The Journal of Immunology

Induction of Broad Cross-Subtype-Specific HIV-1 Immune Responses by a Novel Multivalent HIV-1 Peptide in Cynomolgus Macaques

Ali Azizi,*† David E. Anderson,*¶ Jose´V. Torres,*‡ Andrei Ogrel,* Masoud Ghorbani,* Catalina Soare,*† Paul Sandstrom,§ Jocelyne Fournier,§ and Francisco Diaz-Mitoma1*†

One of the major obstacles in the design of an effective vaccine against HIV-1 is its antigenic variation, which results in viral escape from the immune system. Through a bioinformatics approach, we developed an innovative multivalent HIV-1 vaccine comprised of a pool of 176 lipidated and nonlipidated peptides representing variable regions of Env and Gag proteins. The potency and breadth of the candidate vaccine against a panel of HIV-1 subtypes was evaluated in nonhuman primate (cynomolgus macaques) and humanized mouse (HLA-A2.1) models. The results demonstrate strong immunogenicity with both breadth (humoral and cellular immunity) and depth (immune recognition of widely divergent viral sequences) against heterologous HIV-1 subtypes A–F. The Journal of Immunology, 2008, 180: 2174–2186.

ntigenic variation has thwarted HIV vaccine develop- In this study, we report on the development of a novel HIV-1 ment for over two decades (1, 2). By design, the major- peptide vaccine based on combined prediction algorithms, HLA A ity of the current vaccine strategies target specific polymorphism in human loci, and molecular modeling. More than epitopes of one HIV strain; however, it is important for a vaccine 200 HIV-1 genome sequences were chosen from the HIV Los to have the ability to induce an immune response against a range Alamos sequence database and their epitopes were analyzed. We of HIV variants. synthesized many variants of several epitopes, representing the The role of neutralizing Abs (Nabs)2 in controlling HIV-1 in- variable regions of Env and Gag that were either lipidated or non- fection is undeniable, but to date, only a limited number of Abs lipidated. The presence of lipidated peptide variants served both to with neutralization capabilities against primary isolates have been elicit a CTL response and to activate innate immunity due to TLR2 identified (3–6). The crystallographic structure of gp120 indicates recognition of the lipid moiety present on the peptides (12–14). To that the protein is covered by carbohydrates which facilitate viral evaluate the relative merits of with lipidated, non- escape from Nabs (7, 8). The genetic variability in HIV-gp120 lipidated, or both forms of our peptides, in terms of inducing cel- between groups M, N, and O also affects the induction of Nabs and lular immunity, groups of HLA-A2.1 mice were immunized with by guest on September 30, 2021. Copyright 2008 Pageant Media Ltd. allows the virus to escape immune recognition triggered by hu- lipidated and nonlipidated peptide variants alone or in combina- moral responses directed against gp120 epitopes (9, 10). The chal- tion. As controls, mice were either immunized with adjuvant alone lenge associated with inducing Nabs against primary isolates in or left unvaccinated entirely. The group of mice that received a light of the efficacy of T cells in reducing viral loads in HIV-1- mixture of 5 nonlipidated and 7 lipidated peptide mixtures, com- infected individuals has encouraged focus on cell-mediated im- prising a total of 176 unique peptide variants, showed a higher mune responses (11). level of specific antiviral CD8ϩ T cell responses to heterologous HIV-1 subtypes A–D than did mice immunized with only lipidated or nonlipidated peptides. Based on these results obtained in mice, *Variation Biotechnologies, Ottawa, Ontario, Canada; †Division of Virology and Mo- six macaques were immunized with the mixture of lipidated and http://classic.jimmunol.org lecular Immunology, Department of Pathology and Laboratory Medicine, University of Ottawa and Children’s Hospital of Eastern Ontario Research Institute, Ottawa, nonlipidated peptides. The breadth of cellular and humoral immu- Ontario, Canada; ‡Department of Medical Microbiology and Immunology, Davis nity induced in vaccinated macaques was measured against heter- § School of Medicine, University of California, Davis, CA 95616; Division of HIV, ologous HIV-1 subtypes. Results from this study demonstrated that Public Health Agency of Canada, Ottawa, Ontario, Canada; and ¶Center for Neuro- logic Diseases, Brigham and Women’s Hospital, Harvard Medical School, Boston, the multivalent HIV-1 candidate vaccine was able to generate a MA 02115 broad cell-mediated immune response against a wide range of het- Received for publication April 10, 2007. Accepted for publication November erologous HIV-1 subtypes A–F. Immunophenotypic characteris- Downloaded from 29, 2007. tics of specific T cells in immunized macaques indicated an up- The costs of publication of this article were defrayed in part by the payment of page regulation in memory CD4ϩ, CD8ϩ, and double-positive (DP) charges. This article must therefore be hereby marked advertisement in accordance ϩ ϩ with 18 U.S.C. Section 1734 solely to indicate this fact. CD4 CD8 T cell subsets. The candidate vaccine was also able to 1 Address correspondence and reprint requests to Dr. Francisco Diaz-Mitoma, Divi- induce a relatively high level of IgG and IgA Ab binding and sion of Virology and Molecular Immunology, Research Institute, Children’s Hospital moderate neutralizing activity against T cell line-adapted (TCLA) of Eastern Ontario, 401 Smyth, Ottawa, Ontario, K1H 8L1, Canada. E-mail address: and primary HIV-1 isolates. To investigate whether the immune [email protected] response induced after with the HIV-1 peptides could 2 Abbreviations used in this paper: Nab, neutralizing Ab; DP, double positive; TCLA, T cell line adapted; TFA, trifluoroacetic acid; rVV, recombinant vaccinia virus; ICS, lead to protective immunity, vaccinated HLA-A2.1 mice were intracellular cytokine staining; FCS, forward scatter; SSC, side scatter; SI, stimulation challenged and a significant reduction in viral titers was revealed. index; SFC, spot-forming cell; EM, effector memory; CM, central memory; SP, single These findings indicate that the selected multivalent peptides in the positive; RLU, relative luminescence unit; SHIV, simian HIV; aa, amino acid. vaccine are able to induce broad immunogenicity against various Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 worldwide HIV-1 isolates.

www.jimmunol.org The Journal of Immunology 2175 by guest on September 30, 2021. Copyright 2008 Pageant Media Ltd. http://classic.jimmunol.org Downloaded from 2176 INDUCTION OF CROSS-SUBTYPE-SPECIFIC HIV-1 IMMUNE RESPONSES

