WO1993003766A1

WO1993003766A1 - Multiple antigen peptides for use as hiv vaccines

Info

Publication number: WO1993003766A1
Application number: PCT/US1992/006688
Authority: WO
Inventors: James P. Tam; Albert T. Profy
Original assignee: Repligen Corporation; The Rockefeller University
Priority date: 1991-08-13
Filing date: 1992-08-11
Publication date: 1993-03-04

Abstract

In general, the invention features a multiple antigenic peptide system including a dentritic core and a peptide, wherein the peptide includes the sequence IGPGR (SEQ ID NO: 3), and the multiple antigen peptide system, when injected into a mammal, is capable of eliciting an immune response.

Description

MULTIPLE ANTIGEN PEPTIDES FOR USE AS HIV VACCINES Background of the Invention The field of the invention is vaccines for prevention and treatment of HIV infection.

Highly specific and immunogenic antigens are preferred as vaccines. While the i munogenicity of an antigen can be increased by coupling a protein carrier to the antigen, this approach has several drawbacks. First, if the carrier is large, significant humoral immune response can be directed against the carrier rather than the antigen. Second, a large carrier can suppress humoral response to the antigen. Finally, the coupling of an antigen to a protein carrier can alter the immunogenic determinants of the antigen.

Multiple antigen peptide systems (MAPS) are designed to overcome the problems observed with conventional protein carriers. Most MAPS are composed of several peptide antigens covalently linked to a branching, dendritic core composed of bifunctional units (e.g., lysines) . Thus, a cluster of antigenic epitopes form the surface of a MAPS and a small matrix forms its core. As a result, the core is not immunogenic. MAPS have been used to prepare experimental vaccines against hepatitis (Tam et al., Proc. Natl . Acad. Sci . USA 86:9084, 1989), malaria (Tam et al. , J. Exp. Med. 171:299, 1990), and foot-and-mouth disease. A further advantage of MAPS is that they are chemically unambiguous. This allows different epitopes, such as B cell and T cell epitopes, to be arranged and a particular arrangement and stoichiometry.

European Patent Application 89200145.4 describes a process for preparing MAPS by reacting a branched structure based on an a ino acid such as lysine with a separately synthesized antigenic compound.

European Patent Application 89301288.0 describes peptides (CRIKQIINMWQEVGKAMYAPPISGQIRC (SEQ ID NO: 1), QSVEINCRTPNNNTRKSIRIQRGPGRAFVTIGK (SEQ ID NO: 2), and analogs thereof) which are specifically immunoreactive with antibodies to HIV and suggests that MAPS which include these peptides can be used for immunization to prevent HIV infection. Hart et al. (J. Immunol . , 145:2677, 1990) report that a synthetic peptide construct which includes amino acids 428-443 and 303-321 of HIV-I-III_B envelope protein gpl20, when used as a carrier-free immunogen in primates, can induce a high titer of neutralizing anti-HIV antibodies and can induce T cell proliferative response against native HIV-I gpl20.

Palker et al. (Immunology 142:3612, 1989) describes the use of a 16 amino acid T cell epitope from HIV-I-III_B fused to a synthetic peptide which includes a type-specific neutralizing determinant of a particular HIV-I strain (III_B, MN or RF) to immunize goats. Both T cells and B cells responded to epitopes within the type- specific neutralizing determinant.

PCT Application PCT/US90/02039 discloses multiple antigen peptide systems in which a large number of each of T cell and B cell malarial antigens are bound to the functional groups of a dendritic core molecule.

Summary of the Invention In general, the invention features a multiple antigenic peptide system including a dendritic core and a peptide, wherein the peptide includes the se uence IGPGR (SEQ ID NO: 3) , and the multiple antigen peptide system, when injected into a mammal, is capable of eliciting an immune response. In a preferred embodiment, the peptide includes a pair of six amino acid sequences flanking the sequence IGPGR (SEQ ID NO: 3) , the flanking sequences taken together having at least 36% ho ology to the pair of six amino acid sequences flanking the sequence IGPGR within the V3 loop of HIV-I-MN prototype virus.

The V3 loop sequence of the gpl20 envelope protein of HIV-I-MN includes the 35 amino acids of HIV-I-MN from the invariant cysteine at position 303 to the invariant cysteine at position 3-8, inclusive. The HIV-I-MN prototype virus is defined by a particular amino acid subsequence within the V3 loop region of the gpl20 envelope protein having the sequence KRKRIHIGPGRAFYTTK (SEQ ID NO: 4) . (Amino acid sequences are presented in the standard single-letter code throughout.)

In another preferred embodiment the peptide includes the sequence KRKRIHIGPGRAFYTTK (SEQ ID NO: 4).

In a preferred embodiment, the multiple antigenic peptide system includes a T cell epitope. In more preferred embodiments, the T cell epitope is covalently linked in tandem to the peptide; the T cell epitope includes the sequence QIINMWQEVGKAMYA (SEQ ID NO: 5) . By "T cell epitope" is meant a peptide capable of eliciting a proliferative T cell response. Preferably, the T cell epitope is at least seven amino acids long.

