Liver-Directed but Not Muscle-Directed AAV-Antibody Gene Transfer Limits Humoral Immune Responses in Rhesus Monkeys

A number of publications have described the use of adeno-associated virus (AAV) for the delivery of anti-HIV and anti-simian immunodeficiency virus (SIV) monoclonal antibodies (mAbs) to rhesus monkeys. Anti-drug antibodies (ADAs) have been frequently observed, and long-term AAV-mediated delivery has been inconsistent. Here, we investigated different AAV vector strategies and delivery schemes to rhesus monkeys using the rhesus monkey mAb 4L6. We compared 4L6 immunoglobulin G1 (IgG1) delivery using the AAV1 versus the AAV8 serotype with a cytomegalovirus (CMV) promoter and the use of a muscle-specific versus a liver-specific promoter. Long-term expression levels of 4L6 IgG1 following AAV8-mediated gene transfer were comparable to those following AAV1-mediated gene transfer. AAV1-mediated gene transfer, using a muscle-specific promoter, showed robust ADAs and transiently low 4L6 IgG1 levels that ultimately declined to below detectable levels. Intravenous AAV8-mediated gene transfer, using a liver-specific promoter, also resulted in low levels of delivered 4L6 IgG1, but those low levels were maintained in the absence of any detectable ADAs. Booster injections using AAV1-CMV allowed for increased 4L6 IgG1 serum levels in animals that were primed with AAV8 but not with AAV1. Our results suggest that liver-directed expression may help to limit ADAs and that re-administration of AAV of a different serotype can result in successful long-term delivery of an immunogenic antibody

A recombinant herpesviral vector containing a near-full-length SIVmac239 genome produces SIV particles and elicits immune responses to all nine SIV gene products

The properties of the human immunodeficiency virus (HIV) pose serious difficulties for the development of an effective prophylactic vaccine. Here we describe the construction and characterization of recombinant (r), replication-competent forms of rhesus monkey rhadinovirus (RRV), a gamma-2 herpesvirus, containing a near-full-length (nfl) genome of the simian immunodeficiency virus (SIV). A 306-nucleotide deletion in the pol gene rendered this nfl genome replication-incompetent as a consequence of deletion of the active site of the essential reverse transcriptase enzyme. Three variations were constructed to drive expression of the SIV proteins: one with SIV\u27s own promoter region, one with a cytomegalovirus (cmv) immediate-early promoter/enhancer region, and one with an RRV dual promoter (p26 plus PAN). Following infection of rhesus fibroblasts in culture with these rRRV vectors, synthesis of the early protein Nef and the late structural proteins Gag and Env could be demonstrated. Expression levels of the SIV proteins were highest with the rRRV-SIVcmv-nfl construct. Electron microscopic examination of rhesus fibroblasts infected with rRRV-SIVcmv-nfl revealed numerous budding and mature SIV particles and these infected cells released impressive levels of p27 Gag protein ( \u3e 150 ng/ml) into the cell-free supernatant. The released SIV particles were shown to be incompetent for replication. Monkeys inoculated with rRRV-SIVcmv-nfl became persistently infected, made readily-detectable antibodies against SIV, and developed T-cell responses against all nine SIV gene products. Thus, rRRV expressing a near-full-length SIV genome mimics live-attenuated strains of SIV in several important respects: the infection is persistent; \u3e 95% of the SIV proteome is naturally expressed; SIV particles are formed; and CD8+ T-cell responses are maintained indefinitely in an effector-differentiated state. Although the magnitude of anti-SIV immune responses in monkeys infected with rRRV-SIVcmv-nfl falls short of what is seen with live-attenuated SIV infection, further experimentation seems warranted

Development of an anti-CAR antibody response in SIV-infected rhesus macaques treated with CD4-MBL CAR/CXCR5 T cells