Materials and Methods obtained from National Institutes of Health AIDS Research and Reference Vaccine design, synthesis, isolation, and analysis Reagent Program and used in intracellular cytokine staining (ICS) and ELISPOT assays. vP1286 expresses the MN (subtype B) Env, vT173 ex- We examined 21 subtype A, 128 subtype B, 51 subtype C, 17 subtype D, presses the 94UG114.1 (subtype D) Env, vT176 expresses the 90CF402.1 and 33 subtype E sequences (Los Alamos Database) in the formulation of (subtype E) Env, vBD3 expresses the 89.6 (subtype B) Env, vT198R ex- our vaccine. Vaccine formulations are based on five variable epitopes in presses the 92UG037.1 (subtype A) Env, vT234 expresses the 93BR02 HIV-1 Env and two variable epitopes in HIV-1 Gag (see Fig. 1A). All (subtype F) Env, vABT 408 expresses the 92UG037.1 (subtype B) Env, peptides were synthesized by automated solid-phase F-moc chemistry on a vT331 expresses the 96ZM651.8 (subtype C) Env/Gag/Pol, and vABT489 Pioneer Peptide Synthesizer (Applied Biosystems). All protected amino acid expresses the IIIB (subtype B) Env/Gag/Pol, and empty vT 1170 as a (aa) and resins were purchased from Novabiochem (EMD Biosciences). control. Reagents and palmitic acid were obtained from Sigma-Aldrich. All sol- The recombinant proteins HIV-1 IIIB gp160 (subtype B) and HIV-1 MN vents were obtained from OmniSolv (EMD Chemicals). gp120 (subtype B) were purchased from Advanced Biotechnologies. The Briefly, peptides were assembled by stepwise approach using a four recombinant protein HIV-1 CM gp120 (subtype E) was purchased from times excess of amino acids on a Rink-Amide NovaGel resin. Equimolar Protein Sciences. The recombinant HIV-1 gp120 proteins subtype C amount of amino acids were added at multiple positions with variability (96ZM651), HIV-1 gp120 subtypes A/E (93TH975), HIV-1gp120 subtype within an epitope. For purposes of lipidation, a portion of each of gp120- B (SF162), and HIV-1 peptides corresponding to subtypes A–D were ob- derived peptide formulations, and the entire amount of Gag-derived pep- tained from the National Institutes of Health AIDS Research and Reference tides were elongated by tripeptide linker, Lys-Ser-Ser. N-terminal Lys was Reagent Program. double lipidated at its two free amino groups using a 10-fold excess of palmitic acid for 12 h. Following completion of peptide synthesis, peptides Proliferation assays were cleaved from the solid-phase support and the protective groups were PBMCs from animals were resuspended at 2 ϫ 106 cells/ml in RPMI 1640 removed with trifluoroacetic acid (TFA) containing “mixture” (TFA/water/ containing 10% FCS, 50 ␮M 2-ME, and 100 U/ml penicillin/streptomycin. phenol/thioanisole/triisopropylsilane (83:5:5:5:2)) for 3 h. Crude peptides ␮ ϫ 5 ϫ A 100- l aliquot containing 2 10 cells was added to each well of a were precipitated in cold ether, centrifuged at 1000 g for 5 min, and the 96-well plate. The recombinant proteins HIV-1 IIIB gp160 (subtype B), ether was removed. The extraction procedure was repeated three times, and HIV-1 MN gp120 (subtype B), HIV-1 CM gp120 (subtype E), HIV-1 samples were finally dried under nitrogen gas. Nonlipidated peptide mix- 96ZM651 gp120 (subtype C), HIV-1 93TH975 gp120 (subtypes A/E), and tures were eluted using H2O/CH3CN solvent system on reverse-phase C18 HIV-1 SF162 gp120 (subtype B) were added (1 ␮g/ml) to each well in column (Vydac), and all lipidated peptide formulations were collected by triplicate. Cells were also stimulated with pooled HIV-1 Env peptides. As size-exclusion chromatography on Sephadex LH-20 in DMSO. Organic a positive control, cells were also stimulated with PMA and ionomycin solvents were reduced to a minimum, and the peptides were then resus- (Sigma-Aldrich). After 5 days of culture, 1 ␮Ci [3H]thymidine (Amer- pended in water and finally lyophilized. sham) was added to each well. Following 16 h of incubation, cells were Analytical reverse-phase-HPLC was conducted using a Vydac C18 column ϫ harvested onto glass-fiber filter mats and thymidine incorporation was mea- (4.6 300 mm; Mandel Scientific) at 214 nm. Peptides were eluted with a sured with a Microbeta beta counter (Wallac). Results were expressed as 0–100% gradient over 60 min at flow rate of 1 ml/min using 0.1% TFA in stimulation index calculated by dividing the mean cpm of cells incubated H2O and 0.1% TFA in CH3CN as solvents. All peptides were characterized by with HIV-1 proteins by the cpm of cells incubated with medium alone. MALDI-TOF mass spectrometry using a Voyager-DE Biospectrometry Work- station (Applied Biosystems), equipped with delayed ion extraction. All ex- ELISPOT assay pected peptide masses were identified on mass spectra. PBMCs were stimulated with 2 PFU of rVVs expressing HIV-1 genes from Animal studies and immunization subtypes A–F or empty rVV as a control (National Institutes of Health AIDS Research and Reference Reagent Program) for 90 min. After wash- Twelve cynomolgus macaques serologically negative for SIV were main- ing, 2.5 ϫ 105 cells were added to plates coated with anti-monkey IFN-␥ tained in accordance with the institutional animal care protocols (ACC (Mabtech). After 40 h at 37°C, cells were removed, washed with PBS plus by guest on September 30, 2021. Copyright 2008 Pageant Media Ltd. 03025) of Health Canada. Fifty micrograms of each of the five nonlipidated 0.05% Tween 20, and incubated with 1 ␮g/ml biotinylated anti-monkey (80 variants) and seven lipidated (96 variants) formulations were resuspended IFN-␥ (Mabtech) for2hatroom temperature. After washing, streptavidin- in sterile saline and mixed at a 1:1 ratio with Montanide ISA-51 adjuvant. The ALP-PQ (Mabtech) in PBS plus 0.5% FCS was added and incubated for vaccine was injected into six animals (nos. 079, 012, 059, 020, 011, and 013) 1 h at room temperature. The plates were washed as above and developed ϳ s.c. 2 cm from the inguinal area in the anterior aspect of the thigh, close to with 100 ␮l/well 5-bromo-4-chloro-3-indolyl phosphate/NBT alkaline the draining inguinal lymph nodes. Animals were vaccinated at months 0, 1, 3, phosphatase (Moss) for 20 min at room temperature. The reaction was 7, and 10. Another six animals were left unvaccinated because in a previous stopped by rinsing the plates with tap water. The numbers of spots were experiment, no adaptive immune response difference between unvaccinated counted and expressed as the mean count Ϯ SD. Anti-mouse IFN-␥ Ab and montanide alone group was observed (15). (mAb AN18; Mabtech) and biotinylated anti-mouse IFN-␥ Ab (mAb R4- Six- to 8-wk-old humanized female C57BL/6-TgN (HLA-A2.1) mice 6A2-biotin; Mabtech) were used in mice ELISPOT assay. which contain the full-length human HLA-A2.1 gene were purchased from http://classic.jimmunol.org The Jackson Laboratory and housed in the animal facility at University of Intracellular cytokine staining Ottawa in accordance with the Children’s Hospital of Eastern Ontario-68- approved protocol. Four groups (n ϭ 4) of mice (adjuvant alone, lipidated PBMCs from vaccinated and unvaccinated macaques were stimulated with peptides, nonlipidated peptides, and a mixture of peptides) were immu- 2 PFU of rVVs expressing HIV-1 genes from subtypes A–F or empty rVV nized four times s.c. at the base of the tail. Each mouse received a total of as a control (National Institutes of Health AIDS Research and Reference 120 ␮g of immunogen (1:1 ratio of Montanide ISA-51 adjuvant to peptides Reagent Program). Cells were washed after 90 min and suspended in RPMI in PBS) per vaccination. Ten days after the last immunization, the mice 10 and incubated for 16–18 h in monensin (GolgiStop; BD Biosciences).

