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A longer acting, removable implant for HIV prevention has the potential to improve uptake of HIV pre-exposure prophylaxis (PrEP) by removing the need for daily adherence to an oral tablet, reducing potential side effects, and eliminating concerns about residual drug following injections. To end the HIV epidemic, we must understand the needs and preferences of groups most affected by HIV (e.g., men who have sex with men; MSM), and the physicians who prescribe PrEP to them. This article describes a discrete choice experiment to estimate the preference share for the implant within a competitive context of other PrEP products (including the oral tablet, dissolvable implant, and injection) and evaluate the impact of potential implant attributes. Physicians who had prescribed oral PrEP (n = 75) and MSM at risk for HIV (n = 175) completed a web-based survey that prompted decision-making about PrEP product preferences. The findings from both physicians and MSM demonstrated that the removable implant could capture a meaningful portion of the preference share, making it feasible to advance in the development pipeline as an important addition to the biomedical HIV prevention toolkit. Among MSM, specifically, the cost of treatment was the most important attribute impacting product preference. Our findings inform implant developers and future payers (e.g., commercial manufacturers, insurance companies) about specific device attributes that will likely affect MSM's willingness to use and physicians' willingness to prescribe this HIV prevention strategy.

Long-acting delivery modalities of HIV pre-exposure prophylaxis (PrEP), such as subdermal implants, are in development. To facilitate end-user uptake and sustained use, it is critical to understand potential consumers' and physician prescribers' preferences about, interest in, and relative importance of different implant features. The ordered identification of these key attributes allows implant developers to incorporate this feedback into product design, which theoretically improves acceptability, feasibility, and user experience with the device. In this study, n = 75 PrEP-prescribing physicians and n = 143 men having sex with men (MSM) at risk for HIV completed web-based surveys that directly compared the importance of eight to nine different implant features, respectively. Conjoint analysis determined the importance of these features, relative to each other. Implants presented in the study were well received, with a majority of physicians and MSM indicating that they were likely to recommend or use them. The implant was perceived as unique, reliable, and convenient, as well as able to deliver better compliance. The attributes most critical to the adoption of the implant among physicians and MSM were (1) the chance of becoming infected with HIV while on implant treatment, (2) the length of protection and size of the implant, and (3) the side effect advantages over current PrEP oral pill treatment. Some concerns about the implant included side effects and the product's safety (among MSM) and the cost or insurance coverage level for the implant (both physicians and MSM). There was also some resistance to the implantation procedure itself.

The study described herein is a continuation of our work in which we developed a methodology to identify small foci of transduced cells following rectal challenge of rhesus macaques with a non-replicative luciferase reporter virus. In the current study, the wild-type virus was added to the inoculation mix and twelve rhesus macaques were necropsied 2-4 days after the rectal challenge to study the changes in infected cell phenotype as the infection progressed. Relying on luciferase reporter we noted that both anus and rectum tissues are susceptible to the virus as early as 48h after the challenge. Small regions of the tissue containing luciferase-positive foci were further analyzed microscopically and were found to also contain cells infected by wild-type virus. Phenotypic analysis of the Env and Gag positive cells in these tissues revealed the virus can infect diverse cell populations, including but not limited to Th17 T cells, non Th17 T cells, immature dendritic cells, and myeloid-like cells. The proportions of the infected cell types, however, did not vary much during the first four days of infection when anus and rectum tissues were examined together. Nonetheless, when the same data was analyzed on a tissue-specific basis, we found significant changes in infected cell phenotypes over the course of infection. For anal tissue, a statistically significant increase in infection was observed for Th17 T cells and myeloid-like cells, while in the rectum, the non-Th17 T cells showed the biggest temporal increase, also of statistical significance.IMPORTANCEMen who have sex with men are at the highest risk of acquiring HIV via receptive anal intercourse. Understanding what sites are permissive to the virus, and what the early cellular targets are is critical for development of effective prevention strategies to control HIV acquisition amid receptive anal intercourse. Our work sheds light on the early HIV/SIV transmission events at the rectal mucosa by identifying the infected cells and highlights the distinct roles that different tissues play in virus acquisition and control.

HIV-1 (HIV) infects CD4 positive T cells, the gradual depletion of which can lead to the onset of Acquired Immunodeficiency Syndrome (AIDS) in the absence of antiretroviral therapy (ART). Several forms of cell death have been shown to be involved in HIV-mediated killing of either directly infected or bystander cells. It is still unknown, however, why some cells survive HIV infection and persist as part of the latently infected reservoir that reliably causes recurrent viremia upon ART cessation. Improved understanding of the mechanisms of HIV-mediated cell death could inform innovations designed to clear the latent reservoir. “Death Induced by Survival gene Elimination” (DISE) is an RNA interference (RNAi)-based mechanism that kills cells through short (s)RNAs with toxic 6mer seeds (pos. 2-7 of sRNA). These toxic seeds target reverse complementary seed matches in the 3’UTR of mRNA transcripts to decrease expression of hundreds of genes that are critical for cell survival. In most cells under normal conditions, highly expressed cell-encoded non-toxic microRNAs (miRNAs) block access of toxic sRNAs to the RNA-induced silencing complex (RISC) that mediates RNAi, promoting cell survival. We now report that infection of cells with HIV results in RISC-loading of an HIV-encoded miRNA, v-miRNA HIV-miR-TAR-3p, which kills cells by DISE through a noncanonical (pos. 3-8) 6mer seed. In addition, cellular RISC bound sRNAs shift to lower seed viability. Both these effects also occur with latent HIV provirus reactivation in J-Lat cells, a well-established cell model of HIV latency. Cells lacking expression of miRNA biogenesis genes Drosha, Dicer and Exportin 5 cannot produce protective miRNAs and therefore do not block RISC loading of the v-miRNA HIV-miR-TAR-3p. These mutant cells, as well as cells lacking expression of the RISC component Ago2, are hypersensitive to cell death via DISE induced by HIV infection. More precise targeting of the balance between protective and cytotoxic sRNAs could specifically and transiently increase silencing of cell survival genes to increase DISE. This could be a new addition to a “shock and kill” strategy to enhance depletion of the provirus reservoir during suppressive ART.

