Note: Descriptions are shown in the official language in which they were submitted.
W092/21377
PCT/SE92/003/3
10~61
Peptides for Use in Induction of T Cell
Activation A~ainst HIV-1
This application is a continuati~n-in-part of
pending U.S. patent application Serial No. 07/571,080,
~iled August 22, 1~0.
Back~round_ _f the I~vention
¦ ~ AIDS and AIDS-related disorders ~ARC) are caused
by a retrovirus, the human immunodeficiency virus
. (HIV). Barre-Sinoussi et al., "Isolation of a
~ T-Lymphotropic Retrovirus from a Pati~nt at Risk for
i 5 Acquired Immune Deficiency Syndrome (~IDS), Science,
¦ 220:868 (1983); and Gallo et al., ~'Frequent Detection
¦ and Isolation of Cytopathic Retroviruses (HTL~-III)
From Patients ~ith AIDS and at Risk for AIDS", Science,
224:500 (1984)- O
Li~e most ~iruses, HIV often elicits the produc-
tion of ~eutralizing antibodies. Unlike many other
viruses and other infectious agents for which infection
l~ads to protective immunity, however, HIV sp~ific
antibodies are insu~ficient to halt the progression of
15 the disease. Therefore, in the case o~ HIV, ia vaccine
that elicits the immunity of natural in~ection could
prove to be ineffective. In fact, vaccines prepared
from the HIV protein gpl60 appear to provide little
i ~ unity to HIV infection although they eli~it neu-
2Q tralizing antibodies. The failure to produce an
~ ;: effective anti-HIV vaccine has led to the prediction
¦~ that an e~fective~ vaccine~will not be availablé until
the end of the l990~s. ~
The HIV genome has been well charact~rized. Its
3 25 approximately lOKb enc~des:sequences that contain
,` ~
.
i ~ :
WO92/21377 PCT/SE92/003/~ '
2~ o996~ -2- (
regulatory segments for HIV replication as well as the
qaq, pol and env genes coding for the core proteins,
, the reverse transcriptase-protease-endonucle~e, and
j the internal and ex~ernal envelope glycoproteins
respectively.
The HIV env gene encodes the in~racellular glyco-
I protein, gplS0, which is normally processed by prot,~,o-
I lytic cleavage to form gpl20, the external viral gly-
I coprotein, and gp41, the viral transmembrane glyco-
¦ 10 protein. The gp120 protein remains associated with HIV
I virions by virtue of noncovalent interactions with
! gp41. These noncovalent interactions are weak, conse-
¦ quently most of the gpl20 is released from cells and~
virions in a soluble form.
Previous studies have shown that the proteins
encoded by the g~g and especially the env regions of
the HIV-l genome are immunogenic since antibodies to
the products of the aq and env genes are found in the
sera of HIV infected, AIDS and ARC patients.
It has previously been shown t~ some antibodies
obtained from sera of ~IDS and ARC patients, as well a~
asymptomatic individuals infected with the virus are
speci~ic to gp120 and gpl60. Occasionally these anti~
bodi~s are neutralizingl The envelope glycoproteins
. 25 axe the HIV-1 antiqen most consisten~ly recognized ~y
antibodies in ~IDS and ARC patient sera. Allan et al.,
1 "Major Glycoprotein Antigens that Induce Antibodies in
Z! , j AIDIS Patients are~Encoded by H~L~ ', Science, I ...
. 228:1~91-1094 ~1985); and Barin et al., "Virus ~nvelope
. 30 Pro~ein of HTLV-III Represents Major Target Antigen for
I Antibodies in AIDS Patient~", Science, 228:1094-1096
¦ (Ig85). In addition/ antibodies in patient sera also
~:: recogni2e epitopes of the.viral core proteins encoded
by the ~ gene.
: ImmunQlogically important HIV-1 antigens for use
: in diagnosis and as potential vaccine compositions have
~- W~9~/21377 PCT/SE92/00373
.
1~ _3_
2109961
been prepared by cloning portions o the HIV-l genome
in various expression systems such as bacteria, y~a5t
~r ~accinia. Cabradilla et al., "Serodiagnosis of
~ntibodies to the Human AIDS Retrovirus With a
Bacterially Synthesized env Polypeptide", Bio-
technology, 4:128-133 (1986); and Chang et al.,
"Detection of Antibodies to Human T~Cell Lymphotropic
Virus-III (~TLV-III) With an I~munoassay Employing a
Recombinant Escherichia coli - Derived Viral Antigenic
Peptide", Biotechnology, 3:905-909 (1985). HTV-1
antigens produced by reaombinant DNA methods, however,
must still be exhaustively puri~ied to avoid adverse
reactions upon vaccination and falsa positive reactions
in ELISA assays due to any antibody reaativity to
antigen~ of the expression system whiah may contaminate
the ~IV-l antigen preparation. Also~ denaturation of
HIV-1 antigens during purification may destroy impor-
tant antigen activity. Preparation of proteins from
intact viruses ~an also result in contamination by the
virus. o
S~veral publications have presented data showing
immunologic reactivity of sel~cted synthetic peptides
corr~sponding to portions of the antigenic pro~eins of
HIV-lc In one study, ~ peptide having the amino acid
sequence Tyr-A~p-~rg-Pro-Glu-Gly-Ile-Glu-Glu~Glu-
Gly-Gly-Glu-Arg-Asp-Ar~-Asp-Arg-Ser-Gly~Cy~ whi~h
corresponds to amino acid residu~s 735-752 of HIV-1 was
synthesized. Kennedy et al., "Antiserum to a Synthetic
Peptide ~ecognizas the HTLV-III En~elope Glycoprotein",
Science, 2~1:1556-1559 (1956). This peptide, derived
~rom a portion of gp41, was u~ed to immunize rabbits in
an attempt to elicit a neutralizing antibody response
to HIV-l. Furthermore, s~veral sera from AID~ patients
known ~o contain anti-gp41 antibodies were weakly
r~active wi~h thi~ peptide, thus indicating that this
peptide contains at least one epitope recognized, to
:
. ~ :
W092~1377 PCT/SE92/00373
~, i os ~
. some extent, by antibodies to native gpl60/gp41.
I However, this peptide has not been shown to elicit
¦ neutralizing antibodies in mammals other than rabbits
nor has it been suggested for use as a human vaccine~
Longitudinal studies conducted on cohorts of
HIV-infected individuals have indicated that a stable
clinical condition is associated with presence of high
¦ titers of neutralizing an~ibodies agains~ the envelope
glycoprotein gpl20 of HIV and especially against a
specific segment of ei~ht amino acids. Ranki et al.,
"Neutralizing Antibodies in HIV (HTLV-III) Infection:
Correlatlion with Clinical Outcome and Antibody Response
i Against Di~ferent Viral Proteins", Clin. Exp. Immunol.,
69:231 (1987); and Narx (1989).
I 15 Achieving protective immunity against HIV is
¦ likely to lie on the induc~ion of gp120 specific
neutrali~ing antibodies. Marx, "New Hope on the AIDS
Vaccine Front", Science, 244:1254 (1989). Potent T
cell help might also be critical to promote the gen-
eration and the expansion of virus-s~ecific cytotoxic
T cells. Reinherz and Schlossman, "Th~ Characteriza-
tion and Function of Human Immunoregulatory T Lympho-
cyte Subset~", Immunol~ Today, 2:69 (1981~; Burns
et al, I'Thymus Dependence of Viral Antig~ns", Nature,
..25~:654 (1975); and Askonas et al., "Cytotoxic T-memor~
Cells in Virus Infection and the Specificity of Helper
T Cells"J Immu~ology, 45:79 (1982). To be durable and
broad, protective immunity should rely on induction of
, immunologic memory to structurally co~ser~ed antigenic
¦ 30 moieties comprising epitopes displaying limited MHC
l~ restriction for T helpe~ cell recognition. Askonas
!~ et al. ~1982).
¦ Since production of ~ntibodies, including neu-
tral i2ing antibodies, by B cells is critically depen-
~, 35 dent on cognate T cell help, and antigenic determinants
recognized by T cells are oft~n distinct from the ones
W092~21377 PCTtSE92/00373
. ~5~ 2 lQ~ 9 ~ 1
recognized by B cells, identification of antigenic
moieties recognized by T cells (so-called "T cell
epitopes"), is important when considering vaccination
strategies based on appropriate combinations of T and B
cell epitopes.
It would therefore be useful in the treatment and
prevention of AIDS and ARC to have an HIV vaccine cap-
able of producinq neutralizing antibodies and con-
comitantly eliciting T cell help.
Most antigenic d~terminants recognized by
T cells are composed of continuous stretches of
peptides. Streitcher et al., "Antigen Conformation
Determines Processing Requirements for T-cell
Activation", Proc. Natl. Acad. Sci. U.S.A., 79:472~
(1982); DeLisi and Berzofsky, "T Cell Antigen Sites
Tend to be Amphipathic Structures'i, Proc. Natl. Acad.
Sci. U.S.A., 82:7048 (1985); and Margalit et al.,
"Prediction of Immunodominant Helper T Cell ~ntigenic
Sites From Primary Sequence", J. I~munol., 138:2213
(1987). B and T cell recognition si~es are often
located in diff~rent regions of a complex antigen.
Milich et al., "Nonoverlapping T and B Cell ~eter-
minants on an Hepatitis B Antigen pre-S ( 2 ) Region
Synthetic Peptide" ~ J. Exp. Med., ~6.4 : 532 (1986
Within ~he functional T cell repertoire, T helper~
c~115, T cytotoxic cells, and T suppressor cells,
appear to racognize structurally distinct determinants.
Kræych et al-, "Induction of Helper and Suppr~ssor
T Cells by Nonoverlapping Determinants on the Large
Protein Antigen, ~-galactosidase", FASEB J., 2:141
(1988). This functional separation may have important
bearing on the development o~ vaccines, since
.
particular determinants recognized by ~ suppressor
cells may b~ ~blated:resulting in important ~enefits
for immunogenicity.
