No viral evolution in the lymph nodes of simian immunodeficiency virus-infected rhesus macaques during combined antiretroviral therapy.

To elucidate the mode of viral persistence in primate lentivirus-infected individuals during combination antiretroviral therapy (cART), four simian immunodeficiency virus 239-infected monkeys were treated with cART for 1 year. The viral env genes prepared from total RNA extracted from the mesenteric lymph nodes collected at the completion of therapy were assessed by single genome amplification. Analyses of nucleotide substitutions and phylogeny revealed no viral evolution during cART.

Lymph nodes harbor viral reservoirs that cause rebound of plasma viremia in SIV-infected macaques upon cessation of combined antiretroviral therapy.

Attempts to find a cure for HIV infection are hindered by the presence of viral reservoirs that resist highly active antiretroviral therapy. To identify the properties of these reservoirs, four SIV239-infected Rhesus macaques were treated with combined antiretroviral therapy (cART) for 1 year. While plasma viral RNA (vRNA) was effectively suppressed, a systemic analysis revealed that vRNA was distributed in the following order: lymphatic tissues>lungs and intestine>other tissues. Histochemistry yielded no cells with viral signals. To increase the chance of detection, two additional SIV-infected animals were treated and analyzed on Day 10 after the cessation of cART. These animals exhibited similar vRNA distribution patterns to the former animals, and immunohistochemistry revealed Nef-positive T lymphocytes predominantly in the follicles of mesenteric lymph nodes (MLNs). These data suggest that lymphatic tissues, including MLNs, contain major cellular reservoirs that cause rebound of plasma viremia upon cessation of therapy.

Major histocompatibility complex class I-restricted cytotoxic T lymphocyte responses during primary simian immunodeficiency virus infection in Burmese rhesus macaques.

Major histocompatibility complex class I (MHC-I)-restricted CD8(+) cytotoxic T lymphocyte (CTL) responses are crucial for the control of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication. In particular, Gag-specific CTL responses have been shown to exert strong suppressive pressure on HIV/SIV replication. Additionally, association of Vif-specific CTL frequencies with in vitro anti-SIV efficacy has been suggested recently. Host MHC-I genotypes could affect the immunodominance patterns of these potent CTL responses. Here, Gag- and Vif-specific CTL responses during primary SIVmac239 infection were examined in three groups of Burmese rhesus macaques, each group having a different MHC-I haplotype. The first group of four macaques, which possessed the MHC-I haplotype 90-010-Ie, did not show Gag- or Vif-specific CTL responses. However, Nef-specific CTL responses were elicited, suggesting that primary SIV infection does not induce predominant CTL responses specific for Gag/Vif epitopes restricted by 90-010-Ie-derived MHC-I molecules. In contrast, Gag- and Vif-specific CTL responses were induced in the second group of two 89-075-Iw-positive animals and the third group of two 91-010-Is-positive animals. Considering the potential of prophylactic vaccination to affect CTL immunodominance post-viral exposure, these groups of macaques would be useful for evaluation of vaccine antigen-specific CTL efficacy against SIV infection.

Cutting edge: T cells monitor N-myristoylation of the Nef protein in simian immunodeficiency virus-infected monkeys.

The use of the host cellular machinery is essential for pathogenic viruses to replicate in host cells. HIV and SIV borrow the host-derived N-myristoyl-transferase and its substrate, myristoyl-CoA, for coupling a saturated C(14) fatty acid (myristic acid) to the N-terminal glycine residue of the Nef protein. This biochemical reaction, referred to as N-myristoylation, assists its targeting to the plasma membrane, thereby supporting the immunosuppressive activity proposed for the Nef protein. In this study, we show that the host immunity is equipped with CTLs capable of sensing N-myristoylation of the Nef protein. A rhesus macaque CD8(+) T cell line was established that specifically recognized N-myristoylated, but not unmodified, peptides of the Nef protein. Furthermore, the population size of N-myristoylated Nef peptide-specific T cells was found to increase significantly in the circulation of SIV-infected monkeys. Thus, these results identify N-myristoylated viral peptides as a novel class of CTL target Ag.

