HIV-1 Tat immunization restores immune homeostasis and attacks the HAART-resistant blood HIV DNA: results of a randomized phase II exploratory clinical trial.

BACKGROUND: The phase II multicenter, randomized, open label, therapeutic trial (ISS T-002, Clinicaltrials.gov NCT00751595) was aimed at evaluating the immunogenicity and the safety of the biologically active HIV-1 Tat protein administered at 7.5 or 30 µg, given 3 or 5 times monthly, and at exploring immunological and virological disease biomarkers. The study duration was 48 weeks, however, vaccinees were followed until the last enrolled subject reached the 48 weeks. Reported are final data up to 144 weeks of follow-up. The ISS T-002 trial was conducted in 11 clinical centers in Italy on 168 HIV positive subjects under Highly Active Antiretroviral Therapy (HAART), anti-Tat Antibody (Ab) negative at baseline, with plasma viremia <50 copies/mL in the last 6 months prior to enrollment, and CD4(+) T-cell number ≥200 cells/µL. Subjects from a parallel observational study (ISS OBS T-002, Clinicaltrials.gov NCT0102455) enrolled at the same clinical sites with the same criteria constituted an external reference group to explore biomarkers of disease. RESULTS: The vaccine was safe and well tolerated and induced anti-Tat Abs in most patients (79%), with the highest frequency and durability in the Tat 30 µg groups (89%) particularly when given 3 times (92%). Vaccination promoted a durable and significant restoration of T, B, natural killer (NK) cells, and CD4(+) and CD8(+) central memory subsets. Moreover, a significant reduction of blood proviral DNA was seen after week 72, particularly under PI-based regimens and with Tat 30 µg given 3 times (30 µg, 3x), reaching a predicted 70% decay after 3 years from vaccination with a half-life of 88 weeks. This decay was significantly associated with anti-Tat IgM and IgG Abs and neutralization of Tat-mediated entry of oligomeric Env in dendritic cells, which predicted HIV-1 DNA decay. Finally, the 30 µg, 3x group was the only one showing significant increases of NK cells and CD38(+)HLA-DR(+)/CD8(+) T cells, a phenotype associated with increased killing activity in elite controllers. CONCLUSIONS: Anti-Tat immune responses are needed to restore immune homeostasis and effective anti-viral responses capable of attacking the virus reservoir. Thus, Tat immunization represents a promising pathogenesis-driven intervention to intensify HAART efficacy.

The presence of anti-Tat antibodies in HIV-infected individuals is associated with containment of CD4+ T-cell decay and viral load, and with delay of disease progression: results of a 3-year cohort study.

BACKGROUND: Tat is a key HIV-1 virulence factor, which plays pivotal roles in virus gene expression, replication, transmission and disease progression. After release, extracellular Tat accumulates in tissues and exerts effects on both the virus and the immune system, promoting immune activation and virus spreading while disabling the host immune defense. In particular, Tat binds Env spikes on virus particles forming a virus entry complex, which favors infection of dendritic cells and efficient transmission to T cells via RGD-binding integrins. Tat also shields the CCR5-binding sites of Env rendering ineffective virus neutralization by anti-Env antibodies (Abs). This is reversed by the anti-Tat Abs present in natural infection or induced by vaccination. FINDINGS: Here we present the results of a cohort study, showing that the presence of anti-Tat Abs in asymptomatic and treatment-naïve HIV-infected subjects is associated with containment of CD4+ T-cell loss and viral load and with a delay of disease progression. In fact, no subjects with high anti-Tat Ab titers initiated antiretroviral therapy during the three years of follow-up. In contrast, no significant effects were seen for anti-Env and anti-Gag Abs. The increase of anti-Env Ab titers was associated with a reduced risk of starting therapy only in the presence of anti-Tat Abs, suggesting an effect of combined anti-Tat and anti-Env Abs on the Tat/Env virus entry complex and on virus neutralization. CONCLUSIONS: Anti-Tat immunity may help delay HIV disease progression, thus, targeting Tat may offer a novel therapeutic intervention to postpone antiretroviral treatment or to increase its efficacy.

