TZA, a Sensitive Reporter Cell-based Assay to Accurately and Rapidly Quantify Inducible, Replication-competent Latent HIV-1 from Resting CD4+ T Cells.

The latent HIV-1 viral reservoir in resting CD4+ (rCD4+) T cells represents a major barrier to an HIV-1 cure. There is an ongoing effort to identify therapeutic approaches that will eliminate or reduce the size of this reservoir. However, clinical investigators lack an assay to determine whether or not a decrease in the latent reservoir has been achieved. Therefore, it is critical to develop assays that can reproducibly quantify the reservoir size and changes therein, in participant's blood during a therapeutic trial. Quantification of the latent HIV viral reservoir requires a highly sensitive, cost-effective assay capable of measuring the low frequency of rCD4+ T cells carrying functional provirus. Preferably, such an assay should be such that it can be adopted for high throughput and could be adopted under conditions for use in large-scale clinical trials. While PCR-based assays are commonly used to quantify pro-viral DNA or intracellular RNA transcript, they cannot distinguish between replication-competent and defective proviruses. We have recently published a study where a reporter cell-based assay (termed TZA or TZM-bl based quantitative assay) was used to quantify inducible replication-competent latent HIV-1 in blood. This assay is more sensitive, cost-efficient, and faster than available technology, including the quantitative viral outgrowth assay or the Q-VOA. Using this assay, we show that the size of the inducible latent HIV-1 reservoir in virally suppressed participants on ART is approximately 70-fold larger than previous estimates. We describe here in detail an optimized method to quantitate latently infected cells using the TZA.

Type 1-programmed dendritic cells drive antigen-specific latency reversal and immune elimination of persistent HIV-1.

BACKGROUND: Despite the success of antiretroviral therapy (ART), latent HIV-1 continues to persist in a long-lived population of resting memory CD4+ T cells within those who are infected. Finding a safe and effective means to induce latency reversal (LR) during ART to specifically expose this latent HIV-1 cellular reservoir for immune elimination has been a major barrier to a functional cure. METHODS: In this study, we test the use of antigen-presenting type 1-polarized, monocyte-derived dendritic cells (MDC1) generated from chronic HIV-1-infected individuals on ART as a means to induce HIV-1 latency reversal in autologous CD4+ T cells harboring replication-competent provirus. We use the same MDC1 for ex-vivo generation of autologous HIV-1 antigen-specific CD8+ cytotoxic T cells (CTL) and test their effector responses against the MDC1-exposed HIV-1- infected CD4+ T cell targets. FINDINGS: MDC1 presentation of either HIV-1 or cytomegalovirus (CMV) antigens to CD4+ T cells facilitated HIV-1 LR. This antigen-driven MDC1-mediated LR was sharply diminished with blockade of the CD40L/CD40 'helper' signaling pathway. Importantly, these antigen-presenting MDC1 also activated the expansion of CTL capable of killing the exposed HIV-1-infected targets. INTERPRETATION: Inclusion of virus-associated MHC class II 'helper' antigens in MDC1-based HIV-1 immunotherapies could serve both as a targeted means to safely unmask antigen-specific CD4+ T cells harboring HIV-1, and to support CTL responses that can effectively target the MDC1-exposed HIV-1 cellular reservoir as a functional cure strategy. FUND: This study was supported by the NIH-NAID grants R21-AI131763, U01-AI35041, UM1-AI126603, and T32-AI065380.

Neisseria gonorrhoeae uses cellular proteins CXCL10 and IL8 to enhance HIV-1 transmission across cervical mucosa.

