The Nucleocapsid Protein of SARS-CoV-2, Combined with ODN-39M, Is a Potential Component for an Intranasal Bivalent Vaccine with Broader Functionality.

Despite the rapid development of vaccines against COVID-19, they have important limitations, such as safety issues, the scope of their efficacy, and the induction of mucosal immunity. The present study proposes a potential component for a new generation of vaccines. The recombinant nucleocapsid (N) protein from the SARS-CoV-2 Delta variant was combined with the ODN-39M, a synthetic 39 mer unmethylated cytosine-phosphate-guanine oligodeoxynucleotide (CpG ODN), used as an adjuvant. The evaluation of its immunogenicity in Balb/C mice revealed that only administration by intranasal route induced a systemic cross-reactive, cell-mediated immunity (CMI). In turn, this combination was able to induce anti-N IgA in the lungs, which, along with the specific IgG in sera and CMI in the spleen, was cross-reactive against the nucleocapsid protein of SARS-CoV-1. Furthermore, the nasal administration of the N + ODN-39M preparation, combined with RBD Delta protein, enhanced the local and systemic immune response against RBD, with a neutralizing capacity. Results make the N + ODN-39M preparation a suitable component for a future intranasal vaccine with broader functionality against Sarbecoviruses.

Broad humoral immunity generated in mice by a formulation composed of two antigens from the Delta variant of SARS-CoV-2.

Due to the rapid development of new variants of SARS-CoV-2 as well as the real threat of new coronavirus zoonosis events, the development of a preventive vaccine with a broader scope of functionality is highly desirable. Previously, we reported the functionality of a nasal formulation containing the nucleocapsid protein and the receptor-binding domain (RBD) of the spike protein of the Delta variant of SARS-CoV-2 combined with the ODN-39M adjuvant. This combination induced cross-reactive immunity in mucosal and systemic compartments at the sarbecovirus level. In the present study, we explored the magnitude of the immunity generated in BALB/c mice by the same formulation with alum added as an additional adjuvant, to enhance the humoral immunity against the two antigens. Animals were immunized with three doses of the bivalent formulation, administered by subcutaneous route. Humoral immunity was tested by ELISA, and the neutralizing capacity of the resulting antibodies (Abs) was evaluated using a surrogate test and a vesicular stomatitis virus (VSV) pseudovirus-based assay. Cell-mediated immunity was also investigated using an IFN-γ ELISpot assay. High levels of antibodies against both antigens (N and RBD) were obtained upon immunization. Anti-RBD Abs with neutralizing capacity reacted with the RBD of three SARS-CoV-2 variants tested, including Omicron. Abs recognizing the nucleocapsid proteins of SARS-CoV-1 and the SARS-CoV-2 Delta and Omicron variants were also detected. Taken together, these results suggest that this bivalent formulation could be an attractive component of a pancorona vaccine able to broaden the scope of humoral immunity against both antigens. This will be particularly important for the reinforcement of immunity in previously vaccinated and/or infected populations.

Cross-Reactive Profile Against Two Conserved Coronavirus Antigens in Sera from SARS-CoV-2 Hybrid and Vaccinated Immune Donors.

Since the beginning of the pandemic, the pre-existing immunity against SARS-CoV-2 has been postulated as one possible cause of asymptomatic infections. Later, various works reported that pre-existing immune response against the two structural conserved antigens: S2 subunit and the nucleocapsid protein, were associated to some level of asymptomatic profile in infected individuals. To explore the Ab background against these two antigens, in the context of vaccine-elicited and hybrid (natural infection plus vaccination induced) immunity of SARS-CoV-2, in this work, we tested sera from inactivated vaccine-immunized donors and from vaccinated and subsequent natural infected donors upon the Omicron variant wave in Guangdong province, China. Serum samples were collected from 27 COVID-19 convalescent, 25 SARS-CoV-2 vaccinated, and 10 negative donors. The IgG cross-reactivity response against these two antigens from another relevant human coronavirus (HCoV) was also evaluated. The findings indicate that IgG response against S2 and N protein was particularly higher in sera with hybrid immunity. The cross-reactive Abs were more significant against SARS-CoV-1, while a wide cross-reactivity was detected for N antigen for one human Alpha coronavirus HCoV-229E even in the negative control samples. The presence of cross-reactive Abs against the two conserved antigens N and S2, particularly in the context of hybrid immunity, could pave the way for future boosted vaccines carrying these conserved regions.