José Maria Marcelino

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-2 Genetic Evolution in Patients with Advanced Disease Is Faster than That in Matched HIV-1 Patients

ABSTRACT The objective of this study was to estimate and compare the evolutionary rates of HIV-2 and HIV-1. Two HIV-2 data sets from patients with advanced disease were compared to matched HIV-1 data sets. The estimated mean evolutionary rate of HIV-2 was significantly higher than the estimated rate of HIV-1, both in the gp125 and in the V3 region of the env gene. In addition, the rate of synonymous substitutions in gp125 was significantly higher for HIV-2 than for HIV-1, possibly indicating a shorter generation time or higher mutation rate of HIV-2. Thus, the lower virulence of HIV-2 does not appear to translate into a lower rate of evolution.

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.

Use of a New Dual-Antigen Enzyme-Linked Immunosorbent Assay To Detect and Characterize the Human Antibody Response to the Human Immunodeficiency Virus Type 2 Envelope gp125 and gp36 Glycoproteins

ABSTRACT A dual-antigen enzyme-linked immunosorbent assay specific for human immunodeficiency virus type 2 (HIV-2) envelope proteins, ELISA-HIV2, was developed with two new recombinant polypeptides, rpC2-C3 and rgp36, derived from the HIV-2 envelope. The diagnostic performance was determined with HIV-2, HIV-1, and HIV-1/2 samples. Both polypeptides showed 100% specificity. Clinical sensitivity was 100% for rgp36 and 93.4% for rpC2-C3. ELISA-HIV2 may be used for the specific diagnosis and confirmation of HIV-2 infection.

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.

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.

An ancestral HIV-2/simian immunodeficiency virus peptide with potent HIV-1 and HIV-2 fusion inhibitor activity.

Objectives:To produce new fusion inhibitor peptides for HIV-1 and HIV-2 based on ancestral envelope sequences. Methods:HIV-2/simian immunodeficiency virus (SIV) ancestral transmembrane protein sequences were reconstructed and ancestral peptides were derived from the helical region 2 (HR2). The activity of one ancestral peptide (named P3) was examined against a panel of HIV-1 and HIV-2 primary isolates in TZM-bl cells and peripheral blood mononuclear cells and compared to T-20. Peptide secondary structure was analyzed by circular dichroism. Resistant viruses were selected and resistance mutations were identified by sequencing the env gene. Results:P3 has 34 residues and overlaps the N-terminal pocket-binding region and heptad repeat core of HR2. In contrast to T-20, P3 forms a typical &agr;-helical structure in solution, binds strongly to the transmembrane protein, and potently inhibits both HIV-2 (mean IC50, 63.8 nmol/l) and HIV-1 (11 nmol/l) infection, including T-20-resistant isolates. The N43K mutation in the HR1 region of HIV-1 leads to 120-fold resistance to P3 indicating that the HR1 region in transmembrane glycoprotein is the target of P3. No HIV-2-resistant mutations could be selected by P3 suggesting that the genetic barrier to resistance is higher in HIV-2 than in HIV-1. HIV-1-infected patients presented significantly lower P3-specific antibody reactivity compared to T-20. Conclusion:P3 is an HIV-2/SIV ancestral peptide with low antigenicity, high stability, and potent activity against both HIV-1, including variants resistant to T-20, and HIV-2. Similar evolutionary biology strategies should be explored to enhance the production of antiviral peptide drugs, microbicides, and vaccines.

Envelope-specific antibody response in HIV-2 infection: C2V3C3-specific IgG response is associated with disease progression.