i.v. injected Abs have demonstrated protection against simian HIV infection in rhesus macaques, paving the way for the Antibody Mediated Prevention trial in which at-risk individuals for HIV received an i.v. infusion of the HIV broadly neutralizing Ab VRC01. However, the time needed for these Abs to fully distribute and elicit protection at mucosal sites is still unknown. In this study, we interrogate how long it takes for Abs to achieve peak anatomical levels at the vaginal surface following i.v. injection. Fluorescently labeled VRC01 and/or Gamunex-C were i.v. injected into 24 female rhesus macaques (Macaca mulatta) with vaginal tissues and plasma acquired up to 2 wk postinjection. We found that Ab delivery to the vaginal mucosa occurs in two phases. The first phase involves delivery to the submucosa, occurring within 24 h and persisting beyond 1 wk. The second phase is the delivery through the stratified squamous epithelium, needing ∼1 wk to saturate the stratum corneum. This study has important implications for the efficacy of immunoprophylaxis targeting pathogens at the mucosa.

The ability to successfully develop a safe and effective vaccine for the prevention of HIV infection has proven challenging. Consequently, alternative approaches to HIV infection prevention have been pursued, and there have been a number of successes with differing levels of efficacy. At present, only two oral preexposure prophylaxis (PrEP) products are available, Truvada and Descovy. Descovy is a newer product not yet indicated in individuals at risk of HIV-1 infection from receptive vaginal sex, because it still needs to be evaluated in this population. A topical dapivirine vaginal ring is currently under regulatory review, and a long-acting (LA) injectable cabotegravir product shows strong promise. Although demonstrably effective, daily oral PrEP presents adherence challenges for many users, particularly adolescent girls and young women, key target populations. This limitation has triggered development efforts in LA HIV prevention options. This article reviews efforts supported by the Bill & Melinda Gates Foundation, as well as similar work by other groups, to identify and develop optimal LA HIV prevention products. Specifically, this article is a summary review of a meeting convened by the foundation in early 2020 that focused on the development of LA products designed for extended delivery of tenofovir alafenamide (TAF) for HIV prevention. The review broadly serves as technical guidance for preclinical development of LA HIV prevention products. The meeting examined the technical feasibility of multiple delivery technologies, in vivo pharmacokinetics, and safety of subcutaneous (SC) delivery of TAF in animal models. Ultimately, the foundation concluded that there are technologies available for long-term delivery of TAF. However, because of potentially limited efficacy and possible toxicity issues with SC delivery, the foundation will not continue investing in the development of LA, SC delivery of TAF products for HIV prevention.

Long-acting antiretroviral implants could help protect high-risk individuals from HIV infection. We describe the design and testing of a long-acting reservoir subcutaneous implant capable of releasing cabotegravir for several months. We compressed cabotegravir and excipients into cylindrical pellets and heat-sealed them in tubing composed of hydrophilic poly(ether-urethane) -. The implants have a 47 mm lumen length, 3.6 mm outer diameter, and 200 μm wall thickness. Four cabotegravir pellets were sealed in the membrane, with a total drug loading of 274 ± 3 mg. In vivo, the implants released 348 ± 107 μg/day (median value per implant, N = 41) of cabotegravir in rhesus macaques. Five implants generated an average cabotegravir plasma concentration of 373 ng/ml in rhesus macaques. The non-human primates tolerated the implant without gross pathology or microscopic signs of histopathology compared to placebo implants. Cabotegravir plasma levels in macaques dropped below detectable levels within two weeks after the removal of the implants.

Although vaccination efforts have expanded, there are still gaps in our understanding surrounding the immune response to SARS-CoV-2. Measuring IgG Fc glycosylation provides insight into an infected individual's inflammatory state, among other functions. We set out to interrogate bulk IgG glycosylation changes from SARS-CoV-2 infection and vaccination, using plasma from mild or hospitalized COVID-19 patients, and from vaccinated individuals. Inflammatory glycans are elevated in hospitalized COVID-19 patients and increase over time, while mild patients have anti-inflammatory glycans that increase over time, including increased sialic acid correlating with RBD antibody levels. Vaccinated individuals with low RBD antibody levels and low neutralization have the same IgG glycan traits as hospitalized COVID-19 patients. In addition, a small vaccinated cohort reveals a decrease in inflammatory glycans associated with peak IgG concentrations and neutralization. This report characterizes the bulk IgG glycome associated with COVID-19 severity and vaccine responsiveness and can help guide future studies into SARS-CoV-2 protective immunity.

Understanding the interplay between commensals, pathogens, and immune cells in the skin and mucosal tissues is critical to improve prevention and treatment of a myriad of diseases. While high-parameter flow cytometry is the current gold standard for immune cell characterization in blood, it is less suitable for mucosal tissues, where structural and spatial information is lost during tissue disaggregation. Immunofluorescence overcomes this limitation, serving as an excellent alternative for studying immune cells in mucosal tissues. However, the use of immunofluorescent microscopy for analyzing clinical samples is hampered by a lack of high-throughput quantitative analysis techniques. In this chapter, we describe methods for sectioning, staining, and imaging whole sections of human foreskin tissue. We also describe methods to automate immune cell quantification from immunofluorescent images, including image preprocessing and methods to quantify both circular and irregularly shaped immune cells using open-source software.

