Abstract
The role of vaccine-induced anti-V2 antibodies (Abs) was tested in three protection experiments in rhesus macaques. In an experiment using immunogens similar to those in the RV144 vaccine trial, (Anti-Env) nine rhesus macaques were co-immunized with gp16092TH023 DNA and SIV gag, gp120A244 and gp120MN proteins. In two V2-focused experiments (Anti-V2 and Anti-V2 Mucosal), nine macaques in each group were immunized with V1V292TH023 DNA, V1V2A244 and V1V2CasaeA2 proteins, and cyclic V2CaseA2 peptide. DNA and protein immunogens, formulated in Adjuplex, were given at 0, 4, 12 and 20 weeks, followed by intrarectal SHIVBaL.P4 challenges. Peak plasma viral loads (PVL) of 106–107 copies/mL developed in all nine sham controls. Overall, PVL was undetectable in one-third of immunized macaques, and two animals tightly controlled the virus with the Anti-V2 Mucosal vaccine strategy. In the Anti-Env study, antibodies that captured or neutralized SHIVBaL.P4 inversely correlated with PVL. Conversely, no correlation with PVL was found in the Anti-V2 experiments with non-neutralizing plasma Abs that only captured virus weakly. Titers of Abs against eight V1V2 scaffolds and cyclic V2 peptides were comparable between controllers and non-controllers as were ADCC and ADCVI activities against SHIV-infected target cells and phagocytosis of gp120-coated beads. The Anti-Env experiment supports the role of vaccine-elicited neutralizing and non-neutralizing Abs in control of PVL. However, the two V2-focused experiments did not support a role for non-neutralizing V2 Abs alone in controlling PVL, as neither ADCC, ADCVI nor phagocytosis correlated inversely with heterologous SHIVBaL.P4 infection.
Keywords: HIV vaccine, V2 antibody, V1 antibody, HIV neutralizing antibody, virus capture antibody, ADCC, ADCVI, phagocytosis, mucosal antibodies, SHIV challenge, immune correlates, V1V2 fusion proteins, cyclic V2 peptides
Introduction
In the RV144 HIV vaccine trial, which achieved a modest efficacy of 31.2%, the only immune response associated with a reduction in HIV-1 infection was a high level of plasma anti-V2 antibodies (V2 Abs) (1). This result suggested that V2 Abs contribute to protection against HIV-1 infection, although the protection mechanism remains unknown. Efforts to replicate the efficacy of the RV144 trial findings using similar vaccines in the setting of clade C immunogens in the HVTN 702 clinical trial failed to show differences in HIV acquisition between the vaccine and placebo arms (2). Thus, understanding the role of specific V2-directed envelope (Env) Abs in protection remains a crucial question.
V2 Abs mainly target Tier 1 viruses and mediate several antiviral functions that may contribute to protection, whereas in the RV144 clinical trial, 89% of infections occurred with Tier 2 CRF01_AE strains (3). Neutralizing Abs (NAbs) are critical for protection against many viruses, including HIV-1 (4, 5). Tier-1 neutralizing V2 monoclonal Abs (mAbs) have comparable neutralizing capacity as V3 mAbs and some CD4bs mAbs. These three types of Abs are commonly induced by natural HIV infection and experimental vaccines, but are not broadly neutralizing and rarely neutralize Tier 2 HIV-1 (6–10).
Nonetheless, V2 Abs can mediate effector functions through the Fc fragment in vitro, such as antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent cellular cytotoxicity (ADCC), and these functions potentially modulate infection in vivo (11). A study comparing the capacity of mAbs directed to different regions of HIV-1 Env for mediating phagocytosis revealed that V2 mAbs exhibited comparable activity to mAbs against V3 and the CD4bs (12). Immunization of rabbits and rhesus macaques using V1V2 scaffold proteins elicited V2 Abs that infrequently mediated phagocytosis depending on the animal group and immunogen tested (13, 14). ADCC activity mediated by V2 mAbs was limited in most studies (9, 15, 16). Vaccine-induced V2 Abs in rhesus macaques were able to mediate ADCC sporadically (14). The contribution of phagocytosis and ADCC to protection in several rhesus macaque challenge experiments is still under investigation and rarely correlated with virus acquisition (17–19).
A unique function of V2 mAbs is the ability to bind to the α4β7 integrin-binding site in the V2 region and inhibit the interaction between α4β7 integrin and gp120 or V2 peptide in vitro (20, 21). Other findings suggested that V2 Abs in vivo may block viral adhesion and subsequent infection of Th17 cells expressing CD4, CCR5, and α4β7 integrin (22, 23). This concept was tested in three experiments in rhesus macaques and one clinical trial in humans that showed an anti-α4β7 mAb could not sustain the suppression of viral load upon discontinuation of all treatments (24–27). Furthermore, in one of these studies, two rhesus V2 mAbs that blocked binding of SIV Env to α4β7 did not control SIV infection in rhesus macaques (26).
We tested the protective capacity of a single human V2 mAb (830A) by passive transfer to rhesus macaques with subsequent mucosal SHIVBaL.P4 challenges. In that study, plasma viral load (PVL) and viral DNA in PBMCs and lymphoid tissues were reduced, but the number of infected animals was not reduced compared to controls (16). These results are comparable to three other passive immunization experiments in rhesus macaques that showed non-neutralizing Abs specific to CD4bs, C1 region of gp120, and against gp41 may reduce PVL, but unlike experiments with broadly neutralizing monoclonal Abs (bNAbs), cannot protect against virus infection (28–30).
Here, we performed three separate protection experiments in rhesus macaques to address whether vaccine-induced V2 Abs in rhesus macaques can control a heterologous Tier 1 SHIVBaL.P4 challenge. The first experiment, termed Anti-Env, was intended to determine whether co-immunization with a DNA/protein regimen could mimic the correlation between V2 Abs and the reduced risk of infection reported in the RV144 vaccine trial. The second and third experiments, termed Anti-V2 and Anti-V2 Mucosal, tested the ability of V2-focused Abs induced systemically and mucosally, respectively, to protect against intrarectal challenge. The studies showed that V2 Abs did not independently control SHIVBaL.P4 infection. Neither V2 Ab binding titers nor functional activities of phagocytosis, ADCC or antibody-dependent cell-mediated virus inhibition (ADCVI) correlated with plasma viral load or SHIVBaL.P4 acquisition.
Material and Methods
Ethics Statement.
Experiments in this study were performed in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, the Office of Animal Welfare, and the U.S. Department of Agriculture. All experiments on animals have been reviewed and approved by the Institutional Animal Care and Use Committee at the Oregon Health and Science University (OHSU) and the New York University School of Medicine, New York, NY. The American Association accredits the Oregon National Primate Research Center (ONPRC) for Accreditation of Laboratory Animal Care.
Animals.
A total of thirty-six rhesus macaques (Maccaca mulatta), comprised of twenty-two females and 14 males, between 3 and 9.7 years of age were housed at the ONPRC in Beaverton, OR and used in the study. All animals were free of cercopithicine herpesvirus 1, D-type simian retrovirus, simian T-lymphotropic virus type 1, and SIV infection at the start of the study. Macaques were genotyped for MHC class I alleles Mamu-A*01, -A*02, B*01, -B*08, and -B*17 (Table I).
Table I.
