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

BACKGROUND: This 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 reactivity RESULTS: The 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. CONCLUSION: The 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.

Coreceptor Usage by HIV-1 and HIV-2 Primary Isolates: The Relevance of CCR8 Chemokine Receptor as an Alternative Coreceptor

The human immunodeficiency virus replication cycle begins by sequential interactions between viral envelope glycoproteins with CD4 molecule and a member of the seven-transmembrane, G-protein-coupled, receptors' family (coreceptor). In this report we focused on the contribution of CCR8 as alternative coreceptor for HIV-1 and HIV-2 isolates. We found that this coreceptor was efficiently used not only by HIV-2 but particularly by HIV-1 isolates. We demonstrate that CXCR4 usage, either alone or together with CCR5 and/or CCR8, was more frequently observed in HIV-1 than in HIV-2 isolates. Directly related to this is the finding that the non-usage of CXCR4 is significantly more common in HIV-2 isolates; both features could be associated with the slower disease progression generally observed in HIV-2 infected patients. The ability of some viral isolates to use alternative coreceptors besides CCR5 and CXCR4 could further impact on the efficacy of entry inhibitor therapy and possibly also in HIV pathogenesis.

HIV-2 Integrase Polymorphisms and Longitudinal Genotypic Analysis of HIV-2 Infected Patients Failing a Raltegravir-Containing Regimen

To characterize the HIV-2 integrase gene polymorphisms and the pathways to resistance of HIV-2 patients failing a raltegravir-containing regimen, we studied 63 integrase strand transfer inhibitors (INSTI)-naïve patients, and 10 heavily pretreated patients exhibiting virological failure while receiving a salvage raltegravir-containing regimen. All patients were infected by HIV-2 group A. 61.4% of the integrase residues were conserved, including the catalytic motif residues. No INSTI-major resistance mutations were detected in the virus population from naïve patients, but two amino acids that are secondary resistance mutations to INSTIs in HIV-1 were observed. The 10 raltegravir-experienced patients exhibited resistance mutations via three main genetic pathways: N155H, Q148R, and eventually E92Q - T97A. The 155 pathway was preferentially used (7/10 patients). Other mutations associated to raltegravir resistance in HIV-1 were also observed in our HIV-2 population (V151I and D232N), along with several novel mutations previously unreported. Data retrieved from this study should help build a more robust HIV-2-specific algorithm for the genotypic interpretation of raltegravir resistance, and contribute to improve the clinical monitoring of HIV-2-infected patients.

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.

Epidemiologia Molecular, Evolução Viral e Perfil de Resistência aos Antirretrovirais em uma População de Pacientes Infectados por Hiv-1 e/ou Hiv-2 Residentes em Diferentes Ilhas do Arquipélago de Cabo Verde

O diagnóstico sorológico da infecção pelo HIV-1 e HIV-2 teve início em Cabo Verde em 1987, mas pouco se sabe a respeito da diversidade genética desses vírus nessas ilhas, localizadas na costa Ocidental Africana. Neste estudo, caracterizamos a epidemiologia molecular do HIV-1 e HIV-2 em Cabo Verde, analisamos a origem dos principais clados de HIV introduzidos no país e descrevemos a ocorrência de mutações de resistência aos antirretrovirais (DRM) em indivíduos virgens de tratamento (ARTn) e pacientes em tratamento (ARTexp) oriundos das diferentes ilhas. Amostras de sangue, dados sociodemográfico e clínico-laboratoriais foram obtidos de 221 indivíduos HIV positivos entre 2010-2011. As amostras foram sequenciadas na região da polimerase (1300 pares de bases) e análises filogenéticas e de bootscan foram realizadas para a subtipagem viral. Os algoritmos disponibilizados nos sites Stanford HIV Database e HIV-GRADE e.V. Algorithm Homepage foram utilizados para avaliar a existência de DRM em pacientes positivos para HIV-1 e HIV-2, respectivamente. Os estudos evolutivos e filogeográficos foram realizados através do programa BEAST. Entre os 221 pacientes analisados, sendo 169 (76,5%) HIV-1, 43 (19,5%) HIV-2 e 9 de (4,1%) co-infectados pelo HIV-1 e pelo HIV-2, 67% eram do sexo feminino. As medianas de idade foram de 34 (IQR = 1-75) e 47 (IQR = 12-84) para o HIV-1 e HIV-2, respectivamente. A infecção pelo HIV-1 é causada pelo subtipo G (36,6%), CRF02_AG (30,6%), subtipo F1, (9,7%), URFs (10,4%), subtipo B (5,2%), CRF05_DF (3,0%), subtipo C (2,2%), CRF06_cpx (0,7%), CRF25_cpx (0,7%) e CRF49_cpx (0,7%), e todas as infecções por HIV-2 pertencem ao grupo A. De acordo com as análises filogeográficas e de origem do HIV, estima-se que o HIV-2 foi o primeiro tipo viral introduzido em Cabo Verde e possui relações filogenéticas com sequências referências de Portugal. O HIV-1 entrou no país mais tarde, primeiramente pelo subtipo G, evidenciando relações com sequências da África Central e de Portugal. Transmissão de DRM (TDRM) foi observada em 3,4% (2/58) de pacientes HIV-1 ARTn (1,7% NRTI, NNRTI 1,7%), mas não entre os infectados com HIV-2. Entre os pacientes ARTexp, DRM foi observada em 47,8% (33/69) dos infectados pelo HIV-1 (37,7% NRTI, NNRTI 37,7%, 7,4% de PI, 33,3% para duas classes) e 17,6% (3/17) nos infectados pelo HIV-2 (17,6%, 11,8% NRTI PI, 11,8% para ambas as classes). Este estudo indica que Cabo Verde tem um cenário epidemiológico molecular complexo e único dominado pelo HIV-1 subtipo G, CRF02_AG e F1 e HIV-2 grupo A, sendo esse o primeiro tipo viral introduzido em Cabo Verde. A ocorrência de TDRM e o nível relativamente elevado de DRM entre os pacientes tratados constituem uma preocupação, pelo que o monitoramento contínuo dos pacientes em ARTexp, incluindo genotipagem são políticas públicas a serem implementadas.