SUMMARYCyclic GMP-AMP synthase (cGAS) is a primary sensor of aberrant DNA that governs an innate immune signaling cascade, leading to the induction of the type-I interferon response. We have previously identified polyglutamine binding protein 1, PQBP1, as an adaptor molecule required for cGAS-mediated innate immune response of lentiviruses, including the human immunodeficiency virus 1 (HIV-1), but dispensable for the recognition of DNA viruses. HIV-1- encoded DNA is synthesized as a single copy from its RNA genome, and is subsequently integrated into the host chromatin. HIV-1 then produces progeny through amplification and packaging of its RNA genome, thus, in contrast to DNA viruses, HIV-1 DNA is both transient and of low abundance. However, the molecular basis for the detection and verification of this low abundance HIV-1 DNA pathogen-associated molecular pattern (PAMP) is not understood. Here, we elucidate a two-factor authentication strategy that is employed by the innate immune surveillance machinery to selectively respond to the low concentration of PAMP, while discerning these species from extranuclear DNA molecules. We find that, upon HIV-1 infection, PQBP1 decorates intact viral capsid, which serves as a primary verification step for the viral nucleic acid cargo. As the reverse transcription and capsid disassembly initiate, cGAS protein is then recruited to the capsid in a PQBP1-dependent manner, enabling cGAS molecules to be co-positioned at the site of PAMP generation. Thus, these data indicate that PQBP1 recognition of the HIV-1 capsid sanctions a robust cGAS-dependent response to a limited abundance and short-lived DNA PAMP. Critically, this illuminates a molecular strategy wherein the modular recruitment of co-factors to germline encoded pattern recognition receptors (PRRs) serves to enhance repertoire of pathogens that can be sensed by the innate immune surveillance machinery.

Infection with the novel coronavirus, SARS-CoV-2, results in pneumonia and other respiratory symptoms as well as pathologies at diverse anatomical sites. An outstanding question is whether these diverse pathologies are due to replication of the virus in these anatomical compartments and how and when the virus reaches those sites. To answer these outstanding questions and study the spatiotemporal dynamics of SARS-CoV-2 infection a method for tracking viral spread in vivo is needed. We developed a novel, fluorescently labeled, antibody-based in vivo probe system using the anti-spike monoclonal antibody CR3022 and demonstrated that it could successfully identify sites of SARS-CoV-2 infection in a rhesus macaque model of COVID-19. Our results showed that the fluorescent signal from our antibody-based probe could differentiate whole lungs of macaques infected for 9 days from those infected for 2 or 3 days. Additionally, the probe signal corroborated the frequency and density of infected cells in individual tissue blocks from infected macaques. These results provide proof of concept for the use of in vivo antibody-based probes to study SARS-CoV-2 infection dynamics in rhesus macaques.

We have previously described polyglutamine-binding protein 1 (PQBP1) as an adapter required for the cyclic GMP-AMP synthase (cGAS)-mediated innate response to the human immunodeficiency virus 1 (HIV-1) and other lentiviruses. Cytoplasmic HIV-1 DNA is a transient and low-abundance pathogen-associated molecular pattern (PAMP), and the mechanism for its detection and verification is not fully understood. Here, we show a two-factor authentication strategy by the innate surveillance machinery to selectively respond to the low concentration of HIV-1 DNA, while distinguishing these species from extranuclear DNA molecules. We find that, upon HIV-1 infection, PQBP1 decorates the intact viral capsid, and this serves as a primary verification step for the viral nucleic acid cargo. As reverse transcription and capsid disassembly initiate, cGAS is recruited to the capsid in a PQBP1-dependent manner. This positions cGAS at the site of PAMP generation and sanctions its response to a low-abundance DNA PAMP.

Anti-HIV broadly neutralizing antibodies (bNAbs) may favor development of antiviral immunity by engaging the immune system during immunotherapy. Targeting integrin α4β7 with an anti-α4β7 monoclonal antibody (Rh-α4β7) affects immune responses in SIV/SHIV-infected macaques. To explore the therapeutic potential of combining bNAbs with α4β7 integrin blockade, SHIVSF162P3-infected, viremic rhesus macaques were treated with bNAbs only (VRC07-523LS and PGT128 anti-HIV antibodies) or a combination of bNAbs and Rh-α4β7 or were left untreated as a control. Treatment with bNAbs alone decreased viremia below 200 copies/ml in all macaques, but seven of eight macaques (87.5%) in the bNAbs-only group rebounded within a median of 3 weeks (95% CI: 2 to 9). In contrast, three of six macaques treated with a combination of Rh-α4β7 and bNAbs (50%) maintained a viremia below 200 copies/ml until the end of the follow-up period; viremia in the other three macaques rebounded within a median of 6 weeks (95% CI: 5 to 11). Thus, there was a modest delay in viral rebound in the macaques treated with the combination antibody therapy compared to bNAbs alone. Our study suggests that α4β7 integrin blockade may prolong virologic control by bNAbs in SHIVSF162P3-infected macaques.

Human immunodeficiency virus (HIV) vaccines have not been successful in clinical trials. Dimeric IgA (dIgA) in the form of secretory IgA is the most abundant antibody class in mucosal tissues, making dIgA a prime candidate for potential HIV vaccines. We coupled Positron Emission Tomography (PET) imaging and fluorescent microscopy of 64Cu-labeled, photoactivatable-GFP HIV (PA-GFP-BaL) and fluorescently labeled dIgA to determine how dIgA antibodies influence virus interaction with mucosal barriers and viral penetration in colorectal tissue. Our results show that HIV virions rapidly disseminate throughout the colon two hours after exposure. The presence of dIgA resulted in an increase in virions and penetration depth in the transverse colon. Moreover, virions were found in the mesenteric lymph nodes two hours after viral exposure, and the presence of dIgA led to an increase in virions in mesenteric lymph nodes. Taken together, these technologies enable in vivo and in situ visualization of antibody-virus interactions and detailed investigations of early events in HIV infection., Author summary Vaccines provide protection in humans by eliciting the production of antibodies when exposed to a specific pathogen. Currently, an effective human immunodeficiency virus (HIV) vaccine does not exist. Since the beginning of the HIV epidemic, approximately 38 million people have died, creating the need to develop an HIV vaccine. The most common antibody in the organs that are exposed to HIV is dimeric IgA (dIgA). Here, we used multiple imaging techniques to determine how HIV travels throughout the colon once introduced into the body and how dIgA influences HIV movement in the rectum. We found that dIgA increased the amount of HIV found in the colon, the distance it travelled, and the depth into tissues that HIV penetrated. dIgA also increased the amount of HIV in the mesenteric lymph nodes two hours after viral exposure. Our study shows these imaging technologies can be used to examine interactions between viruses and antibodies in early HIV infection in the natural context of the anatomy and physiology of the rhesus macaque model.