MHC class I alleles in rhesus macaquesa
| ID | Mamu-A1*001:g | Mamu-A1*002:g | Mamu-B*001:g | Mamu-B*008:g | Mamu-B*017:g |
|---|---|---|---|---|---|
| Anti-Env | group | ||||
| 32222 | -- | -- | -- | -- | -- |
| 32248 | -- | -- | -- | -- | -- |
| 32669 | -- | POS | POS | -- | -- |
| 32672 | -- | POS | POS | -- | -- |
| 30300 | -- | -- | -- | -- | -- |
| 33615 | -- | -- | -- | -- | -- |
| 33617 | -- | -- | -- | -- | -- |
| 33620 | -- | -- | -- | -- | -- |
| 33653 | -- | -- | -- | -- | -- |
| 30262 | -- | -- | POS | -- | -- |
| 32061 | -- | -- | POS | -- | -- |
| 32661 | -- | -- | -- | -- | -- |
| Anti-V2 | group | ||||
| 27063 | -- | -- | -- | -- | -- |
| 32037 | -- | -- | POS | -- | -- |
| 33788 | -- | -- | -- | -- | -- |
| 33863 | -- | -- | -- | -- | -- |
| 34151 | -- | -- | -- | -- | -- |
| 31467 | POS | -- | -- | -- | -- |
| 33607 | -- | -- | -- | -- | -- |
| 34149 | -- | -- | -- | -- | -- |
| 34209 | -- | -- | -- | -- | -- |
| 30740 | -- | -- | -- | -- | -- |
| 32195 | -- | -- | POS | -- | -- |
| 34186 | -- | -- | -- | -- | -- |
| Anti-V2 | Mucosal | group | |||
| 28891 | -- | nt | nt | -- | -- |
| 29709 | -- | nt | nt | -- | -- |
| 30555 | -- | nt | nt | -- | -- |
| 30741 | -- | nt | nt | -- | -- |
| 33820 | -- | nt | nt | -- | -- |
| 34630 | -- | nt | nt | -- | -- |
| 34653 | -- | nt | nt | -- | -- |
| 30383 | -- | nt | nt | -- | -- |
| 34578 | POS | nt | nt | -- | -- |
| 27870 | -- | nt | nt | -- | -- |
| 32282 | -- | nt | nt | -- | -- |
| 36204 | -- | nt | nt | -- | -- |
MHC class I alleles were genotyped by sequence-specific PCR at the Macaque Genetics Core, OHSU, Beaverton, OR. Non-controllers (red); controllers (blue); sham vaccine controls (black); nt = not tested.
Generation of the 92TH023/VC10014_F8 gp160 vaccine construct and V1V2 DNA.
The motif-optimized (MO) 92TH023/VC10014_F8 gp160 DNA chimera was generated by in-frame cloning of 92TH023 gp120 with VC10014_F8 gp41. The gp120 DNA sequence of HIV envelope 92TH023 (GenBank accession number EF553537; https://www.ncbi.nlm.nih.gov/nuccore/EF553537) was first MO with the Robins–Krasnitz algorithm as previously described (31, 32) and then synthesized (Blue Heron, Bothell, WA). Using NheI and BmgbI restriction sites, replacement cloning was performed to swap the MO VC10014_F8 gp120 with the MO 92TH023 gp120 fragment so that MO 92TH023 gp120 was cloned in frame with MO VC10014_F8 gp41 in the pEMC* expression vector; the isolation and cloning of HIV envelope VC10014_F8 (GenBank accession number KJ698264; https://www.ncbi.nlm.nih.gov/nuccore/KJ698264) were described in (33). The DNA sequence and in-frame cloning of MO 92TH023/VC10014_F8 gp160 were confirmed by Sanger sequencing. The MO 92TH023/VC10014_F8 gp160 chimera was functional since it produced an infectious pseudovirus by complementing the pSG3ΔEnv backbone DNA plasmid (34). V1V292TH023 DNA with additional nucleotides coding for five amino acids (AA) at each end was produced in the same laboratory and used for the two protection experiments, Anti-V2 and Anti-V2 Mucosal.
Immunogens.
Two recombinant gp120 proteins, gp120A244 (CRF01_AE) and gp120MN (clade B) were used for co-immunization with DNA in the first protection experiment (Anti-Env). The recombinant gp120A244 was produced using a plasmid provided by Dr. X-H. Liao, Duke University, and modified by an N-terminal 11-amino-acid deletion (Δ11) and addition of an HSV gD protein-and derived tag (gD). For the Anti-V2 and Anti-V2 Mucosal experiments, V1V2 scaffolds, V1V2A244-2F5K and V1V2Case A2-2F5K, were produced as described (35) and cyclic V2Case A2 peptide was produced by Biopeptide Co, Inc, San Diego, CA.
Recombinant proteins, V1V2 proteins and V2 peptides.
Three recombinant proteins, gp120MN, gp120BaL and V1V2Case A2-gp70 (clade B), were purchased from Immune Technology Corp., New York, NY. V1V2ZM109-1FD6, V1V2A244-2F5K and V1V21086-tags (clade C) were produced as previously described (35). Biotinylated cyclic peptides, V2BaL, V2CaseA2 (clade B), V2230 (CRF02_AG), V2A244 (clade AE) and V192TH023 (clade AE) were purchased from Biopeptide Co., Inc., San Diego, CA.
Animal immunization.
Macaques were co-immunized at weeks 0, 4, 12, and 20 with 36 μg of HIV DNA and 36μg of SIV gag delivered intradermally (ID) and soluble proteins delivered intramuscularly (IM). The recombinant proteins were delivered along the shaved abdomen and one or more proteins delivered in the quadriceps. The Anti-Env group received gp16092TH023 DNA plus gp120A244 and gp120MN recombinant proteins. In the Anti-V2 and Anti-V2 Mucosal groups, animals were co-immunized with V1V292TH023 DNA (ID) and V1V2A244-2F5K, V1V2Case A2-2F5K proteins with cyclic V2Case A2 peptide, 50 μg each formulated in adjuvant (Adjuplex). The proteins were delivered IM in the Anti-V2 group and by intranasal atomized spray in the Anti-V2 Mucosal group (Fig. 1). The DNA immunogens were delivered via the Particle Mediated Epidermal Delivery (PMED) Gene Gun, a machine that shoots gold “bullets” consisting of 1-micron beads with dried DNA on the surface (PowderJect XR1, developed by PowderMed, Ltd., Oxford, UK). The bullets were shot under the abdomen’s skin to target inguinal lymph nodes nearby or gut-associated lymphoid tissue (GALT). During the same procedure, we injected protein immunogens (approximately 0.1 ml volume) into the quadriceps or sprayed with an atomizer into the nasal cavity.
FIGURE 1. Experimental vaccine and SHIV challenge regimens.
Three groups of nine rhesus macaques were co-immunized with HIV DNA plasmid and soluble protein. At each immunization 36 μg of HIV DNA and 36μg of SIV gag was delivered epidermally (ID) via PMED gene gun (PowderMed, Oxford, UK) plus 100 μg of each soluble protein or peptide delivered either by intramuscular injection (IM) or intranasal atomized spray (IN) formulated with Adjuplex adjuvant as shown. DNA and protein immunogens are described for each group. Three control animals were added to each group that received a single empty plasmid sham vaccine at week 20. SHIVBaL.P4 was administered intrarectally as indicated to all animals starting at week 22, and viral loads in plasma samples (PVL), tested in duplicate, were monitored weekly until necropsy at week 35. All controls were infected after two virus challenges in the Anti-Env group and after five challenges in the Anti-V2 and Anti-V2 Mucosal groups.
Protection study.
Protection experiments were performed on three groups, nine animals each, of immunized rhesus macaques at the Oregon National Primate Research Center, Beaverton, OR. The three control groups of three macaques each were immunized with vector DNA only (sham) to serve as a control group for virus infection. The challenge virus was propagated in PHA-stimulated macaque PBMC and titrated in vivo. A stock of SHIVBaL.P4 was obtained through collaboration with Dr. Sampa Santra, Beth Israel Deaconess Medical Center, Boston, MA. The SHIVBaL.P4 isolate was propagated in human PBMCs to generate a large volume stock for rhesus challenge experiments. The mean genetic diversity of Env determined by single genome sequencing in this stock is 0.3%, with a maximum diversity of 0.7% (29).
Animals were challenged with SHIVBaL.P4 two weeks after the fourth immunization at week 22 of the experiment. The virus stock was diluted and dosed at TCID50 of ~6 × 105 based on the in vivo titration data provided by Dr. Santra. Two repeated intrarectal (IR) exposures given one week apart resulted in a productive infection of all three sham vaccinated animals in the Anti-Env group. Five repeated IR exposures were required to infect controls for the Anti-V2 and Anti-V2 Mucosal experiments.
Detection of virus in plasma, PBMCs and lymph nodes.
Detection and quantification of SIV gag viral RNA (vRNA) to monitor the onset of infection were performed as described (16). Briefly, viral loads in plasma were measured by quantitative reverse transcription-PCR (RT-PCR) (36), whereas viral loads in PBMC DNA were quantitated by PCR using Fast Advanced Mastermix on an Applied Biosystems QuantStudio 6 Flex instrument (Life Technologies, Carlsbad, CA). The detection of vRNA and DNA in lymph nodes was determined by ultrasensitive nested quantitative PCR and RT-PCR methods (37).
