Nicholas J. Maness

Vaccine-Induced Cellular Immune Responses Reduce Plasma Viral Concentrations after Repeated Low-Dose Challenge with Pathogenic Simian Immunodeficiency Virus SIVmac239

ABSTRACT The goal of an AIDS vaccine regimen designed to induce cellular immune responses should be to reduce the viral set point and preserve memory CD4 lymphocytes. Here we investigated whether vaccine-induced cellular immunity in the absence of any Env-specific antibodies can control viral replication following multiple low-dose challenges with the highly pathogenic SIVmac239 isolate. Eight Mamu-A*01-positive Indian rhesus macaques were vaccinated with simian immunodeficiency virus (SIV) gag, tat, rev, and nef using a DNA prime-adenovirus boost strategy. Peak viremia (P = 0.007) and the chronic phase set point (P = 0.0192) were significantly decreased in the vaccinated cohort, out to 1 year postinfection. Loss of CD4+ memory populations was also ameliorated in vaccinated animals. Interestingly, only one of the eight vaccinees developed Env-specific neutralizing antibodies after infection. The control observed was significantly improved over that observed in animals vaccinated with SIV gag only. Vaccine-induced cellular immune responses can, therefore, exert a measure of control over replication of the AIDS virus in the complete absence of neutralizing antibody and give us hope that a vaccine designed to induce cellular immune responses might control viral replication.

The High-Frequency Major Histocompatibility Complex Class I Allele Mamu-B*17 Is Associated with Control of Simian Immunodeficiency Virus SIVmac239 Replication

ABSTRACT Particular HLA alleles are associated with reduced human immunodeficiency virus replication. It has been difficult, however, to characterize the immune correlates of viral control. An analysis of the influence of major histocompatibility complex class I alleles on viral control in 181 simian immunodeficiency virus SIVmac239-infected rhesus macaques revealed that Mamu-B*17 was associated with a 26-fold reduction in plasma virus concentrations (P < 0.001). Mamu-B*17 was also enriched 1,000-fold in a group of animals that controlled viral replication. Even after accounting for this group, Mamu-B*17 was associated with an eightfold reduction in plasma virus concentrations (P < 0.001). Mamu-B*17-positive macaques could, therefore, facilitate our understanding of the correlates of viral control.

Vaccine-Induced Cellular Responses Control Simian Immunodeficiency Virus Replication after Heterologous Challenge

ABSTRACT All human immunodeficiency virus (HIV) vaccine efficacy trials to date have ended in failure. Structural features of the Env glycoprotein and its enormous variability have frustrated efforts to induce broadly reactive neutralizing antibodies. To explore the extent to which vaccine-induced cellular immune responses, in the absence of neutralizing antibodies, can control replication of a heterologous, mucosal viral challenge, we vaccinated eight macaques with a DNA/Ad5 regimen expressing all of the proteins of SIVmac239 except Env. Vaccinees mounted high-frequency T-cell responses against 11 to 34 epitopes. We challenged the vaccinees and eight naïve animals with the heterologous biological isolate SIVsmE660, using a regimen intended to mimic typical HIV exposures resulting in infection. Viral loads in the vaccinees were significantly less at both the peak (1.9-log reduction; P < 0.03) and at the set point (2.6-log reduction; P < 0.006) than those in control naïve animals. Five of eight vaccinated macaques controlled acute peak viral replication to less than 80,000 viral RNA (vRNA) copy eq/ml and to less than 100 vRNA copy eq/ml in the chronic phase. Our results demonstrate that broad vaccine-induced cellular immune responses can effectively control replication of a pathogenic, heterologous AIDS virus, suggesting that T-cell-based vaccines may have greater potential than previously appreciated.

Gag- and Nef-specific CD4+ T cells recognize and inhibit SIV replication in infected macrophages early after infection

The precise immunological role played by CD4+ T cells in retroviral infections is poorly defined. Here, we describe a new function of these cells, the elimination of retrovirus-infected macrophages. After experimental CD8+ cell depletion, elite controlling macaques with set-point viral loads ≤500 viral RNA copies/mL mounted robust Gag- and Nef-specific CD4+ T cell responses during reestablishment of control with ≥54% of all virus-specific CD4+ T cells targeting these 2 proteins. Ex vivo, these simian immunodeficiency virus (SIV)-specific CD4+ T cells neither recognized nor suppressed viral replication in SIV-infected CD4+ T cells. In contrast, they recognized SIV-infected macrophages as early as 2 h postinfection because of presentation of epitopes derived from virion-associated Gag and Nef proteins. Furthermore, virus-specific CD4+ T cells displayed direct effector function and eliminated SIV-infected macrophages. These results suggest that retrovirus-specific CD4+ T cells may contribute directly to elite control by inhibiting viral replication in macrophages.

From biomedicine to natural history research: EST resources for ambystomatid salamanders

BackgroundEstablishing genomic resources for closely related species will provide comparative insights that are crucial for understanding diversity and variability at multiple levels of biological organization. We developed ESTs for Mexican axolotl (Ambystoma mexicanum) and Eastern tiger salamander (A. tigrinum tigrinum), species with deep and diverse research histories.ResultsApproximately 40,000 quality cDNA sequences were isolated for these species from various tissues, including regenerating limb and tail. These sequences and an existing set of 16,030 cDNA sequences for A. mexicanum were processed to yield 35,413 and 20,599 high quality ESTs for A. mexicanum and A. t. tigrinum, respectively. Because the A. t. tigrinum ESTs were obtained primarily from a normalized library, an approximately equal number of contigs were obtained for each species, with 21,091 unique contigs identified overall. The 10,592 contigs that showed significant similarity to sequences from the human RefSeq database reflected a diverse array of molecular functions and biological processes, with many corresponding to genes expressed during spinal cord injury in rat and fin regeneration in zebrafish. To demonstrate the utility of these EST resources, we searched databases to identify probes for regeneration research, characterized intra- and interspecific nucleotide polymorphism, saturated a human – Ambystoma synteny group with marker loci, and extended PCR primer sets designed for A. mexicanum / A. t. tigrinum orthologues to a related tiger salamander species.ConclusionsOur study highlights the value of developing resources in traditional model systems where the likelihood of information transfer to multiple, closely related taxa is high, thus simultaneously enabling both laboratory and natural history research.