SARS-CoV-2 is a respiratory borne pathogenic beta coronavirus that is the source of a worldwide pandemic and the cause of multiple pathologies in man. The rhesus macaque model of COVID-19 was utilized to test the added benefit of combinatory parenteral administration of two high-affinity anti-SARS-CoV-2 monoclonal antibodies (mAbs; C144-LS and C135-LS) expressly developed to neutralize the virus and modified to extend their pharmacokinetics. After completion of kinetics study of mAbs in the primate, combination treatment was administered prophylactically to mucosal viral challenge. Results showed near complete virus neutralization evidenced by no measurable titer in mucosal tissue swabs, muting of cytokine/chemokine response, and lack of any discernable pathologic sequalae. Blocking infection was a dose-related effect, cohorts receiving lower doses (6, 2 mg/kg) resulted in low grade viral infection in various mucosal sites compared to that of a fully protective dose (20 mg/kg). A subset of animals within this cohort whose infectious challenge was delayed 75 days later after mAb administration were still protected from disease. Results indicate this combination mAb effectively blocks development of COVID-19 in the rhesus disease model and accelerates the prospect of clinical studies with this effective antibody combination.

There are several long-acting biomedical HIV prevention products in the development pipeline, including injections and implanted medication delivery devices (IMDDs). It is critical to understand concerns and preferences on the use of these products in populations that shoulder a disproportionate burden of the HIV epidemic, such as transgender women. This will allow researchers and public health professionals to construct interventions tailored to the needs of these women to promote optimal use of these tools. In studies of other biomedical HIV prevention products (e.g., oral PrEP) it is clear that transgender women have unique concerns related to the use of these strategies. This may have an impact on this group's uptake and sustained use of longacting HIV prevention products. This study conducted four focus groups with N = 18 transgender women in New York City to understand their concerns and preferences on long-acting PrEP injections and IMDDs. Findings showed that participants were overwhelmingly positive about long-acting HIV prevention strategies, though they had some apprehensions. Overall, participants felt that injections and IMDDs could help address adherence challenges, and that transgender-specific needs should be addressed during clinical trials. Also, there were concerns related to injection or IMDD logistics, concerns about injections' or IMDDs' presence in the body, and familiarity with these products affected participants' opinions on them. Findings from this work can be used to inform protocols, measures, materials, and adherence interventions in future initiatives for transgender women using PrEP injections or IMDDs.

This paper grows our understanding about PrEP knowledge in transgender women (TW) to improve PrEP-focused education/outreach. Research took place in New York City. We conducted four focus groups in English or Spanish (N = 18). Discussions focused on participants' perceptions and knowledge of oral PrEP. Most participants knew that PrEP is efficacious and requires consistent use. However, some participants were skeptical of medications; others acknowledged that false assumptions about PrEP exist among TW. Most TW in our focus groups were informed about PrEP through clinics or community-based organizations. Some participants felt that messages about medications were oversimplified, and wanted more information.

Understanding the earliest events of HIV sexual transmission is critical to develop and optimize HIV prevention strategies. To gain insights into the earliest steps of HIV rectal transmission, including cellular targets, rhesus macaques were intra-rectally challenged with a single-round SIV-based dual reporter that expresses luciferase and iRFP670 upon productive transduction. The vector was pseudotyped with the HIV-1 envelope JRFL. Regions of tissue containing foci of luminescent, transduced cells were identified macroscopically using an in vivo imaging system, and individual transduced cells expressing fluorescent protein were identified and phenotyped microscopically. This system revealed that anal and rectal tissues are both susceptible to transduction 48 hours after the rectal challenge. Detailed phenotypic analysis revealed that on average, 62% of transduced cells are CCR6+ T cells—the vast majority of which express RORγT, a Th17 lineage-specific transcription factor. The second most common target cells were immature dendritic cells at 20%. These two cell types were transduced at the rates that are four to five times higher than their relative abundances indicate. Our work demonstrates that Th17 T and immature dendritic cells are preferential initial targets of HIV/SIV rectal transmission.IMPORTANCEMen and women who participate in unprotected receptive anal intercourse are at high risk for acquiring HIV. While in vitro data have developed a framework for understanding HIV cell tropism, the initial target cells in the rectal mucosa have not been identified. In this study, we identify these early host cells by using an innovative rhesus macaque rectal challenge model and methodology, which we previously developed. Thus, by shedding light on these early HIV/SIV transmission events, this study provides a specific cellular target for future prevention strategies.

Innate responses during acute HIV infection correlate with disease progression and pathogenesis. However, limited information is available about the events occurring during the first hours of infection in the mucosal sites of transmission. With an ex vivo HIV-1 challenge model of human colorectal tissue we assessed the mucosal responses induced by R5- and X4-tropic HIV-1 isolates in the first 24 h of exposure. Microscopy studies demonstrated virus penetration of up to 39 μm into the lamina propia within 6 h of inoculation. A rapid, 6 h post-challenge, increase in the level of secretion of inflammatory cytokines, chemokines, interferon- γ (IFN-γ), and granulocyte-macrophage colony-stimulating factor (GM-CSF) was observed following exposure to R5- or X4-tropic isolates. This profile persisted at the later time point measured of 24 h. However, exposure to the X4-tropic isolate tested induced greater changes at the proteomic and transcriptomic levels than the R5-tropic. The X4-isolate induced greater levels of CCR5 ligands (RANTES, MIP-1α and MIP-1β) secretion than R5-HIV-1. Potential drugs candidates for colorectal microbicides, including entry, fusion or reverse transcriptase inhibitors demonstrated differential capacity to modulate these responses. Our findings indicate that in colorectal tissue, inflammatory responses and a Th1 cytokine profile are induced in the first 24 h following viral exposure.

