Sex Differences in Human Immunodeficiency Virus Persistence and Reservoir Size During Aging.

BACKGROUND: Sex differences in human immunodeficiency virus (HIV) reservoir dynamics remain underexplored. METHODS: Longitudinal samples from virally suppressed midlife women (n = 59, median age 45 years) and age-matched men (n = 31) were analyzed retrospectively. At each time point, we measured sex hormones (by means of enzyme-linked immunosorbent assay) and cellular HIV DNA and RNA (by means of digital droplet polymerase chain reaction). Number of inducible HIV RNA+ cells, which provides an upper estimate of the replication-competent reservoir, was quantified longitudinally in a different subset of 14 women, across well-defined reproductive stages. Mixed-effects models included normalized reservoir outcomes and sex, time since antiretroviral therapy (ART) initiation, and the sex-by-time interaction as predictors. RESULTS: At ART initiation, women and men had median (interquartile range [IQR]) CD4+ T-cell counts of 204/µL (83-306/µL) versus 238/µL (120-284/µL), respectively; median ages of 45 (42-48) versus 47 (43-51) years; and median follow-up times of 79.2/µL (60.5-121.1/µL) versus 66.2/µL (43.2-80.6/µL) months. We observed a significant decline of total HIV DNA over time in both men and women (P < .01). However, the rates of change differed significantly between the sexes (P < .01), with women having a significantly slower rate of decline than men, more pronounced with age. By contrast, the levels of inducible HIV RNA increased incrementally over time in women during reproductive aging (P < .01). CONCLUSIONS: In contrast to men, in whom the HIV reservoir steadily declines with aging, the HIV reservoir in women is more dynamic. Total HIV DNA (including intact and defective genomes) declines more slowly in women than in men, while the inducible HIV RNA+ reservoir, which is highly enriched in replication-competent virus, increases in women after menopause.

Early Antiretroviral Therapy Is Associated with Lower HIV DNA Molecular Diversity and Lower Inflammation in Cerebrospinal Fluid but Does Not Prevent the Establishment of Compartmentalized HIV DNA Populations.

Even when antiretroviral therapy (ART) is started early after infection, HIV DNA might persist in the central nervous system (CNS), possibly contributing to inflammation, brain damage and neurocognitive impairment. Paired blood and cerebrospinal fluid (CSF) were collected from 16 HIV-infected individuals on suppressive ART: 9 participants started ART <4 months of the estimated date of infection (EDI) ("early ART"), and 7 participants started ART >14 months after EDI ("late ART"). For each participant, neurocognitive functioning was measured by Global Deficit Score (GDS). HIV DNA levels were measured in peripheral blood mononuclear cells (PBMCs) and CSF cell pellets by droplet digital (dd)PCR. Soluble markers of inflammation (sCD163, IL-6, MCP-1, TNF-α) and neuronal damage (neurofilament light [NFL]) were measured in blood and CSF supernatant by immunoassays. HIV-1 partial C2V3 env deep sequencing data (Roche 454) were obtained for 8 paired PBMC and CSF specimens and used for phylogenetic and compartmentalization analysis. Median exposure to ART at the time of sampling was 2.6 years (IQR: 2.2-3.7) and did not differ between groups. We observed that early ART was significantly associated with lower molecular diversity of HIV DNA in CSF (p<0.05), and lower IL-6 levels in CSF (p = 0.02), but no difference for GDS, NFL, or HIV DNA detectability compared to late ART. Compartmentalization of HIV DNA populations between CSF and blood was detected in 6 out of 8 participants with available paired HIV DNA sequences (2 from early and 4 from late ART group). Phylogenetic analysis confirmed the presence of monophyletic HIV DNA populations within the CSF in 7 participants, and the same population was repeatedly sampled over a 5 months period in one participant with longitudinal sampling. Such compartmentalized provirus in the CNS needs to be considered for the design of future eradication strategies and might contribute to the neuropathogenesis of HIV.

Evaluation of the Aptima HIV-1 Quant Dx Assay for HIV-1 RNA Quantitation in Different Biological Specimen Types.

