Cheila Rocha

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.

Potent and Broadly Reactive HIV-2 Neutralizing Antibodies Elicited by a Vaccinia Virus Vector Prime-C2V3C3 Polypeptide Boost Immunization Strategy

ABSTRACT Human immunodeficiency virus type 2 (HIV-2) infection affects about 1 to 2 million individuals, the majority living in West Africa, Europe, and India. As for HIV-1, new strategies for the prevention of HIV-2 infection are needed. Our aim was to produce new vaccine immunogens that elicit the production of broadly reactive HIV-2 neutralizing antibodies (NAbs). Native and truncated envelope proteins from the reference HIV-2ALI isolate were expressed in vaccinia virus or in bacteria. This source isolate was used due to its unique phenotype combining CD4 independence and CCR5 usage. NAbs were not elicited in BALB/c mice by single immunization with a truncated and fully glycosylated envelope gp125 (gp125t) or a recombinant polypeptide comprising the C2, V3, and C3 envelope regions (rpC2-C3). A strong and broad NAb response was, however, elicited in mice primed with gp125t expressed in vaccinia virus and boosted with rpC2-C3. Serum from these animals potently neutralized (median 50% neutralizing titer, 3,200) six of six highly divergent primary HIV-2 isolates. Coreceptor usage and the V3 sequence of NAb-sensitive isolates were similar to that of the vaccinating immunogen (HIV-2ALI). In contrast, NAbs were not reactive on three X4 isolates that displayed major changes in V3 loop sequence and structure. Collectively, our findings demonstrate that broadly reactive HIV-2 NAbs can be elicited by using a vaccinia virus vector-prime/rpC2-C3-boost immunization strategy and suggest a potential relationship between escape to neutralization and cell tropism.

The role of the humoral immune response in the molecular evolution of the envelope C2, V3 and C3 regions in chronically HIV-2 infected patients

BackgroundThis study was designed to investigate, for the first time, the short-term molecular evolution of the HIV-2 C2, V3 and C3 envelope regions and its association with the immune response. Clonal sequences of the env C2V3C3 region were obtained from a cohort of eighteen HIV-2 chronically infected patients followed prospectively during 2–4 years. Genetic diversity, divergence, positive selection and glycosylation in the C2V3C3 region were analysed as a function of the number of CD4+ T cells and the anti-C2V3C3 IgG and IgA antibody reactivityResultsThe mean intra-host nucleotide diversity was 2.1% (SD, 1.1%), increasing along the course of infection in most patients. Diversity at the amino acid level was significantly lower for the V3 region and higher for the C2 region. The average divergence rate was 0.014 substitutions/site/year, which is similar to that reported in chronic HIV-1 infection. The number and position of positively selected sites was highly variable, except for codons 267 and 270 in C2 that were under strong and persistent positive selection in most patients. N-glycosylation sites located in C2 and V3 were conserved in all patients along the course of infection. Intra-host variation of C2V3C3-specific IgG response over time was inversely associated with the variation in nucleotide and amino acid diversity of the C2V3C3 region. Variation of the C2V3C3-specific IgA response was inversely associated with variation in the number of N-glycosylation sites.ConclusionThe evolutionary dynamics of HIV-2 envelope during chronic aviremic infection is similar to HIV-1 implying that the virus should be actively replicating in cellular compartments. Convergent evolution of N-glycosylation in C2 and V3, and the limited diversification of V3, indicates that there are important functional constraints to the potential diversity of the HIV-2 envelope. C2V3C3-specific IgG antibodies are effective at reducing viral population size limiting the number of virus escape mutants. The C3 region seems to be a target for IgA antibodies and increasing N-linked glycosylation may prevent HIV-2 envelope recognition by these antibodies. Our results provide new insights into the biology of HIV-2 and its relation with the human host and may have important implications for vaccine design.

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.