Objective: To examine the unspecific and envelope-specific IgA and IgG responses in acute and chronic HIV-2 infection. Methods: Twenty-eight chronically infected adults and two children with perinatal infection were studied. Total plasma concentrations of IgA and IgG were determined by nephelometry. IgA and IgG reactivity against the immunodominant region in gp36 and the C2V3C3 region in gp125 was tested with the enzyme-linked immunosorbent assay (ELISA)–HIV-2 assay. Clonal sequences of the C2V3C3 env region were obtained for most patients. Results: Total plasma IgG concentration, but not IgA, was significantly higher than normal in HIV-2 patients and correlated inversely with CD4+ T-cell counts. Seroconversion to gp36 occurred during the first year of life in both infants. The infant with rapid disease progression did not elicit C2V3C3-specific antibodies. Most chronically infected patients produced plasma IgG1, IgG3 and IgA antibodies against gp36 and C2V3C3. Lack of C2V3C3-specific IgG response in two patients was associated with a major antigenic change in the V3 region. In longitudinal analysis, there was a significant inverse association between the C2V3C3-specific IgG antibody response and the number of CD4+ T cells. Conclusion: HIV-2 promotes an early, strong and broad gp36 and C2V3C3-specific IgG and IgA response. Increase in the IgG response against the envelope C2V3C3 region is associated with increased loss of CD4+ T cells in chronically infected patients. These results provide further support for the immune protective role of the C2V3C3 envelope region during HIV-2 infection and have direct implications for HIV-2 diagnosis, clinical management and pathogenesis.

Resistance to antibody neutralization in HIV-2 infection occurs in late stage disease and is associated with X4 tropism

Objectives:To characterize the nature and dynamics of the neutralizing antibody (NAb) response and escape in chronically HIV-2 infected patients. Methods:Twenty-eight chronically infected adults were studied over a period of 1–4 years. The neutralizing activity of plasma immunoglobulin G (IgG) antibodies against autologous and heterologous primary isolates was analyzed using a standard assay in TZM-bl cells. Coreceptor usage was determined in ghost cells. The sequence and predicted three-dimensional structure of the C2V3C3 Env region were determined for all isolates. Results:Only 50% of the patients consistently produced IgG NAbs to autologous and contemporaneous virus isolates. In contrast, 96% of the patients produced IgG antibodies that neutralized at least two isolates of a panel of six heterologous R5 isolates. Breadth and potency of the neutralizing antibodies were positively associated with the number of CD4+ T cells and with the titer and avidity of C2V3C3-specific binding IgG antibodies. X4 isolates were obtained only from late stage disease patients and were fully resistant to neutralization. The V3 loop of X4 viruses was longer, had a higher net charge, and differed markedly in secondary structure compared to R5 viruses. Conclusion:Most HIV-2 patients infected with R5 isolates produce C2V3C3-specific neutralizing antibodies whose potency and breadth decreases as the disease progresses. Resistance to antibody neutralization occurs in late stage disease and is usually associated with X4 viral tropism and major changes in V3 sequence and conformation. Our studies support a model of HIV-2 pathogenesis in which the neutralizing antibodies play a central role and have clear implications for the vaccine field.

Memory B-cell depletion is a feature of HIV-2 infection even in the absence of detectable viremia

Objective:Memory B-cell loss has long been recognized as an important contributor to HIV immunodeficiency. HIV-2 infection, which is characterized by a slow rate of progression to AIDS and reduced to undetectable viremia, provides a unique model to investigate B-cell disturbances. Design and methods:B-cell subsets were evaluated in 38 HIV-2-infected individuals, along with markers of T-cell activation and serum levels of immunoglobulins and a major B-cell homeostatic cytokine, B-cell activating factor (BAFF). Untreated HIV-1-infected and seronegative control individuals were studied in parallel. Statistical analysis was performed using Mann–Whitney tests and Spearmans correlations. Results:We found that HIV-2 was associated with significant depletion of both unswitched (CD27+IgD+) and switched (CD27+IgDneg) memory B-cells that directly correlated with T-cell activation, even in individuals with undetectable plasma viral load. Nevertheless, the presence of detectable viremia, even at low levels, was associated with significant memory B-cell loss and higher BAFF levels. Moreover, these alterations were not recovered by antiretroviral-therapy, as treated HIV-2-infected patients showed more pronounced B-cell disturbances, possibly related to their extended length of infection. Conclusion:These first data regarding B-cell imbalances during HIV-2 infection show that, irrespective of viremia, prolonged HIV infection leads to irreversible damage of memory B-cell homeostasis.