In other preferred embodiments, the dendritic core includes lysine; the dendritic core is tetravalent.

In another preferred embodiment, the peptide is between 10 and 40 amino acids long. In other preferred embodiments, the peptide includes the sequence HIGPGR (SEQ ID NO: 6) ; the peptide includes the sequence IHIGPGR (SEQ ID NO: 7) , the peptide includes the sequence RIHIGPGR (SEQ ID NO: 8) ; the peptide includes the sequence IGPGRA (SEQ ID NO: 9) ; the peptide includes the sequence IGPGRAF (SEQ ID NO: 10) ; the peptide includes the sequence KRKRIHIGPGRAFYTTKN (SEQ ID NO. 11) .

In a related aspect, the invention features a method of immunizing a mammal to inhibit HIV infection. The method includes administering to the mammal the multiple antigen peptide system described above.

In related aspect, the invention features a method for eliciting an immune response against HIV in a mammal. The method includes administering to the mammal the multiple antigen peptide system described above.

In another related aspect, the invention features a vaccine which includes an immunologically effective amount of the multiple antigen peptide system described above. In yet another related aspect, the invention features a method for generating antibodies. The method includes administering to a mammal an antibody-generating amount of the multiple antigen peptide system described above. Multiple antigen peptide system (MAPS) is the commonly used name for a molecule composed of two or more, usually identical, antigenic molecules covalently attached to a dendritic core which is composed of bifunctional units. The dendritic core molecule is a branching molecule in which a first bifunctional unit is linked to two additional bifunctional units each of which may be attached to two additional bifunctional units to form a third generation molecule. This pattern may be repeated any number of times to form higher generation molecules. For each molecule the number of free functional groups is equal to 2ⁿ, where n is equal to the generation of the molecule. A third generation molecule thus has 8 free functional groups which can be attached to 8 peptides. Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

Detailed Description The drawings are first briefly described.

Figure 1 is a schematic representation of several tetravalent MAPS and the sequences of the peptides attached to the lysine core. T cell epitopes are represented by the shaded rectangles in the schematic drawings; B cell epitopes are represented by unshaded rectangles. The amino acid sequences of peptides B1-B9 and T are listed.

Figure 2 is a pair of graphs which depict the results of ELISA assays used to measure mouse antisera binding to HIV-I-III_B peptides (panel A) and HIV-I-III_B gpl20 (panel B) . Antisera were raised using Bl peptide (filled circles) , B2 peptide (open circles) , and B3 peptide (filled squares) . Antisera, serially diluted, were tested for their binding activity to wells coated either with 5 μg of the same peptide used to raise the antisera (panel A) or 0.1 /g of recombinant gpl20 (panel B) . Goat anti-mouse IgG was used as secondary antibody. The mean absorbance (405 nm) is plotted as a function of the reciprocal dilution of antisera. Figure 3 is a graph which depicts the results of ELISA assays used to measure mouse antisera binding to peptide B4T. Antisera were obtained after three intraperitoneal immunizations with MAP-B4T (solid circles) or B4T peptide (open squares) . The mean absorbance (405 nm) is plotted as a function of the reciprocal dilution of antisera.

HIV-I-MN and HIV-I-MN Viral Variant Peptides in MAPS In the experiments described below MAPS which include peptides derived from the V3 loop of the HIV-I external envelope protein (gpl20) were used to raise antisera in mice, rabbits and guinea pigs. The V3 loop of gpl20 includes all of the amino acids from cysteine 303 to cysteine 338 of HIV-I. In intact gpl20 a disulfide bond between these two cysteines forms a loop (V3 loop) . The V3 loop represents one of the most variable regions of the envelope protein. Among various HIV-I isolates the sequence of the V3 loop varies by as much as 50%. Despite this variability a relatively conserved GPGR sequence lies at the tip of the loop. This is flanked on both sides by more variable strain- specific sequences. Analysis of the amino acid sequences of the V3 loop of 245 different HIV-I isolates revealed that the V3 loop sequence of HIV-I-MN differs from the consensus at only 6 of 35 amino acid positions (La Rosa et al. Science 249:932, 1990).

Multiple antigen peptide system (MAPS) is the commonly used name for a combination antigen/antigen carrier that is composed of two or more, usually identical, antigenic molecules covalently attached to a dendritic core which is composed of bifunctional units. The dendritic core of a multiple antigen peptide system can be composed of lysine molecules. For example, a lysine is attached via peptide bonds through each of its amino groups to two additional lysines. This second generation molecule has four free amino groups each of which can be covalently linked to an additional lysine to form a third generation molecule with eight free amino groups. A peptide may be attached to each of these free groups to form an octavalent multiple peptide antigen. Alternatively, the second generation molecule having four free amino groups can be used to form a tetravalent MAPS, i.e. , a MAPS having four peptides covalently linked to the core. Many other molecules, including aspartic acid and glutamic acid, can be used to form the dendritic core of a multiple peptide antigen system. The dendritic core, and the entire MAPS may be conveniently synthesized on a solid resin using the classic Merrifield synthesis procedure.

