28 anti-HBs. The latter conjugate administered with Freund's
29 adjuwant (2) appeared poorly immunogenic. S(135-155) linked
to glutaraldehyde treated liposomes containing stearylamine
-78-
1 (conjugate 11) induced levels of anti-HBs comparable to
2 those elicited by those elicited by conjugates with KLIi or
3 LPH (Neurath et al., 1982, s_upra). Incorporation of
4 sphingomyelin and. or lipid A, components reported to enhance
the antigenicity of haptens inserted into liposomal
6 membranes (Yasuda, T., Dancey, G.F. and Kinsky, S.C.,
7 "Immunogenicit.y Of Liposomal Model rlembranes In Mice:
8 Dependence On Phospholipid Composition", Proceedings Of The
9
National Academy Of Sciences, 74, 1234-1236 (1977)), into
the liposomes (conjugates 13, 15a, 16) failed to enhance
11
anti-HBs, responses.
12
Conjugates (18 and 19) prepared by linking
13 .
S(135-155) to glutaraldehyde-treated liposomes through an
14
ethylenediamine bridge rather than directly, had the
capacity to induce anti-HBs but a considerable variability
16
in response between individual rabbits was observed.
17
S(135-155) before or after oxidation and
18
subsequently linked to stearyl-amine-containing liposomes
19
(not fixed with glutaraldehyde; preparations 20 and 21) or
to stearic acid-containing liposomes (22) induced low levels
21
22
of anti-S-135--155 and no measurable anti-HBs.
23 S(1_~5-155) linked directly to microspheres of
24 polyglutaraldehyde (preparations 23 and 24) induced a
Primary anti-IIBs response. However, the level of anti-HBs
26 decreased in i:he course of immunization. Anti-HBs was un-
27 detectable in sera collected two weeks after the third
28 immunization. S(135-155) linked to these microspheres
29 through ~ -am:Lno-caproic acid (25) and 1-cysteine 126)
-79-
_-__- __LL __. _-____ _ _e _.-
1 bridges, respectively, either failed (25) or was marginally
2 efficient (26) in eliciting anti-HBs.
3 S(13!5-155)-KLH or LPH conjugates elicited a
4 primary anti-H)Bs response but the level of anti-IiBs failed
to increase in sera of rabbits after additional antigen
6 doses (Neurath et al., 1982 s_upra). With the conjugates
7 described above, generally, a decrease of anti-HBs levels
8 was observed four or six weeks after primary immunization
9
(Fig. 9B), but exceptions were observed in a minority of
rabbits (panel 5, Fig. 9A). This declining trend was
11
uniformly reversed when RAT was inserted into liposomal
12
membranes together with S(135-155) (for example Fig. 9C and
13
Fig. 9D).
14
The immunogenicity of haptens inserted into
liposomal membranes depends on the phospholipid composition
16
of the liposomes and seemed to be inversely related to the
17
fluidity of these membranes (Yasuda et al., 1977 supra;
18
Dancey, G.F., Yasuda, T, and Kinsky, S.C. ,"Effect Of
19
Liposomal Model Membrane Composition On Immunogenicity", The
Journal Of Immunology, 120, 1109-1113 (1978)).
21 "-'
Treatment of stearylamine-containing liposomes
22
with glutaraldehyde was found to provide reactive groups
23
24 suitable for linking of synthetic peptides and at the same
time increases the rigidity of the lipid membranes. Such
26 liposomes, especially when containing carrier function
27 enhancing RAT sites (Alkan, S.S., Nitecki, D.E. and Goodman,
28 J~W~. "Antigen. Recognition And the Immune Response: The
29 Capacity of 1-Tryosine-Azobenzenearsonate To Serve As A
Carrier For A Macromolecular Hapten", The Journal Of
-80-
1 Immunology,
107, 353-358,
(1971),
and Alkan,
S.S., Williams,
2 E.B., Nitecki
D"E. and
Goodman,
J.W.. "Antigen
Recognition
3 And the Immune
Response.