Binding assays.
Macaque plasma samples were screened against antigens using a standard ELISA as described (38). Briefly, V1V2 scaffold proteins were coated directly onto plastic plates at a concentration of 1 μg/mL, while biotinylated cyclic V2 and V1 peptides were immobilized on streptavidin-coated plates (StreptaWell plates, Roche). The plates were washed and blocked with assay diluent (PBS containing 2.5% bovine serum albumin and 7.5% fetal bovine serum) and then incubated with plasma samples diluted eight times ranging from 1:10 to 1:30,000. After washing, the bound Abs were detected by incubation with alkaline phosphatase-conjugated goat anti-human IgG (γ specific) (Southern Biotech) followed by washing and adding substrate to develop color. Mucosal secretions, buccal and vaginal, were tested by ELISA for Abs that bind to gp120BaL, gp120MN and V1V21086-tags; the second antibody was peroxidase-conjugated goat anti-human IgG (γ specific) (Jackson Immuno Research).
Virus capture assay.
Virus capture assays were carried out according to Gach et al. (39). Briefly, 96 well half-area plates (Corning) were coated with 250 ng per well of a goat anti-monkey IgG gamma chain-specific antibody (Rockland) and incubated overnight at 4°C. Wells were washed and subsequently blocked with 5% nonfat dry milk in PBS. After blocking, plasma samples (50 μL of 1:100 dilution per well) were incubated for one hour at 37°C before adding SHIVBaL.P4 (1 ng p27/well) for another four hours at 37°C. Plates were finally washed five times with PBS and TZM-bl reporter cells were added (1 × 104 cells per well) supplemented with 15 μg/mL of DEAE-dextran. After three days of incubation at 37°C, cells were washed, lysed, and analyzed for luminescence (BioTek) after adding luciferase assay reagent (Promega). Capture assays were performed twice in duplicate wells.
Neutralization assay.
The neutralizing activities of plasma Abs against SHIVBaL.P4 and nine other pseudotyped viruses and the SVA-MLV negative control pseudovirus were tested using the standard TZM-bl cell assay (38, 40, 41). Briefly, plasma samples were heat-inactivated and titrated at 3-fold dilutions starting at 1:50 and then pre-incubated with the virus at an input of 200 TCID50 for 1 h. The virus/plasma Abs mixtures were then incubated for 48 h with TZM-bl cells. Virus infectivity was determined by measuring luciferase activity in cell lysates. The 50% inhibitory dilution (ID50) was determined that resulted in a 50% reduction in relative light units (RLU) compared to wells with the virus only, after the subtraction of cell control RLUs (8, 41). All plasma samples were tested in duplicate. A pool of the pre-immune plasma was used as a negative control. Neutralization dose-response curves were fitted by non-linear regression.
Antibody-dependent cellular cytotoxicity (ADCC).
The assay was performed as described by Hessell et al. (16), and adapted from Alpert et al. (42). Briefly, NKR24 target cells were infected with SHIVSF162p3 and incubated for 3 to 4 days before the start of the assay to achieve an infection titer at least five times over the background. Infected target cells were combined with KHYG-1 rhCD16 cells, derived from the CD16-negative human NK cell line KHYG-1, at an effector-to-target cell (E:T) ratio of 5:1, and added to the assay plate along with serially diluted macaques plasma samples, and positive and negative mAb controls that define 100% and 0% RLU were included. The assay mixture was incubated for 8 h, after which the Bright-Glo luciferase substrate reagent (Promega) was used to read activity. The data were quantified by 50% ADCC titers and AUC values for ADCC.
Antibody-dependent cellular phagocytosis (ADCP).
Phagocytosis activity was measured as previously described (43) with minor modifications. In short, HIV-1 gp120BaL was biotinylated with a Biotin-XX microscale protein labeling kit (Thermo Fisher Scientific, Waltham, MA) and incubated with a 100-fold dilution of 1 μg yellow-green streptavidin-fluorescent beads (Thermo Fisher Scientific) overnight at 4°C in the dark. A 1:25, 1:50, 1:100 and 1:200 dilution of plasma samples from each macaque was added to 400,000 THP-1 cells, plated in a U-bottom 96-well plate. The bead-gp120 mixture was further diluted 5-fold in R10 media, and 50 μL was added to the cells. The mix was incubated for 3 h at 37°C and fixed with 70 μL of 2% paraformaldehyde. Fluorescent bead uptake by THP-1 cells was assessed using a BD Biosciences LSRII flow cytometer (BD Biosciences, San Jose, CA). The phagocytic score of each sample was calculated by multiplying the percentage of bead-positive cells (frequency) by the degree of phagocytosis measured as mean fluorescence intensity (MFI) and dividing by 106. Values were normalized to background values (cells and BSA coated beads) by dividing the phagocytic score of the test sample by the phagocytic score of the background sample.
Antibody-dependent cell-mediated virus inhibition (ADCVI) assay.
ADCVI assays were performed as previously described (44). In brief, heat-inactivated plasma samples were initially diluted 1:25 with medium (final dilution of 1:100) before adding SHIVBaL.P4-infected CEM.NKr-CCR5 target cells and freshly isolated human peripheral mononuclear effector cells at an effector/target ratio of 10:1. After three days of incubation, Abs were removed by washing wells three times with PBS. Four days later, cell-free supernatants were collected and analyzed by a SIV p27 ELISA. Each plasma sample was assayed in triplicate, and the ADCVI activity (percent ADCVI) of week 14 and week 22 samples was calculated relative to the respective week 0 samples. ADCVI assays were repeated at least twice.
Envelope sequencing of plasma breakthrough SHIVBaL.P4.
Plasma viruses from nine non-controllers from two protection studies, one sham animal, and the challenge virus (SHIVBaL.P4) were sequenced in the envelope protein region to analyze sites of immune pressure in the breakthrough viruses in comparison to controls. For each analyzed animal, sequence analysis was performed for the virus bulk sequence and five clones (pCR4 TOPO). Amplification, cloning into pCR4 TOPO and sequencing was based on an established protocol (45) with minor changes. Briefly, vRNA was extracted from the plasma sample using the QIAamp mini kit (Qiagen Inc., Valencia, CA). Reverse transcription was performed using primer SHIV 10231 rev (5’- ACAGAGCGAAATGCAGTGATATT-3’) and GoScript (Promega) Reverse Transcriptase according to the manufacturer’s instructions. Nested PCRs were performed over the full env region, according to HIV-1 HXB2 region 6225–8802 (~2600 bp) using PrimeSTAR GXL DNA polymerase (Clontech) with primers EnvB (5’-AGAAAGAGCAGAAGACAGTGGCA-3’) and SHIV 10231 rev in 1st Round PCR, and platinum Taq polymerase (Invitrogen, Carlsbad, CA) with newly designed primers SHIV Env1 (5’-ATGAGAGTGACGGAGATCAGGA-3’) and SHIV 8496 rev (5’-ACCCATATTGTAGGTAGGCTCG-3’) in 2nd Round PCR. Bulk sequencing was performed with SHIV Env1, SHIV 8496 rev, For15, Rev15, and/or Rev 17 (46). Additionally, PCR products were cloned into the pCR4 TOPO cloning vector (Life Technologies, Carlsbad, CA) and transformed into One Shot TOP10 competent E. coli. Plasmids were sequenced for the insert portion using universal primers M13F, M13R, and For15. Sequence analysis and alignment were performed using SeqMan Pro (DNASTAR), MEGA5.2 with Muscle (47) and MegAlign (DNASTAR).
Statistical analysis.
The 50% titers of plasma Abs and area under the curves (AUC) for two groups of animals, controllers and non-controllers of SHIVBaL.P4 infection, were compared by the nonparametric Mann-Whitney tests. Group comparison significance was determined by One-way ANOVA. The nonparametric Spearman’s test with P values (two-tailed) and linear regression were used to assess the correlation between the results of neutralization, capture Abs, AUC and PVL. A log-rank test was used to analyze the Kaplan-Meier curves and evaluate differences between controllers and non-controllers. Fisher’s exact test was used to determine the significance of controllers in vaccinated animals. The statistical analyses were performed using GraphPad Prism.