T cells expressing a simian immunodeficiency (SIV)-specific chimeric antigen receptor (CAR) and the follicular homing molecule, CXCR5, were infused into antiretroviral therapy (ART) suppressed, SIV-infected rhesus macaques to assess their ability to localize to the lymphoid follicle and control the virus upon ART interruption. While the cells showed evidence of functionality, they failed to persist in the animals beyond 28 days. Development of anti-CAR antibodies could be responsible for the lack of persistence. Potential antigenic sites on the anti-SIV CAR used in these studies included domains 1 and 2 of CD4, the carbohydrate recognition domain (CRD) of mannose-binding lectin (MBL), and an extracellular domain of the costimulatory molecule, CD28, along with short linker sequences. Using a flow cytometry based assay and target cells expressing the CAR/CXCR5 construct, we examined the serum of the CD4-MBL CAR/CXCR5-T cell treated animals to determine that the animals had developed an anti-CAR antibody response after infusion. Binding sites for the anti-CAR antibodies were identified by using alternative CARs transduced into target cells and by preincubation of the target cells with a CD4 blocking antibody. All of the treated animals developed antibodies in their serum that bound to CD4-MBL CAR/CXCR5 T cells and the majority were capable of inducing an ADCC response. The CD4 antibody-blocking assay suggests that the dominant immunogenic components of this CAR are the CD4 domains with a possible additional site of the CD28 domain with its linker. This study shows that an anti-drug antibody (ADA) response can occur even when using self-proteins, likely due to novel epitopes created by abridged self-proteins and/or the self-domain of the CAR connection to a small non-self linker. While in our study, there was no statistically significant correlation between the ADA response and the persistence of the CD4-MBL CAR/CXCR5-T cells in rhesus macaques, these findings suggest that the development of an ADA response could impact the long-term persistence of self-based CAR immunotherapies

AIDS virus–specific CD8+ T lymphocytes against an immunodominant cryptic epitope select for viral escape

Cryptic major histocompatibility complex class I epitopes have been detected in several pathogens, but their importance in the immune response to AIDS viruses remains unknown. Here, we show that Mamu-B*17+ simian immunodeficiency virus (SIV)mac239-infected rhesus macaques that spontaneously controlled viral replication consistently made strong CD8+ T lymphocyte (CD8-TL) responses against a cryptic epitope, RHLAFKCLW (cRW9). Importantly, cRW9-specific CD8-TL selected for viral variation in vivo and effectively suppressed SIV replication in vitro, suggesting that they might play a key role in the SIV-specific response. The discovery of an immunodominant CD8-TL response in elite controller macaques against a cryptic epitope suggests that the AIDS virus–specific cellular immune response is likely far more complex than is generally assumed

Non-neutralizing Antibodies May Contribute to Suppression of SIVmac239 Viremia in Indian Rhesus Macaques

The antiviral properties of broadly neutralizing antibodies against HIV are well-documented but no vaccine is currently able to elicit protective titers of these responses in primates. While current vaccine modalities can readily induce non-neutralizing antibodies against simian immunodeficiency virus (SIV) and HIV, the ability of these responses to restrict lentivirus transmission and replication remains controversial. Here, we investigated the antiviral properties of non-neutralizing antibodies in a group of Indian rhesus macaques (RMs) that were vaccinated with vif, rev, tat, nef, and env, as part of a previous study conducted by our group. These animals manifested rapid and durable control of viral replication to below detection limits shortly after SIVmac239 infection. Although these animals had no serological neutralizing activity against SIVmac239 prior to infection, their pre-challenge titers of Env-binding antibodies correlated with control of viral replication. To assess the contribution of anti-Env humoral immune responses to virologic control in two of these animals, we transiently depleted their circulating antibodies via extracorporeal plasma immunoadsorption and inhibition of IgG recycling through antibody-mediated blockade of the neonatal Fc receptor. These procedures reduced Ig serum concentrations by up to 80% and temporarily induced SIVmac239 replication in these animals. Next, we transferred purified total Ig from the rapid controllers into six vaccinated RMs one day before intrarectal challenge with SIVmac239. Although recipients of the hyperimmune anti-SIV Ig fraction were not protected from infection, their peak and chronic phase viral loads were significantly lower than those in concurrent unvaccinated control animals. Together, our results suggest that non-neutralizing Abs may play a role in the suppression of SIVmac239 viremia

Macaques vaccinated with live-attenuated SIV control replication of heterologous virus

An effective AIDS vaccine will need to protect against globally diverse isolates of HIV. To address this issue in macaques, we administered a live-attenuated simian immunodeficiency virus (SIV) vaccine and challenged with a highly pathogenic heterologous isolate. Vaccinees reduced viral replication by ∼2 logs between weeks 2–32 (P ≤ 0.049) postchallenge. Remarkably, vaccinees expressing MHC-I (MHC class I) alleles previously associated with viral control completely suppressed acute phase replication of the challenge virus, implicating CD8+ T cells in this control. Furthermore, transient depletion of peripheral CD8+ lymphocytes in four vaccinees during the chronic phase resulted in an increase in virus replication. In two of these animals, the recrudescent virus population contained only the vaccine strain and not the challenge virus. Alarmingly, however, we found evidence of recombinant viruses emerging in some of the vaccinated animals. This finding argues strongly against an attenuated virus vaccine as a solution to the AIDS epidemic. On a more positive note, our results suggest that MHC-I–restricted CD8+ T cells contribute to the protection induced by the live-attenuated SIV vaccine and demonstrate that vaccine-induced CD8+ T cell responses can control replication of heterologous challenge viruses