Humoral
And Cellular
Immune
4 P,esponses To Sma 11 Mono- And Bifunctional Antigen
Molecules",
The Journal
Of Experimental
Medicine,
135,
6 1228-1246 (1972)), are a
, promising tool for preparing fully
7
synthetic immunogens for eliciting anti-viral antibodies.
8
9
TABLE 1
List of cross-linkers and carriers used
11 for the preparation
of S(135-155)
conjugates
12
13 (1) Poly-D-lysine (mol. weight 3-7 x 104)
14 (2) 1 + N-Acetylmuramyl-L-alanyl-D-isoglutamine (MDP)
(3) - 2
16 (4) 1,4-diaminobutane
1~ (5) 1,6-diaminohexane
18 (6) 1,8-diaminooctane
19 (7) 1,10-d.iaminodecane
(g) 1,12-diaminododecane
21 (g) Oxidized S(135-155) linked to LPH
22 (10) Oxidized S(135-155) linked to poly-L-valine
23 (11) Liposomes containing stearylamine, and treated
24 with glutaraldehyde
(12) - 11 = L-tyrosine-azobenzene-p-arsonate (RAT)
26 (13) - 11 + Sphingomyelin (from bovine brain)
27 (14) - 13 + RAT
28 (15a) - 11 + Lipid A
2 g ( 15 ) - 15 a -+ RAT
-81-
1340'~~~
1 (16) - 13 + Lipid A
2 (17) - 16 + RAT
3 (18) - 11 treated with ethylenediamine
4 (19) - 18 + RAT
(20) - Li~posomes containing stearylamine reacted
6 with oxidized S(135-155) (see 9)
7 (21) - 20 except S(135-155) was oxidized after
attachment to liposomes
8
(22) Stea:ric acid containing liposomes
9
(23) Polyglutaraldehyde micropheres
(24) - 23 + RAT
11
(25) - 23 treated with ~ -aminocaproic acid
12
(26) - 23 treated with L-cysteine
13
14
Example 8
A peptide pre-S (12-32) (subtype adw2) was
16
synthesized according to the procedure described hereinabove
17
in Example 2. The free peptide, the peptide linked to
18
19 glutaraldehyde cross-linked liposomes (~RAT groups)
(according to t:he procedure described above in Example 7) as
21 well as the peptide linked to KLH were used to immunize
22 rabbits. The corresponding antibodies recognized not only
23 the peptide, but also HBsAg and HBV. In view of the above,
24 this peptide is believed quite useful for a vaccine against
hepatitis B virus, and as the basis of useful fIBV
26 diagnostics based on either the peptide itself (to detect
27 anti-HBV response in infected or immunized individuals), or
28 on peptide antibodies to detect hepatitis H antigens.
29
-82-
. . 1340r1~~
1 Example 9
2 A peptide pre-S (117-134) (subtype adw2) was
3 synthesized according to the procedure described hereinabove
4 in Example 2.
6 Example 10
7
A rabbit was immunized with the peptide pre-S
8
(117-134) prepared according to Example 9 and linked to a
9
carrier according to the procedure of Example 7. Such
immunization was conducted according to the procedure
11
described hereinabove in Example 3 and was found to produce
12
antibodies in i:he serum of the rabbit so innoculated.
13
However, the antibody titers were substantially less than
14
those observed for the use of pre-S (120-145) and pre-S
'
(12-32) .
16
17
Example 11
18
The immune response in rabbits to each of two
19
synthetic peptides corresponding to residues 120-145 and
12-32 of the translational product of the pre-S gene of HBV
21
22 DNA (subtype af~w2) was tested. Peptide pre-S (120-145) was
23 prepared according to Example 2 and peptide pre-S (12-32)
24 was prepared according to Example 8. Their sequences are:
MQ~'~NSTAFHQTLQDPRVRGLYLPAGG (pre-S (120-145)) and
26 MGTNLSVPNPLGFFPDHQLDP (pre-S (12-32)). For immunization,
27 the peptides were used in free form, employing alum or
28 Freund's adjuvant, oz- linked to carriers, i.e., keyhole
29 lympet hemocyanin (KLH) and cross-linked liposomes,
respectively. The liposomes were prepared as described in
-83-
1340r155
1 Example 7.
2 The best results were obtained with peptides
3 covalently linked to the surface of liposomes (see Fig.lO).