Results
Vaccine/challenge experiments in rhesus macaques
Three groups of nine animals each were co-immunized at weeks 0, 4, 12, and 20: intradermally (ID) with HIV and SIV gag DNA, and either intramuscularly (IM) or mucosally with a nasal spray (IN) of soluble proteins and peptide. All proteins were formulated with Adjuplex adjuvant. The first experiment (Anti-Env) was designed to loosely mimic the RV144 vaccine with macaques co-immunized with chimeric gp16092TH023 DNA, plus gp120A244 and gp120MN proteins. The Anti-Env group served as a positive control for two vaccine groups designed to induce only V1V2 Abs (Anti-V2 and Anti-V2 Mucosal). The Anti-V2 and Anti-V2 Mucosal groups were co-immunized with V1V292TH023 DNA (ID) plus V1V2-scaffold proteins V1V2A244-2F5K, V1V2CaseA2-2F5K, and the cyclic V2CaseA2 peptide. These groups differed only in the route of protein immunogen delivery, IM (Anti-V2) or IN (Anti-V2 Mucosal). The V2 peptide was included with the last two immunizations to enhance the induction of Abs against V2 linear antigens (Fig. 1). Controls for each group consisted of three non-human primates (NHP), each receiving an empty plasmid sham vaccine at week 20. Controls were subsequently challenged simultaneously with vaccinated animals. SHIVBaL.P4 at a median tissue culture infectious dose (TCID50) of ~6 × 105 was administered intrarectally (IR) to all animals starting at week 22. We designed these studies under the criterion that challenges would cease when all controls were infected, and we followed this strategy for all three groups. Based on previous experiments, the SHIV BaL stock at the dilution used here was expected to infect sham-vaccinated controls after 3–4 IR challenges. Surprisingly, only a single challenge was required to infect controls and one vaccinated macaque in the Anti-Env experiment. Therefore, challenges were stopped after two challenges. We were unable to run all three experiments concurrently, and the Anti-V2 and Anti-V2 Mucosal V2 groups received five SHIV challenges before all controls were infected (Fig. 1).
PVL was determined weekly by measuring SIV gag vRNA. Peak PVL reached 106 to 107 copies/mL in control animals 2–3 weeks post the first challenge in the Anti-Env group. In the Anti-V2 and Anti-V2 Mucosal groups, peak PVL was recorded 3–5 weeks post the first challenge. The reason for the variation in the number of challenges required to infect the controls between the Anti-Env group and the Anti-V2 groups was undetermined. The virus stock SHIVBaL.P4 TCID50 was re-checked on three separate occasions during repeated challenges and found to be comparable.
In the Anti-Env group, five of nine (55%) vaccinated animals controlled viremia. PVL was undetectable in four of these five animals except for a single blip (a signal that appears at a single time point) of 68–360 vRNA copies/mL (30300, 33615, 33620, 33653). Three PVL blips were detected in the plasma of the fifth Anti-Env group controller (33617) ranging from 140 to 1200 vRNA copies/mL (Fig. 2A). PVL was undetectable in four of nine animals (44%) in the Anti-V2 group (31467, 33607, 34149, 34209) and two of nine animals (22%) in the Anti-V2 Mucosal groups, indicating control of viremia in these animals (Fig. 2A). Cell-associated SIV gag DNA was also undetectable in PBMCs from the vaccinated macaque controllers (Fig. 2B) and axillary, mixed mesenteric, iliosacral, and inguinal lymph nodes collected at necropsy (Fig. 2C).
FIGURE 2. Virus loads in plasma, PBMC and tissue.
(A) SIV gag viral RNA in plasma samples (PVL) of all 36 macaques was followed longitudinally. PVL is graphed for each macaque individually based on the time of first detection of virus during the 16 weeks of repeated SHIV IR challenges. (B) PBMC were isolated from all 36 macaques and used to quantify cell-associated virus (CAVL), and (C) tissue associated virus was measured in duplicate in axillary, mixed mesenteric, iliosacral and inguinal lymph nodes at necropsy and reported as SIV gag DNA per 1×106 cells. The SIV vDNA was significantly increased in the Anti-V2 and Anti-V2 Mucosal groups compared to the Anti-Env group (***p<0.0001). Unique symbols are associated with each animal and repeated in each panel. Vaccinated animals with either undetectable or low transient PVL were considered as controllers and are shown with blue symbols. Vaccinated animals with continuing PVL were considered non-controllers and have red symbols. Sham-vaccinated control animals have black symbols. (D) Adapted Kaplan-Meier curves reflect the rate of detectable PVL occurring in vaccinated macaques compared to sham-vaccinated controls in each group at each SHIV challenge. The percent of controllers, designated by no detected PVL or transient blips during repeated SHIV challenges and through the time of necropsy in each group, was 56% (Anti-Env), 44% (Anti-V2), and 22% (Anti-V2 Mucosal). Anti-Env (p=0.0068), Anti-V2 (NS), Anti-V2 Mucosal (NS) by Log-rank (Mantel-Cox) analysis.
A comparison by group of the mean of all tissue-associated virus underscores the substantial reduction in virus distribution provided by the Anti-Env vaccine. In the draining lymph nodes tested, the increased SIV vDNA per 1×106 cells examined in the Anti-V2 and Anti-V2 Mucosal groups was highly significant compared to the Anti-Env group (p<0.0001) (Fig. 2C). The tissue-associated virus was comparable in the Anti-V2 and Anti-V2 Mucosal groups (p=0.2242), but it was significantly higher in sham controls compared to all vaccinated macaques (p<0.0001).
A Kaplan-Meier analysis of productive infections occurring in non-controllers after each SHIV IR challenge revealed that only the Anti-Env vaccine resulted in a significant difference between controllers (56%, p=0.0068) and non-controllers. For the Anti-V2 and Anti-V2 Mucosal groups, (44%, p=0.0497 and 22% p=0.3703), respectively, of controllers remained after the number of SHIV challenges required to infect controls (Fig. 2D).
Macaque MHC class I alleles
We genotyped class I MHC Mamu-A and B alleles in all 36 rhesus macaques used in the study to define whether the genetic background may impact SHIV infection progression (Table I). Four MHC Mamu-A alleles were detected: Mamu-A1*001:g, associated with reduced loss of CD4+T cells and longer survival (48), was detected in two controllers (31467 and 34578), and Mamu-A1*002:g, associated with longer survival (49), was detected in two non-controllers (32669 and 32672). Mamu-B*001:g, which has not been associated with elite controllers (50), was present in six animals: three non-controllers (32669, 32672, 32037), and three sham animals (30262, 32661, 32195) (Table I). Mamu-B*008:g and Mamu-B*017:g alleles were not detected in any animals. In summary, two alleles that may suppress SHIV infection, Mamu-A1*001:g and Mamu-A1*002:g, were present in both controllers and non-controllers, whereas Mamu-B*001:g was present in sham controls with both high (32661) and low PVL (30262) (Table I). Based on these observations, we consider it unlikely that these three MHC alleles influenced PVL.
Sites of immune pressure in Env revealed by mutations in plasma breakthrough viruses from non-controllers
The identities of point mutations in the envelope region in plasma viruses from immunized and non-controllers can reveal the selection pressure from Abs induced by vaccine on the challenge virus. Accordingly, we obtained plasma samples at peak PVL (weeks 25–29) from nine non-controllers from the Anti-Env and Anti-V2 groups and from sham-vaccinated controls. The Anti-V2 Mucosal samples were not included. We aligned the env gene sequences with those from SHIVBaL.P4 challenge virus (Table II). The majority of the point mutation sites (18 of 23) were located in the conserved regions of gp120, C1 to C4, with two mutations in V1 and V2 each and one in the V3 region (Table II). V254L in the C2 region of gp120 was the most common mutation site in or near the CD4bs; it was present in five out of nine animals and presumably induced by anti-CD4bs Abs.
Table II.
