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  • A61K39/00—Medicinal preparations containing antigens or antibodies
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  • C12N2740/10011—Retroviridae
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  • C12N2740/00011—Details
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  • C12N2740/16011—Human Immunodeficiency Virus, HIV
  • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
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  • C12N2740/16211—Human Immunodeficiency Virus, HIV concerning HIV gagpol
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  • C12N2740/16334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

• This invention relates to a vaccination process, which involves the simultaneous or consecutive use of a priming antigen, in this case the glycoprotein from a virus, such as HIV, SIV or any lentivirus capable of inducing AIDS in its natural host, or from an HTLV-I or HTLV-II type retrovirus, and an amplifying composition comprised of synthetic oligopeptides, which are free or bound to. a carrier molecule and in which the oligopeptides correspond to the neutralization epitopes for this same glycoprotein.
• a priming antigen in this case the glycoprotein from a virus, such as HIV, SIV or any lentivirus capable of inducing AIDS in its natural host, or from an HTLV-I or HTLV-II type retrovirus
• an amplifying composition comprised of synthetic oligopeptides, which are free or bound to.
• a carrier molecule and in which the oligopeptides correspond to the neutralization epitopes for this same glycoprotein also relates to
• An effective vaccine composition against viruses must produce rapid neutralization of the viruses in order to prevent the viruses from possibly protecting themselves in a latent provirus form within the chromosomes of resting cell or from finding refuge in the cellular or tissue compartment where they would be beyond the reach of the immune system.
• virus envelope glycoprotein does not make it possible to obtain a fully protective immune response.
• the virus envelope glycoprotein does not produce a sufficient level - neutralizing antibodies in order to provide protection against infection. Accordingly, there exists a need in the art for a method of inducing a sufficient level of neutralizing antibodies against virus infection in a host susceptible to the infection by the virus. In addition, there exists a need in the art for a pharmaceutical composition for use in the method.
• An object of this invention is to reinforce the immunogenicity of at least one envelope glycoprotein of a virus by combining the glycoprotein with at least one peptide, and preferably at different times a group of peptides, derived from the sequence of the envelope glycoprotein and corresponding to virus-neutralization epitopes, i.e. corresponding to amino acid sequences involved in the production of neutralizing antibodies in the host to which they are administered.
• this invention provides a method of enhancing the immunogenicity of an envelope glycoprotein of a virus in a host and a composition for use in this method.
• the method comprises administering to the host at least one envelope glycoprotein of the virus and at least one peptide derived from the amino acid sequence of the envelope glycoprotein.
• the peptide comprises at least one virus- neutralization epitope.
• the envelope glycoprotein and the peptide are administered in an amount sufficient to induce neutralizing antibodies in the host.
• the invention provides a composition for enhancing the immunogenicity of an envelope glycoprotein of a determined virus, wherein the composition comprises as a combined preparation for simultaneous, separate, or sequential use:
• composition is intended to comprise combined preparation in which the components - in this case the envelope glycoprotein and the peptide or peptides derived from the envelope glycoprotein - can be presented in a mixture or can be presented side-by- side and therefore be applied simultaneously, separately or at intervals, to the host.
• the peptide(s) present in the composition can be maintained separated from other components in order to be administered sequentially to booster the immunogenic reaction which is primed with the envelope glycoprotein.
• the invention provides a composition which comprises the above envelope glycoprotein and peptide providing the envelope glycoprotein is present in an amount sufficient for priming the induction of neutralizing antibodies in a host to which it is administered, and the at least one peptide is in an amount sufficient to enhance the induction of persistent neutralizing antibodies in the host to which it is administered.
• the invention concerns the use of at least one of the above described peptides for enhancing the immunogenicity of an envelope glycoprotein of a virus, when this glycoprotein is administered to a host to induce neutralizing antibodies.
• composition of the invention can be used for the preparation of an immunotherapeutic drug.
• the composition is administered to seropositive people in order to increase the level of neutralizing antibodies and accordingly to enable a control of the virus.
• This invention also provides a composition for vaccinating a host against infection by a virus.
• the composition comprises at least one envelope glycoprotein of the virus in an amount sufficient for priming vaccination in a host to which the envelope glycoprotein is administered.
• the composition also contains at least one peptide derived from the amino acid sequence of the envelope glycoprotein.
• the peptide comprises at least one virus-neutralization epitope of the glycoprotein.
• the composition contains the peptide an amount sufficient to enhance the induction of persistent neutralizing antibodies in the host.
• Peptides and envelope glycoproteins can be combined under conditions allowing them to interact by non-covalent physical combination or by covalent chemical bonding.
• a priming vaccination is achieved by injections of envelope glycoprotein, with protective immunity being subsequently enhanced by the injection of immunogenic peptides corresponding to the neutralization epitopes.
• Fig. 1 depicts anti-HIV antibody level measured by ELISA (Genetic Systems Kit) in chimpanzee FUNFACE (C-339) and a control (C-519). The results are shown as serum ELISA titre (lsdilution giving positive response) versus time. Time zero in the Figure corresponds to the day of the first booster with inactivated HIV. The animal was challenged at 70 weeks (arrow) .
• Fig. 2 depicts neutralizing antibody level in chimpanzees FUNFACE (dark circles) and ROBERT (open circles) in response to the injection of a KLH-BRU peptide conjugate (arrows).
• the animals were inoculated at 0, 3, and 19 weeks (arrows) and challenged at 24 weeks.
• Fig. 3 depicts anti-HIV antibody levels measured by ELISA in chimpanzee ROBERT (C-433). The results are shown as serum ELISA titre (l:dilution giving positive response) versus time. Time zero corresponds to the day of the first antigen injection (gpl ⁇ Oenv, p27nef, p23vif, and pl ⁇ gag) . The animal was challenged at 84 weeks (arrow).
• Fig. 4 depicts neutralization of HIV-1 BRU as a function of the serum dilution in chimpanzees JOJOTOO (499), IRA (151), and HENRY II (531) at time tO ([•]) and at 2 weeks ([] ) and 5 weeks ( ⁇ ) after a third inoculation of free peptides.
• Fig. 5 depicts neutralization of HIV-1 BRU (dotted curves) and HIV-1 ARV-2 (solid curves) as a function of the serum dilution in chimpanzee JOJOTOO (C-499) at time tO ([.]) and after the third inoculation of free peptides ( ⁇ ).
• Fig. 6 shows total HIV-1-specific antibody titers for chimpanzees C-339 (A), C-433 (B), and C-499 (C). At the indicated times, chimpanzees were inoculated with various immunogens (see Table 1) or challenged with HIV-1. Titers are defined as the reciprocal of the highest dilution of serum that was positive using an HIV-1 ERA kit (Genetic Systems) .
• Fig. 7 depicts neutralizing antibody titers in serum from C-339, C-433 and C-499 during immunization with HIV-1 antigens.