4 Immunization with KLH conjugates resulted in a high anti-KLH
response (endpoint titers of 1/5,000,000 by radio-
6 immunoassay), apparently causing low booster responses to
the peptides. On the other hand, much lower antibody
8 responses (approximately 1/103) to RAT groups were detected,
9
when RAT-containing liposomes were used as carriers.
Antibodies to liposomes (lacking RAT) were undetectable.
11
This suggests that liposomes are the carrier of choice for
12
immunization with synthetic peptides.
13
14
Example 12
To establish whether or not antigenic determinants
16
corresponding to pre-S gene coded sequences are
17
preferentially present on HBV particles, the reaction of
18
antisera raised against HBV particles with the two synthetic
19
peptides analogues of the pre-S protein was tested. The
maxium dilutio~ns of this antiserum at which antibodies
21
22 reacting with the synthetic peptides were still detectable
23 were: approximately 1/62,500 (1/2 x 106 with tests utilizing
24 1251-labeled protein A instead of labeled second
antibodies), a,nd approximately 1/2,560 for peptides
26 pre-S(120- .145) and pre-S(12-32), respecti~ly (see Fig. 11).
~~"~, 2~ The antiserum (adsorbed on HBsAg-Sepharose to remove
28 antibodies to S-protein) did not react with synthetic
29 peptide analogwes of the S-protein, peptide (309-329) of the
env gene product (S(135-155)), peptide (222-239) of the env
gene product (S(48-65)) and peptide (243-253) of the env
-84-
1~4Q~~
1 gene product (S(69-79)) and was, therefore, specific for
2 pre-S gene coded sequences. In comparison, the dilution
3 endpoints of a:ntisera prepared against the homologous
4 peptides were .approximately 1/300,000 and approximately
1/80,000 for anti-pre-S(120-145) (see Fig. 11) and
6 anti-pre-S(12-32) (data not shown), respectively.
7 The synthetic peptides were recognized also by
8 antibodies (Ig~G and IgM) in sera of individuals who had just
recovered from acute hepatitis B, and by rabbit antibodies
against a fusion protein between chloramphenicol
11
acetyltransferase and a portion of pre-S protein expressed
12
in E, coli (see Fig. 11).
13
On the other hand, humans vaccinated with
14
pepsin-treaded HBsAg (M. R. Hilleman, E.B. Buynak, W.J.
McAleer, A.A. McLean, P.J. Provost, A.A. Tytell, in Viral
16
hepatitis, 1981 International Symposium, W. Szmuness, H.J.
17
Alter, J.E. Maynard, Eds. (Franklin Institute Press,
18
Philadelphia, PA, 1982), pp. 385-397) or with HBsAg produced
19
in yeast (devoid of pre-S gene coded sequences; W.J.
McAleer, E.B. Buynak, R.F. Maigetter, D.E. Wambler, W.J.
