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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.

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.

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.

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.

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.

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 ra...

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.

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

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.

Understanding vaginal and rectal HIV transmission and protective cellular and molecular mechanisms is critical for designing new prevention strategies, including those required for an effective vaccine. The determinants of protection against HIV infection are, however, poorly understood. Increasing evidence suggest that innate immune defenses may help protect mucosal surfaces from HIV transmission in highly exposed, uninfected subjects 1. More recent studies suggest that systemically administered type 1 interferon protects against simian immunodeficiency virus infection of macaques 2. Here we hypothesized that topically applied type 1 interferons might stimulate vaginal innate responses that could protect against HIV transmission. We therefore applied a recombinant human type 1 interferon (IFN-β) to the vagina of rhesus macaques and vaginally challenged them with pathogenic simian/human immunodeficiency virus (SHIV). Vaginal administration of IFN-β resulted in marked local changes in immune cell phenotype, increasing immune activation and HIV coreceptor expression, yet provided significant protection from SHIV acquisition as interferon response genes (IRGs) were also upregulated. These data suggest that protection from vaginal HIV acquisition may be achieved by activating innate mucosal defenses.

Non-invasive bioluminescent imaging (NIBLI) of HIV-1 infection dynamics allows for real-time monitoring of viral spread and the localization of infected cell populations in living animals. In this report, we describe full-length replication-competent GFP and Nanoluciferase (Nluc) expressing HIV-1 reporter viruses from two clinical transmitted / founder (T/F) strains: TRJO.c and Q23.BG505. By infecting humanized mice with these HIV-1 T/F reporter viruses, we were able to directly monitor longitudinal viral spread at whole-animal resolution via NIBLI at a sensitivity of as few as 30–50 infected cells. Bioluminescent signal strongly correlated with HIV-1 infection and responded proportionally to virus suppression in vivo in animals treated daily with a combination antiretroviral therapy (cART) regimen. Longitudinal NIBLI following cART withdrawal visualized tissue-sites that harbored virus during infection recrudescence. Notably, we observed rebounding infection in the same lymphoid tissues where infection was first observed prior to ART treatment. Our work demonstrates the utility of our system for studying in vivo viral infection dynamics and identifying infected tissue regions for subsequent analyses., Author summary Non-invasive bioluminescent imaging (NIBLI) in small animals allows for in vivo longitudinal imaging of infection spread and pathogenesis. We have taken advantage of the small luciferase reporter protein, Nanoluciferase (Nluc), to generate a replication-competent HIV-1 reporter virus to allow for NIBLI of viral infection in humanized mice. NIBLI via Nluc enabled us to directly visualize longitudinal spreading patterns before, during, and after interruption of daily doses of combined antiretroviral therapy (cART). We observed that rebounding infection often emerged in tissue regions originally associated with infected cells prior to cART treatment. Thus, Nluc-based NIBLI of HIV-1 infection can be used as an experimental tool to study events involved in viral dissemination and spread from initial sites of infection to draining lymphoid tissues before and after cART suppression, as well as locate infected tissues for subsequent cellular characterization of HIV-1 infected cells.

We have recently shown that MUC16, a component of the glycocalyx of some mucosal barriers, has elevated binding to the G0 glycoform of the Fc portion of IgG. Therefore, IgG from patients chronically infected with human immunodeficiency virus (HIV), who typically exhibit increased amounts of G0 glycoforms, showed increased MUC16 binding compared to uninfected controls. Using the rhesus macaque simian immunodeficiency virus SIVmac251 model, we can compare plasma antibodies before and after chronic infection. We find increased binding of IgG to MUC16 after chronic SIV infection. Antibodies isolated for tight association with MUC16 (MUC16-eluted antibodies) show reduced FcγR engagement and antibody-dependent cellular cytotoxicity (ADCC) activity. The glycosylation profile of these IgGs was consistent with a decrease in FcγR engagement and subsequent ADCC effector function, as they contain a decrease in afucosylated bisecting glycoforms that preferentially bind FcγRs. Testing of the SIV antigen specificity of IgG from SIV-infected macaques revealed that the MUC16-eluted antibodies were enriched for certain specific epitopes, including regions of gp41 and gp120. This enrichment of specific antigen responses for fucosylated bisecting glycoforms and the subsequent association with MUC16 suggests that the immune response has the potential to direct specific epitope responses to localize to the glycocalyx through interaction with this specific mucin. IMPORTANCE Understanding how antibodies are distributed in the mucosal environment is valuable for developing a vaccine to block HIV infection. Here, we study an IgG binding activity in MUC16, potentially representing a new IgG effector function that would concentrate certain antibodies within the glycocalyx to trap pathogens before they can reach the underlying columnar epithelial barriers. These studies reveal that rhesus macaque IgG responses during chronic SIV infection generate increased antibodies that bind MUC16, and interestingly, these MUC16-tethered antibodies are enriched for binding to certain antigens. Therefore, it may be possible to direct HIV vaccine-generated responses to associate with MUC16 and enhance the antibody’s ability to mediate immune exclusion by trapping virions within the glycocalyx and preventing the virus from reaching immune target cells within the mucosa. This concept will ultimately have to be tested in the rhesus macaque model, which is shown here to have MUC16-targeted antigen responses.