Downloaded from were euthanized and cell-mediated immune response was measured. Sixteen hours after incubation, the cells were washed with 3 ml of PBS plus 2% FCS plus 0.01% azide and surface-stained for 15 min with anti-human Recombinant vaccinia viruses (rVV), recombinant proteins, CD3-PerCP-Cy 5.5 and CD4-R-PE or CD8-R-PE (BD Biosciences). The and peptides cells were washed as above and fixed according to the manufacturer’s instructions (BD Biosciences). PBMC were permeabilized with Cytoperm The following recombinant vaccinia viruses (vP1286, vT173, vT176, (BD Biosciences) and stained with anti-human perforin-FITC (isotype vBD3, vT198R, vT234, vABT 408, vT331, vABT489, and vT1170) were mouse IgG2b) or IFN-␥-FITC (isotype mouse IgG1) for half an hour at

FIGURE 1. Characterization and synthesis of a peptide-based vaccine. A, HIV peptide sequences contained in the vaccine candidate. A total of 176 peptide species, representing five variable epitopes in HIV-1 env and two variable epitopes in HIV-1 Gag (aa 131–154, 159–185, 307–324, 388–410, and 456–471 in Env, and aa 369–384 and 470–498 in Gag, based on the B.US.SF2 sequence). B, HPLC histograms of two independent syntheses of one of the five nonlipidated vaccine formulations. This formulation contained 16 anticipated peptide variants. C, Mass spectrum demonstrates the ability to identify 16 unique peptide variants contained within a vaccine formulation. Arrows indicate each of the 16 peptide variants. Double arrows over some peaks indicate the presence of two peptide variants of identical m.w. Additional adjacent peaks represent the isotopic pattern for 13C, which occurs in synthesized peptides analyzed by mass spectrometry. The Journal of Immunology 2177

stimulated with the rVV control was 0.6–1.5%. The percentages of specific CD8ϩ/IFN-␥ were calculated by subtracting nonspecific CD8ϩ/IFN-␥ cell frequencies from those stimulated with the rVV control. The percentages of specific CD4ϩ/perforin were calculated by subtracting nonspecific CD4ϩ/ perforin cell frequencies from those stimulated with the rVV control.

Ki67 expression and T cell phenotype PBMCs from vaccinated and unvaccinated animals were isolated and stim- ulated with a pool of HIV-1 peptides. Sixteen to 20 h after incubation, cells were washed with PBS, 2% FCS, 0.01% azide, and then surface-stained with anti-human CD3-allophycocyanin-Cy7 and CD4-PerCP mAbs. The cells were fixed, permeabilized (BD Biosciences), and stained with the FIGURE 2. HIV-1-specific T cell immune responses detected by IFN-␥ human Ki67-PE-labeled mAb (isotype mouse IgG1, k) or with isotype control (BD Biosciences). The frequency of Ki67ϩ T cells was analyzed ELISPOT assay expressed as SFC per million splenocytes in immunized using a FACSCanto flow cytometer (BD Biosciences). PBMCs were also mice. Four mice per group were immunized four times s.c. with the can- stimulated with or without the pool of HIV-1 peptides and the proportions didate HIV-1 (lipidated, nonlipidated, or mixture of peptides) or of central and effector memory CD4 and CD8 T cells were assessed by the adjuvant alone. Ten days after the last immunization, the mice were eu- following Abs: CD95-FITC, CD28-PE, CD4-PerCP, CD8-PE-Cy7, CD3- thanized and ELISPOT assay was performed. The data are the mean Ϯ SD allophycocyanin-Cy7, and IFN␥-allophycocyanin (cynomolgus, rhesus, .(Statistically and baboon reactivity, isotype mouse IgG1, k; BD Biosciences ,ء .values of duplicate samples from four mice in each group significant differences with p Յ 0.01–0.05. Ab binding Total IgG and IgA titers were determined by ELISA as described previ- 4°C. After washing, the cells were analyzed by FACScan (BD Bio- ously (16). Briefly, 96-well EIA/RIA TM Stripwell plates (Corning) were sciences). The data were reanalyzed using the WinMDI version 2.8 soft- coated overnight at 4°C with HIV-1 recombinant proteins or the pool of ware package (J. Trotter, The Scripps Institute, San Diego, CA). The back- HIV-1 peptides at a concentration of 0.3 ␮g/well. The plates were washed ground in the negative cells was 0.1–0.4%. The background in cells six times with 0.5% Tween 20/PBS, and blocked with 1% BSA (fraction

ϩ by guest on September 30, 2021. Copyright 2008 Pageant Media Ltd. FIGURE 3. HIV-1-specific CD8 T cell response detected by ICS in the immunized macaques. PBMCs from vaccinated and control animals were stimulated with a pool of HIV-1 pep- tides, rVVs expressing HIV-1 pro- teins from subtypes A–F or empty rVV. After stimulation, cells were surface stained for CD3ϩ and CD8ϩ, as well as stained intracellularly for http://classic.jimmunol.org IFN-␥ and analyzed by flow cytom- etry. The results were analyzed with the WinMDI program. A, Dot plots show results from individual repre- sentative macaque. B, Percentage of CD8ϩ/IFN-␥ to HIV-1 subtypes A–F is shown in each vaccinated macaque Downloaded from after subtraction of values from empty vaccinia. No specific CD8ϩ T cell re- sponse has been observed in control animals. ND, Not detected; Nd, not determined. 2178 INDUCTION OF CROSS-SUBTYPE-SPECIFIC HIV-1 IMMUNE RESPONSES

FIGURE 4. HIV-1 specific CTL responses detected by IFN-␥ ELISPOT assay expressed as SFC per 1,000,000 PBMCs in immunized macaques. PBMCs from vaccinated and control animals were stimulated with rVVs expressing HIV-1 genes from subtypes A–F or empty rVV. The number of SFC induced was calculated by subtraction of SFC of cells stimulated with control rVV from SFC of cells stimulated with rVV-HIV-1. The data are shown as the mean Ϯ .Statistically significant difference with p Յ 0.01–0.05 ,ء .SD

V)/PBS. Serial dilutions of cynomolgus plasma or saliva in 1% BSA (frac- homogenizer, centrifuged, and the supernatant was collected. Viral load tion V)/PBS were incubated overnight at 4°C. Plates were washed before was determined by performing plaque assays using the homogenated or- the addition of a 1/2000 dilution of peroxidase-labeled affinity purified goat gans. Homogenate dilutions ranging from 101 to 109 were used to inoculate anti monkey IgG or IgA (Kirkegaard & Perry Laboratories). After washing, HeLa cells, which were incubated at 37°C for 3 h. Cells were washed once color was developed with O-phenylenediamine dihydrochloride (Sigma- and covered with 2 ml of IMDM containing 10% FCS and 0.5% agarose. Aldrich). The color reaction was stopped with 1 N HCl and absorbance was The plates were left at room temperature for 30 min and transferred to 37°C by guest on September 30, 2021. Copyright 2008 Pageant Media Ltd. read at 490 nm with an ELISA plate reader (Bio-Rad). for 7 days. The plates were fixed overnight with 4% paraformaldehyde, stained with 0.05% neutral red in PBS, rinsed with tap water, and left Neutralization assays inverted at room temperature to dry before counting. Plasma from immunized macaques was collected in EDTA-coated tubes and neutralization assays were performed at Duke University (17) and Statistical analysis Monogram Biosciences (18) as previously described. Briefly, HEK-293 For flow cytometry analysis, lymphocytes were gated on forward (FSC) vs cells were cotransfected with an HIV Env expression vector and a genomic side scatter (SSC) and 30,000–50,000 events were collected. CD8ϩ, HIV vector which contains a luciferase gene in place of Env. Viral stocks CD4ϩ, and DP CD4ϩCD8ϩ T cells were identified based on SSC and were collected from the HEK-293 cells and incubated for 18 h with serial positive surface expression of CD3. Cells were then gated based on CD8ϩ, dilutions of heat-inactivated animal plasma (1/10 initial dilution, 3-fold CD4ϩ, CD8ϩCD4ϩ T cells vs perforin or IFN-␥. Furthermore, cells were http://classic.jimmunol.org serial dilutions). After incubation, the virus/plasma mixture was used to ϩ ϩ ϩ gated based on CD28 and CD95 expression to define memory T cell sub- infect CXCR4 CCR5 /CD4 /U87 cells. Virus infectivity was determined populations. Analysis was performed with the FACSCanto analyzer. Ab- 3 days later by measuring the amount of luciferase activity expressed in solute numbers were calculated by multiplying the percentage determined infected cells. Neutralizing activity was reported as the concentration or on flow cytometry by the total lymphocyte count determined from a CBC dilution of each plasma required to inhibit the virus by 50% (IC50). The analyzer. For the cell proliferation assay, results were expressed as stim- assay control for nonspecific inhibition was HIV pseudotyped with the ulation index (SI) calculated by dividing the mean cpm of cells incubated non-HIV amphoMuLV Env. Plasma titers that were at least three times with HIV-1 proteins by the cpm of cells incubated with medium alone.