21
Millur, M.R. Hilleman, Nature (London), 307, 178 (1984)) did
22
not develop detectable antibodies recognizing either of the
23
24 two synthetic peptides. On the other hand, 7 out of 12
individuals wh,o received a vaccine consisting of intact
26 HBsAg developed these antibodies.
27
28 Example 13
2g ~ua~otitativa aspects of the immunological cross-
reactivity between pre-S gene coded sequences exposed on HBV
-85-
1340r~~~
1 particles (or on HBsAg) and the synthetic peptide analogues
2 were tested. 'The peptides were conjugated to
3 ~-galactosidase, and the inhibitory effect of free peptides,
4 HBV and HBsAg, respectively, on the formation of immune
complexes containing the enzyme-conjugated peptide was
6 studied. Results shown in Fig. 12 indicate that HBV, at
7 sufficient concentrations, inhibited completely the reaction
8 between anti-pre-S(120-1451 and pre-S(120-145)-
-galactosidase. HBsAg had < 1/5 of the inhibitory activity
corresponding to HBV. The inhibitory activity of
11
pepsin-treated HBsAg was ~ 1/1,000 of the activity
12
corresponding to intact HBsAg. These results indicate the
13
absence in the anti-pre-S(120-145) serum of a subpopulation
14
of antibodies which recognize the synthetic peptide but not
the native protein. Such antibody subpopulations are
16
observed in many other antisera raised against synthetic
17
peptide analogues of viral proteins. The concentration of
18
free peptide sufficient for approximately 50$ inhibition of
19
the reaction of pre-S(120-145)-~-galactosidase with
anti-pre-S(120-145) is approximately 1/100 of that for HBV
21
on a weight basis (see Fig. 11). However, since the
22
molecular weight of pre-S(120-145) is approximately 3 kD and
23
24 the molecular weight of HBV protein components reacting with
anti-pre-S(120-145) (representing a minor (G 20~) portion of
26 the total HBV mass) is between approximately 33 and
27 approximately 67 kD, the molar concentrations of F~BV and
28 pre-S(120-145) required for this degree of inhibition are
29 approximately the same. This indicates that the antigenic
determinants on the peptide analogue and on the
-86-
1340'~5~
1 corresponding segment of the HBV envelope proteins) are
2 structurally closely related.
3
4 Exam le 14
A peptide pre-S (94-117) (subtype adw2) was
6 synthesized according to the procedure described hereinabove
7 in Example 2.
8
9
Example 15
A rabbit was immunized with the peptide pre-S
11
(94-117) prepared according to Example 14 and linked to a
12
carrier according to the procedure of Example 7. Such
13
immunization was conducted according to the procedure
14
described hereinabove for Example 3 and was found to produce
antibodies in the serum of the rabbit so inoculated.
16
However, the antibody titers were substantially less than
17
those observed. for the use of pre-S (120-145) and pre-S
18
(12-32).
19
Example 16
21
A peptide pre-S (153-171) (subtype adw2) was
22
synthesized according to the procedure described hereinabove
23
in Example 2.
24
26 Exam le 17
A rabbit was immunized with the peptide pre-S
27
28 (153-171) prepared according to Example 16 and linked to a
29 carrier according to the procedure of Example 7. Such
immunization was conducted according to the procedure
-87-
- ~.~~~'~~5
1 described hereinabove for Example 3 and was found to produce
2 antibodies in the serum of the rabbit so innoculated.
3 However, the antibody titers were substantially less than
4 those observed four the use of pre-S (120-145) and pre-S
(12-32) .
6
7 Example 18
8 A peptide pre-S (1-21) (subtype adw2) was
9
synthesized according to the procedure described hereinabove
in Example 2.
11
12
Example 19
13
A rabbit was immunized with the peptide pre-S
14
(1-21) prepared according to Example 18 and linked to a
carrier according to the procedure of Example 7. Such
16
immunization was conducted according to the procedure
17
described hereina.bove for Example 3 and was found to produce
18
antibodies in they serum of the rabbit so innoculated.
19
However, the antibody titers were substantially less than
those observed for the use of pre-S (120-145) and pre-S
21
( 12-3 2 ) .
22
23
24 Example 20
A peptide pre-S (32-53) (subtype adw2) was
26 synthesized according to the procedure described hereinabove
27 in Example 2.
28
29
-88-
G
1340r~ ~~
1 Example 21
2 A rabbit was immunized with the peptide pre-S
3 (32-53) prepared according to Example 20 and linked to a
4 carrier according to the procedure of Example 7. Such
immunization was conducted according to the procedure
6 described hereinabove for Example 3 and was found to produce
7 antibodies in th.e serum of the rabbit so innoculated.
