Encouraging results using the SHIV-rhesus macaque model have led to a number of ongoing clinical trials . However, a mucosally transmissible R5 SHIV clade C (SHIV-C) had been unavailable to test vaccine efficacy against HIV clade C (HIV-C). Here, we used two newly developed SHIV-C variants, SHIV-1157ip and SHIV-1157ipd3N4 , viruses that originated from SHIV-1157i, a chimera containing the SHIV-vpu+ backbone (expressing simian immunodeficiency virus (SIV) mac239 gag and nef) and incorporating most of gp120 plus the entire extracellular and transmembrane domains of gp41 of HIV1157i, a recently transmitted virus isolated from a Zambian infant. SHIV-1157ip, the early biological isolate, was obtained after passage through five rhesus macaques during peak viremia, an adaptation strategy we devised to avoid selecting neutralization escape viruses. Three of the five monkeys developed AIDS, and a late virus, SHIV-1157ipd, was isolated from one of them. A molecular clone was derived using the 5′ half of SHIV-vpu+ and the 3′ half of SHIV-1157ipd with an extra NF-κB binding site in the long terminal repeats. The resulting SHIV-1157ipd3N4 is highly replication competent and exclusively R5-tropic.
We developed these SHIV-C strains to evaluate candidate vaccines for use in infants against mucosal HIV-C transmission. Earlier, we showed strong containment of SHIV clade B (SHIV-B) replication in macaques immunized as infants solely with multimeric clade B gp160 [4–6]. We therefore incorporated multimeric clade C gp160 into a protein vaccine that also contained SIV Gag–Pol particles and HIV Tat. We deliberately mismatched SHIV-C env and the Env immunogen, which was derived from HIV1084i  isolated from a postnatally infected infant who was part of the same patient cohort at Lusaka Hospital as infant 1157i, the source of SHIV-C env. We reasoned that primate vaccine efficacy testing should approximate the real-life situation where human AIDS vaccine recipients will likely encounter HIV strains differing from those used to generate vaccines, although they may be exposed to strains circulating in the local community .
Indian-origin rhesus monkeys (Macaca mulatta) were housed at the Yerkes National Primate Research Center (YNPRC), Atlanta, Georgia, USA, a facility fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). All procedures were approved by the Animal Care and Use Committees of Emory University and the Dana-Farber Cancer Institute.
DNA expression plasmids were prepared using the pJW4303 backbone. The pJW-gp160-1084i plasmid encoding env derived from the pediatric isolate, HIV1084i , has been described . Codon-optimized gene fragments of SIVmac239 gag–pro (1.8 kb) and SHIV89.6P tat (0.3 kb) were amplified by PCR from multiple annealed overlapping oligonucleotide primers (100 bp each) . Each fragment was cloned between the NheI and BamHI sites downstream of a tissue plasminogen signal peptide sequence under the control of the cytomegalovirus immediate early promoter and upstream of a bovine growth hormone transcriptional termination sequence of the mammalian expression plasmid pJW4303. For all inoculations, plasmids were purified using EndoFree Giga Prep Kit from Qiagen (Valencia, California, USA). Animals receiving DNA were inoculated intradermally with 250 μg of each plasmid (total 750 μg DNA) at each time point; control animals (Groups 3 and 3a) received 750 μg of empty pJW4303 DNA plasmid vector. For protein inoculations, HIV-C gp160 was prepared from recombinant vaccinia virus infected BSC-40 cells as described previously . SIV Gag–Pol particles were prepared essentially as described [12–14] using an early-late synthetic promoter and HIV Tat was purchased from Advanced Bioscience Laboratories, Inc. (Kensington, Maryland, USA). Each protein was administered at 200 μg/inoculation.
ELISA for antibody binding titers
Microtiter plates were coated with HIV1084i gp160 (in 0.05 M Na2CO3, pH 9.6) overnight and blocked with 3% BSA/0.5% fetal calf serum (FCS) in phosphate-buffered saline (PBS). Serial dilutions of serum samples were prepared in blocking buffer and added to wells. After 2 h incubation at 37°C, wells were washed and alkaline phosphatase (AP)-conjugated rabbit antimonkey IgG (Sigma, St. Louis, Missouri, USA) was added. After an additional 1 h incubation at 37°C, AP substrate p-nitrophenyl phosphate (Sigma) was added and color development measured in an ELISA reader (Dynex Technologies, Chantilly, Virginia, USA). Antibody titers were calculated as reciprocal serum dilution giving optical density (OD) readings > 5 standard deviations above background as calculated using prebleed serum at the same dilutions.