Downloaded from higher than those observed against the negative control virus (aMLV) were Results of ICS, Ab titer, and lymphocyte proliferation were expressed as considered positive. Other control viruses were NL43 and JRCSF, a lab- mean Ϯ SD. The t test was applied for the statistical analysis of the data. oratory-adapted X4-tropic strain and a neutralization-resistant R5-tropic primary isolate, respectively. HIV-1-positive serum (Z23) with a broad neutralizing capacity was used as a control. Results Vaccine design and synthesis Challenge study To elicit immunity capable of recognizing divergent stains of HIV, ϭ Six- to 8-wk-old HLA-A2.1 mice (n 4) were immunized with a mixture we used a novel approach to solid-phase peptide synthesis to ob- of lipidated and nonlipidated peptides. A total of 120 ␮g of vaccine were resuspended in sterile saline and mixed at a 1:1 ratio with Montanide tain degenerative peptide mixtures representing major antigenic ISA-51 adjuvant. The vaccine was injected s.c. with 1-mo interval between variants present in the five hypervariable regions of the HIV-1 each immunization. Four weeks after the last immunization, mice were gp120 as well as two variable epitopes present in Gag. Individual 7 challenged i.p. with 2 ϫ 10 PFU of a recombinant vaccinia virus express- peptides ranged from 15 to 27 aa in length. The complete vaccine ing Gag/Pol/Env proteins from a subtype B variant of HIV-1. Mice were monitored for body weight, sickness, and abnormalities. On day 5, mice contained 7 lipidated and 5 nonlipidated peptide mixtures (96 lipi- dated variants and 80 nonlipidated variants) comprising a total of were euthanized with CO2 gas and the ovaries, the right lung, and spleens were dissected. Ovaries were homogenated separately using a glass Teflon 176 unique peptide variants (Fig. 1A). At the completion of each The Journal of Immunology 2179

FIGURE 5. T cell proliferation results in macaques immunized with the candidate vaccines. PBMCs were cultured and stimulated with the recombinant proteins HIV-1 MN gp120 (subtype B), HIV-1 IIIB gp160 (subtype B), HIV-1 CM gp120 (subtype E), HIV-1 96ZM651 gp120 (subtypes C), HIV-1 SF162 gp120 (subtype B), and HIV-1 93TH975 gp120 (subtypes A/E). Five days later, [3H]thymidine was added and after 16 h the incorporated radioactivity was measured in harvested cells. Results were expressed as SI calculated by dividing the mean cpm of cells incubated with HIV-1 proteins by the cpm of cells incubated with medium alone. Each bar represents SI from an individual immunized macaque. The dotted line indicates the cutoff value which is a SI of 2.

synthesis reaction, either 8 or 16 peptide variants were expected to 10. Macaques were bled 10–14 days after each vaccination and the be present due to incorporation of 2 aa in the growing peptide immunogenicity of the vaccine was compared with control chain at three or four defined positions. To ensure consistency, macaques. HPLC was performed on each batch of peptides. A representative HPLC histogram of two different batches is shown in Fig. 1B. A multivalent HIV-1 peptide vaccine elicits a broad Using mass spectrometry, we were able to identify the appropriate cross-subtype T cell immune response in nonhuman primates number of unique peptide species with their anticipated m.w. (Fig. Cross-subtype reactivity of specific CD8ϩ T cell immune response 1C). was determined with ICS and ELISPOT assays. PBMCs from im- A portion of each of the seven peptide mixtures was lipidated munized macaques were cultured and stimulated with a panel of with palmitic acid. Two of the peptide mixtures, which represent recombinant vaccinia viruses representing the most predominant variable epitopes located in Gag, were lipidated entirely (Fig. 1A). HIV-1 subtypes A–F. After stimulation, cells were surface stained The lipidated form of the peptide variants served two functions: to for CD3 and CD8, as well as stained intracellularly for IFN-␥ and elicit CTL responses and to activate innate immunity during vac- analyzed by flow cytometry. Following each immunization with cination (19–21). Indeed, we vaccinated mice with nonlipidated, lipidated, or a combination of both lipidated and nonlipidated pep- tide mixtures, and evaluated cellular immunity using an IFN-␥ by guest on September 30, 2021. Copyright 2008 Pageant Media Ltd. ELISPOT assay (Fig. 2). We observed that immunization with a combination of both lipidated and nonlipidated peptides induced more robust cellular immunity than did lipidated or nonlipidated peptides alone.

Vaccination strategy Based on the results in mice, we chose a vaccine formulation con- taining both lipidated and nonlipidated peptides to test the immune response induced in nonhuman primates. Six cynomolgus ma- http://classic.jimmunol.org caques were immunized with seven lipidated and five nonlipidated peptide mixtures at a 1:1 ratio with Montanide ISA-51 adjuvant. The vaccine was administered five times at months 0, 1, 3, 7, and

Table I. PBMCs from immunized macaques were cultured and

Downloaded from stimulated with the HIV-1 pool peptides after the first, third, fourth, and fifth vaccinationa

SI in Immunized Macaques No. of Positive Number of SI/No. of Tested Vaccination 013 011 079 059 020 012 Macaques FIGURE 6. Measurement of cytotoxic CD4ϩ T cell activity in immu- 1 0/6 nized macaques. Two weeks after the last immunization, PBMCs from 2 NdNdNdNdNdNd Nd vaccinated and control animals were stimulated with rVVs expressing 3 6 9 2 7 4.5 5/6 HIV-1 proteins from subtypes A–F or empty rVV. After stimulation, cells 4 6 5.8 8 6 5 8.5 6/6 ϩ ϩ 5 2 4.5 4.6 4 8.5 12 6/6 were surface stained for CD3 and CD4 , as well as stained intracellularly for perforin and analyzed by flow cytometry. Frequency of perforin-posi- a Five days later, [3H]thymidine was added and after 16 h the incorporated ra- tive CD4ϩ T cell was detected after stimulation with HIV-1 subtypes B, C, dioactivity was measured in harvested cells. SI was calculated by dividing the mean and E and not other HIV-1 subtypes. The data are shown as the mean Ϯ ء cpm of cells incubated with HIV-1 peptides by the cpm of cells incubated with medium alone. SI was equal to the cutoff value (dash line) after the first vaccination. SD. , A significant difference between the immunized group and control Nd, Not determined. (p Յ 0.05). 2180 INDUCTION OF CROSS-SUBTYPE-SPECIFIC HIV-1 IMMUNE RESPONSES