HIV prevention method preferences were evaluated among 512 U.S. men who have sex with men (MSM; median age: 22 years). Approximately 90 % consistently preferred one option across pairwise comparisons of condoms, daily oral pre-exposure prophylaxis (PrEP), and long-acting PrEP delivered via either an injectable or one of two types of PrEP implants differing in visibility. Condoms were most frequently preferred (33.8 %), followed by non-visible implants (21.5 %), and oral PrEP (17.0 %); HIV risk was reported by more choosing implants. In a follow-up question comparing the four PrEP options only, daily oral pills and non-visible implants were most frequently preferred (35.5 and 34.3 %, respectively), followed by injections (25.2 %) and visible implants (4.3 %). An inductive, open-coding approach determined that convenience, duration of protection, and privacy were the most commonly cited reasons for a PrEP method choice, and associated with self-report of HIV risk. Tailoring PrEP product development to privacy and other concerns important to those at highest HIV risk may improve HIV prevention.

The difficulty in detecting rare infected cells immediately after mucosal HIV transmission has hindered our understanding of the initial cells targeted by the virus. Working with the macaque-simian immunodeficiency virus (SIV) vaginal challenge model, we developed methodology to identify discrete foci of SIV (mac239) infection 48 hours after vaginal inoculation. We find infectious foci throughout the reproductive tract, from labia to ovary. Phenotyping infected cells reveals that SIV has a significant bias for infection of CCR6+ CD4+ T cells. SIV infected cells expressed the transcriptional regulator RORγt confirming that the initial target cells are specifically of the Th17 lineage. Furthermore, we detect host responses to infection, as evidenced by apoptosis, cell lysis, and phagocytosis of infected cells. Thus, our analysis identifies Th17 lineage CCR6+ CD4+ T cells as primary targets of SIV during vaginal transmission. This opens new opportunities for interventions to protect these cells and prevent HIV transmission.

Pre-exposure prophylaxis (PrEP) with oral Truvada(®) prevents HIV infection. However, the adherence to pill taking required for efficacy has sparked interest in developing new antiretroviral delivery systems that decrease such demands. Long-acting formulations, such as injections and implants, represent promising options that require less frequent adherence. It is important, however, that development of these new modalities be driven by understanding of the value seen in them by target users to maximize their uptake. To identify the key product features that impact user acceptance, we used a three-phase marketing research approach. In this study, we describe the results of the first-phase, qualitative focus group research performed in Chicago and San Francisco that explored subjective perceptions of oral versus alternative PrEP modalities among men having sex with men (MSM) and medical practitioners caring for MSM. Data revealed that potential value in long-acting PrEP lies more in simplifying the lives of users rather than in making them more confident in their adherence. The results provide an important guidance for designing and promoting these future long-acting products to enhance their contribution to increasing the current limited uptake of PrEP that will better stem the HIV epidemic.

In a mature, infectious HIV-1 virion, the viral genome is housed within a conical capsid core comprised of the viral capsid (CA) protein. The CA protein, and the structure into which it assembles, facilitate virtually every step of infection through a series of interactions with multiple host cell factors. This review describes our understanding of the interactions between the viral capsid core and several cellular factors that enable efficient HIV-1 genome replication, timely core disassembly, nuclear import and the integration of the viral genome into the genome of the target cell. We then discuss how elucidating these interactions can reveal new targets for therapeutic interactions against HIV-1.

OBJECTIVE We sought to investigate the effects of HIV infection on the vaginal microbiota and associations with treatment and demographic factors. We thus compared vaginal microbiome samples from HIV-infected (HIV+) and HIV-uninfected (HIV-) women collected at two Chicago area hospitals. DESIGN We studied vaginal microbiome samples from 178 women analyzed longitudinally (n = 324 samples) and collected extensive data on clinical status and demographic factors. METHODS We used 16S rRNA gene sequencing to characterize the bacterial lineages present, then UniFrac, Shannon diversity, and other measures to compare community structure with sample metadata. RESULTS Differences in microbiota measures were modest in the comparison of HIV+ and HIV- samples, in contrast to several previous studies, consistent with effective antiretroviral therapy. Proportions of healthy Lactobacillus species were not higher in HIV- patients overall, but were significantly higher when analyzed within each hospital in isolation. Rates of bacterial vaginosis were higher among African-American women and HIV+ women. Bacterial vaginosis was associated with higher frequency of HIV+. Unexpectedly, African-American women were more likely to switch bacterial vaginosis status between sampling times; switching was not associated with HIV+ status. CONCLUSION The influence of HIV infection on the vaginal microbiome was modest for this cohort of well suppressed urban American women, consistent with effective antiretroviral therapy. HIV+ was found to be associated with bacterial vaginosis. Although bacterial vaginosis has previously been associated with HIV transmission, most of the women studied here became HIV+ many years before our test for bacterial vaginosis, thus implicating additional mechanisms linking HIV infection and bacterial vaginosis.