• Titers are the reciprocal of the highest dilution of serum that gave 90% reduction in number of syncytia formed by CEM-SS cells (Nara, P.L., Hatch, W.C., Dunlop, N.M., Robey, W.G., Arthur, L.O., Gonda, M.A. & Fischinger, P.J. (1987) AIDS Res. Human Retroviruses 3, 283-302.) when compared to that obtained with control serum from a naive chimpanze .
• Fig. 8 shows PCR analysis of DNA from PBMC and lymph node tissue obtained 6 months after challenge of chimpanzees C-339 and C-433 with HIV-1.
• Lane 1 0.5 ⁇ g of 0X174 DNA cleaved with Haelll as molecular weight markers.
• Lanes 2-7 positive controls for sensitivity, each containing tenfold fewer molecules of pHXB2 cleaved with Xbal than the previous sample, starting with 3000 molecules in lane 2.
• Each sample was amplified in the presence of 1 ⁇ g DNA (the amount of DNA in 1.5 x 10 cells) from an uninfected control chimpanzee, C-519.
• One negative control sample (lane
• C-487 was an HIV-1 infected chimpanzee, used as a positive control.
• Fig. 9 depicts immunoblot analysis of antibodies to specific HIV-1 proteins following immunization and challenge of chimpanzees C-433, C-339 and C-499.
• Serum samples were diluted 1:200 and tested with a commercial kit (Diagnostics Pasteur) .
• sera were collected one month prior to challenge (marked by arrow) and then at 4 week intervals.
• Molecular weights of HIV-1 proteins are shown for positive control serum.
• Fig. 10 shows anti-gpl60 ELISA titers in Rhesus monkeys treated according to the invention.
• Fig. 11 shows anti-V3 BRU antibody titers in Rhesus monkeys treated according to the invention.
• Fig. 12 shows the serum neutralization of cell-to-cell transmission.
• Fig. 13 shows the antibody titer in chimpanzees, after cell-free HIV challenge.
• Fig. 14 Schedule of immunization.
• Purified recombinant gpl ⁇ O and PND oligopeptide (V3) were infected at the times indicated in the presence of either alum, IFA or SAF-1.
• Fig. 15 Time course of antibody response in the 3 groups of monkeys as measured by ELISA.
• Panel A Anti-gpl60 response.
• Panel B anti-V3 response.
• Geometric mean antibod titers in groups A (0), B ([]) and C ( ⁇ ) were computed from the data in tables 1 and 2 and plotted as a function of tim of immunization.
• Fig. 16 Time course of neutralizing antibody response in animals 57, 59 (group B) , 61 and 64 (group C) . Titers have been expressed as the reciprocal of the dilution of serum giving 50% reduction of syncitium formation.
• Figs. 17 and 18 Correlation between anti-PND ELISA titers and H V-1 neutralizing antibody titers.
• Panel A titers at 5 months; panel B: titers at 7 months.
• 0 group A []: group B;
• ⁇ group C. Best Mode for Carrying Out the Invention
• synthetic peptides live recombinant vaccinia virus (W) expressing HIV antigens, native or recombinant gpl20 or gpl60 envelope antigens, and inactivated whole virus.
• W live recombinant vaccinia virus
• One chimpanzee, C-339 was immunized initially with four injections (at 0, 1, 2 and 6 months) of 250 ⁇ g of formalin and betapropiolactone-inactivated whole HIV mixed with SAF-1 using a concentration of 1 mg threonyl MDP.
• the animal developed high HIV ELISA titers (1:200,000, using the ELAVIA kit from Diagnostic Pasteur with a cutoff of 0.1) and showed strong reactivity by Western blot to gpl ⁇ O, gpl20, and gp41 env. and to p55, p40, p25, and p!8 ⁇ ag.
• Vaccinia virus W-1163 can be made using the procedures described by Kieny et al. , Protein Engineering 2.:219-226 (1988).
• the antigen was purified by sequential lectin and cation-exchange chromatography, then was injected I.D. at multiple sites of the chest (125-150 ⁇ g per injection) with a human dose of BCG. This was followed by 3 successive I.M. injections of the antigen formulated with SAF. ELISA and neutralizing Ab titers were followed on routinely; however, both remained unchanged during and after this course of immunizations.
• HIV neutralizing Ab are primarily directed against the type-specific, hypervariable loop from the V3 region of gpl20. Therefore, using bis-diazobenzidine, a 25-mer oligopeptide with the sequence of that loop 4-(YNTRKSIRIQRGPGRAFVTIGKIGN) from the HIV-I BRU (Illb) strain was cross-linked to KLH. C-339 was injected with the peptide-carrier conjugate in the presence of SAF (300 ⁇ g of peptide) at 0, 3, and 19 weeks. No increase in ELISA titer was observed, but sustained neutralizing Ab titers were obtained following the second injection. The animal was challenged on week 26 (see below), together with another chimpanzee, ROBERT, C-433, that had undergone a parallel, albeit distinct, course of immunization.
• Chimpanzee C-433 had been primed with W-1139, a W recombinant expressing the same uncleaved version of gpl ⁇ Oenv as W-1163, but containing the transmembrane domain.
• Vaccinia virus W-1139 can be made using the procedures described by Kieny et al., Protein Engineering 2 ⁇ 219-226
• V3 peptide-KLH conjugate according to the same immunization protocol, as C-339.
• the neutralizing Ab titer of C-433 was immediately boosted several fold and the animal was challenged in parallel with C-339.
• the two chimpanzees were challenged using a titrated virus stock (III B stock, lot No. 40) from the National
• the stock which contained 10 TCID50/ml, was diluted 1:100, and 1 ml of the dilution was injected I.v. into both of the immunized animals.
• the chimp was used in this experiment. The chimp
• ID50 of this virus stock was equivalent to 4 TCID50, and in two experiments, injection of chimpanzees with 40 TCID50 resulted in the appearance of detectable virus in PBL as early as 2 weeks after injection and was followed by seroconversion at 4 weeks.
• C-433 with 100 TCID50 was not followed by detectable increases in antibody titers during the 24 weeks that have elapsed since time of challenge.
• C-433 has not developed anti-p25 ⁇ a ⁇ Ab
• C-339 has not developed anti-p27nef
• neutralization epitopes is taken to mean, in the case of HIV-1, the major virus-neutralization epitope, such as described, among others, by Putney et al. in 1986 (Science 234:1392-1395) and by Rusche et al. in 1988 (Proc. Natl. Acad. Sci. USA 85:3198- 3202), for which the sequence corresponds approximately to amino acids 296 to 331 of the HIV-1 envelope glycoprotein as described in the work of Myers et al. (Human Retroviruses and AIDS 1989, Los Alamos, Natl. Lab).
