A minor population of macrophage-tropic HIV-1 variants is identified in recrudescing viremia following analytic treatment interruption.

HIV-1 persists in cellular reservoirs that can reignite viremia if antiretroviral therapy (ART) is interrupted. Therefore, insight into the nature of those reservoirs may be revealed from the composition of recrudescing viremia following treatment cessation. A minor population of macrophage-tropic (M-tropic) viruses was identified in a library of recombinant viruses constructed with individual envelope genes that were obtained from plasma of six individuals undergoing analytic treatment interruption (ATI). M-tropic viruses could also be enriched from post-ATI plasma using macrophage-specific (CD14) but not CD4+ T cell-specific (CD3) antibodies, suggesting that M-tropic viruses had a macrophage origin. Molecular clock analysis indicated that the establishment of M-tropic HIV-1 variants predated ATI. Collectively, these data suggest that macrophages are a viral reservoir in HIV-1-infected individuals on effective ART and that M-tropic variants can appear in rebounding viremia when treatment is interrupted. These findings have implications for the design of curative strategies for HIV-1.

Peripheral T Follicular Helper Cells Are the Major HIV Reservoir within Central Memory CD4 T Cells in Peripheral Blood from Chronically HIV-Infected Individuals on Combination Antiretroviral Therapy.

UNLABELLED: In this study, we examined the peripheral blood (PB) central memory (TCM) CD4(+) T cell subsets designated peripheral T follicular helper cells (pTfh cells) and non-pTfh cells to assess HIV permissiveness and persistence. Purified pTfh and non-pTfh cells from healthy HIV-negative donors were tested for HIV permissiveness using green fluorescent protein (GFP)-expressing HIV-1NL4-3/Ba-L, followed by viral reactivation using beads coated with anti-CD3/anti-CD28 monoclonal antibodies. The role of pTfh cells in HIV persistence was analyzed in 12 chronically HIV-1 infected patients before and 48 weeks after initiation of raltegravir-containing combination antiretroviral therapy (cART). Total cellular HIV-1 DNA and episomes containing two copies of the viral long terminal repeat (2LTR circles) were analyzed in using droplet digital PCR in the purified pTfh and non-pTfh cells. Activation-inducible HIV p24 expression was determined by flow cytometry. Results indicate that pTfh cells, in particular PD1(+) pTfh cells, showed greater permissiveness for HIV infection than non-pTfh cells. At week 48 on cART, HIV DNA levels were unchanged from pre-cART levels, although a significant decrease in 2LTR circles was observed in both cell subsets. Inducible HIV p24 expression was higher in pTfh cells than in non-pTfh cells, with the highest frequencies in the PD1(+) CXCR3(-) pTfh cell subset. Frequencies of HLADR(+) CD38(+) activated CD4 T cells correlated with 2LTR circles in pTfh and non-pTfh cells at both time points and with p24(+) cells at entry. In conclusion, among CD4 TCM cells in PB of aviremic patients on cART, pTfh cells, in particular the PD1(+) CXCR3(-) subset, constitute a major HIV reservoir that is sustained by ongoing residual immune activation. The inducible HIV p24 assay is useful for monitoring HIV reservoirs in defined CD4 T cell subsets. IMPORTANCE: Identification of the type and nature of the cellular compartments of circulating HIV reservoirs is important for targeting of HIV cure strategies. In lymph nodes (LN), a subset of CD4 T cells called T follicular helper (Tfh) cells are preferentially infected by HIV. Central memory (TCM) CD4 T cells are the major cellular reservoir for HIV in peripheral blood and contain a subset of CD4 TCM cells expressing chemokine receptor CXCR5 similar in function to LN Tfh cells termed peripheral Tfh (pTfh) cells. We found that the circulating pTfh cells are highly susceptible to HIV infection and that in HIV-infected patients, HIV persists in these cells following plasma virus suppression with potent cART. These pTfh cells, which constitute a subset of TCM CD4 T cells, can be readily monitored in peripheral blood to assess HIV persistence.