Radiation recall dermatitis in course of epidemic Kaposi's sarcoma.

Radiation recall dermatitis is an acute, rare skin reaction confined to previously irradiated areas that can be triggered by chemotherapeutic drugs (generally doxorubicin and taxanes), which are administrated after radiotherapy. We describe this case report to discuss the timing of the different choice of treatments of progressive Kaposis's sarcoma (KS) disease. KS, the neoplastic disease associated with HHV-8 infection, is still the most commonly diagnosed malignancy in HIV-1 patients, even if its incidence dramatically declined in the highly active antiretroviral therapy (HAART) era. The cutaneous form of disease generally improves with HAART alone or in association with local treatment (cryotherapy, radiotherapy, intralesion chemotherapy), whereas disseminated and/or progressive disease needs to be treated with systemic chemotherapy. In selected patients with progressive disease, systemic and local therapeutic options should be associated. We report a case of a 30-year-old HIV-1-positive man, affected by epidemic cutaneous and mucosal KS, who received several cycles of chemotherapy in succession with radiotherapy and other chemotherapy treatments for disease progression. After 7 months, the end of the last rechallenge with chemotherapy, the patient presented cutaneous painful and ulcerated lesions on the same skin areas previously irradiated.

Impact of pre-therapy viral load on virological response to modern first-line HAART.

BACKGROUND: We tested whether pre-HAART viraemia affects the achievement and maintenance of virological success in HIV-1-infected patients starting modern first-line therapies. METHODS: A total of 1,430 patients starting their first HAART (genotype-tailored) in 2008 (median; IQR: 2006-2009) were grouped according to levels of pre-HAART viraemia (≤ 30,000, 30,001-100,000, 100,001-300,000, 300,001-500,000 and > 500,000 copies/ml). The impact of pre-therapy viraemia on the time to virological success (viraemia ≤ 50 copies/ml) and on the time to virological rebound (first of two consecutive viraemia values > 50 copies/ml after virological success) were evaluated by Kaplan-Meier curves and Cox regression analyses. RESULTS: Median pre-HAART viraemia was 5.1 log10 copies/ml (IQR 4.5-5.5), and 53% of patients had viraemia > 100,000 copies/ml. By week 48, the prevalence of patients reaching virological success was > 90% in all pre-HAART viraemia ranges, with the only exception of range > 500,000 copies/ml (virological success = 83%; P < 0.001). Higher pre-HAART viraemia was tightly correlated with longer median time to achieve virological success. Cox multivariable estimates confirmed this result: patients with pre-HAART viraemia > 500,000 copies/ml showed the lowest hazard of virological undetectability after adjusting for age, gender, pre-HAART CD4+ T-cell count, transmitted drug resistance, calendar year and third drug administered (adjusted hazard ratio [95% CI]: 0.27 [0.21, 0.35]; P < 0.001). Pre-HAART viraemia > 500,000 copies/ml was also associated with higher probability of virological rebound compared with patients belonging to lower viraemia strata at weeks 4, 12 and 24 (P = 0.050). CONCLUSIONS: At the time of modern HAART, and even though an average > 90% of virological success, high pre-HAART viraemia remains an independent factor associated with delayed and decreased virological success. Patients starting HAART with > 500,000 copies/ml represent a significant population that may deserve special attention.

The genotypic false positive rate determined by V3 population sequencing can predict the burden of HIV-1 CXCR4-using species detected by pyrosequencing.