Neutralizing antibody assays
Neutralizing antibody titers were measured using a viral infectivity assay of TZM-bl cells expressing the luciferase gene under control of the HIV promoter (obtained through the AIDS Research and Reference Reagent Program (ARRRP), Division of AIDS, National Institute of Allergy and Infectious Disease, National Institute of Health, Bethesda, Maryland, USA) [15,16], with minor modifications. In brief, virus grown in rhesus monkey peripheral blood mononuclear cells (PBMC) was incubated for 1 h at 37°C with pooled sera at the indicated dilutions in Dulbecco's modified essential medium (DMEM) + 10% FCS (D10), then added to TZM-bl cells (plated at 5 × 103/well of a flat-bottom 96-well plate 18 h previously) in the presence of DEAE-dextran (40 μg/ml). After 24 h, immune serum containing-TZM-bl culture medium was replaced with fresh D10. On day 2, Bright-Glo luciferase assay substrate was added according to the manufacturer's directions (Promega, Madison, Wisconsin, USA) and luciferase activity measured in a luminometer (Perkin-Elmer, Boston, Massachusetts, USA). Due to the limitations in the amount of blood that could be collected from the infant macaques, preimmune sera were not always available. As such, percentage neutralization was calculated relative to luciferase activity level in negative control serum samples consisting of virus + pooled sera from five naive rhesus macaques to offset potential virus proliferation inhibitors/and or enhancers occasionally present in macaque serum. Neutralizing antibody titers were estimated as the reciprocal serum dilution giving 50% inhibition of virus replication.
Interferon-γ ELISPOT analysis
Multiscreen-IP plates (Millipore, Bedford, Massachusetts, USA) were coated with purified mouse antihuman interferon (IFN)-γ (BD Biosciences-Pharmigen, San Diego, California, USA) overnight at 4°C, then blocked with 10% FCS in RPMI-1640 for 2 h at room temperature. PBMC were isolated from whole blood by centrifugation in heparinized CPT tubes (BD Biosciences-Pharmigen) and washed three times prior to assay. For antigen-specific T-cell stimulation, overlapping 20-mer SIVmac239 Gag and 15-mer HIV Tat peptides representing the complete protein sequences were obtained through ARRRP. Cells were incubated overnight at 105 cells/well in assay medium consisting of RPMI-1640 containing 10% FCS, L-glutamine, penicillin-streptomycin and 2-mercaptoethanol (5 × 10−5 M) and with pooled peptides of SIVmac239 Gag (pools #1, #2 and #3 consisting of peptides 1–42, 43–84 and 85–125, respectively). Biotinylated mouse monoclonal antihuman IFN-γ (Mabtech, Mariemont, Ohio, USA), diluted in 0.5% FCS/PBS was added to wells for 1 h at room temperature. Plates were washed with 0.05% Tween 20/PBS, horseradish peroxidase-conjugated streptavidin (BD Biosciences Pharmingen) was added and after an additional 1 h incubation, the plate was developed with AEC chromogen substrate solution (BD Biosciences). Assays were done in duplicate and background levels were determined by incubation of cells without peptides. IFN-γ secreting cells were enumerated using an Immunospot ELISPOT reader (CTL, Cleveland, Ohio, USA).
Viruses and challenges
SHIV-1157ip and SHIV-1157ipd3N4 were recently described . Virus stocks of each were prepared from concanavalin A-stimulated (5 μg/ml) naive rhesus PBMC in RPMI-1640 plus 15% FCS, penicillin/streptomycin, L-glutamine and rIL-2 (10 U/ml) and TNF-α (10 ng/ml for generating the SHIV-1157ipd3N4 stock only).
Viral load and statistical measurements
Plasma vRNA loads were assessed by quantitative RT–PCR for SIV gag sequences (QiaAmp RNA Blood Mini-Kit, Qiagen)  Peak viremia levels were compared by two-sided Wilcoxon rank-sum test.
