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.

Seroreactivity to HIV-1 V3 subtypes A to H peptides of Argentinian HIV-positive sera.

Serologic assays could be useful for determining circulating subtypes in different geographic regions. A total of 175 serum samples from the same number of Argentinian HIV-infected patients from Buenos Aires and Rosario were tested against a panel of peptides representing V3 consensus subtypes A through H. A V3 peptide enzyme immunoassay was used for screening the sera. Most sera were reactive with peptides representing subtypes B (58.28%), F (13.14%), and A (8.57%). Cross-reactivity between the remainder of the peptides was observed. Genotypes of eight patients from Rosario were determined and compared with serotyping. Results showed that seven of eight genotyped patients reacted with their respective consensus B peptide and one reacted with consensus B and F. V3 peptide serology proved to be useful for determining HIV-1 clades circulating in Argentina.

PIP: 175 serum samples were collected from 175 HIV-infected Argentineans in Buenos Aires and Rosario during 1987-95, for testing against a panel of peptides representing V3 consensus HIV-1 subtypes A through H. A V3 peptide enzyme immunoassay was used to screen the sera. 58.28% of the sera were infected with HIV-1 subtype B, 13.14% with subtype F, 8.57% with subtype A, 4% with subtype H, 2.85% with subtype D, 2.28% with subtype G, and 1.71% with subtype C. Some cross-reactivity between peptides was observed. Peripheral blood mononuclear cells (PBMCs) were obtained from 8 HIV-infected subjects from Rosario for use in determining genotypes. 7 of the 8 genotyped patients reacted with their respective consensus B peptide and 1 reacted with consensus B and F. V3 peptide serology proved useful in determining which HIV-1 clades are circulating in Argentina.

Immunodominant glycoprotein 41 epitope identified by seroreactivity in HIV type 1-infected individuals.

A highly conserved gp41 epitope (amino acid sequence ELDKWA) has been described to elicit antibodies neutralizing a broad variety of HIV-1 strains. We analyzed the antibody reactivity of HIV-1-infected individuals from Argentina and Sweden to overlapping synthetic peptides covering aa647-684 of HIV-1 MN gp41. An immunodominant antigenic determinant was discovered in the C-terminal region adjacent to the ELDKWA sequence. Most of the sera from both Argentina and Sweden reacted only with peptides representing this region. Two other patterns of reactivity were also observed: some sera reacted only with peptides spanning the central region of gp41, including the ELDKWA sequence; other sera reacted with both the central and C-terminal regions. Differences in reactivity were noted between Argentinian and Swedish sera in terms of peptides covering the central region. In addition, to determine the amino acids essential for antibody binding, seroreactivity against a set of substitution peptides covering the immunodominant epitope was studied. Results obtained indicated that carboxyl amino acids WNWFDI close to the ELDKWA sequence were the most important for antibody binding. Ability of sera, tested for peptide reactivity, to neutralize HIV-1 was also analyzed. High antibody reactivity to the central region was frequently found in sera with high neutralizing titers. This observation was not seen when seroreactivity to peptides spanning the C-terminal region was analyzed. These results suggest that the immunodominant epitope C terminal to the ELDKWA sequence is not involved in inducing neutralizing antibodies.

Analysis of antibody specificity against the third variable region of the envelope glycoprotein gp120 of HIV-1 in plasma from HIV-1-positive individuals residing in Brazil.

1. Antibody specificity for the principal neutralization domain (PND) of the human immunodeficiency virus type 1 (HIV-1) was studied in plasma from 122 HIV-1-infected individuals residing in Brazil. 2. Using 8 overlapping sequential pentadecapeptides corresponding to the third variable region (V3) of 5 different HIV-1 isolates in an enzyme-linked immunosorbent assay (ELISA), a preferential recognition of the peptides with amino acid sequences corresponding to the HIV-1 isolates IIIB and MN (maximal reactivities of 60-70%) compared to the isolates SC, WMJ-2 or RF (maximal reactivities below 60%) was observed. 3. A difference was observed in the overall reactivity pattern to HIV-1 SC peptides of plasma collected from individuals residing in the Brazilian states of Rio de Janeiro and Bahia. However, a statistically significant increased recognition by Bahian plasma was only observed for the HIV-1 SC C55 peptide. 4. The mean CD4/CD8 ratio of the group of plasma with an isolate-restricted recognition of peptides (0.522 +/- 0.074) was significantly lower than that of the total group of plasma (1.00 +/- 0.18).

