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着进展，并作出了无与伦比的研究努力，以制定在受感染个人中根除病毒的治疗战略。然而，该领域越来越多的人认为，用现有的治疗方案完全根除病毒是极不可能的。因此，一种不需要抗逆转录病毒治疗的免疫介导病毒学控制的方法代表了一个更现实的目标，即使艾滋病毒储存库保持不受干扰。在这方面，ECs自然控制艾滋病毒复制的能力证明了在感染者中实现无ART病毒学缓解的可行性。先前的研究已经证实，内皮细胞携带相对较低的艾滋病毒DNA负担，并表现出强大的艾滋病毒特异性免疫反应。然而，很少有研究直接比较内皮细胞和接受抗逆转录病毒治疗(CA)的慢性感染者体内艾滋病毒储备库的多种成分的组成和频率。因此，我们对9个EC和22个CA的免疫学和病毒学参数进行了全面的分析，以解决这个问题。利用基于聚合酶链式反应的分析方法，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CD_4+T细胞中HIVDNA总量(P&lt；0.0001)、细胞相关HIVRNA(P&lt；0.0001)、完整前病毒DNA(P=0.0026)和缺陷(5和3)HIVDNA(P&lt；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细胞中可诱导的病毒相关HIV RNA(IvRNA)和复制能力病毒的水平。与上述数据相反的是，两组之间的ivRNA(P=0.4187)和复制能力(P=0.9303)HIV水平没有显著差异。值得注意的是，3个EC的CD4+T细胞中的HIV感染性负荷非常低。经反复尝试，EC-6和EC-7均未检测到传染性病毒(245×106个CD4+T细胞中有1个)。在评估HIV特异性免疫应答时，EC组多功能(CD107a+干扰素+肿瘤坏死因子-+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细胞可能表达足够的病毒抗原。EC-4和EC-6的完整DNA负载量相对较高(分别为75和105拷贝/106细胞)。因此，我们对IVRNA和完整的前病毒DNA(EC-4)进行了纵向测量，并对分选的CD4+T细胞亚群(EC-6)的HIV DNA进行了近全长(NFL)测序。尽管有禽流感，但EC-4的ivRNA和完整DNA水平随着时间的推移而增加，这可能预示着最终失去对病毒学的控制。在EC-6中，绝大多数完整的DNA存在于过渡性记忆和效应记忆的CD4+T细胞亚群中。通过NFL单基因组扩增，我们从EC-6的效应记忆CD4+T细胞中获得了88个序列。虽然所有克隆都具有完整的HIV包膜，但90%(第1组)的克隆显示GAG起始密码子突变和pol1基因缺失。此外，8%(第2组)的GAG基因起始点有缺失，2%(第3组)的GAG/Pol1基因有较大的缺失。所有三个组都在主要剪接供体位置和二聚化起始序列上存在缺失。这些数据表明，经完整前病毒DNA分析(IPDA)鉴定为完整的79个克隆(第1组)是复制缺陷的，进一步解释了EC-6中未检测到的传染性艾滋病毒。总而言之，我们的数据表明，需要对复制能力强的艾滋病毒和全基因组测序进行直接测量，以准确评估感染个体的病毒库的组成和程度。 最近爆发的口水痘给弱势群体和医学界带来了一系列独特的挑战。报告的大多数病例发生在与男性发生性行为的男性身上，包括那些风险特别高的人。关于HIV感染者感染MPox后免疫学和HIV病毒学变化的动态数据很少。为此，我们评估了一例HIV感染女性在轻度自限性mpox前后的几个免疫学和HIV储存库参数，该女性的HIV血浆病毒血症被抑制。首先，我们对研究参与者在感染羊痘之前、期间和之后的外周血淋巴细胞(B和T细胞)和血浆生物标记物进行了免疫学分析。MPOX后循环中总B细胞显著减少(8.7%)，同时浆母细胞(PBS)增加，随后B细胞总数和PBS在第4周恢复正常。进一步分析PBS发现MPOX前以IgA亚型为主，这与健康献血者和血浆病毒血症检测不到的HIV感染者的免疫球蛋白稳态分布一致。在mpox之后，循环中的PBS发生了强烈而持久的向免疫球蛋白的转变，这与诱导对病毒抗原的免疫反应是一致的。在感染过程中，T细胞亚群比例和CD4+、CD8+T细胞表型标志也发生了变化。有趣的是，在MPOX后，CD38+HLA-DR+CD8+T细胞水平显著增加。此外，血浆生物标志物包括颗粒酶B、穿孔素、RANTES、CCL3、CXCL10、IL-2ra和PD-L1的水平在感染MPOX时显著增加，但不包括IL-6。最后，我们检查了MPOX对在研究参与者的高度浓缩的CD4+T细胞中携带完整的前病毒DNA的HIV储存库的大小的影响。在完整的前病毒或缺陷的艾滋病毒DNA水平上没有明显的变化，这表明一个轻微的mpox感染病例，虽然诱导了强烈的免疫反应，但不会导致该参与者持久的艾滋病毒储存库的大小发生任何实质性的变化。

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.