• peptides corresponding to those known as minor neutralization epitopes characterized by the fact that they belong to conserved regions of the envelope glycoprotein, and that they induce antibodies capable of neutralizing, at relatively low titers, several different isolate ⁇ of the virus under consideration, for example several different isolates of HIV- 1, or even different isolates of HIV-1, and also of HIV-2.
• An example of a minor epitope can be found in the work of Chanh et al. in 1986 (The EMBO Journal, 5_:3065-3071) and in that of Evans et al. in 1989 (Nature 339:385-388U or Almond et al. in "Retroviruses of human AIDS and related animal disease," M. Girard and L. Valette, Foundation Marcel Merieux, Lyon, 1990, in press).
• Immunogenic peptides of major and minor neutralization epitopes are preferably mixed with each other to ensure the greatest possible protection. They can be administered in the free state, not coupled to a carrier molecule. They can also be combined with a sequence of amino acids having one or preferably several T-epitopes from one or several structural or non-structural proteins of the same retrovirus or a retrovirus immunologically cross-reactive with the former, particularly such as described in French patent application of Girard-Gluckman-Bahraoui, No. 89.11044 of August 18, 1989.
• immunogenic peptides corresponding to neutralization epitopes are chemically coupled to sequences of amino acids corresponding to T-epitopes.
• the peptides are coupled to a carrier molecule which bears the desired T-epitopes, by allowing them to react, for example, with a bifunctional reagent or any other coupling agent desired.
• any protein coded for by the viral genome can be used (in the case of HIV, the proteins produced by tat, rev, vif. pol. vpr, vox, vpu. gag, env, or net genes), or other (protein-type) molecules, such as HBs antigen, HBc antigen, tetanus toxoid, hemocyanin, human albumin, or poiypeptides (for example polylysine) or appropriate lipopeptides.
• the priming effect of the envelope glycoproteins appears after the first one or few injections of vaccine, and the amplification effect due to peptides immediately afterward.
• an object of the invention is to use a first antigen, in this case the several envelope glycoproteins of each of the retrovirus serotypes under consideration, which has the effect of- priming the response of the immune system; and a second antigen, in this case the synthetic peptides corresponding to major and also possibly minor neutralization epitopes of the different serotypes of the virus under con ⁇ ideration, for vaccination (preferably consecutively, but in a mixture, if necessary) with the purpose of amplifying and consolidating the initial response, particularly through induction of long-lasting, high-titer neutralizing antibodies.
• This invention makes it possible to induce immunity that persists as long as about six months and even as long as one year or more.
• glycoproteins u ⁇ ed to prime the response of the immune system are preferably whole molecules as obtained before possible cleavage.
• gpl ⁇ O is preferable to gpl20, and the same is true for other retroviru ⁇ e ⁇ .
• This allows anti-gp41 antibodies in particular to be induced, which is a favorable sign in virus carriers (Klasse et al., Proc. Natl. Acad. Sci. USA, 85:5225-5229) .
• the peptides constituting the "amplifier” can be free or physically bound (especially by hydrophobic bonding) or chemically bound (especially by covalent bonding) to carrier molecules. They can also be associated with other peptides corresponding to T-epitopes, or even to peptides, lipopeptides, glycopeptides, aliphatic chains, fatty acids, or any combination of these capable of stimulating the immune system and/or specifically targeting the "amplifier” peptides to antigen-presenting cells.
• a particularly advantageous presentation of peptides corresponding to HIV neutralization epitopes is to bind them, preferably by covalent chemical bonding, to an aliphatic sequence, particularly as described in 1989 by Deres et al. (Nature 342:561-564).
• the amplifying peptide ⁇ presented in this way can induce not only a B-cell response, but also a CTL CD8 response, restricted HLA Class I, as described by Takanashi et al. in 1988 (Proc. Natl. Acad. Sci. USA 85:3105-3109).
• the virus has a high degree of antigenic variability, as in the case of HIV-1 and HIV-2, it is necessary to use as priming antigen not just one, but several envelope glycoproteins with different sequences, each sequence corresponding to an isolate or group of isolates of the virus under consideration, so as to obtain as many priming phenomena as desired, since each is specific for a single isolate or group of isolates.
• the amplifying peptides are composed of the mixture of neutralization peptides of each of the isolates under consideration, as indicated below.
• a preparation of HIV-1 amplifier peptides according the invention is characterized by the fact that it contain at least one of the sequences or one part of the sequences described below in one letter amino acid code:
• the priming and amplifying antigens are preferably administered intramuscularly to a host, such as a primate, and especially a human. Following are typical immunization schedules that can be employed for gpl ⁇ O and peptides of HIV gpl ⁇ O Peptides (months) (months).
• these immunization schedules are merely representative and that the schedules can be varied to obtain the optimum response in the host.
• the amounts of the priming and amplifying antigens can be varied. For example, about 150 ⁇ g of gpl60 in Syntex SAF-1 adjuvant can be administered as indicated, followed by administration of the peptides in amounts of typically 100 ⁇ g of each peptide.
• the relative proportions of the peptides involved can vary according to the desired final proportions of each peptide in the final preparation.
• these proportions will be adjusted as a function of the immunogenicity of each peptide and the number of functional groups carried by each one, which are capable of entering into the conjugation reaction with complementary functional groups, at least when these peptides are coupled to a carrier molecule «
• the injection of amplifying peptides is replaced by the administration of particles, virus, or bacteria, which are recombinant ⁇ expressing the neutralization epitope of the virus under consideration on their surface and/or during their multiplication and in this way are capable of inducing neutralizing antibodies against said retrovirus: HBc antigen particles; HBs antigen particles; bacteria expressing the neutralization epitope in surface or cytoplasmic proteins, such as, for example, the lamB receptor; picorna virus chimeras, such as, for example, poliovirus-HIV chimeras; poxvirus recombinants; adenovirus recombinants or adenovirus chimeras, etc.
• this administration can be carried out in the form of live vaccine administered orally (for example, chimeras constructed from Sabin polioviru ⁇ strains or from human adenoviruses, or from attenuated strains of Salmonella, Shioella.
• enterobacteria or from any organism, virus, yeast, bacteria capable of inducing an immune response after oral administration
• parenteral route for example, recombinant poxvirus
• inactivated vaccine by the parenteral route for example, chimeras constructed from the Mahoney strain of polioviru ⁇ , or inert particles of HBsAg or HBcAg
• the antigen envelope glycoprotein
• the antigen which is injected for the priming of the vaccination, i.e., the envelope glycoprotein of the virus, is presented under the form of particles such ISCOM (Immune Stimulating Complex, comprising an association of an antigenic protein with a glycoside Quil A) or liposomes.
• ISCOM Immuno Stimulating Complex
• the priming antigen and/or the peptide can be also associated with live recombinant microorganisms, such as viruses or bacteria (for instance the poxvirus or BCG: Bacile de Calmette Gerin) or any live vaccine modified to express the envelope glycoprotein or the peptide derived therefrom.
