RESUMO
Two chimeric synthetic peptides incorporating immunodominant sequences from HTLV-II virus were synthesized. Monomeric peptides P2 and P3 represent sequences from transmembrane protein (gp21) and envelope protein (gp46) of the virus. The peptide P2 is a gp21 (370-396) sequence and the peptide P3 is a gp46 (178-205) sequence. Those peptides were arranged in a way that permits one to obtain different combinations of chimeric peptides (P2-GG-P3 and P3-GG-P2), separated by two glycine residues as spacer arms. The antigenic activity of these peptides was evaluated by UltramicroEnzyme-linked immunosorbent assay (UMELISA) by using panels anti-HTLV-II-positive sera (n = 11), anti-HTLV-I/II-positive sera (n = 2), HTLV-positive (untypeable) serum samples (n = 2), and anti-HTLV-I-positive sera (n = 22), while specificity was evaluated with anti-HIV-positive samples (n = 19) and samples from healthy blood donors (n = 30). The efficacy of the chimeric peptides in solid-phase immunoassays was compared with the monomeric peptides and a mixture of the monomeric peptides. Higher sensitivity was observed for chimeric peptide Q5 assay. Those results may be related to a higher peptide adsorption capacity to the solid surface and for epitope accessibility to the antibodies. This chimeric peptide would be very useful for HTLV-II diagnostic.
Assuntos
Produtos do Gene env/imunologia , Anticorpos Anti-HTLV-II/sangue , Antígenos HTLV-II/química , Vírus Linfotrópico T Tipo 2 Humano/imunologia , Proteínas Oncogênicas de Retroviridae/imunologia , Sequência de Aminoácidos , Ensaio de Imunoadsorção Enzimática , Produtos do Gene env/química , Produtos do Gene env/genética , Antígenos HTLV-II/genética , Infecções por HTLV-II/diagnóstico , Infecções por HTLV-II/imunologia , Vírus Linfotrópico T Tipo 2 Humano/genética , Humanos , Epitopos Imunodominantes/química , Epitopos Imunodominantes/genética , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Oncogênicas de Retroviridae/química , Proteínas Oncogênicas de Retroviridae/genética , Produtos do Gene env do Vírus da Imunodeficiência HumanaRESUMO
A study by Hall et al. (J Virol 1992;66:2456-2463; Ref. 11) has suggested the existence of two closely related molecular subtypes of HTLV-II, which were tentatively designated HTLV-IIa and HTLV-IIb. To confirm this nucleotide sequence analysis of 986 bp of the env gene region encoding the entire surface glycoprotein, gp46, and the amino terminus of the transmembrane glycoprotein, gp21, of 10 HTLV-II isolates was carried out. The results clearly established the existence of two subtypes and demonstrated a 4.3% divergence in sequence in this region. Analysis of other gene regions of the provirus, including the pol (1544 bp), gag (448 bp), and the entire LTR (743 bp) of two representative isolates of each subtype, showed a sequence divergence of 3.8 to 5.7%, with greatest divergence occurring in the LTR. In addition to single nucleotide changes, the gag regions encoding the structural protein, p19, of the HTLV-IIb isolates were also found to have a 66-bp deletion that would be expected to result in a p19 protein having a 22-amino acid deletion in the carboxy-terminus region. Attempts to exploit this to differentiate the two subtypes serologically were unsuccessful in that recombinant p19 proteins of both subtypes were found to be antigenically cross-reactive. The finding of two molecular subtypes of HTLV-II may have important implications for a better understanding of the biological and pathogenic properties of the virus, and will be useful in characterizing the viruses present in endemic foci in American Indian populations.
Assuntos
Vírus Linfotrópico T Tipo 2 Humano/genética , Sequência de Aminoácidos , Sequência de Bases , DNA Viral , Produtos do Gene env/genética , Produtos do Gene gag/genética , Genes env , Genes gag , Genes pol , Antígenos HTLV-II/genética , Humanos , Dados de Sequência Molecular , Sequências Repetitivas de Ácido Nucleico , Proteínas Oncogênicas de Retroviridae/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana , Produtos do Gene gag do Vírus da Imunodeficiência HumanaRESUMO
Monoclonal antibodies (MAbs) raised against human T-cell lymphotropic virus type I (HTLV-I) recognized five distinct antigenic domains of viral env gene-encoded proteins. By using recombinant env proteins and synthetic peptides as mapping antigens, it was determined that the most immunogenic region represented a central portion of the retroviral surface protein (domain 2; amino acids 165 to 191). However, only a single MAb was able to react strongly with native viral proteins. This antibody (clone 6C2) was directed to an epitope within domain 4 (amino acids 210 to 306) of the retroviral env gene and reacted with envelope proteins in both HTLV-I and HTLV-II, as determined by immunoprecipitation, solid-phase binding, and immunoblotting. No reactivity against envelope components of other human retroviruses, including human immunodeficiency virus types 1 and 2, was present. Flow cytometry data demonstrated that MAb 6C2 reacted with cell lines chronically infected with HTLV-I or HTLV-II and also with surface antigens expressed on fresh adult T-cell leukemia cells, following up-regulation with interleukin-2. By a chemiluminescence immunoassay procedure, picogram amounts of viral surface protein could be detected in the unconcentrated supernatants of HTLV-infected cell lines and in diagnostic cultures. Levels of env and gag proteins released by cells into culture supernatants were not directly related to percent expression of cell surface viral-coat proteins. Further, the molar ratio of p19 to gp46 in conditioned media varied from strain to strain, possibly reflecting differences in viral assembly or packaging mechanisms. MAb 6C2 will be of value in characterizing the biochemical and immunological behavior of retroviral env gene proteins and in studying the interaction of HTLV-I and HTLV-II with their receptors.
Assuntos
Antígenos de Deltaretrovirus/análise , Produtos do Gene env/análise , Produtos do Gene env/imunologia , Imunoensaio/métodos , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Antígenos de Deltaretrovirus/genética , Epitopos/genética , Produtos do Gene env/genética , Genes env , Antígenos HTLV-I/análise , Antígenos HTLV-I/genética , Antígenos HTLV-II/análise , Antígenos HTLV-II/genética , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , Medições Luminescentes , Camundongos , Dados de Sequência Molecular , Peptídeos/genética , Peptídeos/imunologiaRESUMO
55 HTLV-I (human T-cell lymphotropic virus) and 45 HTLV-II carriers, confirmed by HTLV-type specific polymerase chain reaction (PCR), were distinguished by western blot assays with recombinant HTLV I or II envelope glycoproteins. Recombinant protein (RP) B1 contains aminoacids 166-201 from HTLV-I exterior glycoprotein gp46 and was reactive with HTLV-I samples only. RP-IIB, which contains aminoacids 96-235 from HTLV-II exterior glycoprotein gp52, was reactive with all HTLV-II samples. 39 patients (86.6%) had high reactivity by densitometry. Of 55 HTLV-I samples, 35 (65.5%) had antibody reactivity to RP-IIB, but only 1 (1.8%) had high reactivity by densitometry. RP B1 and IIB western blot assays may replace the PCR test in diagnosis of HTLV infection.