FIGURE 7. Immunophenotype characterization of memory blood CD4ϩ, CD8ϩ, and DP CD4ϩCD8ϩ T cells. A, Two weeks after the final immuni- zation, PBMCs from control and immunized macaques were stimulated with HIV-1 pool peptides. Cells were gated on FSC vs SSC scatter. CD8ϩ, CD4ϩ, and DP CD4ϩCD8ϩ T cells were identified based on SSC and positive surface expression of CD3. Furthermore, cells were gated based on CD28ϩ and CD95ϩ expression to define memory T cell subpopulations. B, The absolute numbers of memory CD4ϩ, CD8ϩ, and DP CD4ϩCD8ϩ T cells were calculated by multiplying the percentage determined on flow cytometry by the total lymphocyte count determined from a CBC analyzer. The data are shown as the by guest on September 30, 2021. Copyright 2008 Pageant Media Ltd. mean Ϯ SD. Statistically significant difference between groups is indicated by their p values.

the candidate HIV-1 vaccine, cellular immunity was analyzed. No- from three subtype B variants, one subtype E variant, one subtype tably, five of six immunized macaques showed a high frequency of C variant, and one subtype A/E variant. Five days after stimula- cross-subtype IFN-␥-secreting CD8ϩ T cells to all HIV-1 subtypes tion, [3H]thymidine was added and 16 h later, cells were harvested A–F after the last immunization (Fig. 3). To confirm the results and thymidine incorporation was measured. Two weeks after the obtained by ICS, ELISPOT analysis was conducted using PBMCs fifth immunization, two macaques showed response to variants from immunized macaques. PBMCs from vaccinated and unvac- from all three subtypes. One animal responded to variants from http://classic.jimmunol.org cinated animals were stimulated with rVVs expressing HIV-1 subtypes B and E, and two additional animals responded to mul- genes from subtypes A–F or empty rVV. The number of spot- tiple subtype B variants (Fig. 5). One of the immunized macaques forming cells (SFC) induced was calculated by subtraction of SFC responded weakly to the Env proteins tested at this time point, but of cells stimulated with rVV-HIV-1 from SFC from cells stimu- did respond to subtypes B and E when assessed at an earlier time lated with control rVV. PBMCs from immunized animals pro- point (4 wk after the third immunization, data not shown). In ad- duced a statistically significant increase in the number of spots dition, proliferative immune responses to HIV-1 pool peptides Downloaded from detected in response to stimulation with heterologous subtypes were also detected in five of six vaccinated macaques after the A–F compared with unstimulated cells (Fig. 4). These results dem- third immunization. All six macaques developed proliferative re- onstrate that our multivalent vaccine is capable of eliciting a broad sponses to the pooled HIV-1 peptides after the fourth immuniza- ϩ cross-specific CD8 T cell response to HIV-1 variants despite tion; however, the level of T cell proliferation was decreased in MHC diversity between animals and significant amino acid se- four and increased in two animals (Table I). quence differences in tested HIV-1 isolates. It has previously been shown that CD4ϩ T cells with cytotoxic activity are expanded during chronic viral (22), and can Induction of proliferative immune response and cytotoxic ϩ be induced with vaccination (23). Analysis of PBMCs 2 wk after CD4 T cell activity by multivalent HIV-1 peptide regimen the last immunization demonstrated a high frequency of perforin- in macaques positive cytotoxic CD4ϩ T cells when stimulated with HIV-1 sub- Proliferation of CD4ϩ T cells plays a crucial role in regulation of types B, C, and E (Fig. 6). None of the immunized animals showed both humoral and cellular immune responses by expansion of Ag- a perforin-positive CD4ϩ T cell response to HIV-1 subtypes A, D, stimulated B and CD8 T cells, respectively. PBMCs from immu- or F. Perforin-positive cells were not detected at the later 6 wk time nized macaques were stimulated with HIV-1 Env proteins derived point, consistent with the notion that these cells are a terminally The Journal of Immunology 2181

differentiated, effector population of Th cells that are lost over time (24). Immunophenotype characterization of specific memory cells after vaccination with HIV-1 peptide regimen The induction of long-lived specific cell-mediated immune re- sponses is a critical aspect in the development of an effective vac- cine. Memory T cells play an important role in jeopardizing viral replication and in providing long-term immunity (25–27). There- fore, it was of interest to measure the induction of these cells in response to immunization. We characterized specific HIV-1 mem- ory T cell responses after the final immunization. The surface markers CD28 and CD95 have been used to distinguish different populations of memory T cells (28, 29). Immunophenotype char- acteristics of specific memory cells were evaluated upon stimula- tion with specific HIV-1-pooled peptides. As indicated in Fig. 7B, the candidate vaccine was able to generate a significantly higher level of effector (EM) and central (CM) memory DP CD4ϩCD8ϩ T cell in immunized macaques compared with control animals. In addition, specific EM cells were increased in both single-positive (SP) CD4ϩ and CD8ϩ T cells after the last immunization. No significant difference in SP CM CD4ϩ and CD8ϩ T cells was detected between immunized and control macaque groups. FIGURE 8. HIV-1-specific total IgG Ab titers in immunized macaques. Previous studies have shown that the Ki67 molecule plays im- A, The 96-well plates were coated with rHIV-1 Env proteins MN, SF162, portant roles in cellular survival and proliferation. Notably, it is not IIIB, CM, and HIV-1 pool peptide. Two weeks after the last immunization, macaque sera were serially diluted in wells and absorbance was read at 490 expressed in resting cells. Thus, this marker can be used to monitor nm with an ELISA plate reader. Results are shown as mean concentration cell cycle progression. We evaluated the expression of Ki67 pre- (n ϭ 6). B, Sera from macaques immunized at 0, 1, 3, 7, and 10 were immunization and after the first, third, and fifth immunization in collected at the indicated time points and tested for the recombinant pro- macaques. Four animals (079, 012, 0120, and 059) had higher T teins HIV-1 MN, 96ZM651, CM, and 93TH975. Responses are reported cell frequencies of Ki67 expression after the last vaccination. for dilution of 1/100 for each sample. The mean OD among the animals There was no expression of Ki67 in animal 011 before or after (n ϭ 6) at each time point is shown. vaccination (data not shown). The assay was not performed in animal 013 due to lack of available PBMCs. Induction of cross-subtype humoral immunity in CxCR4 coreceptors. Neutralizing Ab activity was based on the