During the last decade, there was a marked increase in the development of tools and techniques to study the molecular mechanisms of the HIV replication cycle by using fluorescence microscopy. Researchers often apply the fusion of tags and fluorophores to viral proteins, surrogate proteins, or dyes to follow individual virus particles while they progress throughout infection. The inclusion of such fusion motifs or surrogates frequently disrupts viral infectivity or results in a change of the wild-type phenotype. Here, we detail the construction and functional characterization of two new constructs where we fused fluorescent proteins to the N-terminus of HIV-1 Integrase. In the first, IN is recruited into assembling particles via a codon optimized Gag to complement other viral constructs, while the second is fused to a Gag-Pol expression vector fully capable of integration. Our data shows that N-terminal tagged IN is functional for integration by both recovery of integration of catalytically inactive IN and by the successful infectivity of viruses carrying only labeled IN. These tools will be important to study the individual behavior of viral particles and associate such behavior to infectivity.

In Sub-Saharan Africa, young women 15–24 years of age account for nearly 30% of all new HIV infections, however, biological and epidemiological factors underlying this disproportionate infection rate are unclear. In this study, we assessed biological contributors of SIV/HIV susceptibility in the female genital tract (FGT) using adolescent (n = 9) and adult (n = 10) pigtail macaques (PTMs) with weekly low-dose intravaginal challenges of SIV. Immunological variables were captured in vaginal tissue of PTMs by flow cytometry and cytokine assays. Vaginal biopsies were profiled by proteomic analysis. The vaginal microbiome was assessed by 16S rRNA sequencing. We were powered to detect a 2.2-fold increase in infection rates between age groups, however, we identified no significant differences in susceptibility. This model cannot capture epidemiological factors or may not best represent biological differences of HIV susceptibility. No immune cell subsets measured were significantly different between groups. Inflammatory marker MCP-1 was significantly higher (adj p = .02), and sCD40L trended higher (adj p = .06) in vaginal cytobrushes of adults. Proteomic analysis of vaginal biopsies showed no significant (adj p

In Sub-Saharan Africa, young women 15–24 years of age account for nearly 30% of all new HIV infections, however, biological and epidemiological factors underlying this disproportionate infection ra...

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the coronavirus disease 2019 (COVID-19) with innate and adaptive immune response triggered in such patients by viral antigens. Both convalescent plasma and engineered high affinity human monoclonal antibodies have shown therapeutic potential to treat COVID-19. Whether additional antiviral soluble factors exist in peripheral blood remain understudied. Herein, we detected circulating exosomes that express the SARS-CoV-2 viral entry receptor angiotensin-converting enzyme 2 (ACE2) in plasma of both healthy donors and convalescent COVID-19 patients. We demonstrated that exosomal ACE2 competes with cellular ACE2 for neutralization of SARS-CoV-2 infection. ACE2-expressing (ACE2+) exosomes, but not the ACE2-negative controls, blocked the binding of the viral spike (S) protein RBD to ACE2+ cells in a dose dependent manner, which was 400- to 700-fold more potent than that of vesicle-free recombinant human ACE2 extracellular domain protein (rhACE2). As a consequence, exosomal ACE2 prevented SARS-CoV-2 pseudotype virus tethering and infection of human host cells at a 50–150 fold higher efficacy than rhACE2. A similar antiviral activity of exosomal ACE2 was further demonstrated to block wild-type live SARS-CoV-2 infection. Of note, depletion of ACE2+ exosomes from COVID-19 patient plasma impaired the ability to block SARS-CoV-2 RBD binding to host cells. Furthermore, a dramatic increase in plasma ACE2+ exosome levels were detected in patients with severe COVID-19 pathogenesis. Our data demonstrate that ACE2+ exosomes can serve as a decoy therapeutic and a possible innate antiviral mechanism to block SARS-CoV-2 infection.

Anti-HIV broadly neutralizing antibodies (bNAbs) may favor development of antiviral immunity by engaging the immune system during immunotherapy. Targeting integrin α(4)β(7) with an anti-α(4)β(7) monoclonal antibody (Rh-α(4)β(7)) affects immune responses in SIV/SHIV-infected macaques. To explore the therapeutic potential of combining bNAbs with α(4)β(7) integrin blockade, SHIV(SF162P3)-infected, viremic rhesus macaques were treated with bNAbs only (VRC07–523LS and PGT128 anti-HIV antibodies) or a combination of bNAbs and Rh-α(4)β(7) or were left untreated as a control. Treatment with bNAbs alone decreased viremia below 200 copies/ml in all macaques, but seven of eight macaques (87.5%) in the bNAbs-only group rebounded within a median of 3 weeks (95% CI: 2 to 9). In contrast, three of six macaques treated with a combination of Rh-α(4)β(7) and bNAbs (50%) maintained a viremia below 200 copies/ml until the end of the follow-up period; viremia in the other three macaques rebounded within a median of 6 weeks (95% CI: 5 to 11). Thus, there was a modest delay in viral rebound in the macaques treated with the combination antibody therapy compared to bNAbs alone. Our study suggests that α(4)β(7) integrin blockade may prolong virologic control by bNAbs in SHIV(SF162P3)-infected macaques.