The search for a cure for HIV infection has highlighted the need for increasingly sensitive and precise assays to measure viral burden in various tissues and body fluids. We describe the application of a standardized assay for HIV-1 RNA in multiple specimen types. The fully automated Aptima HIV-1 Quant Dx assay (Aptima assay) is FDA cleared for blood plasma HIV-1 RNA quantitation. In this study, the Aptima assay was applied for the quantitation of HIV RNA in peripheral blood mononuclear cells (PBMCs; n = 72), seminal plasma (n = 20), cerebrospinal fluid (CSF; n = 36), dried blood spots (DBS; n = 104), and dried plasma spots (DPS; n = 104). The Aptima assay was equivalent to or better than commercial assays or validated in-house assays for the quantitation of HIV RNA in CSF and seminal plasma. For PBMC specimens, the sensitivity of the Aptima assay in the detection of HIV RNA decayed as background uninfected PBMC counts increased; proteinase K treatment demonstrated some benefit in restoring signal at higher levels of background PBMCs. Finally, the Aptima assay yielded 100% detection rates of DBS in participants with plasma HIV RNA levels of ≥35 copies/ml and 100% detection rates of DPS in participants with plasma HIV RNA levels of ≥394 copies/ml. The Aptima assay can be applied to a variety of specimens from HIV-infected subjects to measure HIV RNA for studies of viral persistence and cure strategies. It can also detect HIV in dried blood and plasma specimens, which may be of benefit in resource-limited settings.

Replication of Human Herpesviruses Is Associated with Higher HIV DNA Levels during Antiretroviral Therapy Started at Early Phases of HIV Infection.

UNLABELLED: Asymptomatic replication of human herpesviruses (HHV) is frequent in HIV-infected men and is associated with increased T-cell activation and HIV disease progression. We hypothesized that the presence of replication of cytomegalovirus (CMV) and Epstein-Barr virus (EBV) (the most frequently detected HHV) might influence HIV DNA decay during antiretroviral therapy (ART). We investigated 607 peripheral blood mononuclear cell (PBMC) samples from 107 CMV-seropositive, HIV-infected men who have sex with men, who started ART within a median of 3 months from their estimated date of infection (EDI) and were monitored for a median of 19 months thereafter. Levels of HIV, CMV, and EBV DNA and cellular HIV RNA were measured by droplet digital PCR (ddPCR) for each time point. Using a general linear mixed-effect regression model, we evaluated associations between the presence of detectable CMV DNA and EBV DNA levels and HIV DNA decay and cellular HIV RNA levels, while adjusting for peak HIV RNA, nadir CD4(+)count, CD4/CD8 ratio, CMV IgG levels, time from EDI to ART initiation, time from ART initiation to virologic suppression, detectable CMV DNA pre-ART, and age. The presence of intermittent CMV DNA in PBMC during ART was significantly associated with slower decay of HIV DNA (P= 0.011) but not with increased cellular HIV RNA transcription or more detectable 2-long terminal repeat circles. Higher levels of EBV DNA were also associated with higher levels of HIV DNA (P< 0.001) and increased unspliced cellular HIV RNA transcription (P= 0.010). These observations suggest that replication of HHV may help maintain a larger HIV DNA reservoir, but the underlying mechanisms remain unclear. IMPORTANCE: Over three-fourths of HIV-infected men have at least one actively replicating human herpesvirus (HHV) in their mucosal secretions at any one time. Cytomegalovirus (CMV) and Epstein-Barr virus (EBV) are the most common, and although it is often asymptomatic, such CMV and EBV replication is associated with higher levels of immune activation and HIV disease progression. We hypothesized that HHV-associated activation of HIV-infected CD4(+)T cells might lead to increased HIV DNA. This study found that detectable CMV in blood cells of HIV-infected men was associated with slower decay of HIV DNA even during antiretroviral therapy (ART) that was started during early HIV infection. Similarly, levels of EBV DNA were associated with higher levels of HIV DNA during ART. If this observation points to a causal pathway, interventions that control CMV and EBV replication may be able to reduce the HIV reservoir, which might be relevant to current HIV cure efforts.

Absence of detectable HIV-1 viremia after treatment cessation in an infant.

An infant born to a woman with human immunodeficiency virus type 1 (HIV-1) infection began receiving antiretroviral therapy (ART) 30 hours after birth owing to high-risk exposure. ART was continued when detection of HIV-1 DNA and RNA on repeat testing met the standard diagnostic criteria for infection. After therapy was discontinued (when the child was 18 months of age), levels of plasma HIV-1 RNA, proviral DNA in peripheral-blood mononuclear cells, and HIV-1 antibodies, as assessed by means of clinical assays, remained undetectable in the child through 30 months of age. This case suggests that very early ART in infants may alter the establishment and long-term persistence of HIV-1 infection.

