HIV-1 binding and neutralizing antibodies of injecting drug users.

Previous studies have demonstrated a stronger seroreactivity against some synthetic peptides responsible for inducing neutralizing antibodies in injecting drug users (IDU) compared to that of individuals sexually infected with HIV-1 (S), but the effectiveness in terms of the neutralizing ability of these antibodies has not been evaluated. Our objective was to study the humoral immune response of IDU by determining the specificity of their antibodies and the presence of neutralizing antibodies. The neutralization capacity against the HIV-1 isolate MN (genotype B), the primary HIV-1 isolate 95BRRJ021 (genotype F), and the seroreactivity with peptides known to induce neutralizing antibodies, from the V2 and V3 loops of different HIV-1 subtypes, were analyzed. Seroreactivity indicates that IDU plasma are more likely to recognize a broader range of peptides than S plasma, with significantly higher titers, especially of V3 peptides. Similar neutralization frequencies of the MN isolate were observed in plasma of the IDU (16/47) and S (20/60) groups in the 1:10 dilution. The neutralization of the 95BRRJ021 isolate was more frequently observed for plasma from the S group (15/23) than from the IDU group (15/47, P = 0.0108). No correlation between neutralization and seroreactivity with the peptides tested was observed. These results suggest that an important factor responsible for the extensive and broad humoral immune response observed in IDU is their infection route. There was very little difference in neutralizing antibody response between the IDU and S groups despite their differences in seroreactivity and health status.

Correlation between susceptibility of primary HIV-1 isolates to autologous and heterologous neutralizing antibodies. Hospital Evandro Chagas AIDS Clinical Research Group.

OBJECTIVE: To study the susceptibility of primary HIV-1 isolates towards autologous and heterologous neutralizing antibodies (NAb). DESIGN: Blood was collected and primary HIV-1 isolated from individuals residing in Rio de Janeiro, Brazil, in all phases of disease. METHODS: Primary HIV-1 isolates were incubated with autologous or heterologous plasma and neutralization of infection of freshly pre-stimulated normal human peripheral blood mononuclear cells was assayed in parallel to median infectious dose determinations in the absence of antibodies. Levels of HIV-1 p24 antigen were used for evaluation of viral neutralization. RESULTS: Autologous neutralization (75%) was observed for 13 (52%) out of 25 of the primary HIV-1 isolates, and 15 (71%) out of 21 isolates were susceptible to 75% heterologous neutralization by at least one-half of the heterologous plasma tested. Primary HIV-1 isolates susceptible to autologous NAb showed a higher susceptibility towards neutralization by heterologous NAb than isolates that could not be neutralized by the autologous plasma (P = 0.049). The susceptibility of the primary HIV-1 isolates towards neutralization by heterologous NAb was significantly higher for isolates derived from men (P = 0.001), and for isolates obtained from individuals infected through homo-/bisexual risk behaviour in comparison with those infected through heterosexual HIV-1 transmission (P = 0.03). CONCLUSIONS: Susceptibility of primary HIV-1 isolates to autologous and heterologous neutralization was significantly correlated, indicating that escape mutants may become resistant not only to autologous but also to heterologous NAb.

V3 region polymorphisms in HIV-1 from Brazil: prevalence of subtype B strains divergent from North American/European prototype and detection of subtype F.

Viral DNA sequences were determined over the V3 region of env from 28 infected individuals living in the high HIV-1 prevalence Brazilian cities of Rio de Janeiro and São Paulo. Twenty-six belonged to envelope sequence subtype B, prevalent in North America and Europe, and one was classified as subtype F, found recently in Brazil and in Romania (one appeared to be a B/F recombinant). Octameric sequences at the tip of the subtype B V3 loops were variable and distinct from those prevalent in North America and Europe. The GPGR motif, prevalent in North American/European strains, was found in only 8 (28.5%) sequences, whereas GWGR was found in 12 (43%) and novel sequences in 8 (28.5%). Brazilian subtype B sequences also diverged from the consensus North American/European strains over the remainder of the V3 loop. These results suggest that Brazilian HIV-1 B strains may have important antigenic differences from prototype subtype B strains currently being evaluated for use in HIV vaccines. These results should be taken into account for future vaccine programs in Brazil.

