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Allogeneic transplantation (allo-HCT) has led to the cure of HIV in one individual, raising the question of whether transplantation can eradicate the HIV reservoir. To test this, we here present a model of allo-HCT in SHIV-infected, cART-suppressed nonhuman primates. We infect rhesus macaques with SHIV-1157ipd3N4, suppress them with cART, then transplant them using MHC-haploidentical allogeneic donors during continuous cART. Transplant results in ~100% myeloid donor chimerism, and up to 100% T-cell chimerism. Between 9 and 47 days post-transplant, terminal analysis shows that while cell-associated SHIV DNA levels are reduced in the blood and in lymphoid organs post-transplant, the SHIV reservoir persists in multiple organs, including the brain. Sorting of donor-vs.-recipient cells reveals that this reservoir resides in recipient cells. Moreover, tetramer analysis indicates a lack of virus-specific donor immunity post-transplant during continuous cART. These results suggest that early post-transplant, allo-HCT is insufficient for recipient reservoir eradication despite high-level donor chimerism and GVHD.

We previously showed that envelope (gp160)-based vaccines, used in a live recombinant virus priming and subunit protein boosting regimen, protected macaques against intravenous and intrarectal challenges with the homologous simian immunodeficiency virus SIVmne clone E11S. However, the breadth of protection appears to be limited, since the vaccines were only partially effective against intravenous challenge by the uncloned SIVmne. To examine factors that could affect the breadth and the efficacy of this immunization approach, we studied (i) the effect of priming by recombinant vaccinia virus; (ii) the role of surface antigen gp130; and (iii) the role of core antigens (Gag and Pol) in eliciting protective immunity. Results indicate that (i) priming with recombinant vaccinia virus was more effective than subunit antigen in eliciting protective responses; (ii) while both gp130 and gp160 elicited similar levels of SIV-specific antibodies, gp130 was not as effective as gp160 in protection, indicating a possible role for the transmembrane protein in presenting functionally important epitopes; and (iii) although animals immunized with core antigens failed to generate any neutralizing antibody and were infected upon challenge, their virus load was 50- to 100-fold lower than that of the controls, suggesting the importance of cellular immunity or other core-specific immune responses in controlling acute infection. Complete protection against intravenous infection by the pathogenic uncloned SIVmne was achieved by immunization with both the envelope and the core antigens. These results indicate that immune responses to both antigens may contribute to protection and thus argue for the inclusion of multiple antigens in recombinant vaccine designs.

We explored the relationship between T cell activation signals and dendritic cells (DC) in the replication cycle of immunodeficiency viruses. First we analyzed the effect of two cell cycle inhibitors (mimosine and aphidicolin) on SIV reverse transcription, circularization, and integration in macaque resting T cells stimulated with anti-CD3 mAb at the time of infection. The formation of SIV LTR circles was blocked by the G1 inhibitor mimosine. The G1/S inhibitor aphidicolin neither affected circularization nor integration of SIV DNA. Therefore, the induction of SIV LTR circle production is likely to be mediated by signaling events normally regulating the G1 to S transition. We further characterized DC-dependent HIV-expression in human T cells. We examined the effect of ligating two novel receptors, IPO-3 and Bgp95, on DC-dependent HIV-1 expression. Activation of DCs through IPO-3 receptors, and to a lesser extent Bgp95 ligation, upregulated HIV spread in these cells. The mechanisms by which IPO-3 vs. Bgp95 increase HIV-1 levels appear to be different. In particular, IPO-3 ligation alone on T cells also increased HIV-1 levels. Activation of T cells via defined surface receptors or with DCs is required for establishing HIV/SIV cDNA synthesis in T cells.

Although immunodeficiency viruses can enter resting CD4+ T lymphocytes, activation of T cells is required for complete viral cDNA synthesis and transport of double-stranded viral DNA to the nucleus. Cross-linking T cell receptors (TCRs) on resting CD4+ T cells induces reverse transcription of full-length simian immunodeficiency virus (SIV) genomes, but TCR engagement alone is insufficient to stimulate SIV DNA to move to the nucleus and form long terminal repeat (LTR) circles. Neither ligation of TCR or CD28 receptors nor interleukin 2 (IL-2) alone induces formation of LTR circles; however, the combination of TCR ligation with either CD28 ligation or IL-2 doses. Anti-IL-2 serum inhibits the formation of LTR circles induced by cross-linking CD3 and CD28, but has no effect on the induction of increased viral reverse transcription. Thus, two signals appear to be required for immunodeficiency viruses to move to the T cell nucleus, one from the TCR to promote reverse transcription of the viral genome, the other through an IL-2-dependent process leading to formation of LTR circles.

The relationship between T-cell activation and early events in the replication cycle of simian immunodeficiency virus (SIV) was analyzed in resting T lymphocytes from macaques. We used the polymerase chain reaction to detect an early product of reverse transcription (R/U5) and almost complete viral DNA (long terminal repeat/gag). We found that SIV can enter resting T lymphocytes and initiate replication but that the reverse transcription process is not efficient and proceeds slowly in resting cells. Cross-linking the CD3/T-cell receptor complex with monoclonal antibodies, unlike cross-linking either the CD28 or CD2 accessory receptor and like phorbol myristate acetate, induced a rapid increase in viral R/U5 DNA detected within 3 to 6 h postinfection. Anti-CD3 or phorbol myristate acetate induced replication of full-length viral DNA within 6 to 9 h postinfection, but full-length SIV DNA was not detectable at earlier time points. We then compared various inhibitors of T-cell activation for their effects on viral initiation and complete replication. Cyclosporin A, an inhibitor of a distal step in T-cell activation, blocked anti-CD3-induced T-cell proliferation and completion of SIV DNA replication but had no effect on induced increases in SIV R/U5 DNA. By contrast, initial SIV DNA synthesis was partially blocked by inhibitors of very early steps in T-cell activation, including herbimycin A, an inhibitor of protein tyrosine kinases, and by two different inhibitors of protein kinase C, H7 and staurosporine. Since resting T cells do not efficiently complete SIV DNA synthesis and cyclosporin A can block the formation of complete viral DNA induced in activated T cells, a cellular factor(s) present in activated T cells appears to be required for the formation of full-length SIV DNA.