Role of Viral Reservoirs in the Pathogenesis of HIV Disease

National Institute Of Allergy And Infectious Diseases

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Abstract

HIV remains an uncurable disease despite remarkable advances in the treatment of infection and unparalleled research efforts to develop therapeutic strategies for viral eradication in infected individuals. However, there is a growing sentiment in the field that complete eradication of the virus is highly unlikely with the existing therapeutic options. Therefore, an approach toward immune-mediated virologic control without ART represents a more realistic goal even if the HIV reservoir remains unperturbed. In this regard, the ability of ECs to naturally control HIV replication exemplifies the feasibility of achieving ART-free virologic remission in infected individuals. Prior studies have established that ECs carry relatively low HIV DNA burdens and exhibit robust HIV-specific immune responses. However, there are few studies directly comparing the composition and frequencies of multiple components of HIV reservoirs in ECs to chronically infected individuals receiving ART (CAs). Therefore, we conducted comprehensive analyses of immunologic and virologic parameters in 9 ECs and 22 CAs to address this issue. Using PCR-based assays, we first compared the composition and size of HIV reservoirs in the EC and CA groups. The levels of total HIV DNA (P<0.0001), cell-associated HIV RNA (P<0.0001), intact proviral DNA (P=0.0026), and defective (5 and 3) HIV DNA (P<0.0001) in the CD4+ T cell compartment were significantly lower in the EC group compared to those in the CA group, corroborating previous data. To directly assess the replication competency of infected cells, we measured the levels of inducible virion-associated HIV RNA (ivRNA) and replication-competent virus in CD4+ T cells. Contrary to the above data, there was no significant difference in the levels of ivRNA (P=0.4187) and replication-competent (P=0.9303) HIV between the two groups. Of note, 3 ECs had exceptionally low infectious HIV burdens in their CD4+ T cells. EC-6 and EC-7 had no detectable infectious virus (<1 in 245x106 CD4+ T cells) despite repeated attempts. When HIV-specific immune responses were evaluated, the frequency of polyfunctional (CD107a+IFN-+TNF-+MIP1+) HIV Gag-specific CD8+ T cells were significantly higher in the EC group compared to that of the CA group (P=0.0001), suggesting that ECs may express sufficient viral antigens. Intact DNA burdens were relatively high in EC-4 and EC-6 (75 and 105 copies/106 cells, respectively). Therefore, we conducted longitudinal measurements of ivRNA and intact proviral DNA (EC-4) and near full-length (NFL) sequencing of HIV DNA from a sorted CD4+ T cell subset (EC-6). Despite aviremia, the levels of ivRNA and intact DNA increased over time in EC-4, potentially signaling the eventual loss of virologic control. In EC-6, the vast majority of intact DNA was found in the transitional and effector memory CD4+ T cell subsets. Using NFL single genome amplification, we obtained 88 sequences from the effector memory CD4+ T cells of EC-6. Although all clones had intact HIV env, 90% (Group 1) displayed a gag start codon mutation and a deletion in the pol gene. Additionally, 8% (Group 2) had a deletion at the gag start site and 2% (Group 3) had a large deletion in the gag/pol genes. All three groups had deletions in the major splice donor site and the dimerization initiation sequence. These data demonstrated that the 79 clones (Group 1) identified as intact by the intact proviral DNA assay (IPDA) were replication-defective, further explaining undetectable infectious HIV in EC-6. Collectively, our data suggest that direct measurements of replication-competent HIV and whole genome sequencing need to be performed to accurately assess the composition and extent of viral reservoirs in infected individuals. The recent outbreak of mpox has generated a unique set of challenges for vulnerable populations and the medical community. The majority of reported cases have been in men who have sex with men, including those who are at a particularly increased risk. There are sparse data that pertain to the dynamics of immunologic and HIV virologic changes following mpox infection in HIV-infected individuals. To this end, we evaluated several immunologic and HIV reservoir parameters before and after a mild and self-limited case of mpox in an HIV-infected female whose HIV plasma viremia was suppressed. First, we conducted immunologic analyses of our study participants peripheral blood lymphocytes (B and T cells) and plasma biomarkers before, during, and after her mpox infection. There was a marked decrease (8.7%) in circulating total B cells after mpox, with a concomitant increase in plasmablasts (PBs), followed by normalization of both total B cells and PBs by week 4. Further analysis of PBs revealed a predominantly IgA isotype prior to mpox, consistent with immunoglobulin distribution at steady state in healthy donors and in HIV-infected individuals whose plasma viremia is undetectable. Following mpox, there was a strong and persistent shift to IgG among circulating PBs, consistent with the induction of an immune response to viral antigens. The proportion of T-cell subsets and phenotypic markers of CD4+ and CD8+ T cells also underwent changes during the course of infection. Of interest, there was a dramatic increase in the level of CD38+HLA-DR+ CD8+ T cells following mpox. In addition, levels of plasma biomarkers including granzyme B, perforin, RANTES, CCL3, CXCL10, IL-2Ra, and PD-L1, but not IL-6, increased markedly upon mpox infection. Lastly, we examined the impact of mpox on the size of HIV reservoirs carrying intact proviral DNA in highly enriched CD4+ T cells of the study participant. No significant changes were noted in the level of intact proviral or defective HIV DNA, suggesting that a mild case of mpox infection, while inducing a strong immunologic response, did not lead to any substantial change in the size of the persistent HIV reservoir in this participant.