by guest on September 30, 2021. Copyright 2008 Pageant Media Ltd. immunized macaques serum dilution at which relative luminescence units (RLU) were reduced by 50% compared with virus control wells. Although our To measure Ab titers against various HIV-1 gp120 proteins, both vaccine was designed mainly based on CD4ϩ ϩ pre- and postimmunization plasma samples from the immunized and CD8 T cell macaques were collected, and binding reactivity to recombinant epitopes, neutralizing activity was detected in all immunized ma- HIV-1 Env proteins MN, SF162, IIIB, CM, and the pool of HIV-1 caques against HIV-1 isolates MN, SF162, and SS1196.01 after peptides were determined by ELISA. A significant enhancement of the fifth immunization (Fig. 9). The kinetics of Nab response was the levels of anti-Env or anti-HIV-1 peptide Abs was observed in further tested against 14 HIV-1 primary isolates. Three of six an- all immunized animals (Fig. 8A). In fact, the magnitude of Ab imals showed Nab response to HIV-1 subtype D isolates 92UG024 response augmentation in macaques was even higher than in mice and 93UG059 after the third and fifth vaccination. Neutralizing http://classic.jimmunol.org that were immunized with the same HIV-1 vaccine regimen (data response in macaque 079 against isolate 93UG059 reached a titer not shown). The kinetics of Ab responses in sera of immunized of 1:51 after the third immunization and increased to 1:62 after the animals were also characterized using HIV-1 Env from isolates MN, 96ZM651, CM, and 93TH975 (Fig. 8B). Furthermore, we determined whether the multivalent HIV-1 vaccine was able to augment mucosal immunity. Saliva IgA was Downloaded from tested using HIV-1 subtype B (IIIB and MN) and one subtype E (CM) Env proteins. Three of six macaques (079, 011, and 020) had IgA titers against at least one Env protein; two macaques (013 and 012) had titers against both HIV-1 subtypes B and E Env proteins. HIV-1 Env IgA Ab titers were between 1:50 to 1:300. To determine whether vaccine-induced Abs had functional, bi- ologically relevant neutralizing activity, sera from the vaccinated animals were assessed for their ability to neutralize a panel of both TCLA and primary X4 and R5 viruses. Neutralizing activity of FIGURE 9. Nab responses against TCLA strains of HIV-1. Plasma sera was analyzed at Duke University and Biosciences Monogram from immunized macaques were obtained 2 wk after the last immunization. (former ViroLogic), against a total of 3 TCLA and 14 primary Nab activity was shown based on the plasma dilution at which RLU were isolates of HIV-1 variants, respectively. The principle in all Nab reduced by 50% compared with virus control wells. The data are shown as assays used both, in Duke or Monogram, was based on reductions the mean Ϯ SD. The values for each of the preimmune animals were in viral infectivity in target cells expressing both CCR5 and identical (Ͻ20). 2182 INDUCTION OF CROSS-SUBTYPE-SPECIFIC HIV-1 IMMUNE RESPONSES

Table II. Nab titers against HIV-1 subtypes D (92UG024 and 93UG059) and E (93TH053) in immunized macaquesa

IC50 (1/Dilution)

Number of Number of HIV-1 subtype D HIV-1 subtype D HIV-1 subtype E Macaques Vaccination isolate 92UG024 isolate 93UG059 isolate 93TH053 JRCSF NL43 aMLV

M 011 1 Nd Ͻ10 Ͻ10 Ͻ10 Ͻ10 Ͻ10 3Nd Ͻ10 Ͻ10 Ͻ10 Ͻ10 Ͻ10 5Nd Ͻ10 Ͻ10 Ͻ10 Ͻ10 11 M 013 1 15 Ͻ10 Ͻ10 Ͻ10 Ͻ10 11 3 19 Ͻ10 Ͻ10 17 Ͻ10 Ͻ10 5 31 53 Ͻ10 26 Ͻ10 Ͻ10 M 059 1 12 22 14 12 15 16 3 30 Ͻ10 Ͻ10 19 Ͻ10 Ͻ10 5 39 47 Ͻ10 31 Ͻ10 Ͻ10 M20 1 Ͻ10 11 Ͻ10 Ͻ10 17 Ͻ10 316 22Ͻ10 Ͻ10 Ͻ10 12 514 Ͻ10 Ͻ10 Ͻ10 Ͻ10 Ͻ10 M 012 1 Ͻ10 18 15 14 Ͻ10 12 3 Ͻ10 Ͻ10 22 21 Ͻ10 Ͻ10 545 Ͻ10 12 18 Ͻ10 22 M 079 1 Nd Ͻ10 14 Ͻ10 Ͻ10 Ͻ10 3Nd 51 23 19 12 Ͻ10 5Nd 62 18 21 14 Ͻ10 Z23 201 233 198 306 3727 Ͻ50 Z23 272 133 212 350 4141 Ͻ50 Z23 233 206 178 363 4479 Ͻ50

a Plasma from immunized macaques was obtained 2 wk after the first, third, and fifth vaccination and Nab activity was measured against HIV-1 primary isolates. The Nab titer is reported as the reciprocal of the dilution of plasma that produces 50% inhibition of target cell . A positive Nab assay (bold and underlined) was defined as an Ab titer at least three times higher than the negative control infection with aMLV. The titer of plasma against control strains (aMLV, NL43, and JRCSF) is shown. An HIV-1-positive serum (Z23) with a broad neutralizing capacity was used as a control serum. None of the immunized macaques were able to induce neutralizing activity against HIV-1 subtypes A, B, and C. Nd, Not determined.

fifth immunization. Similarly, macaques 013 and 059 had persist- Challenge of HLA A2.1 mice ing Abs after the third and fifth against HIV-1 sub- Because the vaccine was designed to elicit CTL responses to Env type D isolate 92UG024 (Table II). One of six animals also and Gag proteins of HIV-1, we were unable to evaluate its poten- showed Nab response to HIV-1 subtype E isolate 93TH053 after tial efficacy in the vaccinated monkeys; simian HIV (SHIV) vi- the third vaccination but this response was not detected later. Pri- ruses that could be used in challenge experiments express SIV- by guest on September 30, 2021. Copyright 2008 Pageant Media Ltd. mary isolates from subtypes A–C were not neutralized by any of derived Gag, thereby precluding determination of the potential the macaque sera. efficacy of vaccine-induced cellular immunity against the epitopes in this protein. Moreover, the use of SHIV challenge in the species of macaques is of questionable value. For instance, SHIV-89.6P and SIVmac251 exhibits only a low viral replication in cynomol- gus compared with rhesus macaques (30). Due to the above lim- itations to measure vaccine-induced efficacy, a murine model was used. HLA A2.1 mice were immunized four times with the same vaccine formulation used in the macaques. Four weeks after the http://classic.jimmunol.org last immunization, mice was challenged i.p. with 2 ϫ 107 PFU of recombinant vaccinia virus expressing HIV-1 IIIB (subtype B) Env/Gag/Pol proteins. Five days later, mice were sacrificed and the ovaries were harvested, as this is the organ in which vaccinia pref- erentially replicates. Vaccinia virus titers were evaluated by plaque assay using dilutions of ovary homogenates. As shown in Fig. 10, Downloaded from the group immunized with the HIV-1 multivalent peptides showed more than a 2-log reduction in titer of virus compared with control ( p Ͻ 0.05). Although it is unclear how well efficacy in this chal- lenge model may correlate with nonhuman primates or humans, the data nevertheless indicate that vaccine-induced immunity was able to recognize and eliminate processed viral Ags in vivo. FIGURE 10. Challenge study in mice with rVV-expressing HIV-1 IIIB (subtype B). HLA A2.1 mice were immunized with the HIV-1 candidate ϫ 7 vaccine, or adjuvant alone and challenged i.p. with 2 10 PFU of re- Discussion combinant vaccinia virus expressing HIV-1 IIIB Env/Gag/Pol proteins af- ter the last immunization. Five days later, mice were sacrificed and the The unprecedented HIV-1 mutation rate and resulting antigenic ovaries were harvested and assayed for vaccinia virus titer by plaque assay heterogeneity among viruses circulating throughout the world on HeLa cell lines, stained with crystal violet, and plaques counted at each poses a significant challenge to vaccine development (31). Several dilution. Viral loads were significantly lower in vaccinated mice compared strategies are currently being explored to circumvent the extensive with control group (p Ͻ 0.05). The data are shown as the mean Ϯ SD. antigenic variability of HIV-1. An immense deal of interest has The Journal of Immunology 2183

