Avaliação da imunogenicidade de diferentes formas alélicas da proteí­na recombinante PvAMA-1expressa em Pichia pastoris: impacto da diversidade antigênica / Evaluation of the immunogenicity of different allelic forms of PvAMA-1 protein expressed in Pichia pastoris: impact of antigenic diversity

A malária é um problema de saúde pública no Brasil e no mundo. Em 2016, o número de casos estimado pela Organização Mundial de Saúde foi de 216 milhões. Plasmodium falciparum é a espécie mais prevalente e responsável pelo maior número de mortes no mundo, sobretudo no continente africano. Por outro lado, o Plasmodium vivax é conhecido por sua ampla distribuição geográfica, sendo a espécie que predomina nas Américas, incluindo o Brasil. Nos últimos 20 anos, nosso grupo tem gerado e caracterizado diversas proteínas recombinantes baseadas em antígenos imunodominantes de P. vivax que podem servir como base para o desenvolvimento de uma vacina contra malária. Entre os antígenos de merozoítas, uma das principais proteínas em estudo pelo nosso grupo é o Antígeno 1 de Membrana Apical de P. vivax (PvAMA-1), caracterizado previamente como altamente imunogênico em infecções naturais e em camundongos imunizados, na presença de diferentes adjuvantes. O objetivo do presente estudo foi investigar o efeito da diversidade antigênica dessa proteína no reconhecimento por anticorpos específicos e na indução de imunidade contra o parasita. Para isso, foram geradas seis novas proteínas representando diferentes alelos descritos na natureza: PvAMA-1-Belem, PvAMA-1-Sal-I, PvAMA-1-Chesson-I, PvAMA-1-SK0814-apical, PvAMA-1-Indonesia-XIX e PvAMA-1-PNG_62_MU. As proteínas recombinantes foram expressas em leveduras Pichia pastoris e purificadas em duas etapas cromatográficas. Em seguida, as imunizações em camundongos C57BL/6 foram realizadas com as proteínas administradas de forma isolada, ou em combinação, na presença do adjuvante agonista de TLR3 (Poly I:C). Por ELISA, observamos que todas as formulações foram capazes de induzir anticorpos IgG contra as proteínas homólogas e heterólogas, o que sugere que a diversidade antigênica entre as formas alélicas não compromete o reconhecimento. Os dados gerados no presente trabalho sugerem que uma formulação contendo mistura de diferentes alelos representando a proteína AMA-1 pode ser explorada para o desenvolvimento de uma vacina de ampla cobertura contra o P. vivax

Malaria is a public health problem in Brazil and throughout the world. In 2016, the World Health Organization estimated there were 216 million cases of malaria. Plasmodium falciparum is the most prevalent species and is responsible for the largest number of deaths, especially in the African continent. However, Plasmodium vivax is known for its wide geographic distribution, being the species that prevails in the Americas, including Brazil. In the last 20 years, our group has generated and characterized several recombinant proteins based on immunodominant antigens of P. vivax that can serve as a basis for the development of a malaria vaccine. Among the merozoite antigens, one of the main proteins studied by our group is P. vivax apical membrane antigen-1 (PvAMA-1), previously characterized as highly immunogenic in natural infections and immunized mice, in the presence of different adjuvants. The objective of this study was to investigate the effect of antigenic diversity of this protein in the recognition of specific antibodies and the induction of immunity against the parasite. For this, six new proteins were generated representing different alleles described in nature: PvAMA-1-Belem, PvAMA-1-Sal-i, PvAMA-1-Chesson-i, PvAMA-1-SK0814-apical, PvAMA-1-Indonesia-XIX, and PvAMA-1-PNG_62_MU. Recombinant proteins were expressed in Pichia pastoris yeast and purified by two chromatographic stages. Then, C57BL/6 mice were immunized with these proteins administered in isolation or in combination, in the presence of the TLR3 agonist adjuvant, Poly I:C. Using an enzyme-linked immunosorbent assay, we observed that all formulations induced IgG antibodies against homologous and heterologous proteins. This indicates that antigenic diversity between allele forms does not compromise recognition. This finding suggests that a formulation containing a mixture of different alleles representing the PvAMA-1 protein can be exploited for developing of a wide coverage vaccine against P. vivax

Phenotypic and antigenic variation of Mycoplasma Gallisepticum vaccine strains

Cepas vacinais de Mycoplasma gallisepticum, F e TS-11, foram examinadas quanto às suas variacões fenotípicas e antigênicas, por SDS-PAGE e através de dois métodos sorológicos (inibicão da hemaglutinacão e imunoeletroforese). A análise densitométrica das bandas obtidas nos géis de poliacrilamida mostrou pequena variabilidade fenotípica entre as amostras, sendo a banda peptidica de 75 kDa detectada apenas na amostra vacinal F. Anticorpos policlonais produzidos em galinha foram utilizados nos ensaios sorológicos para estudar a variabilidade antigênica das amostras. Houve elevada reatividade cruzada entre as amostras e os anticorpos homólogos e heterólogos. A característica mais evidente foi a resposta específica da banda peptídica de 75 kDa da vacina F ao anticorpo homólogo.

Immunity comparison of both epidemic influenza virus type-A strains circulating in Brazil, in 1991 and 1995

Influenza is a viral disease characterized by a high level of virus dissemination among susceptible hosts - fowls, mammalians and humans - in epidemic periods. Antigenic variations in the viral hemagglutinin (HA) are epidemiologically important. A single radial hemolysis (SRH) method was carried to compare type-A (H3N2) viral strains isolated in the city of São Paulo, State of São Paulo, Brazil, in different periods of time (1991 and 1995). Stored sera sample collected in 1993 from 60 individuals, before and two weeks after their vaccination against A/SP/1/91 influenza virus, were evaluated by this method. Antibody levels evaluated by SRH showed no significant differences between population vaccinated with...

