Assessment of SARS-CoV-2 immune escape by analyzing the immune response of patients hospitalized with moderate or severe COVID-19 during the wave of variant infections Omicron

• Grant number: 23/07287-5
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Effective date (Start): August 01, 2023
• Effective date (End): July 31, 2024
• Field of knowledge: Biological Sciences - Microbiology - Applied Microbiology
• Principal Investigator: Luiz Mário Ramos Janini
• Grantee: Marcia Duarte Barbosa da Silva
• Host Institution: Escola Paulista de Medicina (EPM). Universidade Federal de São Paulo (UNIFESP). Campus São Paulo. São Paulo , SP, Brazil
• Associated research grant: 20/08943-5 - Investigation of the hosts' induced elements in response to the immunisation with ChAdOx1 nCOV-19 vaccine in a Phase III Clinical Trial, AP.TEM

Abstract

This project will be developed jointly with the Biobank of the Federal University of São Paulo - UNIFESP and will have 100 samples of serum, plasma and leukocyte concentrate from of patients who were hospitalized at the Hospital Universitário São Paulo (HUHSP) with COVID-19 moderate or severe during the wave of infection with the Omicron variant of SARS-CoV-2 (and subvariants) and are available at Biobanco UNIFESP. In these samples the cellular immune response will be analyzed and humoral activity, in addition to the profile of cytokines in the serum of patients during infection. These data will be correlated with the vaccination profiles of each of the patients, comorbidities and vaccination status in the time of admission due to COVID-19. With these results, it is expected to evaluate the levels of protection and possible immune escape from circulating SARS-CoV-2 variants and subvariants currently facing the first generation vaccines used in the immunization of the population. With that, expect find elements that allow the improvement of future therapeutic and prophylactic mechanisms, such as the development of more effective vaccines that prevent the patient from being hospitalized in as a result of COVID-19. The quantitative real-time PCR technique is considered the gold standard for monitoring gene expression due to its high sensitivity and specificity and rapid quantification of expression genetics. In order to obtain reliable data, the expression levels of the genes of interest must be normalized using two or more reference genes. Ideally, the reference genes must show stable expression and minimal variability between samples and under different conditions experimental. Therefore, prior to a gene expression quantification study, it is recommended to carrying out an experiment to determine and validate the most stable reference genes for each sample, population and disease studied. Recent studies suggest that SARS-CoV-2 infection represses not only the innate antiviral response, but also alters the epigenetic regulation of the cell host and the expression of reference genes widely cited in the scientific literature (Kee et al., 2022; Kumar et al., 2022). Furthermore, it is important to mention that SARS-CoV-2 manifests a spectrum of illnesses ranging from asymptomatic to moderate and moderate to severe, each with different clinical characteristics, which may invalidate gene expression studies in positive samples for SARS-CoV-2. Given the above problem, this project aims to: 1. Evaluate the specific cellular immune response to peptides from the Spike (S) protein of SARS-CoV-2 in PBMCs from leukocyte concentrates of hospitalized patients with COVID-19 moderate or severe; 2. Evaluate the profile of cytokines in the plasma of patients hospitalized with moderate or severe COVID-19 serious; 3. Assess the ability to neutralize hospitalized patients with moderate or severe COVID-19 against the different variants of SARS-CoV-2. 4. Correlate the data obtained in the steps above with possible comorbidities and status vaccination of patients at the time of hospitalization due to COVID-19 in order to assess the immune escape from currently circulating SARS-CoV-2 variants (subvariants). 5. Evaluate the expression stability of the main reference genes cited in the literature - ACTB, B2M, GAPDH, 18SRNAr and RPL13A - and establish which of them are the most stable to be used in the normalization of the expression of target genes from positive samples for SARSCoV- two. (AU)