PROBLEM: Neisseria gonorrhoeae (NG) infection has been shown to increase sexual transmission of HIV-1. However, the mechanism of NG-induced enhanced HIV-1 transmission is unknown. METHODS: (a) The cervical tissues were exposed to NG, and cytokine induction was monitored by measuring cytokine proteins in culture supernatants and cytokine mRNAs in tissues. (b) Transcription and replication of HIV-1 in TZM-bl, U1, and ACH2 cells were measured by Beta-Gal activity and p24 proteins in the supernatant, respectively. (c) HIV-1 transmission was assayed in an organ culture system by measuring transmitted HIV-1 in supernatant and HIV-1 gag mRNA in the tissues. (d) Transcriptome analysis was done using second generation sequencing. RESULTS: (a) NG induced membrane ruffling of epithelial layer, caused migration of CD3+ cells to the intraepithelial region, and induced high levels of inflammatory cytokines IL-1ß and TNF-α. (b) NG-induced supernatants (NGIS) increased HIV-1 transcription, induced HIV-1 from latently infected cells, and increased transmission of HIV-1 across cervical mucosa. (c) Transcriptome analysis of the epithelial layer of the tissues exposed to NG, and HIV-1 showed significant upregulation of CXCL10 and IL8. IL-1ß increased the induction of CXCL10 and IL-8 expression in cervical mucosa with a concomitant increase in HIV-1 transmission. CONCLUSION: We present a model in which IL-1ß produced from cervical epithelium during NG exposure increases CXCL10 and IL8 in epithelia. This in turn causes upon HIV-1 infection, the migration of HIV-1 target cells toward the subepithelium, resulting in increased HIV-1 transcription in the sub-mucosa and subsequent enhancement of transmission across cervical mucosa.

Inhibitors of Signaling Pathways That Block Reversal of HIV-1 Latency.

Signaling pathways play a key role in HIV-1 latency. In this study, we used the 24ST1NLESG cell line of HIV-1 latency to screen a library of structurally diverse, medicinally active, cell permeable kinase inhibitors, which target a wide range of signaling pathways, to identify inhibitors of HIV-1 latency reversal. The screen was carried out in the absence or presence of three mechanistically distinct latency-reversing agents (LRAs), namely, prostratin, panobinostat, and JQ-1. We identified inhibitors that only blocked the activity of a specific LRA, as well as inhibitors that blocked the activity of all LRAs. For example, we identified 12 inhibitors targeted toward protein kinase C or downstream kinases that blocked the activity of prostratin. We also identified 12 kinase inhibitors that blocked the reversal of HIV-1 latency irrespective of the LRA used in the screen. Of these, danusertib, an Aurora kinase inhibitor, and PF-3758309, a PAK4 inhibitor, were the most potent. The 50% inhibitory concentrations in the 24ST1NLESG cells ranged from 40 to 147 nM for danusertib (selectivity indices, >150) and from 0.1 to 1 nM for PF-3758309 (selectivity indices, >3,300). Both danusertib and PF-3758309 inhibited latency reversal in CD4+ T cells isolated from HIV-1-infected donors. Collectively, our study describes a chemical approach that can be applied to elucidate the role of signaling pathways involved in LRA activity or the maintenance of HIV-1 latency and also identifies inhibitors of latent HIV-1 reactivation that could be used with antiretroviral therapy to reduce residual viremia.

Human immunodeficiency virus infection induces lymphoid fibrosis in the BM-liver-thymus-spleen humanized mouse model.

A major pathogenic feature associated with HIV infection is lymphoid fibrosis, which persists during antiretroviral therapy (ART). Lymphoid tissues play critical roles in the generation of antigen-specific immune response, and fibrosis disrupts the stromal network of lymphoid tissues, resulting in impaired immune cell trafficking and function, as well as immunodeficiency. Developing an animal model for investigating the impact of HIV infection-induced lymphoid tissue fibrosis on immunodeficiency and immune cell impairment is critical for therapeutics development and clinical translation. Said model will enable in vivo mechanistic studies, thus complementing the well-established surrogate model of SIV infection-induced lymphoid tissue fibrosis in macaques. We developed a potentially novel human immune system-humanized mouse model by coengrafting autologous fetal thymus, spleen, and liver organoids under the kidney capsule, along with i.v. injection of autologous fetal liver-derived hematopoietic stem cells, thus termed the BM-liver-thymus-spleen (BLTS) humanized mouse model. BLTS humanized mouse model supports development of human immune cells and human lymphoid organoids (human thymus and spleen organoids). HIV infection in BLTS humanized mice results in progressive fibrosis in human lymphoid tissues, which was associated with immunodeficiency in the lymphoid tissues, and lymphoid tissue fibrosis persists during ART, thus recapitulating clinical outcomes.

