T-cell immune responses against Env from CRF12_BF and subtype B HIV-1 show high clade-specificity that can be overridden by multiclade immunizations.

BACKGROUND: The extreme genetic diversity of the human immunodeficiency virus type 1 (HIV-1) poses a daunting challenge to the generation of an effective AIDS vaccine. In Argentina, the epidemic is characterized by the high prevalence of infections caused by subtype B and BF variants. The aim of this study was to characterize in mice the immunogenic and antigenic properties of the Env protein from CRF12_BF in comparison with clade B, employing prime-boost schemes with the combination of recombinant DNA and vaccinia virus (VV) vectors. METHODOLOGY/PRINCIPAL FINDINGS: As determined by ELISPOT from splenocytes of animals immunized with either EnvBF or EnvB antigens, the majority of the cellular responses to Env were found to be clade-specific. A detailed peptide mapping of the responses reveal that when there is cross-reactivity, there are no amino acid changes in the peptide sequence or were minimal and located at the peptide ends. In those cases, analysis of T cell polifunctionality and affinity indicated no differences with respect to the cellular responses found against the original homologous sequence. Significantly, application of a mixed immunization combining both clades (B and BF) induced a broader cellular response, in which the majority of the peptides targeted after the single clade vaccinations generated a positive response. In this group we could also find significant cellular and humoral responses against the whole gp120 protein from subtype B. CONCLUSIONS/SIGNIFICANCE: This work has characterized for the first time the immunogenic peptides of certain EnvBF regions, involved in T cell responses. It provides evidence that to improve immune responses to HIV there is a need to combine Env antigens from different clades, highlighting the convenience of the inclusion of BF antigens in future vaccines for geographic regions where these HIV variants circulate.

Dendritic cell immunotherapy for HIV infection: from theory to reality.

Knowledge concerning the immunology of dendritic cells (DCs) accumulated over the last few decades and the development of methodologies to generate and manipulate these cells in vitro has made their therapeutic application a reality. Currently, clinical protocols for DC-based therapeutic vaccine in HIV-infected individuals show that it is a safe and promising approach. Concomitantly, important advances continue to be made in the development of methodologies to optimize DC acquisition, as well as the selection of safe, immunogenic HIV antigens and the evaluation of immune response in treated individuals.

Effect of promoters on cellular immune response induced by recombinant fowlpox virus expressing multi-epitope polypeptides from HIV-1.

HIV stimulates strong immune CD8(+) cytotoxic T lymphocytes (CTL) response in infected people, despite causing an immunodeficiency. It has been demonstrated that this response could be very important for the control of the virus. We have shown previously that a recombinant fowlpox virus (rFWPV), expressing the multi-epitope polypeptide (MEP) from HIV-1 TAB9, induces strong and protective Th1 and CTL responses in Balb/c mice. Here, we have studied the CTL response against MEPs TAB9 and CR3 after immunizing with rFWPVs, where these genes are under the control of a strong synthetic early/late promoter or the 7.5 kDa promoter from vaccinia virus. TAB9 expression was increased by more than 9-fold using the strong promoter, which was translated into a two times increase in CTL response. The overall expression of CR3 was already ten times higher when compared with TAB9 with the 7.5 kDa promoter, but the use of a stronger promoter showed no effect either on the expression or CTL response. Moreover, rFWPV expressing TAB9 induced a stronger CTL response than those expressing CR3, measured as the number of interferon- gamma -secreting splenocytes, in spite of its lower antigen expression levels. These results suggest that the capacity of a stronger promoter to increase the MEP expression and/or CTL response against their epitopes is highly dependent on the nature of the polypeptide used.

Induction of a strong HIV-specific CD8+ T cell response in mice using a fowlpox virus vector expressing an HIV-1 multi-CTL-epitope polypeptide.

Recombinant avipoxvirus vectors are attractive candidates for use in vaccination strategies for infections such as human immunodeficiency virus type 1 (HIV-1), where induction of a CD8+ T cell response is thought to be an important component of protective immunity. Here, we report the expression of a multiepitope polypeptide (TAB9) composed of the central 15 amino acids of the V3 loop from six different isolates of HIV-1 in a fowlpox virus (FWPV) vector, and the use of this vector (FPTAB9LZ) to induce strong HIV-specific CD8+ T cell responses in mice. In animals immunized twice intravenously with FPTAB9LZ, almost 2% of the CD8+ T cells in the spleen were shown to produce IFN-gamma in response to stimulation with HIV-1 peptides 1 week after the second immunization. The most dominant response was to the HIV-1 IIIB peptide. A strong HIV-specific response was also induced by intraperitoneal immunization of mice with FPTAB9LZ, whilst subcutaneous immunization elicited a weaker response. Intraperitoneal immunization with FPTAB9LZ was also shown to provide protection against challenge with a recombinant vaccinia virus expressing antigens, including those in TAB9. These results confirm the potential of FWPV vectors for use in HIV vaccination strategies.

