Infection of semen-producing organs by SIV during the acute and chronic stages of the disease

Background

Although indirect evidence suggests the male genital tract as a possible source of persistent HIV shedding in semen during antiretroviral therapy, this phenomenon is poorly understood due to the difficulty of sampling semen-producing organs in HIV+ asymptomatic individuals.

Methodology/Principal Findings

Using a range of molecular and cell biological techniques, this study investigates SIV infection within reproductive organs of macaques during the acute and chronic stages of the disease. We demonstrate for the first time the presence of SIV in the testes, epididymides, prostate and seminal vesicles as early as 14 days post-inoculation. This infection persists throughout the chronic stage and positively correlates with blood viremia. The prostate and seminal vesicles appear to be the most efficiently infected reproductive organs, followed by the epididymides and testes. Within the male genital tract, mostly T lymphocytes and a small number of germ cells harbour SIV antigens and RNA. In contrast to the other organs studied, the testis does not display an immune response to the infection. Testosteronemia is transiently increased during the early phase of the infection but spermatogenesis remains unaffected.

Conclusions/Significance

The present study reveals that SIV infection of the macaque male genital tract is an early event and that semen-producing organs display differential infection levels and immune responses. These results help elucidate the origin of HIV in semen and constitute an essential base to improving the design of antiretroviral therapies to eradicate virus from semen.     

Semen-specific genetic characteristics of human immunodeficiency virus type 1 env

Human immunodeficiency virus type 1 (HIV-1) in the male genital tract may comprise virus produced locally in addition to virus transported from the circulation. Virus produced in the male genital tract may be genetically distinct, due to tissue-specific cellular characteristics and immunological pressures. HIV-1 env sequences derived from paired blood and semen samples from the Los Alamos HIV Sequence Database were analyzed to ascertain a male genital tract-specific viral signature. Machine learning algorithms could predict seminal tropism based on env sequences with accuracies exceeding 90%, suggesting that a strong genetic signature does exist for virus replicating in the male genital tract. Additionally, semen-derived viral populations exhibited constrained diversity (P < 0.05), decreased levels of positive selection (P < 0.025), decreased CXCR4 coreceptor utilization, and altered glycosylation patterns. Our analysis suggests that the male genital tract represents a distinct selective environment that contributes to the apparent genetic bottlenecks associated with the sexual transmission of HIV-1.     

Impact of collection method on assessment of semen HIV RNA viral load

Background

The blood HIV RNA viral load is the best-defined predictor of HIV transmission, in part due to ease of measurement and the correlation of blood and genital tract (semen or cervico-vaginal) viral load, although recent studies found semen HIV RNA concentration to be a stronger predictor of HIV transmission. There is currently no standardized method for semen collection when measuring HIV RNA concentration. Therefore, we compared two collection techniques in order to study of the impact of antiretroviral therapy on the semen viral load.

Methodology/Principal Findings

Semen was collected by masturbation from HIV-infected, therapy-naïve men who have sex with men (MSM) either undiluted (Visit 1) or directly into transport medium (Visit 2). Seminal plasma was then isolated, and the HIV RNA concentration obtained with each collection technique was measured and corrected for dilution if necessary. Collection of semen directly into transport medium resulted in a median HIV RNA viral load that was 0.4 log10 higher than undiluted samples.

Conclusions/Significance

The method of semen collection is an important consideration when quantifying the HIV RNA viral load in this compartment.     

Modeling the role of macrophages in HIV persistence during antiretroviral therapy

