RESUMO
The gag gene of HIV-1 encodes a single open reading frame of 55 kDa that contains three subdomains: the matrix domain (p17), the capsid domain (p24) and the nucleocapsid domain (p15). The p24 and p17 proteins have a predominant a-helical structure and perform important functions throughout thevirallife-cycle. The determination of gag-specific antibodies is important because declining titers of these antibodies herald clinical deterioration.In this work we present the results obtained on immunoreactiviy of synthetic peptides that mimic immunogenic a-helical regions of p24 and p17. The influence on the immunoreactivity of structural modifications in native sequences, including the addition of non immunogenic side chains: AAAC- and -CAAA on both side of minimal epitopes was evaluated in indirect and competitive enzymeimmunoassays. The conformational characteristcs to the peptides were analysed by circular dichroism and these results were correlated with that obtained in the immunoassays. It was shown that the reactivity of peptides mimicking short a-helical regions of p24 and p17 is improved by adding short non immunogenic chains on both N- and C- terminus. These modifications enhanced the immobilization of the peptides onto the solid support and allowed more accesibility to the minimal epitopes byspecific antibodies, in solution.
El gen gag del VIH-1 codifica una región de 55kDA que contiene tres subdominios: matriz (p17), cápside (p24) y nucleocápside (p15). Las proteínas p24 y p17 tienen una estructura predominante helicoidal y cumplen un rol importante en el ciclo de vida del virus. En este trabajo presentamos los resultados de inmunorreactividad de péptidos sintéticos que imitan regiones helicoidales de p24 y p17. Utilizando enzimoinmunoensayos se evaluó la influencia de modificaciones en las secuencias nativas sobre la capacidad de reconocimiento de anticuerpos específicos en solución y en fase sólida, incluyendo el agregado de cadenas no inmunogénicas en ambos extremos de los epitopes mínimos. La conformación de los péptidos se determinó por dicroísmo circular y los resultados se correlacionaron con los de inmunorreactividad. Se observó que la capacidad de reconocimiento de anticuerpos por péptidos pequeños que imitan estructuras helicoidales de p24 y p17 mejoró con el agregado de cadenas no inmunogénicas en ambos extremos de los epitopes. Estas modificaciones mejoran la inmovilización sobre las superficies sólidas y permiten una mayor accesibilidad de los anticuerpos a los epitopes mínimos en solución.
Assuntos
Humanos , Reações Antígeno-Anticorpo , Ensaio de Imunoadsorção Enzimática/métodos , Produtos do Gene gag/imunologia , Anticorpos Anti-HIV/imunologia , Antígenos HIV/imunologia , /imunologia , HIV-1 , Mimetismo Molecular , Fragmentos de Peptídeos/imunologia , Proteínas Virais/imunologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Dicroísmo Circular , Produtos do Gene gag do Vírus da Imunodeficiência Humana , Produtos do Gene gag/química , Anticorpos Anti-HIV/isolamento & purificação , Antígenos HIV/química , /química , Infecções por HIV/sangue , Infecções por HIV/imunologia , Epitopos Imunodominantes/química , Epitopos Imunodominantes/imunologia , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Fragmentos de Peptídeos/síntese química , Soluções , Proteínas Virais/químicaRESUMO
The gag gene of HIV-1 encodes a single open reading frame of 55 kDa that contains three subdomains: the matrix domain (p17), the capsid domain (p24) and the nucleocapsid domain (p15). The p24 and p17 proteins have a predominant alpha-helical structure and perform important functions throughout the viral life-cycle. The determination of gag-specific antibodies is important because declining titers of these antibodies herald clinical deterioration. In this work we present the results obtained on immunoreactiviy of synthetic peptides that mimic immunogenic alpha-helical regions of p24 and p17. The influence on the immunoreactivity of structural modifications in native sequences, including the addition of non immunogenic side chains: AAAC- and -CAAA on both side of minimal epitopes was evaluated in indirect and competitive enzyme immunoassays. The conformational characteristcs to the peptides were analysed by circular dichroism and these results were correlated with that obtained in the immunoassays. It was shown that the reactivity of peptides mimicking short alpha-helical regions of p24 and p17 is improved by adding short non immunogenic chains on both N- and C-terminus. These modifications enhanced the immobilization of the peptides onto the solid support and allowed more accessibility to the minimal epitopes by specific antibodies, in solution.
