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CCR5-tropic simian/human immunodeficiency viruses (SHIV) with clade C transmitted/founder envelopes represent a critical tool for the investigation of HIV experimental vaccines and microbicides in nonhuman primates, although many such isolates lead to spontaneous viral control post infection. Here, we generated a high-titer stock of pathogenic SHIV-C109p5 by serial passage in two rhesus macaques (RM) and tested its virulence in aged monkeys. The co-receptor usage was confirmed before infecting five geriatric rhesus macaques (four female and one male). Plasma viral loads were monitored by reverse transcriptase-quantitative PCR (RT-qPCR), cytokines by multiplex analysis, and biomarkers of gastrointestinal damage by enzyme-linked immunosorbent assay. Antibodies and cell-mediated responses were also measured. Viral dissemination into tissues was determined by RNAscope. Intravenous SHIV-C109p5 infection of aged RMs leads to high plasma viremia and rapid disease progression; rapid decrease in CD4 + T cells, CD4 + CD8 + T cells, and plasmacytoid dendritic cells; and wasting necessitating euthanasia between 3 and 12 weeks post infection. Virus-specific cellular immune responses were detected only in the two monkeys that survived 4 weeks post infection. These were Gag-specific TNFα+CD8+, MIP1β+CD4+, Env-specific IFN-γ+CD4+, and CD107a+ T cell responses. Four out of five monkeys had elevated intestinal fatty acid binding protein levels at the viral peak, while regenerating islet-derived protein 3α showed marked increases at later time points in the three animals surviving the longest, suggesting gut antimicrobial peptide production in response to microbial translocation post infection. Plasma levels of monocyte chemoattractant protein-1, interleukin-15, and interleukin-12/23 were also elevated. Viral replication in gut and secondary lymphoid tissues was extensive.IMPORTANCESimian/human immunodeficiency viruses (SHIV) are important reagents to study prevention of virus acquisition in nonhuman primate models of HIV infection, especially those representing transmitted/founder (T/F) viruses. However, many R5-tropic SHIV have limited fitness in vivo leading to many monkeys spontaneously controlling the virus post acute infection. Here, we report the generation of a pathogenic SHIV clade C T/F stock by in vivo passage leading to sustained viral load set points, a necessity to study pathogenicity. Unexpectedly, administration of this SHIV to elderly rhesus macaques led to extensive viral replication and fast disease progression, despite maintenance of a strict R5 tropism. Such age-dependent rapid disease progression had previously been reported for simian immunodeficiency virus but not for R5-tropic SHIV infections., Competing Interests: The authors declare no conflict of interest.

Defensins are antimicrobial peptides important for mucosal innate immunity. They exhibit a broad spectrum of activity against bacteria, viruses, and fungi. Levels of α-defensins are elevated at the genital mucosa of individuals with sexually transmitted infections (STIs). Somewhat paradoxically, human α-defensin 5 and 6 (HD5 and HD6) promote human immunodeficiency virus (HIV) infectivity, and contribute to STI-mediated enhancement of HIV infection in vitro. Specific amino acid residues of HD5 and HD6 that are crucial for antimicrobial activities have been characterized previously; however, the key determinants of defensins responsible for enhancement of HIV infectivity are not known. Here, we have identified residues of HD5 and HD6 that are required for enhancement of HIV attachment and infection. Most of these residues are involved in hydrophobicity and self-association of defensins. Specifically, we found that mutant defensins L16A-HD5, E21me-HD5, L26A-HD5, Y27A-HD5, F2A-HD6, H27W-HD6, and F29A-HD6 significantly lost their ability to promote HIV attachment and infection. L29A mutation also reduced HIV infection-enhancing activity of HD5. Additionally, a number of mutations in charged residues variably affected the profile of HIV attachment and infectivity. One HD5 charged mutation, R28A, notably resulted in a 34-48% loss of enhanced HIV infectivity and attachment. These results indicate that defensin determinants that maintain high-ordered amphipathic structure are crucial for HIV enhancing activity. In a comparative analysis of the mutant defensins, we found that for some defensin mutants enhancement of HIV infectivity was associated with the reverse transcription step, suggesting a novel, HIV attachment-independent, mechanism of defensin-mediated HIV enhancement., Competing Interests: Authors declare no conflicts of interest.