Multiple antigen peptide systems have many advantages as antigen carrier systems. Their exact structure and composition is known; the ratio of antigen to carrier is quite high; and several different antigens, e.g., a B cell epitope and a T cell epitope, may be attached to a single dendritic core. When both a B cell epitope and a T cell epitope are present it is preferable that they are linked in tandem on the same functional group of the dendritic core. Alternatively the T cell epitope and the B cell epitope may be on separate branches of the dendritic core. Preferably, the T cell epitope is a helper T cell epitope; however a cytotoxic T cell epitope may also be used. Useful T cell epitopes may be derived from the HIV-I envelope protein. However, it is not necessary that the B cell epitope and the T cell epitope both be derived from the HIV-I gpl20 envelope protein. T cell epitopes from different HIV-I proteins (e.g., those encoded by the nef , gag, tat, rev, vif, pol , vpr, vpu , or vpx genes), different retrovirus, or unrelated organisms (e.g., malarial antigens or tetanus toxoid) may be used. T cell epitopes can be identified by a T cell proliferation assay (described herein below) . Multiple antigen peptide systems and methods for their preparation are described more fully in PCT Application WO 90/11778, and European Patent Application 89200145.4 both of which are hereby incorporated by reference. In the experiments described herein MAPS that include peptides derived from the V3 loop of the gpl20 protein of HIV-I-MN are shown to raise potent antisera. Accordingly, vaccines which employ HIV-I-MN MAPS are expected to be particularly useful for generating an immune response in humans.

Also described below are experiments which demonstrate that the addition of a T cell epitope often increases the immunogenicity of HIV-I MAPS. Peptides derived from the V3 loop of HIV-I-MN are capable of raising broadly neutralizing antibodies. Such antibodies can block infection of cultured cells by a wide range of HIV-I strains (PCT Patent Application PCT/US90/03157, hereby incorporated by reference) . Accordingly, MAPS which employ peptides derived from the V3 loop of HIV-I-MN are expected to generate similarly broadly neutralizing antibodies. Experimental Procedures

The experiments described herein were performed according to the procedures described below.

Animals Outbred CD-I mice and New Zealand White rabbits were purchased from Charles River Laboratories (Wilmington, MA) . Outbred Dunkin-Hartley guinea pigs were raised and immunized by Hazelton Biotechnologies Company (Denver, PA) .

Synthesis of peptides Synthetic peptides were prepared manually by a stepwise solid-phase peptide synthesis (Tam, Proc. Nat 'l . Acad . Sci . USA 85:5409, 1988; Merrifield, Science 232:341, 1986) on t-butoxycarbonyl (Boc)-Ala-OCH₂-Pam resin (Mitchell et al., J. Am . Chem .

Soc. 98:7357) or p-alkoxybenzyl alcohol resin. The mono- epitope peptides were synthesized by Boc-benzyl chemistry. The di-epitope peptides were synthesized by Fmoc-tertbutyl chemistry. The coupling was mediated with DCC/1-hydroxybenzotriazole in dimethylformamide. After completion of synthesis, each MAP-resin was treated with a deprotecting reagent to remove N-protecting groups and cleaved with low-high hydrofloride (Boc-chemistry; Tam et al., J^". Am Chem . Soc. 105:6441, 1983) or 95% trifluoroacetiσ acid (Fmoc-chemistry) . The peptide was extracted in 8M urea in 0.1M Tris-HCl, pH 8, then dialyzed several times and lyophilized. All MAPS gave satisfactory amino acid analysis. Preparation of MAPS The preparation of MAPS is described by Posnett et al. (J. Biol . Chem . 263:17179, 1988) and Tam et al. (Proc. Nat 'l . Acad. Sci . USA 85:8409, 1988). The process essentially employs conventional solid-phase peptide synthesis as described by Merrifield (J^". Am. Chem . Soc. 85:2149, 1963). Briefly, a resin coupled to a t-butoxycarbonyl-substituted (Boc-substituted) amino acid is reacted with 50% trifluoroacetic acid to remove Boc and the resulting salt is neutralized with diisopropoylethylamine. The first part of the lysine core is then added by reacting the amino acid-coupled resin with Boc-Lys(Boc) in dimethylformamide followed by reaction with dicyclohexylcarbodiimide and CH₂C1₂- A second such synthetic cycle yields a branched tetrapeptide on which four peptides may be synthesized using conventional solid-phase synthesis techniques. It is also possible to separately synthesize the lysine core and the peptides and then couple the peptides to the core in a subsequent step (European Patent Application 89200145.4) .