Preformulation and Vaginal Film Formulation Development of Microbicide Drug Candidate CSIC for HIV prevention.

PURPOSE: 5-chloro-3-[phenylsulfonyl] indole-2-carboxamide (CSIC) is a highly potent non-nucleoside reverse transcriptase inhibitor (NNRTI) of HIV-1 which has been shown to have a more desirable resistance profile than other NNRTIs in development as HIV prevention strategies. This work involves generation of preformulation data for CSIC and systematic development of a cosolvent system to effectively solubilize this hydrophobic drug candidate. This system was then applied to produce a polymeric thin film solid dosage form for vaginal administration of CSIC for use in prevention of sexual acquisition of HIV. METHODS: Extensive preformulation, formulation development, and film characterization studies were conducted. An HPLC method was developed for CSIC quantification. Preformulation tests included solubility, crystal properties, stability, and drug-excipient compatibility. Cytotoxicity was evaluated using both human epithelial and mouse macrophage cell lines. Ternary phase diagram methodology was used to identify a cosolvent system for CSIC solubility enhancement. Following preformulation evaluation, a CSIC film formulation was developed and manufactured using solvent casting technique. The developed film product was assessed for physicochemical properties, anti-HIV bioactivity, and Lactobacillus biocompatibility during 12-month stability testing period. RESULTS: Preformulation studies showed CSIC to be very stable. Due to its hydrophobicity, a cosolvent system consisting of polyethylene glycol 400, propylene glycol, and glycerin (5:2:1, w/w/w) was developed, which provided a uniform dispersion of CSIC in the film formulation. The final film product met target specifications established for vaginal microbicide application. CONCLUSIONS: The hydrophobic drug candidate CSIC was successfully formulated with high loading capacity in a vaginal film by means of a cosolvent system. The developed cosolvent strategy is applicable for incorporation of other hydrophobic drug candidates in the film platform.

Novel assay reveals a large, inducible, replication-competent HIV-1 reservoir in resting CD4+ T cells.

Although antiretroviral therapy can suppress HIV-1 infection to undetectable levels of plasma viremia, integrated latent HIV-1 genomes that encode replication-competent virus persist in resting CD4+ T cells. This latent HIV-1 reservoir represents a major barrier to a cure. Currently, there are substantial efforts to identify therapeutic approaches that will eliminate or reduce the size of this latent HIV-1 reservoir. In this regard, a sensitive assay that can accurately and rapidly quantify inducible, replication-competent latent HIV-1 from resting CD4+ T cells is essential for HIV-1 eradication studies. Here we describe a reporter cell-based assay to quantify inducible, replication-competent latent HIV-1. This assay has several advantages over existing technology in that it (i) is sensitive; (ii) requires only a small blood volume; (iii) is faster, less labor intensive, and less expensive; and (iv) can be readily adapted into a high-throughput format. Using this assay, we show that the size of the inducible latent HIV-1 reservoir in aviremic participants on therapy is approximately 70-fold larger than previous estimates.

Effect of prolonged freezing of semen on exosome recovery and biologic activity.