Episomal viral cDNAs identify a reservoir that fuels viral rebound after treatment interruption and that contributes to treatment failure.

Viral reservoirs that persist in HIV-1 infected individuals on antiretroviral therapy (ART) are the major obstacle to viral eradication. The identification and definition of viral reservoirs in patients on ART is needed in order to understand viral persistence and achieve the goal of viral eradication. We examined whether analysis of episomal HIV-1 genomes provided the means to characterize virus that persists during ART and whether it could reveal the virus that contributes to treatment failure in patients on ART. For six individuals in which virus replication was highly suppressed for at least 20 months, proviral and episomal genomes present just prior to rebound were phylogenetically compared to RNA genomes of rebounding virus after therapy interruption. Episomal envelope sequences, but not proviral envelope sequences, were highly similar to sequences in rebounding virus. Since episomes are products of recent infections, the phylogenetic relationships support the conclusion that viral rebound originated from a cryptic viral reservoir. To evaluate whether the reservoir revealed by episomal sequence analysis was of clinical relevance, we examined whether episomal sequences define a viral population that contributes to virologic failure in individuals receiving the CCR5 antagonist, Vicriviroc. Episomal envelope sequences at or near baseline predicted treatment failure due to the presence of X4 or D/M (dual/mixed) viral variants. In patients that did not harbor X4 or D/M viruses, the basis for Vicriviroc treatment failure was indeterminate. Although these samples were obtained from viremic patients, the assay would be applicable to a large percentage of aviremic patients, based on previous studies. Summarily, the results support the use of episomal HIV-1 as an additional or alternative approach to traditional assays to characterize virus that is maintained during long-term, suppressive ART.

A therapeutic HIV-1 vaccine reduces markers of systemic immune activation and latent infection in patients under highly active antiretroviral therapy.

HIV infection is characterized by chronic immune activation and the establishment of a pool of latently infected cells. Antiretroviral therapy (ART) can suppress viral load to undetectable levels in peripheral blood by standard measure, however immune activation/chronic inflammation and latent infection persist and affect quality of life. We have now shown that a novel therapeutic HIV vaccine consisting of replication-defective HIV (HIVAX), given in the context of viral suppression under ART, can reduce both immune activation/chronic inflammation and latent infection. Immune activation, as measured by percent of CD8 + HLA-DR + CD38 + T cells, approached levels of healthy controls at week 16 following vaccination. Reduced immune activation was accompanied by a reduction in pro-inflammatory cytokines and peripheral α4ß7 + plasmacytoid DC (a marker of mucosal immune activation). Levels of both HIV-1 DNA and 2-LTR circles were reduced at week 16 following vaccination, suggesting HIVAX can impact HIV-1 latency and reduce viral replication. Surprisingly, reduced immune activation/chronic inflammation was accompanied by an increase in the percent of memory CD4 + T cells expressing markers PD-1 and TIM-3. In addition, evaluation of HIV-1 Gag-specific CD4 + T cells for expression of 96 T cell related genes pre- and post-therapy revealed increased expression of a number of genes involved in the regulation of immune activation, T cell activation, and antiviral responses. Overall this study provides evidence that vaccination with HIVAX in subjects under long term antiviral suppression can reduce immune activation/chronic inflammation and latent infection (Clinicaltrials.gov, identifier NCT01428596).

Twenty-four mini-pool HCV RNA screening outside a blood transfusion setting: results of a 2-year prospective study.