Purpose of review Recently, studies have suggested a role for Fc-mediated effector functions in viremic control of HIV infection and blocking HIV acquisition. Although progress has been made in identifying the mechanisms responsible for regulating various innate functions, minimal research has been performed concerning macrophage-specific phagocytosis and antiviral effects. Recent findings Of what research has been performed, phagocytosis has been identified as a possible key player in antiviral functions during initial infection, offering protection at the HIV mucosal entry sites. Recent research has also highlighted the importance of various antibody characteristics, such as polymorphism, immunoglobulin subclass, and glycan structure on those effector functions modulated. Lastly, despite recent failures in HIV vaccine trials, the RV144 Thai trial illustrated 31.2% efficacy against heterosexual infection. When these protective results were looked at in depth, vaccine-induced antibodies were increased when infection rates decreased, suggesting that HIV might be neutralized through receptor-mediated effector mechanisms, including phagocytosis. Importantly, these data instilled the awareness that the identification of protective immune correlates is imperative to successfully develop vaccine strategies. Summary In this review, we address the antiviral mechanisms of phagocytosis, focusing on complement-mediated phagocytosis and Fc-receptor-mediated antibody-dependent cellular phagocytosis in relation to HIV transmission and infection.

Antibody capacity to recognize infectious virus is a prerequisite of many antiviral functions. We determined the infectious virion capture index (IVCI) of different antibody specificities. Whereas broadly neutralizing antibodies (bNAbs), except for an MPER bNAb, selectively captured infectious virions, non-bNAbs and mucosal human immunodeficiency virus type 1 (HIV-1)-positive IgG captured subsets of both infectious and noninfectious virions. Infectious virion capture was additive with a mixture of antibodies, providing proof of concept for vaccine-induced antibodies that together have improved capacity to recognize infectious virions.

The presence of multiple inflammatory cytokines predicts the failure of a topical 1% tenofovir microbicide gel to protect women against HIV infection.

Progestin-only long-acting reversible contraceptives (LARCs) are increasingly popular among women seeking contraception; however, recent epidemiological studies suggest that systemically administered medroxyprogesterone acetate (MPA) may increase HIV acquisition. In order to determine the exact mechanisms underlying increases in transmission specific to MPA use and to test safer, alternative contraceptive progestin types and delivery methods, in vitro modeling studies must be performed. To achieve this, it is imperative that accurate hormone concentrations be utilized when modeling progestin-mediated outcomes, as the down-stream effects are dose-dependent. The local concentrations of progestins to which the lower female genital tract tissues are exposed after initiation of LARCs are unknown, but they likely differ from peripheral concentrations, dependent upon the progestin type and delivery method. Here, we measured in vivo endocervical and plasma concentrations of (1) systemically-delivered depo MPA (DMPA), (2) levonorgestrel (LNG) delivered via intrauterine system (IUS) and (3) etonogestrel (ETG) delivered via vaginal ring in women who recently initiated contraception treatment. Levels of ETG and LNG in cervical secretions were 100-200 fold higher than plasma levels. In contrast, measurable MPA levels were approximately 10-fold higher in plasma compared to cervical secretions. These results will inform the design of accurate in vitro studies on the influence of progestins on epithelial cells, tissue explants, and peripheral blood cells, to be able to better predict in vivo outcomes. Subsequent observations will aid in determining how MPA might influence HIV acquisition and may facilitate identification of optimal progestin-containing LARC alternatives for women at high risk for HIV infection.

Cervical and vaginal epithelia are primary barriers against human immunodeficiency virus type I (HIV-1) entry during male-to-female transmission. Cervical mucus (CM) is produced by the endocervix and forms a layer locally as well as in the vaginal compartment in the form of cervicovaginal mucus (CVM). To study the potential barrier function of each mucus type during HIV-1 transmission, we quantified HIV-1 mobility in CM and CVM ex vivo using fluorescent microscopy. Virions and 200-nm PEGylated beads were digitally tracked and mean squared displacement was calculated. The mobility of beads increased significantly in CVM compared to CM, consistent with the known decreased mucin concentration of CVM. Unexpectedly, HIV-1 diffusion was significantly hindered in the same CVM samples in which bead diffusion was unhindered. Inhibition of virus transport was envelope-independent. Our results reveal a previously unknown activity in CVM that is capable of impeding HIV-1 mobility to enhance mucosal barrier function.

HIV-1-infected individuals with protective HLA class I alleles exhibit better control of viremia and slower disease progression. Virus control in these individuals has been associated with strong and potent HIV-1-specific cytotoxic-T-lymphocyte (CTL) responses restricted by protective HLA alleles, but control of viremia also occurs in the presence of selected CTL escape mutations. CTL escape mutations restricted by protective HLA class I molecules are frequently located in the conserved p24 Gag sequence of HIV-1 that encodes the conical capsid core and have been suggested to reduce viral replication capacity. In this study, the consequences of well-described CTL-associated p24 Gag sequence mutations for HIV-1 capsid stability were assessed using a cyclosporine (CsA) washout assay. The frequently occurring HLA-B57- and HLA-B27-associated CTL escape mutations T242N and R264K resulted in delayed capsid uncoating, suggesting modulation of capsid stability. The described compensatory mutations L268M and S173A observed in R264K viruses reconstituted the capsid-uncoating half-time. Interestingly, capsid stability was correlated with infectivity. Taken together, these data demonstrate that CTL-driven escape mutations within p24 Gag restricted by protective HLA class I alleles have a significant impact on capsid stability that might contribute to the persistent control of viral replication observed despite viral escape from CTL responses. IMPORTANCE Sequence mutations within p24 Gag selected by CTL responses restricted by protective HLA class I alleles have been associated with reduced viral fitness. However, the precise mechanisms underlying the reduced viral replication capacity and lower viral loads associated with these mutations remain unclear. Here, we demonstrate that dominant HLA-B27-associated CTL escape mutations within HIV-1 capsid lead to enhanced capsid rigidity, providing a possible mechanism for the reduced viral fitness of these variants.