8 However, the antibody titers were substantially less than
9
those observed f:or the use of pre-S (120-145) and pre-S
( 12-3 2 ) .
11
12
Example 22
13
A peptide pre-S (57-73) (subtype adw2) was
14
synthesized according to the procedure described hereinabove
in Example 2.
16
17
Example 23
18
A rabbit was immunized with the peptide pre-S
19
157-73) prepared according to Example 22 and linked to a
carrier according to the procedure of Example 7. Such
21
immunization was conducted according to the procedure
22
described herei:nabove for Example 3 and was found to produce
23
antibodies in the serum of the rabbit so innoculated.
24
~iowever, the antibody titers were substantially less than
26 those observed for the use of pre-S (120-145) and pre-S
27 (12-32).
28
29
_gg_
134~r1~'~
1 Example 24
2
DetE:ction of anti-pre-S protein antibodies in
3 human sera using synthetic peptides.
4 As discussed above, antibodies recognizing
synthetic peptide analogues of the pre-S protein were
6 detected in sera of humans during recovery from hepatitis B
7 (Fig. 11). Tree time course of development of antibodies
recognizing pre-S(120-145) in a selected patient is shown in
Fig. 13.
Anti-pre-S protein antibodies are detected in
11
human sera early during acute hepatitis type B. IgM
12
antibodies recognizing the peptides were detected during
13
HBsAg antigenE;mia before antibodies to the S-protein
14
(anti-HBs) or to hepatitis B core antigen (anti-HBc) were
detectable. After development of the latter two antibodies,
16
the level of antibodies with anti-pre-S specificity
17
declined. Variations of this pattern of anti-pre-S
18
development among patients with hepatitis B were observed.
19
In some cases, antibodies recognizing the synthetic peptides
were present even before HBsAg was detected in plasma, or
21
when HBsAg never appeared in blood and the only marker for
22
hepatitis H was anti-HBc and later anti-HBs.
23
24 Antibodies to pre-S(120-145) were measured by RIA.
Similar results were obtained by assaying antibodies to
26 Pre-S(12-32). HBsAg, anti-HBs and antibodies to hepatitis B
27 core antigen (anti-IiBc) were assayed using commercial test
28 kits (Abbot Laboratories, North Chicago, Illinois). The
29 broken line at the end of bars corresponding to the
different markers of HBV infection indicates positivity at
-90-
~.~ you ~~
1 the termination of surveilance. Antibody titers represent
2 the highest dilution of serum at which radioactivity counts
corresponding to the specimens divided by counts
4 corresponding to equally diluted control serum were > 2.1.
Humans vaccinated with pepsin-treated HBsAg
6 (Hilleman, M.P:., Buynak, E.B., McAleer, W.J., McLean, A.A.,
7 Provost, P.J. & Tytell, A.A. in Viral Hepatitis, 1981
International Sympsosium (eds. Szmuness, W., Alter, H.J. &
9
Maynard, J.E.) 385-397 (Franklin Institute Press,
philadelphia, PA, 1982)1, (pepsin treatment removes all
11
anti-pre-S (120-145) reactive material) , or with IiBsAg
12
produced in yeast (devoid of pre-S gene coded sequences
13
(McAleer, W.J. Buynak, E.B. Maigetter, R.Z., Wambler, D.E.,
14
Miller, W.J., Hillemann, M.R. Nature, (London), 307, 178-180
(1984); did not develop detectable antibodies recognizing
16
either of the two synthetic peptides. On the other hand, 7
17
out of 12 individuals who received a vaccine consisting of
18
intact HHsAg (McAul:iffe, V.J., Purcell, R.H., Gerin, J.L. &
19
Tyeryar, F.J. in Viral Hepatitis (eds Szmuness, W., Alter,
H.J. & Maynarcl, J.E.) 425-435, Franklin Institute Press,
21
22 Philadelphia, PA) developed these antibodies. These 7
individuals also had the highest antibodiy response to the
23
24 S-Protein, as measured by the AUSAB test (Abbott),
suggesting that a lack of detectable response to the pre-S
26 Protein was due to the sensitivity limits of the test. In
27 this respect, it is of importance that the hepatitis B
28 vaccine heretofore used, the production of which involves
29 pepsin treatment of HBsAg, although highly efficient in
aPParently healthy :individuals, has had low immunogencity
-91-
1 and no protective effect in hemodialysis patients (Stevens,
2 C.E., Alter, H'.J., Taylor, P.E., Zang, E.A., Harley, E.J. &
3 Szmuness, W., N. Engl. J. Med., 311, 496-501 (1984)). Other
4 vaccines produ~~ced without pepsin treatment do not seem to
have this defect (Desmyter, J. in Viral Hepatitis and Liver
6 Disease (eds G'yas, G.N., Dienstag, J.L. & Hoofnagle, J.), in
7 press Grune arid Stratton, Orlando, Fl. 1984).