HIV preferentially infects activated CD4+ T cells. Current antiretroviral therapy cannot eradicate the virus. Viral infection of other cells such as macrophages may contribute to viral persistence during antiretroviral therapy. In addition to cell-free virus infection, macrophages can also get infected when engulfing infected CD4+ T cells as innate immune sentinels. How macrophages affect the dynamics of HIV infection remains unclear. In this paper, we develop an HIV model that includes the infection of CD4+ T cells and macrophages via cell-free virus infection and cell-to-cell viral transmission. We derive the basic reproduction number and obtain the local and global stability of the steady states. Sensitivity and viral dynamics simulations show that even when the infection of CD4+ T cells is completely blocked by therapy, virus can still persist and the steady-state viral load is not sensitive to the change of treatment efficacy. Analysis of the relative contributions to viral replication shows that cell-free virus infection leads to the majority of macrophage infection. Viral transmission from infected CD4+ T cells to macrophages during engulfment accounts for a small fraction of the macrophage infection and has a negligible effect on the total viral production. These results suggest that macrophage infection can be a source contributing to HIV persistence during suppressive therapy. Improving drug efficacies in heterogeneous target cells is crucial for achieving HIV eradication in infected individuals.

     

Genital mucosal transmission of simian immunodeficiency virus: animal model for heterosexual transmission of human immunodeficiency virus.

An animal model for the heterosexual transmission of human immunodeficiency virus (HIV) was developed by the application of simian immunodeficiency virus (SIV) onto the genital mucosas of both mature and immature, male and female rhesus macaques. Virus preparations were infused into the vaginal vaults or the urethras (males) of the animals through a soft plastic pediatric nasogastric feeding tube. The macaques that were infected by this route (six males and nine females) developed SIV-specific antibodies, and SIV was isolated from peripheral mononuclear cells of all seropositive animals. One male and one female infected by this route developed severe acquired immunodeficiency syndrome-like disease with retroviral giant-cell pneumonia. As few as two inoculations of cell-free SIV containing 50 50% tissue culture infective doses induced persistent viremia. Cell-free virus preparations were capable of producing infection by the genital route. Much higher doses of virus were required to transmit SIV by this route than are required for transmission by intravenous inoculation. Thus, it appears that the mucous membranes of the genital tract act as a barrier to SIV infection. Spermatozoa and seminal plasma were not required for the genital transmission of SIV. Rarely, SIV was recovered from mononuclear cells in semen and vaginal secretions. The SIV-rhesus macaque model is suitable for assessing the role of cofactors in heterosexual transmission of HIV and will be useful for testing the effectiveness of spermicides, pharmacologic agents, and vaccines in preventing the heterosexual transmission of HIV.     

Zika virus persistence in the male macaque reproductive tract

Zika virus (ZIKV) is unique among mosquito-borne flaviviruses in that it is also vertically and sexually transmitted by humans. The male reproductive tract is thought to be a ZIKV reservoir; however, the reported magnitude and duration of viral persistence in male genital tissues vary widely in humans and non-human primate models. ZIKV tissue and cellular tropism and potential effects on male fertility also remain unclear. The objective of this study was to resolve these questions by analyzing archived genital tissues from 51 ZIKV-inoculated male macaques and correlating data on plasma viral kinetics, tissue tropism, and ZIKV-induced pathological changes in the reproductive tract. We hypothesized that ZIKV would persist in the male macaque genital tract for longer than there was detectable viremia, where it would localize to germ and epithelial cells and associate with lesions. We detected ZIKV RNA and infectious virus in testis, epididymis, seminal vesicle, and prostate gland. In contrast to prepubertal males, sexually mature macaques were significantly more likely to harbor persistent ZIKV RNA or infectious virus somewhere in the genital tract, with detection as late as 60 days post-inoculation. ZIKV RNA localized primarily to testicular stem cells/sperm precursors and epithelial cells, including Sertoli cells, epididymal duct epithelium, and glandular epithelia of the seminal vesicle and prostate gland. ZIKV infection was associated with microscopic evidence of inflammation in the epididymis and prostate gland of sexually mature males, pathologies that were absent in uninfected controls, which could have significant effects on male fertility. The findings from this study increase our understanding of persistent ZIKV infection which can inform risk of sexual transmission during assisted reproductive therapies as well as potential impacts on male fertility.     