Table of contents A1 Introduction to the 2nd synchronicity forum of GHRI/CHVI-funded Canadian and African HIV prevention and vaccine teams O1 Voluntary medical male circumcision for prevention of heterosexual transmission of HIV in adult males in Soweto: What do indicators and incidence rate show? Hillary Mukudu, Neil Martinson, Benn Sartorius O2 Developing a peer-led community mobilization program for sex workers in Soweto: HIV risk and demographics Jenny Coetzee, Janan Dietrich, Kgaugelo Mokgatswana, Rachel Jewkes, Glenda E. Gray O3 Salient beliefs about adherence: A qualitative survey conducted as part of the demonstration study on "treatment as prevention" (TasP) and "pre-exposure prophylaxis" (PrEP) among female sex workers (FSWS) in Cotonou, Benin Marylène Dugas, Luc Béhanzin, Fernand A. Guédou, Marie-Pierre Gagnon, Michel Alary O4 Relative perception of risk as a driver of unsafe sexual practices among key populations: Cases of fisherfolk and women and their partners involved in multiple sexual partnerships in Uganda Rwamahe Rutakumwa, Martin Mbonye, Thadeus Kiwanuka, Sarah Nakamanya, Richard Muhumuza, Winfred Nalukenge, Janet Seeley O5 Exploring the acceptability of new biomedical HIV prevention technologies among MSM, adolescents and heterosexual adults in South Africa Millicent Atujuna, Melissa Wallace, Ben Brown, Linda Gail Bekker, Peter A. Newman O6 HIV-susceptible target cells in foreskins after voluntary medical male circumcision in South Africa Rushil Harryparsad, Abraham J. Olivier, Heather B. Jaspan, Douglas Wilson, Janan Dietrich, Neil Martinson, Hillary Mukudu, Nonhlanhla Mkhize, Lynn Morris, Gianguido Cianci, Minh Dinh, Thomas Hope, Jo-Ann S. Passmore, Clive M. Gray O7 HIV-1 proteins activate innate immune responses via TLR2 heterodimers Bethany M. Henrick, Xiao-Dan Yao, Kenneth L. Rosenthal, the INFANT Study Team O8 Characterization of an innate factor in human milk and mechanisms of action against HIV-1 Bethany M. Henrick, Xiao-Dan Yao, Anna G. Drannik, Alash’le Abimiku, Kenneth L. Rosenthal, the INFANT Study Team O9 Secretor status and susceptibility to HIV infections among female sex workers in Nairobi, Kenya Nadia Chanzu, Walter Mwanda, Julius Oyugi, Omu Anzala O10 Natural Killer cell recall responsiveness to Gag-HIV-1 peptides of HIV-1 exposed but uninfected subjects are associated with peripheral CXCR6+ NK cell subsets Moustapha Mbow, Sabelle Jallow, Moussa Thiam, Alberta Davis, Assane Diouf, Cheikh T. Ndour, Moussa Seydi, Tandakha N. Dieye, Souleymane Mboup, Martin Goodier, Eleanor Rilley, Assan Jaye O11 Profiles of resistance: Local innate mucosal immunity to HIV-1 in commercial sex workers Xiao-Dan Yao, RW. Omange, Bethany M. Henrick, Richard T. Lester, Joshua Kimani, T. Blake Ball, Francis A. Plummer, Kenneth L. Rosenthal O12 Early antiretroviral therapy and pre-exposure prophylaxis for HIV prevention among female sex workers in Cotonou, Benin: A demonstration project Luc Béhanzin, Fernand A. Guédou, Nassirou Geraldo, Ella Goma Mastétsé, Jerôme Charles Sossa, Marcel Djimon Zannou, Michel Alary O13 Building capacity for HIV prevention trials: Preliminary data from a Nigerian cohort of HIV exposed sero-negatives (HESN) Sophia Osawe, Evaezi Okpokoro, Felicia Okolo, Stephen Umaru, Rebecca Abimiku, Sam Audu, Pam Datong, Alash’le Abimiku O14 Equipping healthcare professionals with skills required for the conduct of clinical trials in an effort to build capacity. Lessons learned Jacquelyn Nyange, Joyce Olenja, Gaudensia Mutua, Walter Jaoko, Gloria Omosa-Manyonyi, Bashir Farah, Maureen Khaniri, Omu Anzala O15 Educational technology to support active learning for HIV researchers and planners Anne Cockcroft, Kendra Tonkin, Indu Girish, Puna Mhati, Ashley Cunningham, Neil Andersson O16 From Lake Kivu (Rwanda) and Lake Malawi (Tanzania) to the shores of Lake Victoria (Uganda): Strengthening laboratory capacity through Good Clinical Laboratory Practice training Bashir Farah, Jackton Indangasi, Walter Jaoko, Gaudensia Mutua, Maureen Khaniri, Jacquelyn Nyange, Omu Anzala O17 Rilpivirine and etravirine resistance mutations in HIV-1 subtype C infected patients on a non-nucleoside reverse transcriptase inhibitor-based combination antiretroviral therapy in Botswana Thabo Diphoko, Simani Gaseitsiwe, Victoria Maiswe, Thato Iketleng, Dorcas Maruapula, Keabetswe Bedi, Sikhulile Moyo, Rosemary Musonda, Mark Wainberg, Joseph Makhema, Vladimir Novitsky, Richard Marlink, Max Essex O18 From home-based HIV testing to initiation of treatment: The AIDS Support Organization (TASO) Experience with Home-based HIV Counselling and Testing (HBHCT) among Adolescents in Uganda, 2005-2011 Stephen Okoboi, Livingstone Ssali, Sam Kalibala, Josephine Birungi, Aggrey Egessa, Jonathan Wangisi, Lyavala Joanne Okullu, Celestin Bakanda, Francis Obare41 O19 Feasibility study on using real time medication monitoring among HIV infected and Tuberculosis patients in Kilimanjaro, Tanzania I. Marion Sumari-de Boer, Hadija H. Semvua, Jossy van den Boogaard, Krisanta W. Kiwango, Kennedy M. Ngowi, Pythia T. Nieuwkerk, Rob E. Aarnoutse, Ireen Kiwelu, Eva Muro, Gibson S. Kibiki O20 Deaths still among sero-discordant cohort in Nigeria despite Access to treatment Ruth Datiri, Grace Choji, Sophia Osawe, Evaezi Okpokoro, Felicia Okolo, Stephen Umaru, Rebecca Abimiku, Samuel Audu, Pam Datong, Alash’le Abimiku O21 Therapeutic HIV-1 vaccine trials in