WO92/21377 PCT/SE92/0037
~ 6- f
2l09961
AIDS and ARC are associated with progressive
imp~irment o~ CD4~ T cells, and increased suscep-
tibility to opportunistic infectionsO In this respect,
HIV-infected persons show decreased T helper cell
activity for polyclonal B cell differentiation and
decreased T cell proliferative responses to antigens
and mitogens associated with an early loss of CD29~
memory T cells. Terpstra et al.l "Longitudinal Study
of Leukocyte Functions in Homosexual Men Seroconverted
for HIV: Rapid and Persistent Loss of B Cell Function
After HIV Infection", Eur. ~. Immun~l., 19:667 (1989);
Fahey et al., I'Quantitative Changes in T Helper or
Suppressor/Cytotoxic Lymphocyte Subsets that Distin-
guish Acquired Immune Deficiency Syndrome From Other
Immune Subsets Disorders", JANA, 76:95 tl984); Shearer
et al., "Functional T Lymphocyte Immune Deficiency in a
Population of Homosexual Men Who do not Exhibit
Symptoms of Acquired Immune Deficiency Syndrome", J.
Clin. Inve t., 7~:496-506 ~1984); Giorgi et al~, "Early
Effects of HIV on CD4 Lymphocytes ~ vivo", J.
Immunol., 138:3725 (1987); and van Noesels et al. t
"Functional and Ph~notypic EYidence for a Selective
Los~ of Memory T Cells in Asymptomatic H~man Immuno-
de~icienc~ Viru -in~ected Men", 86:293 (1990~. .
,~ 25 The use o~ synthetic peptides as artificial T ce~l
recognition sites in the composition o~ candidate
subunit vaccines, offers attractive prospects. In this
regard, the pQssibility to educate T,helper cell~ with
~ynth~tic peptides for the developmant of subsequent
antibody responses against overlapping and non-
:~ overlapping B cell (antibody) recognition sites has
: been documented in several experimen~al systems.
Streitcher et al. (1982); DeLisi and Berzovsky (1985);
and Milich et al~, "A Single 10-residue PreS(1) Peptide
Can Prime T Cell Help for Antibody Production to
~; Multiple Epitopes Within the pre-S(1), pre-S~2), and S
,
W~'9V~1377 PCT/SE92/00373
~ _7_ . .
21099~1
regions of HBsAg", J. Immunol., 138:4457 (1987). It
has now been found that peptid25 derive~ from two
regions o~ the HIV genome elicit T cell activation.
These peptides are also capable of inducing the
production of neutra1izing antibodies to HIV-1.
Summa~y o~ the Invention
In accordanc~ with the present invention, novel
peptides corresponding to epitopes of HIV-1 gpl20
protein and analogues and homologs thereof are
provided. These peptides can b~ uti}ized alo~e or in
combination, uncoupled or coupled to other molecules or
, ~ubstrates. The peptide~ are useful in eliciting T
cell activation, immunization against HIV infection,
induction of a heightened i~mune response to HIV and in
production of polyclonal and monoclonal antibodies.
Forty synthetic peptides corre~ponding to the
entire primary seguence of ~he envelope gp120 of the
human immunodeficiency virus type I (HIV-1) ware
examined for their ability to induceOantibody formation
and/or T cell activation anti~ody formation was
determined by m~a~uring the amount of peptide-specific
antibody formed. T cell activation was measured by the
ability of the peptides to induce in vitro
prolifarative responses and/or IL-2 production when
~ 25 add~d to cultures o~ unfractionated, T cell enriched,
¦ and/or ~D4+ T cell enriched peripheral blood
mononuclear cells (PBMC) from immune~ monk~ys. Among ~ ~ .
~our major areas of T cell recognition identified~ two
I novel T cell activating regions were identified, both
;~ 30 of which ~ere also found to be capable of inducing, 1n
` vivo, the production of neutralizing antibodies to
HIV-1. one of these two nov8l areas corresponds to a
high1y conser~ed region of HIV-1 gp-120, the other area
being located to~a va~iable region of gp-120.
~: 35 Recognition of the latter variable region does not
. ~ .
~i
,
WO9~/2t377 PCTtSE92/00373
21~96`1j -8- ~
appear to be restricted by MHC polymorphism, since all
of six monkeys immunized with corresponding peptides
were found to display in vitro proliferative responses
to thesei peptides. The results of peptides thus have
great utility for the deivelopment of synthetic subunit
AIDS vaccines.
Brie~ Desc~iption_of the ~rawinqs
Fig. 1 is a graph depicting ln vitro prolifeirative
responses of monkey peripheral blood mononuclear cells
(PBMC) to recall peptide after two and/or three peptide
immunizations.
Fig. 2 is a graph depicting in vi~ro proli~erative
responses of monkey PB~C to half-overlapp~ng peptides.
Detailed DescriP~ion o~ the Inventi~n
~ vaccine against AIDS, if an efficient one is to
be found, is likely to contain components that are
capable of inducing T helper cell activity to cognate B
ce}ls committed to the prcduction ofHIV neutralizing
antibodies.
The present inve~tion provides peptides, some of
which have previousl~ been found to elicit production
of HIV neutralizing antibodies by primate subjects and
all o~ ~hich ha~ now.been found to have the surprisinq
property o~ eliciting T cell activation. The peptides
correspond to regions of the gpl20 protein with amino
acid aoordinates as defined by Kennedy et al.,
"Antiserum to a Synthetic Peptide Recognizes the HTLV-
III Envelope ~lycoprotei.n", Science, 231:1556-1559
(1986). The peptides of the present invention are
tarmed gpl20-11 (amino acid coordinates 141-164~,
gp120-12 (amino acid coordinates 151-176), gpl20-13
(amino acid coordinates 164-192), gpl20-16 (amino acid
coordinates 205-230) and gpl20-19 (amino acid coor-
dinates 247-269), ypl20-29 (amino acid coordinates 366-
~ 4~ 92~21377 PC~rtSE92/00373
-9- s~ g 6 1
~ .
389) and gpl~0-30 (amino acid coordinates 377-400).
The peptides of ~he present invention have been des-
cribed for use as immunogens in vaccine compositions
,l and to elicit polyclonal or monoclonal antibody pro-
ductions in United States patent application Serial
Number 07/589,422 filed Sept. 27, 1990 which is
incorporated herein by reference.
Four topographically ralated groups of peptides
derived ~rom gp120 ha~e now been identified which
display T cell activating properties. Two of the gp120
regions found to-elicit T cell:actîvation are similar
to previo~sly identified T celI epitopes. Bolognesi,
"HIV Antibodies and AIDS:Design", AIDS 3:Slll-S118
(1989). The present study indicates that recognition
of ~he area defined by amino acid coordinates 295-343
by immune T cells may be submitted to strong MHC-
restriction as one of the 2 monkeys injected with
peptide ~p120-25 failed to respond to that particular
peptide n yit~o. The T cell antigenic determinants in
this area seem to be~more or less e~clusively located
within each of the immunizing peptides as none of the
overlapping peptides gave rise to i~ vitro prolifera-
t~ive responses.~ Nowever, PRMC isolated from monkeys
i~unixed with peptide gpl20-24 secreted IL-2 when
cultured in the presence~of peptide gpl20-25, indica-
.
:~ tlng the existen~e~:of a~minor ep:itope shared by these
, two peptides.
The~region~ofigpl2Q corresponding ~o amino acid ~ ~ .coordinates 295 ~to 343 (peptides gp120-23, gp120-24 and
gpl20-25),:is~similar to~a region ~(amino acid coordi-
nates 301 to 338) which has previously~een shown by
other investigators~to contain~a major T cell recogni-
tion ~site ~amino acid coordinates ~303-337) whose
se~uence encompasses that of the neutraIizing loop.
35~ Bolognesi (I989); Javaherian :et al., "Principal
Neutralizing Domain :of the: Humi3n Immunodeficiency Virus
W O 9~t21377 PCT/SE92/00373
21~ Sl lo - `
Type 1 Envelope Protein", Proc. Natl. Acad. Sci. USA,
86:676B (1989); and Rusohe et al., "Antibodies That Inhibit
Fusion of Human Immunodeficiency Virus infec~d Cells Bind
a 24-amino Acid Sequence of the Viral Envelope, gpl20",
Proc. Natl. Acad. Sci. USA, 85:3198 (1988).
The area of gp-120 located between amino acid coordi-
nates 409 and 466 ~peptides gpl20-33, gpl20-34, gpl20-35 and
gpl20-36), as described ~n the Examples presented below, was
ound to have p~tent T cell activating properties. The area
between amino acid coordinate. 409 och 466 has previoucly
- been shown to accommodate T cell activa~ing domainsO Bolog-
nesi (1989). In this area, two T cell epitopes have been
identlfied, one between amino acid coordinates 410 and 429,
and one betwaen am~no acid coordinates 428 and 443. The
latter area largely overlaps with ~he CD4-bind~ng site
(amino ac~d aoordinates 420-463) of gpl20, the ma~n site of
virus attachment on permissive T cells.
Several ~ther epitopas with T cell activating pro-
pertles have now been identified inadiscrete areas o the
S~pl20 molecule. Thus, peptide gpl20-4 ( amino acid cc~ordina-
tes 53-74 ), gpl20-5 ( amino acid coordinates 64-89 ), gpl20-8
( amino acid coordinates 100-126 ), gp 12C)-7 ( am~ no acid
coordinates 218-247) and peptide gpl20-21 (amino acid coor-
dinates 269-295 ), and at least on of their overlapping
peptides, were shown to be capable of inducing ~ cell re-
sponses in vitro. However, both of the monlceys immunized
with peptide gpl20-21, when retested five months after the
lastlboos~er dose, hade lost their in vi~ro T cell responsi
veness to recall peptlde. Thus, not every peptide capable of
eliciting
.
Sg~BSl~ TE~ S~EE~li'
~ I WVr92/21377 . PCl /SE92/00373
-11- 2109g61
T cell activation is suitable for use in treatment and
prevention of AIDS.
Surprisingly,:peptide gpl20 19, has now been shown
to have T cell immunogenic properties as defined ~y in
vitro proliferative responses of simian PBMC to cognate
peptide. Additionally, PBMC from a monkey immunized
with OVA-conjugated peptide:gpl20-16 has now been fo~nd
to secrete I~2 a~ter n vitr~o ~expOi6ure to peptide
gp120-16. Peptide gpl20-16 thexefore represents an
additional novel T cell epitope.
