Control of simian immunodeficiency virus replication by vaccine-induced Gag- and Vif-specific CD8+ T cells.

For development of an effective T cell-based AIDS vaccine, it is critical to define the antigens that elicit the most potent responses. Recent studies have suggested that Gag-specific and possibly Vif/Nef-specific CD8(+) T cells can be important in control of the AIDS virus. Here, we tested whether induction of these CD8(+) T cells by prophylactic vaccination can result in control of simian immunodeficiency virus (SIV) replication in Burmese rhesus macaques sharing the major histocompatibility complex class I (MHC-I) haplotype 90-010-Ie associated with dominant Nef-specific CD8(+) T-cell responses. In the first group vaccinated with Gag-expressing vectors (n = 5 animals), three animals that showed efficient Gag-specific CD8(+) T-cell responses in the acute phase postchallenge controlled SIV replication. In the second group vaccinated with Vif- and Nef-expressing vectors (n = 6 animals), three animals that elicited Vif-specific CD8(+) T-cell responses in the acute phase showed SIV control, whereas the remaining three with Nef-specific but not Vif-specific CD8(+) T-cell responses failed to control SIV replication. Analysis of 18 animals, consisting of seven unvaccinated noncontrollers and the 11 vaccinees described above, revealed that the sum of Gag- and Vif-specific CD8(+) T-cell frequencies in the acute phase was inversely correlated with plasma viral loads in the chronic phase. Our results suggest that replication of the AIDS virus can be controlled by vaccine-induced subdominant Gag/Vif epitope-specific CD8(+) T cells, providing a rationale for the induction of Gag- and/or Vif-specific CD8(+) T-cell responses by prophylactic AIDS vaccines.

Association of major histocompatibility complex class I haplotypes with disease progression after simian immunodeficiency virus challenge in burmese rhesus macaques.

Nonhuman primate AIDS models are essential for the analysis of AIDS pathogenesis and the evaluation of vaccine efficacy. Multiple studies on human immunodeficiency virus and simian immunodeficiency virus (SIV) infection have indicated the association of major histocompatibility complex class I (MHC-I) genotypes with rapid or slow AIDS progression. The accumulation of macaque groups that share not only a single MHC-I allele but also an MHC-I haplotype consisting of multiple polymorphic MHC-I loci would greatly contribute to the progress of AIDS research. Here, we investigated SIVmac239 infections in four groups of Burmese rhesus macaques sharing individual MHC-I haplotypes, referred to as A, E, B, and J. Out of 20 macaques belonging to A(+) (n = 6), E(+) (n = 6), B(+) (n = 4), and J(+) (n = 4) groups, 18 showed persistent viremia. Fifteen of them developed AIDS in 0.5 to 4 years, with the remaining three at 1 or 2 years under observation. A(+) animals, including two controllers, showed slower disease progression, whereas J(+) animals exhibited rapid progression. E(+) and B(+) animals showed intermediate plasma viral loads and survival periods. Gag-specific CD8(+) T-cell responses were efficiently induced in A(+) animals, while Nef-specific CD8(+) T-cell responses were in A(+), E(+), and B(+) animals. Multiple comparisons among these groups revealed significant differences in survival periods, peripheral CD4(+) T-cell decline, and SIV-specific CD4(+) T-cell polyfunctionality in the chronic phase. This study indicates the association of MHC-I haplotypes with AIDS progression and presents an AIDS model facilitating the analysis of virus-host immune interaction.

Evaluation of chimeric antigen receptor T cell therapy in non-human primates infected with SHIV or SIV.

Achieving a functional cure is an important goal in the development of HIV therapy. Eliciting HIV-specific cellular immune responses has not been sufficient to achieve durable removal of HIV-infected cells due to the restriction on effective immune responses by mutation and establishment of latent reservoirs. Chimeric antigen receptor (CAR) T cells are an avenue to potentially develop more potent redirected cellular responses against infected T cells. We developed and tested a range of HIV- and SIV-specific chimeric antigen receptor (CAR) T cell reagents based on Env-binding proteins. In general, SHIV/SIV CAR T cells showed potent viral suppression in vitro, and adding additional CAR molecules in the same transduction resulted in more potent viral suppression than single CAR transduction. Importantly, the primary determinant of virus suppression potency by CAR was the accessibility to the Env epitope, and not the neutralization potency of the binding moiety. However, upon transduction of autologous T cells followed by infusion in vivo, none of these CAR T cells impacted either acquisition as a test of prevention, or viremia as a test of treatment. Our study illustrates limitations of the CAR T cells as possible antiviral therapeutics.