Exosomes are important vehicles of intercellular communication that shape host responses to physiologic, tumorigenic, and pathogenic conditions. The composition and function of exosomes are dynamic and depends on the state and condition of the cellular source. In prior work, we found that semen exosomes (SE) from healthy donors who do not use illicit drugs potently inhibit HIV-1. Following semen donation, specimens are either used immediately or frozen for use at a later time. It has been shown that short-term freezing of semen has no effect on SE-mediated HIV-1 inhibition. However, the effect of illicit drugs and prolonged freezing on SE bioactivity is unknown. Here, we show preservation of SE physical properties, (morphology, concentration, intensity/size) irrespective of illicit drug use or duration of semen freezing. Interestingly, illicit drugs and prolonged freezing decreased the levels of SE-bound CD63/CD9 and acetylcholinesterase activity respectively. Furthermore, we show differential effects of illicit drug use and prolonged freezing on SE-mediated HIV-1 inhibition. Our results highlight the importance of the source of SE and condition of semen storage on SE content and function. In-depth evaluation of donor drug-use and duration of semen storage on SE cargo and bioactivity will advance our understanding of SE composition and function.

Identification of the transcripts associated with spontaneous HCV clearance in individuals co-infected with HIV and HCV.

BACKGROUND: Infection with human immunodeficiency virus (HIV) influences the outcome and natural disease progression of hepatitis C virus (HCV) infection. While the majority of HCV mono-infected and HCV/HIV co-infected subjects develop chronic HCV infection, 20-46% of mono- and co-infected subjects spontaneously clear HCV infection. The mechanism underlying viral clearance is not clearly understood. Analysis of differential cellular gene expression (mRNA) between HIV-infected patients with persistent HCV infection or spontaneous clearance could provide a unique opportunity to decipher the mechanism of HCV clearance. METHODS: Plasma RNA from HIV/HCV co-infected subjects who cleared HCV and those who remained chronically infected with HCV was sequenced using Ion Torrent technology. The sequencing results were analyzed to identify transcripts that are associated with HCV clearance by measuring differential gene expression in HIV/HCV co-infected subjects who cleared HCV and those who remained chronically infected with HCV. RESULTS: We have identified plasma mRNA, the levels of which are significantly elevated (at least 5 fold, False Discovery Rate (FDR) <0.05) before HCV infection in subjects who cleared HCV compared to those who remained chronically infected. Upon further analysis of these differentially expressed genes, before and after HCV infection, we found that before HCV infection 12 genes were uniquely upregulated in the clearance group compared to the chronically infected group. Importantly, a number of these 12 genes and their upstream regulators (such as CCL3, IL17D, LBP, SOCS3, NFKBIL1, IRF) are associated with innate immune response functions. CONCLUSIONS: These results suggest that subjects who spontaneously clear HCV may express these unique genes associated with innate immune functions.

Placental Mechanics in the Zika-Microcephaly Relationship.

How the Zika virus (ZIKV) accesses the embryo remains unknown. In this issue, Quicke et al. (2016) use an in vitro model of the human placenta to show that placental macrophages are more permissive to ZIKV infection than trophoblasts, which may be refractory to infection (Bayer et al., 2016).

Effective Cytotoxic T Lymphocyte Targeting of Persistent HIV-1 during Antiretroviral Therapy Requires Priming of Naive CD8+ T Cells.

UNLABELLED: Curing HIV-1 infection will require elimination of persistent cellular reservoirs that harbor latent virus in the face of combination antiretroviral therapy (cART). Proposed immunotherapeutic strategies to cure HIV-1 infection include enhancing lysis of these infected cells by cytotoxic T lymphocytes (CTL). A major challenge in this strategy is overcoming viral immune escape variants that have evaded host immune control. Here we report that naive CD8(+) T cells from chronic HIV-1-infected participants on long-term cART can be primed by dendritic cells (DC). These DC must be mature, produce high levels of interleukin 12p70 (IL-12p70), be responsive to CD40 ligand (CD40L), and be loaded with inactivated, autologous HIV-1. These DC-primed CD8(+) T cell responders produced high levels of gamma interferon (IFN-γ) in response to a broad range of both conserved and variable regions of Gag and effectively killed CD4(+) T cell targets that were either infected with the autologous latent reservoir-associated virus or loaded with autologous Gag peptides. In contrast, HIV-1-specific memory CD8(+) T cells stimulated with autologous HIV-1-loaded DC produced IFN-γ in response to a narrow range of conserved and variable Gag peptides compared to the primed T cells and most notably, displayed significantly lower cytolytic function. Our findings highlight the need to selectively induce new HIV-1-specific CTL from naive precursors while avoiding activation of existing, dysfunctional memory T cells in potential curative immunotherapeutic strategies for HIV-1 infection. IMPORTANCE: Current immunotherapeutic approaches aim to enhance antiviral immunity against the HIV-1 reservoir; however, it has yet to be shown whether T cells from persons on cART can recognize and eliminate virus-infected cells. We show that in persons on cART a personalized medicine approach using their dendritic cells to stimulate their naive T cells induces potent effector CTL in vitro that recognize and eradicate HIV-1-infected CD4(+) T cells. Additionally, we show that the same stimulation of existing memory T cells results in cytokine secretion but limited effector function. Our study demonstrates that the naive T cell repertoire can recognize persistent HIV-1 during cART and supports immunotherapy strategies for an HIV-1 cure that targets naive T cells, rather than existing, dysfunctional, memory T cells.

