Perpétua Gomes

Human immunodeficiency virus type 2 (HIV‐2) in Portugal: Clinical spectrum, circulating subtypes, virus isolation, and plasma viral load

The human immunodeficiency virus type 2 (HIV‐2) is responsible for 4.5% of AIDS cases in Portugal. Six HIV‐2 subtypes have been described so far, subtype A being proposed as more pathogenic than the rest. The relationship between the clinical status and levels of both cellular and plasma HIV‐2 viraemia is not well known, nor their modifications under antiretroviral therapy. Thirty‐two consecutive HIV‐2 infected persons (17 men, 15 women) attending two different hospitals in Lisbon in 1997 were enrolled prospectively in the study. All but 4 individuals most likely acquired the infection through heterosexual contact. More than half of the study population was of African origin, mainly from Guinea‐Bissau. Eleven (34.4%) patients had developed clinical manifestations included within the B or C groups of the CDC classification system for HIV infection, with the rest being asymptomatic. Half of the population was undergoing antiretroviral treatment at the time of the study. HIV‐2 subtypes were investigated using a new Nef‐based restriction fragment length polymorphism (RFLP) method that allows differentiation of the main two variants, A and B. Plasma viral load was quantified using a new quantitative competitive reverse transcriptase polymerase chain reaction (QcRT‐PCR) procedure as well as the Amp‐RT assay. Virus isolation was attempted from peripheral blood mononuclear cells. All but one person carried HIV‐2 subtype A. Plasma viraemia examined by QcRT‐PCR was measurable in 15 (50%) of 30 subjects, yielding in all instances values below 20,000 HIV‐2 RNA copies per ml. Plasma RT activity could be detected in only 10 (33%) of 30 subjects, a rate much lower than that seen in HIV‐1 infection. Virus was isolated from 16 (53.3%) of 30 patients. A significant correlation was found between CD4+ counts, clinical status, rate of virus isolation, and plasma viral load by both QcRT‐PCR and Amp‐RT. In conclusion, HIV‐2 subtype A is the predominant variant circulating in Portugal among both natives and immigrants. A lower cellular and plasma viral load with respect to HIV‐1 was seen in persons without immunosuppression, from whom the rate of virus recovery was extremely low. J. Med. Virol. 61:111–116, 2000.

Protease mutation M89I/V is linked to therapy failure in patients infected with the HIV-1 non-B subtypes C, F or G

Objective:To investigate whether and how mutations at position 89 of HIV-1 protease were associated with protease inhibitor (PI) failure, and what is the impact of the HIV-1 subtype. Methods:In a database containing pol nucleotide sequences and treatment history, the correlation between PI experience and mutations at codon 89 was determined separately for subtype B and several non-B subtypes. A Bayesian network model was used to map the resistance pathways in which M89I/V is involved for subtype G. The phenotypic effect of M89I/V for several PIs was also measured. Results:The analysis showed that for the subtypes C, F and G in which the wild-type codon at 89 was M compared to L for subtype B, M89I/V was significantly more frequently observed in PI-treated patients displaying major resistance mutations to PIs than in drug-naive patients. M89I/V was strongly associated with PI resistance mutations at codons 71, 74 and 90. Phenotypically, M89I/V alone did not confer a reduced susceptibility to PIs. However, when combined with L90M, a significantly reduced susceptibility to nelfinavir was observed (P < 0.05) in comparison with strains with L90M alone. Conclusions:The results of the present study show that M89I/V is associated with PI experience in subtypes C, F and G but not in subtype B. M89I/V should be considered a secondary PI mutation with an important effect on nelfinavir susceptibility in the presence of L90M.

Quality control assessment of human immunodeficiency virus type 2 (HIV-2) viral load quantification assays: results from an international collaboration on HIV-2 infection in 2006.

ABSTRACT Human immunodeficiency virus type 2 (HIV-2) RNA quantification assays used in nine laboratories of the ACHIEV2E (A Collaboration on HIV-2 Infection) study group were evaluated. In a blinded experimental design, laboratories quantified three series of aliquots of an HIV-2 subtype A strain, each at a different theoretical viral load. Quantification varied between laboratories, and international standardization of quantification assays is strongly needed.

Major depletion of plasmacytoid dendritic cells in HIV-2 infection, an attenuated form of HIV disease.