OBJECTIVE: The false-positive rate (FPR) is a percentage-score provided by Geno2Pheno-algorithm indicating the likelihood that a V3-sequence is falsely predicted as CXCR4-using. We evaluated the correlation between FPR obtained by V3 population-sequencing and the burden of CXCR4-using variants detected by V3 ultra-deep sequencing (UDPS) and Enhanced-Sensitivity Trofile assay (ESTA). METHODS: 54 HIV-1 B-subtype infected-patients (all maraviroc-naïve), with viremia >10,000copies/ml, were analyzed. HIV-tropism was assessed by V3 population-sequencing, UDPS (considering variants with >0.5% prevalence), and ESTA. RESULTS: By UDPS, CCR5-using variants were detected in 53/54 patients, irrespective of FPR values, and their intra-patient prevalence progressively increased by increasing the FPR obtained by V3 population-sequencing (rho = 0.75, p = 5.0e-8). Conversely, the intra-patient prevalence of CXCR4-using variants in the 54 patients analyzed progressively decreased by increasing the FPR (rho = -0.61; p = 9.3e-6). Indeed, no CXCR4-using variants were detected in 13/13 patients with FPR>60. They were present in 7/18 (38.8%) patients with FPR 20-60 (intra-patient prevalence range: 2.1%-18.4%), in 5/7 (71.4%) with FPR 10-20, in 4/6 (66.7%) with FPR 5-10, and in 10/10(100%) with FPR<5 (intra-patient prevalence range: 12.1%-98.1%). CONCLUSIONS: FPR by V3 population-sequencing can predict the burden of CXCR4-using variants. This information can be used to optimize the management of tropism determination in clinical practice. Due to its low cost and short turnaround time, V3 population-sequencing may represent the most feasible test for HIV-1 tropism determination. More sensitive methodologies (as UDPS) might be useful when V3 population-sequencing provides a FPR >20 (particularly in the range 20-60), allowing a more careful identification of patients harboring CXCR4-using variants.

Anal cytological abnormalities and epidemiological correlates among men who have sex with men at risk for HIV-1 infection.

BACKGROUND: The incidence of anal cancer, a Human Papillomavirus (HPV)-related neoplasia, has been increasing in recent decades, mainly in men who have sex with men (MSM). Cytological changes of the anal epithelium induced by HPV can be detected through an anal pap smear. This study aimed to evaluate the prevalence and epidemiological correlates of anal cytological abnormalities among relatively young MSM at risk for HIV-1 infection, to help clarify whether or not this population deserves further investigation to assess the presence of anal cancer precursor lesions. METHODS: MSM were recruited among attendees of a large STI clinic for a HIV-1 screening program. Anal samples, collected with a Dracon swab in PreservCyt, were used both for liquid-based cytology and HPV testing by the Linear Array HPV Genotyping Test. Data regarding socio-demographic characteristics and sexual behavior were collected in face-to-face interviews. RESULTS: A total of 346 MSM were recruited (median age 32 years). Overall, 72.5% of the individuals had an anal HPV infection, with 56.1% of them being infected by oncogenic HPV genotypes. Anal cytological abnormalities were found in 29.8% of the cases (16.7% ASC-US and 13.1% L-SIL). Presence of ASC-US+ was strongly associated with infection by any HPV type (OR = 4.21, 95% CI: 1.97-9.23), and particularly by HPV 16 and/or 18 (OR = 5.62, 95% CI: 2.33-13.81). A higher proportion of ASC-US+ was found in older MSM, in those with a higher number of lifetime partners and in those with a history of ano-genital warts. However, none of these variables or the others analyzed showed any significant association with abnormal cytological findings. CONCLUSIONS: The presence of anal cytological abnormalities in about one third of the recruited MSM and their strong association with HPV infection, in particular that caused by HPV 16 and/or 18, might provide a further complement to the data that now support the introduction of HPV vaccination among MSM to protect them from the development of HPV-associated diseases. Additional studies are needed to determine whether and how screening for anal cancer precursor lesions should be performed in younger MSM.

Comparative analysis of drug resistance among B and the most prevalent non-B HIV type 1 subtypes (C, F, and CRF02_AG) in Italy.