AIDS virus-specific CD8+T lymphocytes against an immunodominant cryptic epitope select for viral escape

Cryptic major histocompatibility complex class I epitopes have been detected in several pathogens, but their importance in the immune response to AIDS viruses remains unknown. Here, we show that Mamu-B*17+ simian immunodeficiency virus (SIV)mac239-infected rhesus macaques that spontaneously controlled viral replication consistently made strong CD8+ T lymphocyte (CD8-TL) responses against a cryptic epitope, RHLAFKCLW (cRW9). Importantly, cRW9-specific CD8-TL selected for viral variation in vivo and effectively suppressed SIV replication in vitro, suggesting that they might play a key role in the SIV-specific response. The discovery of an immunodominant CD8-TL response in elite controller macaques against a cryptic epitope suggests that the AIDS virus–specific cellular immune response is likely far more complex than is generally assumed

Broadly Neutralizing Human Anti-HIV Antibody 2G12 Is Effective in Protection against Mucosal SHIV Challenge Even at Low Serum Neutralizing Titers

Developing an immunogen that elicits broadly neutralizing antibodies (bNAbs) is an elusive but important goal of HIV vaccine research, especially after the recent failure of the leading T cell based HIV vaccine in human efficacy trials. Even if such an immunogen can be developed, most animal model studies indicate that high serum neutralizing concentrations of bNAbs are required to provide significant benefit in typical protection experiments. One possible exception is provided by the anti-glycan bNAb 2G12, which has been reported to protect macaques against CXCR4-using SHIV challenge at relatively low serum neutralizing titers. Here, we investigated the ability of 2G12 administered intravenously (i.v.) to protect against vaginal challenge of rhesus macaques with the CCR5-using SHIVSF162P3. The results show that, at 2G12 serum neutralizing titers of the order of 1∶1 (IC90), 3/5 antibody-treated animals were protected with sterilizing immunity, i.e. no detectable virus replication following challenge; one animal showed a delayed and lowered primary viremia and the other animal showed a course of infection similar to 4 control animals. This result contrasts strongly with the typically high titers observed for protection by other neutralizing antibodies, including the bNAb b12. We compared b12 and 2G12 for characteristics that might explain the differences in protective ability relative to neutralizing activity. We found no evidence to suggest that 2G12 transudation to the vaginal surface was significantly superior to b12. We also observed that the ability of 2G12 to inhibit virus replication in target cells through antibody-mediated effector cell activity in vitro was equivalent or inferior to b12. The results raise the possibility that some epitopes on HIV may be better vaccine targets than others and support targeting the glycan shield of the envelope

Allogeneic Lymphocytes Persist and Traffic in Feral MHC-Matched Mauritian Cynomolgus Macaques

Thus far, live attenuated SIV has been the most successful method for vaccinating macaques against pathogenic SIV challenge; however, it is not clear what mechanisms are responsible for this protection. Adoptive transfer studies in mice have been integral to understanding live attenuated vaccine protection in models like Friend virus. Previous adoptive transfers in primates have failed as transferred cells are typically cleared within hours after transfer.Here we describe adoptive transfer studies in Mauritian origin cynomolgus macaques (MCM), a non-human primate model with limited MHC diversity. Cells transferred between unrelated MHC-matched macaques persist for at least fourteen days but are rejected within 36 hours in MHC-mismatched macaques. Cells trafficked from the blood to peripheral lymphoid tissues within 12 hours of transfer.MHC-matched MCM provide the first viable primate model for adoptive transfer studies. Because macaques infected with SIV are the best model for HIV/AIDS pathogenesis, we can now directly study the correlates of protective immune responses to AIDS viruses. For example, plasma viral loads following pathogenic SIV challenge are reduced by several orders of magnitude in macaques previously immunized with attenuated SIV. Adoptive transfer of lymphocyte subpopulations from vaccinated donors into SIV-naïve animals may define the immune mechanisms responsible for protection and guide future vaccine development