been focused on the induction of specific immunity based on con- of all 20 amino acids at any single position within an epitope. This served regions of HIV-1; however, none of these approaches were somewhat limited diversity exists despite immune pressure target- able to induce a broadly cross-reactive immunity and only a lim- ing these regions. In this study, several peptide variants were de- ited breadth of reactivity was raised against HIV-1 variants signed to overcome the genetic diversity by providing frequently (32–35). occurring amino acid substitutions of each epitope. The synthetic ϩ Previous studies have demonstrated that CD8 T cell function is peptide variants first were tested in HLA-A2.1 mice alone or in inversely correlated with viral load and is associated with protec- combination. The outcome of the preliminary experiments in mice tion and disease (36, 37). In an interesting study, Rowland-Jones led us to immunize macaques with a mixture of 176 peptides, ϩ et al. (38) showed the presence of a high frequency of CD8 T cells representing major variants known or predicted to be present in the in HIV-resistant sex workers in Nairobi. These results concluded hypervariable regions of HIV-1. To quantify cross-subtype CD8ϩ ϩ that effective cross-subtype CD8 T cells could be responsible for T cell recognition, PBMCs from immunized and control macaques protection against constant HIV exposure and infection. were analyzed for IFN-␥ production in response to HIV-1 subtypes Advances in MHC-peptide binding studies and characterization from global geographical regions. The multivalent vaccine was of resulting antiviral immune responses has initiated a new era in able to elicit a broad cross-subtype CD8ϩ T cell response as mea- vaccine design. Using lipidated peptide immunogens is one of sev- sured by ICS. To verify these results, the cross-subtype reactivity eral strategies in the improvement of immunogenicity that triggers of CD8ϩ T cells was further characterized by the ELISPOT assay. TLR pathways. Jackson et al. (39) demonstrated that lipid moieties These results also revealed a striking cross CD8ϩ T cell response present on peptides prolongs the duration of Ag presentation, en- toward heterologous HIV-1 subtypes in all vaccinated animals. hances cytosolic uptake of peptide immunogens, activates innate These results are significant as HIV-1 amino acid sequences within immunity due to TLR2 agonist effect, and differentiates nonacti- a subtype may differ by up to 20% and between subtypes by up to vated B cells into Ig-secreting plasma cells. In another study, Es- 35% within the regions that our vaccine targets. puelas et al. (40) studied the effect of lipopeptide analogues incor- To pursue the question of whether the vaccine regimen elicited porated into liposomes on the maturation of human DCs. They CD4ϩ Th immune responses in macaques, a proliferation assay found that slight modifications in the peptide moiety of lipopep- was performed using Env proteins from different HIV-1 variants as tides have an immense impact on cell surface markers such as stimulating Ags. Proliferative immune responses were detected in CD80, CD83, CD86, and HLA-DR on human DCs. In a landmark five to six immunized macaques against HIV-1 Env from two or study by Gahery et al. (41), a candidate HIV-1 vaccine comprising more distinct variants. The responses to Env proteins were differ- six lipidated peptides (Nef, Gag, and Env) were administered to ent in each macaque, for instance, two of the macaques responded highly active antiretroviral therapy-treated patients. The breadth better to subtypes C and E than other subtypes. The heterogeneity and magnitude of HIV-1-specific cell-mediated immune responses of these responses may be because macaques are outbred and have were measured and notably, some patients were able to induce new ϩ diverse MHC polymorphisms and carry different TCR repertoires. CD4 and CD8 T cell responses after vaccination. ϩ This may also explain variability in the response of vaccinated It is believed that all CD8 T cells are not identical in terms of animals to the panel of recombinant Env proteins. MHC diversity their ability to eliminate virus-infected cells. Essential factors, and TCR variability are also challenges to HIV vaccine develop- such as avidity between TCR-MHC/peptide, frequency of effective

by guest on September 30, 2021. Copyright 2008 Pageant Media Ltd. ment in the human population (49). CTL activity, or structural constrains on the epitope region, play ϩ ϩ Consistent with induction of a reactive Th cell repertoire, CD4 imperative roles in CD8 T cell effectiveness (42, 43). There are T cells were further analyzed for secretion of perforin. Previous unanswered questions about the immune response generated from ϩ vaccines targeting conserved epitopes, which may not be immu- studies showed that perforin-positive cytotoxic CD4 T cells nodominant and whether the frequency of specific immune cells is could contribute to the breadth of immune response by forming not sufficient to control viral infection. In a peptide vaccine study, pores in membrane of target cells that facilitate the entry of gran- zymes and consequently initiate an apoptotic cascade pathway (50, eight rhesus macaques were immunized with seven lipidated pep- ϩ tides comprising SIV-Nef and Gag proteins. Specific CD8ϩ T cells 51). Analysis of perforin-positive cytotoxic CD4 T cells 2 wk were detected in seven animals and two of eight animals demon- after the fifth immunization demonstrated an increased frequency http://classic.jimmunol.org strated a multispecific CTL immune response (44). These animals to HIV-1 subtypes B, C, and E but not A, D, or F. Interestingly, perforin-positive CD4ϩ T cells were not detected at the later 6 wk plus three control macaques were further challenged with SIVmac ϩ 251. The three control animals died on month 4 after infection. All time point, indicating that cytotoxic CD4 T cells are time depen- vaccinated macaques became infected, but survived after week 35 dent and that their population decreases over time (52). postchallenge, having a 2- to 3-log decrease in viral load compared In the quest for an effective vaccine, memory T cells may be with the control animals (45). imperative to viral destruction and providing long-term immunity Downloaded from Unlike conserved regions, hypervariable regions of HIV-1 con- (53–56). CM and EM T cells are recognized as two main popula- tain highly immunogenic regions encompassing critical B and T tions of memory T cells with different migration patterns (57). In ϩ ϩ ϩ cell epitopes. Although the highly variable HIV regions allow the particular, CM T cells expressing CD28 , CD95 , CCR7 , and virus to escape from the immune response, targeting the immuno- L-selectin, which are homing on lymph nodes, whereas EM T cells dominant epitopes in hypervariable regions may prove to be more do not express CD28 or CCR7 and home to peripheral tissues (58, effective in the induction of immunity with greater depth compared 59). The mechanism behind the generation of memory T cells is with the vaccines targeting conserved areas (46–48). not understood; however, the type and potency of Ags may influ- In this study, we demonstrate the ability of a polyvalent peptide- ence the quantity and differentiation of memory T cells produced based vaccine targeting the variable regions of Gag and Env pro- in response to Ags (59). In this study, an extensive analysis of teins to elicit broadly reactive T cell responses in immunized ma- specific memory T cell lymphocytes was performed in control vs caques. Although some amino acid positions within an epitope are vaccinated macaques after the last immunization. Our results in- quite variable, limits exist in the variation. For example, 5 or fewer dicated that effector memory SP CD4ϩ and CD8ϩ T cells ( p Ͻ amino acids comprise the majority of amino acids present at any 0.05 and 0.008 respectively) are up-regulated after vaccination. In given variable position, and there is little evidence for the presence addition, we found that central (CD28ϩCD95ϩ) and effector 2184 INDUCTION OF CROSS-SUBTYPE-SPECIFIC HIV-1 IMMUNE RESPONSES