Vaccination and prechallenge immune responses
Five macaques were given the multigenic protein-only vaccine (Group 1, Fig. 1), Group 2 (five animals) received a more conventional bimodal DNA prime/protein boost regimen, and Group 3 (controls) received empty vector DNA. DNA vaccination consisting of codon-optimized plasmids encoding SIV gag–protease, HIV tat and heterologous HIV1084i clade C env started on postnatal day 1–5 (Group 2, Fig. 1); another DNA immunization was given at week 6. Group 2 was rested and then given soluble protein boosts consisting of SIV Gag–Pol particles [12–14], HIV Tat and HIV1084i gp160. At this time, age-matched Group 1 animals were vaccinated with the viral proteins.
Two weeks after the final protein inoculation, all groups were challenged orally with heterologous SHIV-1157ip. At this time IFN-γ ELISPOT analysis of PBMC (Table 1) showed strong SIV Gag-specific reactivity in three Group 1 animals (RLu-9, RAt-9 and RSr-9); RSr-9 also had strong HIV Tat-specific ELISPOT responses. Two animals from Group 2 (RHy-9 and RRz-9) demonstrated strong SIV Gag-specific and HIV Tat-specific ELISPOT responses. No specific reactivity was observed in any controls (Group 3). All 10 animals from Groups 1 and 2 had high-titer binding antibodies to homologous HIV1084i gp160, a relatively neutralization resistant virus (Table 1). Only one animal of Group 1 had measurable anti-HIV1084i nAb activity. Importantly, 9 of 10 vaccinees had significant nAb activity to the heterologous challenge virus, SHIV-1157ip. Controls (Group 3) showed no anti-Env-C antibodies. These data demonstrate that multimeric HIV-C gp160 (as part of a multigenic protein vaccine) or DNA priming/protein boosting induced cross-reactive nAb to SHIV-C in macaques.
Low-dose oral challenge with early SHIV-1157ip
Oral SHIV-1157ip challenge employed a relatively low dose (3.7 50% animal infectious doses (AID50); (Fig. 2a and b) in an attempt to approximate the lower HIV-C inocula that are likely involved in milk-borne HIV-C transmission . Three animals from Group 1 (RAt-9, RSr-9 and RNu-9) remained virus-free and two animals from Group 2 (RJy-9 and RBa-10) were only transiently viremic with viral RNA (vRNA) loads of 1465 and 267 vRNA copies/ml of plasma, respectively (Fig. 2a and b). At this virus dose, 4 of 5 controls became systemically infected (Fig. 2c).
High-dose intrarectal challenge with late SHIV-1157ipd3N4
Next, we sought to test whether vaccinees with no or transient infection after low-dose challenge might be protected from high-dose challenge with the more virulent SHIV-1157ipd3N4. Thus, animals that remained uninfected or had < 1000 vRNA copies/ml plasma were rechallenged intrarectally with SHIV-1157ipd3N4. This included three animals from Group 1 (RAt-9, RSr-9 and RNu-9), one animal from Group 2 (RBa-10), and one from control Group 3 (RAr-9) (Fig. 2d and e). After rechallenge with SHIV-1157ipd3N4, the nonvaccinated animal, RAr-9, had a peak plasma viremia level of 7.7 × 107 vRNA copies/ml (Fig. 2e); similarly (Fig. 2e), eight additional age-matched, nonvaccinated controls that received the same dose of SHIV-1175ipd3N4 intrarectally (Group 3a, Fig. 1) had peak plasma viremia levels ranging from 1.3 × 107 to 7.1 × 107 vRNA copies/ml. All controls, that is animals of Group 3a plus RAr-9, rapidly became viremic with vRNA loads ranging from 103 to 105 copies/ml at 1 week postexposure. In contrast, only one of four previously vaccinated monkeys had detectable virus by week 1 after SHIV-1157ipd3N4 exposure. Among the vaccinees that became infected, peak plasma viremia levels ranged from 3.3 × 106 to 7.4 × 106 copies/ml, that is a log lower than controls (Fig. 2d; P = 0.003, rechallenged vaccinees versus controls). Importantly, one vaccinee of Group 1 given protein only, RAt-9, had no evidence of infection after rechallenge with high-dose SHIV-1157ipd3N4. RNA isolated from a peripheral lymph node biopsy of RAt-9 at week 10 post SHIV-1157ipd3N4 rechallenge was also negative by RT–PCR analysis. These results indicate that protein immunization alone can confer complete protection against a sequential low-dose/high-dose mucosal challenge with heterologous R5 SHIV-C.