Denmark and Guinea-Bissau Fomsgaard A, Karlsson I, Jensen KJ, Jensen SS, Leo-Hansen C, Jespersen S, Da Silva Té D, Rodrigues CM, da Silva ZJ, Janitzek CM, Gerstoft J, Kronborg G, the WAPHIR Group O22 Willingness to participate in a HIV vaccine Trial among HIV exposed sero-negative (HESN) persons in Jos, Nigeria Evaezi Okpokoro, Sophia Osawe, Ruth Daitiri, Grace Choji, Stephen Umaru, Felicia Okolo, Pam Datong, Alash'le Abimiku O23 Clinical research volunteers’ perceptions and experiences of screening for enrolment at KAVI-Institute of Clinical Research, Kenya Nyariki Emily, Olenja Joyce, Lorway R. Robert, Anzala Anzala O24 Gut microbiome, HIV-exposure, and vaccine responses in South African infants Katie Viljoen, Jerome Wendoh, Elvis Kidzeru, Ulas Karaoz, Eoin Brodie, Gerrit Botha, Nicola Mulder, Clive Gray, William Cameron, Alain Stintzi, Heather Jaspan, for the INFANT study team O25 Analysis of HIV pol diversity in the concentrated HIV epidemic in Saskatchewan Paul N. Levett, David Alexander, Naveed Gulzar, Prabvir S. Grewal, Art F. Y. Poon, Zabrina Brumme, P. Richard Harrigan, James I. Brooks, Paul A. Sandstrom, Stryker Calvez, Stephen E. Sanche, Jamie K. Scott P1 Evaluating a HIV vaccine research community engagement programme at two HIV prevention research centres in the Western Cape Leslie Swartz, Ashraf Kagee, Anthea Lesch, Zuhayr Kafaar, Anneliese De Wet P2 Validating HIV acquisition risk score using a cohort HIV exposed sero-negative persons in a discordant relationship in Jos, Nigeria, West Africa Evaezi Okpokoro, Sophia Osawe, Ruth Daitiri, Grace Choji, Stephen Umaru, Felicia Okolo, Pam Datong, Alash'le Abimiku P3 Bridging the gap between adults and adolescents and youth adults (AYA) – Employing a youth-centred approach to investigate HIV risk among AYA in Soweto and Durban, South Africa Janan Dietrich, Tricia Smith, Laura Cotton, Stefanie Hornschuh, Martin van der Watt, Cari L. Miller, Glenda Gray, Jenni Smit, Manjeetha Jaggernath, Thumbi Ndung’u, Mark Brockman, Angela Kaida, on behalf of the AYAZAZI study teams P4 Neighbours to sex workers: A key population that has been ignored Maureen Akolo, Joshua Kimani, Prof Larry Gelmon, Michael Chitwa, Justus Osero P5 Young women’s access to structural support programmes in a district of Botswana Anne Cockcroft, Nobantu Marokoane, Leagajang Kgakole, Boikhutso Maswabi, Neo Mpofu, Umaira Ansari, Neil Andersson P6 Voices for action from peri-urban Ugandan students, teachers and parents on HIV/STI prevention: Qualitative research results Nakinobe Elizabeth, Miiro George Mukalazi, Zalwango Flavia, Nakiyingi-Miiro Jessica, Kaleebu Potiano P7 Engaging Social Media as an education tool on the fly: The use of Facebook for HIV and Ebola prevention and awareness amongst adolescents in Uganda John Ross Semwanga, Emily Nyanzi, Saidat Namuli Musoke, Elizabeth Nakinobe, George Miiro, Edward Katongole Mbidde, Tom Lutalo, Pontiano Kaleebu P8 Circulating HIV-1 subtypes among sexual minority populations in Zambia Ray Handema, Graham P. Chianzu P9 The Development of HIV Bio-bank resource management to support clinical trial and Intervention research: WAPHIR experience Moussa Thiam, Diabou Diagne-Gueye, Mame K. Ndiaye, Moustapha Mbow, Birahim P. Ndiaye, Ibrahima Traore, Mamadou C. Dia, Gilleh Thomas, Coumba Tour-Kane, Souleymane Mboup, Assan Jaye P10 Capacity building for clinical trials as a novel approach for scaling up HIV prevention research initiatives in East Africa: achievements and challenges Emily Nyanzi, Edward Katongole Mbidde, Pontiano Kaleebu, Juliet Mpendo, Joshua Kimani, Josephine Birungi, Winnie Muyindike, Andrew Kambugu P11 Community and media perspective of research; an advocacy workshop on HIV prevention research Hachizovu Sebastian, Handema Ray, Chaponda Mike, Kabuya Jean Bertin, Mulenga Modest P12 Development of a quantitative HIV-1 and HIV-2 real time PCR (qRT-PCR) viral load assay Moussa Thiam, Omar Janha, Alberta Davis, Alfred Amambua-Ngwa, Davis C. Nwakanma, Souleymane Mboup, Assan Jaye P13 Differential effects of sex in a West African Cohort of HIV-1, HIV-2 and HIV-1/2 dual infected patients: Men are worse off Sanne Jespersen, Bo Langhoff Hønge, Joakim Esbjörnsson, Candida Medina, David Da Silva TÉ, Faustino Gomes Correira, Alex Lund Laursen, Lars Østergaard, Andreas Andersen, Peter Aaby, Christian Erikstrup, Christian Wejse, for the Bissau HIV Cohort study group P14 HIV-infected adolescents in transition from pediatric to adult HIV care in Dakar, Senegal: sample characteristics and immunological and virological profiles Siry Dieye, Moussa Sarr, Haby Sy, Helene D Mbodj, Marianne Ndiaye, Amy Ndiaye, Seydi Moussa, Assan Jaye, Souleymane Mboup100 P15 Molecular characterization of vertically transmitted HIV-1 among children born to HIV-1 seropositive mothers in Northern Tanzania Balthazar M. Nyombi, Elichilia R. Shao, Innocent B. Chilumba, Sikhulile Moyo, Simani Gaseitsiwe, Rosemary Musonda P16 Breast-fed HIV-1 exposed infants play catch up. A preliminary report Pam Datong, Bucky Inyang, Sophia Osawe, Abel Izang, Chundung Cole, Felicia Okolo, Bill Cameron, Kenneth Rosenthal, Clive Gray, Heather Jaspan, Alash’le Abimiku, the INFANT study team P17 The frequency of N348I mutation in patient failing combination antiretroviral treatment In Botswana Boitumelo Seraise, Kerstin Andrea-Marobela, Sikhulile Moyo, Rosemary Musonda, Joseph Makhema, Max Essex, Simani Gaseitsiwe

