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.

The group sizes for experiments with mice, hamsters and human samples were determined by power calculations statistical analysis.
No data were excluded from the analysis.
The experiments were repeated at least twice, if the statistical significance was P<0.05. All attempts at replication were successful.
In mice, hamsters and human experiments we used age and sex-matched individual in the control groups.
Except for the histopathology description that was performed by two independent observers, we didn't find the need for blinding analysis in the other experiments because data was objective and phenotypes were very distinct. Note that full information on the approval of the study protocol must also be provided in the manuscript.

Human research participants
Policy information about studies involving human research participants Population characteristics

Recruitment
Ethics oversight Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry
Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.

Methodology
Sample preparation

Vero E6 cells (ATCC CRL-1586)
Cells were low passage cells from ATCC, which authenticates them. Cell morphology and growth was consistent with Vero-E6.
Cells were frequently tested for mycoplasma contamination and were negative. None.
The study did not include samples collected from the field.
The protocols for animal experiments were approved by Fundação Oswaldo Cruz and Universidade Federal de São Paulo Ethics. Commission on Animal Use (CEUA) LW 25/20 and 105/2020, respectively.
Population characteristics are detailed in table 1. Included subjects were older than 18 years of age and no older than 70 years of both sexes. In Brazil there is a great diversity in genetic origin including African Americans, Asians, South American Indians, Mestizos and Caucasians of European origin, thus our cohort was representative of such diversity. Participants included did not reported any health condition or current treatment considered as a potential bias. We did not anticipate any biases influencing the cohort composition.
All participants were invited to answer a questionnaire and donate blood based on inclusion criteria of each category. Vaccinated participants had complete vaccination with Coronavac within 60 days after booster. Unvaccinated healthy donors (HD) where RT-PCR and antibody test negative, and reported never having had COVID-19. They have not received any vaccine or displayed respiratory symptoms up to 30 days before sampling. Convalescents were unvaccinated before infection and diagnosed by RT-PCR. Potential bias includes gender distribution, since all vaccinated volunteers were health workers that have a higher representation of females. It is also important to notice that all vaccinated volunteers were health workers, whereas HD and convalescent included both health workers and non-health workers. Nevertheless, we believe these potential biases have none or minor effect on results and do not impact on our conclusions.
This study was performed under protocols reviewed and approved by the Ethical Committees on Human Experimentation from Fundação Hospitalar do Estado de Minas Gerais (FHEMIG) -CAAE: 43335821.4.0000.5119.