Brief Report: Association Between Low HIV-1 DNA and Western Blot Reactivity to HIV-1 Pol in Chronically Infected Individuals.

BACKGROUND: The aim of the study was to evaluate whether negative HIV-1 pol on Western blot (WB) was associated with low HIV-DNA in adults with chronic HIV-1 infection and suppressive antiretroviral therapy. METHODS: Cross-sectional parent study of the APACHE trial, conducted in subjects with chronic infection, HIV-1 RNA <50 copies/mL for ≥10 years, no residual viremia for ≥5 years and CD4 >500 cells/µL screened for HIV-1 DNA. HIV-1 DNA was quantified in peripheral blood mononuclear cells (PBMCs) by real-time polymerase chain reaction and HIV-1 serostatus was tested by HIV Blot 2.2 WB assay. Multivariate logistic regression was used to determine factors associated with low HIV-1 DNA. RESULTS: We evaluated 96 patients: 78 (81%) and 18 (19%) subjects with HIV-1 DNA ≥100 copies/10 PBMCs and with HIV-1 DNA <100 copies/10 PBMCs, respectively. Median age was 32.5 (25.3-38.9), and 61 (64%) were men; moreover, we reported that nadir CD4 was 253 (167-339) cells/µL and HIV-RNA <50 copies/mL for 11.7 (10.6-14.0) years. At multivariate analysis, higher nadir CD4 [adjusted odds ratio (AOR) [95% confidence interval (CI) 1.35 (95% CI: 1.03 to 1.76), P = 0.029], longer years of HIV-1 RNA <50 copies/mL [AOR (95% CI) 2.98 (95% CI: 1.25 to 7.10), P = 0.014], a R5-tropic virus [AOR (R5 vs. non-R5) 0.20 (95% CI: 0.04 to 0.96), P = 0.044], and negative HIV-1 pol [AOR 6.59 (95% CI: 1.47 to 29.54), P = 0.014] were associated with low HIV-1 DNA. CONCLUSIONS: In patients with chronic HIV-1 infection and suppressive antiretroviral therapy, negative HIV-1 pol on WB was associated with low HIV-1 DNA as well as higher nadir CD4, longer years of HIV-1 RNA <50 copies/mL, and a R5-tropic virus.

Single-Cell Characterization of Viral Translation-Competent Reservoirs in HIV-Infected Individuals.

HIV cure efforts are hampered by limited characterization of the cells supporting HIV replication in vivo and inadequate methods for quantifying the latent viral reservoir in patients receiving antiretroviral therapy. We combine fluorescent in situ RNA hybridization with detection of HIV protein and flow cytometry, enabling detection of 0.5-1 gag-pol mRNA(+)/Gag protein(+)-infected cells per million. In the peripheral blood of untreated persons, active HIV replication correlated with viremia and occurred in CD4 T cells expressing T follicular helper cell markers and inhibitory co-receptors. In virally suppressed subjects, the approach identified latently infected cells capable of producing HIV mRNA and protein after stimulation with PMA/ionomycin and latency-reversing agents (LRAs). While ingenol-induced reactivation mirrored the effector and central/transitional memory CD4 T cell contribution to the pool of integrated HIV DNA, bryostatin-induced reactivation occurred predominantly in cells expressing effector memory markers. This indicates that CD4 T cell differentiation status differentially affects LRA effectiveness.

Declining prevalence of HIV-1 drug resistance in antiretroviral treatment-exposed individuals in Western Europe.

HIV-1 drug resistance represents a major obstacle to infection and disease control. This retrospective study analyzes trends and determinants of resistance in antiretroviral treatment (ART)-exposed individuals across 7 countries in Europe. Of 20 323 cases, 80% carried at least one resistance mutation: these declined from 81% in 1997 to 71% in 2008. Predicted extensive 3-class resistance was rare (3.2% considering the cumulative genotype) and peaked at 4.5% in 2005, decreasing thereafter. The proportion of cases exhausting available drug options dropped from 32% in 2000 to 1% in 2008. Reduced risk of resistance over calendar years was confirmed by multivariable analysis.

Recent HIV-1 infection contributes to the viral diffusion over the French territory with a recent increasing frequency.

OBJECTIVE: To analyse the contribution of primary human immunodeficiency virus type 1 (HIV-1) infection (PHI) to the French viral epidemic. METHODS: HIV-1 pol sequences included 987 PHI from the French ANRS PRIMO cohort between 1999 and 2010 and were analysed using a population-based phylogenetic approach. Clinical features, risk factors, sexual behaviour and drug resistance for clustered and nonclustered transmission events were ascertained. RESULTS: Viruses from 125 (12.7%) of PHI cosegregated into 56 transmission chains, with increasing frequency during the last years (10.2% before 2006 versus 15.2% of clusters in 2006-2010, p = 0.02). The mean number of patients per cluster was 2.44. Compared to unique PHI, clusters involved more often men, infected through homosexual intercourse, of young age, with a high number of casual sexual partnerships and frequent previous HIV serological tests. Resistant strains were found in 16.0% and 11.1% of clusters and unique PHI, respectively (p = 0.11). Overall, 34% (n = 9) clusters included patients followed in French regions far apart, involving 13 clusters with at least one Parisian patient. CONCLUSIONS: PHIs are a significant source of onward transmission, especially in the MSM population. Recently infected people contribute to the spread of the viral epidemic throughout the French territory. Survey of transmitted drug resistance and behavioural characteristics of patients involved into clustered PHI may help to guide prevention and treatment interventions.

