Abstract: Upon HIV transmission, some patients develop AIDS in only a few months, while others remain disease free for 20 or more years. This variation in the rate of disease progression is poorly understood and has been attributed to host genetics, host immune responses, co-infection, viral genetics, and adaptation. Here, we develop a new "relaxed-clock" phylogenetic method to estimate absolute rates of synonymous and nonsynonymous substitution through time. We identify an unexpected association between the synonymous substitution rate of HIV and disease progression parameters. Since immune activation is the major determinant of HIV disease progression, we propose that this process can also determine viral generation times, by creating favourable conditions for HIV replication. These conclusions may apply more generally to HIV evolution, since we also observed an overall low synonymous substitution rate for HIV-2, which is known to be less pathogenic than HIV-1 and capable of tempering the detrimental effects of immune activation. Humoral immune responses, on the other hand, are the major determinant of nonsynonymous rate changes through time in the envelope gene, and our relaxed-clock estimates support a decrease in selective pressure as a consequence of immune system collapse.
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.
Abstract: HIV-2 sequence divergence and evolution in vivo has not been well characterized so far. To investigate the extent of HIV-2 genetic diversity and better understand how HIV-2 evolves in vivo, env C2-C3 nucleotide sequences were obtained from the plasma and PBMCs virus populations of four HIV-2 patients with different infection periods. Phylogenetic analysis showed that three patients were infected with subtype A HIV-2 and the remaining patient was infected with a divergent HIV-2 that could not be genotyped. Virus populations from the plasma and PBMCs clustered together in all patients suggesting that there is continuous and unrestricted virus flow between plasma and PBMCs. HIV-2 genetic diversity was not correlated with CD4+ cell counts and plasma viral load. There was a direct association between the period of infection and genetic divergence of virus populations both in the env C2-C3 and V3 regions such that higher genetic diversity was observed in long-term infected patients. In three patients, the average frequency of synonymous substitutions (dS) was significantly higher than the nonsynonymous substitutions (dN) whereas in the fourth patient the dN/dS ratio approached the unity. These data demonstrate that negative selective pressure determines the evolution of the HIV-2 env C2-C3 region in vivo. Our results suggest that throughout HIV-2 infection low virus adaptation to strong selective pressures (e.g. immune pressure) promotes the predominance of a few optimally adapted forms.
Abstract: To investigate which HIV-1 genetic forms are circulating in Angola, we have determined the gag and/or env genotypes of 48 isolates from patients living in Cabinda and Luanda provinces. The following subtypes were identified: A1 (18 samples, 38%), C (7, 15%), H (5, 10%), J (3, 6%), G (2, 4%), A2 (2, 4%), F1 (1, 2%), and D (1, 2%). The env gene fragment was untypable in one sample. Discordant subtype classifications in the gag and env genes were found in eight (17%) samples. There were six different recombination patterns (gag/env): A1/H (3, 6%), A1/G (1, 2%), C/A2 (1, 2%), F1/B (1, 2%), G/B (1, 2%), and G/H (1, 2%). The A1/H recombinant may represent a new circulating recombinant form. The marked genetic heterogeneity of HIV-1 in Angola has important implications for vaccine development.
Abstract: The study of true seronegative HIV-1 infections may have important implications for the diagnosis and prevention of HIV-1 infection. The case of an AIDS patient with persistently negative HIV serology is described. Genetic and phylogenetic analysis indicated that she was infected with A2 subsubtype HIV-1 transmitted by her seropositive and asymptomatic sexual partner. The clinical and serological discordant results suggest the presence of an immunological deficiency that prevents the formation of HIV-1-specific antibodies.
Abstract: HIV-2 infection was diagnosed in two patients 15 and 24 years of age and, thereafter, in their mothers. Epidemiological data suggested that vertical transmission was the most probable mode of infection in both patients (Mota-Miranda A, et al.: AIDS 2001;15:2460-2461). Phylogenetic analysis of env C2-C3 sequences from the patients and their mothers was used in an attempt to confirm or exclude the events of perinatal HIV-2 transmission. Sequences from each putative transmission pair formed monophyletic clusters in phylogenetic analysis, a clear indication of common ancestry. Interpatient nucleotide distances increased with the period of infection being consistent with long-term infection. In conclusion, the results are consistent with an epidemiological linkage between the viruses infecting each mother-child pair and support the occurrence of perinatal HIV-2 infection in both cases. Application of similar phylogeny methods to other suspected transmission cases may permit a better understanding of the epidemiology and molecular evolution of HIV-2.
Abstract: In the present study, we sought to define the importance of serum IgA (sIgA)-mediated immunity in HIV-2 infection. Serum samples from a total of 29 HIV-2-infected patients from Guinea-Bissau (n = 20) and Portugal (n = 9) were studied. Samples from seronegative individuals were used as controls. Antibody reactivity to native and recombinant envelope glycoproteins as well as peptides representing various regions of the envelope glycoproteins was investigated. Furthermore, the capacity of purified IgA to neutralize the HIV-2(SBL6669) strain was tested. All serum samples showed IgA reactivity against whole HIV-2 antigen. Twenty-eight out of 29 IgA samples (96%) reacted with native HIV-2 gp125, 26/29 (90%) with recombinant gp105, and 29/29 (100%) with recombinant gp36. When using peptides, the most prominent IgA reactivity was seen against the peptide representing aa 644-658 of the transmembranous protein gp36, to which 72% of the sera reacted. Purified sIgA antibodies showed neutralizing effects against HIV-2(SBL6669) in 17/29 cases (59%). This work describes the HIV-2-specific sIgA antigenic response. Moreover, our findings show, for the first time, that sIgA may play a role in the in vitro neutralization of HIV-2.
