Inês Bártolo

Evaluation of the Clinical Sensitivities of Three Viral Load Assays with Plasma Samples from a Pediatric Population Predominantly Infected with Human Immunodeficiency Virus Type 1 Subtype G and BG Recombinant Forms

ABSTRACT The viral load assays AMPLICOR HIV-1 Monitor Test 1.5, Nuclisens HIV-1 QT, and Quantiplex HIV RNA 3.0 (bDNA) were evaluated for their abilities to quantify human immunodeficiency virus type 1 (HIV-1) RNA in 64 plasma samples from 21 children infected in Portugal. The children were infected with HIV-1 subtypes A1, B, F1, G, and BG recombinant virus. AMPLICOR v1.5 and Quantiplex v3.0 detected all samples, and there was a good correlation of results between the two kits. Thirty-eight specimens containing HIV-1 subtype B, G, or recombinant BG, could not be detected by Nuclisens HIV-1 QT. We also evaluated the new Retina HIV-1 assay on 21 samples that were HIV-1 positive; Retina HIV-1 failed to detect 5 of 11 subtype G specimens. AMPLICOR v1.5 and Quantiplex v3.0 assays may be used for HIV-1 RNA quantification in Portugal, whereas an improvement in sensitivity for subtype G and recombinant BG is required for Nuclisens HIV-1 QT and Retina HIV-1.

Highly divergent subtypes and new recombinant forms prevail in the HIV/AIDS epidemic in Angola: new insights into the origins of the AIDS pandemic.

Angola, located in South-Western Africa, has a remarkably low HIV/AIDS prevalence in the adult population (3.7%). It is bordered in the North by the Democratic Republic of Congo (DRC) and Republic of Congo that are at the origin of human HIV-1 infections. It is, therefore, likely that HIV-1 strains circulating in Angola are genetically diverse and representative of the origin of the HIV/AIDS epidemic. The aim of this work was to investigate in detail the genetic diversity and molecular epidemiology of HIV-1 in Angola. Almost 400 sequences were obtained from the gag (p17), pol (PR and RT) and/or env (C2C3) genes of 159 HIV-1 infected patients living in eight provinces of Angola (Benguela, Cabinda, Cuanza Norte, Luanda, Lunda Norte, Malange, Uíge, and Zaire) and their genotype was determined by phylogenetic analyses. Gene regions representing all HIV-1 group M clades were found as well as unclassifiable sequences. In env and pol (RT), two groups of sequences forming distinct sub-clusters within the subtype A radiation were found and may define new A5 and A6 sub-subtypes. Recombinant forms were found in almost half (47.1%) of the patients of which 36.0% were second-generation recombinants. Fifty-eight different patterns of recombination were found. The A subtype, including CRF02_AG, was represented in most recombinant viruses. Epidemiological data suggests that the AIDS epidemic in Angola has probably started as early as 1961, the major cause being the independence war, and spread to Portugal soon thereafter. The extraordinary degree of HIV-1 group M genetic diversity and evolution in Angola may pose unprecedented challenges to diagnostic, treatment and prevention of HIV-1 infection.

Baseline susceptibility of primary HIV-2 to entry inhibitors.

BACKGROUND The baseline susceptibility of primary HIV-2 to maraviroc (MVC) and other entry inhibitors is currently unknown. METHODS The susceptibility of 19 HIV-2 isolates obtained from asymptomatic and AIDS patients and seven HIV-1 clinical isolates to the fusion inhibitors enfuvirtide (ENF) and T-1249, and to the coreceptor antagonists AMD3100, TAK-779 and MVC, was measured using a TZM-bl cell-based assay. The 50% inhibitory concentration (IC(50)), 90% inhibitory concentration (IC(90)) and dose-response curve slopes were determined for each drug. RESULTS ENF and T-1249 were significantly less active on HIV-2 than on HIV-1 (211- and 2-fold, respectively). AMD3100 and TAK-779 inhibited HIV-2 and HIV-1 CXCR4 tropic (X4) and CCR5 tropic (R5) variants with similar IC(50) and IC(90) values. MVC, however, inhibited the replication of R5 HIV-2 variants with significantly higher IC(90) values (42.7 versus 9.7 nM; P<0.0001) and lower slope values (0.7 versus 1.3; P<0.0001) than HIV-1. HIV-2 R5 variants derived from AIDS patients were significantly less sensitive to MVC than variants from asymptomatic patients, this being inversely correlated with the absolute number of CD4(+) T-cells. CONCLUSIONS T-1249 is a potent inhibitor of HIV-2 replication indicating that new fusion inhibitors might be useful to treat HIV-2 infection. Coreceptor antagonists TAK-779 and AMD3100 are also potent inhibitors of HIV-2 replication. The reduced sensitivity of R5 variants to MVC, especially in severely immunodeficient patients, indicates that the treatment of HIV-2-infected patients with MVC might require higher dosages than those used in HIV-1 patients, and should be adjusted to the disease stage.

HIV-1 genetic diversity and transmitted drug resistance in health care settings in Maputo, Mozambique.