IFN-ε is a unique type I IFN that is not induced by pattern recognition response elements. IFN-ε is constitutively expressed in mucosal tissues, including the female genital mucosa. Although the direct antiviral activity of IFN-ε was thought to be weak compared with IFN-α, IFN-ε controls Chlamydia muridarum and herpes simplex virus 2 in mice, possibly through modulation of immune response. We show here that IFN-ε induces an antiviral state in human macrophages that blocks HIV-1 replication. IFN-ε had little or no protective effect in activated CD4 + T cells or transformed cell lines unless activated CD4 + T cells were infected with replication-competent HIV-1 at a low MOI. The block to HIV infection of macrophages was maximal after 24 hours of treatment and was reversible. IFN-ε acted on early stages of the HIV life cycle, including viral entry, reverse transcription, and nuclear import. The protection did not appear to operate through known type I IFN-induced HIV host restriction factors, such as APOBEC3A and SAMHD1. IFN-ε-stimulated immune mediators and pathways had the signature of type I IFNs but were distinct from IFN-α in macrophages. IFN-ε induced significant phagocytosis and ROS, which contributed to the block to HIV replication. These findings indicate that IFN-ε induces an antiviral state in macrophages that is mediated by different factors than those induced by IFN-α. Understanding the mechanism of IFN-ε-mediated HIV inhibition through immune modulation has implications for prevention., Competing Interests: The authors have declared that no conflict of interest exists.

Alpha-defensins, including human neutrophil peptides 1-3 (HNP1-3) and human defensin 5 (HD5), are elevated at the genital mucosa in individuals with sexually transmitted infections (STIs). The presence of STIs is associated with an increased risk of human immunodeficiency virus (HIV) transmission, suggesting there may be a role for defensins in early events of HIV transmission. HD5 has been demonstrated to contribute to STI-mediated increased HIV infectivity in vitro. HNPs exhibit anti-HIV activity in vitro. However, increased levels of HNPs have been associated with enhanced HIV acquisition and higher viral load in breast milk. This study found that HNP1, but not HD5, significantly disrupted epithelial integrity and promoted HIV traversal of epithelial barriers. Linear HNP1 with the same charges did not affect epithelial permeability, indicating that the observed effect of HNP1 on the epithelial barrier was structure dependent. These results suggest a role for HNP1 in STI-mediated enhancement of HIV transmission.

Human immunodeficiency virus (HIV) primary infection occurs at mucosa tissues, suggesting an intricate interplay between the microbiome and HIV infection. Recent advanced technologies of high-throughput sequencing and bioinformatics allow researchers to explore nonculturable microbes, including bacteria, virus, and fungi, and their association with diseases. HIV/simian immunodeficiency virus infection is associated with microbiome shifts and immune activation that may affect the outcome of disease progression. In this review, the authors focus on microbiome in HIV infection at various mucosal compartments. Understanding the relationship between microbiome and HIV may offer insights into development of better strategies for HIV prevention and treatment., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: We have previously shown that human defensin 5 (HD5) promotes HIV infectivity in both primary CD4+ T cells and HeLa cells expressing CD4 and CCR5. HD5 is induced in response to sexually transmitted infections (STIs) such as Chlamydia trachomatis and Neisseria gonorrhoeae, suggesting it plays a role in STI-mediated enhancement of HIV transmission. In contrast to our findings, a recent study reports that HD5 has an anti-HIV effect in primary CD4+ T cells under serum-deprived conditions. To resolve these apparently contradictory observations, we investigated experimental parameters that might contribute to contrasting effects of HD5., Results: Serum-deprived culture conditions were associated with anti-HIV activity. In contrast to the dependence of the HIV enhancing effect on HD5 structure, the anti-HIV activity in serum-deprived primary CD4+ T cells was independent of HD5 structure as the linear peptide [Abu] HD5 exhibited similar anti-HIV activity. Under serum deprived conditions, HD5 blocked CD4-receptor-independent HIV-1vsv infection before or after viral entry. We found that HD5 and its linear form induced significant cell death in primary CD4+ T cells under serum-deprived culture conditions. HD5-mediated apoptosis was observed as early as 2 h after addition of defensins to serum-deprived primary CD4+ T cells. In contrast to primary CD4+ T cells, HD5 did not induce cytotoxicity and promote HIV infectivity of HeLa-CD4-CCR5 cells under serum-deprived conditions., Conclusions: These results indicate that under serum-deprived culture conditions HD5 is toxic for primary CD4+ T cells, warranting caution in data interpretation.

