CRISPR Genome Editing and the Study of Chagas Disease.

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, is an illness that affects 6-8 million people worldwide and is responsible for approximately 50,000 deaths per year. Despite intense research efforts on Chagas disease and its causative agent, there is still a lack of effective treatments or strategies for disease control. Although significant progress has been made toward the elucidation of molecular mechanisms involved in host-parasite interactions, particularly immune evasion mechanisms, a deeper understanding of these processes has been hindered by a lack of efficient genetic manipulation protocols. One major challenge is the fact that several parasite virulence factors are encoded by multigene families, which constitute a distinctive feature of the T. cruzi genome. The recent advent of the CRISPR/Cas9 technology represented an enormous breakthrough in the studies involving T. cruzi genetic manipulation compared to previous protocols that are poorly efficient and required a long generation time to develop parasite mutants. Since the first publication of CRISPR gene editing in T. cruzi, in 2014, different groups have used distinct protocols to generated knockout mutants, parasites overexpressing a protein or expressing proteins with sequence tags inserted in the endogenous gene. Importantly, CRISPR gene editing allowed generation of parasite mutants with gene disruption in multi-copy gene families. We described four main strategies used to edit the T. cruzi genome and summarized a large list of studies performed by different groups in the past 7 years that are addressing several mechanisms involved with parasite proliferation, differentiation, and survival strategies within its different hosts.

Evaluation of an RBD-nucleocapsid fusion protein as a booster candidate for COVID-19 vaccine.

Despite successful vaccines and updates, constant mutations of SARS-CoV-2 makes necessary the search for new vaccines. We generated a chimeric protein that comprises the receptor-binding domain from spike and the nucleocapsid antigens (SpiN) from SARS-CoV-2. Once SpiN elicits a protective immune response in rodents, here we show that convalescent and previously vaccinated individuals respond to SpiN. CD4+ and CD8+ T cells from these individuals produced greater amounts of IFN-γ when stimulated with SpiN, compared to SARS-CoV-2 antigens. Also, B cells from these individuals were able to secrete antibodies that recognize SpiN. When administered as a boost dose in mice previously immunized with CoronaVac, ChAdOx1-S or BNT162b2, SpiN was able to induce a greater or equivalent immune response to homologous prime/boost. Our data reveal the ability of SpiN to induce cellular and humoral responses in vaccinated human donors, rendering it a promising candidate.

Differential requirement of neutralizing antibodies and T cells on protective immunity to SARS-CoV-2 variants of concern.

The current COVID-19 vaccines protect against severe disease, but are not effective in controlling replication of the Variants of Concern (VOCs). Here, we used the existing pre-clinical models of severe and moderate COVID-19 to evaluate the efficacy of a Spike-based DNA vaccine (pCTV-WS) for protection against different VOCs. Immunization of transgenic (K18-hACE2) mice and hamsters induced significant levels of neutralizing antibodies (nAbs) to Wuhan and Delta isolates, but not to the Gamma and Omicron variants. Nevertheless, the pCTV-WS vaccine offered significant protection to all VOCs. Consistently, protection against lung pathology and viral load to Wuhan or Delta was mediated by nAbs, whereas in the absence of nAbs, T cells controlled viral replication, disease and lethality in mice infected with either the Gamma or Omicron variants. Hence, considering the conserved nature of CD4 and CD8 T cell epitopes, we corroborate the hypothesis that induction of effector T-cells should be a main goal for new vaccines against the emergent SARS-CoV-2 VOCs.

Promotion of neutralizing antibody-independent immunity to wild-type and SARS-CoV-2 variants of concern using an RBD-Nucleocapsid fusion protein.

Both T cells and B cells have been shown to be generated after infection with SARS-CoV-2 yet protocols or experimental models to study one or the other are less common. Here, we generate a chimeric protein (SpiN) that comprises the receptor binding domain (RBD) from Spike (S) and the nucleocapsid (N) antigens from SARS-CoV-2. Memory CD4+ and CD8+ T cells specific for SpiN could be detected in the blood of both individuals vaccinated with Coronavac SARS-CoV-2 vaccine and COVID-19 convalescent donors. In mice, SpiN elicited a strong IFN-γ response by T cells and high levels of antibodies to the inactivated virus, but not detectable neutralizing antibodies (nAbs). Importantly, immunization of Syrian hamsters and the human Angiotensin Convertase Enzyme-2-transgenic (K18-ACE-2) mice with Poly ICLC-adjuvanted SpiN promotes robust resistance to the wild type SARS-CoV-2, as indicated by viral load, lung inflammation, clinical outcome and reduction of lethality. The protection induced by SpiN was ablated by depletion of CD4+ and CD8+ T cells and not transferred by antibodies from vaccinated mice. Finally, vaccination with SpiN also protects the K18-ACE-2 mice against infection with Delta and Omicron SARS-CoV-2 isolates. Hence, vaccine formulations that elicit effector T cells specific for the N and RBD proteins may be used to improve COVID-19 vaccines and potentially circumvent the immune escape by variants of concern.

Avaliação do papel do receptor ß2-adrenérgico durante a infecção pelo vírus influenza em modelo murino

A infecção pelo vírus influenza pode provocar desde infecções assintomáticas, quanto casos graves de infecção respiratória, sendo frequentemente mortais, sobretudo em idosos. Nesse sentido, a infecção pelo vírus influenza ocasiona cerca de cinco milhões de casos graves no mundo, resultando em cerca de 500.000 mortes por ano. A gravidade da infecção está relacionada tanto com a resposta imune do hospedeiro quando à virulência do patógeno. Nesse contexto, o sistema imune deverá atuar buscando a restauração da homeostase. Para isso, sua coordenação de forma sistêmica e, sobretudo, ao nível do trato respiratório é fundamental para a resolução das lesões causadas pela infecção. Um dos mecanismos chave que coordenam as ações da resposta imune é o Sistema Nervoso Simpático (SNS). Desse modo, o presente estudo teve como objetivo investigar a relevância e a integração da sinalização adrenérgica e o sistema imune por meio receptor ß2-AR durante infecção pelo vírus influenza A, adaptado a camundongo. Camundongos ß2 KO foram empregados como modelo de infecção com o vírus influenza adaptado a camundongos: A/PR8/34. Após 6 dias de infecção foi avaliada a mecânica pulmonar, contagem celular e proteínas no BALF, carga viral pulmonar e sobrevivência e peso. Embora não tenha sido observada diferença na carga viral, os animais ß2 KO apresentaram aumento do número de linfócitos e redução do número de neutrófilos no lavado bronco alveolar (BALF) e maior comprometimento das funções ventilatórias dos pulmões dos camundongos, quando comparado aos animais do grupo controle. Curiosamente, ainda que não haja diferença na perda de peso entre os animais ß2 KO em relação ao grupo controle, os animais do grupo ß2 KO apresentaram maior mortalidade quando inoculados com uma dose letal do vírus PR8. Desta forma, os achados obtidos são sugestivos de que o ß2-AR é relevante para mecanismos de ventilação pulmonar e possuem papel importante no recrutamento de células do sistema imune durante a resposta inflamatória no tecido pulmonar durante infecção pelo vírus influenza e finalmente, para a sobrevivência dos animais infectados.