8
Example 25
RIA Tests of Preparations Containing IIBV-specific
11
proteins
12
Antibodies to the S-protein were removed from
13
rabbit anti-serum against HBV particles by affinity
14
chromatography (Neurath, A.R., Trepo, C., Chen, M., Prince,
A.M., J. Gen. Virol., _30, 277-285 (1976) - See Fig. 14. The
16
tested antigens were: HBV particles and tubular forms (m,
17
e); approximately 20 nm spherical particles of HBsAg isolated
18
from plasma (o,~ ); and the latter particles treated with
19
pepsin (1 mg/ml HBsAg, 50Jug/ml pepsin in 0.1 M glycine-HC1,
pH 2.2, 2 hours at 37°C) (O). The RIA tests were performed
21
as described in Neurath, A.R., Kent, S.B.H., Strick, N.,
22
Science, 224, 392-395 (1984). The concentration of HBsAg
23
24 S-Protein was adjusted to the same level in all preparations
tested as based on RIA tests (AUSRIA, Abbot Laboratories).
26 HBV Particles (contaminated with tubular forms of HBsAg)
27 were concentrated from serum approximately 100x by
28 centrifugation for 4 hours at 25,000 rpm in a Spinco 35
29 rotov The concentrate.(2 ml) was layered over a
discontinuous gradient consisting of. 11 ml of each 20, 10
-92-
1 and 5o sucrose (w/w) in 0.14 M NaCl-0.01 M Tris-0.02$ NaN3,
2 pH 7.2 (TS) anal centrifuged for 16 hours at 25,000 rpm in a
Spinco rotor SW 27. The final pellet was resuspended in TS.
4 HBV particles were recognized much more
efficiently than purified approximately 22 nm spherical
6 particles in P:IA tests based on polystyrene beads coated
7 with either anti-pre-S(120-145) or with rabbit antibodies to
8
HBV particles. Treatment of HBsAg with pepsin, a step used
9
in preparing some current hepatitis B vaccines, resulted in
an approximately 103-fold decrease in reactivity with
11
anti-pre-S(120-145)» HBsAg from vaccines derived either from
12
infected plasma (Hilleman, M.R., et al, 1982) supra), or
13
produced in yeast McAleer et al (1984), supra), had ~
14
1/5,000 of the: reactivity of intact HBsAg in these tests.
In reverse tests, beads coated with HBsAg, with
16
HBV particles, with pepsin-treated HBsAg, or with HBsAg
17
corresponding to the vaccines mentioned above were utilized.
18
IgG antibodies. (from different rabbit antisera to pre-S
19
sequences) rea~,cting with the beads were assayed based on the
subsequent attachment of labeled anti-rabbit IgG. Positive
21
results using anti-pre-S(120-145) were obtained only with
22
beads coated with intact HBsAg or with HBV particles.