In recent years, increasing numbers of patients infected with HIV-1 non-B subtypes have been treated with modern antiretroviral regimens. Therefore, a better knowledge of HIV drug resistance in non-B strains is crucial. Thus, we compared the mutational pathways involved in drug resistance among the most common non-B subtypes in Italy (F, C, and CRF02_AG) and the B subtype. In total, 2234 pol sequences from 1231 virologically failing patients from Central Italy were analyzed. The prevalence of resistance mutations in protease and reverse transcriptase between non-B and B subtypes has been evaluated. Among patients treated with nucleoside/nucleotide reverse transcriptase inhibitors (NRTI) and with thymidine analogues (TA) experience, TAMs1 M41L and L210W were less prevalent in CRF02_AG, while TAMs2 T215F and K219E were more prevalent in the F subtype. In NRTI-treated patients having experience with abacavir, didanosine, tenofovir, or stavudine the K65R mutation was mostly prevalent in the C subtype. In non-NRTI (NNRTI)-treated patients infected by the C subtype the prevalence of K103N was lower than in patients infected with other subtypes, while the prevalence of Y181C and Y188L was higher compared to subtype B. The prevalence of Y181C was higher also in subtype F as compared to subtype B. In patients treated with protease inhibitors, L89V was predominantly found in CRF02_AG, while the TPV resistance mutation T74P was predominantly found in the C subtype. Some differences in the genotypic drug resistance have been found among patients infected with B, C, F, and CRF02_AG subtypes in relationship to treatment. These results may be useful for the therapeutic management of individuals infected with HIV-1 non-B strains.

Prevalence, genotype diversity and determinants of anal HPV infection in HIV-uninfected men having sex with men.

BACKGROUND: Anal human papillomavirus (HPV) infection is very common in men having sex with men (MSM), but the available data on its burden and characteristics mainly concern HIV-infected individuals. OBJECTIVES: This study aimed to assess the prevalence, spectrum of genotypes, and determinants of the anal HPV infection in metropolitan HIV-1 uninfected MSM. STUDY DESIGN: A cohort of 258 MSM (median age 32 years, IQR 26-39) enrolled at an STI Clinic was screened for anal HPV infection using a highly sensitive PCR-based genotyping method. Medical history and behavioral data were collected. RESULTS: Overall, 74.8% of the MSM were HPV-positive, with 56.2% of the participants being infected by high-risk (HR) types. A multiple infection was detected in 65.3% of the HPV-positive MSM, with up to 10 different HPV types detected in the same sample. A broad spectrum of infecting HPV types was observed, with 36 different types found overall and HPV16 representing the most common type (17.8%). The lifetime and recent number of sexual partners as well as having receptive anal sex were significantly associated with the anal HPV infection, confirming the role of sexual behavior in risk of HPV infection. However, neither younger age at first intercourse nor inconsistent use of condom was significantly associated with the infection. CONCLUSIONS: The present findings highlight the need to create a more significant awareness about the risk of anal HPV infection among HIV-uninfected MSM and warrant the investigation of possible anal intraepithelial lesions, particularly in view of the increasing anal cancer incidence in high-risk populations.

'Sentinel' mutations in standard population sequencing can predict the presence of HIV-1 reverse transcriptase major mutations detectable only by ultra-deep pyrosequencing.

OBJECTIVES: This proof-of-concept study aimed to identify whether mutations considered not yet relevant for drug resistance (but located at key drug-resistance positions) can act as 'sentinels' of minority resistant variants in HIV-1 drug-naive patients. METHODS: We focused our attention on three reverse transcriptase (RT) mutations (T69S, L210M and K103R) easily detected by standard population sequencing [i.e. the genotypic resistance test (GRT)]. Ultra-deep pyrosequencing (UDPS) of HIV-1 RT was performed using GS-FLX Roche, on plasma RNA from 40 drug-naive patients infected with HIV-1 subtype B without primary resistance detected by GRT. Only RT drug resistance mutations detected at >0.1% in both forward and reverse directions were considered. Associations between GRT sentinel mutations and UDPS drug resistance were assessed using Fisher's exact test. RESULTS: UDPS detected drug resistance mutations in 18/40 drug-naive patients. Patients carrying HIV-1 strains with T69S and L210M by GRT showed a trend to greater infection by minority drug-resistant variants than control patients infected by HIV-1 without these mutations (5/10 and 7/10 versus 3/10; P = not significant). No association was found for K103R by GRT. Notably, T69S and L210M (but not K103R or control viruses) were associated with GRT minority drug-resistant variants with a prevalence >1% (3/10 and 4/10 versus 0/20 in K103R and controls; P = 0.03 and P = 0.008, respectively). Moreover, the presence of L210M or T69S viruses by GRT significantly correlated with that of minority thymidine analogue mutations by UDPS (6/20 patients carrying HIV-1 strains with T69S/L210M versus 0/20 patients carrying HIV-1 having K103R or none of these mutations; P = 0.03). CONCLUSIONS: This proof-of-concept study suggests the existence of genetic markers, detectable by routine testing, potentially acting as sentinel mutations of minority drug resistance. Their identification may help in the selection of patients at high risk of resistance in reservoirs without the necessity of using UDPS.