Sites of immune pressure in Env revealed by mutations in plasma breakthrough viruses from non-controllersa
| Anti - | Env | Anti - | V2 | Sham | |||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| AA (HXB2) | Site | 32222 | 32248 | 32669 | 32672 | 34151 | 27063 | 32037 | 33788 | 33863 | 30740 |
| 22 | C1 | L/P,4 | |||||||||
| 24 | C1 | I/M | |||||||||
| 65 | C1 | V/E | |||||||||
| 130 | C1 | N/Y | |||||||||
| 143 | V1 | S/G | |||||||||
| 147 | V1 | G/E | |||||||||
| 168 | V2 | K/E | |||||||||
| 189/90 | V2 | NN (--) | |||||||||
| 229 | C2 | K/R | K/R | ||||||||
| 254 | C2 | V/L | V/L | V/L | V/L | V/L | |||||
| 287 | C2 | Q/H | |||||||||
| 289 | C2 | N/S,4 | |||||||||
| 290 | C2 | E/K | |||||||||
| 295 | C2 | N/S | |||||||||
| 312 | V3 | G/E | |||||||||
| 338 | C3 | D/N | |||||||||
| 375 | C3 | S/G | |||||||||
| 433 | C4 | A/T | |||||||||
| 839 | gp41 | A/T | A/T | ||||||||
The point mutations in the envelope protein of the breakthrough viruses were determined by analysis of the virus bulk sequence and five clones. The mutations in all six sequences from each plasma virus are shown in the table. The most common point mutation V/L occurred in five animals. The point mutations in the sham animal were present in 4 of 6 sequences.
Immune pressure on the V2 region was minimal, indicating the mutations were induced by de novo Abs to the SHIVBaL.P4 during infection. The deletions at positions 189/190 were detected in the hypervariable part of the V2 (Anti-Env, 32672) that could be induced exclusively by homologous Abs elicited by SHIVBaL.P4. Abs to the challenge virus also probably led to the point mutation K168E in the Anti-V2 experiment, and an additional 11 mutations outside of the V2 in the Anti-V2 study, although it is possible that Abs elicited by the V1V2 vaccine against V2 could have resulted in this mutation. Mutation sites in C1 and C2 in sham controls likely reflect the other constant region changes in the Anti-Env and Anti-V2 groups and generally supports the conclusion that the mutations found were essentially induced by Ab responses to the challenge virus (Table II).
Binding of plasma antibodies to V1V2 scaffold proteins and cyclic V2 peptides
We measured the binding of titrated plasma samples collected at week 22, the time of the first challenge (TOC), from each group, Anti-Env, Anti-V2 and Anti-V2 Mucosal, against four V1V2 scaffold proteins and four biotinylated cyclic V2 (cV2) peptides (Supplementary Fig. 1). Midpoint plasma Ab titers were binned and analyzed by three methods. First, individual animal responses against each V1V2 scaffold protein representing clades B, C, and AE (V1V2CaseA2-gp70, V1V21086-tags, V1V2ZM109-1FD6 and V1V2A244-2F5K) were compared by the group. Ab titers in the Anti-V2 group were significantly higher compared to titers in the Anti-Env group and the Anti-V2 Mucosal group (**p<0.01) (Fig. 3A). Next, group responses were compared against each V1V2 scaffold protein. Ab titers in the Anti-V2 group were consistently highest among the groups against all of the V1V2 proteins tested. Ab titers to V1V2CaseA2-gp70 and V1V21086-tags in all Anti-V2 macaques exceeded 1000 and were significantly higher than Ab titers in either the Anti-Env or Anti-V2 Mucosal groups (***p<0.001) (Fig. 3B). V1V2 scaffold proteins induced comparable titers in the Anti-Env and Anti-V2 Mucosal groups. The lowest titers in all groups were against the V1V2ZM109-1FD6 scaffold protein.
FIGURE 3. Binding of plasma antibodies against V1V2 scaffold proteins and cyclic V2 peptides and comparison of Ab titers in controllers versus non-controllers.
(A, B) The 50% maximum binding titers of plasma Abs against four V1V2 scaffold proteins, and (C, D) four cyclic V2 peptides compared by vaccine group. Titers were determined from the binding curves shown in Supplementary Fig. 1. All plasma dilutions were tested in duplicate. P values are shown *p<0.05, **p<0.01 and ***p<0.001. (E ) Comparison of 50% titers of plasma V1V2 and (F) cV2p Abs in controllers (blue) versus non-controllers (red). Nonparametric Mann-Whitney test. NS – not significant. Unique animal symbols are consistent with Fig. 2.
We determined Ab titers from serially diluted plasma against four cV2 peptides (cV2CaseA2, cV2230, cV2A244, cV2BaL) for individual animals in each group (Fig. 3C). Similar to the pattern seen with responses against the V1V2 scaffold proteins, binding Ab titers to each cV2 peptide were significantly higher in the Anti-V2 group compared to the other two groups, (Fig. 3D). Overall, the titers of plasma Abs binding to V1V2 scaffold proteins and cV2 peptides were comparable between controllers and non-controllers (Fig. 3E, 3F) except for the Anti-Env group titers against cV2230 peptide (p=0.016, Fig. 3F).
One of the cV2 peptides we tested matches the V2BaL sequence that is also present in the SHIVBaL.P4 challenge stock. When compared across groups as AUC, V2BaL Ab titers were highest in the Anti-V2 group compared to the Anti-Env (p=0.0353) or Anti-V2 Mucosal (p=0.0005) groups (Supplementary Fig. 2A), but no difference was seen between controllers and non-controllers in either group (Supplementary Fig. 2B). To evaluate whether the Ab titers we measured against the cV2 BaL peptide could have been induced after challenge and during infection, we analyzed early and late PVL timepoints in both Anti-V2 groups. However, we found no correlations between PVL and titers of plasma Abs against the cV2BaL peptide in the Anti-V2 and Anti-V2 Mucosal groups (Supplementary Fig. 2C, 2D). We conclude from the lack of an inverse relationship between anti-V2 Ab titers and PVL and from a lack of difference in V2 Abs titers between controllers and non-controllers that plasma Abs binding to V1V2 proteins and V2 peptides likely did not contribute to control of viremia during SHIVBaL.P4 infection.
Plasma antibodies against cyclic V192TH023 peptide
The V1 region encoded by gp16092TH023 DNA was present in the Anti-Env vaccine, and V1V292TH023 DNA was present in both the Anti-V2 and Anti-V2 Mucosal vaccines. In order to determine if anti-V1 Abs were induced, we tested titrated plasma samples against a biotinylated cyclic homologous V192TH023 peptide, given that the V1 region is exceptionally hypervariable (Fig. 4). The binding activities varied greatly among the groups with virtually no detectable anti-V1 Abs in the Anti-Env group (Fig. 4A). Thus, no control of SHIVBaL.P4 can be attributed to anti-V1 Abs in the Anti-Env group. When analyzed as the area under the curve (AUC), the difference in titers of anti-V192TH023 binding Abs in the Anti-V2 group compared to either the Anti-Env or Anti-V2 Mucosal groups was highly significant (p<0.0001, Fig. 4D). However, we found no differences between controllers and non-controllers in either group, suggesting that anti-V192TH023 Abs were not responsible for preventing SHIVBaL.P4 infection or viral control in any group (Fig. 4E).
FIGURE 4. Binding of plasma antibodies to cyclic V192TH023 peptide.
(A) Plasma samples from week 22 (time of challenge) from Anti-Env, (B) Anti-V2 and (C) Anti-V2 Mucosal group (each group contains plasma samples from nine animals) were titrated at 1:10 to 1:30,000 dilution and tested by ELISA against biotinylated cV192TH023 peptide. All plasma samples were tested in duplicate. The area under the curve (AUC) of binding Ab titers are compared by group against (D) cV192TH023 peptide and (E ) for controllers (blue) versus non-controllers (red). Data shown as median with interquartile range. Nonparametric Mann-Whitney test, NS - not significant.
Anti-V2 antibodies in mucosal secretions
Buccal and vaginal secretions were collected and samples from all three groups at TOC tested against experiment-relevant antigens. We used buccal and vaginal secretions to avoid interference with multiple IR challenges. The Anti-Env group samples were tested by ELISA for IgG binding Abs against gp120BaL and gp120MN, and samples from the Anti-V2 and Anti-V2 Mucosal groups were tested against gp120BaL and V1V21086-tags (Fig. 5). Due to the limitations of sample volume, IgA Abs were not tested. IgG Ab binding activity against gp120BaL and gp120MN was much stronger in titrated buccal and vaginal fluids of Anti-Env vaccinated macaques than the Anti-V2 groups, especially in vaginal secretions (compare Fig. 5A–B, 5G–H to 5C–F). Commensurate with V1V2 scaffold data in plasma samples, Abs were present in secretion fluids from the Anti-V2 group that strongly reacted with V1V21086-tags (Fig. 5I–J). Noticeably, buccal and vaginal IgG Abs in secretions were detectable from only two animals in the Anti-V2 Mucosal group (Fig. 5K–L). Across groups and irrespective of the antigen tested, IgG Abs from either buccal or vaginal secretions were comparable between controllers and non-controllers (Fig. 5M–5R), indicating that Abs against gp120 and V2 in mucosal secretions were not controlling the SHIVBaL.P4 infection.