Comparative analysis of measures of viral reservoirs in HIV-1 eradication studies.

HIV-1 reservoirs preclude virus eradication in patients receiving highly active antiretroviral therapy (HAART). The best characterized reservoir is a small, difficult-to-quantify pool of resting memory CD4(+) T cells carrying latent but replication-competent viral genomes. Because strategies targeting this latent reservoir are now being tested in clinical trials, well-validated high-throughput assays that quantify this reservoir are urgently needed. Here we compare eleven different approaches for quantitating persistent HIV-1 in 30 patients on HAART, using the original viral outgrowth assay for resting CD4(+) T cells carrying inducible, replication-competent viral genomes as a standard for comparison. PCR-based assays for cells containing HIV-1 DNA gave infected cell frequencies at least 2 logs higher than the viral outgrowth assay, even in subjects who started HAART during acute/early infection. This difference may reflect defective viral genomes. The ratio of infected cell frequencies determined by viral outgrowth and PCR-based assays varied dramatically between patients. Although strong correlations with the viral outgrowth assay could not be formally excluded for most assays, correlations achieved statistical significance only for integrated HIV-1 DNA in peripheral blood mononuclear cells and HIV-1 RNA/DNA ratio in rectal CD4(+) T cells. Residual viremia was below the limit of detection in many subjects and did not correlate with the viral outgrowth assays. The dramatic differences in infected cell frequencies and the lack of a precise correlation between culture and PCR-based assays raise the possibility that the successful clearance of latently infected cells may be masked by a larger and variable pool of cells with defective proviruses. These defective proviruses are detected by PCR but may not be affected by reactivation strategies and may not require eradication to accomplish an effective cure. A molecular understanding of the discrepancy between infected cell frequencies measured by viral outgrowth versus PCR assays is an urgent priority in HIV-1 cure research.

HIV type 1 (HIV-1) proviral reservoirs decay continuously under sustained virologic control in HIV-1-infected children who received early treatment.

BACKGROUND: Early initiation of combination antiretroviral therapy (cART) to human immunodeficiency virus type 1 (HIV-1)-infected infants controls HIV-1 replication and reduces mortality. METHODS: Plasma viremia (lower limit of detection, <2 copies/mL), T-cell activation, HIV-1-specific immune responses, and the persistence of cells carrying replication-competent virus were quantified during long-term effective combination antiretroviral therapy (cART) in 4 perinatally HIV-1-infected youth who received treatment early (the ET group) and 4 who received treatment late (the LT group). Decay in peripheral blood mononuclear cell (PBMC) proviral DNA levels was also measured over time in the ET youth. RESULTS: Plasma viremia was not detected in any ET youth but was detected in all LT youth (median, 8 copies/mL; P = .03). PBMC proviral load was significantly lower in ET youth (median, 7 copies per million PBMCs) than in LT youth (median, 181 copies; P = .03). Replication-competent virus was recovered from all LT youth but only 1 ET youth. Decay in proviral DNA was noted in all 4 ET youth in association with limited T-cell activation and with absent to minimal HIV-1-specific immune responses. CONCLUSIONS: Initiation of early effective cART during infancy significantly limits circulating levels of proviral and replication-competent HIV-1 and promotes continuous decay of viral reservoirs. Continued cART with reduction in HIV-1 reservoirs over time may facilitate HIV-1 eradication strategies.

Decreased HIV type 1 transcription in CCR5-Δ32 heterozygotes during suppressive antiretroviral therapy.

Individuals who are heterozygous for the CCR5-Δ32 mutation provide a natural model to examine the effects of reduced CCR5 expression on human immunodeficiency virus (HIV) persistence. We evaluated the HIV reservoir in 18 CCR5-Δ32 heterozygotes and 54 CCR5 wild-type individuals during suppressive antiretroviral therapy. Cell-associated HIV RNA levels (P=.035), RNA to DNA transcriptional ratios (P=.013), and frequency of detectable HIV 2-long terminal repeat circular DNA (P=.013) were significantly lower in CD4+ T cells from CCR5-Δ32 heterozygotes. Cell-associated HIV RNA was significantly correlated with CCR5 surface expression on CD4+ T cells (r2=0.136; P=.002). Our findings suggest that curative strategies should further explore manipulation of CCR5.