• live recombinant microorganisms such as viruses or bacteria (for instance the poxvirus or BCG: Bacile de Calmette Gerin) or any live vaccine modified to express the envelope glycoprotein or the peptide derived therefrom.
• the envelope glycoprotein and/or the peptide derived therefrom can also be presented by inactivated particles, for instance viral particles, such as the HIV virus or a part of this virus, or particles without genome.
• viral particles such as the HIV virus or a part of this virus
• particles without genome have been described to produce vaccine by Haffar 0. et al., Journal of Virology, 64*2653-2659 (1990)
• These particles can be called HIV-like particles in the case of HIV virus: for the purpose of the invention they do not contain the complete HIV genome, but they enable the exposition at their surface of the virus components of the composition of the invention.
• the envelope glycoprotein antigen is combined in a mixture with other antigens.
• the priming antigen is the HIV envelope glycoprotein
• one or several antigens such as gag net, vif, pol, GPG or GLG antigens, can be combined with it, as they can be combined with the peptides of the composition
• the invention also comprises the compositions above described, wherein the env glycoprotein is replaced by or associated with a fragment thereof.
• This fragment has advantageously more than 50 amino acids and is characterize in that it has the immunogenic properties of the glycoprotein in the context of the invention.
• the invention also concerns monoclonal or polyclonal antibodies, which recognize the glycoprotein and/or peptides of the composition. These antibodies can be associated in a mixture and used, for instance, for serotherapeutic purposes.
• EXAMPLE 1 Immunization of a chimpanzee with HIV-1 BRU and the glycoprotein of this isolate; amplification of the response with a BRU env oligopeptide coupled to KLH.
• Chimpanzee 339 (FUNFACE) was first immunized with three injections at one month intervals of 250 ⁇ g of purified HIV- 1 BRU virus, inactivated by treatment with 0.025 percent formalin for 48 hour ⁇ at 30°C and 0.025 percent betapropiolactone for 30 minutes at 37°C, combined with Syntex adjuvant containing 1 mg/ml threonyl-MDP in an emulsion of 5 percent squalane and 2.5 percent pluronic polymer. These injections were followed by a first booster at 7 months and a second booster one year later.
• the animal then received five injections of BRU virus envelope glycoprotein (gpl60) purified from supernatant of BHK-21 cell cultures infected with a vaccinia virus recombinant (strain Wenv 1163) having a genome for which genetic recombination techniques were used to insert the sequences of HIV-1 BRU coding for gpl ⁇ Oenv modified through oligonucleotide site-directed mutagenesis to eliminate the sequences involved in gpl20/gp41 cleavage and from which the transmembrane hydrophobic zone was deleted, as described in Kieny et al. in 1988 (Prot. Engineering 2. s 219-226).
• gpl60 BRU virus envelope glycoprotein purified from supernatant of BHK-21 cell cultures infected with a vaccinia virus recombinant (strain Wenv 1163) having a genome for which genetic recombination techniques were used to insert the sequences of HIV-1 BRU coding for g
• the purified protein was used in an amount of 125-150 ⁇ g per intramuscular injection in the presence of Syntex adjuvant.
• the culture medium of BHK cells infected with W-1163 was concentrated by precipitation with ammonium ⁇ ulfate, then with trichloracetic acid, and the glycoprotein was then purified by three succe ⁇ sive runs of affinity chromatography over lentil lectin, ion exchange over cation-exchange resin, and high-performance liquid chromatography (HPLC). The recombinant gpl ⁇ O obtained in this way is 95 percent pure.
• the level of antibodies induced in response to injections of inactivated virus was not changed appreciably by the injection of gpl60.
• the animal was given 300 ⁇ g of preparation of synthetic peptide having the sequence Y N T R K S I R I Q R G P G R A F V T I G K I G N corresponding to the neutralization epitope of the BRU i ⁇ olate, the tyrosine residue (Y) being coupled to hemocyanine (KLH) with bis(diazobenzidine) and combined with Syntex adjuvant.
• the injection was repeated once three weeks later, then a second time at 19 weeks.
• FUNFACE was then challenged at 26 weeks, by administering an intravenous injection of 1 ml of a 1:100 dilution, or 100 TCID50 of an HIV-1 stock titrating 10 4 TCID50/ml, kindly provided by Larry Arthur (NCI, Frederick). This stock 040 was titered on two occasion ⁇ in the chimpanzee, which allowed Arthur et al. to determine that its ID50 for the chimpanzees was 4 TCID50.
• the chimpanzee FUNFACE demonstrated apparently total protection against infection with 100 TCID50 of the stock 040 virus, because at up to six months after the challenge injection, no viru ⁇ wa ⁇ detected in his lymphocytes (as measured either by gene amplification with pol and gag probes, or by coculture with human lymphocytes and assay of reverse transcriptase in 100,000 x g pellets obtained from culture supernatants) and at six months, there was no anti- HIV anamnestic response as measured by ELISA or by Western blot (Table 2) and no anti-nef antibody detectable by Western blot.
• EXAMPLE 2 Immunization of a chimpanzee with recombinant antigens env, gag, net, and vif of HIV-1; amplification of the response by a BRU env oligopeptide coupled to KLH.
• Chimpanzee 433 (ROBERT) was first primed with three o consecutive scarifications of 2 x 10 PFU of a recombinant vaccinia virus (Wenv 1139) expressing the gpl ⁇ Oenv of HIV-1 BRU, then by the intravenous administration of his own lymphocytes which previously had been infected .in vitro by the recombinant virus Wenv 1139 and fixed in formaldehyde. The animal then received three consecutive intramuscular injections at one month intervals, then three boosters at 33, 38, and 40 weeks and a last booster at 66 weeks consisting of a mixture of 125-150 ⁇ g of each of the following antigens combined with Syntex adjuvant: gpl ⁇ Oenv.
• Robert was then challenged in parallel with FUNFACE, by the intravenous inoculation of 100 TCID50 of the same stock 040 of HIV-1 virus from NCI as in the previous example.
• total protection against infection appears to have been obtained as judging from the absence of virus in the animal's lymphocytes and the negativity of the PCR six months after challenge and by the absence of anti-p25oag and anti- p27nef antibodie ⁇ , as well as the absence of anamnestic anti- HIV response as measured by ELISA or by Western blot six months after challenge.
• Table 4 shows the same absence of anamnestic effect on the anti-gpl60 and anti-BRU neutralization epitope.
• EXAMPLE 3 Immunization of a chimpanzee with gpl60env and p!8gag of HIV-1 antigens; amplification with HIV-1 env peptides not coupled to a carrier molecule.