Therapeutic immunization with HIV-1 Tat reduces immune activation and loss of regulatory T-cells and improves immune function in subjects on HAART.

UNLABELLED: Although HAART suppresses HIV replication, it is often unable to restore immune homeostasis. Consequently, non-AIDS-defining diseases are increasingly seen in treated individuals. This is attributed to persistent virus expression in reservoirs and to cell activation. Of note, in CD4(+) T cells and monocyte-macrophages of virologically-suppressed individuals, there is continued expression of multi-spliced transcripts encoding HIV regulatory proteins. Among them, Tat is essential for virus gene expression and replication, either in primary infection or for virus reactivation during HAART, when Tat is expressed, released extracellularly and exerts, on both the virus and the immune system, effects that contribute to disease maintenance. Here we report results of an ad hoc exploratory interim analysis (up to 48 weeks) on 87 virologically-suppressed HAART-treated individuals enrolled in a phase II randomized open-label multicentric clinical trial of therapeutic immunization with Tat (ISS T-002). Eighty-eight virologically-suppressed HAART-treated individuals, enrolled in a parallel prospective observational study at the same sites (ISS OBS T-002), served for intergroup comparison. Immunization with Tat was safe, induced durable immune responses, and modified the pattern of CD4(+) and CD8(+) cellular activation (CD38 and HLA-DR) together with reduction of biochemical activation markers and persistent increases of regulatory T cells. This was accompanied by a progressive increment of CD4(+) T cells and B cells with reduction of CD8(+) T cells and NK cells, which were independent from the type of antiretroviral regimen. Increase in central and effector memory and reduction in terminally-differentiated effector memory CD4(+) and CD8(+) T cells were accompanied by increases of CD4(+) and CD8(+) T cell responses against Env and recall antigens. Of note, more immune-compromised individuals experienced greater therapeutic effects. In contrast, these changes were opposite, absent or partial in the OBS population. These findings support the use of Tat immunization to intensify HAART efficacy and to restore immune homeostasis. TRIAL REGISTRATION: ClinicalTrials.gov NCT00751595.

Population dynamics of HIV-1 subtype B in a cohort of men-having-sex-with-men in Rome, Italy.

A recent increase in HIV diagnoses among men-having-sex-with-men (MSM) has been shown by surveillance data from Europe and Italy, and new approaches to inferring viral population dynamics from heterochronously sampled gene sequences have been developed. The aim of this study was to reconstruct the epidemiological history of HIV-1 subtype B in a homogeneous group of Italian MSM using a coalescent-based Bayesian framework. A total of 125 HIV-1 subtype B pol sequences were analyzed using Bayesian methods and a relaxed molecular clock to reconstruct their dated phylogeny and estimate population dynamics. At least 10 epidemiological clusters of 3-9 isolates were identified: half including the largest clades originated in the early 1990s and the other half radiated from 1999. Demographic analysis showed that the HIV epidemic grew in accordance with a logistic model characterized by a rapid exponential increase in the effective number of infections (r = 1.54 year) starting from the early 1980s and reaching a plateau 10 years later. Our data suggest that the HIV B epidemic entered our MSM population through multiple transmission chains about 20 years later than in other Western European country. Epidemiological clusters originating in the early 2000s suggest a recent re-emergence of HIV in Italian MSM.

Secondary integrase resistance mutations found in HIV-1 minority quasispecies in integrase therapy-naive patients have little or no effect on susceptibility to integrase inhibitors.