(CD28ϪCD95ϩ) DP memory CD4ϩCD8ϩ T cells ( p ϭ 0.0043 ally, the natural mechanism of these chimeric viruses limits their and 0.0087, respectively) are increased in vaccinated animals. relevance in appraisal of CTL-based vaccines. Pathogenic Expression of Ki67 is important in differentiation between rest- SHIV89.6P has been shown to induce a severe loss of CD4ϩ T ing and activated T cells. Furthermore, induced Ki67 may correlate cells, while such a rapid loss of circulating CD4ϩ T cells is un- with cell cycle progression and IL-2 secretion. Thus, we further usual in HIV-1 individuals (68). Unlike SHIV89.6P, some patho- analyzed the kinetic expression of Ki67 before and after the first, genic SIV strains such as SIVmac251 or SIVsmE660 do not down- third, and fifth vaccination. Expression of Ki67 by T cells was regulate circulating CD4ϩ T cells and replicate highly in the detected in four of five animals. The results demonstrate that our chronic phase of disease, again being different from the HIV-1 candidate vaccine may be able to generate specific memory T cell mechanism of pathogenesis (69). Mechanisms of chimeric viruses immune responses that may contribute to long-term protection. are dependent on the species of macaques. For instance, viral rep- To address whether our vaccine induced a similar depth and lication of SHIV-89.6P and SIVmac251 in cynomolgus is lower breadth in the humoral response, the binding Ab titer and neutral- than either Chinese or Indian rhesus macaques and CD4ϩ T cell izing activity was characterized in immunized macaques. Two loss is lower and survival is longer in cynomolgus and Chinese weeks after each vaccination, plasma was collected and Ab titer rhesus macaques than Indian rhesus macaques (70). Due to these was measured against a panel of Env proteins and a mixture of difficulties in macaques challenge, a mouse model was chosen to HIV-1 peptides. All animals showed a substantial level of IgG Ab evaluate efficacy of the vaccine. HLA-A2.1 mice were immunized titer to the selected immunogens after the second immunization as with the vaccine, followed by challenge with a recombinant vac- measured by ELISA. Given that HIV infection predominantly oc- cinia virus expressing HIV-1 IIIB Env/Gag/Pol proteins. The re- curs via the mucosal route, there is interest in developing candidate sults indicate that vaccine-induced immunity was able to recognize HIV vaccines that can elicit mucosal immunity (60–62). To study and eliminate processed viral Ags in vivo. Taken together, these this phenomenon, saliva from immunized macaques was collected results indicate that our multivalent peptide vaccine is able to in- and anti-Env IgA Ab titer was measured against proteins from duce moderate humoral but broadly reactive cellular immune re- distinct HIV-1 subtypes. Interestingly, mucosal immunity was de- sponses with both breadth and depth against HIV-1 variants from tected in immunized animals even though the candidate vaccine different global geographical regions. This approach is now set to was administered s.c. undergo the first human on HIV-1-infected individu- Induction of Nabs is also important in an effective vaccine. We als in Ottawa to examine its safety and immunogenicity. first determined whether Nab directed against TCLA strains of HIV was detectable. Positive neutralizing activity was observed Acknowledgments against HIV-1 MN, SF162, and SS1196 isolates. We also mea- We acknowledge Drs. Julia Hurwitz and Katrina Gee for critical review of sured Nabs raised against 14 HIV-1 primary isolates from subtypes this manuscript. We thank Dr. David Montefiori at Duke University and the A–E. Notably, three of six immunized macaques developed neu- staff in Monogram Biosciences for measuring neutralizing activity in tralization activity against HIV-1 subtype D isolates 92UG024 and HIV-1 variants. We thank Susan Aucoin and Rita Frost for their technical 93UG059. One macaque was also able to induce neutralizing ac- assistance. We thank the National Institutes of Health AIDS Research and Reference Reagent program for providing the HIV-1 reagents. tivity against HIV-1 subtype E (93TH053) after the third vaccina- tion. HIV-1 primary isolates from subtypes A–C were not neutral-

by guest on September 30, 2021. Copyright 2008 Pageant Media Ltd. Disclosures ized by any of the macaque plasma. The Env peptides incorporated Drs. Francisco Diaz-Mitoma, Ali Azizi, Andrei Ogrel, and Masoud Ghor- in our vaccine formulation included the variable regions (V1 to bani are full time employees of Variation Biotechnologies Inc. (VBI), the V5). Except for an area of 4 aa (IGPG) in the tip of the V3 loop, sponsor of the research. Their compensation with VBI includes both salary which is conserved between HIV-1 subtypes (63), most other pep- and stock options. Dr. Francisco Diaz-Mitoma is also cross-appointed at tide regions demonstrated variability. To compare the similarity the Children’s Hospital of Eastern Ontario and the University of Ottawa. between our peptide sequences with known B cell epitopes in the Catalina Soare is a Ph.D. candidate at the University of Ottawa. She is HIV-1 isolates demonstrating neutralization, HIV-1 B cell epitopes employed part-time by VBI and is compensated for her efforts at an hourly sequences were searched in the Los Alamos database. We found rate. She holds no other interest in VBI. Drs. David E. Anderson and Jose that 5 aa (FYKDL) in the V2 peptide and 5 aa (RKSIR) in the V3 V. Torres are co-founders of VBI. They hold an ownership. http://classic.jimmunol.org peptide sequence are known B cell epitopes; however, it is not clear whether these sequences have been associated with neutral- References 1. Levy, J. A. 2006. HIV pathogenesis: knowledge gained after two decades of izing anti-HIV-1 activity. research. Adv. Dent. Res. 19: 10–16. To our knowledge, this is the first time that Nabs are demon- 2. Gallo, R. C. 2005. The end or the beginning of the drive to an HIV-preventive strated against HIV-1 isolates using linear hypervariable epitopes vaccine: a view from over 20 years. Lancet 366: 1894–1898. 3. Srivastava, I. K., J. B. Ulmer, and S. W. Barnett. 2004. Neutralizing antibody as immunogens. Because little is known about the kinetics of the responses to HIV: role in protective immunity and challenges for vaccine design. Downloaded from antiviral immune response with this immunization approach, ma- Expert Rev. Vaccines 3: S33–S52. caques were immunized five times. In contrast to the T cell re- 4. Li, M., F. Gao, J. R. Mascola, L. Stamatatos, V. R. Polonis, M. Koutsoukos, G. Voss, P. Goepfert, P. Gilbert, K. M. Greene, et al. 2005. Human immunode- sponse plateau observed after the third vaccination, Nab titers in- ficiency virus type 1 env clones from acute and early subtype B infections for creased after the fourth and fifth booster immunizations. standardized assessments of vaccine-elicited neutralizing antibodies. J. Virol. 79: 10108–10125. We were not able to evaluate the potential efficacy of vaccine by 5. Sarmati, L., G. d’Ettorre, E. Nicastri, L. Ercoli, I. Uccella, P. Massetti, SIV challenge due to highly divergent sequences between the Env S. G. Parisi, V. Vullo, and M. Andreoni. 2001. Neutralizing antibodies against genes of HIV-1 and SIV. Recent studies showed that chimeric autologous human immunodeficiency virus type 1 isolates in patients with in- creasing CD4 cell counts despite incomplete virus suppression during antiretro- SHIV strains are not an appropriate model to test the efficacy of viral treatment. Clin. Diagn. Lab. Immunol. 8: 822–824. HIV-1 vaccines and that the results have the potential to be mis- 6. Locher, C. P., R. M. Grant, E. A. Collisson, G. Reyes-Teran, T. Elbeik, interpreted (64–67). To acquire virulence in nonhuman primates, J. O. Kahn, and J. A. Levy. 1999. Antibody and cellular immune responses in breakthrough infection subjects after HIV type 1 glycoprotein 120 vaccination. SHIV strains have been passaged several times through animals. AIDS Res. Hum. Retroviruses 15: 1685–1689. These passages results in numerous alterations in the SHIV Env 7. Kwong, P. D., R. Wyatt, S. Majeed, J. Robinson, R. W. Sweet, J. Sodroski, and W. A. Hendrickson. 2000. Structures of HIV-1 gp120 envelope glycoproteins protein compared with the HIV-1 Env protein, and accordingly from laboratory-adapted and primary isolates. Structure Fold. Des. 8: limit their use as a challenge model for HIV vaccines. Addition- 1329–1339. The Journal of Immunology 2185

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