The usefulness of 24 mini-pool hepatitis C virus (HCV) RNA screening was evaluated in a 2-year prospective study carried out on a total of 6432 consecutive anti-HCV negative specimens in a routine diagnostic laboratory setting. A total of 268 mini-pools were tested using an automated commercial PCR assay for qualitative detection of HCV RNA, with a lower limit of detection of 50 IU/ml. Eighteen (0.28%) anti-HCV negative/HCV RNA positive serum samples obtained from 12 patients (all intravenous drug users), were detected. Ten patients responded to an invitation for follow-up testing. Five, three and one patient seroconverted in the first, second and third follow-up sample, respectively. One patient had not seroconverted by the end of the study period. The interval between the first HCV RNA positive sample and the first anti-HCV positive samples was 24-192 days. The costs of detecting a single anti-HCV negative/HCV RNA positive sample and a single anti-HCV negative/HCV RNA positive patient using the 24 mini-pool HCV RNA screening strategy were estimated to be around euro 643 and 965, respectively. It was shown that screening for HCV infection using the 24 mini-pool HCV RNA screening strategy can also be both useful and cost effective outside a blood transfusion setting.

Prevalence of antiretroviral drug resistance mutations and HIV-1 non-B subtypes in newly diagnosed drug-naïve patients in Slovenia, 2000-2004.

In order to estimate the prevalence and patterns of antiretroviral drug resistance mutations in drug-naïve HIV-1 infected patients in Slovenia, and the prevalence of non-B subtypes, a retrospective study was conducted on a cohort, representing 87% of the total of newly diagnosed HIV-1 infected patients, in a 5 year period (2000-2004). Protease (PR) and reverse transcriptase (RT) sequences were determined in 77 newly diagnosed HIV-1 patients. Non-B subtypes were present in 18% of the population tested. Transmitted drug resistance was identified as in the CATCH study: the presence of primary PR and RT gene mutations according to the IAS-USA mutation list including the revertant mutations in codon 215 and excluding mutations on the RT positions 44 and 118. The estimated prevalence of transmitted resistance mutations was 3.9%. Namely, three out of 77 patients had mutations associated with resistance to NRTIs: one patient carried M184V in association with A62V, while a revertant mutation T215D was found in two patients. No transmitted drug resistance to NNRTIs or PIs was detected. However, to score the expected response to therapy using the REGA and the Stanford algorithms, we also took into account secondary PR mutations and additional RT mutations. Reduced response to some therapeutic options was predicted in five patients (6.5%). In conclusion, testing the vast majority of all newly diagnosed HIV-1 patients in the last 5 years in Slovenia uncovered a relatively high prevalence of non-B subtypes and a low prevalence of transmitted drug resistance.

Genetic variability of gag and env regions of HIV type 1 strains circulating in Slovenia.

The aim of this study was to investigate the genetic diversity of HIV-1 strains circulating in Slovenia. Proviral DNA isolated from peripheral blood mononuclear cells (PBMCs) of 20 randomly selected HIV-1-infected individuals was classified into subtypes by sequence-based phylogenetic analysis of the env (C2V3) and gag (p24) regions of the viral genome. The phylogenetic tree based on env C2V3 sequences showed that 15 of the 20 samples were subtype B, two A1, one F1, one CRF01_AE, and one CRF02_AG. The phylogenetic analysis of the gag gene yielded identical results expect for one sample that had a discordant subtype; it was identified as subtype A1 in the env and AE in the gag region. Our study confirmed that although subtype B predominates, other subtypes and circulating recombinant forms (CRFs) are also present in Slovenia. The high intrasubtype genetic diversity of subtype B sequences suggests a multiple introduction of subtype B strains into Slovenia.

HIV-1 subtype B epidemic and transmission patterns in Slovenia.

In the present study the epidemic of human immunodeficiency virus type 1 (HIV-1) subtype B in Slovenia during the 10-year period was investigated using phylogenetic analysis of pol gene sequences. 119 pol sequences generated on samples dated from January 1996 to December 2005 were retrieved from the database of Slovenian HIV/AIDS Reference Laboratory. The phylogenetic analysis revealed 14 potentially significant transmission clusters (bootstrap value > or = 98%), comprising 34 HIV-1 strains. The vast majority of clustered individuals were men (91%), and of them, 79% were men who have sex with men. Factors significantly associated with clustering were: recent infection (HIV-1 infection during or after year 2003), diagnosis of primary HIV-1 infection, higher CD4 cell count and acquiring HIV-1 infection in Slovenia. Recent subtype B HIV-1 infections are the important driving force of current HIV-1 epidemic in Slovenia.