Repeated Intravaginal Inoculation with Cell-Associated Simian Immunodeficiency Virus Results in Persistent Infection of Nonhuman Primates

The goal of the present study was to develop a nonhuman primate model of intravaginal human immunodeficiency virus (HIV) transmission with cell-associated virus. Reproductively mature, cycling cynomolgus macaques with or without chemically induced, transient ulcers of the lower female reproductive tract repeatedly received challenge with a variable amount of in vitro simian immunodeficiency virus mac239-infected peripheral blood mononuclear cells. Persistent viremia was established with surprisingly few infectious lymphocytes containing physiologically relevant quantities of cell-associated virus. This model will be indispensable for the testing of vaccines and topical agents that are aimed toward the prevention of heterosexual transmission of HIV.

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.

Comprehensive Immunological Evaluation Reveals Surprisingly Few Differences between Elite Controller and Progressor Mamu-B*17-Positive Simian Immunodeficiency Virus-Infected Rhesus Macaques

ABSTRACT The association between particular major histocompatibility complex class I (MHC-I) alleles and control of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication implies that certain CD8+ T-lymphocyte (CD8-TL) responses are better able than others to control viral replication in vivo. However, possession of favorable alleles does not guarantee improved prognosis or viral control. In rhesus macaques, the MHC-I allele Mamu-B*17 is correlated with reduced viremia and is overrepresented in macaques that control SIVmac239, termed elite controllers (ECs). However, there is so far no mechanistic explanation for this phenomenon. Here we show that the chronic-phase Mamu-B*17-restricted repertoire is focused primarily against just five epitopes—VifHW8, EnvFW9, NefIW9, NefMW9, and envARFcRW9—in both ECs and progressors. Interestingly, Mamu-B*17-restricted CD8-TL do not target epitopes in Gag. CD8-TL escape variation occurred in all targeted Mamu-B*17-restricted epitopes. However, recognition of escape variant peptides was commonly observed in both ECs and progressors. Wild-type sequences in the VifHW8 epitope tended to be conserved in ECs, but there was no evidence that this enhances viral control. In fact, no consistent differences were detected between ECs and progressors in any measured parameter. Our data suggest that the narrowly focused Mamu-B*17-restricted repertoire suppresses virus replication and drives viral evolution. It is, however, insufficient in the majority of individuals that express the “protective” Mamu-B*17 molecule. Most importantly, our data indicate that the important differences between Mamu-B*17-positive ECs and progressors are not readily discernible using standard assays to measure immune responses.

CD8+ T cell recognition of cryptic epitopes is a ubiquitous feature of AIDS virus infection.

ABSTRACT Vaccines designed to elicit AIDS virus-specific CD8+ T cells should engender broad responses. Emerging data indicate that alternate reading frames (ARFs) of both human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) encode CD8+ T cell epitopes, termed cryptic epitopes. Here, we show that SIV-specific CD8+ T cells from SIV-infected rhesus macaques target 14 epitopes in eight ARFs during SIV infection. Animals recognized up to five epitopes, totaling nearly one-quarter of the anti-SIV responses. The epitopes were targeted by high-frequency responses as early as 2 weeks postinfection and in the chronic phase. Hence, previously overlooked ARF-encoded epitopes could be important components of AIDS vaccines.

CD8+ and CD4+ cytotoxic T cell escape mutations precede breakthrough SIVmac239 viremia in an elite controller

BackgroundVirus-specific T cells are critical components in the containment of immunodeficiency virus infections. While the protective role of CD8+ T cells is well established by studies of CD8+ T cell-mediated viral escape, it remains unknown if CD4+ T cells can also impose sufficient selective pressure on replicating virus to drive the emergence of high-frequency escape variants. Identifying a high frequency CD4+ T cell driven escape mutation would provide compelling evidence of direct immunological pressure mediated by these cells.ResultsHere, we studied a SIVmac239-infected elite controller rhesus macaque with a 1,000-fold spontaneous increase in plasma viral load that preceded disease progression and death from AIDS-related complications. We sequenced the viral genome pre- and post-breakthrough and demonstrate that CD8+ T cells drove the majority of the amino acid substitutions outside of Env. However, within a region of Gag p27CA targeted only by CD4+ T cells, we identified a unique post-breakthrough mutation, Gag D205E, which abrogated CD4+ T cell recognition. Further, we demonstrate that the Gag p27CA-specific CD4+ T cells exhibited cytolytic activity and that SIV bearing the Gag D205E mutation escapes this CD4+ T cell effector function ex vivo.ConclusionsCumulatively, these results confirm the importance of virus specific CD8+ T cells and demonstrate that CD4+ T cells can also exert significant selective pressure on immunodeficiency viruses in vivo during low-level viral replication. These results also suggest that further studies of CD4+ T cell escape should focus on cases of elite control with spontaneous viral breakthrough.