Diversity of mosaic structures and common ancestry of human immunodeficiency virus type 1 BF intersubtype recombinant viruses from Argentina revealed by analysis of near full-length genome sequences.

The findings that BF intersubtype recombinant human immunodeficiency type 1 viruses (HIV-1) with coincident breakpoints in pol are circulating widely in Argentina and that non-recombinant F subtype viruses have failed to be detected in this country were reported recently. To analyse the mosaic structures of these viruses and to determine their phylogenetic relationship, near full-length proviral genomes of eight of these recombinant viruses were amplified by PCR and sequenced. Intersubtype breakpoints were analysed by bootscanning and examining the signature nucleotides. Phylogenetic relationships were determined with neighbour-joining trees. Five viruses, each with predominantly subtype F genomes, exhibited mosaic structures that were highly similar. Two intersubtype breakpoints were shared by all viruses and seven by the majority. Of the consensus breakpoints, all nine were present in two viruses, which exhibited identical recombinant structures, and four to eight breakpoints were present in the remaining viruses. Phylogenetic analysis of partial sequences supported both a common ancestry, at least in part of their genomes, for all recombinant viruses and the phylogenetic relationship of F subtype segments with F subtype viruses from Brazil. A common ancestry of the recombinants was supported also by the presence of shared signature amino acids and nucleotides, either unreported or highly unusual in F and B subtype viruses. These results indicate that HIV-1 BF recombinant viruses with diverse mosaic structures, including a circulating recombinant form (which are widespread in Argentina) derive from a common recombinant ancestor and that F subtype segments of these recombinants are related phylogenetically to the F subtype viruses from Brazil.

Chimeric synthetic peptide as antigen for immunodiagnosis of HIV-1 infection.

One chimeric peptide incorporating antigenic sequences from the gp41 transmembrane region (peptide H-18) and the gp120 envelope region (peptide H-15) corresponding to amino acids (587-617) on gp41 and (495-516) on gp120 of human immunodeficiency virus (HIV 1) was synthesized. Both sequences were separated by two glycine residues. This peptide was evaluated as antigen in an ultramicro-enzyme-linked immunosorbent assay (UMELISA) with samples derived from HIV-1 (n = 30) with different titers of antibodies and healthy blood donors (n = 30). The results were compared to plates coated with monomeric peptides and to plates coated with two monomeric peptides together. Results demonstrated that monomeric peptides gp41 (H-18) and gp120 (H-15) were good as antigens with samples that present antibodies to these regions. The chimeric peptide was the most antigenic. Those results may be related to the peptide structure, adsorption to the solid surface, and epitope accessibility to the antibodies. This chimeric peptide would be very useful for HIV-1 diagnostics.

Identification of human immunodeficiency virus type 1 subtypes B and F B/F recombinant and dual infection with these subtypes in Argentina.

DNA sequences encoding the third variable region (V3) of human immunodeficiency virus type-1 (HIV-1) envelope glycoprotein gp120 were obtained from 18 infected individuals residing in different regions of Argentina. Proviral DNA representing the env V3 region was obtained by PCR from uncultured peripheral blood mononuclear cells (PBMC) and genetic heterogeneity was examined by phylogenetic analysis. Sequences representing the gag p17 region were also obtained for a subset of these samples. Moreover, 1 sample that it was not possible to classify according to initial phylogenetic analysis was further analysed by molecular cloning of both V3 and p17 regions. Phylogenetic analysis according to different methodologies were performed comparing obtained sequences with a set of reference sequences representing previously characterized HIV-1 subtypes. The recombinant identification program (RIP) was used to study the presence of possible recombinant sequences. Phylogenetic analysis demonstrated that viruses representing both subtypes B and F are circulating among HIV-1 infected individuals in Argentina. In addition, RIP analysis showed that an initially unclassified sequence exhibited similarities to subtypes B and F in different fragments of the V3 region. Separate phylogenetic analysis of each of these fragments revealed divergent clustering, suggesting that this sequence harbours a point of recombination within the V3 loop. Interestingly, we also identified a dually infected individual with viruses belonging to subtypes B and F, as demonstrated by molecular cloning analysis of the env V3 and the gag p17 regions. Taken together, our study shows that both subtypes B and F are circulating in different regions of Argentina. Moreover, the data presented here show that dual infections with subtypes B and F can occur, and consequently B/F recombinant sequences are arising in the region.

Epitope mapping, V-region DNA sequence, and neutralizing Fab fragments of two monoclonal antibodies against the HIV-1 V3 loop.