Clinical features and virologic characteristics of primary and early HIV-1 infection in Slovenian patients.

Analysis of time trends in newly diagnosed HIV-1 infected patients in Slovenia over a 10-year period (1996-2005) showed an increase in the number of newly diagnosed HIV-1 infected patients in 2004 and 2005 as well as increase in the number of newly diagnosed patients with primary/early HIV-1 infection. A retrospective analysis was performed in order to evaluate the clinical, epidemiological, laboratory and virological parameters of primary/early HIV-1 infection presenting with or without acute retroviral syndrome (ARS). Primary/early HIV-1 infection was diagnosed in 33 (19.5%) out of 169 newly diagnosed HIV-1 infected patients during the 10-year period. Most patients experienced ARS, the most commonly reported symptoms being fever, malaise and pharyngitis, followed by rash and lymphadenopathy. Median CD4 cell count was 415 cells/mm3, median CD8 cell count was 865 cells/mm3 and median HIV-1 viral load at the time of diagnosis was 5.1 loglo copies/mL. The increase in the number of newly diagnosed HIV-1 infected patients may be in part due to increased awareness among clinicians of the possibility of ARS, and the possibility of increased awareness of symptoms of ARS among persons at high risk of infection.

False-positive result of a confirmatory human immunodeficiency virus line immuno assay in an apparently healthy individual--a case report.

A case of a false-positive result of human immunodeficiency virus (HIV) confirmatory immunoblot-based assay is described. Repeatedly borderline reactive anti-HIV screening enzyme immunoassay result obtained in a local hospital resulted in directing the sample to the Slovenian HIV/AIDS Reference Laboratory. In the Reference Laboratory, both anti-HIV screening assays and confirmatory Western blot were negative, while a confirmatory test INNO-LIA HIV I/II Score (Innogenetics, Ghent, Belgium) was anti-HIV-1 positive due to sgp120 and gp41 reactivity. The results of serological testing of the second sample obtained three weeks later were completely identical, while in the third sample obtained 5 months later, seroreversion was observed. Due to a negative dynamics in anti-HIV serological profile and repeatedly negative results of the molecular tests for HIV-1 and HIV-2, HIV infection was excluded and the results of test INNO-LIA HIV I/II Score were finally interpreted as false positive.

Distribution of human papillomavirus genotypes in plucked eyebrow hairs from Slovenian males with genital warts.

In the present study, the presence and distribution of human papillomaviruses (HPVs) in the plucked eyebrow hairs obtained from 49 Slovenian male patients with genital warts were investigated. Using polymerase chain reaction (PCR) with three sets of degenerate primers targeting all known HPV genotypes, HPV DNA was found in 31 (63.3%) of 49 eyebrow hair samples. Epidermodysplasia verruciformis (EV) associated HPV specific Ma/Ha nested PCR system detected HPVs in 27 (55.1%) and CPI/CPIIs primers that amplify the majority of cutaneous/EV HPV genotypes in 20 (40.8%) of 49 samples tested. The CPI/CPIIg PCR specific for E1 open reading frame of genital HPVs showed the presence of HPV DNA in 10 (20.4%) of 49 specimens. Direct sequencing of the Ha PCR products showed the presence of three putative new HPV genotypes, named SIBX1, SIBX2 and SIBX3. Similarly, three potential new HPV genotypes, SIBX4, SIBX5 and SIBX6, were detected by sequencing CPI/CPIIs PCR products. In total, at least 24 different HPV genotypes were detected in 31 HPV DNA positive samples of plucked eyebrow hairs. The results of our study showed that the use of a combined degenerate primer PCR approach considerably improves the HPV DNA detection over individual primer sets and greatly improves the detection of different HPV genotypes in the plucked eyebrow hairs.