Two novel areas with ~n vitro T cell activating
- properties have now been identified. Peptides gp120-
11, gpl20-12 and gpl20-13, corr sponding to amino acid
coordinates 141 to 192, induce the most potent in vitro
proliferative responses, with SI values exceeding some-
~; times 20 in cultures of:T;cells from monkeys immunized -~
with carresponding OVA-conjugated:peptides. The fact
that six out of six monkeys from;~an outbred population,
~: ~ r~sponded to peptide~s gpl20-11, gpl20-12 and gp120-13
strongly indicate~ that~recognition,of~this; ona ~ T
cel} epitope area~by:~simian T cells~is not:under strict
~:~ MHC control. Accordingly, at least~;three distinct epi-
~: : topes~have now been recognized:~y immune~m~nkey PBMC,
:one shared~by peptides~;gplZO-ll and~gpl20-1~2:, one
~: 25 ~shared by~peptides~:~gpl20-12~and~ gpl20-13 and one addi-
tional epitope~within:~peptide gpl20-13.~ In:addition,
~ prolifèrative responses to peptides gpl20-lZ
;~ and gp120-13 have now been demonstrated in.~cultures of j,
CD2 :T ~iells,~ an~ also ~D4~ T:cells, initiated as late
3~ : as:fi~e months~af~ter~the second immu:n~ization indicating
the~presence~oP~memory~T~helper (CD4+)~ cell activating
:epitopes ~in~hat~`area. ~
`: Another~novel~area~ldentified~in~this study,
includes~peptides~gpl2V-29 and gpl2~0-30~ ~-am1no acid ~
3;5~ co~ord~inates~36G:to~400)~which~1nduced T;cell reisponses :
ln~ 3 out of ~4:~monkeys. ~Recognition of this area
SUBSTITUTE: SHEET
W092/21377 PCT/SE92/00373
r
210996i
by immune T ceills appears to be also under limited MHC
restriction, or to involve epitope~s) associated with
polymorphic MHC determinants. At least two epitopes
would be expected within this area as the responses did
S not always overlap.
Importantly, among the novel T cell activating
areas that have now been identified, three peptides
were also found to be capable of inducing, in ViVQ, the
production of neutralizing antibodies against HIV-l.
Thus, sera obtained from all monkeys immunized with
peptide gp120-12, peptide gpl20-16 and peptide gp120-19
inhibited n vitro HIV induced p-24 antigen release and
syncytia formation by human permiissive T cell lines
exposed to HIV-l virions of the corresponding (BRU)
isolate. Further, peptide gpl20-12 is derived from a
partly conserved region of gpl20 and is associated with
a site recognized by neutralizing antibodies. Peptide
gp120-16 represents a highly conserved area of gp120
within all 14 dif~erent isolates investigated. The
efficiency of the peptides, derived ~rom a conserved
~ region of HIV, at inducing the production of HIV-
- neutralizing antibodies as well as at triggexing a
T cell response is n~eworthy. Peptides gpl20-12 and
gpl20-16 are thus the preferred embodiments of the
present invention. - ~
~ Less than 10% of HIV infected individuals produce
`; antibodies capable of recognizing peptides gpl20-12,
gpl20-15, gpl20-16 and gpl20-19. Since antibodies are
generated in response to immunization with these pep~
tides it is possible to;induce an increase in the
repertoire o~ neutralizing antibody producing B cells
~;~ in~HIV posiitive~individuals. ~ ~
Proteins contain a number of a~ntigenic deter-
minants or epitopes which;are the regions of the
proteins comprising the~recognition and binding sites
for specific antibodies. An epitope contains a
` ~
I WV92/2l377 PCT/SE92/0031~
' -13- 21~961
sequence of 6 to 8 amino acids. Epitopes can be either
aontinuous wherein the sequence of 6-8 amino acids are
linear or discontinuous in which case the amino acids
are brouqht together by the three dimensional folding
of the protein. Even though an epitope constitutes
only a relatively few am~ino acids, its reactivity with
an antibody may be influenced by the amino acids in the
protein which surround the~epitope.
Studies aimed at mapping antigenic sites or
lO epitopes of~ proteins have been aided by the use of ''
synthetic peptides corresponding to various regions of
- the proteins of interest. Lerner et al., in, The
Biology of Immunological Disease: A Hospital Practice "`'
800k, (Dixon and Fisher, eds.) pp. 331-338 (1983); and ~'`
Lerner, Adv. Immunol., 36:1 (1984). In addition to
~ .,
their usefulness~in~epitope mapping studies, synthetic ;~
peptides, if encompassing major~antigenic determinants `'~
of a~ protein, hàve potential as vaccines and diagnostic
; reagents. Van Regenmortel, Ann. Inst. Pasteur Virol.,
20 ~ 137E~:49~7-528 (1986~); and~Van Regenm4~tel,~ Laboratory
Techniques~i~n Bioc~emistry and Molecular BioIogy,
Buroden~and Van~Knippènburg~eds. Vol.;l9, Synthetic '~'
Peptides as Antig'ens,~Elsevier;ISBN~0-444-80974 0
~ Synthetic peptides-have several advantages with
regard~to specif;ic~antibody~production and reactivity. ''
The~exact~sequence of~the synthesized~peptide can be
selected from the amino acid sequence~of the protein as , '
determined~by amino acid sequencing of the protein or ''
30~ the~predicted amino~acid~sequence~determined from the
DNA~seguence~ encoding~the~protein.~ The use o'f specific
synthetic~peptidè-s~el~iminates~the~need'~for the full-
length~protein~in~`vaccination;~and the~production of or
assay for~anti~odies.~ Furthermore,~the sQlid phase ~?~'
3~5~ pèptide~ synthetic~techniques~of~Merrifield and co-
v~rke~s~ailov for~essent~ally;~unllmited quantities of
W092/21377 . PCT/SE92/00373
2 1 0g9 61. -14- (_
the synthesized peptide of interest to be chemically
produced. Erickson and Merrifield in The Proteins, 3rd
Edit., Vol. 2, Academic Press, New York, Chapter 3
(1976). The availability of automated peptide syn-
S thesizers has ~urther advanced such techniques.
Although a variety of criteria can be used to
predict antiqenic regions o~ proteins, peptides corres-
ponding to such regions may not always be useful as
vaccines. For example, antigenicity may be lnst
because the peptide is not in the proper spatial
orientation to be recognized by antibodies which react
with the protein. It has also been ~ound that certain
peptides derived from type C retroviruses and HIV act
as immune-suppressive a~ents much as~does HIV itself.
15 Cianciolo et al., J. Im~unol~, 124:2900-290S (1~80);
and Cianciolo et a~., Proc. Natl. Acad. Sci. USA,
230:453-455 (1985). Peptides such as these, which have
a deleterious effect on the patient, would not be
suitable for use as vaccines.
Furthermore, as is particularl~ evident with HIV-l
and HIV-2, there is significant genetic variability
within each of these two virus groups leading to many
serotypes, or isolates, of the viruses. This has put a
significant constraint on choosing a region of a pro-
~ 25 tein from which to derive a peptide for use in formu-
: lating immunogens. However, certain immunodominant
portions of HIV-1 and H$V-2 proteins have been found to
be Ir~latively invariant. Synthetic peptides may also
be Xey to viral vaccines in that they may induce an
~: 30 immune response against type common sequences not nor-
mally immunogenic in the native molecule. These other~
wise~ silent epltopas may be of broad protecti~e speci-
: ficity. Stevard et al., Immunol. Today, 8:51-58
; ~ (1987)~. Several experimental vaccines have been for-
; 35 mulated with the~aim:of preventing infection in those
people who are~likely to be exposed to the virus.
W0~2~21377 PCT~SE92tO0373
, -15- ~
2109961`
Berman et a~., "Protection of Chimpanzees from
Infection by HIV-l After Vaccination With Recom~inant
Glycoprotein gpl20 bu~ Not gpl60", Nature 345:622-625
(1990)~
A number of neutralization epitopes on gpl20 have
been found and defined by several investigators, for an
over~iew see Bolognesi, AIDS (1989) 3(suppl 1):
S111-sll8. In his overview Bolognesi refers to four
different virus neutralization epitopes with the ~-
~ollowing amino acid coordinates:~254-274, 303-337,
458-484 and 491-523. The peptide with amino acid
- coordinates 254-274 was used to immuniæe rabbits and
the resulting antiserum was found to neutralize HIV-l
; as descri~ed above. Ho et al., Science, 239:1021-1023
~5 t1988). `
The peptides encompassed by the invention comprise
amino acid sequences eaah containing at least one con-
tinuous ~lineax) epitope that elicits production of
: activated T cells in the host in addition to eliciting
; 20~: the production of HIV speci~ic antib ~ ies.
The invention thus`encompasses immunogenic pep-
tides corresponding to regions of HIV gp120 protein
: encoded by the envelope gene of HIV-1 HTLV III-B
described by Muesing et al., "Nucleic Acid Stxucture
~: 25 and Expre~sion of~the~Hu~an AIDS/Ly~phadenopathy
: : : Retrovirus", Nature, 3~13:450-458 (193:5). The nucleo-
: tide sequence is given in Genbank Release 63 under the
name HIVPV22. The invention further encompasses func-
tional~ly equi~àlent~variants of the peptides which do
not significantly affect th~ im~unogen:ic properties of
; the peptides. For~instance~,:co~servative~substitution
of:amino acid residues, one or~a~few~amino acid dela-
: tions or~additions,~ and~substituti:on:of amino acidresidues:by amino acid analogues are within the scope
of~the~invention. ;~
WO92/~1377 PCT/SE92/00373
~ 9;61 -16~
Homologs are peptides which have conservatively
substituted amino acid resi~ues and peptides derived
from c~rresponding regions of differen~ HIV isolates.
Amino acids which can be conservatively substituted for
S one another include but are not limited ~o: glycine/
alanine; valine/isoleu~ine/leucine; asparagine/
glutamine; a~part~c acidlglutamic acid; serins/
threonine; lysinelarginine; and phenylalar.ine/tyrosine.