It may be desirable to use peptides which have been circularized prior to attachment to the dendritic backbone. This circularization can be accomplished via disulfide bond formation between cysteines present in the peptide. Such an arrangement is particularly desirable for peptides derived from the V3 loop of gpl20 since the sequence in this region forms a loop in the intact virus. Immunization procedure The animals were injected four times, at two week intervals, using complete Freund's adjuvant (Sigma, St. Louis, MO) for the first injection, and incomplete Freund's adjuvant for the booster injections. Mice (five for each peptide) received intraperitoneally 50 μg of the peptide for each injection. Guinea pigs (three for each group) received subcutaneously 100 μg of the peptides the first and second injections and 50 μg for the last two injections. Rabbits (two for each peptide) were injected intradermally with 400 μg (first injection, day 0) and 200 μg (second injection, day 14) and subsequently intramuscularly with 200 μg of the peptides at day 28 and day 42. The animals were bled immediately before each injection. The antisera used for the reported experiments were obtained fifteen days after the last injection.

ELISA Assays Mouse and rabbit antisera were analyzed by standard direct ELISA using flat-bottomed microplates (Maxisorp, Nunc, Denmark) coated with 5 g/well of each peptide or 0.1 μg/well of purified recombinant gpl20 (Repligen, Cambridge, MA) . The assays of the guinea pig sera were performed using plates coated with the 24 amino acid peptides, RP135 (III_B) , RP139 (RF) , and RP142 (MN) (Rusche et al., Proc . Nat 'l Acad Sci . USA 85:3198, 1988). The plates were blocked for 90 min at 37°C with the diluent buffer (PBS + 1% calf serum) . Incubation with antisera, serially diluted in the same buffer, was carried out for 2 hr at 37°C and was followed by three washes with 0.05% Tween 20 in PBS. Phosphatase- conjugated goat secondary antibody (Sigma) , diluted 1:1000, was then added for 2 hr at 37°C. After an additional three washes, the substrate p-nitrophenyl phosphate (1 mg/ l; Sigma) in diethanolamine buffer (pH 9.8) was added, and the bound secondary antibody was detected at 405 nm. The antibody titer was calculated as the reciprocal of the antiserum dilution giving the half maximal response. The optical density obtained with preimmunization sera was always less than 0.1 units. The recombinant virus expressing the env gene is used to infect CD4+ cells (e.g., CEM, M0LT4, or SUP-T1 cells) . The HIV envelope protein presented on the surface of these cells will bind to the cell surface receptor, CD4, resulting in the fusion of the cells and the formation of giant multinucleated cells called syncytia. Syncytium formation can be assayed in the presence or absence of antiserum at a series of dilutions. The number of syncytia that are formed are quantified at an appropriate time post-infection. The preferred MAPS are those which raise antisera that inhibits syncytia formation even when the antisera is substantially diluted.

In the experiments described below CD4-positive CEM cells (Accession Number CCL119, American Type Culture Collection, Rockville MD) were infected with recombinant vaccinia viruses expressing full length gpl60 at a multiplicity of infection of 1. For HIV-I-III_B and HIV-I-RF, the recombinant virus expressed the entire envelope gene. For HIV-I-MN the recombinant virus expressed the V3 region of HIV-I-MN inserted into the gpl60 of HIV-I-III_B as described by Scott et al. (Proc. Nat 'l . Acad. Sci . USA 87:8597, 1990). Immune sera were added to the cultures 1 hr post-infection and syncytia were counted 24 hr post-infection. The fusion inhibition titer for each immune serum is defined as the reciprocal of the dilution which reduces the number of syncytia to 10% of the number observed in the presence of a normal serum control.