The goal of this study was to explore the presence of integrase strand transfer inhibitor (InSTI) resistance mutations in HIV-1 quasispecies present in InSTI-naïve patients and to evaluate their in vitro effects on phenotypic susceptibility to InSTIs and their replication capacities. The RT-RNase H-IN region was PCR amplified from plasma viral RNA obtained from 49 HIV-1 subtype B-infected patients (21 drug naïve and 28 failing highly active antiretroviral therapy [HAART] not containing InSTIs) and recombined with an HXB2-based backbone with RT and IN deleted. Recombinant viruses were tested against raltegravir and elvitegravir and for replication capacity. Three-hundred forty-four recombinant viruses from 49 patients were successfully analyzed both phenotypically and genotypically. The majority of clones were not phenotypically resistant to InSTIs: 0/344 clones showed raltegravir resistance, and only 3 (0.87%) showed low-level elvitegravir resistance. No primary resistance mutations for raltegravir and elvitegravir were found as major or minor species. The majority of secondary mutations were also absent or rarely present. Secondary mutations, such as T97A and G140S, found rarely and only as minority quasispecies, were present in the elvitegravir-resistant clones. A novel mutation, E92G, although rarely found in minority quasispecies, showed elvitegravir resistance. Preexisting genotypic and phenotypic raltegravir resistance was extremely rare in InSTI-naïve patients and confined to only a restricted minority of secondary variants. Overall, these results, together with others based on population and ultradeep sequencing, suggest that at this point IN genotyping in all patients before raltegravir treatment may not be cost-effective and should not be recommended until evidence of transmitted drug resistance to InSTIs or the clinical relevance of IN minor variants/polymorphisms is determined.

The preventive phase I trial with the HIV-1 Tat-based vaccine.

The native HIV-1 Tat protein was chosen as vaccine candidate for phase I clinical trials based on its role in the natural infection and AIDS pathogenesis, on the association of Tat-specific immune response with the asymptomatic stage as well as on its sequence conservation among HIV clades. A randomized, double blind, placebo-controlled phase I study (ISS P-001) was conducted in healthy adult volunteers without identifiable risk of HIV infection. Tat was administered 5 times monthly, subcute in alum or intradermic alone at 7.5 microg, 15 microg or 30 microg, respectively (ClinicalTrials.gov identifier: NCT00529698). Vaccination with Tat resulted to be safe and well tolerated (primary endpoint) both locally and systemically. In addition, Tat induced both Th1 and Th2 type specific immune responses in all subjects (secondary endpoint) with a wide spectrum of functional antibodies that are rarely seen in natural infection, providing key information for further clinical development of the Tat vaccine candidate.

Phase I therapeutic trial of the HIV-1 Tat protein and long term follow-up.

A randomized, double blind, placebo-controlled phase I vaccine trial based on the native Tat protein was conducted in HIV-infected asymptomatic individuals. The vaccine was administered five times subcute with alum or intradermally without adjuvant at 7.5microg, 15microg or 30microg doses, respectively. The Tat vaccine was well tolerated both locally and systemically and induced and/or maintained Tat-specific T helper (Th)-1 T-cell responses and Th-2 responses in all subjects with a wide spectrum of functional anti-Tat antibodies, rarely seen in HIV-infected subjects. The data indicate the achievement of both the primary (safety) and secondary (immunogenicity) endpoints of the study.

Incidence of human herpesvirus 8 (HHV-8) infection among HIV-uninfected individuals at high risk for sexually transmitted infections.