Homologous peptides are considered to be within the
10 scope of the invention i~ they are recognized by :`
antibodies which recognize the peptides designated
gp120-12, gpl20-16 and gpl20-19 the sequences of which
are shown below. Further, all homologous peptides
corresponding to the peptides of the present invention
but derived ~rom different HIV isolates are also
~ncompassed by the scope o~ this invention.
The invention ~lso er.compasses polymers of one or
more of ~he peptides, and peptide analogues or homologs
are within the scope of the invention. Also within the
20 scope of this invention are peptides of fewer amino :~
acid residues than the peptides but which encompass one
or more immunogenic epitopes present in any one of the
pepti~es and thus retain the immunogenic properties of
the ba e peptide~
The in~ention further encompa~ses ~unctionally
equivalant ~riants of the peptides which do not
significantly affect the antigenic or T cell ~ctivating
.~ properties o~ the peptides. For instance, ~arious
analogues, or peptidomimetics, are known in the art and
~can be used to replace one or more o~ the amino acids
in the peptides. Analogues are defined:as peptides
:~ which are functionally equivalent to the peptides of
the present invention but which contain certain non-
natur~lly occurring or modified amino;acid residues~ ;
Additionally, polymers of one or more of the peptides
are:within t~e scope of~the invention.
:: :
.~ W092/21377 PC~/SE92/00373
-17- 210~9
The use o~ peptide analogues can result in pep~
tides with increased activity, that are less sensitive
to enzymatic d~igradation, and which are more selective.
A suitable proline analogue is 2-aminocyclopentane
carboxylic acid (~A~c) which has been shown to increase
activity of a native peptide more than 20 times.
Mierke et a~ orphiceptin Analogs Containing 2-
aminocyclopentane Carboxylic A~id as a Peptidomimetic
~or Proline", Int. J. Peptide Protein Res., 35:35-45
(1990). See also Portoghese et al., "Design of
Peptidomimetic S Opioid Receptor Antagonists Using the
Message-Address Concept", J. Med. Chem., 33:1714-1720
~1~90); and Goodman et al., "Peptidomimetics:
Synthesis, Spectroscopy, and Computer Simulations~',
Biopolymers, 26:S25-S32 ~1987)~
The peptides were synthesized by known solid phase
peptide synthesis techniques. Merrifield and Barany,
The Peptides: Analysis, Synthesis, Biology, Vol. 1,
Gross and Meinenhofer, eds., Academic Press, New York,
Chap. 1 ~1980). The synthecis also:~llows for one or
more amino acids not correspondin~ to th~i original
protein ~equence to be added to~the amino or carboxyl
terminus of the peptide. Such extra amino acids are
useful *or coupling the peptides to another peptide~ to
a large carrier prot2in or to a solld support~ ~mino
acids that are useful for these purposes include but
are not limited to tyrosine, lysine, glutamic acid,
`i aspartic acid, cysteine and derivati~es:thereiof~ l
Additional protein modiflcation techn1ques may be us~id,
e.y., NH2-acetylation or COOH-terminal amidation, to
:~ pro~ide additional means for coupling the peptides to
: ano~her protein~or peptide molecule or a support~.
Procedures for coupling p~ptides to each other, carrier
proteins and solid supports are well known in thei art.
Peptides containing th~ abovei-mentioned extra amino
acid residues eithèr carboxy or amino terminaliy,
WO92~21377 PCT/SE92/00373
2~99C~ -18~
uncoupled or coupled to a carrier or solid support are
consequently within the scope of the invention. Refer-
ence to the peptides of the present invention encom-
passes all of the embodiments discussed herein.
An alternatiYe metho~ of vaccine production is to
use molecular biology techniques to produce a fusion
protein containing one or more of the peptides of the
present invention and a hlghly immunogenic protein.
For instance, fusion proteins containing the antigen of
interest and the B subunit of cholera toxin have been
shown to induce an immune response to the antig~n of
interest. Sanchez et al., "Recombinant System For
Overexpression of Cholera Toxin B Subunit in Vibrio
aholerae as a Basis for Vaccine Development", iroc.
Natl. Acad. Sci. USA, 86:481-485 (1989). It is thus
implicit ih tha present invention that vaccine con-
structs based on appropriate constructi~ns of B and ~-
T cell epitopes ~used to a carrier protein like cholera
toxin would represent important. benefits in
vaccination.
The novel peptide~ amino acid sequences are set
forth below and in Table~2. The amino acid~residues
are derived from the nucleotide sequence previously
described by Kennedy et al. (1986). The peptides may
conkain either an amido or-carboxy group at their
car~oxy termini.
g~l20-11
X~Ser~Ser-Ser-Gly-Arg-Met-Ile-Met-Glu-Lys-Gly-Glu-Ile-
Lys-Asn-Cys-Ser-Phe-Asn~lle-Ser-Thr-Ser-Y-Z
gp120-12
X-Gly-~lu-Ile-Lys-Asn-Cys-Ser-Phe-Asn-Ile-Ser-Thr-
Ser-I~le-Arg~Gly-Lys-Val-Gln-Lys-Glu-Tyr-Ala-Phe-Phe-Y-Z
:~ : :: :
WO92/~1377 PCT/SE92/00373
-19- 210!~96l ~
gp120-13
X-Ile-~rg-Gly-~ys-Val-Gln-Lys-Glu-Tyr-Ala-Phe-Phe-Tyr-
Lys~Leu-Asp-Ile-Ile-Pro-Ile-Asp-Asn-Asp Thr--Thr-Ser-
Tyr-Thr-Y-Z
gpl20-16
X-Pro-Lys-Val-Ser-Phe-Glu-Pro-Ile-Pro-Ile ~is-Tyr-Cys-
Ala-Pro-Ala-Gly-Ph~-Ala-Ile-Leu-Ly~-Cys-Asn-Asn-Y-Z
gpl20-19
X-Thr-His-Gly-Ile-Arg-Pro-Val-Val-SerThr-Gln-Leu- ;Leu-Leu-Asn Gly-Ser-~eu-Ala-Glu-Glu-Glu-Y-Z
`
gp120-29
X-Gly-Asp-Pro-Glu-Ile-Val-Thr-His-Ser-Phe-Asn-Cys-Gly-
Gly-Glu-Phe-Phe-Tyr-Cys-Asn-Ser-Thr-Gln-Y Z
gpl20-30
X-Cys-Gly-Gly-Glu-Phe-Phe-Tyr-Cys-Asn-Ser-Thr-Gln Leu-
Phe-Asn-Ser-Thr-Trp-Phe-Asn-Ser-Thr-~ p-Y-Z
wherein X is either a hydrogen ato~ of the amino
t~rminal NH2 group of the peptide or an additional amino
acid ~eing selected to facilitate coupling of the
pep~ide to a carri:er; Y is absen~ or Cys; and Z is the
caxboxyl group of the carboxy terminal amino acid or an
amido group. The amino acid abbreviations used are
defined in T?ble 2~
In addition to eliciting T cell activati~n,
several of the peptides are useful as:vaccines to
protect against future infection by HIV or to heighten
the immune response to HIV in subjects already i~fected
by NIV. Al~hough any human subject could be vaccinated
with khe peptides, the most suitable ~ubjects are
people at ri~k for HIV infection~ Such subjects
include but are not limited to homosexuals,
W092/2l377 PCT/SE92/0037~
210996i ~
prostitutes, intravenous drug users, hemophiliacs and
those in the medical professions who have contact with
patients or biological samples. The invention also
provides monoclonal and polyclonal antibodie~ which
sp~cifically ~ecognize the peptides. The invention
further provides antibodies produced in response to
vaccination with the peptides which neutralize HIV.
In the preferred embodiment of the present Inven-
tion, the peptides are formulated into compositions for
use as immunogens. These immunogens can be used as
vaccines in mammals including humans or to elicit
T cell activation and/or production of polyclonal and
monoclonal antibodies in animals. For formulation of~
suçh compositions, an amount sufficient to elicit
T cell activation of at least one of the peptides
(about 1-500 ~g) is admixed with a physiologically
acceptable carrier suitable for administration to
mammals including humans~ ~
The peptid~s may be covalently a~tached to each
other, to other peptides, to a proté~n carrier or to
other carrierC, incorporated into liposomes or other
such vesicl~s, andjor mixed with an adjuvant or
adsorbent as is known in the vaccine art. For
instance, the peptide or peptides can be mixed with
immunostimulating~complexes as deæc~ibed by Takahashi
; et al., "Induction of CD8+ Cytotoxic T Cells by
Immunization With Purifiad ~IV-1 Envelope Protein and
; ISCOMS", Nature; 344: 873-875 (1990).l Alternati~ely, i ~ _
the p~ptides are uncoupled and merely admixed with a
: 30 physiologically:acceptable carrier such as normal
saline or a buffering compound sui~able for admin-
~ ~ istration to mammals includi~g humans.
::~ : As with all immunogenic composi:~ions for eliciting
antibodies, ~he immunogenically effective amounts of
~: 35 ~he peptides of:the~invention must ~e determinedempirically. Factors to be considered include the
`
` WO92/21377 PCT/SE92/00373
r
-21- 2109~61
immunogenicity of the native peptide, whether or not
the peptide will be complexed with or covalently
attaahed to an adjuvant or carrier protein or other ~:
carrier and route of administration for the compo- :
sition, i.e. intravenous, intramuscular, subcutaneous,
etc., and the number of immunizing doses to be admin-
is~ered. Such factors are known in the vaccine art and
it is w~ll within the skill of immunologists to make
such determinations without undue experimentation.
Tha invention is further illustrat~d by the
following specific examples which are not intended in
any way to limit the scope of the invention. In order ,`
to determine T cell activation, PBMC from monkeys .
immunized with OVA-conjugated HIV gpl20 peptides were
lS tes~ed for their ability to produce IL-2 and/or ~o
proliferate when exposed ln vitro to recall (immuniz-
ing), overlapping, and non overlapping peptide(s).
,,
}:xamPle 1 ':,
Animals Used in Subsequent ~xamples
Cynomolgus monkeys (~s~g~ ~ascic~arls) were ~.
given 3 intramuscular doses of ovalbumin (OVA)-
conjugated peptides (~ee below), three weeks apart,
each dose consisting of lOO ~g of ovalbumin coupled
peptide emulsified ~n Freund's complete (first dose) or
incomplete (booster doses) adjuvant.