Antisera can also be assessed using a viral neutralization assay. In this assay viral reverse transcriptase activity is used as a measure of viral activity. Dilutions of antiserum are incubated with HIV and are than added to HIV susceptible CD4+ cells. Such an assay is described by Robey et al., Proc . Nat 'l . Acad. Sci . USA 83:7023, 1986; Popovic et al., Science 224:497, 1984; and Robert-Guroff, Nature 316:72, 1985). PEPSCAN Antigen domains can be identified by pepscan analysis as described by Geyson et al. (J. Immunol . Methods 102:259, 1987). Briefly, overlapping peptides derived from the sequence of a peptide to be analyzed can be synthesized on the tips of polyethylene rods. The rods are then assembled into a holder with the format of a microtiter plate. All the subsequent reactions can be carried out at the tips of the rods using a microtiter plate. Nonspecific binding can be avoided by the incubation for 1 hr at room temperature with diluent buffer. The rods are then incubated with diluted antiserum for 16 hr at 4°C, washed 4 times in 0.05% Tween 20 in PBS and incubated with a secondary antibody, (e.g., goat anti-mouse or anti-rabbit IgG) coupled to alkaline phosphatase for 1 hr at room temperature. The presence of the conjugate antibody on the tips can then be detected by reaction with substrate solution. Syncytia Inhibition Assay A recombinant vaccinia virus syncytium inhibition assay can be used to assess the effectiveness of antisera raised using the MAPS herein described. This assay can be performed using cells infected with a vaccinia virus expressing an HIV env gene rather than actual HIV infected cells. Construction of a recombinant vaccinia virus capable of expressing the full-length HIV envelope gene from a vaccinia virus promoter is described in EP Publication No. 0 243 029, hereby incorporated by reference. Antibody Response to Mono-Epitope MAPS Nine peptides from the V3 regions of HIV-I isolates III_B, RF and MN were incorporated into tetravalent MAPS (prepared as described above) . These MAPS are designated MAP Bl through B9 to indicate that they include a B cell epitope. Referring to Fig. 1, parallel groups of three peptides with chain lengths spanning from 11 to 24 residues were synthesized in MAPS format for each isolate. Tetravalent MAPS were prepared since they have been shown to be as effective as the octavalent MAPS (Tam et al., J. Exp. Med. 171:299, 1990). Mice, rabbits and guinea pigs were immunized with one of the nine mono-epitope MAPS. The antisera were analyzed for reactivity against both the immunizing peptide (Fig. 2, panel A) and gpl20 (Fig. 2, panel B) in an ELISA assay.

Referring to Fig. 2, panel A, ELISA assays demonstrated that antisera titers in mice were closely related to the length of the III_B peptide used for the immunization, with the MAPS using the longest peptide (Bl, amino acids 308-331) inducing the strongest response, followed by the MAPS using the intermediate peptide (B2, amino acids 312-328) which elicited a reduced reactivity, while the MAPS using the shortest peptide (B3, amino acids 315-325) was completely non- immunogenic. The same pattern of ELISA reactivity was observed against native gpl20 protein (Fig. 2, panel B) . The good response elicted by the Bl MAPS suggests that the Bl peptide, derived from the HIV-I-III_B sequence, contains a T helper cell determinant. Evidently, this epitope is completely lost when the peptide is reduced to only 11 amino acids as in the B3 peptide. This was confirmed by the induction of specific proliferative response in the lymph nodes of mice immunized with the Bl peptide and not with the B3 peptide (described in detail below) . The presence of a T cell epitope within this portion of the V3 loop in a III_B peptide (residues 303- 321) was observed by others in goats (Hart et al., J. Immunol . 145:2677, 1990). Moreover (Table 1), there was no substantial antibody production in mice against the two other series of peptides, RF (B4-B6) and MN (B7-B9) , except for a low reactivity in the group immunized with B8 (MN isolate) .

Referring to Table 1, ELISA assay of the rabbit antisera showed that nearly all of the mono-epitope MAPS were able to elicit strong responses; antibody titers varied from the highest value (1.2 x 10⁶) in the antisera of rabbits immunized by the B2 and B8 peptides, to the lowest (2.3 x 10⁴) found in the antisera of rabbits injected with the shortest peptide of the RF series (B6) .

Referring to Table 2, the responses of the guinea pigs were uneven. While all MAPS having a peptide derived from the HIV-I-III_B isolate (B1-B3) produced good responses, only the two longer of the sequences of the RF isolate (B4 and B5) and the MN isolate (B7 and B8) were able to elicit a response.

Table 1: Comparison of mouse and rabbit antibody response to mono-and di-epitope MAPS

Synthetic HAP peptides containing HIV-I B cell or B cell and T cell epitopes from different HIV- i isolates were used to inoculate animals. The resulting immune sera were assayed by ELISA against the immunizing peptide; the antibody titers are presented as the geometric mean (±SEM) of endpoint dilutions corresponding to antisera from five (mice) or two (rabbits) animals.

Table 2: Comparison of guinea pig antibody response to mono-and di-epitope MAPS

ELISA with guinea pig antisera was performed using plates coated with 24 amino acid peptides RP135 (IIIB), RP139 (RF), or RP142 (MN). Titers are the geometric mean (±SEM) of endpoint dilutions of the antisera from three animals.

Antibody Response to Di-Epitope MAPS Mice, rabbits and guinea pigs were immunized with one of the nine di-epitope MAP constructs. Each di- epitope MAPS contains a tandem configuration in which a T-helper cell peptide derived from HIV-I envelope protein was added at the carboxyl-end of each B cell peptide. Accordingly, these MAPS are referred to as MAP BIT through MAP B9T to indicate that they include a T cell epitope in addition to one of the B cell epitope peptides, B1-B9. A 16 amino acid peptide, located in the fourth conserved domain of gpl20 (III_B isolate, residues 429-443) , was selected because it stimulates T helper cell activity in mice (Cease et al., Proc. Nat 'l . Acad . Sci . USA 84:4249), and probably humans (Berzofsky et al.. Nature 334:706, 1988) and goats (Palker et al., J. Immunol . 142:3612, 1989).