Dominant induction of vaccine antigen-specific cytotoxic T lymphocyte responses after simian immunodeficiency virus challenge.

Cytotoxic T lymphocyte (CTL) responses are crucial for the control of human and simian immunodeficiency virus (HIV and SIV) replication. A promising AIDS vaccine strategy is to induce CTL memory resulting in more effective CTL responses post-viral exposure compared to those in natural HIV infections. We previously developed a CTL-inducing vaccine and showed SIV control in some vaccinated rhesus macaques. These vaccine-based SIV controllers elicited vaccine antigen-specific CTL responses dominantly in the acute phase post-challenge. Here, we examined CTL responses post-challenge in those vaccinated animals that failed to control SIV replication. Unvaccinated rhesus macaques possessing the major histocompatibility complex class I haplotype 90-088-Ij dominantly elicited SIV non-Gag antigen-specific CTL responses after SIV challenge, while those induced with Gag-specific CTL memory by prophylactic vaccination failed to control SIV replication with dominant Gag-specific CTL responses in the acute phase, indicating dominant induction of vaccine antigen-specific CTL responses post-challenge even in non-controllers. Further analysis suggested that prophylactic vaccination results in dominant induction of vaccine antigen-specific CTL responses post-viral exposure but delays SIV non-vaccine antigen-specific CTL responses. These results imply a significant influence of prophylactic vaccination on CTL immunodominance post-viral exposure, providing insights into antigen design in development of a CTL-inducing AIDS vaccine.

Evaluation of the immune response and protective effects of rhesus macaques vaccinated with biodegradable nanoparticles carrying gp120 of human immunodeficiency virus.

We previously reported that biodegradable amphiphilic poly(gamma-glutamic acid) nanoparticles (NPs) carrying the recombinant gp120 env protein of the human immunodeficiency virus type 1 (HIV-1) were efficiently taken up by dendritic cells, and induced strong CD8(+) T cell responses against the gp120 in mice. To evaluate gp120-carrying NPs (gp120-NPs) as a vaccine candidate for HIV-1 infection, we vaccinated rhesus macaques with these gp120-NPs and examined the immune response and protective efficacy against a challenge inoculation of simian and human immunodeficiency chimeric virus (SHIV). We found that gp120-NP vaccination induced stronger responses for both gp120-specific cellular and humoral immunity than gp120-alone vaccination. After the challenge inoculation with SHIV, however, the peak value of viral RNA in the peripheral blood was higher in the vaccinated groups, especially the gp120-NP vaccinated group, than naive control group. Higher value of viral load was also maintained in gp120-NP vaccinated group. Furthermore, CD4(+) T cells from the peripheral blood decreased more in the vaccinated groups than the control group. Thus, induced immune responses against gp120 enclosed in NPs were not effective for protection but, conversely enhanced the infection, although the gp120-NPs showed a stronger induction of immune responses against the vaccinated antigen in rhesus macaques. These results support the importance of determining immune correlate of protective immunity for vaccine development against HIV-1 infection.

In vivo analysis of a new R5 tropic SHIV generated from the highly pathogenic SHIV-KS661, a derivative of SHIV-89.6.

Although X4 tropic SHIVs have been studied extensively, they show distinct infection phenotypes from those of R5 tropic viruses, which play an important role in HIV-1 transmission and pathogenesis. To augment the variety of R5 tropic SHIVs, we generated a new R5 tropic SHIV from the highly pathogenic X4 tropic SHIV-KS661, a derivative of SHIV-89.6. Based on consensus amino acid alignment analyses of subtype B R5 tropic HIV-1, five amino acid substitutions in the third variable region successfully changed the secondary receptor preference from X4 to R5. Improvements in viral replication were observed in infected rhesus macaques after two passages, and reisolated virus was designated SHIV-MK38. SHIV-MK38 maintained R5 tropism through in vivo passages and showed robust replication in infected monkeys. Our study clearly demonstrates that a minimal number of amino acid substitutions in the V3 region can alter secondary receptor preference and increase the variety of R5 tropic SHIVs.