Example 2
Pe~ide Synthesis
40 HIV-l gpl20 peptides~Table l), with an addi-
tional carboxy-terminal cysteine residue, were syn-
thesized on solid phase with an Applied Biosystems 430
peptide synthesi~er (Applied Biosystems, Foster City,
: CA, USA) uslng ~he polymer p-methylbenzhydryl amine
resin as solid phase (Peptides Int., Louisville, USA).
W092/21377 PCT/SE92/00373
?~109`9~
All amino acids for use in synthesis contained
t-butylcarbonyl groups (t-Boc) prot~cting the ~-NH2
group and were obtained f~om Novabiochem AG,
Switzerland. Amino acids wi~h ~eactive side chain
groups contained additional protective groups to
prevent unwanted and undesirable side chain reactions.
The individual protected amino acids used in synthe-
sizing all of the peptldes are set forth in Table 1.
TABLE l
10. AMINO ACIDS USED IN PEPTIDES SYNTHESIS
Boc-Ala-OH
Boc-Arg tTos)-OH
Boc-Asn-OH
Boc-Asp ~Obzl)-OH
Boc-~ys (Pmeobzl)-Oh
Boc-Glu ~Obzl)-OH
3Oc;Gln-OH
Boc-Gly-OH
Boc-His-~Tos)-O~I
Boc-Ile-OH^l/2 H2O
Boc-Leu-OH~H20
Boc-Lys (~-CI-Z)-OH (cryst.)
Boc-Met-OH
Boc-Phe-OH
Boc-Pro-OH
Bo~-S~r (Bzl)-OH^DCHA
Boc-Thr (Bzl)-OH
Boc-Trp (Formyl)-OH
Boc-Tyr (2-Br- æ )-OH :
Boc~Val-OH
Tos: Tosyl or p-Toluene sulfonic acid
` I Obzl = Benzyloxy
Pm~obzl = p-Methylbanzyloxy
2-CL~Z = Carbobenzoxy chloride
~: 35 2-Br-Z = Carbobenzoxy bromide
The peptides wexe synthesized using the t-Boc
synthesis protocol as suggested by the manufacturer.
All solvents were ~rom Applied Biosystems and the side
chain protected amino acids used were from Nova Biochem
W~92/21377 PCT/SE92/00373
, -23-
2109961
(Switzerland) and Applied Biosystems. Following e~ch .
amino acid coupling, a sample was taken and a quanti
tative ninhydrin assay was performed. Only if the -
coupling efficiency exceeded 99% for each amino acid
S couple,d was the peptide acc~pted for further
proc,~ssing. Completed peptides were cleaved from the :`
solid phase and amino acid ~ide chains were deprotected
by acidic hydrolysis using anisole and ethanedithiol
(Merak, Germ,any) as scavengers.
After completion of a particular synthesis, the
protecting groups were removed from the synthesized
peptide and the peptide wa~ cleaved from ~he solid
support resin by treatment with trifluorom,thane
sulfonic acid (TFMSA) according to the method des-
cribed by Bergot et al., "Utility of Trifluoromethan~e
Sulfonic Acid as a Cleavage Reagent in Solid Phase
Peptide Synthesis", Applied Biosystems User Bulletin,
Peptide Synthesizer, Issue No. 16, Sept. 2, l9B6. ~Xhe
following is the detailed protocol used.
1. For 1 gram peptide-resin, 3 ~1 Thio-Anisol
1,2-Ethane-Dithiol (2:1) was added as scavenging agent
and the mixture was incubated with continuous stirring
for 10 min. at room temperature. .
2. l'rifluoracetic Acid (TFA), 10 ml, was added
and stirred continuously for 10 min. at room
temperature~
3. TFMSA, 1 ml, was added dropwise with forceful
stlr~ing and reacted for 25 min. at room temperature. I ;
4. Following cleavage, the peptides were pre-
cipitated with and washed with anhydrous ether.
5. The precipitated and washed peptides weredissolved in a small volume of TFA.6. The d~ssolved peptides were again precipitat,ed
and washed as above in step 4 and the precipitate was
dried under a stream of N2.
:.
W092/21377 PCT/SE92/00373
~ 24~
~2l~96~
Prior to use in specific assays,.the peptides can
be further purified, if desired, by reverse phase high
performance liquid chromatography (HPLC). A particu-
larly suitable column for such purification i5 the
reverse-phase ~ydak~ C-18 column using a water (TFA) -
acetonitrile (TFA) gradient to elute the peptides.
Forty peptides were synth~sized having the amino acid
sequences shown in Table 2. `
The amino acid sequences of th~e peptides, 17-29
amino acids long, half overlapping each other and
entirely encompassing gp-120, were obtained from the
HIV-l BRU isolate. Muesing et al., "Nucleic Acid
Structure and Expression o~ the Human AIDS/Lym-
phadenopathy Retrovirus", Nature, 313:450 (1985).
; ~
.
; .
~'
: : ;: '
WO92~21377 PCT~SE92/00373
~- -25- 21 09 ~ 6
...... --- _....... - - - . I
TABLE 2 ~
~ ~ .................. _ ... .... . .. ~
P I Coor~ t~8~* A~i~o ~cid 8QqU~
_ . ,_ , ~ _ _
gpl20-1 1-28 MRVKEKYQHLWRWGTMLGNLMIC
. _ _ . , .
Igpl20-2 22-46 GMhMICSATEKLWVTVYYGVPVWK
gpl20-3 40-64 GVPVWKEATTTL~C~SDAKAYDTE
gpl20-4 53-74 CASDAKAYDT ~ ATHAC
. , . , _
gp120-5 64-89 VHNVWATHACVPTDPNPQEVVLVNV
_ _ . _
gpl20-6 74-100 VPTDPNPQEVVLVNVTENFNNWXNDM
gp120-7 89-116 TENFNMWKNDMVEQMHEDIISLWDQSL
10 gpl20-8 100-126 VEQ ~ EDIISLW~QSLKPCVKLTPLC
- ~
gpl20-9 116~141 _ KPCVKLTPLCVSLKCTDLKNDINTN
gpl20-10 126-151 VS~KCTDLKNDTNTNS9SC~NI~ER
gpl20-11 141-164 SSSGRMIMEKGEIKNCS~NISTS
gpl20-12 151-17~ GEIKNCSFNISTSI~GKVQKEYAFF
~ ~ _
qpl20-13 164-192 IRGXVQKEYAFFYKL,DIIPIDNDTTSYT
_ _ _ _ _
gpl20-14 176-205 YRLDrIPIDNDTTSYTLTSCNTS ITQAC _
gpl20-15 _ 192-218 LTSCNTSVITQACPKVSFEPIPIHYC
gpl2_-16 _ __ 205-230 ~ _ PKVSFEPIPIHYCAPAGFAILKCNN
. _ _~
gp120-17 21~-247 APAGFAILKCNNKTFNGTGPCTNVSTVQC
.. __ _ . ................... ....
: 20 gp120-18 ; 230-257 KTFNGTGPC~NVSTVQCTHGIRP W ST
. ~... .... _~ __ , . . .
:~ gpl20-l9 247-269 THGIRP W STQLLLNGSLAEEE
. ,. ,,, .~ _ .
~ gp120-20 257-282 QLLLNGSLAEEEVVIRSANFTDNAX
_ _ .
I 1 ! i !g~ll20 ~ 21 : 269-2~5 _ W IRSANFTDNAK~IIVQLNQSVE~N _ .
gpl20-22 282-306 TIIVQ~NQSVEINCTRPNNNTRKS
gpl20-23~ 295-320 CTRPNNNTRKSIRIQRGPGRAFVTI _
gpl20-24 306-326 : IRIQRGPGRAFVTIGKIGNMRQAH
. _ _ _ , , . . _ I
: ~ : gpl20-25 ~320-343 ~ GKIGNMRQAHKNISRAKWNNT~X
. . _ , : ' I
~pl20-26 : 326-353 : KNISRAKWNNTLKQIDSKLREQF
. ~
;~ ~gpl20-27 ~ 343-366 : QIDSKLREQFGNNKTIIFKQS~G
30gpl20-28 : 353-377 GNNKTIIFKQSSGGDPEIVTHSFN
__ __ _ _ _
`: : :
~` .
WO 92J21377 PCr/S E92/00373
--26--
2'1 0 9`'~';6~
TABLE 2
. - - _ _ - _ _ _ i
~Qpti~e Coor~ntes~ A~ino Acid l3eque~ce~ ¦
-.,.. .... _. ........ _ _ _ _
gp 20--29 366--389 GDPEIVTHSFNCGGEFFYCNSTQ
_ __
~p120-30 377-400 CG&EFFYCNSTQLFNSTWFNSTW
_ __ _ _
ypl20-31 389-409 LFNSTWFNSTWSTEGSNNTE
_ . _ _
gp120-32 400-417 STEGSNNTEGSDTITLP
gp120-33 409-429 GSDTITLPCRIKQFINMWQE
_ _ .. - 11
gp120--34 417-444 CRIKQFINMWQEVGKAMYAPPISt;QIR _
_ _ ::
gp120-35 429-453 VGRAMY~PPISGQIRCSSNITGLL ~:
~ . ~ '
_ gp120-36 444-466 CSSNITGLLLTRDGGNNNNESE _
9pl20-37 453-4 7 6 L~RDGGNNNNESEIF~PGGGDMR
__ _ , , ,, _
qp120--38 4 66-488 IFRPG~;GDMRDNWRSELY~tYKV
_ . _ _ ___ . _
~120--39 476--497 DNWRS~5LYXY~WKIEPLGVA
_ _ .
gp120-40 488-511 VK}EPLGVAPTKAKRR W QREKR ~:.
t~mino acid abbreviati~ns
,_, -- ~ - - _ _ _ :
~Alanine _ _ Ala A_ Leucine heu L i
. - ~ __ ~ _ i.