Cellular entry of retroviruses is the first critical stage of retroviral replication. Live cell imaging has been utilized to visualize the dynamics, localization, and kinetics of the viral fusion process. Here, we review the different methodologies used for live cell imaging and how the use of these techniques has better elucidated the viral entry process of avian sarcoma and leukosis virus (ASLV) and human immunodeficiency virus type 1 (HIV-1) as well as cell-to-cell transmission of retroviruses. Although some controversies remain, further development of these techniques will provide new insights into the process and dynamics of retroviral fusion in vivo.

Live cell imaging is a valuable technique that allows the characterization of the dynamic processes of the HIV-1 life cycle. Here, we present a method of production and imaging of dual-labeled HIV viral particles that allows the visualization of two events. Varying release of the intravirion fluid phase marker reveals virion fusion and the loss of the integrity of HIV viral cores with the use of live wide-field fluorescent microscopy.

HIV sexual transmission via the male genital tract remains poorly defined. Male circumcision was shown to reduce female-to-male transmission in Africa, providing a clue that the foreskin plays a role in the route of transmission. Scientific data in four categories relating to how the foreskin might affect HIV transmission is summarized: (i) surface area, (ii) microbiologic environment, (iii) HIV-1-susceptible cells, and (iv) tissue structure. The relative contribution of each of these areas is yet unknown, and further studies will be crucial in understanding how male circumcision affects HIV transmission in men.

The decrease in HIV acquisition after circumcision suggests a role for the foreskin in HIV transmission. However, the mechanism leading to protection remains undefined. Using tissue explant cultures we found that Langerhans cells (LCs) in foreskin alter their cellular protein expression in response to external stimuli. Furthermore, we observe that upon treatment with TNF-alpha, tissue-resident LCs became activated and that stimulatory cytokines can specifically cause an influx of CD4+ T-cells into the epithelial layer. Importantly, both of these changes are significant in the inner, but not outer, foreskin. In addition, we find that LCs in the inner foreskin have increased ability to sample environmental proteins. These results suggest differences in permeability between the inner and outer foreskin and indicate that HIV target cells in the inner foreskin have increased interaction with external factors. This increased responsiveness and sampling provides novel insights into the underlying mechanism of how circumcision can decrease HIV transmission.

