Introduction: COVID-19 is an infectious disease caused by SARS-CoV-2, discovered in December 2019. Since then, this highly infectious coronavirus caused a world-wide emergency, with just over 1 year infected more than 100 million of people around the world with almost 2,5 million of deaths Until February 2021, Brazil is third in ranking of infected and second in number of deaths, with more than 9 million of infected and 240 thousand of deaths, respectively. Even with the start of vaccination, the development of therapeutic approaches aimed at reversing severe conditions in patients affected by COVID-19, besides new inputs for diagnosis. Therefore, neutralizing monoclonal antibodies have been emerging as an important alternative to treat cases, with the approval of some of them for emergency use.Objective: To select fragments of human antibodies from patients affected by SARS-COV-2, using the phage display technique.Methodology: Twenty-three individuals with confirmed SARS-CoV-2 were recruited for this study (CEP-CAAE 31368620.0.0000.5262). In order to build a human immune library of antibody fragments, peripheral blood mononuclear cells from donors were isolated by Ficoll gradient. From an RNA template, it was produced a pool of cDNA. Heavy and light chains regions were synthesized by PCR to construct scFv genes by overlaping PCR both with specific primers. The scFvlibrary genes were cloned into pCOMB3XSS vector and transformed into Escherichia coli XL1-blue. To recover scFv the culture was infected with helper phage VCSM13. To select the target, a recombinant protein was immobilized into 96 wells plate and we have obtained a pool of specific phage-scFvs, confirmed by ELISA. Furthermore, single cells were cultivated and the expression of each scFv was analyzed by dot blot and sequenced by SANGER to identify major prevalence of sequences and the most promising clone was assessed by ELISA against SARS-COV-2 antigens. Results:The PCR produced 400 bp amplicons for VH and 350 bp for VL and the overlap PCR generated a 800 bp product for scFvs and after four rounds of biopanning was performed and selected scFv against the target. The evaluation of specificity of scFvs was confirmed by high ODs obtained in ELISA tests using phage-scFv and purified scFv. After the evaluation by dot blot of single cells product, the better clone was sequenced and analysed the directly neutralizing activity by PRNT against virus inactivated particle, but was not show effective. However, the PRNT will be repeated using an approach to obtain a complete antibody from this clone. Conclusion:It was possible to obtain, in record time, an immune library for phage display selection of human fragments. Our panning methodology was successful in selecting specific scFvs fragments against our target, as well as, the whole virus, and could possibly become a tool for diagnosis and treatment for COVID-19

SARS-CoV-2 virus jumped the barrier between species and started to infect humans in Wuhan province, China. The disease has spread quickly and has caused thousands of deaths since the declaration of the Covid-19 pandemic by the WHO. In order to avoid the spread of the disease, mass diagnosis is essential as a strategy for public health, allowing immediate quarantine of infected people. Added to this, the discovery of an effective treatment brings hope to those currently infected. Herein we have developed antibodies against Spike and Nucleocapsid proteins of SARS-CoV-2 which could help the development of antigen capture point-of-care tests and treatment of this illness.

Introduction: Vaccines are the most effective strategy to prevent infectious diseases. COVID-19 is caused by the Sars-CoV-2 virus and it was declared a pandemic disease in March of 2020. Since then, research groups all over the world are working to develop and produce a safe and effective vaccine to avoid deaths and the health system collapse. Now the world is facing the limited amount of vaccine supply, so it is important to have a vaccine produced in Brazil in an attempt to supply the country needs and guarantee future demand following the pandemic period. The Technological Development Division of Bio-Manguinhos/Fiocruz is working on two different strategies to face this problem: a synthetic vaccine based on antigenic peptide epitope selection and a subunit vaccine based on recombinant protein fragments. Objective:The initial objective of this work is to select the best formulations for a COVID-19 vaccine.Methodology: For the synthetic vaccine strategy, we initially performed an in silico epitope prediction for the Spike (S) and Nucleoprotein (N) proteins of Sars-Cov-2, combining bioinformatic methodologies. The epitopes selected have been synthesized by a specialized service, analysed by ELISA for their recognition of patient antibodies or T cells, respectively, and formulated in nanoparticles in the presence of adjuvants. For the subunit vaccine, the genes cloned in pET28a were obtained by a custom gene synthesis service, and the proteins were expressed in prokaryotic system and purified. The recombinant proteins were analysed by western blot and then formulated with adjuvants and inoculated in Balb/C mouse. The immunogenicity was determined by ELISA, the T cell response was determined by ELISPOT and plaque reduction neutralization test (PRNT) was used to quantify the presence of neutralizing antibodies. Results:In its first analysis the epitopes were recognized by confirmed COVID-19 patient´s monoclonal antibodies in ELISA assay and the recombinant proteins antigenicity and identity was demonstrated. The preclinical assay results showed promising candidates and formulations. Histopathology data demonstrated that the vaccine formulations were safe for the animals in the dose utilised, whereas subsequent ELISA and ELISPOT assays demonstrated the immunogenicity of our formulations and the ability to elicit a cellular immune response, making them promising candidates for continuous development. Conclusion:Our initial results show that we have promising candidates for a vaccine development and additional tests have been planned to confirm our preliminary results and advance the product development.

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Cristina da Costa Neves 1 . 1Fiocruz/Bio-Manguinhos; 2Fiocruz/CDTS. Introduction:Cancer is a group of diseases that involve abnormal cell growth, with potential to invade and spread to other parts of the body forming secondary tumors, called metastasis. Neoplasms are the main cause of death in the world, mainly due to the metastasis. In Brazil, there are more than 600.000 new cases in 2018, and breast cancer is the most frequent among Brazilian women. The most promising therapies, at the time, to combat this disease are immunotherapies, which deal with the manipulation of the immune system to better respond against the tumor and eliminate it. Some metastatic cancers, such as breast cancer, are poorly immunogenic and therefore difficult to eradicate. However, specific mutations in tumor proteins can be considered as targets for cancer immunotherapy, since it could be recognized as neoantigens by host T cells, allowing the development of therapeutic vaccines.Objective: The aim of this work is to develop an in silico strategy to identify tumor neoantigens in invasive and noninvasive models of mouse breast tumors, in order to obtain a proof of principle for the use of this methodology in the identification of human epitopes.Methodology: Metastatic (4T1) and non-metastatic (67NR and 168 FARN) mouse lineages were used as models for this study. First, data of RNA seq of these three lineages were selected in one study through the GEO database (NCBI). Using these data, we have selected all genes overexpressed in 4T1 lineage in comparison to the non-metastatic lineages. In order to have data from mutated proteins in 4T1 tumor, we have used the study "Mutated tumor alleles are expressed according to their DNA frequency", which identified mutated genes in these cells. So, we combined the data of overexpression with mutations and, based on the proteins selected, we have performed in silico prediction of T cells epitopes using NetPanMHC and IEDB softwares.Results: For prediction at IEDB site, we obtained 14 possible epitopes generated from 11 over expressed genes when 4T1 is compared to 67NR lineage and 9 epitopes generated from 8 genes in the comparison between 4T1 and 168FARN. From the NetPanMHC site, only MHC class 1 predictions were made due to the restriction of the site, resulting in 7 epitopes from 5 genes comparing 4T1 and 67NR lineages. As a final result, we have identified 22 possible epitopes in total, generated from 18 genes, 13 of which were MHC Class 2 and 9 of MHC Class 1. Conclusion:In this work, it was possible to develop a usefull in silico tool to identify neoantigens from tumors. To identify these neoantigens is important to make them a possible target of the immune system through a much more efficient treatment, once they could be tested in formulations of therapeutic vaccines.