• the first, JOJOTOO received three injections, at one month intervals, of 120-150 ⁇ g of gpl ⁇ Oenv and pl ⁇ gag, purified as described above, and mixed with Syntex adjuvant. This first series of injections was followed by three boosters of the same antigen given at weeks 33, 38, and 40, and a final booster at 14 months. These injections resulted in the appearance of a high antibody level detectable by Western blot and by ELISA starting immediately after the first three injections, although the level of neutralizing antibodies was relatively low, as described below. o
• the second chimpanzee, IRA was immunized with 10 PFU of each of the four recombinant vaccinia virus stocks expressing, respectively, gpl ⁇ Oenv. p55gag. p27nef. and p23vif of HIV-1 BRU. These inoculations given by the intradermal route, did not lead to the appearance of any neutralizing antibody, but a barely significant level ( ⁇ 1:200) of antibody was detectable by Western blot or by ELISA. Chimpanzee IRA was then rested for two years.
• HIV-1 human immunodeficiency virus type 1
• Animals Animals. Animals used in this study were adult male chimpanzees that had been used previously in hepatitis A, B and non-A and non-B experiments. The chimpanzees were maintained at LEMSIP, New York University Medical Center, in biosafety level 3 facilities. All experimental procedures were done according to institutional guidelines for containment of infectious diseases and for humane care and handling of primates (Moor-Jankowski, J. & Mahoney, C.J. (1989) J. Med. Primatol. 18, 1-26).
• PBMC peripheral blood mononuclear cells
• Recombinant gpl ⁇ Oenv was purified from the culture medium of BHK21 cells infected with W-1163, a recombinant vaccinia virus expressing the gpl60env gene modified by site-directed mutagenesi ⁇ to destroy the gp!20/41 cleavage site and to remove the anchor domain of gp41 (Kieny, M.P., Lathe R. , Riviere, Y., Dolt, K., Schmitt, D., Girard, M., Montagnier, L. & Lecocq. J.P. (1988) Prot.
• the challenge inoculum was from a stock of HIV-1 strain HTLV-IIIB (obtained from L. Arthur), which had been titrated in chimpanzees and used in other HIV vaccine challenge studies (Arthur, L.O., Bess, J.W., Waters, D.J., Pyle, S.W., Kelliher, J.C., Nara, P.L., Krohn, K., Robey, W.G. , Langlois, A.J., Gallo, R.C. & Fischinger, P.J.
• the infectivity titer of this HIV-1 stock is considered to be 10 TCID50 per ml and 3 4 x 10 infectious units per ml for chimpanzees.
• the chimpanzees were challenged IV with 1 ml of a 1:100 dilution.
• the challenge inoculum had a titer of greater than 64 immunofluorescent focus-forming units (end-point not reached) for the first aliquot and 170 for the second.
• Neutralization activity in serum samples from immunized chimpanzees was determined by inhibition of syncytia formation in CEM-SS cells, as described (Nara, P.L., Hatch, W.C., Dunlop, N.M. , Robey, W.G., Arthur, L.O., Gonda, M.A. & Fischinger, P.J. (1987) AIDS Res. Human Retroviruses 3, 283-302), or inhibition of immunofluorescent foci in H9 cells.
• PBMC or bone marrow cells obtained as aspirates
• normal human PBMC normal human PBMC
• CD 4-enriched lymphocytes were obtained • from chimpanzee PBMC by separation with magnetic beads to which were attached monoclonal antibodies specific for the CD8 cell-surface antigen (Dynabeads, Robbins Scientific).
• CD+4-enriched cells were stimulated 2 days with ⁇ oncanavalin
• Lymph nods tissue obtained by biopsy was minced with scissors and cultured with human PBMC. All cultures were maintained and monitored for reverse transcriptase activity for 6 week ⁇ before being discarded.
• PCR Polymerase Chain Reaction
• PBMC from C-433 were stimulated with PHA, cultured in medium containing IL-2 and then infected with W-1139 at a multiplicity of infection of 7. Following culture for an additional 16 hours, the PBMC were fixed with 0.8% paraformaldehyde and reinje ⁇ ted into C-433 by the IV route (Zagury, D., Bernard, J., Cheynier, R., Desporte ⁇ , I., Leonard, R., Fouchard, M.
• Chimpanzee C-339 was first immunized on week 33 by IM injection of inactivated HIV (125 ⁇ g viral protein) mixed with SAF-1 (1 mg threonyl muramyl dipeptide), followed by booster inoculations on weeks 37, 41, 62 and 124.
• C-339 was then inoculated with purified gpl60env only " (125 ⁇ g per dose) on weeks 66, 74, 81, 85 and 87.
• the V3 peptide 300 ⁇ g peptide per dose was administered IM on weeks 105, 108 and 126.
• C-339 and C-433 were challenged on week 131 with 100 TCID 50 of HIV-l ⁇ _-.____schreib.
• C-449 was inoculated IM with a mixture of gpl60env, pl ⁇ gag and SAF-1 on weeks 0, 6, 10, 33, 38, 66 and 76.
• week 0 for C-499 corresponds to week 48 for C-433 and C-339.
• a mixture of 21 free V3 peptides (100 ⁇ g each per dose) was administered IM with SAF-1 on weeks 79, 83, 87 and 102.
• C-499 and C-087, a naive control were challenged on week 106 and 100 TCID_ 0 of HIV tj firr V _ ⁇ B -
• V3 loop the third hypervariable region of the external envelope glycoprotein, termed the V3 loop (Putney, S.D., Matthews, T.J., Robey, W.G., Lynn, D.L., Robert-Guroff, M., Mueller, W.T., Langlois, A.L., Ghrayeb, J., Petteway, S.R., Weinhold, K.J., Fischinger, P.J., Wong-Staal, F., Gallo, R.C. & Bolognesi, D.P.
• V3 loop Putney, S.D., Matthews, T.J., Robey, W.G., Lynn, D.L., Robert-Guroff, M., Mueller, W.T., Langlois, A.L., Ghrayeb, J., Petteway, S.R., Weinhold, K.J., Fischinger, P.J., Wong-Staal, F., Gallo
• Neutralizing antibodies to V3 epitopes can, in fact, be added as long as 40 to 60 minutes after virus binds to the cell and still prevent infection (Nara, P.L., (1989) in Vaccines 89, eds. Lerner, R.A., Ginsberg, H., Chanock, R.M. & Brown, F. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) pp. 137- 144). Therefore, to determine whether immunization with the V3 loop would boost neutralizing antibody titers, C-339 was injected with an oligopeptide of 25 amino acids, having the
• C-433 At the time C-433 first received the purified recombinant proteins (48 weeks), a third chimpanzee, C-499, received an IM injection of purified gpl ⁇ Oenv and pl ⁇ ag formulated with SAF-1. C-499 received six booster inoculations of the same antigens, followed by a series of four injections of a mixture of 21 free (unconjugated) V3 peptides (Myers, G. (1990) in Human Retroviruses and AIDS, eds. Myers, G., Jo ⁇ ephs, S.F. , Wong-Staal, F. , Rabson, A.B. , Smith, T.F. & Berzofsky, J.A.
