Salivary, serological, and cellular immune response to the CoronaVac vaccine in health care workers with or without previous COVID-19.

We investigated the anti-SARS-CoV-2 post-vaccine response through serum and salivary antibodies, serum antibody neutralizing activity and cellular immune response in samples from health care workers who were immunized with two doses of an inactivated virus-based vaccine (CoronaVac) who had or did not have COVID-19 previously. IgA and IgG antibodies directed at the spike protein were analysed in samples of saliva and/or serum by ELISA and/or chemiluminescence assays; the neutralizing activity of serum antibodies against reference strain B, Gamma and Delta SARS-CoV-2 variants were evaluated using a virus neutralization test and SARS-CoV-2 reactive interferon-gamma T-cell were analysed by flow cytometry. CoronaVac was able to induce serum and salivary IgG anti-spike antibodies and IFN-γ producing T cells in most individuals who had recovered from COVID-19 and/or were vaccinated. Virus neutralizing activity was observed against the ancestral strain, with a reduced response against the variants. Vaccinated individuals who had previous COVID-19 presented higher responses than vaccinated individuals for all variables analysed. Our study provides evidence that the CoronaVac vaccine was able to induce the production of specific serum and saliva antibodies, serum virus neutralizing activity and cellular immune response, which were increased in previously COVID-19-infected individuals compared to uninfected individuals.

Immunodominant antibody responses directed to SARS-CoV-2 hotspot mutation sites and risk of immune escape.

Introduction: Considering the likely need for the development of novel effective vaccines adapted to emerging relevant CoV-2 variants, the increasing knowledge of epitope recognition profile among convalescents and afterwards vaccinated with identification of immunodominant regions may provide important information. Methods: We used an RBD peptide microarray to identify IgG and IgA binding regions in serum of 71 COVID-19 convalescents and 18 vaccinated individuals. Results: We found a set of immunodominant RBD antibody epitopes, each recognized by more than 30% of the tested cohort, that differ among the two different groups and are within conserved regions among betacoronavirus. Of those, only one peptide, P44 (S415-429), recognized by 68% of convalescents, presented IgG and IgA antibody reactivity that positively correlated with nAb titers, suggesting that this is a relevant RBD region and a potential target of IgG/IgA neutralizing activity. Discussion: This peptide is localized within the area of contact with ACE-2 and harbors the mutation hotspot site K417 present in gamma (K417T), beta (K417N), and omicron (K417N) variants of concern. The epitope profile of vaccinated individuals differed from convalescents, with a more diverse repertoire of immunodominant peptides, recognized by more than 30% of the cohort. Noteworthy, immunodominant regions of recognition by vaccinated coincide with mutation sites at Omicron BA.1, an important variant emerging after massive vaccination. Together, our data show that immune pressure induced by dominant antibody responses may favor hotspot mutation sites and the selection of variants capable of evading humoral response.

Clonagem, expressão recombinante e validação por microarray de proteínas alimentares potencialmente alergênicas / Cloning, recombinant expression and validation by microarray of potentially allergenic food proteins

A maioria das respostas alérgicas a alimentos é mediada por IgE, que pode ser detectada para fins de diagnóstico da alergia alimentar. No entanto, para isso é necessário que alérgenos purificados estejam disponíveis para a elaboração dos diferentes formatos de ensaio, inclusive por microarray, que se constitui em uma ferramenta bastante útil para análise simultânea, e também para a identificação de reatividade cruzada. A esse respeito, é imprescindível ampliar a plataforma de alérgenos que possam ser empregados para a confecção de microarrays. Atualmente, alguns alimentos que constituem objeto de interesse na clínica em função do número de casos de alergia, e sobre os quais as informações a respeito dos alérgenos são escassas, são: abacaxi, mamão, mandioca e manga. Assim, o objetivo desse trabalho foi clonar, expressar e purificar proteínas potencialmente alergênicas de alimentos de importância regional. Após confirmadas por ensaios imunológicos, essas proteínas foram utilizadas na construção e validação de um microarray através de ensaios com os soros de pacientes alérgicos aos alimentos selecionados. Para atingir esse objetivo, foram selecionadas proteínas potencialmente alergênicas coincidentes, apontadas tanto pela similaridade com espécies taxonomicamente mais próximas, quanto pela técnica 2D Western Blotting acoplada à espectrometria de massas. Dezenove proteínas, sendo 4 de abacaxi, 5 de mamão, 6 de mandioca e 4 de manga, foram expressas em Pichia pastoris, purificadas e impressas em um microarray. Após incubar essas proteínas com os soros dos pacientes alérgicos aos alimentos estudados, 18 proteínas mostraram-se potencialmente alergênicas. Além disso, foi observada reatividade cruzada entre proteínas dos alimentos estudados e também em relação ao látex e outros frutos

The majority of allergic reactions to foods is IgE-mediated, which can be detected for the diagnosis of food allergy. However, purified allergens are necessary to produce different kinds of allergy tests, including microarray, which is a useful tool for simultaneous analysis, as well as for the identification of cross-reactivity. In this respect, it is essential to expand the platform of allergens to include them on microarrays. Nowadays, some foods that are object of interest in the clinical area in Brazil and it is necessary a further evaluation about their potential allergens, since there is a limited information about them, are: pineapple, papaya, cassava and mango. Therefore, the aim of this study was cloning, expressing and purifying potentially allergenic proteins of important Brazilian foods. After confirmed by immunological tests, these proteins were used in microarray production and validation by assays with sera from allergic patients to the selected foods. Achieving this goal, matching potentially allergenic proteins were selected, which were identified by comparison among taxonomically closer species (in silico) and 2D Western Blotting coupled with Mass Spectrometry. Nineteen proteins: 4 from pineapple, 5 from papaya, 6 from cassava and 4 from mango were expressed in Pichia pastoris, purified and printed on a microarray. After incubating those proteins with sera from allergic patients to the selected foods, 18 proteins were detected as potentially allergenic. In addition, cross-reactivity was observed among the proteins from the studied foods, and also regarding to the latex and other fruits

Comparative Proteome Analysis of the Tuberous Roots of Six Cassava (Manihot esculenta) Varieties Reveals Proteins Related to Phenotypic Traits.

Cassava (Manihot esculenta Crantz) is a staple food and an important source of starch, and the attributes of its tuberous root largely depend on the variety. The proteome of cassava has been investigated; however, to date, no study has focused on varieties that reveal the molecular basis of phenotypical characteristics. Therefore, we aimed to compare the proteome of the tuberous roots of six cassava varieties that differed in carbohydrates, carotenoids, and resistance to diseases, among other attributes. Two-dimensional gels showed 146 differential spots between the varieties, and the functional roles of some differential proteins were correlated to phenotypic characteristics of the varieties, such as the amount of carbohydrates or carotenoids and the resistance to biotic or abiotic stresses. The results obtained here highlight elements that might help to direct the improvement of new cultivars of cassava, which is an economically and socially relevant crop worldwide.