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Germinal center (GC) CD4+ follicular Th (Tfh) cells are critical for cognate B cell help in humoral immune responses to pathogenic infections. Although Tfh cells are expanded or depleted in HIV/SIV-infected adults, the effects of pediatric HIV/SIV infection on Tfh cells remain unclear. In this study, we examined changes in lymphoid follicle formation in lymph nodes focusing on GC Tfh cells, B cell development, and differentiation in SIV-infected neonatal rhesus macaques (Macaca mulatta) compared with age-matched cohorts. Our data showed that follicles and GCs of normal infants rapidly formed in the first few weeks of age, in parallel with increasing GC Tfh cells in various lymphoid tissues. In contrast, GC development and GC Tfh cells were markedly impaired in SIV-infected infants. There was a very low frequency of GC Tfh cells throughout SIV infection in neonates and subsequent infants, accompanied by high viremia, reduction of B cell proliferation/resting memory B cells, and displayed proinflammatory unresponsiveness. These findings indicate neonatal HIV/SIV infection compromises the development of GC Tfh cells, likely contributing to ineffective Ab responses, high viremia, and eventually rapid disease progression to AIDS.

Material & Method By quantitative real-time/digital PCR, we here longitudinally examined dynamic decay of viral parameters (cell-associated unspliced and multiply spliced viral RNA, episomal 2-LTR circular viral DNA and integrated proviral DNA) in systemic and lymphoid tissues in SIV-infected rhesus macaques on long-term combined antiretroviral therapy (cART, up to 20 months), and evaluated reservoir size and timing of viral rebound after treatment interruption. SIV RNA or DNA copy numbers were normalized and expressed as per 106 cell equivalents. Frequencies and absolute numbers of peripheral CD4+ T cells were analyzed before and after suppressive antiretroviral therapy. Results Our results showed that suppressive ART significantly reduced levels of cell-associated unspliced and multiply spliced SIV RNA/2-LTR DNA to undetectable, yet levels of integrated proviral DNA alone were stably maintained in systemic and lymphoid compartments throughout cART, which correlated with levels of viral replication prior to initiating cART. Following ATI, rapid clonal expansion of cells with integrated provirus were detected, as indicated by undetectable spliced tat/rev RNA and 2-LTR but increased levels of total proviral DNA, which may have contributed to subsequent viral rebound. Cell-associated SIV RNA and DNA levels completely recovered to pre-treatment levels in blood, spleen, lymph node, tonsil and gut-associated lymphoid tissues by 3 months after ATI. Conclusions These findings suggest that discriminating integrated HIV proviral DNA from non-integrated or defective proviruses in tissues are useful biomarkers for monitoring HIV persistence and latency for treatment and cure strategies.

Background The chimeric simian/human immunodeficiency viruses (SHIVs), carrying HIV envelope glycoproteins from transmitted founder (T/F) viruses, are valuable as a challenge viruses in nonhuman primate models for validating HIV-1 vaccines, assessing viral reservoirs and latency, and developing functional “cure” strategies in HIV infection. This study comprehensively investigated immunological changes, viral replication and reservoir size in deep tissues of SHIV-CH848-infected rhesus macaques on antiretroviral therapy (cART). Material & Method A total of 10 rhesus macaques (Macaca mulatta, RMs) were intravenously (i.v.)inoculated with SHIV-CH848, containing vpu-env(gp140) sequence from T/F HIV-1 subtype C strain CH848. At 3 months post SHIV infection, 5 animals received cART for 6 months, and remaining 5 animals as untreated controls. Blood, lymph node and rectal biopsies were collected to examine plasma viral load, cell-associated viral RNA/DNA and host immune responses. Results SHIV-CH848 inoculation resulted in rapid viral replication, reaching peak viremia at 14dpi. SHIV-CH848 primary infection significantly depleted peripheral and mucosal CD4+ T cells. Also, broadly neutralizing antibodies were also not elicited up to 15 months post infection. Despite gradual recovery of CD4+ T cells after cART, the levels of integrated viral DNA in tissues remained stable throughout 6-months of cART, and rapid viral rebound was observed after ART interruption. Conclusions These findings suggest that the new SHIV strain, SHIV-CH848, is able to maintain persistent viral replication in macaques, and establish long-term viral reservoirs/latency in tissues, serving as a valuable model for HIV research.