Proteasome inhibition interferes with Gag polyprotein processing, release, and maturation of HIV-1 and HIV-2

Retrovirus assembly and maturation involve folding and transport of viral proteins to the virus assembly site followed by subsequent proteolytic cleavage of the Gag polyprotein within the nascent virion. We report that inhibiting proteasomes severely decreases the budding, maturation, and infectivity of HIV. Although processing of the Env glycoproteins is not changed, proteasome inhibitors inhibit processing of Gag polyprotein by the viral protease without affecting the activity of the HIV-1 viral protease itself, as demonstrated by in vitro processing of HIV-1 Gag polyprotein Pr55. Furthermore, this effect occurs independently of the virus release function of the HIV-1 accessory protein Vpu and is not limited to HIV-1, as proteasome inhibitors also reduce virus release and Gag processing of HIV-2. Electron microscopy analysis revealed ultrastructural changes in budding virions similar to mutants in the late assembly domain of p6gag, a C-terminal domain of Pr55 required for efficient virus maturation and release. Proteasome inhibition reduced the level of free ubiquitin in HIV-1-infected cells and prevented monoubiquitination of p6gag. Consistent with this, viruses with mutations in PR or p6gag were resistant to detrimental effects mediated by proteasome inhibitors. These results indicate the requirement for an active proteasome/ubiquitin system in release and maturation of infectious HIV particles and provide a potential pharmaceutical strategy for interfering with retrovirus replication.

A epidemiologia da infeção por HIV-2

Dissertação para obtenção do grau de Mestre no Instituto Superior de Ciências da Saúde Egas Moniz

Expression and biochemical analysis of the entire HIV-2 gp41 ectodomain: determinants of stability map to N- and C-terminal sequences outside the 6-helix bundle core

The folding of HIV gp41 into a 6-helix bundle drives virus-cell membrane fusion. To examine the structural relationship between the 6-helix bundle core domain and other regions of gp41, we expressed in Escherichia coli, the entire ectodomain of HIV-2(ST) gp41 as a soluble, trimeric maltose-binding protein (MBP)/gp41 chimera. Limiting proteolysis indicated that the Cys-591-Cys-597 disulfide-bonded region is outside a core domain comprising two peptides, Thr-529-Trp-589 and Val-604-Ser-666. A biochemical examination of MBP/gp41 chimeras encompassing these core peptides; indicated that the N-terminal polar segment, 521-528, and C-terminal membrane-proximal segment, 658-666, cooperate in stabilizing the ectodomain. A functional interaction between sequences outside the gp41 core may contribute energy to membrane fusion. (C) 2004 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

Greater CD8+ TCR heterogeneity and functional flexibility in HIV-2 compared to HIV-1 infection

Virus-specific CD8+ T cells are known to play an important role in the control of HIV infection. In this study we investigated whether there may be qualitative differences in the CD8+ T cell response in HIV-1- and HIV-2-infected individuals that contribute to the relatively efficient control of the latter infection. A molecular comparison of global TCR heterogeneity showed a more oligoclonal pattern of CD8 cells in HIV-1- than HIV-2-infected patients. This was reflected in restricted and conserved TCR usage by CD8+ T cells recognizing individual HLA-A2- and HLA-B57-restricted viral epitopes in HIV-1, with limited plasticity in their response to amino acid substitutions within these epitopes. The more diverse TCR usage observed for HIV-2-specific CD8 T cells was associated with an enhanced potential for CD8+ expansion and IFN- production on cross-recognition of variant epitopes. Our data suggest a mechanism that could account for any possible cross-protection that may be mediated by HIV-2-specific CD8+ T cells against HIV-1 infection. Furthermore, they have implications for HIV vaccine development, demonstrating an association between a polyclonal, virus-specific CD8+ T cell response and an enhanced capacity to tolerate substitutions within T cell epitopes.

APOBEC3 host factors modulate viral production and infectivity of HIV-2

Poster presented at the 15th European AIDS Conference/EACS. Barcelona, 21-24 October 2015.

HIV-2 viral production and infectivity are affected by APO3 host factors

Poster presented at the 7th Postgraduate iMed.ULisboa Students Meeting. Faculty of Pharmacy, Universidade de Lisboa, 15-16 July 2015.

Development of water-soluble polyanionic carbosilane dendrimers as novel and highly potent topical anti-HIV-2 microbicides

This is the author’s version of a work that was accepted for publication in Nanoscale.