Incomplete IgG response to HIV-1 proteins and low avidity levels in recently converted HIV patients treated with early antiretroviral therapy.

OBJECTIVES: To evaluate the evolution of antibody avidity and Western blot reactivity in recently infected HIV-1 subjects and to study the impact of highly active antiretroviral therapy (HAART) on avidity maturation of HIV-1-specific immunoglobulin G (IgG) in patients with recent HIV-1 infection. METHODS: Thirty-six HIV-1 seroconverters were enrolled in this study and followed longitudinally over 24 months to evaluate if the administration of antiretroviral therapy during primary infection affects Western blot reactivity and the evolution of antibody avidity. The patients were divided into two groups; group A consisted of 19 HIV-1-untreated patients who did not receive any drug treatment during our follow-up period; group B consisted of 17 subjects who were treated early with an association of two nucleoside reverse transcriptase inhibitors (NRTI) and one non-nucleoside reverse transcriptase inhibitor (NNRTI) within 3 months after seroconversion. RESULTS: At diagnosis, Western blot analysis and avidity index (mean value) were exactly matched in untreated and treated patients; subsequently, however, a significantly lower reactivity to HIV-1 pol and gag proteins and a lower avidity index (mean values) were observed in HAART-treated patients up until the end of the follow-up period. CONCLUSIONS: The impaired production and maturation of the humoral immunological response in antiretroviral-treated patients might be related to a rapid suppression of HIV replication, driven by HAART. These results could have important implications in understanding the complex mechanism of the immune response during HIV infection.

Young pregnant women living with HIV/AIDS in Criciuma, Southern Brazil, are infected almost exclusively with HIV type 1 clade C.

Southern Brazil has the highest prevalence rate of AIDS in the country and is the only region in the Americas where HIV-1 C prevails. Metropolitan areas and harbor cities have been evaluated, but limited information is available for small towns and specific populations. We studied women attending the obstetric outpatient clinic of Criciuma, State of Santa Catarina in 2007 to evaluate the molecular epidemiology of HIV-1 among pregnant women living with HIV/AIDS. Forty-two cases had partial pol gene sequenced and additional partial gag and/or env genes from nine women. HIV subtyping was evaluated by phylogenetic methods and antiretroviral (ARV) drug resistance mutations (DRMs) at the Stanford Database. DRMs to one or more ARV class was observed in 20/42, 48% of cases, with 15/41, 37% with viral load <500 copies/ml. Subtype C at pol was identified in 33/42, 78.6% (95% CI: 64-89%), C mosaics (CB, CF) in 2, 4.8% (95% CI: 0.8-19%), F in 4, 9.5% (95% CI: 3-21%), and B in 3, 7.1% (95% CI: 1.8-18%). Discordance in concatenated gag/pol/env or intraregion mosaic was observed in 1/9, 11% of HIV-1 C genomes. The proportion of HIV-1 C in this study is the highest rate described in the Americas. Molecular surveillance in specific populations is instrumental for a better understanding of the Brazilian HIV epidemic.

Molecular characterization of the human immunodeficiency virus type 1 among children in Lima, Peru.

In Peru, there is a lack of information on molecular analysis in pediatric human immunodeficiency virus (HIV) infection. At present, the mother-to-child transmission rate is estimated at approximately 2-4%. The objective of this study was to assess the molecular epidemiology of HIV-1 in infected children. Children with suspected or confirmed pulmonary tuberculosis were evaluated at two public hospitals between 2002 and 2007. Whole blood samples were obtained from 90 HIV-positive children, who were confirmed to be positive by enzyme-linked immunosorbent assay and Western blot. The specimens were subjected to envelope heteroduplex mobility assay (env HMA) followed by gag and pol gene region sequence analysis. Subtype B was found in 88 (98%) of 90 children and 2 (2%) children were subtype BF recombinants. This is the first report of recombinant HIV strains in HIV-infected children in Peru. Understanding the origin, diversity, and spread of HIV strains worldwide will be necessary for the development of an effective vaccine that targets pediatric populations throughout the world.

Comparative analysis of polymorphisms in the HIV type 1 pol gene in the proviral DNA and viral RNA in the peripheral compartment.