As summarized in Table 1, addition of a T cell peptide in the di-epitope MAPS constructs used for the immunization substantially increased mouse immune response. When mice were injected with the longest di- epitope MAP of the III_B series (BIT MAPS) , the immune response was only slightly higher than that induced by the corresponding mono-epitope MAPS (Bl MAPS) , probably because a T-helper epitope is present in the Bl sequence. However, a much improved antibody titer was observed in the mice injected with B2T MAPS than in those receiving the intermediate-length mono-epitope B2 MAP. In contrast to the response elicited by B3 MAPS, a very strong antibody induction was observed using B3T MAPS. Similar findings were also observed for RF and MN series MAPS; totally non-immunogenic mono-epitope MAPS, such as B4, B5, B7 and B9, stimulate humoral immune responses in the di-epitope MAPS, B4T, B5T, B7T and B9T.

In rabbits (Table 1) , antisera elicited by each of the di-epitope MAPS showed greater immunoreactivity than antisera raised against the corresponding mono-epitope MAP constructions. The enhancement of immune responses in rabbits were less marked than in mice, since in rabbits the mono-epitope MAP constructs alone were able to elicit strong antibody responses, without the use of an additional T-helper epitope.

Referring to Table 2, in guinea pigs, the addition of a T-helper epitope did not consistently enhance the immune response induced by mono-epitope MAPS in the RF series.

In the one case tested, the comparative immunization of mice with a di-epitope peptide in MAP configuration (MAP-B4T) and the corresponding linear peptide (B4T) revealed that a greater immune response was induced by the tetrameric MAP. Specificity of the Antibody Response

To characterize the specificity of the B cell response to MAPS Bl and BIT, antisera were assayed for their reactivity to B cell epitope peptides and T cell epitope peptides (Table 3) . The mono-epitope Bl MAP induced antisera which reacted with Bl peptide; the di- epitope BIT MAP induced antisera which reacted with the T peptide in addition to the Bl peptide. These results demonstrate that both B and T epitopes are immunogenic and that the T cell epitope also serves as a B cell epitope. Furthermore, these results show that the antisera are specific, since the Bl MAPS induced antisera did not react with the T peptide.

Table 3: Specificity of the Antibody Response to Mono-or Di-Epitope MAPS

Peptide blocking ELISA assays were performed with a dilution of rabbit antisera giving an absorbance between 1.3 and 1.6. The wells were coated with 5 μg of the indicated peptide. The competing peptide was present at 50 g/ml.

Cross-Reactivity of the Antibody Response Among HIV Isolates Rabbit antisera were analyzed for their ability to react with peptides derived from HIV-I isolates other than the ones used to raise them.

Referring to Table 4, there was a moderate level of cross-reactivity between III_B and RF isolates. The antisera to Bl peptide from III_B was able to bind the homologous B4 peptide from RF, although less strongly. Conversely, the B4 antisera reacted with Bl peptide. In contrast, antisera from rabbits immunized with B7 peptide from MN isolate did not react with either Bl or B4 peptides but preferentially only with its own peptide. The cross-reactivity between III_B and RF peptides is likely due to the close homology of the Bl and B4 peptides at their a ino-terminus. Seven consecutive amino acids (position 308-314) , preceding the conserved central tripeptide GPG, are the same in these two sequences. Indeed, analysis of rabbit antisera raised using MAP-B4 showed a strong cross-reactivity to BIT peptide only over the first 8 amino acids of BIT. In contrast, B7 antisera (MN isolate) was far less reactive with Bl peptide. The Bl and B7 peptides share little homology at the amino-end of each sequence. Thus it appears that the observed cross-reactivity results from recognition of related sequences at the amino terminus of the III_B and RF peptides.

Table 4: Crossreactivity of MAP-induced Antisera^a

Anti-B1 MAP sera Anti-B4 MAP sera Anti-B7 MAP sera

B1 peptide 350.000 B4 peptide = 570,000 B7 peptide = 660,000 B4 peptide 30,000 B1 peptide = 41,000 B1 peptide = 25,000 B7 peptide 4,000 B7 peptide = — B4 peptide = — gp120-IIIB 90,000 gp120-IIIB = gp120-IIIB = —-

° Rabbits were inmunized with the indicated MAP, and the immune sera were analyzed for their ability to bind resin-bound peptides or gp!20-III_β- Results are expressed as the geometric mean of several ELISA titers. Proliferative T Cell Response to BIT and B3T MAPS

A T cell poliferation assay was performed using BIT MAPS and B3T MAPS. Lymph node cells from antigen-primed

Balb/c mice were cultured in vitro with the same BT MAPS used for the immunization or with the corresponding B cell epitope MAPS. As shown in Table 5, lymph node cells from mice immunized with BIT MAPS proliferated when cultured with Bl MAPS (which appears to have a helper deteminant) and even more strongly when the BIT MAPS was used for the in vitro stimulation. Further, the B3T

MAPS-primed cells exhibited a high level of proliferation following stimulation with the di-epitope B3T MAPS. In contrast, the B3 peptide did not induce proliferation.