HIV-1 expression within resting CD4+ T cells after multiple doses of vorinostat.

BACKGROUND: A single dose of the histone deacetylase inhibitor vorinostat (VOR) up-regulates HIV RNA expression within resting CD4(+) T cells of treated, aviremic human immunodeficiency virus (HIV)-positive participants. The ability of multiple exposures to VOR to repeatedly disrupt latency has not been directly measured, to our knowledge. METHODS: Five participants in whom resting CD4(+) T-cell-associated HIV RNA (rc-RNA) increased after a single dose of VOR agreed to receive daily VOR Monday through Wednesday for 8 weekly cycles. VOR serum levels, peripheral blood mononuclear cell histone acetylation, plasma HIV RNA single-copy assays, rc-RNA, total cellular HIV DNA, and quantitative viral outgrowth assays from resting CD4(+) T cells were assayed. RESULTS: VOR was well tolerated, with exposures within expected parameters. However, rc-RNA measured after dose 11 (second dose of cycle 4) or dose 22 (second dose of cycle 8) increased significantly in only 3 of the 5 participants, and the magnitude of the rc-RNA increase was much reduced compared with that after a single dose. Changes in histone acetylation were blunted. Results of quantitative viral outgrowth and other assays were unchanged. CONCLUSIONS: Although HIV latency is disrupted by an initial VOR dose, the effect of subsequent doses in this protocol was much reduced. We hypothesize that the global effect of VOR results in a refractory period of ≥ 24 hours. The optimal schedule for VOR administration is still to be defined.

Increase in 2-long terminal repeat circles and decrease in D-dimer after raltegravir intensification in patients with treated HIV infection: a randomized, placebo-controlled trial.

BACKGROUND: The degree to which human immunodeficiency virus (HIV) continues to replicate during antiretroviral therapy (ART) is controversial. We conducted a randomized, double-blind, placebo-controlled study to assess whether raltegravir intensification reduces low-level viral replication, as defined by an increase in the level of 2-long terminal repeat (2-LTR) circles. METHODS: Thirty-one subjects with an ART-suppressed plasma HIV RNA level of <40 copies/mL and a CD4(+) T-cell count of ≥350 cells/mm(3) for ≥1 year were randomly assigned to receive raltegravir 400 mg twice daily or placebo for 24 weeks. 2-LTR circles were analyzed by droplet digital polymerase chain reaction at weeks 0, 1, 2, and 8. RESULTS: The median duration of ART suppression was 3.8 years. The raltegravir group had a significant increase in the level of 2-LTR circles, compared to the placebo group. The week 1 to 0 ratio was 8.8-fold higher (P = .0025) and the week 2 to 0 ratio was 5.7-fold higher (P = .023) in the raltegravir vs. placebo group. Intensification also led to a statistically significant decrease in the D-dimer level, compared to placebo (P = .045). CONCLUSIONS: Raltegravir intensification resulted in a rapid increase in the level of 2-LTR circles in a proportion of subjects, indicating that low-level viral replication persists in some individuals even after long-term ART. Intensification also reduced the D-dimer level, a coagulation biomarker that is predictive of morbidity and mortality among patients receiving treatment for HIV infection.

Associations between virologic and immunologic dynamics in blood and in the male genital tract.

To determine the influence of asymptomatic genital viral infections on the cellular components of semen and blood, we evaluated the associations between the numbers and activation statuses of CD4+ and CD8+ T lymphocytes in both compartments and the seminal levels of cytomegalovirus (CMV), herpes simplex virus (HSV), and human immunodeficiency virus 1 (HIV). Paired blood and semen samples were collected from 36 HIV-infected antiretroviral-naïve individuals and from 40 HIV-uninfected participants. We performed multiparameter flow cytometry analysis (CD45, CD45RA, CD3, CD4, CD8, and CD38) of seminal and blood cellular components and measured HIV RNA and CMV and HSV DNA levels in seminal and blood plasma by real-time PCR. Compared to HIV-uninfected participants, in the seminal compartment HIV-infected participants had higher levels of CMV (P < 0.05), higher numbers of total CD3+ (P < 0.01) and CD8+ subset (P < 0.01) T lymphocytes, and higher CD4+ and CD8+ T lymphocyte activation (RA-CD38+) (P < 0.01). Seminal CMV levels positively correlated with absolute numbers of CD4+ and CD8+ T cells in semen (P < 0.05) and with the activation status of CD4+ T cells in semen and in blood (P < 0.01). HIV levels in semen (P < 0.05) and blood (P < 0.01) were positively associated with T-cell activation in blood. Activation of CD8+ T cells in blood remained an independent predictor of HIV levels in semen in multivariate analysis. The virologic milieu in the male genital tract strongly influences the recruitment and activation of immune cells in semen and may also modulate T-cell immune activation in blood. These factors likely influence replication dynamics, sexual transmission risk, and disease outcomes for all three viruses.