Retrovirus infections in a sample of injecting drug users in Rio de Janeiro City, Brazil: prevalence of HIV-1 subtypes, and co-infection with HTLV-I/II.

BACKGROUND: Retrovirus infections among injecting drug users (IDUs), a core at-risk population for both HIV-1 and HTLV-I/II infections in Brazil, were assessed within an ongoing cooperative research. OBJECTIVE: The study assessed the seroprevalences of HIV-1 and HTLV-I/II infections, as well as the prevalence of HIV-1 subtypes in a sample of IDUs from Rio de Janeiro, Brazil. An attempt to evaluate HIV incidence was carried out using a dual 'sensitive/less sensitive' testing strategy. STUDY DESIGN: Cross-sectional evaluation of 175 IDUs. Serostatus for HIV-1 and HTLV-I/II were established by enzyme-linked immunosorbent assays, and confirmed by western blot. The dual testing strategy aimed to estimate HIV-1 incidence rates. Differentiation between HTLV-I and -II was performed by western blot. DNA samples were polymerase chain reaction amplified by a nested protocol, and HIV-1 subtyping was determined by heteroduplex mobility assay. RESULTS: Forty-six and 29 samples were found to be, respectively, positive for HIV-1 and HTLV-I/II, 15 of them co-infected by both viruses. Among HTLV-I/II-infected patients, 75.9% were infected by HTLV-I. Thirty-one HIV samples were identified as B subtype, with seven of them showing the typical "Brazilian B" pattern in the gp120 V3 loop, and ten were identified as F subtype. The use of less sensitive assays for HIV infection wrongly identified a deeply immunocompromised patient as an incident case. CONCLUSION: Moderately high seroprevalences were found for both HIV-1 and HTLV-I/II infections, HIV-1/HTLV-I co-infections being of special concern. A non-statistically significant higher prevalence of F subtype was observed, when compared with the distribution of F/B subtypes among Brazilian patients from other exposure categories. No recent HIV-1 infections were detected, but a limitation of the "sensitive/less-sensitive" testing strategy was made evident.

Anti-HIV-1 seroreactivity and HIV transmission route[R1]. The HEC/FIOCRUZ AIDS Clinical Research Group.

BACKGROUND: Antibody binding assays carried out by our group have consistently indicated a higher reactivity of sera from male HIV-1 infected individuals. This study was carried out in order to analyze the importance of gender, route of transmission, disease progression and HIV-1 genotype in seroreactivity assays. STUDY DESIGN: Specificity of antibody binding was studied in plasma of 247 HIV-1 seropositive individuals belonging to patient groups of pregnant women, injecting drug users (IDUs) and recent seroconvertors, resident in Rio de Janeiro, RJ. Recognition of synthetic peptides corresponding to antigenically important epitopes in the envelope of HIV-1 (gp41 immunodominant epitope, V3 loop, V2 loop and gp41 735-752 epitope) was determined. RESULTS: The immunodominant gp41 peptide (amino acids 594-613, HIV-1 MN sequence) was recognized by 85% of all plasma tested. Reactivity with the gp41 735-752 peptide and gp120 V2 loop peptides was low but quite variable, being generally more often specific to a Brazilian V2 peptide used than to the HIV-1 MN derived V2 peptide. The overall recognition of the different V3 peptides tested varied from 41 to 76%. Patients with more advanced disease showed a more frequent reactivity with the peptides studied than did asymptomatic patients. Statistically significant differences in peptide recognition were observed by multiple logistic analyses comparing plasma derived from individuals infected by blood or sexual HIV transmission, adjusting for disease progression and gender. Plasma from individuals infected by sexual transmission showed lower peptide recognition than did plasma from individuals infected through HIV positive blood. Association attempts between seroreactivity and genotype indicated that plasma derived from patients infected with HIV-1 of the F subtype showed highest recognition of heterologous V3 peptides, as well as a slightly more frequent recognition of the non-V3 peptides tested. Recognition of homologous peptides was generally higher than recognition of heterologous peptides. Differences were most pronounced between the prototypical HIV-1 B subtype and the Brazilian B" variant of this subtype but almost non-existent between the HIV-1 B and F subtypes. CONCLUSIONS: Individual gender was shown to be a confounder when investigating the relationships of peptide reaction to HIV-1 route of transmission through multivariate statistical methods: patients infected by blood transmission (IDU) present higher frequency of peptide recognition than individuals infected by sexual HIV-1 transmission. Plasma from individuals infected with the B" variant (GWG) of B subtype HIV-1 showed lower heterologous peptide recognition than that from HIV-1 B (GPG) or F infected individuals.