Arginine Arg R :Lysine Lys K
__ ; ~ . . ~
: :~ Aspa~agine Asn : N Methionine Met M .;
,: _ _ __ . ~
. :Aspartic acid Asp : DPhenylalanine Phe F
_. _ ~
~ Cyæteine ~Cys C Proline _ Pro P ..
_ _ _ _ ___
Glutamine Gln : Q Ser:ine Ser S
_ _ . , _ _ I ~;;
Glutamic acid : Glu EThreonine Thr T
. : _ _ . _- . I ;
;~ . GIycine: ~ G . ~Tryptoph~n Trp W
: HistidineHis ~H ~Tyrosine ~yr Y
. _ _. _ I ,.
Isoleucine:Ile I Valine Val V
, . . . . ~ ~ - - . _ .
** As previously described by Kennedy et al. (19~6).
. , .
:
:
:~ . : : . :
I WO92/21377 PCT/SE92/00373
21099.~1
Example 3
Preparation of P~ptides for Immunization
Peptides ac~ording to the presen invention were
covalently coupled ~-o ovalbumin grade V (Sigma, St.
S Louis, MO, U5A) at an approximate lO:1 (peptide:
ovalbumin) molar ratio u ing N-succinimidyl
3-~2-pyridyldithio) propionate ~SPDP), (Pharmacia,
Uppsala, Sweden) as bifunctional linker according to
the manufacturer's instructions (Pharmacia) i.e.
briefly as follows:
Ovalbumin was dissolved in coupling buffer ~0.2 M
NaH2PO4, Ph 8.5~. The dissolved ovalbumin was then run
through a Sephadex G-25M column (Pharmacia, Sweden),
using the same buffer. Protein concentration was
measured at 280 nm and the recovery was determin~d.
SPDP was dissolved in 99.5% ethanol to a final
concentration of 40 mM. SPDP was then added dropwise
to the ovalbumin solution under stirring. The
SPDP~ovalbumin mixture was then left a~ room
temperature for approximately 30 min~tes. The
ovalbumin- SPDP conjugate was separated from
unconjugated SPDP by running the mixture through a
Sephadex G-25M column, using water as sluent. The
degree of ~ubstitution for the ovalbumin-SPDP conjugate
was det~rmined afte~ diluting 50 ~1 conjugate in 2 ml
of water, by measuring the diluted conjugate at 280 nm
and the diluted conjugate plus 100 ~l Dithiothreitol
(DTT~ (Sigma) at 343 nm, in order to determine the
amount to be added to the peptide solution.
. 30 ~inally, the synthetic peptide to be coupled to
the ovalbumin-SPDP conjugate wa~ dissolved in 10%
acetic acid to a final concentration of 1 mg/ml and a
suitable amount of ovalb ~ in~SPD~ conjugate (as
; determined by the subst:itution degree above) was added
and allowed to stand overnight at room temperature.
WO 92t21377 PCr/SE92~00373
2 ~
21~996i
xam~le 4
Immunization Protocols
M. fascicularis were used to generate antibodies.
Prior to the initial peptide injection, a blood sample
S was drawn ~rom the monkeys. This initial blood sample
is termed "preoimmune" ~Tables 5-81 and is u~ed as an
internal control and analyzed simultaneously with
respective immuneserum.
The monkeys were injected with 100 ~g peptide-
SPDP-ovalbumin suspended in 0.5~ml phosphate buffered
saline ~PBS). The monkeys were i~munized
intramuscularly three;tim:es, three weeks apart~ As
adjuvant, 0.5 ml of Freund's complete adjuvant was used
for all initial immunizations and Freund's incompletP
: 15 ad]uvant was use~d~for:booster shots. Two weeks after
~the ~:inal immunization the monkeys were bled by
r~moving a lO ml blood:sample from ~he fossa and
pre-immune:and hyperimmune s~ra were subject to
neutralization assays~a~described in:Example 9.
~ : Exam~le S
Isolàtion~and:~Fractionation of
; ; ~ :; Lv~hocYtes~from~Immuni~zed M. fascicularis
Heparinized~venou~ blood was co}lected from the
emoral vein, at :least~two~weeks~after the~second.~
29~ and/or the third~injections. Periphe~al bIood ~;
mononu~lear cells ~(PBMC) were:obtained by gelatin
,
sedimentation ~f~ollowed ~y~density gradient~
centrifugation by the~following me~hod. A salution of
3~ weight/Yolùme~ gelàtin:~tqèlatin~936,:PB &elatins ~ `~
30~:; UX~LTD,~ G8)~in~Bank~'s~Balan~ed;Salt~Solution was~mixed ~; :
w1th the;~blood~at~a~ 3~rat~io~and~erythrocytes were ~;
allowed:to~sedlment~or~one~hour~at 37C.: The
erythxocyte-free~:supernatant~was~layered onto a
F~icoll~-Hypaque~ cus~hion~:(Pha~rmacia~!;Sweden) and
35~ centr:lfug~ed;for~l;5~minutes~a:t::930~x g, at 20~C.
~i` ~
' WO9~/21377 PCT/S~92/00373
... .
-29- 21099
.
Inter~iace PBMC were washed twice by centrifugation
(500 xg, 20C, 5 min) with i~otonic pho~phate-buffered
saline (PBS, O.Ol M pho~phate buffer in O.lS M NaCl,
pH 7.4).
In some Examples represented below, T cells were
enriched by rosetting with AET-treated sheep red blood
cells as described by Raplan and Clarc, ~'Improved
Rosetting Assay fox Detection of Humian T Lymphocytes",
J. Immunol. Met., 6:131 ~1974) follQwed by density
centri~ugation on Ficoll-Hypaque. The rosetted cells
(nominal T cells) w~re aollected from the pellets and
re~uspended for 20 seconds with distilled water to lyse
sheep red blood cells. Further enrichment into CD4+
T cells was obtained ~y paramagnetic depletion of
CD8+ T cells using microspheres coated with monoclonal
anti-CD8 antibodies ~Dynal AS, Norway), according to
the manufacturer's instruations.
: ~xample 6
LymphQcyte Proliferati~ As~ays
Unfractionated PB~C were resuspended in complete
medium (see below~ and ~ispersed in rou~d-bottomed 96
micro-well plates (Nunc, Denmark) at three different
~: cell densities (2x105, lX~05 and 5x104 cells per well) in
Iscove's msidiumi supplemented with 10% fetal calf ser~m
~ 25 ~ (FC~, Biological Industries, Israel), 3 ~g/ml
: L-Glutamine (Gibco, UK) and 0.1 mg/ml.Gentamycinsulfate
(Esssx~ Lakem~del A8, Sw~den). FraGtionated T cells
(2x105 or 1.2xlOs nominal T cells, or 4x104 CD4'. T cells~
~ : were dispersed in~separate sets of;wells together with
:: ~ 30 4x104 or 2x104 autologous T cell-depleted irradiated
(2500 rad), PBNC as a ~source of accessory cells.
Synthetic peptides were dissolved in dimethylsulfoxide
t20 ~glml) and further diluted in culture mediumi.
; Uncoupled p~ptides were added at different
:: :
WO92/~1377 PCT/SE92~00373
2 ¦ 9 ~ 30
concentrations (lO, l and O.l ~g/ml) to the culture wells.
Concanavalin A (Sigma~ (lO ~g/ml) was added to separa-
te cultur~s as a positive control. Cells, in a final volume
~f 0.2 ml, were incubated for ive days at 37C in a humid
atmosphere with 7.5~ CO2. After four days, 25 ~l of culture
superna~ant were collected from each well and frozen a~
-70C until assayed for IL-2 activity according to the
method described in exampla 5. l6 hours prior ~o the comple-
tlon of the aulture period t 20 ~l culture m~diwm containing
1 ~Ci of ~ 3~] thymidine (Amersham, ~UK) were added to each
~ well. The harvesting and subsequent meaæuremen~ of incorpo-
rated radioactivity was performed on an automated fil~er
cell harvester coupled to an argon activa~ed B-scin~illa~ion
counter (Inotech, Switzerland). Data were expressed as
arithmetic mean stimulating ind~xes (SI), the latter SI
belng defined as the mean ratio of ~3H]-thymidine incorpora-
~ ted in peptide stimulated cultures (mean from thre cul~ures
: 20 divided by corresponding triplicate of con~rol cultures
(unstlmulated). A mean SI of at le,~st 2.4 is considered
positlve. SI values equal to at least 2.4 ~l.e. twice the
sum of the mean plus ~3.3 times the SD of the re~licate
aultures exposed to irrelevant peptides (confidence in~er-
val, p ~ O.OOl, Student's + test)] were aonsidered as:signi-
~ ficantly increased~ :
:~ As: s~en i Figure l, a substantial number (18/40) of
; peptides that were inj:ected:into monkeys in an OVA-substitu-
t~d form, induced in vitro proliferation of PBMC from corre-
i~l30 sponding immune animals. In Figure 1, resu~ts are expressed
as mean SI ~ SD of~all triplicates tested~% SD (if tested on
;~ two monkeys). Black bars indicate a positive result. The
frequency of responding monkey~s) is indicated. ~our major
areas corresponding to ~the additive sequence of 2 to 3
~::35 ~ overlapping peptides were found to accommodate this activi~
: ty. Pepti~es gpl20~ gpl20-12 and gpl20-13
Su B s T1T~ Sl!~FT
W092~21377 PCT/SE92/00373
31 2 lOg 9 6 l
(amino acid ccordinates 141-192) correspond to one such
area. Five ou~ of six monkeys immunized with one of these
three peptides responded ~o recall pepti~e. Another major
area compriseæ pep~ides gpl20-23, gpl20-24 and gpl20-25
(amino acid coordinates 295-343) which induced proliferative
re~p~nses o~ PBMC from at least one out of 2 mo~keys immuni-
zed w~th the corresponding peptide. A thlrd area, comprising
peptides gpl20-29 and gpl20-30, accommodate~ a site(2~ o~
proli~eration inducing activi~y on PBMC from monkeys immuni-
zed with the corresponding OVA-con;uga~ed papt~des. The
~ourth area consists of peptides gpl20-33, gp120-34, gpl20-
35 and gpl20-36 (amino acid aoordinates 4090466) where each
peptide could induce profilerationof P3M~ from at least one
o~ the immunized monkeys.