Ebola virus (EBOV) infects several cell types and while viral entry is known to be pH-dependent, the exact entry pathway(s) remains unknown. To gain insights into EBOV entry, the role of several inhibitors of clathrin-mediated endocytosis in blocking infection mediated by HIV pseudotyped with the EBOV envelope glycoprotein (EbGP) was examined. Wild type HIV and envelope-minus HIV pseudotyped with Vesicular Stomatitis Virus glycoprotein (VSVg) were used as controls to assess cell viability after inhibiting clathrin pathway. Inhibition of clathrin pathway using dominant-negative Eps15, siRNA-mediated knockdown of clathrin heavy chain, chlorpromazine and sucrose blocked EbGP pseudotyped HIV infection. Also, both chlorpromazine and Bafilomycin A1 inhibited entry of infectious EBOV. Sensitivity of EbGP pseudotyped HIV as well as infectious EBOV to inhibitors of clathrin suggests that EBOV uses clathrin-mediated endocytosis as an entry pathway. Furthermore, since chlorpromazine inhibits EBOV infection, novel therapeutic modalities could be designed based on this lead compound.

By using immunofluorescence microscopy to observe and analyze freshly made HIV-1 virions adsorbed onto cells, we found that they are inherently highly infectious, rather than predominantly defective as previously suggested. Surprisingly, polycations enhance titers 20- to 30-fold by stabilizing adsorption and preventing a previously undescribed process of rapid dissociation, strongly implying that infectivity assays for many viruses are limited not only by inefficient virus diffusion onto cells but also by a postattachment race between entry and dissociation. This kinetic competition underlies inhibitory effects of CCR5 antagonists and explains why adaptive HIV-1 mutations overcome many cell entry limitations by accelerating entry.

The vast majority of new HIV infections are acquired via the genital and rectal mucosa. Here, we provide an overview of our current knowledge of how HIV establishes local infection, with an emphasis on viral invasion through the female genital tract. Studies using human explant tissues and in vivo animal studies have improved our understanding of the cellular and molecular pathways of infection; this information could be harnessed to design effective HIV vaccines and microbicides.

Efforts to decrease the spread of HIV worldwide continue at a rapid pace. With the development of new biomedical interventions and findings from pivotal clinical trials, a new framework for short-term and long-term prevention strategies is emerging. It is clear that biomedical-based approaches targeted at the highest risk populations have the greatest potential to have a short-term impact. Unfortunately, challenges with adherence in healthy populations at risk are now well-recognized, and competing health care priorities in the context of fragile delivery infrastructures pose formidable obstacles to implementation. We need better ways to identify high-risk populations, sophisticated understanding of the behavioral parameters that can ensure adherence, and the development of better strategies to provide sustained delivery of preexposure prophylaxis (PrEP). In the long term, we need an effective vaccine--a path that has proven to be rocky. Research facilitating an increased understanding of immune responses and what represents effective responses to prevent HIV acquisition should facilitate progress. While we wait for that time, PrEP offers the best strategy for short-term impact.

Uncoating of Human Immunodeficiency Virus type 1 (HIV-1) and type 2 (HIV-2) conical cores is an important early step for establishment of infection. In Old World Monkey (OWM) cells, the TRIM5α cellular factor potently suppresses an early step of infection by HIV-1. Previously, biochemical studies using whole cell lysates of infected cells revealed that OWM TRIM5α accelerates the uncoating of HIV-1, leading to premature reverse transcription. In the present study, we re-evaluated uncoating kinetics of HIV-1 in the presence of OWM TRIM5α by using an in situ uncoating assay, which allowed us to differentiate productive HIV-1 entry from simple (non-productive) endocytosis. Results showed that the uncoating kinetics of HIV-1 was indeed accelerated in the presence of OWM TRIM5α. Furthermore, we adapted an in situ uncoating assay to HIV-2, which showed wide variations in TRIM5α sensitivity among different isolates. HIV-2 isolate GH123, whose infectivity was suppressed by cynomolgus monkey (CM) TRIM5α, showed accelerated uncoating in the presence of CM TRIM5α. In contrast, mutant HIV-2 ASA, whose infectivity was unaltered by CM TRIM5α, showed no change in uncoating kinetics in the presence of CM TRIM5α. These results confirmed and further extended the previous notion that accelerated uncoating is associated with restriction activity of TRIM5α against lentiviruses.

The intracellular steps involved in viral infection, namely cytoplasmic trafficking and nuclear import, are critical events in the viral life cycle that have lagged behind other areas of viral research. This review examines recent advances in our understanding of these steps for viruses commonly employed as viral gene delivery vectors. Steps governing the cytoplasmic trafficking and nuclear import of Herpes Simplex virus, Human Immunodeficiency virus and Adenovirus are reviewed in this article.