During heterosexual HIV-1 transmission, a genetic bottleneck occurs in the newly infected individual as the virus passes from the mucosa, leading to systemic infection with a single transmitted HIV-1 clone in the recipient. This bottleneck in the recipient has just been described, and the mechanisms involved in this selection process have not been elucidated., Exposure of the genital mucosa to a genetically diverse viral swarm from the donor HIV-1 can result in breakthrough and systemic infection by a single transmitted/founder (TF) virus in the recipient. The highly diverse HIV-1 envelope (Env) in this inoculating viral swarm may have a critical role in transmission and subsequent immune response. Thus, chronic (Envchronic) and acute (Envacute) Env chimeric HIV-1 were tested using multivirus competition assays in human mucosal penile and cervical tissues. Viral competition analysis revealed that Envchronic viruses resided and replicated mainly in the tissue, while Envacute viruses penetrated the human tissue and established infection of CD4+ T cells more efficiently. Analysis of the replication fitness, as tested in peripheral blood mononuclear cells (PBMCs), showed similar replication fitness of Envacute and Envchronic viruses, which did not correlate with transmission fitness in penile tissue. Further, we observed that chimeric Env viruses with higher replication in genital mucosal tissue (chronic Env viruses) had higher binding affinity to C-type lectins. Data presented herein suggest that the inoculating HIV-1 may be sequestered in the genital mucosal tissue (represented by chronic Env HIV-1) but that a single HIV-1 clone (e.g., acute Env HIV-1) can escape this trapped replication for systemic infection. IMPORTANCE During heterosexual HIV-1 transmission, a genetic bottleneck occurs in the newly infected individual as the virus passes from the mucosa, leading to systemic infection with a single transmitted HIV-1 clone in the recipient. This bottleneck in the recipient has just been described (K. Klein et al., PLoS Pathog 14:e1006754, https://doi.org/10.1371/journal.ppat.1006754), and the mechanisms involved in this selection process have not been elucidated. However, understanding mucosal restriction is of the utmost importance for understanding dynamics of infections and for designing focused vaccines. Using our human penile and cervical mucosal tissue models for mixed HIV infections, we provide evidence that HIV-1 from acute/early infection, compared to that from chronic infection, can more efficiently traverse the mucosal epithelium and be transmitted to T cells, suggesting higher transmission fitness. This study focused on the role of the HIV-1 envelope in transmission and provides strong evidence that HIV transmission may involve breaking the mucosal lectin trap.

Background A long-acting implant for HIV pre-exposure prophylaxis (PrEP) is in development in the Sustained Long-Action Prevention Against HIV (SLAP-HIV) trial. This could provide an alternative to oral PrEP. Objective Our mixed methods study aimed to understand (1) users’ experiences with a similar subdermal implant for contraception and (2) factors influencing the likelihood that gay and bisexual men (GBM) would use a proposed PrEP implant. Methods Work was completed in 4 stages. In stage 1, we conducted a scientific literature review on existing subdermal implants, focusing on users’ experiences with implant devices. In stage 2, we reviewed videos on YouTube, focusing on the experiences of current or former contraceptive implant users (as these implants are similar to those in development in SLAP-HIV). In stage 3, individuals who indicated use of a subdermal implant for contraception in the last 5 years were recruited via a web-based questionnaire. Eligible participants (n=12 individuals who liked implants a lot and n=12 individuals who disliked implants a lot) completed in-depth phone interviews (IDIs) about their experiences. In stage 4, results from IDIs were used to develop a web-based survey for HIV-negative GBM to rate their likelihood of using a PrEP implant on a scale (1=very unlikely and 5=very likely) based on likely device characteristics and implant concerns identified in the IDIs. Results In the scientific literature review (stage 1), concerns about contraceptive implants that could apply to the PrEP implants in development included potential side effects (eg, headache), anticipated high cost of the device, misconceptions about PrEP implants (eg, specific contraindications), and difficulty accessing PrEP implants. In the stage 2 YouTube review, individuals who had used contraceptive implants reported mild side effects related to their device. In stage 3, implant users reported that devices were comfortable, unintrusive, and presented only minor discomfort (eg, bruising) before or after insertion and removal. They mainly reported removing or disliking the device due to contraceptive-related side effects (eg, prolonged menstruation). Participants in the stage 4 quantitative survey (N=304) were mainly gay (204/238, 85.7%), white (125/238, 52.5%), cisgender men (231/238, 97.1%), and 42.0% (73/174) of them were on oral PrEP. Not having to take a daily pill increased the likelihood of using PrEP implants (mean 4.13). Requiring >1 device to achieve 1 year of protection (mean range 1.79-2.94) mildly discouraged PrEP implant use. Participants did not mind moderate bruising, a small scar, tenderness, or bleeding after insertion or removal, and an implant with a size slightly larger than a matchstick (mean ratings 3.18-3.69). Conclusions PrEP implants are promising among GBM. Implant features and insertion or removal-related concerns do not seem to discourage potential users. To ensure acceptability, PrEP implants should require the fewest possible implants for the greatest protection duration.