HIV Exposure to the Epithelia in Ectocervical and Colon Tissues Induces Inflammatory Cytokines Without Tight Junction Disruption.

Epithelial cells in human cervical and colonic mucosa do not express HIV receptor. However, HIV transmission occurs across the unbreached epithelia by an unknown mechanism. In this study, the effect of HIV exposure on tight junction (TJ) and cytokine production in ectocervical and colon mucosal epithelia in tissue biopsies was investigated in an organ culture model. After HIV exposure, the distribution patterns and quantities of epithelial TJ and adherens proteins were evaluated by immunofluorescence staining followed by confocal microscopy. Cytokine mRNA in the mucosal epithelia was also evaluated by real-time reverse transcription-polymerase chain reaction (RT-PCR). HIV transmission was evaluated by measuring p24 production in culture supernatant. Our results showed there were no significant changes in the distribution and quantities of epithelial TJ/adherens junction (AJ) proteins after exposure to HIV. However, higher levels of CXCL10 and CXCL11 mRNA expression were detected in HIV-exposed ectocervical epithelia. In case of colon mucosa, higher levels of CXCL10 and IL-6 mRNA expression were detected in HIV-exposed colon mucosa. Our study suggests that HIV induces cytokine production in epithelial cells, which may facilitate HIV transmission by recruiting HIV target cells in the submucosal region. Furthermore, HIV transmission may not occur through epithelial TJ/AJ disruption.

Acute complications following intracavitary high-dose-rate brachytherapy in uterine cancer.

PURPOSE: Almost 30% of malignancies in women of developing countries are gynecological and brachytherapy is an integral part of management of these patients. Reports of complications (both acute and late) of high-dose-rate (HDR) intracavitary brachytherapy are sparse in world literature due to relatively small number of gynecological malignancies, particularly in advanced stage, in developed countries. High-dose-rate brachytherapy is gaining popularity in developing countries due to scientific and economic reasons. Here we are reporting our experience regarding acute complications of intracavitary brachytherapy (events occurring within 30 days of insertion needing hospitalization or death) and their causes to improve the quality of management, so that the already low incidence of acute complications can be further reduced. MATERIAL AND METHODS: From February 2004 to December 2012, a total of 1947 patients with uterine cancer were treated by HDR intracavitary brachytherapy in the Department of Radiotherapy, of a tertiary cancer centre of a developing country, 86% of them were cervical cancer and 14% endometrial cancer. Excluding the post-operative patients, a total of 4285 insertions were done in 1527 patients with intact uterus (eligible for analysis) and acute complications were analyzed. RESULTS: Out of 4285 intracavitary brachytherapy insertions in gynecological malignancy patients, only 12 mortality and 239 morbidity instances needing hospitalization were documented and most of them were in cervical carcinoma patients. CONCLUSIONS: Our results have indicated that acute complications can be minimized by pre-treatment management of co-morbidities, decreasing the time of operative lithotomy position and bed rest, avoidance of 'conscious sedation' in selected cases etc. Routine post insertion CT scan if done in all patients in all insertions, then only, uterine perforations can be detected early and prompt management can reduce both the mortality and morbidity to a great extent.