HIV-1 Populations in Semen Arise through Multiple Mechanisms

HIV-1 is present in anatomical compartments and bodily fluids. Most transmissions occur through sexual acts, making virus in semen the proximal source in male donors. We find three distinct relationships in comparing viral RNA populations between blood and semen in men with chronic HIV-1 infection, and we propose that the viral populations in semen arise by multiple mechanisms including: direct import of virus, oligoclonal amplification within the seminal tract, or compartmentalization. In addition, we find significant enrichment of six out of nineteen cytokines and chemokines in semen of both HIV-infected and uninfected men, and another seven further enriched in infected individuals. The enrichment of cytokines involved in innate immunity in the seminal tract, complemented with chemokines in infected men, creates an environment conducive to T cell activation and viral replication. These studies define different relationships between virus in blood and semen that can significantly alter the composition of the viral population at the source that is most proximal to the transmitted virus.     

Viral RNA Levels and env Variants in Semen and Tissues of Mature Male Rhesus Macaques Infected with SIV by Penile Inoculation

HIV is shed in semen but the anatomic site of virus entry into the genital secretions is unknown. We determined viral RNA (vRNA) levels and the envelope gene sequence in the SIVmac 251 viral populations in the genital tract and semen of 5 adult male rhesus monkeys (Macaca mulatta) that were infected after experimental penile SIV infection. Paired blood and semen samples were collected from 1–9 weeks after infection and the monkeys were necropsied eleven weeks after infection. The axillary lymph nodes, testes, epididymis, prostate, and seminal vesicles were collected and vRNA levels and single-genome analysis of the SIVmac251 env variants was performed. At the time of semen collection, blood vRNA levels were between 3.09 and 7.85 log10 vRNA copies/ml plasma. SIV RNA was found in the axillary lymph nodes of all five monkeys and in 3 of 5 monkeys, all tissues examined were vRNA positive. In these 3 monkeys, vRNA levels (log10 SIVgag copies/ug of total tissue RNA) in the axillary lymph node (6.48±0.50) were significantly higher than in the genital tract tissues: testis (3.67±2.16; p<0.05), epididymis (3.08±1.19; p<0.0001), prostate (3.36±1.30; p<0.01), and seminal vesicle (2.67±1.50; p<0.0001). Comparison of the SIVmac251 env viral populations in blood plasma, systemic lymph node, and genital tract tissues was performed in two of the macaques. Visual inspection of the Neighbor-Joining phylograms revealed that in both animals, all the sequences were generally distributed evenly among all tissue compartments. Importantly, viral populations in the genital tissues were not distinct from those in the systemic tissues. Our findings demonstrate striking similarity in the viral populations in the blood and male genital tract tissues within 3 months of penile SIV transmission.     

Semen-derived amyloid fibrils drastically enhance HIV infection

Sexual intercourse is the major route of HIV transmission. To identify endogenous factors that affect the efficiency of sexual viral transmission, we screened a complex peptide/protein library derived from human semen. We show that naturally occurring fragments of the abundant semen marker prostatic acidic phosphatase (PAP) form amyloid fibrils. These fibrils, termed Semen-derived Enhancer of Virus Infection (SEVI), capture HIV virions and promote their attachment to target cells, thereby enhancing the infectious virus titer by several orders of magnitude. Physiological concentrations of SEVI amplified HIV infection of T cells, macrophages, ex vivo human tonsillar tissues, and transgenic rats in vivo, as well as trans-HIV infection of T cells by dendritic or epithelial cells. Amyloidogenic PAP fragments are abundant in seminal fluid and boost semen-mediated enhancement of HIV infection. Thus, they may play an important role in sexual transmission of HIV and could represent new targets for its prevention.     

Patterns of residual HIV-1 RNA shedding in the seminal plasma of patients on effective antiretroviral therapy

Background

More and more HIV-1-infected men on effective antiretroviral treatment (ART) have unprotected sex in order to procreate. The main factor influencing transmission is seminal HIV shedding. While the risk of HIV transmission is very low, it is difficult to assess in individuals. Nevertheless, it should be quantified.