Polyfunctional CD4+ T-cell induction in neutralizing antibody-triggered control of simian immunodeficiency virus infection.

Rapid depletion of memory CD4(+) T cells and delayed induction of neutralizing antibody (NAb) responses are characteristics of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infections. Although it was speculated that postinfection NAb induction could have only a limited suppressive effect on primary HIV replication, a recent study has shown that a single passive NAb immunization of rhesus macaques 1 week after SIV challenge can result in reduction of viral loads at the set point, indicating a possible contribution of postinfection NAb responses to virus control. However, the mechanism accounting for this NAb-triggered SIV control has remained unclear. Here, we report rapid induction of virus-specific polyfunctional T-cell responses after the passive NAb immunization postinfection. Analysis of SIV Gag-specific responses of gamma interferon, tumor necrosis factor alpha, interleukin-2, macrophage inflammatory protein 1beta, and CD107a revealed that the polyfunctionality of Gag-specific CD4(+) T cells, as defined by the multiplicity of these responses, was markedly elevated in the acute phase in NAb-immunized animals. In the chronic phase, despite the absence of detectable NAbs, virus control was maintained, accompanied by polyfunctional Gag-specific T-cell responses. These results implicate virus-specific polyfunctional CD4(+) T-cell responses in this NAb-triggered virus control, suggesting possible synergism between NAbs and T cells for control of HIV/SIV replication.

Real-Time Killing Assays to Assess the Potency of a New Anti-Simian Immunodeficiency Virus Chimeric Antigen Receptor T Cell.

The success of chimeric antigen receptor (CAR) T cell therapies for treating leukemia has resulted in a booming interest for the technology. Expression of a CAR in T cells allows redirection of their natural cytolytic activity toward cells presenting a specific designated surface antigen. Although CAR T cell therapies have thus far shown promising results mostly in B cell malignancy trials, interest in their potential to treat other diseases is on the rise, including using CAR T cells to control human immunodeficiency virus infection. The assessment of CAR T cell potency toward specific targets in vitro is a critical preclinical step. In this study, we describe novel assays that monitor the cytotoxicity of candidate CAR T cells toward simian immunodeficiency virus (SIV) infected CD4 T cells. The assays involve live cell imaging using a fluorescence microscopy system that records in real time the disappearance or appearance of targets infected with SIV carrying a fluorescent protein gene. The assays are highly reproducible, and their rapid turn around and reduced cost present a significant advance regarding the efficient preclinical evaluation of CAR T cell constructs and are broadly applicable to potential human diseases that could benefit from CAR T cell therapy.

Gag-specific cytotoxic T-lymphocyte-based control of primary simian immunodeficiency virus replication in a vaccine trial.

Gag-specific cytotoxic T lymphocytes (CTLs) exert strong suppressive pressure on human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication. However, it has remained unclear whether they can actually contain primary viral replication. Recent trials of prophylactic vaccines inducing virus-specific T-cell responses have indicated their potential to confer resistance against primary SIV replication in rhesus macaques, while the immunological determinant for this vaccine-based viral control has not been elucidated thus far. Here we present evidence implicating Gag-specific CTLs as responsible for the vaccine-based primary SIV control. Prophylactic vaccination using a Gag-expressing Sendai virus vector resulted in containment of SIVmac239 challenge in all rhesus macaques possessing the major histocompatibility complex (MHC) haplotype 90-120-Ia. In contrast, 90-120-Ia-positive vaccinees failed to contain SIVs carrying multiple gag CTL escape mutations that had been selected, at the cost of viral fitness, in SIVmac239-infected 90-120-Ia-positive macaques. These results show that Gag-specific CTL responses do play a crucial role in the control of wild-type SIVmac239 replication in vaccinees. This study implies the possibility of Gag-specific CTL-based primary HIV containment by prophylactic vaccination, although it also suggests that CTL-based AIDS vaccine efficacy may be abrogated in viral transmission between MHC-matched individuals.

A high throughput lentivirus sieving assay identifies neutralization resistant Envelope sequences and predicts in vivo sieving.