Cellular viral reservoirs are rapidly established upon HIV/SIV infection, which persist throughout viral infection, even under long-term antiretroviral therapy (ART). Recent studies report α4β7 blockade in vivo could preserve mucosal CD4+ T cells, and control persistent viral infection in SIV-infected macaques. In this study, we investigated the effects of early administration with α4β7 antagonist (antibody against α4β7 or small-molecule dual inhibitor TR-14035) for the prevention of infection, and for reducing cellular reservoirs in systemic and lymphoid tissues post SIV infection. Rhesus macaques (Macaca mulatta, RMs) were intravenously (i.v.) inoculated with 100 TCID50 SIVmac251, then administrated a single i.v. dose of anti-α4β7mAb one week prior to SIV inoculation and another 3 weeks post SIV inoculation, or orally treated with TR-14035 for two weeks beginning on the day of SIV inoculation. Our results indicated that early α4β7 blockade did not result in viral containment or reduction of the reservoir size post SIV infection. Plasma viral loads and cell-associated SIV DNA/RNA levels in blood and lymphoid tissues in treated animals were similar to untreated cohorts. Interestingly, TR-14035, a dual inhibitor for α4β7 and α4β1, resulted in persistently higher viremia throughout infection, and more rapid disease progression when administrated in the acute stage of SIV infection. Conclusions These findings suggest that α4β7 blockade alone may not effectively control viral infection, replication, and dissemination and reservoir establishment in HIV/SIV infection. Its usage for potential preventive or/and therapeutic strategies in combined with ART requires further investigation.

Suppression of dendritic cell (DC) function in HIV-1 infection is thought to contribute to inhibition of immune responses and disease progression, but the mechanism of this suppression remains undetermined. Using the rhesus macaque model, we show B7-H1 (programmed death [PD]-L1) is expressed on lymphoid and mucosal DCs (both myeloid DCs and plasmacytoid DCs), and its expression significantly increases after SIV infection. Meanwhile, its receptor, PD-1, is upregulated on T cells in both peripheral and mucosal tissues and maintained at high levels on SIV-specific CD8+ T cell clones in chronic infection. However, both B7-H1 and PD-1 expression in SIV controllers was similar to that of controls. Expression of B7-H1 on both peripheral myeloid DCs and plasmacytoid DCs positively correlated with levels of PD-1 on circulating CD4+ and CD8+ T cells, viremia, and declining peripheral CD4+ T cell levels in SIV-infected macaques. Importantly, blocking DC B7-H1 interaction with PD-1+ T cells could restore SIV-specific CD4+ and CD8+ T cell function as evidenced by increased cytokine secretion and proliferative capacity. Combined, the results indicate that interaction of B7-H1–PD-1 between APCs and T cells correlates with impairment of CD4+ Th cells and CTL responses in vivo, and all are associated with disease progression in SIV infection. Blockade of this pathway may have therapeutic implications for HIV-infected patients.

Multiple modes of suppressive mechanisms including IL-10 are thought to be implicated in CD4+CD25+ regulatory T (Treg) cell-mediated suppression. However, the cellular source, role, and molecular mechanism of IL-10 in Treg cell biology remain controversial. We now studied the interaction between Treg cells and APCs. We demonstrate that Treg cells, but not conventional T cells, trigger high levels of IL-10 production by APCs, stimulate APC B7-H4 expression, and render APCs immunosuppressive. Initial blockade of B7-H4 reduces the suppressive activity mediated by Treg cell-conditioned APCs. Further, APC-derived, rather than Treg cell-derived, IL-10 is responsible for APC B7-H4 induction. Therefore, Treg cells convey suppressive activity to APCs by stimulating B7-H4 expression through IL-10. Altogether, our data provide a novel cellular and molecular mechanism for Treg cell-mediated immunosuppression at the level of APCs, and suggest a plausible mechanism for the suppressive effect of IL-10 in Treg cell-mediated suppression.