Chlamydia trachomatis infection is one of the most prevalent bacterial STIs in the USA and worldwide, and women with C. trachomatis infection are at increased risk of acquiring HIV. Because immune activation at the genital mucosa facilitates HIV/SIV infection, C. trachomatis-mediated cytokine induction may contribute to increased HIV transmission in asymptomatic women. To begin to elucidate the mechanisms, we longitudinally analyzed profiles of innate immune factors and HIV infectivity in genital secretions from anatomically specific sites in asymptomatic women during C. trachomatis infection and post-antibiotic treatment. We found higher levels of cytokines and chemokines in endocervical secretions than vaginal secretions. Compared with the convalescent state, G-CSF, IL-1α, and RANTES were elevated in endocervical secretions, IFN-γ and TNF-α were elevated in vaginal secretions, and IFNγ, IL-1β, and MIP1-α were elevated in cervicolavage fluid (CVL), before adjustment of multiple comparisons. Elevated endocervical levels of IP-10 and MCP-1 were associated with the use of hormonal contraception in infected women after successful treatment, suggesting the role of hormonal contraception in inflammation independent of STIs. Importantly, soluble factors found in endocervical secretions during infection enhanced HIV infectivity while no difference in HIV infectivity was found with vaginal secretions or CVL during infection or at convalescence. Taken together, the profiles of immune mediators and in vitro HIV infectivity indicate that the endocervical and vaginal mucosa are immunologically distinct. Our results underscore the importance of considering anatomical site and local sampling methodology when measuring mucosal responses, particularly in the presence of C. trachomatis infection., (Copyright © 2013 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.)

TLR2 activation plays a crucial role in Neisseria gonorrheae-mediated enhancement of HIV infection of resting CD4(+) T cells. We examined signaling pathways involved in the HIV enhancing effect of TLR2. TLR2 but not IL-2 signals promoted HIV nuclear import; however, both signals were required for the maximal enhancing effect. Although TLR2 signaling could not activate T cells, it increased IL-2-induced T cell activation. Cyclosporin A and IkBα inhibitor blocked TLR2-mediated enhancement of HIV infection/nuclear import. PI3K inhibitor blocked HIV infection/nuclear import and T cell activation and exerted a moderate inhibitory effect on cell cycle progression in CD4(+) T cells activated by TLR2/IL-2. Blockade of p38 signaling suppressed TLR2-mediated enhancement of HIV nuclear import/infection. However, the p38 inhibitor did not have a significant effect on T cell activation or TCR/CD3-mediated enhancement of HIV infection/nuclear import. The cell cycle arresting reagent aphidicolin blocked TLR2- and TCR/CD3-induced HIV infection/nuclear import. Finally, cyclosporin A and IκBα and PI3K inhibitors but not the p38 inhibitor blocked TLR2-mediated IκBα phosphorylation. Our results suggest that TLR2 activation enhances HIV infection/nuclear import in resting CD4(+) T cells through both T cell activation-dependent and -independent mechanisms.

Background: Concurrent sexually transmitted infections (STIs) increase the likelihood of HIV transmission. The levels of defensins are frequently elevated in genital fluids from individuals with STIs. We have previously shown that human defensins 5 and 6 (HD5 and HD6) promote HIV entry and contribute to Neisseria gonorrhoeae-mediated enhancement of HIV infectivity in vitro. In this study, we dissect the molecular mechanism of the HIV enhancing effect of defensins., Results: HD5 and HD6 primarily acted on the virion to promote HIV infection. Both HD5 and HD6 antagonized the anti-HIV activities of inhibitors of HIV entry (TAK 779) and fusion (T-20) when the inhibitors were present only during viral attachment; however, when these inhibitors were added back during viral infection they overrode the HIV enhancing effect of defensins. HD5 and HD6 enhanced HIV infectivity by promoting HIV attachment to target cells. Studies using fluorescent HIV containing Vpr-GFP indicated that these defensins enhanced HIV attachment by concentrating virus particles on the target cells. HD5 and HD6 blocked anti-HIV activities of soluble glycosaminoglycans including heparin, chondroitin sulfate, and dextran sulfate. However, heparin, at a high concentration, diminished the HIV enhancing effect of HD5, but not HD6. Additionally, the degree of the HIV enhancing effect of HD5, but not HD6, was increased in heparinase-treated cells. These results suggest that HD5 and haparin/heparan sulfate compete for binding to HIV., Conclusions: HD5 and HD6 increased HIV infectivity by concentrating virus on the target cells. These defensins may have a negative effect on the efficacy of microbicides, especially in the setting of STIs.