Abasic phosphorothioate oligomers inhibit HIV-1 reverse transcription and block virus transmission across polarized ectocervical organ cultures.

In the absence of universally available antiretroviral (ARV) drugs or a vaccine against HIV-1, microbicides may offer the most immediate hope for controlling the AIDS pandemic. The most advanced and clinically effective microbicides are based on ARV agents that interfere with the earliest stages of HIV-1 replication. Our objective was to identify and characterize novel ARV-like inhibitors, as well as demonstrate their efficacy at blocking HIV-1 transmission. Abasic phosphorothioate 2' deoxyribose backbone (PDB) oligomers were evaluated in a variety of mechanistic assays and for their ability to inhibit HIV-1 infection and virus transmission through primary human cervical mucosa. Cellular and biochemical assays were used to elucidate the antiviral mechanisms of action of PDB oligomers against both lab-adapted and primary CCR5- and CXCR4-utilizing HIV-1 strains, including a multidrug-resistant isolate. A polarized cervical organ culture was used to test the ability of PDB compounds to block HIV-1 transmission to primary immune cell populations across ectocervical tissue. The antiviral activity and mechanisms of action of PDB-based compounds were dependent on oligomer size, with smaller molecules preventing reverse transcription and larger oligomers blocking viral entry. Importantly, irrespective of molecular size, PDBs potently inhibited virus infection and transmission within genital tissue samples. Furthermore, the PDB inhibitors exhibited excellent toxicity and stability profiles and were found to be safe for vaginal application in vivo. These results, coupled with the previously reported intrinsic anti-inflammatory properties of PDBs, support further investigations in the development of PDB-based topical microbicides for preventing the global spread of HIV-1.

The CD8 antiviral factor (CAF) can suppress HIV-1 transcription from the long terminal repeat (LTR) promoter in the absence of elements upstream of the CATATAA box.

BACKGROUND: The CD8 Antiviral Factor (CAF) suppresses viral transcription from the HIV-1 Long Terminal Repeat (LTR) promoter in a non-cytolytic manner. However, the region on the LTR upon which CAF acts is unknown. Our objective was to determine the region on the LTR upon which CAF acts to suppress HIV-1 transcription. METHODS: Serial deletions of the LTR from the 5' end and inactivating point mutations were made. RESULTS: Serial deletions of the LTR from the 5' end indicated the importance of a short ~120 bp segment, containing the 3 SpI sites, CATA box (used by HIV-1 instead of the TATA box) and TAR region, in the suppressive process. Introduction of deletions or inactivating point mutations in the SpI sites or deletion of the TAR region did not abolish CAF-mediated transcriptional suppression. Yet, CAF-mediated transcriptional suppression was still retained in the HIV-1 CATA-TAR segment. CONCLUSION: CAF is able to suppress transcription from the LTR lacking all the elements upstream of the CATA box. Our results suggest that the HIV-1 CATA box may be responsible for CAF-mediated suppression of transcription from the HIV-1 LTR.

Dendritic cells restore CD8+ T cell reactivity to autologous HIV-1.