BACKGROUND: Experimental evidence suggests that neutralizing antibodies could constitute an important factor in the control of AIDS progression and that the V3 loop of gp120 constitutes the main target for such purposes. We have previously developed two neutralizing murine monoclonal antibodies (Mabs) against the V3 region of the HIV-1 MN strain. OBJECTIVES: To characterize those Mabs in terms of fine specificity and DNA sequence of their V regions and to study if Fab fragments retain their neutralizing potential in vitro. STUDY DESIGN: A set of 12-mer alanine substituted peptides were employed for epitope mapping using two ELISA procedures: (1) indirect, with each peptide bound to polystyrene plates, and (2) competitive, with co-incubation of peptides and Mabs in solution. The V regions of both Mabs were PCR amplified from cDNA and their nucleotide sequences were determined. Finally, Fab fragments of Mab 10F10 were generated and their neutralizing capacity against the MN isolated was assessed. RESULTS: We first restricted the minimal length of the epitopes recognized by 2C4 and 10F10 to the 12-mer peptide KRIHIGPGRAFY. The core of the epitopes recognized by Mabs 2C4 and 10F10 were IHIGP-R and IHIG-R, respectively. While substitution of proline in position 7 completely abolished the binding of 2C4, it only reduced that of 10F10 by 50%. Finally, Fab fragments of Mab 10F10 were still able to neutralize the HIV-1 MN strain in vitro. CONCLUSION: This subtle distinction in the fine mapping of the epitope recognized by Mabs 2C4 and 10F10 should correspond to three amino acid differences that we found in the heavy chain V-regions. The Fab fragments of Mab 10F10 retained the neutralizing capacity. This indicates that HIV neutralization by anti V3 Mabs is an Fc independent process.

V3 peptide binding pattern and HIV-1 transmission route in Rio de Janeiro.

To characterize antibody binding to a panel of V3 loop peptides representing diverse HIV-1 neutralization epitopes, 149 HIV-1 infected individuals from Rio de Janeiro (RJ) were investigated. Results were analyzed with respect to risk factors for infection and other epidemiological and clinical data. Peptide reactivity was not associated with sex, clinical status, CD4 counts, antigenemia or beta 2-microglobulin serum level. A segregation of peptide reactivity according to route of infection was encountered. This finding suggests that more then one viral strain may be circulating in RJ, in subjects with different risk factors for HIV-1 infection. An investigation of prevalent HIV-1 genotypes, serotypes and immunotypes may be of importance for the design and selection of potential vaccines to be used in Brazil as well as for the selection of populations to be included in future vaccine efficacy trials.

PIP: HIV-1 is highly polymorphic both genetically and antigenically. The HIV-1 principal neutralizing determinant (PND) is located in the third hypervariable domain (V3 loop) of gp120. The V3 loop is an important epitope for neutralization, viral tropism, host range, and syncytium inducing capability. An epitope localized at its tip is the major binding site for type-specific neutralizing antibodies, and anti-V3 loop antibodies have protective capabilities. Enzyme immunoassays based upon synthetic peptides derived from known V3 loop sequences have been developed. 149 serum samples were collected during August-December 1991 from HIV-infected outpatients attending a prospective cohort study of HIV infection in Rio de Janeiro in a study to characterize antibody binding to a panel of V3 loop peptides representing diverse HIV-1 neutralization epitopes. 134 subjects acquired HIV through sexual intercourse, while 15 reported parenteral risk factors such as IV drug use or blood transfusion. Peptide reactivity was not associated with sex, clinical status, CD4 counts, antigenemia, or B2-microglobulin serum level, and a segregation of peptide reactivity according to route of infection was encountered. Study findings suggest that more than one viral strain may be circulating in Rio de Janeiro, in subjects with different risk factors for HIV-1 infection. An investigation of prevalent HIV-1 genotypes, serotypes, and immunotypes is warranted with regard to vaccine research, development, and use in Brazil.

Evaluation of a dipstick method for the detection of human immunodeficiency virus infection.

Serology has been a fundamental tool to prevent post-transfusional infection with human immunodeficiency virus (HIV) and for epidemiological surveys, the first step to attempt control of the pandemia. Enzyme immunoassay is in widespread use. Nevertheless, simpler methods are needed in many countries, where laboratory facilities and trained personnel are limited, and HIV prevalence is high. The evaluation of a simple and noninstrumented HIV antibody test is presented here. The test employs synthetic antigens of HIV-1 and HIV-2 attached to the teeth of a polystyrene comb, which fit into the wells of standard microtiter plates where samples are diluted. Captured antibodies are developed with colloidal gold-labeled Protein A. Three seroconversion panels plus 662 samples were tested, including HIV-1 and HIV-2-infected individuals, normal blood donors, and a noninfected baby born to a seroreactive mother. When compared with enzyme-linked immunosorbent assay (ELISA) and Western blot, the dipstick showed 100% sensitivity and 98.7% specificity. The simplicity of result evaluations and excellent reagent stability make the dipstick suitable for small blood banks and for epidemiological surveys.