Objectives:To characterize HIV-1 diversity and transmitted drug resistance in persons with access to care and treatment in Maputo, Mozambique. Methods:Samples were collected in 2002-2004 from 144 drug-naive patients attending public hospitals and private clinics. Plasma viremia, CD4, and CD8 cell counts were determined for each patient. The Stanford Algorithm was used for resistance genotyping on pol sequences. Subtyping was done by phylogenetic analysis. Results:Most patients had high viral load (mean, 5.0 log copies/mL) and low CD4 cell counts (median, 260 CD4 cells/μL). Protease and/or reverse transcriptase sequences were obtained from 104 (72%) samples. Patients harbored subtypes C (80.8%), G (3.8%), CRF37_cpx (6.7%), untypable (U) (1.0%), and recombinant strains (7.7%) comprising the A, C, D, F, and U clades. There were no major protease inhibitor resistance mutations. Mutations conferring resistance to the nucleoside/nucleotide reverse transcriptase inhibitors and/or nonnucleoside reverse transcriptase inhibitors were found in 4 (4/68; 5.9%) patients. Phylogenetic analysis suggested an imported origin for 2 resistant variants. Conclusions:The HIV-1 epidemic in Maputo is evolving rapidly in genetic complexity due to the recent introduction of all major subtypes and recombinant forms. Continued surveillance of drug resistance in treated and untreated populations is needed to prevent further transmission of HIV drug-resistant variants and maximize the efficacy of antiretroviral therapy in Maputo.

Origin and epidemiological history of HIV-1 CRF14_BG.

Background CRF14_BG isolates, originally found in Spain, are characterized by CXCR4 tropism and rapid disease progression. This study aimed to identify the origin of CRF14_BG and reconstruct its epidemiological history based on new isolates from Portugal. Methodology/Principal Findings C2V3C3 env gene sequences were obtained from 62 samples collected in 1993–1998 from Portuguese HIV-1 patients. Full-length genomic sequences were obtained from three patients. Viral subtypes, diversity, divergence rate and positive selection were investigated by phylogenetic analysis. The molecular structure of the genomes was determined by bootscanning. A relaxed molecular clock model was used to date the origin of CRF14_BG. Geno2pheno was used to predict viral tropism. Subtype B was the most prevalent subtype (45 sequences; 73%) followed by CRF14_BG (8; 13%), G (4; 6%), F1 (2; 3%), C (2; 3%) and CRF02_AG (1; 2%). Three CRF14_BG sequences were derived from 1993 samples. Near full-length genomic sequences were strongly related to the CRF14_BG isolates from Spain. Genetic diversity of the Portuguese isolates was significantly higher than the Spanish isolates (0.044 vs 0.014, P<0.0001). The mean date of origin of the CRF14_BG cluster was estimated to be 1992 (range, 1989 and 1996) based on the subtype G genomic region and 1989 (range, 1984–1993) based on the subtype B genomic region. Most CRF14_BG strains (78.9%) were predicted to be CXCR4. Finally, up to five amino acids were under selective pressure in subtype B V3 loop whereas only one was found in the CRF14_BG cluster. Conclusions CRF14_BG emerged in Portugal in the early 1990 s soon after the beginning of the HIV-1 epidemics, spread to Spain in late 1990 s as a consequence of IVDUs migration and then to the rest of Europe. CXCR4 tropism is a general characteristic of this CRF that may have been selected for by escape from neutralizing antibody response.

Evolutionary and Structural Features of the C2, V3 and C3 Envelope Regions Underlying the Differences in HIV-1 and HIV-2 Biology and Infection

Background Unlike in HIV-1 infection, the majority of HIV-2 patients produce broadly reactive neutralizing antibodies, control viral replication and survive as elite controllers. The identification of the molecular, structural and evolutionary footprints underlying these very distinct immunological and clinical outcomes may lead to the development of new strategies for the prevention and treatment of HIV infection. Methodology/Principal Findings We performed a side-by-side molecular, evolutionary and structural comparison of the C2, V3 and C3 envelope regions from HIV-1 and HIV-2. These regions contain major antigenic targets and are important for receptor binding. In HIV-2, these regions also have immune modulatory properties. We found that these regions are significantly more variable in HIV-1 than in HIV-2. Within each virus, C3 is the most entropic region followed by either C2 (HIV-2) or V3 (HIV-1). The C3 region is well exposed in the HIV-2 envelope and is under strong diversifying selection suggesting that, like in HIV-1, it may harbour neutralizing epitopes. Notably, however, extreme diversification of C2 and C3 seems to be deleterious for HIV-2 and prevent its transmission. Computer modelling simulations showed that in HIV-2 the V3 loop is much less exposed than C2 and C3 and has a retractile conformation due to a physical interaction with both C2 and C3. The concealed and conserved nature of V3 in the HIV-2 is consistent with its lack of immunodominancy in vivo and with its role in preventing immune activation. In contrast, HIV-1 had an extended and accessible V3 loop that is consistent with its immunodominant and neutralizing nature. Conclusions/Significance We identify significant structural and functional constrains to the diversification and evolution of C2, V3 and C3 in the HIV-2 envelope but not in HIV-1. These studies highlight fundamental differences in the biology and infection of HIV-1 and HIV-2 and in their mode of interaction with the human immune system and may inform new vaccine and therapeutic interventions against these viruses.