Apart from these ma~or areas, five peptides, i.e.
peptides gpl20-4 (amino acid coordinates 53-74), gp 120-5
(am~no acid coordinates 64-89), gpl20-17 (amino acid coordi-
natas 218-2~7)and gpl20-21 (amino ac~d coordinates 269-295),
we~ shown to induce in Yi tro profi;erative re~ponses when
added to PBMC from monkeys immunized wlth ~he corresponding
OV~-a~n~ ugated peptide.
Peptides found to be capable of inducing a prolifera-
tive r~sponse of PBMC rom monkeys immunized with the corre-
. sponding OVA-coupled peptide were ~aassayed on~PBMC ~rom at
least three other monkeys immunized with a non-c~gnate OVA-
coupled peptide. Peptides gpl20-4, gpl2013 and ~pl2V-34
i~duced prolifera~lon o~ PBMC from 1 ou~ of 3 monkeys and
peptide gpl20-30 in 1 monkey out o~ 7 (SI ranging between
2.0 and 2.5) while~the other peptides :~ailed to induce any
signi~icant proliferative responses.
Pep~ides capable:of inducing a prol~ferative response
in one or two~immunized monkeys were retest~d after ~he
~hird immunization. On this occastion, the in vitro pro-
liferative responses of:PBMC from immune
SUBSTlr~E SHEET
WO92/21377 PCT/SE92/00373
21 09 g 6 ~ 32 (~
monkeys to each of 2 peptides containing a sequence half
overlapping with the imm~nizing pep~ide were also evaluated.
As seen in Figure 2, PBMC from both monkeys immunized
with OVA-con~gated peptide gpl~0-ll responded also in vitro
to peptide gpl20-l2, ~ut none of the peptide gpl20-12-immu
nized monkeys responded to peptide gpl20-ll. Cells were
obtained two waaks after ~h~ third immunization. The pepti-
des te~ted were selected on the basis of in vitro responsi-
ven~ss to the immunizing peptide a~ter two immunizations. In
- Figure 2, res~lts are expressed as mean SI of all triplica-
tes tested % SD (if tested on ~wo monkeys). Rlack bars
indiaate a positive result.
Similarly, monkeys immunized with peptide gpl20-l2
responded to peptide gpI20-13 but none o~ ~he peptide gp120-
13 immunlzed monkeys responded to peptide gpl20-12. In the
next area o~ in vitro profilerative activity, i.a. peptides
gpl20-23, gpl~0-24 and gpl20-25, none~of ~he overlapping
peptides induced in vitro profilerataion of PBMG from any of
the monkeys immunized ~ith OVA-con~ugated peptid2s. The same
holds true ~or PBMC ~rom monkeys immunized with peptides
gpl20-29 and gpl20-30 (OVA-conjugated) as Il~ response to
overlapping pept~des is achieved. In ~he area consisting of
peptides ~pl20-33, gpl20-34, and gpl20-35, none of the
overlapping peptides induced in vitro prol~feration of P~MC
from any of the monkeys immunized with OVA-con~ugated pepti-
des. PBMC ~rom the other monkeys were also nega~ive in this
rèspect. Of the other epitopes identifiedr only PBMC from
the pe2tide gp120-4 immunized monkey responded in vitro to
an overlapping peptida, i.e. to peptide gpl20-5.
The profilerat1ve responses of different cell frac-
tions from two monkeys (immuniz~d with pep~ide gpl20-l2 or
pepelde gpl20-13) were examined. As seen
,
S ~ ~ ~ TIT W ~ S ~ F~T
WO92/21377 PCT/SE92/00373
- ~33~ 21~ 99 6 i
;
in Table 3, enrichment of CD2+ T cells increased ~he
proliferative response of PBMC obtained from bo~h
monkeys when cultured in the presence o~ immunized (but
uncoupled) peptide. ~lso, existing responses ~o
overlapping peptides remàined relati~ely ~ons~ant.
After a further depletion of CD8+ T cells, th~ CD4+
T cell enriched fractions (containing 9 to 18% of the
original CD2+ T cell fractions) still proliferated in
response to incubation with immunized peptide.
However, the CD4+ T cell enriched fraction from the
peptide gpl20-12 immunized monkey did not proliferate
in response to any of the overlapping peptides.
.
: _ ` TABLE 3 _
, ~ ~ ~
IN VI~RO PROLIFERATIVE RESPONSES TO RECAI.L I .
PEPTIDES OF TOTA~ PBMC AS WELL AS ~D2' AND CD4~ ¦ :
ENRIC~ED FRACTIONS OF CELLS OBTAINED FROM l
MONKEYS 5 MONTHS AFTER IMMnNIZATION WITH I
:: :~YA-CONJUGATED PEPTIDES
_ ~ Stimu}ation~ndex (SI) of
, . ~
CD2~ I CD4+
Immunized In ~E~ Total Enriched ¦ Enriched
Peptlde Peptide PBMC Fraction ¦ Fraction
i ~ _ _. . _
gp120-12 gpl20-11 2.7 2.8 1.1
; gpl~0-12 2.5 8.6 2.6 I, . ~ - ~ ~ _
gpl20-13 `3.9 3.6 1.4
.: ` ._. ~, ~ . _ _
__ I ~- i
. qp120-13 -gpl20-12; 1.~0 ~ O.4 O.8
1: ~ 1 ~, , . _
gp120-13 2.5 2.9 ~.6
: ~ .gpl20-I4 2.1 2.4 4~0
. ~
: :: : : Xn Table 3, the various columns were obtained as
follows. Immunized peptide: 1~0 ~g o~ OVA-conjugated
~ ZS peptide was immunized~at three occasions in Freund's
,~ :: ::
W O 92~21377 P ~ /SE92~00373
`'; ' '
2109~6i
.,.
complete (lst dose) or incomplete (boosting doses)
adjuvant;
In vitro peptide: unconjugated peptide;
Total PBMC: mean SI of four triplicates of
different cell ~ensities and peptide concentrations;
CD2+ enriched fraction: 2x105 S~BC-rosetted PBMC
incubated with 4x104 irradiated, non-rosetted cells
together with lO~g/ml of peptideits):
CD4+ enriched fraction: 1.25x105 (peptide gp120-12
immunized monkey) or 4xlO~ (peptide gpl20-13 immunized
monkey) SRBC-rosetted PBMC~urther enriched in CD4+ T
cells by incubating with anti-CD~+ coated beads were
incubated with 2x104 irradiated, non-rosetted cells
together with 10 ~g/ml of peptide(s).
:
Example 7
IL-2 assa~
~: The IL-2 content of individual cell microcultures
: was determihed by the bioassay performed as described
by Gillis et al~, "T Cell Growth Factor: Parameters of
Production and a Quantitative:Microassay for Activity",
J. Immunol., 120:2027 (1978). 8riefly, supernatants '
were added at a final dilution of 1:4 to 104 CTLL-2
~ cells. Cells were incubated for 24 hours at 37C in ;;
; : flat-b~ttomed 96 microwell plates (Nunc, Denmark) in
Iscove's medium supplemented with 10% FCS, 3 ~g/ml
L-Glutamine, 0.1 mg/ml Gentamycinsulfate and 5x105 M
Mercaptoethanol.i Six~hours prior to completion!of
the culture period, l ~Ci of ~3H]-thymidine was added. :
The cells were harvested and [3H~]-thymidine
incorporation was~ determined~as described in Example 6.
IL-2 content in~thè supernatants was determined by
: extrapolation from a standard dose-response curve gen-
erated by culturing CTLL-2 cells in the presence of
~: :
. WO92/21377 PCT/SE92~00373
~35- 21099~1
known amounts of recombinant human IL-2 tGenzyme,
Boston, MA).
As seen in Table 4, several cell culture super-
natants contained detectable amounts of IL-2 in
S cultures of PBMC from monkeys immunized with
0~-conjugated p~ptides gp120-11, gp120-12, gpl~0 13,
gp~20-16, gp120-21, gp120-25, gp120-30 and gp170-34,
fiecreted IL-2 ~ould be detected after in vitro
challenge with the corresponding, uncon jugated,
peptide. The ratio of secreted IL-2 found after 4 days
of n vitro ~ulturing ranged from 0.2 to 1.0 U/ml.
~ Cell culture supernatants of PBMC derived from monkeys
i~munized with peptides gp120011, gp120~12, gp120-13,
gp120-30 and gp120-34 also contained I~-2 after in
it~o expo~ure to one or two of the o~erlapping
peptides. Accordingly, PB~C from a monkey immuniz~d
wi~h peptide gp120-11 secreted detectable levels of
IL-2 in the cell supernatant after 4 days of
stimulation wit~ peptide gpl~0-12, and a monkey
immunized with peptide gpl20-12 secreted detectable
levels of IL-2 after stimulation with peptide ~p120-13.
Cell culture supernatants containing I~-2 were
identified from both PBMC cultures containing
overlapping peptides tpeptides gpl20-12 and gpl20-14)
together with PBMC from a peptide gpl20-13 immunized
monkey and the same holds true for peptides gpl20-33
and gpl20 35 wh~n co-cultured with P~MC from a peptide
lZ0-34 immunized monkey. Finally, PBMC obtained from
a paptide gpl2 0D3 0 immunized monkey secreted detectable
30: amo~nt~ of IL-2 not only when cultured in the presence
o~ peptide gpl20-30, but also when peptide gpl20-29 had
been added to the cultures.
; ~
WO92~21377 PCT/SE92/00373
-36-
210~961
l TABLE 4 l
; ~
¦ IL-;2 CONTENT IN SUPERNATANTS OF PBMC FROM MONKEYS
IMMUNIZED WITH OVA-CONJUGATED PEPTIDES AFTER IN Y!TRO
EXPOSURE TO UNCONJUGATED. RECALL PEPTIDE~S).
I _ _ ~ .