In vivo CD8+ T-cell suppression of siv viremia is not mediated by CTL clearance of productively infected cells.

The CD8+ T-cell is a key mediator of antiviral immunity, potentially contributing to control of pathogenic lentiviral infection through both innate and adaptive mechanisms. We studied viral dynamics during antiretroviral treatment of simian immunodeficiency virus (SIV) infected rhesus macaques following CD8+ T-cell depletion to test the importance of adaptive cytotoxic effects in clearance of cells productively infected with SIV. As previously described, plasma viral load (VL) increased following CD8+ T-cell depletion and was proportional to the magnitude of CD8+ T-cell depletion in the GALT, confirming a direct relationship between CD8+ T-cell loss and viral replication. Surprisingly, first phase plasma virus decay following administration of antiretroviral drugs was not slower in CD8+ T-cell depleted animals compared with controls indicating that the short lifespan of the average productively infected cell is not a reflection of cytotoxic T-lymphocyte (CTL) killing. Our findings support a dominant role for non-cytotoxic effects of CD8+ T-cells on control of pathogenic lentiviral infection and suggest that cytotoxic effects, if present, are limited to early, pre-productive stages of the viral life cycle. These observations have important implications for future strategies to augment immune control of HIV.

Impact of seminal cytomegalovirus replication on HIV-1 dynamics between blood and semen.

The genital tract of individuals infected with human immunodeficiency virus type 1 (HIV-1) is an anatomic compartment that supports local HIV-1 and cytomegalovirus (CMV) replication. This study investigated the association of seminal CMV replication with changes in HIV-1 clonal expansion, evolution and phylogenetic compartmentalization between blood and semen. Fourteen paired blood and semen samples were analyzed from four untreated subjects. Clonal sequences (n = 607) were generated from extracted HIV-1 RNA (env C2-V3 region), and HIV-1 and CMV levels were measured in the seminal plasma by real-time PCR. Sequence alignments were evaluated for: (i) viral compartmentalization between semen and blood samples using Slatkin-Maddison and F(ST) methods, (ii) different nucleotide substitution rates in semen and blood, and (iii) association between proportions of clonal HIV-1 sequences in each compartment and seminal CMV levels. Half of the semen samples had detectable CMV DNA, with at least one CMV positive sample for each patient. Seminal CMV DNA levels correlated positively with seminal HIV-1 RNA levels (Spearman P = 0.05). A trend towards an association between compartmentalization of HIV-1 sequences sampled from blood and semen and presence of seminal CMV was observed (Cochran Q test P = 0.12). Evolutionary rates between semen and blood HIV-1 populations did not differ significantly, and there was no significant association between seminal CMV DNA levels and the frequency of non-unique clonal HIV-1 sequences in the semen. In conclusion, the effects of CMV replication on HIV-1 viral and immunologic dynamics within the male genital tract are not significant enough to perturb evolution or disrupt compartmentalization in the genital tract.

Transplantation of CCR5∆32 Homozygous Umbilical Cord Blood in a Child With Acute Lymphoblastic Leukemia and Perinatally Acquired HIV Infection.