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.
Abstract: Human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) differ in their pathogenic mechanisms as evidenced by lower rate of disease progression, lower transmission rates and lower viral load in peripheral blood for HIV-2. One of the many factors that are involved in these characteristics is the interaction between viral glycoproteins and cellular receptors. The study of these interactions in an HIV-2 model could lead to important conclusions regarding pathogenesis and transmission mechanisms of HIV-2 infection. Here we report the design of a method enabling the construction of recombinant proviral HIV-2 DNAs in a moderate copy number plasmid that allows the analysis of env gene structure and functionality. This method constitutes an important tool for the study of HIV-2 env glycoproteins and for the mappings of genetic determinants of HIV-2 coreceptor usage and CD4-independent interaction. Furthermore, this knowledge will help towards the understanding of the different pathogenic mechanisms of HIV-1 and HIV-2.
Abstract: One anticapsid (p26) mouse monoclonal antibody was developed after immunization with recombinant p26 Gag protein and was tested for reactivity with different HIV-1 and HIV-2 isolates by ELISA and Western blot analysis. This antibody, named R26.1, reacted with all HIV-2 isolates tested and with recombinant p26 proteins, but no HIV-1 isolates. The epitope of antibody R26.1 was mapped to residues 50-71 in the N-terminal domain of the capsid protein, a highly conserved region in all HIV-2 isolates sequenced to date. This monoclonal antibody may be useful for the detection of HIV-2, and for the discrimination between HIV-1 and HIV-2 infections. Likewise, the identified epitope may be useful for the detection of p26 antibodies in HIV-2-infected individuals.
Abstract: A new quantitative-competitive PCR-based human immunodeficiency virus type 2 (HIV-2) proviral DNA assay (QC-PCR) was developed and used to determine the proviral load in HIV-2-infected individuals. Proviral load varied considerably, with means of 1,831 copies per 10(6) peripheral blood mononuclear cells for asymptomatic subjects (n = 19) and 2,587 for AIDS patients (n = 2). HIV-2 viral and proviral loads also varied significantly over time in asymptomatic patients. These data suggest that a high level of virus replication occurs throughout the asymptomatic phase of HIV-2 infection.
Abstract: To demonstrate that human immunodeficiency virus type 2 (HIV-2) mother-to-child transmission exists, HIV-2 isolates were obtained from both an asymptomatic mother (HIV-2 strain ARM), and her child (HIV-2 strain SAR), who had a diagnosis of AIDS. To determine their biological phenotype, primary isolates were used to infect various primary mononuclear cells and cell lines. HIV-2 ARM replicates in primary cells and Jurkat-tat, while HIV-2 SAR infects these cells plus SupT1, which led us to classify HIV-2 ARM as a slow/low virus and HIV-2 SAR as having an intermediate (slow/low-3) phenotype. Molecular analysis of the env region corresponding to gp125 was performed. Viral DNA was cloned, sequenced, and used to construct phylogenetic trees. The DNA sequence analysis demonstrated an overall nucleotide diversity of 7.6%. The results present evidence that the child's strain is more virulent than the mother's strain, which is in agreement with the immunodeficiency of the child. The phylogenetic trees that were constructed demonstrate that the two isolates cluster together, being closer to each other than to any other isolate described until now.
Abstract: In contrast to HIV-1, no studies have been published on the genetic and functional analysis of the envelope gene of primary NSI isolates of HIV-2. However several studies on HIV-1 have shown that NSI strains are the most frequently transmitted strains and probably the most important strains in the pathogenesis of HIV infection. Furthermore, it has been shown that the genetic and biological characteristics of primary isolates of HIV-1 differ widely from those of T-cell-line adapted isolates. Two different polymerase chain reaction (PCR) methods, nested-polymerase chain reaction and overlapp-extension amplification, were used to amplify the envelope genes from a primary non-syncytium-inducing HIV-2 isolate, HIV-2ALI, and from the T-cell line adapted syncytium-inducing isolate, HIV-2ROD. These methods could amplify the complete envelope gene from both viruses. Nested-polymerase chain reaction method was highly sensitive, enabling the amplification of one proviral copy of HIV-2ALI in 10000 peripheral blood mononuclear cells. The use of the methods described herein may help to expand our knowledge on the genetic diversity of HIV-2 as well as on the structure and function of the envelope glycoproteins of primary HIV-2 isolates.
Abstract: We have developed a new non-isotopic polymerase chain reaction (PCR) based method for the detection of the human immunodeficiency virus type 1 (HIV-1) DNA in clinical samples. In this method a two step PCR is used to amplify and label HIV-1 DNA segments by incorporation of digoxigenin-11-dUTP (dig-dUTP). Digoxigenin labelled amplified products are hybridized to membrane immobilized complementary DNA probes. Hybridization is detected non-radioactively by incubating the filters with antidigoxigenin antibody conjugated with alkaline phosphatase followed by a standard phosphatase assay. With this method the detection limit was between 1 and 10 HIV-1 DNA copies in a background of 1 microgram of human genomic DNA. Furthermore, we were able to detect HIV-1 DNA in 41 out of 41 HIV-1 antibody positive individuals while 10 out of 10 HIV-1 seronegative individuals gave consistently negative results. Our data indicate that this simple non-isotopic technique is sensitive and specific for the detection of HIV-1 DNA in clinical samples and can constitute a good alternative to other non-isotopic methods described to date.