Trypanosoma cruzi: antigenic analysis of cloned stocks.

The antigenic constitution of two Trypanosoma cruzi strains and six cloned stocks was determined immunoelectrophoretically. Five to seven antigens common to all of the T. cruzi stocks were found. However, each stock also contained "strain-specific antigens," and five of the six cloned stocks presented "clone-specific antigens." These data demonstrate that a T. cruzi strain is composed of an antigenically heterogeneous population of organisms.

Production of monoclonal antibodies for the identification of a strain of Trypanosoma cruzi.

Mouse monoclonal antibodies (MAb) were produced against antigen from epimastigote forms of the Montalvania 17 strain of Trypanosoma cruzi. Several T. cruzi-specific MAb were obtained, some of which were capable of discriminating between different T. cruzi strains.

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.

Synergistic signal of an anti-hamster T cell monoclonal antibody on antigen-induced T cell proliferation.

1. The use of monoclonal antibodies (mAb) has permitted the identification of T cell surface antigens and the classification of these antigens based upon phenotype and function. Some of these monoclonal antibodies can identify antigens specifically involved in T lymphocyte activation and are also able to induce, under certain conditions, T cell proliferation. 2. We describe a new mAb raised against hamster T cells which binds to a 45-kDa cell surface antigen expressed on 45% of thymic cells and 90% of mature T lymphocytes. This mAb, designated X2VA, alone does not cause T cell proliferation, but increases in a synergistic manner T cell proliferation when these cells are cultured in the presence of specific antigens, or used in mixed lymphocyte reactions. 3. When the X2VA mAb is used as a single signal for the T cells it induces the production of a T cell growth factor, suggesting that the synergist effect observed during antigen-induced T cell proliferation is mediated by one or more cytokines. 4. Our results indicate that the X2VA mAb recognizes an antigen which is expressed during T cell ontogenesis and which is involved in hamster T cell activation.

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).

Production of a monoclonal anti-hamster thymocyte antibody with mitogenic properties.

A monoclonal antibody (MAb), designated H3, with specificity for hamster lymphocytes, was produced by somatic cell hybridization of myeloma Sp 2/0 and spleen cells of Balb/c mice immunized with suspensions of viable hamster thymocytes. The H3 MAb (IgG 3) reacted specifically with hamster thymocyte surface membranes (immunofluorescent assay). The antibody recognized a protein of an approximate molecular weight of 44,000 Daltons in immunoblots of hamster thymocyte extracts. The soluble H3 MAb presented potent mitogenic properties as indicated by the DNA synthesis induced in in vitro hamster lymphocyte cultures.

[Elution of proteins from polyacrylamide gel: description of a simple and economic method]. / Eluição de proteínas de gel de poliacrilamida: descrição de metodologia simples e econômica.

A simplified methodology for the quantitative electroelution of proteins from polyacrylamide gels is described. After staining with Coomassie Brilliant Blue R 250, the identified bands are excised from the gel and the proteins eluted using a procedure developed for use in conventional tube gel electrophoresis equipment.

Human immunodeficiency virus type 1 neutralization by plasma from B or F genotype infected individuals.

