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.

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.