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

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 recombinant influenza virus expressing a segment of the Spike protein. Our hypothesis is that the pathology is largely triggered by an 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, consequent increase in lung injury. 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, cytokine production and lung injury. These studies will focus on 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 evaluate the capacity of the PR8 influenza virus, which 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 animals. Therefore, we believe that we have a bivalent vaccine against influenza and SARS-CoV2. In addition, in previous studies we built an influenza virus that expresses the pneumococcal PspA protein and protects against co-infection with influenza and S. pneumumoniae, 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 animals vaccinated and challenged with SARS-CoV-2, and in some groups, infected concomitantly with competent influenza-replication or pneumococcus. As for the protection mechanism induced by the vaccine, we will study the level of protection in mice deficient in B lymphocytes, CD8 + T lymphocytes and with combined immunodeficiency, using mutant mice for m chain, b2-microglobulin mice and RAG knockouts (KO) mice. 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)