Anti-human immunodeficiency virus type 1 (HIV-1) "binding antibodies" (antibodies capable of binding to synthetic peptides or proteins) occur throughout HIV-1 infection, are high-titered and highly cross-reactive, as confirmed in this study by analyzing plasma from B and F genotype HIV-1 infected individuals. Plasma from individuals infected with clade F HIV-1 displayed the most frequent cross-reactivity, in high titers, while Bbr plasma showed much higher specificity. Similarly, neutralization of a reference HIV-1 isolate (HIV-1 MN) was more frequently observed by plasma from F than B genotype infected individuals. No significant difference was seen in neutralization susceptibility of primary B, Bbr or F clade HIV-1 by plasma from individuals infected with the classical B (GPGR) or F HIV-1, but Bbr (GWGR) plasma were less likely to neutralize the F genotype primary HIV-1 isolates. The data indicate that both B and F genotype derived vaccines would be equally effective against B and F HIV-1 infection, with a slightly more probable effectiveness for F than B genotype. Although the Bbr variant appears to induce a much more specific humoral immune response, the susceptibility in neutralizing the Brazilian HIV-1 B genotype Bbr variant is similar to that observed with the classical B genotype HIV-1.

Vertical human immunodeficiency virus type 1--HIV-1--transmission--a review.

Several factors appear to affect vertical HIV-1 transmission, dependent mainly on characteristics of the mother (extent of immunodeficiency, co-infections, risk behaviour, nutritional status, immune response, genetical make-up), but also of the virus (phenotype, tropism) and, possibly, of the child (genetical make-up). This complex situation is compounded by the fact that the virus may have the whole gestation period, apart from variable periods between membrane rupture and birth and the breast-feeding period, to pass from the mother to the infant. It seems probable that an extensive interplay of all factors occurs, and that some factors may be more important during specific periods and other factors in other periods. Factors predominant in protection against in utero transmission may be less important for peri-natal transmission, and probably quite different from those that predominantly affect transmission by mothers milk. For instance, cytotoxic T lymphocytes will probably be unable to exert any effect during breast-feeding, while neutralizing antibodies will be unable to protect transmission by HIV transmitted through infected cells. Furthermore, some responses may be capable of controlling transmission of determined virus types, while being inadequate for controlling others. As occurrence of mixed infections and recombination of HIV-1 types is a known fact, it does not appear possible to prevent vertical HIV-1 transmission by reinforcing just one of the factors, and probably a general strategy including all known factors must be used. Recent reports have brought information on vertical HIV-1 transmission in a variety of research fields, which will have to be considered in conjunction as background for specific studies.

Prevalence of antibodies against an immunodominant region of the HIV-1 envelope glycoprotein gp41 in sera from HIV-1 infected individuals in Brazil.

PIP: Findings are reported from a study conducted with plasma from HIV-1 positive individuals resident in Brazil to determine whether sera from Brazilian HIV-1 infected individuals react with the gp41 immunodominant epitope to a comparable degree as do North American/European sera. 120 plasma samples were collected at different hospitals in Rio de Janeiro during 1990-93 from HIV-1 seropositive individuals in all stages of the infection and analyzed. The subjects were participating in a multicenter study of heterosexual HIV transmission in the city. An additional 18 plasma from HIV-1 seronegative individuals from the same risk groups and three normal plasma from blood donors were used as controls. It is concluded upon analysis that a diagnostic assay based upon use of a synthetic peptide corresponding to the envelope gp41 immunodominant epitope must be carefully screened with local HIV-1 positive plasma banks before introduction to commercial use. The use of a shorter peptide, however, comprising the sequence CSGKLIC identified as the minimum epitope may increase sensitivity of the assay, as it appears to be most conserved between different HIV-1 isolates. Brazilian HIV-1 isolates should be analyzed to verify if they conform to this rule.^ieng

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

HIV-1 subtyping in Salvador, Bahia, Brazil: a city with African sociodemographic characteristics.