Antiretroviral Drug Resistance Surveillance among Treatment-Naive Human Immunodeficiency Virus Type 1-Infected Individuals in Angola: Evidence for Low Level of Transmitted Drug Resistance

ABSTRACT The prevalence of transmitted human immunodeficiency virus type 1 drug resistance in Angola in 2001 in 196 untreated patients was investigated. All subtypes were detected, along with unclassifiable and complex recombinant strains. Numerous new polymorphisms were identified in the reverse transcriptase and protease. Two (1.6%) unrelated patients harbored nucleoside reverse transcriptase inhibitor- and nonnucleoside reverse transcriptase inhibitor-resistant viruses (mutations: M41L, D67N, M184V, L210W, T215Y or T215F, and K103N). Continued surveillance of drug resistance is required for maximization of ART efficacy in Angola.

Rapid clinical progression to AIDS and death in a persistently seronegative HIV-1 infected heterosexual young man.

Seronegative HIV-1 infection has important implications for diagnosis and prevention. We describe a case of HIV-1 infection without seroconversion and fast progression to AIDS and death. Genetic and phylogenetic analysis indicated that the patient was infected with a CRF14_BG-like strain selectively transmitted by his seropositive sexual partner. The results suggest a massive infection with a highly aggressive CRF14_BG-like strain and/or the presence of an unidentified immunological deficiency that has prevented the formation of HIV-1-specific antibodies.

Predictors of attrition and immunological failure in HIV- 1 patients on highly active antiretroviral therapy from different healthcare settings in Mozambique

In Mozambique, the evaluation of retention in HIV care and ART programmes is limited. To assess rate and predictors of attrition (no retention in care) and HAART effectiveness in HIV-1 infected patients who pay for medication and laboratory testing in Mozambique, we conducted a multicenter survey of HIV-1-infected patients who started HAART during 2002–2006. Cox proportional hazard models were used to assess risk of attrition and of therapy failure. Overall, 142 patients from 16 healthcare centers located in the capital city Maputo were followed-up for 22.2 months (12.1–46.7). The retention rate was 75%, 48% and 37% after one, two and three years, respectively. Risk of attrition was lower in patients with higher baseline CD4 count (P = 0.022) and attending healthcare center 1 (HCC1) (P = 0.013). The proportion of individuals with CD4 count ≤200 cells/µL was 55% (78/142) at baseline and decreased to 6% (3/52) at 36 months. Among the patients with available VL, 86% (64/74) achieved undetectable VL levels. The rate of immunologic failure was 17.2% (95% CI: 12.6–22.9) per 100 person-years. Risk of failure was associated to higher baseline CD4 count (P = 0.002), likely reflecting low adherence levels, and decreased with baseline VL ≥10,000 copies/mL (P = 0.033). These results suggest that HAART can be effective in HIV-1 infected patients from Mozambique that pay for their medication and laboratory testing. Further studies are required to identify the causes for low retention rates in patients with low CD4 counts and to better understand the association between healthcare setting and attrition rate.

HIV-1 Diversity, Transmission Dynamics and Primary Drug Resistance in Angola

Objectives To assess HIV-1 diversity, transmission dynamics and prevalence of transmitted drug resistance (TDR) in Angola, five years after ART scale-up. Methods Population sequencing of the pol gene was performed on 139 plasma samples collected in 2009 from drug-naive HIV-1 infected individuals living in Luanda. HIV-1 subtypes were determined using phylogenetic analysis. Drug resistance mutations were identified using the Calibrated Population Resistance Tool (CPR). Transmission networks were determined using phylogenetic analysis of all Angolan sequences present in the databases. Evolutionary trends were determined by comparison with a similar survey performed in 2001. Results 47.1% of the viruses were pure subtypes (all except B), 47.1% were recombinants and 5.8% were untypable. The prevalence of subtype A decreased significantly from 2001 to 2009 (40.0% to 10.8%, P = 0.0019) while the prevalence of unique recombinant forms (URFs) increased>2-fold (40.0% to 83.1%, P<0.0001). The most frequent URFs comprised untypable sequences with subtypes H (U/H, n = 7, 10.8%), A (U/A, n = 6, 9.2%) and G (G/U, n = 4, 6.2%). Newly identified U/H recombinants formed a highly supported monophyletic cluster suggesting a local and common origin. TDR mutation K103N was found in one (0.7%) patient (1.6% in 2001). Out of the 364 sequences sampled for transmission network analysis, 130 (35.7%) were part of a transmission network. Forty eight transmission clusters were identified; the majority (56.3%) comprised sequences sampled in 2008–2010 in Luanda which is consistent with a locally fuelled epidemic. Very low genetic distance was found in 27 transmission pairs sampled in the same year, suggesting recent transmission events. Conclusions Transmission of drug resistant strains was still negligible in Luanda in 2009, five years after the scale-up of ART. The dominance of small and recent transmission clusters and the emergence of new URFs are consistent with a rising HIV-1 epidemics mainly driven by heterosexual transmission.