Commensal Bacteria Modulate Innate Immune Responses of Vaginal Epithelial Cell Multilayer Cultures

The human vaginal microbiome plays a critical but poorly defined role in reproductive health. Vaginal microbiome alterations are associated with increased susceptibility to sexually-transmitted infections (STI) possibly due to related changes in innate defense responses from epithelial cells. Study of the impact of commensal bacteria on the vaginal mucosal surface has been hindered by current vaginal epithelial cell (VEC) culture systems that lack an appropriate interface between the apical surface of stratified squamous epithelium and the air-filled vaginal lumen. Therefore we developed a reproducible multilayer VEC culture system with an apical (luminal) air-interface that supported colonization with selected commensal bacteria. Multilayer VEC developed tight-junctions and other hallmarks of the vaginal mucosa including predictable proinflammatory cytokine secretion following TLR stimulation. Colonization of multilayers by common vaginal commensals including Lactobacillus crispatus, L. jensenii, and L. rhamnosus led to intimate associations with the VEC exclusively on the apical surface. Vaginal commensals did not trigger cytokine secretion but Staphylococcus epidermidis, a skin commensal, was inflammatory. Lactobacilli reduced cytokine secretion in an isolate-specific fashion following TLR stimulation. This tempering of inflammation offers a potential explanation for increased susceptibility to STI in the absence of common commensals and has implications for testing of potential STI preventatives

HIV pre-exposure prophylaxis for women and infants prevents vaginal and oral HIV transmission in a preclinical model of HIV infection

Approximately 1.5 million HIV-positive women become pregnant annually. Without treatment, up to 45% will transmit HIV to their infants, primarily through breastfeeding. These numbers highlight that HIV acquisition is a major health concern for women and children globally. They also emphasize the urgent need for novel approaches to prevent HIV acquisition that are safe, effective and convenient to use by women and children in places where they are most needed

Role of Semen on Vaginal HIV-1 Transmission and Maraviroc Protection

ABSTRACT We used bone marrow/liver/thymus (BLT) humanized mice to establish the effect of semen on vaginal HIV infection and on the efficacy of topically applied maraviroc. Our results demonstrate that vaginal transmission of cell-free HIV occurs efficiently in the presence of semen and that topically applied maraviroc efficiently prevents HIV transmission in the presence of semen. We also show that semen has no significant effect on the transmission of transmitted/founder viruses or cell-associated viruses

HIV Co-infection Augments EBV-Induced Tumorigenesis in vivo

In most individuals, EBV maintains a life-long asymptomatic latent infection. However, EBV can induce the formation of B cell lymphomas in immune suppressed individuals including people living with HIV (PLWH). Most individuals who acquire HIV are already infected with EBV as EBV infection is primarily acquired during childhood and adolescence. Although antiretroviral therapy (ART) has substantially reduced the incidence of AIDS-associated malignancies, EBV positive PLWH are at an increased risk of developing lymphomas compared to the general population. The direct effect of HIV co-infection on EBV replication and EBV-induced tumorigenesis has not been experimentally examined. Using a humanized mouse model of EBV infection, we demonstrate that HIV co-infection enhances systemic EBV replication and immune activation. Importantly, EBV-induced tumorigenesis was augmented in EBV/HIV co-infected mice. Collectively, these results demonstrate a direct effect of HIV co-infection on EBV pathogenesis and disease progression and will facilitate future studies to address why the incidence of certain types of EBV-associated malignancies are stable or increasing in ART treated PLWH

A long-acting formulation of the integrase inhibitor raltegravir protects humanized BLT mice from repeated high-dose vaginal HIV challenges

Pre-exposure prophylaxis (PrEP) using antiretroviral drugs (ARVs) has been shown to reduce HIV transmission in people at high risk of HIV infection. Adherence to PrEP strongly correlates with the level of HIV protection. Long-acting injectable ARVs provide sustained systemic drug exposures over many weeks and can improve adherence due to infrequent parenteral administration. Here, we evaluated a new long-acting formulation of raltegravir for prevention of vaginal HIV transmission