Retroviruses cause severe diseases in animals and humans, including cancer and acquired immunodeficiency syndromes. To propagate infection, retroviruses assemble new virus particles that contain viral proteins and unspliced vRNA to use as gRNA. Despite the critical requirement for gRNA packaging, the molecular mechanisms governing the identification and selection of gRNA by the Gag protein remain poorly understood. In this report, we demonstrate that the Rous sarcoma virus (RSV) Gag protein colocalizes with unspliced vRNA in the nucleus in the interchromatin space. Using live-cell confocal imaging, RSV Gag and unspliced vRNA were observed to move together from inside the nucleus across the nuclear envelope, suggesting that the Gag-gRNA complex initially forms in the nucleus and undergoes nuclear export into the cytoplasm as a viral ribonucleoprotein (vRNP) complex., Packaging of genomic RNA (gRNA) by retroviruses is essential for infectivity, yet the subcellular site of the initial interaction between the Gag polyprotein and gRNA remains poorly defined. Because retroviral particles are released from the plasma membrane, it was previously thought that Gag proteins initially bound to gRNA in the cytoplasm or at the plasma membrane. However, the Gag protein of the avian retrovirus Rous sarcoma virus (RSV) undergoes active nuclear trafficking, which is required for efficient gRNA encapsidation (L. Z. Scheifele, R. A. Garbitt, J. D. Rhoads, and L. J. Parent, Proc Natl Acad Sci U S A 99:3944–3949, 2002, https://doi.org/10.1073/pnas.062652199; R. Garbitt-Hirst, S. P. Kenney, and L. J. Parent, J Virol 83:6790–6797, 2009, https://doi.org/10.1128/JVI.00101-09). These results raise the intriguing possibility that the primary contact between Gag and gRNA might occur in the nucleus. To examine this possibility, we created a RSV proviral construct that includes 24 tandem repeats of MS2 RNA stem-loops, making it possible to track RSV viral RNA (vRNA) in live cells in which a fluorophore-conjugated MS2 coat protein is coexpressed. Using confocal microscopy, we observed that both wild-type Gag and a nuclear export mutant (Gag.L219A) colocalized with vRNA in the nucleus. In live-cell time-lapse images, the wild-type Gag protein trafficked together with vRNA as a single ribonucleoprotein (RNP) complex in the nucleoplasm near the nuclear periphery, appearing to traverse the nuclear envelope into the cytoplasm. Furthermore, biophysical imaging methods suggest that Gag and the unspliced vRNA physically interact in the nucleus. Taken together, these data suggest that RSV Gag binds unspliced vRNA to export it from the nucleus, possibly for packaging into virions as the viral genome.

Therapeutic strategies are being clinically tested either to eradicate latent HIV reservoirs or to achieve virologic control in the absence of antiretroviral therapy. Attaining this goal will require a consensus on how best to measure the numbers of persistently infected cells with the potential to cause viral rebound after antiretroviral-therapy cessation in assessing the results of cure-directed strategies in vivo. Current measurements assess various aspects of the HIV provirus and its functionality and produce divergent results. Here, we provide recommendations from the BEAT-HIV Martin Delaney Collaboratory on which viral measurements should be prioritized in HIV-cure-directed clinical trials. This work was supported by the NIH-funded BEAT-HIV Martin Delaney Collaboratory to cure HIV-1 infection (1UM1Al126620). LJM is also supported by NIH R01 AI065279, U01 AI065279, R01 DA048728, R01 DA049666, Kean Family Professorship, and the Philadelphia Foundation (Roberts I. Jacobs Fund). M-AM is supported by NIH grants (DK123733, AG062383, NS117458, AI143385, AI129636, and NS106970), The Foundation for AIDS Research (amfAR) impact grant # 109840–65-RGRL, and W.W. Smith Charitable Trust grant # A1901, Wistar Cancer Center Support Grant P30 CA010815–49S2, and the Penn Center for AIDS Research (AI 045008). MJB is supported by The Miguel Servet program funded by the Spanish Health Institute Carlos III (CP17/00179). M. L. Is supported by NIH grants AI117841, AI120008, AI124776, AI130005, AI122377, and AI135940. XGY is supported by NIH grants AI116228, AI078799, HL134539, AI125109, and DA047034. RS supported by AI126603, AI126620 and AI12661, AI094189, 43222 Howard Hughes Medical Institute, and the Bill and Melinda Gates Foundation (OPP1115715). VP supported by AI143567, AI124843. Y-C Ho supported by Yale Top Scholar, Rudolf J. Anderson Fellowship, AI141009, DA047037, AI118402, W.W. Smith AIDS Research Grant, Gilead AIDS Research Grant, Gilead Research Scholar Grant, AI150464, AI094189, AI14868. J.D.E is supported by NIH and the Bill and Melinda Gates Foundation grants 75N93019C00070, AI133706, AI110164, AI141258, AI143411, AI149672, CA206466, DK119945, INV-002704, and OD011092–60, and OPPO1108533. Sí

Most new HIV infections, over 80%, occur through sexual transmission. During sexual transmission, the virus must bypass specific female and male reproductive tract anatomical barriers to encounter viable target cells. Understanding the generally efficient ability of these barriers to exclude HIV and the precise mechanisms of HIV translocation beyond these genital barriers is essential for vaccine and novel therapeutic development. In this review, we explore the mucosal, barriers of cervico-vaginal and penile tissues that comprise the female and male reproductive tracts. The unique cellular assemblies of the squamous and columnar epithelium are illustrated highlighting their structure and function. Each anatomical tissue offers a unique barrier to virus entry in healthy individuals. Unfortunately barrier dysfunction can lead to HIV transmission. How these diverse mucosal barriers have the potential to fail is considered, highlighting those anatomical areas that are postulated to offer a weaker barrier and are; therefore, more susceptible to viral ingress. Risk factors, such as sexually transmitted infections, microbiome dysbiosis, and high progestin environments are also associated with increased acquisition of HIV. How these states may affect the integrity of mucosal barriers leading to HIV acquisition are discussed suggesting mechanisms of transmission and revealing potential targets for intervention.