5 ¦ Immuniz~d In vitro IL-2 Conten~ n ~ulture ;
I Peptide~ Peptideb Supernatants (U/ml)C
;- - - - -- ----- ------- _ _ - ~ .
gpl20~ gpl20-11 . 0.28
_ _ _ gpl20-12 _ 0.26_
gpl20-12 gpl20-12 0.28
. _ _ . _
gpl20-13 0.22
. .__ __ . __ .
gP120-13 gpl2o 12 0.36
. gpl20-13 1.01 '
..... . _ _ ~ . , _
gpl20-14 ~ : 0.~1
, . __ ... . ._ ~_ __ _~
_ gpl20-16 gpl20-16 0.22 _
gpl~o-2lgpl20-21 0.37 _ _ __.
gpl20-22 gpl20-3 0.20
. .
gp120-24gpl20-25 0.21
. ~ . , __. _
gpl20-25gpl20-25 ~ _ 0.2 _
I5 _ gpl20-30 qpl20-29 0.23_
: gpl20-30 0.20
-: ~ _ ~
gpl20-33~ gpl20-34 0.53
gpl20-34gp120-33 0.20
, .. . .... .. . . , , .
gpl20-34 0.27
: gpl20-35 0.25
...... . _
i OVA-conjugated pept~ides. ...
b unconjugated peptides
c I~-2 content of the highest triplicate from
¦ peptide-stimulated PBMC in~vitro.
' : ~
WO 92/2 l 377 2 1 0 9 9 6 1
--3 7--
Example 8
Cells and Virus Sto ks
All neutraliæation tests were pPrformed using H-9
c~lls and ~TLV-lllB virus (originating from R. C. Gallo
and supplied by Dr. William Hall, North Shore ~ospital,
M~nhasset, New York). H-9 cells ~designated Hs NY)
were maintained in RPMI Medium (Gi~co~ supplemented
with 20% ~etal calf serum ~FCS3,
penicillin/streptomycin (PENf STREP 50 ~g/ml each and
without any fungicides). Cells were subcultured at a
dilution of 1:3 every 4 days.
Cells wexe scraped from the plates and pelleted by
centri~ugation at 325 x g. Pelleted cells were
r~3suspended in 1 ml of stock virus previously diluted
1/10 and allowed to adsorb for 60 min at 37C with
frequent stirring. Af~er ad~orption of the virus, the
cells were recentrifuged and resuspended in 10 ml of
RPMI ~ith 20% FCS and l?olybrene (2 ,ug/ml) (giving a
final concentration of sx105 cellslml) and~ incubated at
37 C in 5% C02.
Infected cells were shown to be detectable at 4-5
days post-inf~ction (poi-) by monitoring syncytia
formation, positive cells in immunofluorescence and
p-24 production (assayed by the Abbott p-24 antigen
test). The peak of HIV production was -~een 10 - 15
days p.i. at which time virus was collected. After low
speed centrifugation to remove debris, supernatants
con~aining virusjcollected from infec~ed cells were
frozen in stocks at -sooc. One virus sto~k with
endpoint tit~r of 40~000 50% tissue culture infective
dioses (TCID50~ was used throughout the studies (re~erred
: ~ to as NT3-NT19).
::
:: :: :
: ~:
W092/21377 ~ PCTtSE9~/00373
-38- ~ :
2109961
Exam~le 9
HIV-l Neutralization Assay
Sera containing antibodies that neutralize HTLV
lll-B infectivity were detected by their ability to
prevent HIV l syncytium formation, p-24 antigen
production and decreased number of infected cells as
determined by immunofluorescence markers, compared to
control infections lacking peptide specific antisera.
Stock virus, described in Example 8 was diluted to 100
TCIDso and mixed with serial fourfold dilutions (1/5,
1/20, and 1~80) of complement-inacti~at~d immunasera
obtained from the monkeys immunized as described in
Example 4. As a positi~e ~ontrol, a guinaa pig
hyperimmune serum ~re~erred to as ~SV) with known HIV
neutralizing titer of 1/40 - 11160 was included in all
exp~riments (kindly provided by Prof. B. ~orein, Dept.
~eterinary Virolc~y, BMC, Uppsala, Sweden). After
incubation for 60 min at 37C or 16 hours at 4C, the
serum-virus mixture was added to lx106 H-9 cells and
incubated for another 60 min at 37C.~ Following
incubation, the calls were washed once and plaoed in 24
well multidish plates with~2 ml of growth medium (RPMI,
10~ FCS, 2 ~g polybrene/ml) per well.
Cells were ~xamined under the microscope (magni-
fication x200) for the presence of syncytia on days 5-
12 p.i. Supernatants ~rom infected cells were assayed
for the p~esence cf p-24 antigen according to ~he
manufacturer's instructions ~Abbott ag test HIV.~G-10,
Enzyme Immunoassay for the Detection of Human
Immunodeficiency Virus Type I (HIV-l) Antigen(s) in
Human Serum or Plasma) in tenfold serial dilutions
(1l10 ~ 1/1,000) at 10 days p.i. The results are
present~d as absorbance values at 454 nm with higher
absorbance values indicating higher P-24 antigen
concantration and`hence HIV infection. Serial
dilutions of the supernatants were made so as to detect
~ W092~21377 CT/SE92~00373
210~96~
, _39_
, .
p-24 concentrations in the most accurate range (< 2.0
absorbance units).
~ he number of infected cells were determined at
the end of experiment (usually on day 15 p.i.) by
acetone- fixation of cells on slides adopted for
immunofluorescence (IF). An indirect IF test w~s used
according to s~andard procedures described in Jeansson
et al., "Elimination of Mycoplasmas from Cell Cultures
Utilizing Hyperimmune Sera", Ex. Cell Res., 161:181 18
(1985), with 1/400 dilution hyperimmune sera ~rom
HIV-infected individuals and a fluorescein
~ isothiocyanate (FITC) labeled antihuman IgG antibody
(Bio-Merieux France) diluted 1/100 . ~ables 5-8 show
the results obtained from screening of hyperimmune sera
from monkeys immunized with peptides 1-40~
In Tables 5(A-D)-8 the p24 antigen content of the
~: supernatants was analyzed by ELISA , indiract IF and
syncytia formation as described aboveO The relative
amount of antigeh positive cells i5 depicted as AG POS
cells wherein the percentages are repFesented by:
_ a 0% ~ + = >0-2% ~ +~ - 3-10~ and ++~ 20% where
the percentage interval indicates~the number of antiqen
positive cells,
Table SA (HIVNT3P1.XLS) depicts the results
obta~ned with sera dèrived frQm monkeys immunized wi~h
peptides gpl20-1 - gp120-10. The cells;used were H9 NY
and the virus used was HTLV-IIIB, Batch 18 described in
Example 8. The incubation protocol was (virus plus
serum) incubatian at 37C for one hour.
~able 5B ~HIVNT4Pl.XLS):depicts the results
obtained wi~h sera derived from monkey~ immunized with
peptides gp~20-11~-~ gpl20-20. The cells uced were H9
: : NY and the vlrus used was HTLV-IIIB, Batch 18 described
in Example 8. The incubation protocol~was (Yirus plus
35: serum) i ncubation at 37C:for one: hour.
`: . `~:::
WO 9:2/21377 , , , S . PCI ~SE92/00373
.r~
--4 0--
'~109961
Table 5C (HIVNTSPl.XLS) depicts the results
obtained wit~ sera derived from monkeys immunized with
peptides gpl20-21 - gpl20-30. The cells used were H9
NY, and the virus used was HTLV-IIIB, Batch 18
S described in Example 8. The incubation protocol used
was virus plus serum incubated at 37C for one hour.
Table SD (RIVN~6Pl.XLS) depicts the results
obtained with sera derived from mo~keys immunized with
peptides gpl20-31 - gp120-40. The cells used were H9
NY and the virus used was HTLV-IIIB, Batch 18 describ~d
in Example 8. The incubation protocol was (virus plu5
serum) incubation at 37C for one hour.
Table 6 (HIVTAB4.XLS) shows the results of he
first retest of putative neutralizing antibodies as
determined by the first test (Tables 5A-~). In each
test the virus used was HT~V-IIIB, Batch }8 and the
cells use~ were H9 NY. The first retest results in
rows 1-19 are the results of neutralization test number
5. The incubation protocol was incubation at 37C for
one hour. The first retest results i~ rows 20-32 are
the results of neutralization test number 7. The
incubation protocol was inouba~ion of at 37C for one
hour.
Table 7 tHIVTABS.X~S~ shows second, third and
2S ~ourth rete~t results of the positive peptides. In
each test the virus used was HTLV-IIIB Batch 1~ and the
cells used were H9 NY. The second retest results in
, I i rows 1-4 are the~results of neutralization test number I ;
7. The incubation protocol was incubation at 37C for
one hour. The second retest results in rows 5-13 are
the results o~ neutralization test number 12. The
third retest results are shown rows 14-16 are the
results of neutralization test number 12. The
incubation protocol was incubation at 37C for one
hour. The fourth retest results in rows 17-39 are the
results of neutralizatlon tes~ number 16. The
:` :
.. . . .
~` WO92/21377 ~ 1 Q~ 9~ ~CT~SE92~00373
-41-
incubation protocol was at 4C for 16 hours. The
second retest results in rows 40-53 are the result of
neutralization test 19~ The incubation pro~ocol was
cells plus virus at 4 for 16 hours.
S Table 8 (HIVKOMBP.XLS~ shows the neutralization
assay results with combined hyperimmune sera. Note
that the incubation of:virus and cells was at 4C for
16 hours.
The results depicted in Tables 5(A-D)-8 indicate
that peptides gpl20-12, gpl20-16, and gp120~1~ elicit
the production of HIV neutralizing~antibodies in
primate subjects. The use of the peptides in
vaccination of human subjects is therefore applicable
to prevent infection by HIY or to induce heightened
immune respons~ in subjects already infected by HIV.
' ~ :
:
. : :
.
.
~: :
' r
~, ' : :: ~ `
WO 92~21377 ~ IJ ~"P ~ r~ 42--PCI /SE92/00373
2109g61
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~n ~ _ _ _ __ _ _ __ _ _ _ ~ __
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IJ,~ ~ _ r1 _I O l O ~ t`~ ~ Z ~1 ~ Ir~ ~ a:~ ~ N ~ 7 Ul t`
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WO 92~21 377 42 10~9~ 9 G~/SE92/00373
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WO 92~21377 , ~ 44- PCI`/SE92J00373
2i09~61
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