Role of Viral Reservoirs in the Pathogenesis of HIV Disease

艾滋病毒仍然是一种无法治愈的疾病，尽管在治疗感染方面取得了显著进展，并且在制定根除感染者病毒的治疗策略方面做出了无与伦比的研究努力。然而，该领域越来越多的人认为，以现有的治疗方案完全根除该病毒是极不可能的。因此，即使HIV病毒库保持不受干扰，在没有抗逆转录病毒治疗的情况下进行免疫介导的病毒学控制是一个更现实的目标。在这方面，内皮细胞自然控制艾滋病毒复制的能力证明了在受感染个体中实现无art病毒学缓解的可行性。先前的研究已经确定，ec携带相对较低的HIV DNA负荷，并表现出强大的HIV特异性免疫反应。然而，很少有研究直接比较ECs和接受抗逆转录病毒治疗（CAs）的慢性感染者中HIV病毒库多种成分的组成和频率。因此，我们对9例ECs和22例CAs的免疫学和病毒学参数进行了综合分析，以解决这一问题。使用基于pcr的检测，我们首先比较了EC组和CA组中HIV储存库的组成和大小。与CA组相比，EC组CD4+ T细胞区室中总HIV DNA （P<0.0001）、细胞相关HIV RNA （P<0.0001）、完整前病毒DNA （P=0.0026）和缺陷（5和3）HIV DNA （P<0.0001）水平显著降低，证实了先前的数据。为了直接评估感染细胞的复制能力，我们测量了CD4+ T细胞中诱导病毒相关的HIV RNA （ivRNA）和复制能力病毒的水平。与上述数据相反，两组之间的ivRNA （P=0.4187）和复制能力HIV （P=0.9303）水平无显著差异。值得注意的是，3例ECs CD4+ T细胞的传染性HIV负荷异常低。尽管多次尝试，EC-6和EC-7未检测到感染性病毒（245x106 CD4+ T细胞<1）。当评估HIV特异性免疫应答时，EC组中多功能（CD107a+IFN-+TNF-+MIP1+） HIV gag特异性CD8+ T细胞的频率显著高于CA组（P=0.0001），提示EC可能表达了足够的病毒抗原。完整DNA负荷在EC-4和EC-6中相对较高（分别为75和105拷贝/106个细胞）。因此，我们对CD4+ T细胞亚群（EC-6）的ivRNA和完整的前病毒DNA （EC-4）进行了纵向测量，并对HIV DNA进行了近全长（NFL）测序。尽管病毒血症，但EC-4中ivRNA和完整DNA的水平随着时间的推移而增加，这可能表明最终失去病毒学控制。在EC-6中，绝大多数完整的DNA存在于移行性和效应记忆CD4+ T细胞亚群中。利用NFL单基因组扩增，我们从EC-6的效应记忆CD4+ T细胞中获得88个序列。尽管所有克隆都具有完整的HIV环境，但90%（组1）显示gag启动密码子突变和pol基因缺失。此外，8%（第2组）在gag起始位点有缺失，2%（第3组）在gag/pol基因上有大量缺失。所有三组在主要剪接供体位点和二聚化起始序列上都有缺失。这些数据表明，通过完整原病毒DNA测定（IPDA）鉴定为完整的79个克隆（第1组）存在复制缺陷，进一步解释了EC-6中检测不到感染性HIV的原因。总的来说，我们的数据表明，需要进行复制能力HIV的直接测量和全基因组测序，以准确评估感染个体中病毒库的组成和范围。

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.