The role of core antigen detection in management of hepatitis C: a critical review.

Several assays in research format and two commercial assays for the detection of hepatitis C virus (HCV) core protein or HCV core antigen have been developed in recent years. In order to elucidate the role and significance of HCV core antigen detection in the diagnosis and management of hepatitis C, we reviewed 56 studies published in peer-reviewed journals until September 2004. Evaluations in transfusion settings showed that the HCV core antigen assay detects HCV infection, similarly as nucleic acid techniques (NAT), between 40 and 50 days earlier than the current third generation HCV antibody screening assays. HCV core antigen levels closely track HCV RNA dynamics, and allow clinical monitoring of a patient's therapy, independently of HCV genotype, however, mainly in the samples with HCV RNA levels above 20,000 IU/ml. Considering the lower sensitivity of HCV core antigen detection in comparison to NAT, the HCV core antigen assay is not practical for the determination of the end of treatment response and sustained viral response, but could be useful for the determination of early viral response in the pegylated interferon-alpha and ribavirin treated patients infected with HCV genotype 1. The HCV core antigen detection is a viable tool for study of hepatitis C pathogenesis. The HCV core antigen can be used as a marker of HCV replication in anti-HCV positive individuals in the areas of the world that cannot afford NAT and/or in the settings that are not equipped or competent to perform HCV RNA testing. Because the manufacturer of HCV core antigen assays recently stopped an active marketing of these assays in several countries, it will, unfortunately and probably, never be possible to determine the actual potential and usefulness of HCV core antigen testing in the management of hepatitis C.

HIV controllers with different viral load cutoff levels have distinct virologic and immunologic profiles.

BACKGROUND: The mechanisms behind natural control of HIV replication are still unclear, and several studies pointed that elite controllers (ECs) are a heterogeneous group. METHODS: We performed analyses of virologic, genetic, and immunologic parameters of HIV-1 controllers groups: (1) ECs (viral load, <80 copies/mL); (2) ebbing elite controllers (EECs; transient viremia/blips); and viremic controllers (VCs; detectable viremia, <5000 copies/mL). Untreated noncontrollers (NCs), patients under suppressive highly active antiretroviral therapy (HAART), and HIV-1-negative individuals were analyzed as controls. RESULTS: Total and integrated HIV-1 DNA for EC were significantly lower than for NC and HAART groups. 2-LTR circles were detected in EEC (3/5) and VC (6/7) but not in EC. Although EC and EEC maintain normal T-cell counts over time, some VC displayed negative CD4 T-cell slopes. VC and EEC showed a higher percentage of activated CD8 T cells and microbial translocation than HIV-1-negative controls. EC displayed a weaker Gag/Nef IFN-γ T-cell response and a significantly lower proportion of anti-HIV IgG antibodies than EEC, VC, and NC groups. CONCLUSION: Transient/persistent low-level viremia in HIV controllers may have an impact on immunologic and virologic profiles. Classified HIV controller patients taking into account their virologic profile may decrease the heterogeneity of HIV controllers cohorts, which may help to clarify the mechanisms associated to the elite control of HIV.

Association of single nucleotide polymorphisms in the lens epithelium-derived growth factor (LEDGF/p75) with HIV-1 infection outcomes in Brazilian HIV-1+ individuals.