HIV produces structural, regulatory, and accessory proteins during viral replication in host cells. The accessory proteins include Nef, viral infectivity factor (Vif), viral protein R, and viral protein U or viral protein X. Although these accessory proteins are generally dispensable for viral replication in vitro, they are essential for viral pathogenesis in vivo. Consequently, there has been much interest in understanding how these accessory proteins function because this research may yield new antiviral targets to curb HIV pathogenesis in vivo. Therefore, this review highlights recent advances in understanding the HIV accessory proteins and emphasizes breakthrough insights into the elusive Vif protein and potential new targets for therapeutic intervention.

After viral fusion with the cell membrane, the conical capsid of HIV-1 disassembles by a process called uncoating. We recently utilized the cyclosporine (CsA) washout assay, in which TRIM-CypA-mediated restriction of viral replication is used to detect the state of the viral capsid, to study the kinetics of uncoating in HIV-1-infected cells. Here we have extended this analysis to examine the effects of p24 capsid protein (p24 CA ) mutations and cellular environment on the kinetics of uncoating in infected cells. We found that p24 CA mutations can significantly increase (A92E), delay (E45A and N74D), or have no effect (G94D) on the rate of uncoating and that these alterations are not due to changes in reverse transcription. Inhibition of reverse transcription delayed uncoating kinetics to an extent similar to that of the wild-type virus with all the p24 CA mutant viruses tested. In addition, we observed differences in uncoating in two cell lines, which suggests that the cellular environment can differentially impact the disassembly of wild-type and mutant capsids. Collectively, these experiments suggest that viral and cellular factors are important for the process of uncoating. Finally, these data support the model whereby early steps in reverse transcription facilitate HIV-1 uncoating. IMPORTANCE The HIV-1 capsid is a cone-shaped structure, composed of the HIV-1-encoded protein p24 CA , which contains the viral RNA and other proteins needed for infection. After the virus enters a target cell, this capsid must disassemble by a process called uncoating. Uncoating is required for HIV-1 infection to progress, but the details of how this process occurs is not known. In this study, we used an in vivo assay to examine the uncoating process in HIV-1-infected cells. We determined that p24 CA mutations could increase or decrease the rate of uncoating and that this rate varied in different cell lines. We also found that reverse transcription of the viral RNA altered the process of uncoating before the p24 CA mutations. Collectively, these experiments provide a better understanding of how viral and cellular factors are involved with a poorly understood step in HIV-1 infection.

In addition to the structural proteins required for viral replication HIV expresses several additional proteins. Although these proteins are not required for viral replication in tissue culture systems they are important factors for the development of pathogenesis. These factors known as accessory proteins include the HIV proteins Nef Vpr Vpu and Vif. The accessory proteins are a defining feature of lentiviruses and are necessary for HIV to cause AIDS. Because the accessory proteins play an important role in causing the clinical manifestations of HIV disease they have become potential targets for antiviral therapies and many basic research studies have been designed to gain an understanding of their modes of action. The following discussion highlights a number of important insights that have emerged over the past year into how this important class of HIV protein functions. (excerpt)

Herpes simplex virus genes are predominantly intronless. We identified cis -acting elements in the intronless herpes simplex virus type 1 thymidine kinase (TK) gene that facilitate intron-independent gene expression. TK sequences functionally replaced the hepatitis B virus (HBV) posttranscriptional regulatory element (PRE) by inducing the expression of the intronless HBV surface message. TK also activated the pDM138 assay by inducing the cytoplasmic accumulation of intron-containing RNA. Multiple cis -acting RNA sequences, or subelements, that induce cytoplasmic localization of unspliced RNA were mapped within the TK gene. The presence of multiple RNA subelements within the TK gene is reminiscent of the multiple subelements in the HBV PRE required for the cytoplasmic accumulation of intronless HBV RNAs. Similar to HBV PRE subelements, duplication of a single TK subelement resulted in greater-than-additive increases in activity. A reporter chimera containing a single TK subelement juxtaposed to an HBV PRE subelement demonstrated a commensurate increase in activity. These results suggest that viral intronless genes utilize a similar strategy for intron-independent gene expression that requires multiple cis -acting RNA signals. Furthermore, like HBV PRE-containing RNA, TK cytoplasmic localization is not sensitive to leptomycin B, a drug that inhibits the export of proteins containing nuclear export signals. From this, we conclude that proteins that bind TK and facilitate its cytoplasmic accumulation do not travel through a CRM1-dependent RNA transport pathway.

Human immunodeficiency virus type 1 Rev export depends upon the presence of the nuclear export signal (NES), a leucine-rich stretch of hydrophobic amino acids. Recently, the nuclear NES-binding receptor has been identified as CRM1 or exportin 1. Rev export has been shown to be CRM1 dependent. The function of the atypical NES-containing Rev-like proteins of equine infectious anemia virus (EIAV) and feline immunodeficiency virus (FIV) is inhibited by leptomycin B, a drug that specifically blocks NES-CRM1 interactions. These data suggest that the function of atypical NES-containing proteins is CRM1 dependent. In contrast to the inhibition of EIAV Rev and FIV Rev, the cytoplasmic accumulation of hepatitis B virus (HBV) posttranscriptional regulatory element (PRE)-containing and Mason-Pfizer monkey virus (MPMV) constitutive transport element (CTE)-containing RNAs is not inhibited by leptomycin B treatment. We conclude that the HBV PRE, like the MPMV CTE, functions independently of an NES receptor-exportin 1 interaction.