BACKGROUND A long-acting implant for HIV pre-exposure prophylaxis (PrEP) is in development in the Sustained Long-Action Prevention Against HIV (SLAP-HIV) trial. This could provide an alternative to oral PrEP. OBJECTIVE Our mixed methods study aimed to understand (1) users’ experiences with a similar subdermal implant for contraception and (2) factors influencing the likelihood that gay and bisexual men (GBM) would use a proposed PrEP implant. METHODS Work was completed in 4 stages. In stage 1, we conducted a scientific literature review on existing subdermal implants, focusing on users’ experiences with implant devices. In stage 2, we reviewed videos on YouTube, focusing on the experiences of current or former contraceptive implant users (as these implants are similar to those in development in SLAP-HIV). In stage 3, individuals who indicated use of a subdermal implant for contraception in the last 5 years were recruited via a web-based questionnaire. Eligible participants (n=12 individuals who liked implants a lot and n=12 individuals who disliked implants a lot) completed in-depth phone interviews (IDIs) about their experiences. In stage 4, results from IDIs were used to develop a web-based survey for HIV-negative GBM to rate their likelihood of using a PrEP implant on a scale (1=very unlikely and 5=very likely) based on likely device characteristics and implant concerns identified in the IDIs. RESULTS In the scientific literature review (stage 1), concerns about contraceptive implants that could apply to the PrEP implants in development included potential side effects (eg, headache), anticipated high cost of the device, misconceptions about PrEP implants (eg, specific contraindications), and difficulty accessing PrEP implants. In the stage 2 YouTube review, individuals who had used contraceptive implants reported mild side effects related to their device. In stage 3, implant users reported that devices were comfortable, unintrusive, and presented only minor discomfort (eg, bruising) before or after insertion and removal. They mainly reported removing or disliking the device due to contraceptive-related side effects (eg, prolonged menstruation). Participants in the stage 4 quantitative survey (N=304) were mainly gay (204/238, 85.7%), white (125/238, 52.5%), cisgender men (231/238, 97.1%), and 42.0% (73/174) of them were on oral PrEP. Not having to take a daily pill increased the likelihood of using PrEP implants (mean 4.13). Requiring >1 device to achieve 1 year of protection (mean range 1.79-2.94) mildly discouraged PrEP implant use. Participants did not mind moderate bruising, a small scar, tenderness, or bleeding after insertion or removal, and an implant with a size slightly larger than a matchstick (mean ratings 3.18-3.69). CONCLUSIONS PrEP implants are promising among GBM. Implant features and insertion or removal-related concerns do not seem to discourage potential users. To ensure acceptability, PrEP implants should require the fewest possible implants for the greatest protection duration.

Vertical transmission of human immunodeficiency virus (HIV) can occur in utero, during delivery, and through breastfeeding. We utilized Positron Emission Tomography (PET) imaging coupled with fluorescent microscopy of 64Cu-labeled photoactivatable-GFP-HIV (PA-GFP-BaL) to determine how HIV virions distribute and localize in neonatal rhesus macaques two and four hours after oral viral challenge. Our results show that by four hours after oral viral exposure, HIV virions localize to and penetrate the rectal mucosa. We also used a dual viral challenge with a non-replicative viral vector and a replication competent SHIV-1157ipd3N4 to examine viral transduction and dissemination at 96 hours. Our data show that while SHIV-1157ipd3N4 infection can be found in the oral cavity and upper gastrointestinal (GI) tract, the small and large intestine contained the largest number of infected cells. Moreover, we found that T cells were the biggest population of infected immune cells. Thus, thanks to these novel technologies, we are able to visualize and delineate of viral distribution and infection throughout the entire neonatal GI tract during acute viral infection., Author summary Approximately 1.8 million children are currently living with human immunodeficiency virus (HIV). While mother-to-child HIV transmission can occur in utero and during delivery, it most commonly occurs through breastfeeding, creating the need to understand how the virus moves throughout the body and infects the infant once breast milk is consumed. Here, we used multiple imaging techniques and PCR to determine how HIV distributes throughout the gastrointestinal tract after oral viral exposure and in which tissues and cell types become acutely infected. We found that HIV rapidly spreads throughout and penetrates the entire gastrointestinal tract as early as four hours after exposure. We also found that the intestine contained the largest number of infected cells at 96 hours and that most cells infected were T cells. Our study shows that these imaging technologies allow for the examination of viral distribution and infection in a rhesus macaque model.

Adolescents in Africa are at high risk for HIV infection, other sexually transmitted infections (STIs) and bacterial vaginosis (BV). Since behavior and burden of STIs/BV may influence HIV risk, behavioral risk factors and prevalence of STIs/BV were compared in HIV-seronegative adolescent females (n = 298; 16–22 years) from two South African communities (Soweto and Cape Town). STIs ( Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, Mycoplasma genitalium, herpes simplex virus (HSV)-1, HSV-2, Treponema pallidum, and Haemophilus ducreyi) were detected by multiplex polymerase chain reaction, human papillomavirus (HPV) by Roche Linear Array, and BV by Nugent scoring. Rates of BV (Nugent ≥7; 46.6%) and HPV (66.8%) were high in both communities. Prevalence of C. trachomatis and N. gonorrhoeae were >2-fold higher in Cape Town than Soweto (Chlamydia: 42% [62/149] versus 18% [26/148], p 70% of young women with treatable conditions that could enhance HIV risk would have been missed because they lacked symptoms associated with syndromic management.

A deficient interferon (IFN) response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been implicated as a determinant of severe coronavirus disease 2019 (COVID-19). To identify the molecular effectors that govern IFN control of SARS-CoV-2 infection, we conducted a large-scale gain-of-function analysis that evaluated the impact of human IFN-stimulated genes (ISGs) on viral replication. A limited subset of ISGs were found to control viral infection, including endosomal factors inhibiting viral entry, RNA binding proteins suppressing viral RNA synthesis, and a highly enriched cluster of endoplasmic reticulum (ER)/Golgi-resident ISGs inhibiting viral assembly/egress. These included broad-acting antiviral ISGs and eight ISGs that specifically inhibited SARS-CoV-2 and SARS-CoV-1 replication. Among the broad-acting ISGs was BST2/tetherin, which impeded viral release and is antagonized by SARS-CoV-2 Orf7a protein. Overall, these data illuminate a set of ISGs that underlie innate immune control of SARS-CoV-2/SARS-CoV-1 infection, which will facilitate the understanding of host determinants that impact disease severity and offer potential therapeutic strategies for COVID-19., Graphical abstract, Deficient interferon responses to SARS-CoV-2 infection have been associated with severe COVID-19. Martin-Sancho et al. utilized a gain-of-function screen to identify interferon-stimulated effectors that govern innate immune responses to SARS-CoV-2. These factors could underlie genetic predisposition to severe COVID-19 and can serve as candidates for development of antiviral therapies.