Analysis of antibody specificity against the third variable region of the envelope glycoprotein gp120 of HIV-1 in plasma from HIV-1-positive individuals residing in Brazil

1. Antibody specificity for the principal neutralization domain (PND) of the human immunodeficiency virus type 1 (HIV-1) was studied in plasma from 122 HIV-1-infected individuals residing in Brazil. 2. Using 8 overlapping sequential pentadecapeptides corresponding to the third variable region (V3) of 5 different HIV-1 isolates in an enzyme-linked immunosorbent assay (ELISA), a preferential recognition of the peptides with amino acid sequences corresponding to the HIV-1 isolates IIIB and MN (maximal reactivities of 60-70) compared to the isolates SC, WMJ-2 or RF (maximal reactivities below 60) was observed. 3. A difference was observed in the overall reactivity pattern to HIV-1 SC peptides of plasma collected from individuals residing in the Brazilian states of Rio de Janeiro and Bahia. However, a statistically significant increased recognition by Bahian plasma was only observed for the HIV-1 SC C55 peptide. 4. The mean CD4/CD8 ratio of the group of plasma with an isolate-restricted recognition of peptides (0.522 +/- 0.074) was significantly lower than that of the total group of plasma (1.00 +/- 0.18).

Correlation between anti-V3 peptide and neutralizing antibodies in plasma from HIV-1 infected individuals resident in Brazil.

PIP: A study was conducted of the reactivity of 85 plasma samples from HIV-1 seropositive persons living in Brazil. Five HIV-1 plasma samples had synthetic peptides from the V3 region isolates (IIIB, MN, SC, WMJ-2, and RF). There was a low reactivity (59%) with HIV-1 isolate MN derived PND peptides, while most studies report a 90% or higher reactivity. Brazilian HIV-1 seropositive sera had lower reactivity with MN isolate PND peptides than sera from other countries. Researchers used 5 HIV-1 MN isolate PND peptides from other sources to analyze the same plasma to confirm the study's results. The HIV-1 MN isolate PND peptides had different amino acid sequences, but all had the GPGR top of the V3 loop. 52% of the Brazilian plasma recognized V3 peptides, 55% for 48 peptides, 56% for C52 peptides, and 49% for C53 pep tides. Only 27% of the Brazilian plasma recognized the C54 peptide. There was considerable overlapping reactivity, however. For example, 65% of the V3 MN reactive plasma also recognized the C52 MN peptide and the C53 MN peptide. The overlap between C52 MN and C53 MN was 69%. These findings suggest that, in the Brazilian study population, at least 2 different anti-MN-PND antibody specificities directed against amino acid sequences including the GPGR top of the V3 loop exist (an epitope including PNY[NKRKRIHIGPGR] and the epitope including [KRKRIHIGPGR]AFY). 86% of the plasma reacted with at least 20% of the MN PND peptides, which equals reactivities in other studies. 96% of the Brazilian plasma were positive for neutralizing antibodies based on overlap of peptide recognition and HIV-1 MN isolate neutralization (titers = 1:100-1:52,600). Recognition of the V3 loop of the HIV-1 MN isolate in sera from HIV-1 positive persons in Brazil has two different antibody specificities. Most of the Brazilian plasma recognize the N terminal arm, which consists of the GPGR top of the V3 loop.^ieng

Production of human monoclonal antibodies against HIV-1 peptides.

Protocols were evaluated in an attempt to produce human monoclonal antibodies (HumAb) specific for the human immunodeficiency virus type 1 (HIV-1). The first series of experiments involved in vitro immunization of normal human peripheral blood lymphocytes (PBL) with peptides C57 (HIV-1 strain IIIB clone BH10 gp 120 amino acids 324-338: GNMRQAHCNISRAKW) followed by either fusion to mouse/human heterohybrids or transformation with Epstein Barr virus (EBV). Using the hybridoma technology, three IgM class (lambda light chain) HumAb were obtained. In a parallel study, PBL from two HIV-1-infected patients were immortalized after in vitro stimulation with fragments of the HIV-1 envelope glycoprotein (recombinant gp120, the PB1 fragment of gp120, amino acids 295-473, or the penv9 fragment of gp160, amino acids 474-757). Five IgG class HumAb (three IgG2, lambda; one IgG1, K; one IgG3, lambda) reactive with the antigens used in the in vitro stimulations were obtained.

Production of human monoclonal antibodies against HIV-1 peptides

Protocols were evaluated in an attempt to produce human monoclonal antibodies (HumAb) specific for the human immunodeficiency virus type 1 (HIV-1). The first series of experimentls involved in vitro immunization of normal human peripheral blood lymphocytes (PBL) with peptide C57 (HIV-1 strain IIIB clone BH10 gp 120 amino acids 324-338: GNMRQAHCNISRAKW) followed by either fusion to mouse/human heterolhybrids or transformation with Epstein Barr virus (EBV). Using the hybridoma technology, three IgM class (alfa light chain) Human Ab were obtained. In a parallel study, PBL from two HIV-1 infected patients were immortalized after in vitro stimulation with fragments of the HIV-1 envelope glycoprotein (recombinant gp120 fragment of gp 120, amino acids 295-473, or the penv9 fragment of gp160, amino acids 474-757). Five IgG class Human b (three IgG2m alfa; one IgG1, K; one IgG3, alfa) reactive with the antigens used in the in vitro stimulations were obtained