The lens epithelium-derived growth factor p75 (LEDGF/p75), coded by the PSIP1 gene, is an important host co-factor that interacts with HIV-1 integrase to target integration of viral cDNA into active genes. The aim of this study was to investigate the association of SNPs in the PSIP1 gene with disease outcome in HIV-1 infected patients. We performed a genetic association study in a cohort of 171 HIV-1 seropositive Brazilian individuals classified as rapid progressors (RP, n = 69), typical progressors (TP, n = 79) and long-term nonprogressors (LTNP, n = 23). The exonic SNP rs61744944 and 9 tag SNPs were genotyped. A group of 192 healthy subjects was analyzed to determine the frequency of SNPs and haplotypes in the general population. Linkage disequilibrium (LD) analyses indicated that the SNPs analyzed were not in high LD (r2<0.8). Logistic regression models suggested that patients carrying the T allele rs61744944 (472L) were more likely to develop a LTNP phenotype (OR = 4.98; p = 0.05) as compared to TP group. The same trend was observed when LTNPs were compared to the RP group (OR = 3.26). Results of haplotype analyses reinforced this association, since the OR values obtained for the haplotype carrying allele T at rs61744944 also reflected an association with LTNP status (OR = 6.05; p = 0.08 and OR = 3.44; p = 0.12 for comparisons to TP and RP, respectively). The rare missense variations Ile436Ser and Thr473Ile were not identified in the patients enrolled in this study. Gene expression analyses showed lower LEDGF/p75 mRNA levels in peripheral blood mononuclear cells obtained from HIV-1 infected individuals. However, these levels were not influenced by any of the SNPs investigated. In spite of the limited number of LTNPs, these data suggest that the PSIP1 gene could be associated with the outcome of HIV-1 infection. Further analyses of this gene may guide the identification of causative variants to help predict disease course.

Beta-papillomaviruses in anogenital hairs plucked from healthy individuals.

A total of 150 specimens of anogenital hairs plucked from the scrotal, pubic, and perianal region of 51 immunocompetent healthy male individuals were tested for the presence of beta-papillomaviruses (beta-HPV) using the nested M(a)/H(a) polymerase chain reaction. Beta-HPV were found in a total of 38 (25.3%) of 150 hair samples. According to the sampling sites, beta-HPV were detected in 18/51 (35.3%), 13/50 (26.0%), and 7/49 (14.3%) plucked hair samples obtained from the pubic, scrotal, and perianal region, respectively. The prevalence of beta-HPV in the plucked pubic hairs was significantly higher than in the perianal hairs (P = 0.013). In contrast, the difference in the prevalence of beta-HPV in the pubic and scrotal hairs as well as in scrotal and perianal hairs did not reach statistical significance (P = 0.302 and P = 0.227, respectively). The difference in the lifetime-cumulative sun exposure is the most likely explanation for the differences obtained on beta-HPV prevalence. Beta-HPV genotype HPV-38 was detected most frequently, followed by HPV-36, HPV-15, and HPV-14D. In addition to the beta-HPV recognized officially five partial DNA sequences suggesting putative new HPV genotypes were identified.

Molecular epidemiology of HIV-1 subtypes based on analysis of pol sequences in Slovenia, 1996-2005.

Various studies have demonstrated the increasing prevalence of non-B HIV-1 subtypes in Western Europe. In contrast, knowledge about the molecular epidemiology of HIV-1 in Central and Eastern Europe is limited. The objective of present study was to investigate the HIV-1 molecular diversity as well as time trends in HIV-1 subtype distribution in Slovenia. A retrospective molecular epidemiological survey was conducted on a cohort representing 88% (131/149) of all HIV-1 infected patients diagnosed between January 1996 and June 2005. The study revealed that subtype B is a predominant HIV-1 subtype in Slovenia (110/131; 84%), although a relatively high proportion (21/131; 16%) of non-B subtypes was found. Among them, a high proportion of recombinant (10/21; 48%) and different unclassified strains (8/21; 38%) were identified. Non-B subtype viruses were predominant among heterosexuals (19/21; 90%) and subtype B viruses among men who have sex with men (84/110; 76%). Importantly, 86% (18/21) of patients infected with non-B subtypes were of Slovenian nationality. In contrast to Western European countries, a significant increase (P = 0.015) in the proportion of men who have sex with men was observed recently among newly diagnosed HIV-1 infected patients in Slovenia.