Results

We retrospectively analysed seminal plasma HIV-1 shedding by 362 treated HIV-infected men attending a medically assisted reproduction centre (1998–2013) in order to determine its frequency, the impact of the antiretroviral regimen on HIV shedding, and to identify shedding patterns. The HIV-1 virus loads in 1396 synchronized blood and semen samples were measured, and antiretroviral treatment, biological and epidemiological data were recorded.We detected isolated HIV-1 shedding into the seminal plasma in 5.3% of patients on efficient antiretroviral treatment, but there was no association with the HIV antiretroviral drug regimen or the CD4 cell count. These men had undergone more regimen changes since treatment initiation and had been on the ongoing drug regimen longer than the non-shedding men. The patterns of HIV seminal shedding among patients with undetectable HIV blood virus load varied greatly. HIV seminal shedding can occur as long as 5 years after starting antiretroviral treatment.

Conclusions

The seminal HIV load was used to monitor risk for infertile HIV-infected patients on an assisted reproductive technology program. This can still be recommended for patients who recently (6 months) started ART, or those with a poor history of adherence to ART but may also be usefull for some patients during counselling. Residual HIV seminal shedding is probably linked to breaks in adherence to antiretroviral treatment but local genital factors cannot be ruled out.

     

After 18 months of antiretroviral therapy,total HIV DNA decreases more pronouncedly in patients infected by CRF01_AE than in those infected by subtype B and CRF07_BC

Whether the amount of HIV DNA is associated with the subtype of HIV‐1 after antiretroviral therapy (ART) has not been reported. In the present study, the amount of HIV DNA and RNA and CD4+T counts in blood and semen prior to and after 18 months of ART were compared in 48 patients infected by CRF01_AE, subtype B or CRF07_BC of HIV‐1. Viral RNA was suppressed and CD4 cell count recovery achieved in all patients. The level of HIV DNA were similar before ART; however, patients with CRF01_AE had less HIV DNA after ART than those with subtype B and CRF07_BC infection. According to prediction of co‐receptor usage by Geno2Pheno and PSSM in combination, more than 35.6% of clones for CRF01_AE were predicted as CXCR4‐using before ART, whereas less than 6% of those for subtype B and CRF07_BC were predicted as CXCR4‐using. After 18 months of ART, no CXCR4‐using clones were predicted in any of the subtypes. Despite more HIV RNA and fewer CD4 + T cells in patients with CRF01_AE before therapy, no significant differences (P > 0.05) in viral RNA or CD4 cell counts were observed between the subtypes after 18 months of ART. Thus, 18 months of antiretroviral therapy was more efficient in patients with CRF01_AE. Considering that successful ART dramatically reduces the viral load in both blood and semen, risks of sexual transmission of HIV were reduced, contributing to prevention of rapid spread of HIV among men who have sex with men in the region.

     

Human Immunodeficiency Virus Type 1 Populations in Blood and Semen

Transmission of human immunodeficiency virus type 1 (HIV-1) usually results in outgrowth of viruses with macrophage-tropic phenotype and consensus non-syncytium-inducing (NSI) V3 loop sequences, despite the presence of virus with broader host range and the syncytium-inducing (SI) phenotype in the blood of many donors. We examined proviruses in contemporaneous peripheral blood mononuclear cells (PBMC) and nonspermatozoal semen mononuclear cells (NSMC) of five HIV-1-infected individuals to determine if this preferential outgrowth could be due to compartmentalization and thus preferential transmission of viruses of the NSI phenotype from the male genital tract. Phylogenetic reconstructions of ~700-bp sequences covering the second constant region through the fifth variable region (C2 to V5) of the viral envelope gene revealed distinct variant populations in the blood versus the semen in two patients with AIDS and in one asymptomatic individual (patient 613), whereas similar variant populations were found in both compartments in two other asymptomatic individuals. Variants with amino acids in the V3 loop that predict the SI phenotype were found in both AIDS patients and in patient 613; however, the distribution of these variants between the two compartments was not consistent. SI variants were found only in the PBMC of one AIDS patient but only in the NSMC of the other, while they were found in both compartments in patient 613. It is therefore unlikely that restriction of SI variants from the male genital tract accounts for the observed NSI transmission bias. Furthermore, no evidence for a semen-specific signature amino acid sequence was detected.     