Plasmacytoid dendritic cells (pDC) provide an important link between innate and acquired immunity, mediating their action mainly through IFN-α production. pDC suppress HIV-1 replication, but there is increasing evidence suggesting they may also contribute to the increased levels of cell apoptosis and pan-immune activation associated with disease progression. Although having the same clinical spectrum, HIV-2 infection is characterized by a strikingly lower viremia and a much slower rate of CD4 decline and AIDS progression than HIV-1, irrespective of disease stage. We report here a similar marked reduction in circulating pDC levels in untreated HIV-1 and HIV-2 infections in association with CD4 depletion and T cell activation, in spite of the undetectable viremia found in the majority of HIV-2 patients. Moreover, the same overexpression of CD86 and PD-L1 on circulating pDC was found in both infections irrespective of disease stage or viremia status. Our observation that pDC depletion occurs in HIV-2 infected patients with undetectable viremia indicates that mechanisms other than direct viral infection determine the pDC depletion during persistent infections. However, viremia was associated with an impairment of IFN-α production on a per pDC basis upon TLR9 stimulation. These data support the possibility that diminished function in vitro may relate to prior activation by HIV virions in vivo, in agreement with our finding of higher expression levels of the IFN-α inducible gene, MxA, in HIV-1 than in HIV-2 individuals. Importantly, serum IFN-α levels were not elevated in HIV-2 infected individuals. In conclusion, our data in this unique natural model of “attenuated” HIV immunodeficiency contribute to the understanding of pDC biology in HIV/AIDS pathogenesis, showing that in the absence of detectable viremia a major depletion of circulating pDC in association with a relatively preserved IFN-α production does occur.

New findings in HCV genotype distribution in selected West European, Russian and Israeli regions

BACKGROUND HCV affects 185 million people worldwide and leads to death and morbidities. HCV has a high genetic diversity and is classified into seven genotypes and 67 subtypes. Novel anti-HCV drugs (Direct-Acting-Antivirals) eligibility, resistance and cure rates depend on HCV geno/subtype (GT). OBJECTIVES Analysis of epidemiological information and viral GT from patients undergoing viral genotyping in 2011-2015. STUDY DESIGN Anonymized information from 52 centers was analyzed retrospectively. RESULTS 37,839 samples were included in the study. We show that the GT distribution is similar throughout Western European countries, with some local differences. Here GTs 1 and 2 prevalences are lower and of GT4 higher than in all previous reports. Israel has a unique GT pattern and in South Russia the GT proportions are more similar to Asia. GTs 5 and 6 were detected in very low proportions. Three cases of the recombinant genotype P were reported in Munich (Germany). In addition, we observed that GT proportion was dependant on patientś gender, age and transmission route: GTs 1b and 2 were significantly more common in female, older, nosocomially-infected patients, while GTs 1a, 3 and 4 were more frequent in male, younger patients infected by tattooing, drug consume, and/or sexual practices. In infections acquired by drug consume, GTs 1a (35.0%) and 3 (28.1%) prevailed. In infections related to sexual practices lower proportion of GT3 (14.0%) and higher of GT4 (20.2%) were detected. GT4 was mostly abundant in MSM (29.6%). HIV coinfection was significantly associated with higher proportions GTs 1a and 4 (42.5% and 19.3%, respectively). CONCLUSION Genotype prevalence evolves and correlates to epidemiological factors. Continuous surveillance is necessary to better assess hepatitis C infection in Europe and to take appropriate actions.

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.

Transmission of HIV-2

The original geographical heartlands of HIV-2 infection are believed to be the former Portuguese and French colonies in west and south-central Africa from where the infection spread mainly to Europe with a higher prevalence in Portugal and France. Portugal is the European country with the highest rates of imported HIV-2 infection. The first case of HIV-2 infection was identified in 1987 in an individual from Guinea- Bissau attending Egas Moniz Hospital Lisbon. However in Africa HIV-2 infection has been traced back to 1966 through documented HIV-2 transfusion cases. (excerpt)

An International Collaboration To Standardize HIV-2 Viral Load Assays: Results from the 2009 ACHIEV2E Quality Control Study

ABSTRACT Accurate HIV-2 plasma viral load quantification is crucial for adequate HIV-2 patient management and for the proper conduct of clinical trials and international cohort collaborations. This study compared the homogeneity of HIV-2 RNA quantification when using HIV-2 assays from ACHIEV2E study sites and either in-house PCR calibration standards or common viral load standards supplied to all collaborators. Each of the 12 participating laboratories quantified blinded HIV-2 samples, using its own HIV-2 viral load assay and standard as well as centrally validated and distributed common HIV-2 group A and B standards (http://www.hiv.lanl.gov/content/sequence/HelpDocs/subtypes-more.html). Aliquots of HIV-2 group A and B strains, each at 2 theoretical concentrations (2.7 and 3.7 log10 copies/ml), were tested. Intralaboratory, interlaboratory, and overall variances of quantification results obtained with both standards were compared using F tests. For HIV-2 group A quantifications, overall and interlaboratory and/or intralaboratory variances were significantly lower when using the common standard than when using in-house standards at the concentration levels of 2.7 log10 copies/ml and 3.7 log10 copies/ml, respectively. For HIV-2 group B, a high heterogeneity was observed and the variances did not differ according to the type of standard used. In this international collaboration, the use of a common standard improved the homogeneity of HIV-2 group A RNA quantification only. The diversity of HIV-2 group B, particularly in PCR primer-binding regions, may explain the heterogeneity in quantification of this strain. Development of a validated HIV-2 viral load assay that accurately quantifies distinct circulating strains is needed.

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.

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.