To investigate the prevalence of the HIV-1 subtypes in different populations from Salvador, Bahia, Brazil, blood samples from 72 HIV-1-seropositive injecting drug users (IDUs) and 62 individuals infected sexually were analyzed using the heteroduplex mobility assay (HMA). In the IDU group, 89.5% were classified as subtype B, 3% as subtype F, and 7.5% showed a B/F HMA profile. In the sexual transmission (ST) group, 95% were identified as B subtype, 3.4% showed a B/F profile, and 1.6% a B/C/E HMA profile. All Brazilian samples that showed multiple reactivities in the HMA analysis clustered on sequencing with B North American/ European HIV-1 isolates in the phylogenetic analysis, whereas the F subtypes clustered with F Brazilian HIV-I isolates. Serologic reactivities of IDU's sera were examined using a panel of synthetic V3 loop peptides representative of the different HIV-1 subtypes. No difference in serologic reactivity between F and B subtype plasma could be observed. Predominance of HIV-I subtype B was identified in both study groups, whereas subtype F was detected only among IDUs in a frequency lower than described for other Brazilian regions.

Neutralization susceptibility of B subtype variant B" primary HIV-1 isolates. The HEC/FIOCRUZ AIDS Clinical Research Group.

Susceptibility to autologous and heterologous neutralization of primary human immunodeficiency virus (HIV)-1 isolates belonging to subtype B, to the B"-variant of subtype B or to subtype F from infected individuals residing in Rio de Janeiro was assayed. A lower infectivity of the B"- and F isolates when compared to the classical B-subtype HIV-1 isolates was observed. Comparisons of neutralization susceptibilities were carried out for 19 B-subtype, 11 B"-variant and two F-subtype HIV-1 isolates with plasma from autologous and heterologous samples. Frequency of autologous neutralization was slightly lower for B-subtype isolates in comparison to B"-variant isolates. Heterologous intra-subtype neutralization was significantly lower for B-subtype than for the B"-variant or the F-subtype isolates. While B-subtype isolates were neutralized by most anti-F-subtype plasma, F-subtype isolates, although most susceptible to F-subtype antibodies, were highly susceptible to neutralization by anti-B-subtype antibodies. Cross-neutralization for B"-variant and B-subtype isolates was not as extensive as observed for B- and F-subtype isolates. However, the results presented indicate a quite extensive cross-neutralization between Brazilian HIV-1 isolates.

Vertical HIV-1 transmission: importance of neutralizing antibody titer and specificity.

Neutralization analyses were carried out with plasma from 132 volunteer human immunodeficiency virus (HIV)-1 infected women (76% pregnant, 24% with infants suspected for HIV-1 infection) collected between 1994 and 1998, against autologous and heterologous primary- and the reference HIV-1 MN isolates. A significantly lower percentage of HIV-1 transmissions was observed after 1996, parallel to a more intense antiretroviral treatment of infected pregnant women. HIV-1 isolation was significantly more frequent from peripheral blood mononuclear cells of mothers of infected children than mothers of uninfected children (P = 0.0065). Neutralization of autologous HIV-1 isolates was comparable for HIV-1 transmitters and nontransmitters' plasma, whereas neutralization of the reference isolate HIV-1 MN was more frequent at high titers for pregnant women who did not transmit HIV to their offspring compared to pregnant women who did. Although neutralization of heterologous primary HIV-1 isolates from HIV transmitters and non transmitters by transmitter plasma occurred with similar frequency, neutralization of isolates from transmitters was much more frequent when heterologous plasma from nontransmitters were used. Macrophage-tropic heterologous HIV-1 isolates were neutralized more frequently at higher titers by plasma from nontransmitters than from transmitters. The results obtained indicate that antiretroviral treatment, lack of success of HIV-1 isolation and high titers of antibodies able to neutralize macrophage-tropic viruses appear to be of importance for protection against HIV-1 vertical transmission for the group of patients studied.

Neutralization titres and vertical HIV-1 transmission.

Replication of the human immunodeficiency virus type 1 (HIV-1) isolate MN in CEM cells was less neutralized by the plasma from the mothers of infected children (MIC) in comparison with the plasma from the mothers of uninfected children (MUC). Significantly higher neutralization titres were observed for the sera from MUCs compared with MICs, and only the sera from MUC showed 100% neutralization of the HIV-1 MN strain. We suggest that a simple neutralization assay as described here could be useful in prognostic analyses.