BACKGROUND: Allogeneic hematopoietic cell transplantation (allo-HCT) in a CCR5∆32 homozygous donor resulted in HIV cure. Understanding how allo-HCT impacts the HIV reservoir will inform cure strategies. METHODS: A 12-year-old with perinatally acquired, CCR5-tropic HIV and acute lymphoblastic leukemia underwent myeloablative conditioning and umbilical cord blood (UCB) transplantation from a CCR5∆32 homozygous donor. Peripheral blood mononuclear cells (PBMCs) and the rectum were sampled pre- and post-transplant. The brain, lung, lymph node (LN), stomach, duodenum, ileum, and colon were sampled 73 days after transplantation (day +73), when the patient died from graft-vs-host disease. Droplet digital polymerase chain reaction (ddPCR) and in situ hybridization (ISH) were used detect the HIV reservoir in tissues. CCR5 and CD3 expression in the LN was assessed using immunohistochemistry (IHC). RESULTS: HIV DNA (vDNA) was detected in PBMCs by ddPCR pretransplant but not post-transplant. vDNA was detected by ISH in the rectum at days -8 and +22, and in the LN, colon, lung, and brain day +73. vDNA was also detected in the lung by ddPCR. IHC revealed CCR5+CD3+ cells in the LN postmortem. CONCLUSIONS: HIV was detected in multiple tissues 73 days after CCR5∆32 homozygous UCB allo-HCT despite myeloablative conditioning and complete donor marrow engraftment. These results highlight the importance of analyzing tissue during HIV cure interventions and inform the choice of assay used to detect HIV in tissue reservoirs.

Genetic attributes of cerebrospinal fluid-derived HIV-1 env.

HIV-1 often invades the CNS during primary infection, eventually resulting in neurological disorders in up to 50% of untreated patients. The CNS is a distinct viral reservoir, differing from peripheral tissues in immunological surveillance, target cell characteristics and antiretroviral penetration. Neurotropic HIV-1 likely develops distinct genotypic characteristics in response to this unique selective environment. We sought to catalogue the genetic features of CNS-derived HIV-1 by analysing 456 clonal RNA sequences of the C2-V3 env subregion generated from CSF and plasma of 18 chronically infected individuals. Neuropsychological performance of all subjects was evaluated and summarized as a global deficit score. A battery of phylogenetic, statistical and machine learning tools was applied to these data to identify genetic features associated with HIV-1 neurotropism and neurovirulence. Eleven of 18 individuals exhibited significant viral compartmentalization between blood and CSF (P < 0.01, Slatkin-Maddison test). A CSF-specific genetic signature was identified, comprising positions 9, 13 and 19 of the V3 loop. The residue at position 5 of the V3 loop was highly correlated with neurocognitive deficit (P < 0.0025, Fisher's exact test). Antibody-mediated HIV-1 neutralizing activity was significantly reduced in CSF with respect to autologous blood plasma (P < 0.042, Student's t-test). Accordingly, CSF-derived sequences exhibited constrained diversity and contained fewer glycosylated and positively selected sites. Our results suggest that there are several genetic features that distinguish CSF- and plasma-derived HIV-1 populations, probably reflecting altered cellular entry requirements and decreased immune pressure in the CNS. Furthermore, neurological impairment may be influenced by mutations within the viral V3 loop sequence.

Relative efficacy of T cell stimuli as inducers of productive HIV-1 replication in latently infected CD4 lymphocytes from patients on suppressive cART.

Quantification of cell-associated replication-competent HIV, in blood samples from patients with undetectable plasma viremia, requires specialized culture conditions that include exogenous pan T cell stimulation. Different research groups have used several stimuli for this purpose; however, the relative efficacies of these T cell stimuli to induce productive HIV replication from latently infected cells ex vivo have not been systematically evaluated. To this end, we compared four commonly used T cell stimuli: 1) irradiated allogeneic cells plus phytohaemagglutinin (PHA); 2) PHA alone; 3) phorbol myristate acetate plus Ionomycin; and 4) immobilized αCD3 plus αCD28 antibodies. End-point dilutions of patient CD4 T cells were performed, using virion RNA production to quantify HIV induction. Our results demonstrated that these activation approaches were not equivalent and that antibody cross-linking of CD3 and CD28 membrane receptors was the most effective means to activate HIV replication from a resting cell state, closely followed by stimulation with irradiated allogeneic cells plus PHA.

A Novel Shiga Toxin 1a-Converting Bacteriophage of Shigella sonnei With Close Relationship to Shiga Toxin 2-Converting Pages of Escherichia coli.

In recent studies, strains of non-dysenteriae 1 Shigella (NDS) expressing Shiga toxin have been reported. In this study, we report a novel stx1a-converting bacteriophage of Shigella sonnei associated with travel to Mexico. Phylogenetic comparison between this and other stx-converting phages suggests that toxigenic NDS strains have arisen through separate horizontal transfer events from toxigenic Escherichia coli.