The Semen Microbiome and Its Relationship with Local Immunology and Viral Load in HIV Infection

Semen is a major vector for HIV transmission, but the semen HIV RNA viral load (VL) only correlates moderately with the blood VL. Viral shedding can be enhanced by genital infections and associated inflammation, but it can also occur in the absence of classical pathogens. Thus, we hypothesized that a dysregulated semen microbiome correlates with local HIV shedding. We analyzed semen samples from 49 men who have sex with men (MSM), including 22 HIV-uninfected and 27 HIV-infected men, at baseline and after starting antiretroviral therapy (ART) using 16S rRNA gene-based pyrosequencing and quantitative PCR. We studied the relationship of semen bacteria with HIV infection, semen cytokine levels, and semen VL by linear regression, non-metric multidimensional scaling, and goodness-of-fit test. Streptococcus, Corynebacterium, and Staphylococcus were common semen bacteria, irrespective of HIV status. While Ureaplasma was the more abundant Mollicutes in HIV-uninfected men, Mycoplasma dominated after HIV infection. HIV infection was associated with decreased semen microbiome diversity and richness, which were restored after six months of ART. In HIV-infected men, semen bacterial load correlated with seven pro-inflammatory semen cytokines, including IL-6 (p = 0.024), TNF-α (p = 0.009), and IL-1b (p = 0.002). IL-1b in particular was associated with semen VL (r² = 0.18, p = 0.02). Semen bacterial load was also directly linked to the semen HIV VL (r² = 0.15, p = 0.02). HIV infection reshapes the relationship between semen bacteria and pro-inflammatory cytokines, and both are linked to semen VL, which supports a role of the semen microbiome in HIV sexual transmission.     

gp340 expressed on human genital epithelia binds HIV-1 envelope protein and facilitates viral transmission

During sexual transmission of HIV in women, the first cells likely to be infected are submucosal CD4(+) T cells and dendritic cells of the lower genital tract. HIV is segregated from these target cells by an epithelial cell layer that can be bypassed even when healthy and intact. To understand how HIV penetrates this barrier, we identified a host protein, gp340, that is expressed on genital epithelium and binds the HIV envelope via a specific protein-protein interaction. This binding allows otherwise subinfectious amounts of HIV to efficiently infect target cells and allows this infection to occur over a longer period of time after binding. Our findings suggest a mechanism of viral entry during heterosexual transmission where HIV is bound to intact genital epithelia, which then promotes the initial events of infection. Understanding this step in the initiation of infection will allow for the development of tools and methods for blocking HIV transmission.     

Male Infertility Workup Needs Additional Testing of Expressed Prostatic Secretion and/or Post-Massage Urine

The male factor accounts for almost 50% of infertility cases. Inflammation may reduce semen quality via several pathways, including oxidative stress (OxS). As male infertility routinely is assessed using semen analysis only, the possible presence of non-leukocytospermic asymptomatic inflammatory prostatitis may be overlooked. We compared local and systemic OxS levels in male partners of infertile couples with different inflammation patterns in their genital tract and/or oligospermia.Subjects (n=143) were grouped according to inflammation in their semen, expressed prostatic secretion (EPS), and/or post-massage urine (post-M). Systemic (8-isoprostanes in urine) and local (diene conjugates and total antioxidant capacity in seminal plasma) OxS was measuredThe levels of OxS markers were significantly elevated in both severe inflammation groups – leukocytospermic men and subjects whose inflammation was limited only to EPS and/or post-M. Comparison between oligospermic and non-oligospermic men with genital tract inflammation, and oligozoospermic men with or without inflammation in the genital tract indicated that inflammation but not oligospermia status had significant impact on the measured OxS markers.Hence, a high leukocyte count in prostate-specific materials (EPS, post-M), even in absence of clear leukocytopsermia, is an important source of local and systemic OxS that may be associated with male infertility and affect general health. We suggest including the tests for detection of inflammation of the prostate into the workup of infertile men as was suggested in the WHO 1993 recommendation.     