FIGURE 5. Vaccine-induced antibodies in mucosal secretions.
Buccal and vaginal mucosal secretions collected at the time of challenge (week 22) from each vaccine group were screened by ELISA against gp120BaL (A-F), gp120MN (G, H) and V1V21086-tags (I-L). Vaginal secretions were collected from the nine female animals in Anti-Env group, and from the five animals in Anti-V2 and Anti-V2 Mucosal groups. Mucosal secretions were tested in duplicate. (M-R) Comparison of AUCs between controllers (blue) and non-controllers (red). Nonparametric Mann-Whitney test. NS – not significant. Unique animal symbols are consistent with Fig. 2.
Virus neutralization by plasma antibodies
We measured neutralizing plasma Abs (NAbs) longitudinally throughout the vaccine and challenge phases in each group. Only the Anti-Env vaccine induced NAbs against the challenge virus, SHIVBaL.P4 (Fig. 6A). NAbs were not detected in either the Anti-V2 or Anti-V2 Mucosal groups against any viruses tested (data not shown). In the sham vaccinated control animals challenged concurrently with the Anti-Env group, peak PVL occurred at or near week 24, two weeks post TOC (Fig 2A). In contrast, at this time point, all but one animal (32238) in the Anti-Env group developed 50% neutralizing (ID50) >1,000 against the challenge strain, ranging from 1,077 to 5,837, that inversely correlated with PVL (p=0.0357) (Fig. 6B).
FIGURE 6. Plasma antibody neutralization of pseudotyped viruses.
The 50% inhibitory dilution (ID50) was determined from plasma samples serially diluted and tested for neutralizing activities using the TZM-bl cell assay (duplicate samples, representative of two independent experiments). (A) Anti-Env group plasma longitudinal neutralization of SHIVBaL.P4 is shown over the course of the vaccine/challenge experiment. The arrow denotes the time of challenge (TOC). (B) Inverse correlation between ID50 plasma neutralization and PVL at week 24. (C) Neutralization of a panel of pseudotyped viruses by Anti-Env plasma samples. Virus clades are noted in parenthesis. (D) Comparison of SHIVBaL.P4 plasma neutralizing Abs (NAbs) in controllers (blue) vs non-controllers (red). Nonparametric Mann-Whitney test used for significance. Spearman correlation; r and P values are shown. Unique animal symbols are consistent with Fig. 2.
Neutralization by plasma Abs at week 22 (TOC) was tested against seven other pseudotyped viruses, including the clade C, tier 1 SHIV-1157ipEL. Anti-Env neutralizing titers against SHIV-1157ipEL were similar to those against the challenge virus (Fig. 6C). In addition, the Anti-Env plasma Abs neutralized the HIV-1 Tier 1-pseudotyped viruses SF162, (clade B), Q461d1, Q23env17 (clade A) and DJ263.8 (02_AG); while ZM109 (clade C) was resistant (Fig. 6C). The only Tier 2 virus neutralized was JRCSF (clade B). During the vaccine phase of the experiment, neutralizing titers of controllers and non-controllers were comparable against all viruses tested (Supplementary Fig. 3A). A clear and significant difference is seen between these two sub-groups after challenges begin and continuing through necropsy at week 35 with lower neutralization in the controllers (p=0.0159, Fig. 6D). This difference reflects the extremely low or absent persistent antigen in the controllers, and thus no additional increases in neutralization titers developed. Although only a weak inverse correlation was found between SHIVBaL.P4 NAbs and PVL, it suggests that the vaccine-induced plasma NAbs may account for the lack of viremia in Anti-Env controllers.
The Anti-V2 vaccine consisted of both the V1 and V2 regions of HIV Env (92TH023, A244) that could potentially induce V1 Abs and neutralize viruses homologous to the immunogens. To test whether any Anti-V1 vaccine-specific NAbs were induced, we assayed the plasma samples against HIV-1 Tier 1, 92TH023.6 and Tier 2, CM244.c01 (Table III). Plasma samples from all animals in the Anti-Env group neutralized the Tier 1 virus 92TH023.6 with titers ranging from 592 to 4,277. In contrast, plasma Abs from the Anti-V2 study were very weak against 92TH023.6 and considered non-neutralizing based on the criterion requiring a greater than three-fold increase in activity compared with the SVA-MLV negative control pseudovirus. Anti-Env group plasma weakly neutralized the Tier 2 virus CM244.c01 with titers >50 in only one of the nine animals (Table III) and showed no correlation with SHIVBaL.P4 NAbs (Supplementary Fig. 3C). No CM244.c01 neutralization was detected in any of the animals in the Anti-V2 study (Table III). Binding Abs against the cV192TH023 peptide, V1V2A244 or cV2A244 peptide did not correlate with NAbs against SHIVBaL.P4 (Supplementary Fig. 3B, 3D, 3E, respectively). We conclude that, unlike the Anti-V2 and Anti-V2 Mucosal groups, NAbs against SHIVBaL.P4 induced by the Anti-Env vaccine likely influenced the control of infection.
Table III.
Neutralization activity of plasma antibodies at week 22 against pseudotyped viruses autologous to 92TH023 immunogensa.
| Animal | SVA-MLV | 92TH023.6 | CM244.c01 |
|---|---|---|---|
| Anti-Env | group | ||
| 32222 | <20 | 943 | 27 |
| 32248 | <20 | 592 | 29 |
| 32669 | <20 | 2429 | 75 |
| 32672 | <20 | 1876 | <20 |
| 30300 | 22 | 4277 | 46 |
| 33615 | <20 | 539 | <20 |
| 33617 | <20 | 3138 | 26 |
| 33620 | <20 | 1166 | 27 |
| 33653 | <20 | 1656 | 46 |
| Anti-V2 | group | ||
| 27063 | 37 | 93 | 27 |
| 32037 | 20 | 28 | <20 |
| 33788 | 24 | 43 | 21 |
| 33863 | 33 | 53 | <20 |
| 34151 | 23 | 29 | 25 |
| 31467 | 29 | 33 | 28 |
| 33607 | 25 | 40 | 22 |
| 34149 | 33 | 24 | 22 |
| 34209 | 28 | 42 | 27 |
Values are the serum dilution at which relative luminescence units (RLUs) were reduced by 50% compared to virus control wells (no test sample). Values in light gray boxes are background signal against the MLV-pseudotyped negative control virus. Values in bold black type are considered positive for neutralizing antibody activity based on the criterion of signal >3X the observed background against the SVA-MLV negative control pseudovirus in that sample. Non-controllers (red); controllers (blue).
ADCC and ADCP activities of plasma Abs
We tested macaque plasma samples from the Anti-Env and Anti-V2 groups at week 22 against SHIVSF162P3-infected cells in an ADCC assay using an NK cell line expressing rhesus CD16 as effector cells. Cytotoxic activity is represented by a reduction in relative light units (RLUs) with a plasma dilution resulting in 50% cell death considered the threshold for activity. Plasma Ab ADCC activity varied in each group with only two animals in the Anti-Env group (33617, 33620) and four animals (33788, 33863, 34151, 34209) in the Anti-V2 group reaching the 50% threshold for ADCC activity at the lowest dilutions (Fig. 7A, 7B). When calculated as AUC, there was no difference between non-controllers and controllers in either group (Fig. 7E), and ADCC activity did not correlate with PVL at week 24 (Supplementary Fig. 4A, 4B) or with NAbs against the challenge virus (Supplementary Fig. 4C).
FIGURE 7. ADCC and ADCP activity of plasma antibodies.
(A) Plasma samples collected at week 22 were serially diluted and used to assess ADCC against cells infected with SHIVSF162P3 in Anti-Env and (B) Anti-V2 experiment. A reduction in % RLUs indicates an increase in ADCC activity. Control is plasma from a naïve rhesus macaque. (C, D) Phagocytic activity of plasma Abs tested at four dilutions: 1:25 to 1:200. Phagocytosis indicates a fold increase of phagocytic scores with the gp120BaL-coated beads over beads coated with BSA. (E) ADCC and (F) ADCP AUCs are compared between controllers (blue) and non-controllers (red) in the Anti-Env and Anti-V2 groups. Duplicate samples, representative of two independent tests. Nonparametric Mann-Whitney test.