Assuntos
Reações Antígeno-Anticorpo , Ensaio de Imunoadsorção Enzimática/métodos , Produtos do Gene gag/imunologia , Anticorpos Anti-HIV/imunologia , Antígenos HIV/imunologia , Proteína do Núcleo p24 do HIV/imunologia , HIV-1/imunologia , Mimetismo Molecular , Fragmentos de Peptídeos/imunologia , Proteínas Virais/imunologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Dicroísmo Circular , Produtos do Gene gag/química , Anticorpos Anti-HIV/isolamento & purificação , Antígenos HIV/química , Proteína do Núcleo p24 do HIV/química , Infecções por HIV/sangue , Infecções por HIV/imunologia , Humanos , Epitopos Imunodominantes/química , Epitopos Imunodominantes/imunologia , Dados de Sequência Molecular , Fragmentos de Peptídeos/síntese química , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Soluções , Proteínas Virais/química , Produtos do Gene gag do Vírus da Imunodeficiência HumanaRESUMO
The gag gene of HIV-1 encodes a single open reading frame of 55 kDa that contains three subdomains: the matrix domain (p17), the capsid domain (p24) and the nucleocapsid domain (p15). The p24 and p17 proteins have a predominant alpha-helical structure and perform important functions throughout the viral life-cycle. The determination of gag-specific antibodies is important because declining titers of these antibodies herald clinical deterioration. In this work we present the results obtained on immunoreactiviy of synthetic peptides that mimic immunogenic alpha-helical regions of p24 and p17. The influence on the immunoreactivity of structural modifications in native sequences, including the addition of non immunogenic side chains: AAAC- and -CAAA on both side of minimal epitopes was evaluated in indirect and competitive enzyme immunoassays. The conformational characteristcs to the peptides were analysed by circular dichroism and these results were correlated with that obtained in the immunoassays. It was shown that the reactivity of peptides mimicking short alpha-helical regions of p24 and p17 is improved by adding short non immunogenic chains on both N- and C-terminus. These modifications enhanced the immobilization of the peptides onto the solid support and allowed more accessibility to the minimal epitopes by specific antibodies, in solution.
RESUMO
The serologic diagnosis of human immunodeficiency virus (HIV) infection is currently done by detecting the presence of antibodies against the different antigenic viral proteins through immunoassays and later confirmation by Western blot. Several types of antigens can be used in immunoassays, but recombinant proteins and synthetic peptides are the most frequently used. In this paper, peptides mimicking antigenic regions from p24 (region 196-224), gp41 (region 600-614) and gp120 (region 303-338, Loop V3) proteins of HIV-1 have been used as antigens in an enzyme-linked immunosorbent assay and their reactivity was screened against a panel of positive and negative sera. Six antigenic mixtures containing different amounts of each peptide were prepared, and the one consisting of 1 microgram of gp41-15, 0.5 microgram of p24-1 and 0.5 microgram of gp120-1 per well has shown the best performance to differentiate positive and negative serum samples, with sensitivity and specificity values of 99.18% and 100%, respectively. Considering the potential utilization of this system for screening of HIV infection, it would be relevant to evaluate the additional incorporation of sequences derived from Argentine local circulating viral variants to improve the diagnostic sensitivity of the assay, allowing the development of an ELISA based on specific viral sequences.
Assuntos
Sorodiagnóstico da AIDS , Anticorpos Anti-HIV/sangue , Antígenos HIV/imunologia , Fragmentos de Peptídeos/imunologia , Sequência de Aminoácidos , Western Blotting , Ensaio de Imunoadsorção Enzimática , Antígenos HIV/química , Proteína do Núcleo p24 do HIV/química , Proteína do Núcleo p24 do HIV/imunologia , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/imunologia , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/imunologia , Dados de Sequência Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Sensibilidade e EspecificidadeRESUMO
The serologic diagnosis of human immunodeficiency virus (HIV) infection is currently done by detecting the presence of antibodies against the different antigenic viral proteins through immunoassays and later confirmation by Western blot. Several types of antigens can be used in immunoassays, but recombinant proteins and synthetic peptides are the most frequently used. In this paper, peptides mimicking antigenic regions from p24 (region 196-224), gp41 (region 600-614) and gp120 (region 303-338, Loop V3) proteins of HIV-1 have been used as antigens in an enzyme-linked immunosorbent assay and their reactivity was screened against a panel of positive and negative sera. Six antigenic mixtures containing different amounts of each peptide were prepared, and the one consisting of 1 microgram of gp41-15, 0.5 microgram of p24-1 and 0.5 microgram of gp120-1 per well has shown the best performance to differentiate positive and negative serum samples, with sensitivity and specificity values of 99.18
and 100
, respectively. Considering the potential utilization of this system for screening of HIV infection, it would be relevant to evaluate the additional incorporation of sequences derived from Argentine local circulating viral variants to improve the diagnostic sensitivity of the assay, allowing the development of an ELISA based on specific viral sequences.
RESUMO
The serologic diagnosis of human immunodeficiency virus (HIV) infection is currently done by detecting the presence of antibodies against the different antigenic viral proteins through immunoassays and later confirmation by Western blot. Several types of antigens can be used in immunoassays, but recombinant proteins and synthetic peptides are the most frequently used. In this paper, peptides mimicking antigenic regions from p24 (region 196-224), gp41 (region 600-614) and gp120 (region 303-338, Loop V3) proteins of HIV-1 have been used as antigens in an enzyme-linked immunosorbent assay and their reactivity was screened against a panel of positive and negative sera. Six antigenic mixtures containing different amounts of each peptide were prepared, and the one consisting of 1 microgram of gp41-15, 0.5 microgram of p24-1 and 0.5 microgram of gp120-1 per well has shown the best performance to differentiate positive and negative serum samples, with sensitivity and specificity values of 99.18
, respectively. Considering the potential utilization of this system for screening of HIV infection, it would be relevant to evaluate the additional incorporation of sequences derived from Argentine local circulating viral variants to improve the diagnostic sensitivity of the assay, allowing the development of an ELISA based on specific viral sequences.