• chimpanzees C-433, C-339 and C-499 were challenged by IV inoculation of 100 TCID e n (40 chimpanzee infectious doses) of HIV-1.
• 50% neutralization titers by an immunofluorescence inhibition assay were 1:2000, 1:280-350 and 1:2000, and 90% neutralization titers by a syncytia-. inhibition assay (Nara, P.L., Hatch, W.C, Dunlop, N.M., Robey, W.G., Arthur, L.O., Gonda, M.A. & Fischinger, P.J. (1987) AIDS Res.
• C-087 control animal, C-087.
• Virus was isolated from C-087' ⁇ PBMC at 2 weeks post-inoculation (PI) as well as at all subsequent times, ⁇ howing that a 1:100 dilution of the HIV-1 stock readily infected chimpanzees under our conditions.
• CD8 cells have been shown to suppress virus replication not only in HIV-infected humans (Walker, CM., Moody, D.J., Stites, D.P. & Levy, J.A. (1986) Science 234, 1563-1566; and Tsubota, H., Lord, C.I., Watkin ⁇ , D.I., Morimoto, C. & Letvin, N.L. (1989) J. Exp. Med.
• chimpanzee ⁇ P.N.F., unpubli ⁇ hed data
• SIV-infected macaques Tsubota, H., Lord, C.I., Watkins, D.I., Morimoto, C & Letvin, N.L. (1989) J. Exp. Med. 169, 1421-1434
• chimpanzee PBMC were depleted of CD8 lymphocytes before cultures were established.
• virus recovery from the control animal C-087, virus was not recovered from either total PBMC or CD4 -enriched cells from C-339, C-433, or C-499 at any time during the first 6 months of follow-up.
• inguinal lymph node biopsies were performed on all animals as well as on uninfected and HIV-infected control chimpanzees.
• virus was recovered from the lymph node of the infected control, but not from those of the immunized and challenged chimpanzees (data not shown).
• virus was isolated from C-433 cocultivation of PBMC obtained at 32 weeks and thereafter of bone marrow obtained 37 weeks after challenge.
• C-339 was immunized successive ⁇ ively with inactivated HIV, purified gpl ⁇ O, and the V3 peptide-KLH conjugate.
• C-433 was immunized first with a vaccinia virus-gpl60env recombinant, then with a mixture of purified env, pl ⁇ gag, nef and vif antigens, and finally with the V3 peptide-KLH conjugate.
• the simple ⁇ t immunization regimen was that of C-499; it consisted of purified gpl60env and pl ⁇ ga ⁇ followed by unconjugated V3 peptides.
• the antigens that were common to the three animals were gpl60e ⁇ v, plSgag and the V3 peptide, but their relative importance remains to be determined. Adequate protection might require multiple antigenic determinants found on more than one viral protein, and/or multiple presentations of the same antigenic determinant.
• C-433 di ⁇ played a sustained, strong T-helper cell reactivity to the V3 peptide, while C-339 had only a weak response.
• the responses of C-449 are currently under study. Repeated attempts to detect cytotoxic T lymphocytes (CTL) in PBMC of the vaccinated chimpanzees before, on the day of, and after challenge have failed. It appears, therefore, that the observed protection did not correlate with the T-helper cell or CTL activity. .
• CTL cytotoxic T lymphocytes
• the experiment was carried out in Rhesus macaques (4 animals per lot) using 100 ⁇ g of gpl60 BRU for priming and a mixture of 200 ⁇ g each of V3-BRU (gpl20 amino acid residues 302-335) and V3-MN (same residues) for boosting.
• the animals were bled at monthly intervals and anti-V3 and anti-gp antibody (Ab) titers were determined by ELISA.
• Neutralizing Ab titers were determined by the inhibition of immunofluorescent foci formation assay.
• Anti-gpl60 Ab were measured by ELISA using plaques coated with purified gpl ⁇ O BRU. A fast anti-gpl60 Ab response was observed in the 3 groups of animals (Fig.
• Anti-V3 Ab were measured by ELISA using plaques coated with the BRU peptide.
• the response to V3 was clearly biphasic in all groups, with a ⁇ trong booster effect seen upon injection of the V3 peptide at 3 months (Fig. 11).
• anti-V3 titers increased 10 fold between months 3 and 4 and then plateaued, confirming the remarkable booster effect of a V3 peptide injection in gpl ⁇ O-primed animals. This was observed irrespective of the adjuvant used in the experiment.
• a two-step immunization schedule can be defined as follows: priming: gpl ⁇ O at 0 and 1 month boosting: V3 peptides at 3 months second boosting: gpl ⁇ O + V3 peptides at 6 months.
• the second boost can be placed at a later time, such as 12 months, to increase further the anamnestic response.
• Neutralizing antibody titers - Sera from the 5th and 7th month of immunization were monitored for HIV-1 neutralizing antibodies as described in Material and Methods.
• PBMC peripheral blood mononuclear cells
• chimpanzee C-339 was immunized with various HIV-1 antigens and was subsequently challenged with an intravenous injection of 100 TCID 5Q of cell-free HIV-1. This animal had remained virus negative by multiple criteria and did not develop an anamnestic antibody response to the virus through 40 weeks after challenge. Becau ⁇ e unrelated in vivo studies had indicated that immune stimulation induced increases in HIV-1 expression in long- term infected chimpanzees (), and to insure that C-339 had indeed been protected from infection, we attempted to reactivate or induce detectable expression of putative latent virus by stimulating the animal's immune sy ⁇ tem.
• HIV-1 antigens inactivated HIV-1_ AV _ 1 : recombinant antigens gpl ⁇ Oenv, p25- and pl ⁇ -ga ⁇ ; and peptides representing the V3 immunodominant loop, all formulated with SAF-1).
• PBMC from C-339 when used as indicator cells, would prevent tran ⁇ mi ⁇ ion and replication of virus when cocultivated with PBMC from an HIV-1-infected chimpanzee (C-087).
• PBMC from C-339 were added at a fixed concentration (2-3 x 106 cells/well) to wells of 12-well tissue culture plates.
• C-087' ⁇ PBMC were serially diluted 1:4, and cells from each dilution were added to duplicate wells containing PBMC from C-339 (or normal human or chimpanzee PBMC, as controls), starting with a ratio of 1:1. Culture supernatants were monitored periodically for virus production by reverse transcriptase as ⁇ ay.
• Inhibitory activity was considered to be present in cells from the immunized animals if (i) a larger number of C-087's PBMC were required to yield viru ⁇ -positive cultures within 6 week ⁇ of observation, and (ii) there wa ⁇ a delay in time at which cultures became virus po ⁇ itive, compared with those coculture ⁇ established with PBMC from HIV-1-naive individuals.