The aim of this study was to evaluate the presence of mutations and polymorphisms associated with drug resistance among HIV-1-infected patients in proviral DNA and viral RNA extracted from PBMCs and plasma, respectively, in 34 HIV-1-infected patients (11 naive and 23 receiving HAART). Additional drug resistance mutations were found in only one compartment in 14 of 23 treated patients. Mutations conferring resistance to an additional drug were found in plasma in only 7 of 23 patients. A greater number of differences was found in strains in patients infected for at least more than 9 years, compared to naive patients and patients for whom the time since the first diagnosis was lower (p < 0.02). This study confirms the usefulness of simultaneous testing of different compartments for assessing drug resistance in the pol region and suggests that the heterogeneity observed in different compartments might be increased with time of infection and treatment experience.

Pol bootscanning analysis of HIV type 1 can exhibit unexpected recombinations.

In France the recommendation is to sequence the RT gene of HIV-1 isolates prior to initiation of antiretroviral therapy. The data are routinely used for molecular characterization of the viruses yielding the subtype or CRF of the isolates investigated together with the absence or presence of drug resistance mutations. In this study, we performed bootscanning analysis on the whole pol gene, in which in vitro and in vivo intersubtype recombination has been reported to occur frequently. We showed that out of 15 HIV-1 isolates, two exhibited a recombination unexpected by this routine sequencing method.

Multiple-infection and recombination in HIV-1 within a longitudinal cohort of women.

BACKGROUND: Recombination between strains of HIV-1 only occurs in individuals with multiple infections, and the incidence of recombinant forms implies that multiple infection is common. Most direct studies indicate that multiple infection is rare. We determined the rate of multiple infection in a longitudinal study of 58 HIV-1 positive participants from The Women's Interagency HIV Study with a richer sampling design than previous direct studies, and we investigated the role of recombination and sampling design on estimating the multiple infection rate. RESULTS: 40% of our sample had multiple HIV-1 infections. This rate of multiple infection is statistically consistent with previous studies once differences in sampling design are taken into account. Injection drug use significantly increased the incidence of multiple infections. In general there was rapid elimination of secondary strains to undetectable levels, but in 3 cases a superinfecting strain displaced the initial infecting strain and in two cases the strains coexisted throughout the study. All but one secondary strain was detected as an inter- and/or intra-genic recombinant. Injection drug use significantly increased the rate of observed recombinants. CONCLUSION: Our multiple infection rate is consistent with rates estimated from the frequency of recombinant forms of HIV-1. The fact that our results are also consistent with previous direct studies that had reported a much lower rate illustrates the critical role of sampling design in estimating this rate. Multiple infection and recombination significantly add to the genetic diversity of HIV-1 and its evolutionary potential, and injection drug use significantly increases both.

Characterization of genetically diverse HIV type 1 from a London cohort: near full-length genomic analysis of a subtype H strain.

HIV-1 is characterized by an exceptional level of sequence diversity and a rapid rate of evolution. HIV diversity has implications for reliability of assays designed to detect and monitor infection, pathogenesis, disease progression, response to antiviral therapeutics, resistance pathways, and vaccine development. In the present study, HIV-1 strain diversity was assessed for a small clinical cohort (n = 15) from London, England at risk for infection with non-subtype B strains. Analysis of gag p24, pol IN, and env gp41 IDR revealed the presence of five subtypes (A, B, C, D, H), CRF02_AG, and four unique recombinant forms. Due to the paucity of complete subtype H genomes available, we performed near full-length genome sequence analysis on the candidate subtype H strain, designated as 00GB.AC4001. Phylogenetic analysis revealed that it formed a monophyletic cluster with the three available subtype H reference sequences. Bootscanning analysis confirmed that 00GB.AC4001 represents a new nonrecombinant subtype H genome.

Superinfection of defective human immunodeficiency virus type 1 with different subtypes of wild-type virus efficiently produces infectious variants with the initial viral phenotypes by complementation followed by recombination.

Superinfection rates of human immunodeficiency virus type 1 (HIV-1) have increasingly been leading to more variation in HIV-1, as evidenced by the emergence of circulating recombinant forms (CRFs). We recently reported complementation in a persistently replication-defective subtype B-infected cell clone, L-2, by superinfection with CRF15_01B. The L-2 cells continuously produce immature particles due to a one-base insertion at pol protease. Proviruses in the superinfected cells carried both subtypes and produced particles with a mature morphology. In this study, we examined possible recombination following complementation to generate replication-competent variants by using three cell clones prepared from superinfected L-2 cells. The individual clones predominantly expressed the initial subtype B-derived mature Gag proteins. However, the viral particles carried both subtype B with the mutation and wild-type CRF15_01B at pol, suggesting the generation of virions with heterozygous RNAs. Interestingly, with cell-free passages of the progeny, defective particles disappeared, and were replaced with heterogeneous recombinants in the pol region with sequences derived from CRF15_01B that expressed subtype B phenotype. Thus, even a defective form of persistent HIV-1 can become replication-competent through superinfection-mediated complementation followed by recombination. These findings suggest the significance of long-lived infected cells as recipients for superinfection.