Antigenic relatedness of selected flaviviruses: study with homologous and heterologous immune mouse ascitic fluids

A relacao antigenica de 9 Flavivirus, febre amarela (YF), Wesselsbron (WSL), Uganda S (UGS), Potiskum (POT), West Nile (WN), Banzi (BAN), Zika (ZK), Dengue tipo 1 (DEN-1) e Dengue tipo 2 (DEN-2), foi avaliada por reacao de inibicao da hemaglutinacao cruzada (cross-HI) e reacao de fixacao do complemento cruzada (Cross-CF) entre cada um dos virus e seu fluido ascitico homologo em camundongos. Medias de titulos foram calculadas usando os titulos heterologos e homologos. Reacoes cruzadas CF revelaram maiores variacoes antigenicas entre virus do que reacoes cruzadas HI. Nao houve variacao antigenica significativa entre virus WSL, POT e YF usando cada um dos metodos. Todavia, diferencas definidas da antigenicidade foram observadas entre eles e os virus UGS, BAN e ZK. Nao existiram diferencas significativas entre UGS, BANe ZK ou entre DEN-1 e DEN-2. A relacao sorologica entre Flavivirus e importante para se estabelecer o diagnostico e a epidemiologia destas infeccoes na Africa

Distinct antigenic subtypes of human beta-interferon can be distinguished by neutralization

Different molecular configurations of human beta interferon were titrated with the standard reference antiserum of the National Institutes of Health (NIH) which had been prepared with natural beta fibroblast interferon in order to determine to what extent differences in these configurations would influence the neutralization of the antiviral action of interferon. Neutralization tests were carried out in Vero cells by diluting both interferon and antiserum. Encephalomyocarditis virus was employed as challenge virus. The neutralization titer was considered to have been reached when the effect of eight units of interferon was reduced to one. Two natural beta interferons prepared from fibroblasts and from amniotic membranes gave similar high titers. However, titers were reduced five-fold with recombinant interferons expressed in Escherichia coli, which do not contain carbohydrate, one with the natural sequence and a mutant with a single amino acid substitution (cysteine for serine). The NIH antiserum did not neutralize the effect of a protein fraction from amniotic membranes antigenically different from the human alpha, beta, or gamma interferons but having the biological activity of interferon. We conclude that the carbohydrate moieties of human beta interferons are essential for their recognition by the NIH antiserum and that antibodies specific for human recombinant beta interferon, which does not contain carbohydrate, are needed.

Antigenic relationships of isolates of Bordetella bronchiseptica from swine with atrophic rhinitis

Estudaram-se as relaçöes antigênicas de vinte amostras de Bordetella bronchiseptica recuperadas de suínos com rinite atrófica mediante um teste de soroaglutinaçäo quantitativo. Os títulos dos soros produzidos em coelhos contra cada um dos isolamentos foram determinados usando antígenos homólogos e heterólogos. Doze soros que apresentaram reatividade cruzada foram absorvidas e seus títulos determinados com antígenos heterólogos. Estimaram-se as relaçöes antigênicas entre os isolamentos calculando os índices de reatividade cruzada. Dois isolamentos apresentaram alta reatividade cruzada com outros 16, entretanto só um näo reagiu com o painel utilizado. Concluiu-se que B. bronchiseptica recuperada de suínos é antigenicamente heterogênea

Immunoblot analysis to demonstrate antigenic variability of clinical isolated. Pseudomonas pseudomallei.

Pseudomonas pseudomallei (Ps.ps.) is the causative organism of melioidosis, and is widely distributed in Southeast Asia and Northern Australia. Clinical manifestations range from subclinical infection to fulminant septicemia. To demonstrate the antigenic variability of Ps.ps., 62 clinical isolates from 31 blood, 13 sputum, 9 pus, 3 urine and 6 body fluid culture specimens were studied by SDS-PAGE and immunoblotting. In SDS-PAGE, there were approximately 20 antigenic components with molecular weights ranging from 14 to 66 kilodaltons (KD) which suggested that there was antigenic variability among these 62 clinical isolates of Ps.ps. Attempts to correlate immunoblot profiles with clinical illness or sources of specimens were not successful but 6 common antigens were identified with molecular weight of 17.5, 21, 33, 34, 40 and 45 KD, respectively. Among these antigens, the 45 KD component was recognised by all patients' sera. Thus, the 45 KD protein antigen may be useful for the future approach in immunodiagnosis of melioidosis.

Estudo do comportamento de variantes antigênicas do virus da raiva, isoladas no Brasil, em camundongos / Study on antigenic variants of rabies virus, isolated in Brazil, in mice

A study comparing four antigenic variants of reabies virus, two of them grouped as a dog strain (C/PI and C/rn), and the other two, as a bovine strain (B/RN) and B/AL), isolated in the Northeastern of Brazil, was carried out. All the strains were antigenically identified by the monoclonal antibodies technique. These variants were inoculated into groups of 37 days-old mice, intracerebrally, with 0.03 ml of the viral dilution containing 50 LD50 of virus. The animals were observed for 30 days, considering the clinical observation periods (incubation and illness). The results showed behavioral differences among the four variants, independent of the nucleocapsid antigenic profile