Defective proviruses rapidly accumulate during acute HIV-1 infection.

Although antiretroviral therapy (ART) suppresses viral replication to clinically undetectable levels, human immunodeficiency virus type 1 (HIV-1) persists in CD4(+) T cells in a latent form that is not targeted by the immune system or by ART. This latent reservoir is a major barrier to curing individuals of HIV-1 infection. Many individuals initiate ART during chronic infection, and in this setting, most proviruses are defective. However, the dynamics of the accumulation and the persistence of defective proviruses during acute HIV-1 infection are largely unknown. Here we show that defective proviruses accumulate rapidly within the first few weeks of infection to make up over 93% of all proviruses, regardless of how early ART is initiated. By using an unbiased method to amplify near-full-length proviral genomes from HIV-1-infected adults treated at different stages of infection, we demonstrate that early initiation of ART limits the size of the reservoir but does not profoundly affect the proviral landscape. This analysis allows us to revise our understanding of the composition of proviral populations and estimate the true reservoir size in individuals who were treated early versus late in infection. Additionally, we demonstrate that common assays for measuring the reservoir do not correlate with reservoir size, as determined by the number of genetically intact proviruses. These findings reveal hurdles that must be overcome to successfully analyze future HIV-1 cure strategies.

Quantification of Total and 2-LTR (Long terminal repeat) HIV DNA, HIV RNA and Herpesvirus DNA in PBMCs.

Almost all individuals infected with human immunodeficiency virus (HIV) are also infected with cytomegalovirus (CMV) and Epstein Barr virus (EBV). The aims of our studies have included characterizing and measuring the latent HIV reservoir and understanding the association between asymptomatic replication of CMV (and other herpesvirus, including EBV) and this HIV reservoir (Gianella et al., 2014). This protocol was designed to simultaneously co-extract DNA and RNA from the same peripheral blood mononuclear cell (PBMC) aliquot and quantify HIV, CMV and EBV DNA, as well as HIV RNA using droplet digital PCR (ddPCR). For collection and processing of male genital secretions and quantification of HIV RNA and DNA from seven human herpesviruses from seminal plasma, refer to protocol "Quantification of HIV RNA and Human Herpesvirus DNA in Seminal Plasma" (Vargas-Meneses et al., 2015).

During Stably Suppressive Antiretroviral Therapy Integrated HIV-1 DNA Load in Peripheral Blood is Associated with the Frequency of CD8 Cells Expressing HLA-DR/DP/DQ.

BACKGROUND: Characterising the correlates of HIV persistence improves understanding of disease pathogenesis and guides the design of curative strategies. This study investigated factors associated with integrated HIV-1 DNA load during consistently suppressive first-line antiretroviral therapy (ART). METHOD: Total, integrated, and 2-long terminal repeats (LTR) circular HIV-1 DNA, residual plasma HIV-1 RNA, T-cell activation markers, and soluble CD14 (sCD14) were measured in peripheral blood of 50 patients that had received 1-14 years of efavirenz-based or nevirapine-based therapy. RESULTS: Integrated HIV-1 DNA load (per 10(6) peripheral blood mononuclear cells) was median 1.9 log10 copies (interquartile range 1.7-2.2) and showed a mean difference of 0.2 log10 copies per 10 years of suppressive ART (95% confidence interval - 0.2, 0.6; p = 0.28). It was positively correlated with total HIV-1 DNA load and frequency of CD8(+)HLA-DR/DP/DQ(+) cells, and was also higher in subjects with higher sCD14 levels, but showed no correlation with levels of 2-LTR circular HIV-1 DNA and residual plasma HIV-1 RNA, or the frequency of CD4(+)CD38(+) and CD8(+)CD38(+) cells. Adjusting for pre-ART viral load, duration of suppressive ART, CD4 cell counts, residual plasma HIV-1 RNA levels, and sCD14 levels, integrated HIV-1 DNA load was mean 0.5 log10 copies higher for each 50% higher frequency of CD8(+)HLA-DR/DP/DQ(+) cells (95% confidence interval 0.2, 0.9; p = 0.01). CONCLUSIONS: The observed positive association between integrated HIV-1 DNA load and frequency of CD8(+)DR/DP/DQ(+) cells indicates that a close correlation between HIV persistence and immune activation continues during consistently suppressive therapy. The inducers of the distinct activation profile warrant further investigation.