Evolution of the human immunodeficiency virus type 2 envelope in the first years of infection is associated with the dynamics of the neutralizing antibody response

BackgroundDifferently from HIV-1, HIV-2 disease progression usually takes decades without antiretroviral therapy and the majority of HIV-2 infected individuals survive as elite controllers with normal CD4+ T cell counts and low or undetectable plasma viral load. Neutralizing antibodies (Nabs) are thought to play a central role in HIV-2 evolution and pathogenesis. However, the dynamic of the Nab response and resulting HIV-2 escape during acute infection and their impact in HIV-2 evolution and disease progression remain largely unknown. Our objective was to characterize the Nab response and the molecular and phenotypic evolution of HIV-2 in association with Nab escape in the first years of infection in two children infected at birth.ResultsCD4+ T cells decreased from about 50% to below 30% in both children in the first five years of infection and the infecting R5 viruses were replaced by X4 viruses within the same period. With antiretroviral therapy, viral load in child 1 decreased to undetectable levels and CD4+ T cells recovered to normal levels, which have been sustained at least until the age of 12. In contrast, viral load increased in child 2 and she progressed to AIDS and death at age 9. Beginning in the first year of life, child 1 raised high titers of antibodies that neutralized primary R5 isolates more effectively than X4 isolates, both autologous and heterologous. Child 2 raised a weak X4-specific Nab response that decreased sharply as disease progressed. Rate of evolution, nucleotide and amino acid diversity, and positive selection, were significantly higher in the envelope of child 1 compared to child 2. Rates of R5-to-X4 tropism switch, of V1 and V3 sequence diversification, and of convergence of V3 to a β-hairpin structure were related with rate of escape from the neutralizing antibodies.ConclusionOur data suggests that the molecular and phenotypic evolution of the human immunodeficiency virus type 2 envelope are related with the dynamics of the neutralizing antibody response providing further support for a model in which Nabs play an important role in HIV-2 pathogenesis.

Development of synthetic light-chain antibodies as novel and potent HIV fusion inhibitors.

Objective:To develop a novel and potent fusion inhibitor of HIV infection based on a rational strategy for synthetic antibody library construction. Design:The reduced molecular weight of single-domain antibodies (sdAbs) allows targeting of cryptic epitopes, the most conserved and critical ones in the context of HIV entry. Heavy-chain sdAbs from camelids are particularly suited for this type of epitope recognition because of the presence of long and flexible antigen-binding regions [complementary-determining regions (CDRs)]. Methods:We translated camelid CDR features to a rabbit light-chain variable domain (VL) and constructed a library of minimal antibody fragments with elongated CDRs. Additionally to elongation, CDRs’ variability was restricted to binding favorable amino acids to potentiate the selection of high-affinity sdAbs. The synthetic library was screened against a conserved, hidden, and crucial-to-fusion sequence on the heptad-repeat 1 (HR1) region of the HIV-1 envelope glycoprotein. Results:Two anti-HR1 VLs, named F63 and D104, strongly inhibited laboratory-adapted HIV-1 infectivity. F63 also inhibited infectivity of HIV-1 and HIV-2 primary isolates similarly to the Food and Drug Administration-approved fusion inhibitor T-20 and HIV-1 strains resistant to T-20. Moreover, epitope mapping of F63 revealed a novel target sequence within the highly conserved hydrophobic pocket of HR1. F63 was also capable of interacting with viral and cell lipid membrane models, a property previously associated with T-20s inhibitory mechanism. Conclusion:In summary, to our best knowledge, we developed the first potent and broad VL sdAb fusion inhibitor of HIV infection. Our study also gives insights into engineering strategies that could be explored to enhance the development of antiviral drugs.

miRNA profiling of human naive CD4 T cells links miR-34c-5p to cell activation and HIV replication

Cell activation is a vital step for T‐cell memory/effector differentiation as well as for productive HIV infection. To identify novel regulators of this process, we used next‐generation sequencing to profile changes in microRNA expression occurring in purified human naive CD4 T cells in response to TCR stimulation and/or HIV infection. Our results demonstrate, for the first time, the transcriptional up‐regulation of miR‐34c‐5p in response to TCR stimulation in naive CD4 T cells. The induction of this miR was further consistently found to be reduced by both HIV‐1 and HIV‐2 infections. Overexpression of miR‐34c‐5p led to changes in the expression of several genes involved in TCR signaling and cell activation, confirming its role as a novel regulator of naive CD4 T‐cell activation. We additionally show that miR‐34c‐5p promotes HIV‐1 replication, suggesting that its down‐regulation during HIV infection may be part of an anti‐viral host response.

Antiretroviral drug resistance surveillance among treatment-naive HIV-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.