We also examined ADCP of plasma samples from the Anti-Env and Anti-V2 groups at four dilutions (1:25–1:200), using beads coated with gp120BaL (Fig 7C, 7D). In both studies, Anti-Env and Anti-V2, the phagocytic activity measured by AUC was significantly higher at TOC (week 22) compared to pre-vaccination, day 0 (p=0.0028, p=0.0083, respectively), indicating that the immunization strategy induced Abs capable of mediating phagocytosis (Supplementary Fig. 4D, 4E). AUC of ADCP activity was comparable between controllers and non-controllers in both studies (Fig. 7F), suggesting that Abs mediating phagocytosis did not correlate with SHIVBaL.P4 acquisition. Taken together, the data indicate that neither ADCC nor ADCP was associated with SHIV acquisition or control.
ADCVI activity of plasma antibodies against SHIVBaL.P4-infected target cells
Plasma samples from each group were tested longitudinally throughout the immunizations (weeks 0, 14, 22) at a dilution of 1:100 for ADCVI. We measured higher ADCVI activity in the Anti-Env group than the Anti-V2 or the Anti-V2 Mucosal groups, with the lowest activity seen in the Anti-V2 group (Fig 8A–C). Measured as AUC, the ADCVI activity at week 22 in both the Anti-Env group and the Anti-V2 Mucosal group was significantly higher compared to the Anti-V2 group (p=0.0188, p=0.0012, respectively) (Fig. 8D). When analyzed to compare controllers and non-controllers, ADCVI activity was comparable in all three groups, suggesting that this function was not involved in the control of SHIVBaL.P4 infection (Fig. 8E).
FIGURE 8. ADCVI activity in plasma samples derived from vaccinated rhesus macaques.
(A) Plasma ADCVI activity in Anti-Env, (B) Anti-V2, and (C) Anti-V2 Mucosal experiments tested at preimmunization day 0, week 14 and pre-challenge week 22. (D) Comparison of ADCVI AUC in each group. One-way ANOVA. (E) AUC compared between controllers (blue) and non-controllers (red). Duplicate samples, representative of two independent tests. Nonparametric Mann-Whitney test.
Antibody capture of infectious SHIVBaL.P4
We tested plasma samples collected at weeks 0, 14, and 22 in a virus capture assay using the SHIVBaL.P4 challenge virus (Fig. 9). Capture Abs increased between weeks 14 and 22 (2 weeks after immunizations 3 and 4) in all three groups, and the groups’ comparison reveals that Abs from the Anti-Env group at week 14, 22 captured significantly more virus than the Anti-V2 or Anti-V2 Mucosal groups (p=0.006) (Fig. 9A). Capture Abs in the Anti-V2 and Anti-V2 Mucosal groups were comparable at weeks 14 and 22. At TOC, week 22, the Anti-Env group had significantly more SHIVBaL.P4 capture Abs than the Anti-V2 group (p=0.0028) (Fig. 9B).
FIGURE 9. Capture of infectious SHIVBaL.P4 by plasma antibodies from three protection experiments, Anti-Env, Anti-V2 and Anti-V2 Mucosal.
(A) SHIVBaL.P4 was captured by rhesus macaques’ immune plasma Abs at week 14 and 22 immobilized on the plates by goat anti-monkey IgG (γ) antibody. The infectivity of the captured virus was determined using TZM-bl cells. Capture is reported as relative light units (RLUs) with deduced RLU values of preimmune plasma samples at day 0. (B) RLUs of Abs capturing SHIVBaL.P4 at week 22 from two experiments, (C) Comparison of titers of plasma Abs binding to cV2BaL peptide at week 22; the data are from Supplementary Fig. 1B. (B, C) The ratio of RLUs and titers for Env and V2 antibodies is 31 and 0.4, respectively. (D) Comparison of plasma Abs from controllers (blue) and non-controllers (red) at week 22 capturing SHIVBaL.P4. (E) Level of SHIV capturing Abs, RLUs, at week 22 inversely correlated with PVL at week 26 in Anti-Env experiment, and (F) RLUs at week 14 correlated with ADCVI activity at week 22. Duplicate samples, representative of two independent tests. Nonparametric Mann-Whitney test; NS – not significant. Unique animal symbols are consistent with Fig. 2.
We also compared SHIV BaL.P4 capture Abs to cV2BaL peptide-binding Abs in the Anti-Env and Anti-V2 groups. Our goal was to determine if we could detect a qualitative difference in plasma Abs induced by the Anti-Env vaccine compared to the Anti-V1V2 vaccine. At the TOC, binding Ab titers to cV2BaL peptide were comparable between the Anti-Env and Anti-V2 groups (Fig. 9C). Different assays determined capture and binding Abs. Therefore, to compare the groups more functionally, we calculated a ratio of SHIVBaL.P4 capture Abs to titers of cV2BaL peptide-binding Abs. Titers of cV2BaL binding Abs between the groups were similar with a ratio of 0.4. In contrast, the ratio of capture Abs between the groups was 31, representing a much greater proportion of Anti-Env Abs recognizing Env on infectious virions (Fig. 9B, 9C). These results indicate that anti-V2 Abs in both groups bind to the linear epitope of the cV2BaL peptide equally well, but the groups differ significantly in the ability to capture SHIVBaL.P4 virions.
To evaluate whether the capture Abs in the Anti-Env group influenced SHIV pathogenesis or virus control, we compared vaccine-induced SHIVBaL.P4 capture Abs at TOC (week 22) in each group and compared controllers to non-controllers. Capture Abs in the Anti-Env group were greater in controllers (p=0.0317) (Fig. 9D) and correlated inversely with PVL at week 26 (p=0.0013) (Fig. 9E). In contrast, capture Abs in the Anti-V2 and Anti-V2 mucosal groups were comparable between controllers and non-controllers and no correlation with PVL was observed (data not shown). We found a correlation between Anti-Env capture Abs elicited by the third immunization (samples from week 14) and ADCVI activity at week 22 (p=0.0124, Fig 9F).
In summary, polyclonal virus capturing and NAbs against the challenge virus, SHIVBaL.P4, suggest two immune correlates for the Anti-Env experiment that were not detected in either the Anti-V2 or Anti-V2 Mucosal groups, indicating that V2 Abs acting alone or mediating cellular functions were not associated with control of SHIVBaL.P4 viremia.
Discussion
This study was designed to test whether V1V2 vaccine-induced anti-V2 Abs could protect against heterologous Tier 1 SHIVBaL.P4 challenge in rhesus macaques. The results revealed that a vaccine eliciting neutralizing and virus-capturing Abs was more likely to provide tight virus control than titers of V2 Abs without these functions. No significant difference was observed between titers of V2 Abs to various V1V2 scaffolds and cyclic V2 peptides of controllers and non-controllers in all three groups. Buccal and vaginal secretions were comparable between controllers and non-controllers, and plasma Abs from the Anti-V2 studies did not neutralize the SHIVBaL.P4 virus. SHIVBaL.P4 virions were weakly captured by plasma V2 Abs from the Anti-V2 and Anti-V2 Mucosal groups, and significantly less so than by Abs from the Anti-Env group. ADCC and ADCVI activities against target cells infected with SHIVSF162P3 and ADCP against gp120BaL-coated beads were very modest, with no difference between controllers and non-controllers. A caveat to our ADCC assay is that while the macaque Abs are tested for cytotoxicity against a human NK cell line transduced to express rhesus CD16, it may not fully represent the activity of macaque effector cells in vivo. Nonetheless, we conclude that in macaques, the vaccine-induced V2 Abs in this study could not sufficiently inhibit virus either by neutralization or by Fc-mediated activity. Given that the clinical trial and experiments in rhesus macaques testing the immune response to a mosaic HIV-1 vaccine were comparable (51), our results in macaques suggest that a high level of binding V2 Abs, one of the correlates in the RV144 trial, may be a surrogate marker for another immune response mediating protection from infection as has been proposed in other macaque studies (51–54).