An effective prophylactic vaccine against human immunodeficiency virus (HIV) will likely require a potent antibody response that can neutralize the virus at the mucosal portal of entry. The elicitation of potent broadly-neutralizing anti-sera will be an iterative process, optimizing candidates that only block a fraction of potential viral strains. This effect, termed "sieving", is evidence of a partially efficacious vaccine. Understanding the mechanisms of resistance of the breakthrough viruses is important for improving vaccines. We developed a high-throughput assay that can be used on vaccine-elicited antisera or monoclonal antibodies. Using the SIVsmE660 swarm stock and sera from a large NHP vaccine/challenge study, our in vitro sieving assay identified the same viral subspecies as in the animal study-those with a canonical C1 amino acid variants conferring global neutralization resistance to antibodies. Using a genetically divergent swarm stock, we identified five other amino acid variants that confer global resistance; the C1 mutations in this stock were not selected, also in agreement with in vivo challenge studies. Thus, the in vitro sieving assay can be used with genetically diverse challenge stocks to predict the coverage of a vaccine-elicited sera and possibly inform candidate vaccine development efforts.

Blocking α4ß7 integrin binding to SIV does not improve virologic control.

A study in nonhuman primates reported that infusions of an antibody against α4ß7 integrin, in combination with antiretroviral therapy, showed consistent, durable control of simian immunodeficiency virus (SIV) in rhesus macaques. The antibody used has pleiotropic effects, so we set out to gain insight into the underlying mechanism by comparing this treatment to treatment with non-neutralizing monoclonal antibodies against the SIV envelope glycoprotein that only block α4ß7 binding to SIV Env but have no other host-directed effects. Similar to the initial study, we used an attenuated strain of SIV containing a stop codon in nef. The study used 30 macaques that all began antiretroviral therapy and then were divided into five groups to receive different antibody treatments. Unlike the published report, we found no sustained virologic control by these treatments in vivo.

Immunogenicity of repeated Sendai viral vector vaccination in macaques.

Induction of durable cellular immune responses by vaccination is an important strategy for the control of persistent pathogen infection. Viral vectors are promising vaccine tools for eliciting antigen-specific T-cell responses. Repeated vaccination may contribute to durable memory T-cell induction, but anti-vector antibodies could be an obstacle to its efficacy. We previously developed a Sendai virus (SeV) vector vaccine and showed the potential of this vector for efficient T-cell induction in macaques. Here, we examined whether repeated SeV vector vaccination with short intervals can enhance antigen-specific CD8(+) T-cell responses. Four rhesus macaques possessing the MHC-I haplotype 90-120-Ia were immunized three times with intervals of three weeks. For the vaccination, we used replication-defective F-deleted SeV vectors inducing CD8(+) T-cell responses specific for simian immunodeficiency virus Gag(206-216) and Gag(241-249), which are dominant epitopes restricted by 90-120-Ia-derived MHC-I molecules. All four animals showed higher Gag(206-216)-specific and Gag(241-249)-specific CD8(+) T-cell responses after the third vaccination than those after the first vaccination, indicating enhancement of antigen-specific CD8(+) T-cell responses by the second/third SeV vector vaccination even with short intervals. These results suggest that repeated SeV vector vaccination can contribute to induction of efficient and durable T-cell responses.

Broadening of CD8+ cell responses in vaccine-based simian immunodeficiency virus controllers.

OBJECTIVE: In our prior study on a prophylactic T-cell-based vaccine, some vaccinated macaques controlled a simian immunodeficiency virus (SIV) challenge. These animals allowed viremia in the acute phase but showed persistent viral control after the setpoint. Here, we examined the breadth of postchallenge virus-specific cellular immune responses in these SIV controllers. DESIGN: We previously reported that in a group of Burmese rhesus macaques possessing the MHC haplotype 90-120-Ia, immunization with a Gag-expressing vaccine results in nonsterile control of a challenge with SIVmac239 but not a mutant SIV carrying multiple cytotoxic T lymphocyte (CTL) escape gag mutations. In the present study, we investigated whether broader cellular immune responses effective against the mutant SIV replication are induced after challenge in those vaccinees that maintained wild-type SIVmac239 control. METHODS: We analyzed cellular immune responses in these SIV controllers (n = 8). RESULTS: These controllers elicited CTL responses directed against SIV non-Gag antigens as well as Gag in the chronic phase. Postvaccinated, prechallenge CD8(+) cells obtained from these animals suppressed wild-type SIV replication in vitro, but mostly had no suppressive effect on the mutant SIV replication, whereas CD8(+) cells in the chronic phase after challenge showed efficient antimutant SIV efficacy. The levels of in-vitro antimutant SIV efficacy of CD8(+) cells correlated with Vif-specific CD8(+) T-cell frequencies. Plasma viremia was kept undetectable even after the mutant SIV superchallenge in the chronic phase. CONCLUSION: These results suggest that vaccine-based wild-type SIV controllers can acquire CD8(+) cells with the potential to suppress replication of SIV variants carrying CTL escape mutations.