Table 5: Proliferative T-cell Response Induced by BIT MAPS and B3T MAPS

T cell primed witha Restimulated withb

Index

B1T MAPS

B1 MAPS BIT MAPS

B3T MAPS

B3 MAPS B3T MAPS

. Mice (3 Balb/c per group) were injected with 50/ιg MAPS in Fruend's complete adjuvant. Restimulated with 10/ιg/ml MAPS in virto.

Biological Activity of the Immune Response The biological activity of the rabbit and guinea pig MAPS induced antisera were tested in a syncytium inhibition assay, utilizing CEM cells infected with a recombinant vaccinia virus expressing gpl60 of the III_B, RF or MN strain. Titers obtained in such an assay generally correlate with the ability of sera to neutralize cell free virus in vitro. The results of this assay are presented in Tables 6 and 7 which lists the reciprocal of the antiserum dilution which reduces the number of syncytia formed by 90%. Table 6: MAPS-Induced Rabbit antisera syncytia inhibition titers against different HIV-I isolates^*1 πi„ RF MN —!

B1 = 20, <20 B4 = 40, 40 B7 = 160, 320

B2 = <20, 40 B5 = 80, <20 B8 = 640, 640

B3 = 40, <20 B6 = <20, <20 B9 = 160, <20 B1T = 320, 80 B4T = 160, 160 B7T = 320, 320

B2T = <20, 40 B5T = 40, 40 B8T = 160, 640

B3T = <20, <20 B6T = 160, 80 B9T = <20, 40 ^a Rabbit irrrnune sera were tested for their ability to inhibit HIV-I-1 envelope protein- nduced syncytium formation. Fusion inhibition titer is the reciprocal of the dilution that reduces the runber of syncytia by 90%. Serum from two rabbits in each group was analyzed.

Table 6 demonstrates that the majority of the rabbits developed antisera that inhibited syncytia formation in a culture expressing envelop of the same strain used as an immunogen. The data suggest that inhibitory antibodies are elicited primarily by the di- epitope MAPS for the RF series and to some extent for the III_B series. In rabbits, the T cell epitope does not seem to influence the response of MAPS in the MN series which appears to be significantly greater than the response elicited by MAPS in the III_B and RF series. For instance, in the III_B series, BIT MAP induced an antisera which was better at inhibiting syncytia formation than Bl MAP. A clear correlation between the titer observed in ELISA assay and the syncytium inhibition activity of the antibodies was not evident, since antisera which show very high peptide binding capacity were less effective than others in inhibiting syncytium formation.

Table 7 presents the results of similar syncytium inhibition assays in guinea pigs. These data show that MAPS in the MN series elicit a better immune response than MAPS in the RF or III_B series and that the B8 peptide appears to be the most effective peptide in the MN series. Table 7: MAPS-Induced Guinea Pig antisera syncytia inhibition titers against different HIV-I isolates^**1

^a Guinea pig itαmune sera were tested for their ability to inhibit HIV-I-1 envelope protein- induced syncytium formation. Fusion inhibition titer is the reciprocal of the dilution that reduces the number of syncytia by 90%. Serum from three guinea pigs in each group was analyzed.

Therapy

The MAPS of the invention may be administered by direct injection into the blood stream. They may also be incorporated into polymeric microcapsules (e.g., liposomes) and/or administered with an adjuvant (e.g., alum) . Liposome-encapsulated antigens often elicited a higher antibody titer than non-encapsulated antigens.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANTS: REPLIGEN CORPORATION

THE ROCKEFELLER UNIVERSITY

(ii) TITLE OF INVENTION: MULTIPLE ANTIGEN PEPTIDES FOR USE AS HIV VACCINES

(iii) NUMBER OF SEQUENCES: 21

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Fish & Richardson

(B) STREET: 225 Franklin Street

(C) CITY: Boston

(D) STATE: Massachusetts

(E) COUNTRY: U.S.A.

(F) ZIP: 02110-2804

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: 3.5" Diskette, 1.44 Mb

(B) COMPUTER: IBM PS/2 Model 50Z or 55SX

(C) OPERATING SYSTEM: IBM P.C. DOS (Version 3.30)

(D) SOFTWARE: WordPerfect (Version 5.1)

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 744,281

(B) FILING DATE: 13 August 1992

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Paul T. Clark

(B) REGISTRATION NUMBER: 30,162

(C) REFERENCE/DOCKET NUMBER: 00231/052WO1

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (617) 542-5070

(B) TELEFAX: (617) 542-8906

(C) TELES: 200154

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 1: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: Cys Arg lie Lys Gin lie lie Asn Met Trp Gin Glu Val Gly Lys Ala