We then sought to delineate the immune parameters that may explain the complete protection of RAt-9 at the time of SHIV-1157ipd3N4 rechallenge. At this time, RAt-9 had much higher virus-specific cellular immune reactivity to both SIV Gag (8615 spot-forming cells (SFC)/106 PBMC for all three Gag peptide pools) and HIV Tat (705 SFC/106 PBMC) than the rechallenged vaccinees RBa-10 and RNu-9, which had evidence of viral containment compared to controls (Fig. 3). These two animals had 240 and 550 anti-Gag IFN-γ ELISPOT, respectively, and none against Tat (Fig. 3a). Of note, control monkey RAr-9 that had been exposed orally to SHIV-1157ip without becoming infected had no specific antiviral cellular immunity (Fig. 3a). We then compared total ELISPOT reactivity against all Gag plus Tat peptide pools at the time of the first and second challenges (Fig. 3d). The protected animal, RAt-9, was the only one with a large increase of total ELISPOT activity. Even the transiently infected animal, RBa-10, had fewer total ELISPOT at the time of the second challenge compared to the first. Thus, protection in RAt-9 was associated with a strong boosting effect via the initial virus exposure.
Although the protected animal, RAt-9, had a nAb titer of 1: 160 against the first virus, SHIV-1157ip (Fig. 3b), nAb activity against the rechallenge virus was minimal (Fig. 3c), reflecting the relative neutralization resistance of the late virus, SHIV-1157ipd3N4. However, RAt-9 was the only double-challenged animal that maintained nAb titers during the 6 months between challenges. NAb activity of all the other rechallenged animals was minimal or negative against all viruses tested. These data suggest that virus-specific T-cell immunity was associated with complete protection of RAt-9 from mucosal SHIV-1157ipd3N4 challenge as only low-level nAb activity against the second challenge virus was seen.
The course of the protected monkey sheds light onto the mechanisms responsible for immune protection. The clear-cut boosting of cellular antiviral immunity leads us to postulate that this animal had sub-threshold infection with the live early virus, which was reined in by preexisting antiviral immunity and thus never became overt. This nevertheless led to strong amplification of cellular immunity and the maintenance of nAb titers against the early virus. When this animal then encountered the high dose of the relatively neutralization-resistant late virus, cellular immunity – probably supported by antibody-dependent cellular cytotoxicity (ADCC) – then held SHIV-1157ipd3N4 in check. Of note, this protected animal continues to maintain very high levels of antivirus ELISPOT activity 1 year after challenge.
Our upfront heterologous challenge study based upon locally divergent HIV-C env genes was designed to model repeated human mucosal exposure to evolving strains circulating within the community. Vaccination with multimeric gp160 from a recently transmitted local strain was able to induce nAbs against a heterologous challenge virus. The fact that we observed either viral containment or complete protection is promising, especially when considering the single high-dose (20 AID50) intrarectal challenge with the late virus.
The protection from the initial low-dose SHIV-1157ip challenge in animals immunized solely with soluble viral proteins, including multimeric Env, is consistent with our previous study showing containment of SHIV-B in macaques immunized with multimeric Env only [4–6]; high nAb activity was associated with, but not predictive of virus containment. Several groups have pursued AIDS vaccine strategies based upon multimeric gp140 to maintain conserved, conformational Env determinants in an effort to generate broadly reactive nAbs [19–25]. Our data show that multimeric gp160 is a potent immunogen and can generate protective antiviral immunity when used as part of a more complex protein vaccine.
The authors thank Jane Moon and Bill Sutton for technical assistance Stephanie Ehnert for logistical coordination, and Susan Sharp for the preparation of this manuscript.
Additional authors of the Clade C Program Project include: Ela Shai-Kobiler (Department of Cancer Immunology & AIDS, Dana-Farber Cancer Institute and Harvard Medical School), Jennifer McKenna, Brad Cleveland (University of Washington), and Elizabeth Strobert (Yerkes National Primate Research Center, Emory University).
Sponsorship: This work was supported by NIH grants P01 AI48240 to R.M.R., R.A.R., A.-L.C., S.-L.H., and J.G.E. and RR-00165 providing base grant support to the Yerkes National Primate Research Center.
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