UNLABELLED: Recall T cell responses to HIV-1 antigens are used as a surrogate for endogenous cellular immune responses generated during infection. Current methods of identifying antigen-specific T cell reactivity in HIV-1 infection use bulk peripheral blood mononuclear cells (PBMC) yet ignore professional antigen-presenting cells (APC) that could reveal otherwise hidden responses. In the present study, peptides representing autologous variants of major histocompatibility complex (MHC) class I-restricted epitopes from HIV-1 Gag and Env were used as antigens in gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) and polyfunctional cytokine assays. Here we show that dendritic cells (DC) enhanced T cell reactivity at all stages of disease progression but specifically restored T cell reactivity after combination antiretroviral therapy (cART) to early infection levels. Type 1 cytokine secretion was also enhanced by DC and was most apparent late post-cART. We additionally show that DC reveal polyfunctional T cell responses after many years of treatment, when potential immunotherapies would be implemented. These data underscore the potential efficacy of DC immunotherapy that aims to awaken a dormant, autologous, HIV-1-specific CD8+ T cell response. IMPORTANCE: Assessment of endogenous HIV-1-specific T cell responses is critical for generating immunotherapies for subjects on cART. Current assays ignore the ability of dendritic cells to reveal these responses and may therefore underestimate the breadth and magnitude of T cell reactivity. As DC do not prime new responses in these assays, it can be assumed that the observed responses are not detected without appropriate stimulation. This is important because dogma states that HIV-1 mutates to evade host recognition and that CD8+ cytotoxic T lymphocyte (CTL) failure is due to the inability of T cells to recognize the autologous virus. The results presented here indicate that responses to autologous virus are generated during infection but may need additional stimulation to be effective. Detecting the breadth and magnitude of HIV-1-specific T cell reactivity generated in vivo is of the utmost importance for generating effective DC immunotherapies.

Alterations in cholesterol metabolism restrict HIV-1 trans infection in nonprogressors.

ABSTRACT HIV-1-infected nonprogressors (NP) inhibit disease progression for years without antiretroviral therapy. Defining the mechanisms for this resistance to disease progression could be important in determining strategies for controlling HIV-1 infection. Here we show that two types of professional antigen-presenting cells (APC), i.e., dendritic cells (DC) and B lymphocytes, from NP lacked the ability to mediate HIV-1 trans infection of CD4(+) T cells. In contrast, APC from HIV-1-infected progressors (PR) and HIV-1-seronegative donors (SN) were highly effective in mediating HIV-1 trans infection. Direct cis infection of T cells with HIV-1 was comparably efficient among NP, PR, and SN. Lack of HIV-1 trans infection in NP was linked to lower cholesterol levels and an increase in the levels of the reverse cholesterol transporter ABCA1 (ATP-binding cassette transporter A1) in APC but not in T cells. Moreover, trans infection mediated by APC from NP could be restored by reconstitution of cholesterol and by inhibiting ABCA1 by mRNA interference. Importantly, this appears to be an inherited trait, as it was evident in APC obtained from NP prior to their primary HIV-1 infection. The present study demonstrates a new mechanism wherein enhanced lipid metabolism in APC results in remarkable control of HIV-1 trans infection that directly relates to lack of HIV-1 disease progression. IMPORTANCE HIV-1 can be captured by antigen-presenting cells (APC) such as dendritic cells and transferred to CD4 helper T cells, which results in greatly enhanced viral replication by a mechanism termed trans infection. A small percentage of HIV-1-infected persons are able to control disease progression for many years without antiretroviral therapy. In our study, we linked this lack of disease progression to a profound inability of APC from these individuals to trans infect T cells. This effect was due to altered lipid metabolism in their APC, which appears to be an inherited trait. These results provide a basis for therapeutic interventions to control of HIV-1 infection through modulation of cholesterol metabolism.

Transmission and evolution of hepatitis C virus in HCV seroconverters in HIV infected subjects.

HIV/HCV co-infection provides a model to determine the role of immunity on HCV transmission and evolution. In this study HCV transmission and evolution were evaluated in 6 HCV seroconverters in HIV-infected subjects with a wide range of CD4 cell count. The HCV envelope E1/E2 sequences were analyzed for transmission bottleneck, viral diversity/divergence, immune pressure, and mutations of HLA class I/II restricted epitopes. HCV infection started with transmission bottleneck in all HIV-infected individuals. During the 1.0-2.0 years of infection there was a shift of viral quasispecies in majority of the subjects from one to next visit. However, HCV diversity, divergence, mutations in HLA class I/II restricted and virus neutralizing epitopes were similar in all subjects regardless of CD4 cell count at the time of HCV infection. Our results suggest that HCV transmission and evolution in HIV-infected subjects may not be influenced by host CD4 cell count at the time of infection.