23
24 Anti-pre-S(12-~32) reacted exclusively with HBV-coated beads.
26 Example 26
27 Involvement of pre-S Gene Coded HBV Domains In
28 Attachment to Cell Receptors
. 29 It has been suggested that the 55 C-terminal amino
acids of the pre-S protein mediate the attachment of HBsAg
to human albumin polymerized by glutaraldehyde (pHSA) and
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1 that this attachment plays an essential role in the in vivo
2 adsorption of HBV to hepatocytes (Machida, A. et al,
3 Gastroenterolo~y, 86, 910-918, (1984); Machida, A. et al,
4 Gastroenterolo~, 85, 268-274, (1983). However, there is no
compelling evidence to support the role of the pIiSA-HBV
6 interaction in infection of liver cells by HBV. In
7
addition, both HBsAg containing or lacking these 55 amino
8 acid residues react with pHSA (Fig. 15), albeit the reaction
is enhanced by the presence of the pre-S gene coded
sequences. The RIA tests involved in Fig. 15 were conducted
11
12
13
14
as described in Neurath, A.R., Strick, N. Intervirology, 11,
128-132 (1979) .
To explore directly the reaction of HBsAg with
liver cells, an assay system based on the attachment of
liver cells to insolubilized HBsAg was developed.
16
HBsAg (HBV) was attached to
17
N-N'-p-phenylenedimaleimide-derivatized sulfhydryl cellulose
18
under conditions described for linking of pre-S(120-145), as
19
described above. About 4 mg of HBsAg was linked to 1 g of
the cellulose derivative. A control cellulose derivative
21
22 was prepared by linking bovine serum albumin to the
23 activated matrix. Forty mg of the cellulose derivative
24 suspended in TS containing 10 mg/ml of bovine serum albumin
(TS-BSA) were :mixed with approximately 2 x 106 washed Hep G2
26 human hepatoma cells (see Aden, D.P., Fogel, A., Plotkin,
27 S., Damjanov, .J., Knowles, B.B., Nature (London), 282,
28 615-617 (1979) suspended in TS-BSA and incubated for 30 min
29 at 37°C, followed by 1 hour at 4°C. HeLa cells and Clone 9
normal rat liver cells (American Type Culture Collection)
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1340"t~~
1 were used as controls. The cell-cellulose fixtures were
2 layered on top of 1 ml of 33$ (w/w) Hypaqu~~and centrifu ed
9
~'- 3
for 2 minutes at 3,000 rpm. The cellulose derivative with
4 attached cells pelleted under these conditions. Unattached
cells recovered from the Hypaque-TS-BSA interphase were
6 diluted 5-fold. in TS-BSA and pelleted by centrifugation.
The relative proportion of adsorbed and unadsorbed cells was
8 determined by measurement of lactate dehydrogenase (LDH)
9
activity in ap~proprxate aliquots of cell lysates obtained
after exposure. to the detergent Triton X-100 (5 mg/ml in
11
H20). LDH activity was determined using diagnostic kit No.
12
500 (Sigma).
13
Approximately 80 to 95~ of human hepatoma Hep G2
14
cells (Aden, D~.P. s-upra) attached to immobilized HBsAg in
this assay. T'he attachment of control cells (HeLa, rat
16
17
18
hepatocytes) was in the range of 10 to 20$. About 10$ of
Hep G2 cells attached to control cellulose. In the presence
of anti-pre-S(120-145) and anti-pre-S(12-32) IgG (15 mg/ml),
19
the adsorption of Hep G2 cells to HBsAg-cellulose decreased
to 60 and 30~, respectively. A mixture of both antibodies
21
22 (7~5 mg/ml of IgG each) caused a decrease of cell adsorption
23 to 20~, indist:inguishable from background levels.
24 Normal rabbit IgG, as well as antibodies to the
S-protein (eli.cited by immunization with pepsin-treated
26 HBsAg), failed to diminish the cell attachment, despite high
27 levels of anti.-HBs present in this serum (positive at a 10 6
28 dilution in the AUSAB test).
29 It will be appreciated that the instant
specification and c:Laims are set forth by way of
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1340755
1 illustration and not limitation and that various
2 modifications and changes may be made without departing from
3 the spirit and scope of the present invention.
4
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14
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22
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