Loss of In Vivo Replication Fitness of HIV-1 Variants Resistant to the Tat Inhibitor, dCA

HIV resistance to the Tat inhibitor didehydro-cortistatin A (dCA) in vitro correlates with higher levels of Tat-independent viral transcription and a seeming inability to enter latency, which rendered resistant isolates more susceptible to CTL-mediated immune clearance. Here, we investigated the ability of dCA-resistant viruses to replicate in vivo using a humanized mouse model of HIV infection. Animals were infected with WT or two dCA-resistant HIV-1 isolates in the absence of dCA and followed for 5 weeks. dCA-resistant viruses exhibited lower replication rates compared to WT. Viral replication was suppressed early after infection, with viral emergence at later time points. Multiplex analysis of cytokine and chemokines from plasma samples early after infection revealed no differences in expression levels between groups, suggesting that dCA-resistance viruses did not elicit potent innate immune responses capable of blocking the establishment of infection. Viral single genome sequencing results from plasma samples collected at euthanasia revealed that at least half of the total number of mutations in the LTR region of the HIV genome considered essential for dCA evasion reverted to WT. These results suggest that dCA-resistant viruses identified in vitro suffer a fitness cost in vivo, with mutations in LTR and Nef pressured to revert to wild type

Macrophages sustain HIV replication in vivo independently of T cells

Macrophages have long been considered to contribute to HIV infection of the CNS; however, a recent study has contradicted this early work and suggests that myeloid cells are not an in vivo source of virus production. Here, we addressed the role of macrophages in HIV infection by first analyzing monocytes isolated from viremic patients and patients undergoing antiretroviral treatment. We were unable to find viral DNA or viral outgrowth in monocytes isolated from peripheral blood. To determine whether tissue macrophages are productively infected, we used 3 different but complementary humanized mouse models. Two of these models (bone marrow/liver/thymus [BLT] mice and T cell–only mice [ToM]) have been previously described, and the third model was generated by reconstituting immunodeficient mice with human CD34+ hematopoietic stem cells that were devoid of human T cells (myeloid-only mice [MoM]) to specifically evaluate HIV replication in this population. Using MoM, we demonstrated that macrophages can sustain HIV replication in the absence of T cells; HIV-infected macrophages are distributed in various tissues including the brain; replication-competent virus can be rescued ex vivo from infected macrophages; and infected macrophages can establish de novo infection. Together, these results demonstrate that macrophages represent a genuine target for HIV infection in vivo that can sustain and transmit infection

A long-acting formulation of the integrase inhibitor raltegravir protects humanized BLT mice from repeated high-dose vaginal HIV challenges

Objectives: Pre-exposure prophylaxis (PrEP) using antiretroviral drugs (ARVs) has been shown to reduce HIV transmission in people at high risk of HIV infection. Adherence to PrEP strongly correlates with the level of HIV protection. Long-acting injectable ARVs provide sustained systemic drug exposures over many weeks and can improve adherence due to infrequent parenteral administration. Here, we evaluated a new long-acting formulation of raltegravir for prevention of vaginal HIV transmission. Methods: Long-acting raltegravir was administered subcutaneously to BALB/c, NSG (NOD -scid -gamma) and humanized BLT (bone marrow -liver -thymus) mice and rhesus macaques. Raltegravir concentration in peripheral blood and tissue was analysed. Suppression of HIV replication was assessed in infected BLT mice. Two high-dose HIV vaginal challenges were used to evaluate protection from HIV transmission in BLT mice. Results: Two weeks after a single subcutaneous injection of long-acting raltegravir in BLT mice (7.5 mg) and rhesus macaques (160 mg), the plasma concentration of raltegravir was comparable to 400 mg orally, twice daily in humans. Serum collected from mice 3 weeks post-administration of long-acting raltegravir efficiently blocked HIV infection of TZM-bl indicator cells in vitro. Administration of long-acting raltegravir suppressed viral RNA in plasma and cervico-vaginal fluids of infected BLT mice, demonstrating penetration of active raltegravir into the female reproductive tract. Using transmitted/founder HIV we observed that BLT mice administered a single subcutaneous dose of long-acting raltegravir were protected from two high-dose HIV vaginal challenges 1 week and 4 weeks after drug administration. Conclusions: These preclinical results demonstrated the efficacy of long-acting raltegravir in preventing vaginal HIV transmission

