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Bivalent intranasal vaccine using influenza virus expressing SARS-CoV-2 protein S (spike): protection mechanisms and lung injury

Grant number: 20/05527-0
Support type:Regular Research Grants
Duration: July 01, 2020 - June 30, 2022
Field of knowledge:Biological Sciences - Immunology - Cellular Immunology
Principal Investigator:Ricardo Tostes Gazzinelli
Grantee:Ricardo Tostes Gazzinelli
Home Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
Assoc. researchers:Alexandre de Magalhães Vieira Machado ; João Santana da Silva ; Luciana Benevides ; Luiz Tadeu Moraes Figueiredo
Associated research grant:16/23618-8 - Immunological mechanisms of resistance and disease in malaria, AP.TEM


Our proposal will consist of the evaluation of the mechanisms of the disease in models of pulmonary comorbidities challenged with SARS-CoV-2 and protection induced by a vaccine using a recombinant influenza virus expressing a segment of the Spike protein. We hypothesis is that the pathology triggered by excessive activation of the innate immune response in the airway mucosa and that with the development of the acquired response (IgA and cytotoxic T lymphocytes) induced by vaccination, the replication of the virus is minimized and the activation of cells of the innate immune system is attenuated. It is known that COVID-19 worsens in individuals with pulmonary comorbidities. However, it is not known whether this process involves a weakening of the immune response and an increase in viral load or just a worsening of the inflammatory reaction and, consequently, an increase in lung damage. As a model of comorbidities, we chose models widely used in our laboratories that trigger different pulmonary inflammatory response processes. Specifically, the models used will be infection models with fungus (Paracoccidioides brasiliensis, Pb), bacteria (Streptococcus pneumoniae, pneumococcus), viral (Influenza) and an asthma model, which induce chronic inflammation of the Th17 type and rich in neutrophils, a model of chronic pro-inflammatory inflammation rich in monocytes, acute pro-inflammatory response rich in monocytes and neutrophils and chronic Th2 response rich in eosinophils, respectively. In these models of comorbidities, different parameters will be evaluated, such as viral load, quality of the inflammatory infiltrate, activation of different pathways of the innate immune system, production of cytokines and lung injury. These studies will evaluate the toll-like receptors (TLR), inflammasomes and type I IFN pathways, using MyD88, ASC and type I IFN (IFNAR) deficient mice, respectively. Next, we will assess the ability of the PR8 influenza virus that is defective for multiplication because it does not express neuraminidase. In this virus, the extracellular region of neuroamindase is replaced by the RBD domain of the SARS-CoV-2 Spike protein. Studies carried out by our group have already demonstrated the ability of this neuraminidase-deficient virus to provide 100% protection for challenged mice. Therefore, we believe that we have a bivalent vaccine against influenza and SARS-CoV-2. Besides, in previous studies, we built an influenza virus that expresses the PspA protein of pneumococcus and protects against co-infection with influenza and S. Pneumoniae, the major cause of lethality in patients with influenza and superinfected patients. Therefore, we will evaluate the degree of protection and immunological mechanisms involved in the viral load and inflammation of mice vaccinated and challenged with SARS-CoV-2, and in some groups, infected concomitantly with competent influenza-replication or pneumococcus. As for the protective mechanism induced by the vaccine, we will study the level of protection in mice deficient in B lymphocytes, CD8 T lymphocytes, and combined immunodeficiency, using the mutant mice for ¼ chain, b2-microglobulin mice and RAG knockouts (KO) mice. The focus will be given to the role of neutralizing antibodies and IgA produced in the mucosa, as well as cellular response mediated by CD4 Th1 and CD8 cytotoxic lymphocytes. We believe that these studies will lead to a better understanding of the mechanisms of the disease and protection against influenza and SARS-CoV-2 viruses. (AU)