Sexually transmitted infections (STIs) increase the likelihood of HIV transmission. Defensins are part of the innate mucosal immune response to STIs and therefore we investigated their role in HIV infection. We found that human defensins 5 and 6 (HD5 and HD6) promoted HIV infection, and this effect was primarily during viral entry. Enhancement was seen with primary viral isolates in primary CD4(+) T cells and the effect was more pronounced with R5 virus compared with X4 virus. HD5 and HD6 promoted HIV reporter viruses pseudotyped with vesicular stomatitis virus and murine leukemia virus envelopes, indicating that defensin-mediated enhancement was not dependent on CD4 and coreceptors. Enhancement of HIV by HD5 and HD6 was influenced by the structure of the peptides, as loss of the intramolecular cysteine bonds was associated with loss of the HIV-enhancing effect. Pro-HD5, the precursor and intracellular form of HD5, also exhibited HIV-enhancing effect. Using a cervicovaginal tissue culture system, we found that expression of HD5 and HD6 was induced in response to Neisseria gonorrhoeae (GC, for gonococcus) infection and that conditioned medium from GC-exposed cervicovaginal epithelial cells with elevated levels of HD5 also enhanced HIV infection. Introduction of small interfering RNAs for HD5 or HD6 abolished the HIV-enhancing effect mediated by GC. Thus, the induction of these defensins in the mucosa in the setting of GC infection could facilitate HIV infection. Furthermore, this study demonstrates the complexity of defensins as innate immune mediators in HIV transmission and warrants further investigation of the mechanism by which defensins modulate HIV infection.

SAMMA, a mandelic acid condensation polymer, exhibits a broad antimicrobial activity against several sexually transmitted pathogens including human immunodeficiency virus (HIV). Here we demonstrated that SAMMA suppressed HIV transmission by dendritic cells (DCs), one of the first target cells for primary infection. The greatest inhibitory effect was achieved when SAMMA was present during the co-culture with target cells. The inhibitory effect of SAMMA on DC-mediated HIV transmission was not due to cytotoxicity. Analysis of the level of DC-associated HIV p24 antigen revealed that SAMMA prevented HIV internalization by DCs when the virus was pre-incubated with the compound. In contrast, pre-incubation of DCs with SAMMA followed by wash-off did not affect the amount of cell-associated HIV p24 antigen. In addition, SAMMA blocked HIV glycoprotein-mediated cell-cell fusion. This study suggests that SAMMA prevents HIV infection through multiple mechanisms.

Mammalian defensins are small cationic antimicrobial peptides predominantly found in leukocytes and epithelial cells engaged in host defense. These peptides act as effector molecules in innate immunity as well as regulators in adaptive immunity. Increasing evidence indicates that defensins are effective inhibitors of HIV-1. While the level of defensins in HIV-1 infected individuals has not been determined, neutropenia and neutrophil dysfunction associated with HIV disease progression may result in altered alpha-defensin production. This review provides an overview of the structure and function of defensins, and focuses on the anti-HIV-1 activity of defensins and the mechanism of this activity. Although many questions remain, studying the complex function of defensins in innate immunity against HIV has implications for our further understanding of disease progression and for the development of novel approaches to prevention and therapy.

The article presents the study which examines the HIV-1 infection in human peritoneal macrophages from ascitic fluid. It states that macrophages are cells that are major target by HIV because they support both productive and latent HIV-1 infection. The study demonstrated that peritoneal macrophages (PMs) are rich in ascitic fluid of patients with liver cirrhosis. It indicates that PMs are expressed CD68, a differentiation marker, which displayed phagocytic activity.

α-Defensins are abundant antimicrobial peptides in polymorphonuclear leukocytes and play an important role in innate immunity. We have previously shown that α-defensin-1 can inhibit HIV-1 replication following viral entry. Here we examined the molecular mechanism(s) of α-defensin-1-mediated HIV-1 inhibition. α-Defensin-1 had a direct effect on HIV-1 virions at a low MOI in the absence of serum. The direct effect on HIV-1 virions was abolished by the presence of serum or an increase in virus particles. Studying the kinetics of the HIV life cycle revealed that α-defensin-1 inhibited steps following reverse transcription and integration. Analysis of PKC phosphorylation in primary CD4+ T cells in response to α-defensin-1 indicated that α-defensin-1 inhibited PKC activity. Pretreatment of infected CD4+ T cells with a PKC activator, bryostatin 1, partially reversed α-defensin-1-mediated HIV inhibition. Like α-defensin-1, the PKC isoform-selective inhibitor Go6976 blocked HIV-1 infection in a dose-dependent manner. Furthermore, kinetic studies and analysis of H!V-1 products indicated that α-defensin-1 and Go6976 blocked HIV-1 infection at similar stages in its life cycle, including nuclear import and transcription. Taken together, our studies demonstrate that, in the absence of serum, α-defensin-1 may act directly on the virus, but, in the presence of serum, its effects are on the cell, where it inhibits HIV-1 replication. At least 1 of the cellular effects associated with H!V inhibition is interference with PKC signaling in primary CD4+ T cells. Studying the complex function of α-defensin-1 in innate immunity against H!V has implications for prevention as well as therapeutics. [ABSTRACT FROM AUTHOR]