Serotyping HIV type 1 by antibody binding to the V3 loop: relationship to viral genotype. WHO Network for HIV Isolation and Characterization.

We have investigated whether peptides representing the HIV-1 principal neutralization domain (V3) can be used as antigens in antibody-binding assays to predict the genotypes of the subjects' virus. Serum samples collected from HIV-1-infected subjects from the four WHO-sponsored vaccine evaluation sites (Uganda, Rwanda, Thailand, and Brazil) were characterized by antibody binding to a panel of synthetic V3 peptides that were derived from the consensus sequences of the V3 region of the HIV-1 subgroups according to the env phylogenetic analysis (A-E). An indirect V3 peptide-binding assay was used for primary screening, and a V3 peptide antigen-limiting ELISA was then used as a secondary assay to discriminate cross-reactivity if the screening assay was equivocal. In general, V3 peptide serology could predict HIV-1 genotypes. In sera for which the genotype of the virus was known, peptide assays could predict the correct genotype in approximately 90% of cases for genotypes A, B, C, and E; Ugandan sera of genotype D were more broadly reactive. There was considerable serological cross-reactivity between some HIV-1 genotypes, in particular between A and C, and, to a lesser extent, B and D subtypes. Owing to polymorphism at the crown of the V3 loop, an additional B peptide (B') was required to type Brazilian B genotype sera. These simple assays may help facilitate the determination and distribution of HIV-1 genotypes circulating in populations.

Phylogenetic analysis of gag genes from 70 international HIV-1 isolates provides evidence for multiple genotypes.

OBJECTIVE: To determine the extent of genetic variation among internationally collected HIV-1 isolates, to analyse phylogenetic relationships and the geographic distribution of different variants. DESIGN: Phylogenetic comparison of 70 HIV-1 isolates collected in 15 countries on four continents. METHODS: To sequence the complete gag genome of HIV-1 isolates, build multiple sequence alignments and construct phylogenetic trees using distance matrix methods and maximum parsimony algorithms. RESULTS: Phylogenetic tree analysis identified seven distinct genotypes. The seven genotypes were evident by both distance matrix methods and maximum parsimony analysis, and were strongly supported by bootstrap resampling of the data. The intra-genotypic gag distances averaged 7%, whereas the inter-genotypic distances averaged 14%. The geographic distribution of variants was complex. Some genotypes have apparently migrated to several continents and many areas harbor a mixture of genotypes. Related variants may cluster in certain areas, particularly isolates from a single city collected over a short time. CONCLUSIONS: The genetic variation among HIV-1 isolates is more extensive than previously appreciated. At least seven distinct HIV-1 genotypes can be identified. Diversification, migration and establishment of local, temporal 'blooms' of particular variants may all occur concomitantly.

[Molecular analysis of the principal neutralization epitope (V3 loop) of human immunodeficiency virus type 1 in Argentina]. / Análisis molecular del principal epítope de neutralización ("V3 loop") del virus de la inmunodeficiencia humana tipo 1 de la Argentina.

The main goal of the present paper was to analyze the molecular diversity of the principal neutralizing domain (V3 loop) of the HIV 1 gp120 in samples from patients of Argentina. The study was carried out on a total of 30 HIV 1 positive blood samples, obtained during 1991-1992, belonging to 15 intravenous drug users (group A), 5 homosexual men (group B), 8 children born to HIV 1 positive mothers (group C) and 2 AIDS patients (group D). By using extracted DNA from peripheral blood lymphocytes and from infected cells of the viral isolates in the case of the 2 AIDS patients, the V3 loop region was amplified by means of polymerase chain reaction. Direct sequencing by Sanger methodology was then performed on DNA fragments and nucleotide sequences obtained were translated into the correspondent amino acids. Consensus sequences for each group and a general consensus sequence were established (Table 1). Its alignment with V3 loop amino acid sequences of the major HIV 1 strains isolated worldwide is showed in table 2. Homology analysis between each sequence of the study population and sequence of different HIV 1 isolates showed that most of these samples share high homology with SF2 and BH10 strains. In contrast a low homology was found with JH3 and MN isolated (table 3). The presence of highly conserved amino acid residues as substitutions and insertions was determined in the Argentinian V3 loop sequences giving them a local pattern. The present paper is of great importance for our country, considering that the V3 loop is the main neutralizing domain becoming a major target in the development of HIV 1 vaccine. To our knowledge, this is the first report on the sequencing of the principal neutralizing domain of the Human Immunodeficiency Virus type 1 in Latin America.