• These assays indicated that C-339 had substantial inhibitory activity on week 40, which was before the two booster injections of HIV-1 antigen ⁇ (Figure 2B). Although this inhibitory activity had declined by week 73, enrichment for CD8+ cells by magnetic bead depletion of CD4+ cell ⁇ resulted in complete inhibition of viru ⁇ recovery ( Figure 2C) .
• the apparent enhancement of infection with the CD4+-enriched population of C-339's PBMC probably is a function of the greatly increased number of cells capable of supporting replication of HIV-1.
• a challenge inoculum consisting of PBMC from an HIV-1-infected chimpanzee was believed to most nearly approximate transmission that occurs between, for example, intravenous drug users. Since the minimal infectious dose of HIV-infected cells required for infection of chimpanzees had not been determined, and because of the limited number of available chimpanzees, the dose of the challenge inoculum was selected empirically. This selection was based on the result ⁇ of in vitro titrations of aliquots of the cryopreserved PBMC from chimpanzee C-087, using PHA- ⁇ timulated normal PBMC from both humans and chimpanzees as indicator cells (). From these as ⁇ ays, it was determined
• PBMC This number is a minimum estimate and is based on the as ⁇ umption that one infected cell is sufficient for a culture to become virus positive.
• Virus isolation attempts were performed by cocultivation of PBMC from each animal with
• PBMC peripheral blood mononuclear cells
• HIV-specific antibodies were detected in serum from C-435 initially at 8 weeks after challenge, and titers continued to rise through week 24
• the second chimpanzee, C-447 had been immunized initially with purified recombinant gpl60env, pl8gag, vif, and nef protein ⁇ in SAF-1, and then received booster immunizations with purified gpl ⁇ Oenv and pl ⁇ gag, followed by peptide ⁇ repre ⁇ enting the principal neutralizing determinant (V3 loop) of HIV-1 H _ LV _ IIIB and purified nef protein in SAF-1. Chimpanzee C-447 had not been exposed previously to infectious HIV-1 in any form.
• C-447 and C-499 had fourfold lower Hiv-l EIA antibody titers (1:6400 ver ⁇ u ⁇ 1:25,600), but four- to eight-fold higher neutralizing antibody titer ⁇ (1:256 and 1:512 versus 1:64), compared with those of C-339.
• serum samples from C-447 and C-499 were tested. Serum obtained from C-447 and C-499 on day of challenge inhibited cell-to-cell transmission of HIV-1 by 25% and 52%, respectively.
• HIV-1 was initially detected in PBMC from C-339 (by cocultivation with normal human PBMC) that were obtained 4 weeks after this third HIV-1 challenge, and an increase in HIV-1 EIA antibody titer was observed at 6 weeks after challenge (Figure 1, week 110). Because C-339 had not received a booster immunization or been exposed to HIV-1 for 1 year prior to this second challenge with cell-free HIV-1, the immune respon ⁇ e elicited by vaccination did not persist at a level sufficient to protect against this last exposure to virus. C-339 became infected despite the presence of a stable HIV-1 immune response, and infection was detected relatively soon after the third exposure to virus.
• C-447 had received three booster immunizations with only V3 peptides and Nef protein during an interval 2 to 5 months earlier; these inoculations had resulted in more than a tenfold increase in neutralizing antibody titers, but no detectable increase in HIV-specific EIA antibody titers. That PBMC from C-339 subsequently lost the ability to prevent cell-to-cell transmi ⁇ ion in vitro ⁇ upport ⁇ this pos ⁇ ibility. Irre ⁇ pective of this, it appear ⁇ that neither of the in vitro assay ⁇ , as performed with serum or PBMC, are predictive of protective immunity.
• a vaccine against any pathogen ⁇ hould be one that elicit ⁇ long-lasting immunity following a minimal number of immunizations. While we have observed long-lasting, stable EIA and neutralizing antibody titers in our immunized chimpanzees, these were achieved with a large number of immunizations (no fewer than ?12?) over a minimum of 2 years. These regimens, to say the least, are not practical for use in Western nations, much less in developing countries. Based on studies to date in nonhuman primate models, it appears a ⁇ though immunization against HIV-1 will require at least three inoculations initially and booster inoculations at unspecified intervals.
• the envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1) is made of two moieties that arise by proteolytic cleavage of a large precursor, gpl ⁇ O.
• the exterior surface glycoprotein gpl20 corresponds to the amino- terminal region of gpl ⁇ O, whereas the transmembrane glycoprotein gp41 is derived from its carboxyl-terminal region (1, 2).
• the principal neutralization determinant (PND) for the viru ⁇ ha ⁇ been mapped to the third hyper- variable domain (V3) of gpl20, a conserved cysteine loop located at residue ⁇ 303-338 for ⁇ train IIIB (3-5) (numbering according to ref 6).
• both gpl20 and gp41 carry minor neutralization epitopes (for a review see ref 7).
• the neutralization epitope ⁇ of the V3 loop are of a sequential nature (8, 9) but the 3-D conformation of the loop seems to be important for reactivity (10, 11).
• PNDs from different HIV-1 isolates exhibit extensive sequence divergence (4, 12, 13), which explains why antibodies to the PND neutralize virus infectivity in a type specific manner. These antibodies also inhibit syncitia formation and viru ⁇ spread from cell-to-cell.
• PND-targeted antibodies act at the level of fusion between the virus envelope and the membrane of target cells, or between the membranes of infected and non- infected target cell ⁇ (4, 14).
• chimpanzees that had been primed by hyperimmunization with a variety of HIV-1 antigens, among which. gpl ⁇ O, then boosted with either free or KLH- coupled PND peptides, developed high titers of PND-specific neutralizing antibodies and were protected against subsequent HIV challenge (16).
• the simplest immunization regimen able to induce protective immunity consisted of gpl ⁇ Oenv and pl ⁇ gag followed by unconjugated PND peptides. There is little reason to believe that p!8gag could be involved in protection.
• the minimal protective immunization regimen should consist of gpl ⁇ O followed by boosting with the corresponding PND peptide.
• the efficacy of such a priming- booster immunization schedule is likely to depend on multiple parameters such as dose and physical statu ⁇ of the gpl60 antigen, amount and sequence of the PND peptide ⁇ , number and spacing of the injections, and also on the nature of the adjuvant.
• the efficacy of alum, incomplete Freund adjuvant (IFA), and Syntex adjuvant formulation 1 (SAF-1) (20) were compared in a simplified priming-booster immunization regimen in rhesus monkeys.
• W1163 the recombinant vaccinia viru ⁇ used for the production of gpl ⁇ O from the LAV-1 (LAI) i ⁇ olate of HIV-1 (21) ha ⁇ been described previously (22).
• the gpl ⁇ O gene carried by W1163 wa ⁇ mutagenized at the gpl20-gp41 cleavage site and deleted of the transmembrane domain.