5 10 15

Met Tyr Ala Pro Pro lie Ser Gly Gin lie Arg Cys 20 25

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 2: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Gin Ser Val Glu lie Asn Cys Arg Thr Pro Asn Asn Asn Thr Arg Lys

5 10 15

Ser lie Arg lie Gin Arg Gly Pro Gly Arg Ala Phe Val Thr lie Gly 20 25 30

Lys

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: lie Gly Pro Gly Arg

5

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Lys Arg Lys Arg lie His lie Gly Pro Gly Arg Ala Phe Tyr Thr Thr

5 10 15

Lys

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: Gin lie lie Asn Met Trp Gin Glu Val Gly Lys Ala Met Tyr Ala

5 10 15

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

His lie Gly Pro Gly Arg

5

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: lie His lie Gly Pro Gly Arg

5

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

Arg lie His lie Gly Pro Gly Arg

5

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: lie Gly Pro Gly Arg Ala

5

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7

(B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

He Gly Pro Gly Arg Ala Phe 5

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

Lys Arg Lys Arg He His He Gly Pro Gly Arg Ala Phe Tyr Thr Thr

5 10 15

Lys Asn

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

Asn Asn Thr Arg Lys Ser He Arg He Gin Arg Gly Pro Gly Arg Ala

5 10 15

Phe Val Thr He Gly Lys He Gly 20

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

Lys Ser He Arg He Gin Arg Gly Pro Gly Arg Ala Phe Val Thr He

5 10 15

Gly

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

Arg He Gin Arg Gly Pro Gly Arg Ala Phe Val

5 10 (2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 15: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:

Asn Asn Thr Arg Lys Ser He Thr Lys Gly Pro Gly Arg Val He Tyr

5 10 15

Ala Thr Gly Gin He He Gly 20

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

Thr Arg Lys Ser He Thr Lys Gly Pro Gly Arg Val He Tyr Ala Thr

5 10 15

Gly

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

Ser He Thr Lys Gly Pro Gly Arg Val He Tyr

5 10

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:

Pro Asn Tyr Asn Lys Arg Lys Arg He His He Gly Pro Gly Arg Ala

5 10 15

Phe Tyr Thr Thr Lys Asn He He 20

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 19:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:

Lys Arg Lys Arg He His He Gly Pro Gly Arg Ala Phe Tyr Thr Thr

5 10 15

Lys Asn

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:

Arg He His He Gly Pro Gly Arg Ala Phe Tyr Thr

5 10

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: in He He Asn Met Trp Gin Glu Val Gly Lys Ala Met Tyr Ala

5 10 15

Claims

1. A multiple antigen peptide system comprising a dendritic core covalently attached to a peptide, said peptide including the sequence IGPGR (SEQ ID NO: 3), said multiple antigen peptide system, when injected into a mammal, being capable of eliciting an immune response.

2. The multiple antigen peptide system of claim 1 wherein said peptide includes a pair of six amino acid sequences flanking said sequence IGPGR (SEQ ID NO: 3) , said flanking sequences taken together having at least 36% homology to the pair of six amino acid sequences flanking the sequence IGPGR within the V3 loop of HIV-I- MN prototype virus.

3. The multiple antigen peptide system of claim 1 wherein said peptide includes the sequence

KRKRIHIGPGRAFYTTK (SEQ ID NO: 4).

4. The multiple antigen peptide system of claim 1 further comprising a covalently attached T cell epitope.

5. The multiple antigen peptide system of claim 4 wherein said T cell epitope is covalently linked in tandem to said peptide.

6. The multiple antigen peptide system of claim 4 wherein said T cell epitope includes the sequence QIINMWQEVGKAMYA (SEQ ID NO: 5).

7. The multiple antigen peptide system of claim 1 wherein said dendritic core includes lysine.

8. The multiple antigen peptide system of claim 1 wherein said dendritic core is tetravalent.

9. The multiple antigen peptide system of claim 1 wherein said peptide is between 10 and 40 amino acids long.

10. The multiple peptide antigen system of claim 1 wherein said peptide includes the sequence HIGPGR (SEQ

ID NO: 6) .

11. The multiple antigen peptide system of claim

10 wherein said peptide includes the sequence IHIGPGR (SEQ ID NO: 7) .

12. The multiple antigen peptide system of claim

11 wherein said peptide includes the sequence RIHIGPGR (SEQ ID NO: 8) .

13. The multiple antigen peptide system of claim

12 wherein said peptide includes the sequence IGPGRA (SEQ ID NO: 9) .

14. The multiple antigen peptide system of claim

13 wherein said V3 loop peptide includes the sequence IGPGRAF (SEQ ID NO: 10).

15. The multiple antigen peptide system of claim 14 wherein said V3 loop peptide includes the sequence

KRKRIHIGPGRAFYTTKN (SEQ ID NO: 11) .

16. A vaccine comprising an immunologically effective amount of the multiple antigen peptide system of claim 1.