BACKGROUND: The occurrence of, and risk factors for, HHV-8 infection have yet to be definitively determined, particularly among heterosexual individuals with at-risk behavior for sexually transmitted infections (STI). The objective of this study was to estimate the incidence and determinants of HHV-8 infection among HIV-uninfected individuals repeatedly attending an urban STI clinic. METHODS: Sera from consecutive HIV-uninfected individuals repeatedly tested for HIV-1 antibodies were additionally tested for HHV-8 antibodies using an immunofluorescence assay. To identify determinants of HHV-8 infection, a nested case-control study and multivariate logistic regression analysis were performed. RESULTS: Sera from 456 HIV-uninfected individuals (224 multiple-partner heterosexuals and 232 men who have sex with men (MSM]) were identified for inclusion in the study. The HHV-8 seroprevalence at enrollment was 9.4% (21/224; 95% C.I.: 6.0-14.2%) among heterosexuals with multiple partners and 22.0% (51/232; 95% C.I.: 16.9-28.0%) among MSM. Among the 203 multiple-partner heterosexuals and 181 MSM who were initially HHV-8-negative, 17 (IR = 3.0/100 p-y, 95% C.I.: 1.9 - 4.8) and 21 (IR = 3.3/100 p-y, 95% C.I:.2.1 - 5.1) seroconversions occurred, respectively. HHV-8 seroconversion tended to be associated with a high number of sexual partners during the follow-up among MSM (> 10 partners: AOR = 3.32 95% CI:0.89-12.46) and among the multiple-partner heterosexuals (> 10 partner; AOR = 3.46, 95% CI:0.42-28.2). Moreover, among MSM, HHV-8 seroconversion tended to be associated with STI (AOR = 1.80 95%CI: 0.52-7.96). During the study period the HIV-1 incidence was lower than that of HHV-8 among both groups (0.89/100 p-y among MSM and 0.95/100 p-y among multiple-partner heterosexuals). CONCLUSION: The large difference between the incidence of HHV-8 and the incidence of HIV-1 and other STIs may suggest that the circulation of HHV-8 is sustained by practices other than classical at-risk sexual behavior.

Characterization and structural analysis of novel mutations in human immunodeficiency virus type 1 reverse transcriptase involved in the regulation of resistance to nonnucleoside inhibitors.

Resistance to antivirals is a complex and dynamic phenomenon that involves more mutations than are currently known. Here, we characterize 10 additional mutations (L74V, K101Q, I135M/T, V179I, H221Y, K223E/Q, and L228H/R) in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase which are involved in the regulation of resistance to nonnucleoside reverse transcriptase inhibitors (NNRTIs). These mutations are strongly associated with NNRTI failure and strongly correlate with the classical NNRTI resistance mutations in a data set of 1,904 HIV-1 B-subtype pol sequences from 758 drug-naïve patients, 592 nucleoside reverse transcriptase inhibitor (NRTI)-treated but NNRTI-naïve patients, and 554 patients treated with both NRTIs and NNRTIs. In particular, L74V and H221Y, positively correlated with Y181C, were associated with an increase in Y181C-mediated resistance to nevirapine, while I135M/T mutations, positively correlated with K103N, were associated with an increase in K103N-mediated resistance to efavirenz. In addition, the presence of the I135T polymorphism in NNRTI-naïve patients significantly correlated with the appearance of K103N in cases of NNRTI failure, suggesting that I135T may represent a crucial determinant of NNRTI resistance evolution. Molecular dynamics simulations show that I135T can contribute to the stabilization of the K103N-induced closure of the NNRTI binding pocket by reducing the distance and increasing the number of hydrogen bonds between 103N and 188Y. H221Y also showed negative correlations with type 2 thymidine analogue mutations (TAM2s); its copresence with the TAM2s was associated with a higher level of zidovudine susceptibility. Our study reinforces the complexity of NNRTI resistance and the significant interplay between NRTI- and NNRTI-selected mutations. Mutations beyond those currently known to confer resistance should be considered for a better prediction of clinical response to reverse transcriptase inhibitors and for the development of more efficient new-generation NNRTIs.

Increased HIV incidence among men who have sex with men in Rome.

Among 976 men who have sex with men (MSM) who had undergone repeat HIV testing between 1984 and 2003 at a sexually transmitted infection clinic in Rome, Italy, we observed a dramatic increase in HIV incidence in 2002 and 2003, with the cumulative incidence for 2000-2003 being twice as high as that for 1984-1995, and significantly higher than that for 1996-1999. This trend suggests the need for interventions aimed at encouraging behavioural changes among MSM.