Breast Milk of HIV-Positive Mothers Has Potent and Species-Specific In Vivo HIV-Inhibitory Activity

ABSTRACT Despite the nutritional and health benefits of breast milk, breast milk can serve as a vector for mother-to-child HIV transmission. Most HIV-infected infants acquire HIV through breastfeeding. Paradoxically, most infants breastfed by HIV-positive women do not become infected. This is potentially attributed to anti-HIV factors in breast milk. Breast milk of HIV-negative women can inhibit HIV infection. However, the HIV-inhibitory activity of breast milk from HIV-positive mothers has not been evaluated. In addition, while significant differences in breast milk composition between transmitting and nontransmitting HIV-positive mothers have been correlated with transmission risk, the HIV-inhibitory activity of their breast milk has not been compared. This knowledge may significantly impact the design of prevention approaches in resource-limited settings that do not deny infants of HIV-positive women the health benefits of breast milk. Here, we utilized bone marrow/liver/thymus humanized mice to evaluate the in vivo HIV-inhibitory activity of breast milk obtained from HIV-positive transmitting and nontransmitting mothers. We also assessed the species specificity and biochemical characteristics of milk's in vivo HIV-inhibitory activity and its ability to inhibit other modes of HIV infection. Our results demonstrate that breast milk of HIV-positive mothers has potent HIV-inhibitory activity and indicate that breast milk can prevent multiple routes of infection. Most importantly, this activity is unique to human milk. Our results also suggest multiple factors in breast milk may contribute to its HIV-inhibitory activity. Collectively, our results support current recommendations that HIV-positive mothers in resource-limited settings exclusively breastfeed in combination with antiretroviral therapy. IMPORTANCE Approximately 240,000 children become infected with HIV annually, the majority via breastfeeding. Despite daily exposure to virus in breast milk, most infants breastfed by HIV-positive women do not acquire HIV. The low risk of breastfeeding-associated HIV transmission is likely due to antiviral factors in breast milk. It is well documented that breast milk of HIV-negative women can inhibit HIV infection. Here, we demonstrate, for the first time, that breast milk of HIV-positive mothers (nontransmitters and transmitters) inhibits HIV transmission. We also demonstrate that breast milk can prevent multiple routes of HIV acquisition and that this activity is unique to human milk. Collectively, our results support current guidelines which recommend that HIV-positive women in resource-limited settings exclusively breastfeed in combination with infant or maternal antiretroviral therapy

Ultra-long-acting removable drug delivery system for HIV treatment and prevention

Non-adherence to medication is an important health care problem, especially in the treatment of chronic conditions. Injectable long-acting (LA) formulations of antiretrovirals (ARVs) represent a viable alternative to improve adherence to HIV/AIDS treatment and prevention. However, the LA-ARV formulations currently in clinical trials cannot be removed after administration even if adverse events occur. Here we show an ultra-LA removable system that delivers drug for up to 9 months and can be safely removed to stop drug delivery. We use two pre-clinical models for HIV transmission and treatment, non-human primates (NHP) and humanized BLT (bone marrow/liver/thymus) mice and show a single dose of subcutaneously administered ultra-LA dolutegravir effectively delivers the drug in both models and show suppression of viremia and protection from multiple high-dose vaginal HIV challenges in BLT mice. This approach represents a potentially effective strategy for the ultra-LA drug delivery with multiple possible therapeutic applications