Detection of viral genomes of caprine arthritis-encephalitis virus (CAEV) in semen and in genital tract tissues of male goat

The aim of this study was to determine the infectious status of semen and genital tract tissues from male goat naturally infected with the caprine lentivirus. Firstly, polymerase chain reaction (PCR) was used to detect the presence of CAEV proviral-DNA in the circulating mononuclear cells, semen (spermatozoa and non-spermatic cells), and genital tract tissues (testis, epididymis, vas deferens, and vesicular gland) of nine bucks. RT-PCR was used to detect the presence of CAEV viral RNA in seminal plasma. Secondly, in situ hybridization was performed on PCR-positive samples from the head, body, and tail of the epididymis. CAEV proviral-DNA was identified by PCR in the blood cells of 7/9 bucks and in non-spermatic cells of the seminal plasma of 3/9 bucks. No CAEV proviral-DNA was identified in the spermatozoa fraction. The presence of CAEV proviral-DNA in non-spermatic cells and the presence of CAEV in the seminal plasma was significantly higher (p<0.01) in bucks with PCR-positive blood. Two of the three bucks with positive seminal plasma cells presented with at least one PCR-positive genital tract tissue. Proviral-DNA was found in the head (3/9), body (3/9), and tail (2/9) of the epididymis. In situ hybridization confirmed the presence of viral mRNA in at least one of each of these tissues, in the periphery of the epididymal epithelium. This study clearly demonstrates the presence of viral mRNA and proviral-DNA in naturally infected male goat semen and in various tissues of the male genital tract.     

Dendritic Cell–T-Cell Interactions Support Coreceptor-Independent Human Immunodeficiency Virus Type 1 Transmission in the Human Genital Tract

Worldwide, human immunodeficiency virus (HIV) is transmitted predominantly by heterosexual contact. Here, we investigate for the first time, by examining mononuclear cells obtained from cervicovaginal tissue, the mechanisms whereby HIV type 1 (HIV-1) directly targets cells from the human genital tract. In contrast to earlier findings in mucosal models such as human skin, we demonstrate that the majority of T cells and macrophages but none or few dendritic cells (DC) express the HIV-1 coreceptor CCR5 in normal human cervicovaginal mucosa, whereas all three cell types express the coreceptor CXCR4. To understand the role of coreceptor expression on infectivity, mucosal mononuclear cells were infected with various HIV-1 isolates, using either CCR5 or CXCR4. Unstimulated T cells become rapidly, albeit nonproductively, infected with R5- and X4-tropic variants. However, DC and T cells form stable conjugates which permit productive infection by viruses of both coreceptor specificities. These results indicate that HIV-1 can exploit T-cell-DC synergism in the human genital tract to overcome potential coreceptor restrictions on DC and postentry blocks of viral replication in unactivated T cells. Thus, mononuclear cells infiltrating the genital mucosa are permissive for transmission of both R5- and X4-tropic HIV-1 variants, and selection of virus variants does not occur by differential expression of HIV-1 coreceptors on genital mononuclear cells.     

As Good As It Gets? The Problem of HIV Persistence despite Antiretroviral Drugs

Human immunodeficienty virus (HIV) infection is suppressed but not eliminated by antiretroviral drugs.?Viral?persistence in the face of therapy has been explained by viral latency, lowered effectiveness of drugs?in some anatomical sites and cell types, and cell-to-cell spread. These mechanisms allow for drug-sensitive virus to persist despite treatment. Understanding the persistence mechanism at work at?different times after infection, including the time of initial infection immediately following transmission?when reservoirs are first formed, will reveal if we are at the limit of what can be achieved with the?current therapy paradigm of suppressing ongoing virus replication with drugs. We discuss some of?the possible reasons why HIV persists at different points on the infection timeline, focusing on the?role ongoing replication may have in maintaining the infection despite drugs at early times postexposure.