The level of plasma V2 Abs is associated with an HIV-infected individuals’ general ability to mount an Ab response to Env proteins. The level of V2 Abs in a cohort of chronically HIV-infected subjects strongly correlated with the levels of Abs against gp120 (p<0.0001) and gp41, and the titer of anti-gp120 Abs was significantly lower in patients with deficient anti-V2 Abs than in donors with high titer and cross-reactive V2 Abs (55). Thus, those participants in the RV144 vaccine trial with high V2 Ab levels were likely good immune responders, and other activities could have contributed to the observed protection, e.g., mucosal Abs or secondary correlates such as neutralizing Abs and ADCC.
Several studies have been performed in rhesus macaques to test the efficacy of different vaccine regimens. These studies also analyzed the relationship between the level or titer of V2 Abs and the outcome of viral infection. There have been two classes of protection experiments, the first using HIV-1 Env antigens for vaccination, followed by SHIV challenge, and the second using SIV Env, followed by SIV infection. The two classes have yielded different results regarding the function of V2 Abs. Studies using the HIV Env immunogens and SHIV challenge protocol did not report a correlation between V2 Abs and reduced virus acquisition (17, 18, 51). In another study, using two similar protocols, the association between V2 Abs and outcome was only detected when using vaccines with the replicating simian adenovirus (SAd7) vector, but not with the non-replicating human Ad4 (44).
The second class of protection experiments, using the SIV Env immunogens and SIV challenge protocol, yielded mixed results regarding the correlation between V2 Abs and the risk of virus infection. Correlation between V2 Abs and protection was reported in five studies, including two in which the correlation depended on the adjuvant used or whether the V2 Abs were measured in mucosal secretions or serum (52, 53, 56–58). Four other studies reported no impact of the V2 Abs on virus acquisition (54, 59–61). Several of these studies suggested that V2 Abs could be a surrogate for another immune response (51–54).
One reason for the difference between these two classes of protection experiments might be the higher immunogenicity of the V2 region in SIV relative to HIV-1. This could be related to the pair of V2 cysteine residues at positions 183 and 191 that play a role in stabilizing the Env trimer (62). Vaccination of nonhuman primates with SIVmac239 and SIVsmE660 Env antigens induces much higher V2 Abs than immunization with HIV-1 Env with sequences of A244, TH023, MN, 1086, and ZM651 viruses (63).
Sequencing env genes of plasma viruses from nine non-controllers derived from the Anti-Env and Anti-V2 group animals revealed a number of mutations that resulted from the pressure of de novo Abs developed during SHIVBaL.P4 infection and not from vaccine-elicited Abs (Table II). Our conclusion is based on (i) the presence of 11 point mutations outside of the V2 region in the Anti-V2 group that received only V1V2 immunogens, (ii) mutations from the control animal that was challenged, but not immunized, and (iii) mutations in the V1 region that could be induced only by homologous V1 sequence-specific Abs, while the vaccine was heterologous to the virus challenge.
Ten of 21 (48%) mutations in plasma viruses were located in the C2 domain, and three others in the C3 and C4 domains, most likely elicited by CD4bs and CD4-induced Abs. A range of 1–3 mutations were detected in four regions: C1, V1, V2 and V3. As the CD4bs was mainly under immune pressure, it is interesting to note that it was also under immune pressure determined by sieve analysis of breakthrough HIV-1 in the HVTN 505 clinical trial (64).
There were two mutations in the V2 region. In one virus isolated from a macaque in the Anti-Env study, two residues were deleted from the hypervariable part of the V2 region, NN189/190. This could only have been induced by the homologous anti-SHIVBaL.P4 Abs, but not by Abs induced by immunogens with heterologous sequences. A second mutation was located in the conserved part of the V2 region, K168E. This mutation was most likely induced by immune pressure from the challenge virus in the Anti-V2 study and eventually initiated by the vaccine-induced V2 Abs because all immunogens contain K at Env position 168 (Table II).
The minimal selective pressure of V2 Abs can be explained by their weak ability to bind and capture SHIVBaL.P4 virions. We found that V2 Abs from both the Anti-Env group and the Anti-V2 group bind to the cV2BaL peptide equally well (Fig. 9C), but the Anti-V2 Abs captured significantly fewer SHIVBaL.P4 virions compared to Anti-Env Abs (Fig. 9B). The poor binding of V2 Abs to intact virions is caused by glycans shielding the V2 region and its exceptional flexibility (65, 66). Thus, capturing virions by Abs is probably more biologically relevant than measuring binding to proteins and/or peptides.
These results differ from the sieve analysis of breakthrough viruses in RV144 that detected two amino acid positions at 169 and 181 in V2 that were deemed to be associated with vaccine-induced immune responses (67). First, given that plasma viruses were analyzed at the time of HIV-1 diagnosis, the Abs induced by the infecting virus – not by the vaccine – may have led to these changes in Env sequences, comparably as we discussed above for our study. Second, Tier 1 SHIVBaL.P4 may provide more accessibility for V2 Ab binding than the Tier 2 CRF01-AE in RV144. However, our SHIVBaL.P4 capture assay showed that V2 Abs captured Tier 1 virions poorly, suggesting that capturing Tier 2 viruses could be even weaker, thus resulting in limited pressure on the V2 region (Fig. 9B). Finally, matching a sieve analysis of breakthrough viruses with the titer of plasma V2 Abs would be interesting because chronically HIV-infected individuals harbor a broad range of V2 Abs, including those with no V2 Abs over many years (55).
The study was focused on vaccine-induced potentially protective V2 Abs, and the results showed that control of SHIVBaL.P4 infection was diminished without neutralization or virus capturing capabilities, and virus spread into draining lymph nodes was not different from that of controls. However, several animals in the Anti-V2 and Anti-V2 Mucosal groups controlled the SHIVBaL.P4 challenge. Although we did not test for other immune correlates, it is possible that innate immunity could contribute to viral control during repeated low-dose SHIV challenges. Innate immunity has no antigen specificity or memory, but some stimuli can prime monocytes and macrophages and train the cells to respond to future antigen, contributing to partial protection (68, 69). This hypothesis is supported by data showing that a mucosal vaccine protected several rhesus macaques against Tier 1 SHIVSF162P4 without inducing any gp120 Abs. In that study, immune correlates included innate myeloid cell accumulation, monocytes, and macrophages in the colorectal mucosa that produced TNF-α, IL-6, as well as MIP1α (69). In another study of rhesus macaques immunized with SIV Env immunogens and challenged with SIV, CD14+ innate monocytes were associated with a reduced risk of SIVmac251 acquisition (59).
In summary, the V1V2 immunogens in this study induced non-neutralizing V2 Abs in rhesus macaques that provided no immune correlation within the parameters tested for the six macaques controlling infection in the presence of weak Fc-mediated activities, and poor virus capture against SHIVBaL.P4. The HIV-1 Env immunogens resulted in neutralizing and virus-capturing Abs that correlated with virus control in the majority of animals. The data support the concept that to increase HIV vaccine efficacy, emphasis should continue to favor induction of a broad adaptive immune response, including neutralizing activity, while boosting trained innate immunity.
Supplementary Material
Key points:
Vaccines were designed to induce only V1V2 antibodies in NHP before SHIV challenge.
Vaccine-induced V2 antibodies did not independently control SHIV infection.
Neutralizing and virus capture anti-Env antibodies correlated with SHIV virus control.
Acknowledgments
We thank Lily Liu, Leechun Weng and Flavia Camacho for their technical and administrative assistance. We also thank Dr. David Evans for the NK effector cell line, KHYG-1 rhCD16, and for the target cells, CEM-NKR-CCR5-sLTR-Luc (NKR24) used in the ADCC assay.
The study was supported by NIH grant AI112546 (MKG), P51 OD011092 (ONPRC), and by Quality Biological, Inc. and the Vaccine Research Program, Division of AIDS, NIAID, NIH under contract No. HHSN272201800007C.
Abbreviations used in this article:
- ADCC
antibody-dependent cellular cytotoxicity
- ADCP
antibody-dependent cellular phagocytosis
- ADCVI
antibody-dependent cell-mediated virus inhibition
- AUC
area under the curve
- bNAbs
broadly neutralizing antibodies
- CD4bs
CD4 binding site
- CRF
circulating recombinant form
- Env
envelope
- PVL
plasma viral load
- RLU
relative light units
- SHIV
simian-human immunodeficiency virus
Footnotes
Disclosures
None of the authors declares conflicts of interest.
The online version of this article contains supplemental data.
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