• the antigen was purified from the cell culture medium of W1163-infected BHK-21 cells as described (22-24).
• PND peptide (LAI) was prepared by solid phase synthesis as a 34 amino acid residues peptide with the sequence
• Monkeys were immunized by the I.M. route with 2 injections of 100 ⁇ g recombinant gpl ⁇ O at 1 month interval, followed by 2 injections of 200 ⁇ g PND peptide at 3 and 4 months (see Fig. 1) . All antigens were in final volume of 1 ml.
• Four monkeys (group A) were immunized with the antigens adsorbed to 0.2% aluminium hydroxyde (Superfos), another four animals (group B) with the antigens emulsified in 1 ml IFA (Difco) and the la ⁇ t four (group C) with the antigens emulsified in 1 ml SAF-1 (20) containing 1 mg threonyl-MDP per dose.
• Anti-PND and anti-gpl60 antibody titers were determined by ELISA using microwe11 plaques (Nunc) coated with 0.10 ⁇ g PND peptide or 0.15 ⁇ g gpl ⁇ O per well, respectively. Incubation with the appropriate dilutions of serum was for 1.5 hr at 37 ⁇ C, after which sera were replaced by horseradish peroxydase-labeled rabbit anti-monkey immunoglobulin (Nordic) and incubation wa ⁇ continued for another 1.5 hr at 37°C Bound enzyme activity wa ⁇ measured using orthophenylenediamine (Merck) with 0.03 % hydrogen-peroxide a ⁇ a ⁇ ub ⁇ trate. The reaction was stopped after 30 min.
• End point titers were calculated from a linearized standard curve obtained with a selected pool of positive macaque sera used as an internal standard. Titers obtained correspond approximately to the reciprocal of the highest serum dilution that re ⁇ ulted in an optical den ⁇ ity of at least 0.1.
• Neutralizing antibodies were measured by inhibition of immunofluorescent foci formation on H9-cells or inhibition of synicita formation on CEM-SS cells (26) using a IIIB virus stock. Except where otherwise stated, neutralization titers were defined as the reciprocal of the serum dilution that reduced foci formation by 50% or synicita formation by 90% as compared with control.
• Antibody response ⁇ to whole gpl ⁇ O and to the PND of the same HIV-1 isolate (LAI) as used for immunization, as well as neutralizing antibody responses to HIV-1 IIIB were monitored regularly during the 6 month immunization period and an additional 6 month follow- up.
• LAI HIV-1 isolate
• Anti-gpl60 antibody titers induced by the two initial injections of gpl60 were approximately ten times higher in the IFA and SAF-1 groups than in the alum group (B, C, and A, respectively, table 1 and Fig. 2). These titers increased only slightly in response to the injection ⁇ of PND peptide, and began declining thereafter.
• the six month booster injection with gpl ⁇ O plus peptide led to a 4-10 fold anamnestic antibody response to all three groups, but, again, the gpl60 antibody titer in the alum group remained about one order of magnitude lower than the IFA or SAF-1 groups (Fig. 2A) .
• titers After the peak at 7 months, titers progressively declined but were still significantly elevated at the 12th month bleed, except in the alum group (table 1).
• Anti-PND antibody titers remained low after one injection of gpl ⁇ O and became moderately high after two injections (table 2). They were markedly (3 to more than 20 fold) enhanced by the fir ⁇ t injection of PND peptide, confirming our previous observation in chimpanzees (16). However, no further increase in titer wa ⁇ observed after the second injection of PND peptide (Fig. 2B) . In several animals, that injection wa ⁇ actually followed by a drop in anti-PND titer (table 2). Similarly, the effect of the booster injection at 6 months on the anti-PND titer was of very limited amplitude, after which titers steadily declined.
• Anti-PND titers at 7 months were thus paradoxally lower than those at 4 months. The difference was significant at the 1% level. This suggest ⁇ that too many injection ⁇ of PND peptide could actually lead to ⁇ ome sort of immune paralysis. Anti- PND titers in the alum group remained at all times about one order of magnitude lower than those in the IFA and SAF-1 group and reached low level values at 12 months.
• Neutralizing antibody titers were determined at 5 months (one month after the second injection of PND peptide) and again at 7 months (one month after the 6 month booster). As shown in table 3, the highest neutralizing titers were generated in the IFA group (B) and the SAF-1 group (C). Neutralizing titers in the alum group (A) remained definitely lower and some animals even ⁇ cored negative in that group when mea ⁇ ured at 7 month ⁇ by ⁇ yncitium inhibition assay.
• the aim of the present study was to compare the efficacy of three different adjuvant formulations with respect to their capacity to induce HIV-1 neutralizing antibodies in rhesus macaques in a gpl ⁇ O priming - PND peptide booster immunization schedule limited to 5 injection ⁇ over the course of a 6 month immunization period.
• the data presented here show that high anti-gpl ⁇ O, anti-PND, and neutralizing antibody titers could be raised in respon ⁇ e to such a short regimen of immunization, provided the adjuvant was suitably chosen.
• aluminium hydroxyde was unable to provide help for a strong anti-HIV-1 antibody response.
• the 12 animals used in this study showed a strong, ⁇ ignificant correlation between anti-PND and neutralizing antibody titers at the 5 month ⁇ and 7 months time points, ⁇ ugge ⁇ ting that the majority of the neutralizing antibodies were targeted to the PND, independent of the adjuvant used in the vaccine (Fig. 4).
• a similar correlation was observed in chimpanzees, although not a ⁇ strict a ⁇ observed here (unpublished observations).
• Nara PL Neutralization of HIV-1 : a binding /po ⁇ t- binding event mediated through the g ⁇ l20 immunodominant epitope.
• Retroviruses of human and related animal diseases M, Girard and L Valette (eds) Fondation Marcel Merieux, Lyon 1988, pp 138-150.
• Girard M The challenge of HIV vaccines. Vaccine 1991; in press.
• Kieny MP Lathe R, Riviere Y, Dott K, Schmitt D, Girard M, Montagnier L, and Lecocq JP: Improved antigenicity of the HIV env protein by cleavage site removal Protein Engineering 1988;2:219-225.
• Schmitt D Dezutter-Dambuyant C, Hanau D, Schmitt DA, Kolbe HV, Kieny MP, Cazenave JP, and Thivolet J: Les prot ⁇ ines d'enveloppe du VIH ⁇ ont fixee ⁇ par les cellules de Langerhans epidermique ⁇ INE ⁇ ⁇ ur un ⁇ ite de fixation qui differe du site port6 par la molecule CD4, et s'internali ⁇ ent par une "endocytose par recepteurs”. C R Acad Sci (Paris) 1989;309(III):269-275.
• the envelope glycoprotein of human immunodeficiency virus type 1 is a dimer of 125-kilodalton subunits stablilized through interactions between their gp41 domains. J Virol 1991;65:3797-3803.
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