Iris Valdes^*,1, Laura Lazo¹, Edith Suzarte¹, Karem Cobas¹, Yusleidi Pérez¹, Rocío Garateix¹, Enrique Noa², Rubén Amaya¹, Dionne Casillas¹, Elias Nelson¹, Yinet Cartaya¹, Monica Bequet¹, Julio C. Aguilar¹, Carlos A. Guzmán³, Gerardo Guillen¹

¹Center for Genetic Engineering and Biotechnology (CIGB), Avenue 31, P.O. Box 6162, Havana, Cuba

²Civilian Defense Scientific Research Center, Carretera de Jamaica y Autopista Nacional, San José de las Lajas, Mayabeque, Cuba

³Department of Vaccinology and Applied Microbiology, Helmholtz Centre for Infection Research (HZI), Germany

* Corresponding author: Iris Valdes, Vaccine Department, Center for Genetic Engineering and Biotechnology (CIGB), Ave 31, P.O. Box 6162, Havana 6, 10 600, Cuba.

Received: 06 January 2026; Accepted: 14 January 2026; Published: 26 February 2026

Article Information

Citation: Iris Valdes, Laura Lazo, Edith Suzarte, Karem Cobas, Yusleidi Pérez, Rocío Garateix, Enrique Noa, Rubén Amaya, Dionne Casillas, Elias Nelson, Yinet Cartaya, Monica Bequet, Julio C. Aguilar, Carlos A. Guzmán, Gerardo Guillen. A Nasal Subunit Vaccine Candidate Based on The Viral Antigens RBD and N Combined with C-Di- AMP as an Approach to Enhance Specific Immunity to SARS-Cov-2. Archives of Microbiology and Immunology. 9 (2026): 09-21.

DOI: 10.26502/ami.936500234

Abstract

Keywords

SARS-CoV-2; cyclic di-adenosine monophosphate (c-di-AMP); intranasal vaccination; cell-mediated immunity; neutralizing antibody; mucosal immunity

Article Details

Introduction

The coronavirus disease 2019 (COVID-19) outbreak caused by the highly transmissible and pathogenic coronavirus, the SARS-CoV-2 has resulted in 772 million documented infections and 7 million deaths worldwide [1]. According to WHO data, there are even many asymptomatic cases of COVID-19 that can silently amplify these statistics [2, 3]. Nowadays, the specific immunity through vaccination remains the single best strategy to control the COVID-19 disease and prevent mortality. However, less than half of the world's population has access to one dose of COVID-19 vaccines [4]. The Spike-protein (S-protein) and the receptor-binding domain (RBD) of the S-protein of SARS-CoV-2 have been used as the main antigens for vaccine design to elicit mainly neutralizing antibodies [5, 6]. Though, the S protein shows considerable sequence variations among SARS-CoV-2 variants of concern, resulting in these variants being able to evade the neutralizing antibody response generated in vaccinated individuals [7, 8]. While the levels of neutralizing antibodies elicited by vaccination or natural infection are taken as the main predictors of protective immunity [9], recently evidences for the function of T cells in mediating immunity to SARS-CoV-2 have accumulated [10]. Hence there is a need for next generation vaccines that are able to protect against multiple variants and encompass additional antigens that provide a broader protection [11-14]. In this sense, the nucleocapsid (N) viral protein is more conserved and stable among of many coronaviruses with 90% amino acid homology and fewer mutations over time [15]. This protein, highly immunogenic and expressed abundantly during natural infection [16], is a representative antigen for the T-cell response in a vaccine setting, inducing SARS-specific T-cell proliferation and cytotoxic activity [15, 17]. For that, N antigen has been tested in several vaccine platforms and formulations with encouraging results against SARS-CoV-2 [14, 18-23].

Parenteral vaccination is the most used method for vaccine administration, leading to elicitation of antigen-specific systemic cellular and humoral immunity. During the COVID-19 pandemic, real world evidence showed that this approach successfully reduced severe forms of disease and death tolls. However, parenteral vaccines have a modest effect in the prevention of infection and subsequent horizontal transmission to susceptible hosts. In contrast, mucosal vaccination effectively stimulates both systemic and mucosal immune responses [24, 25]. This local mucosal response at the portal of entry is critical to provide early protection against viral infection. In addition, it has the advantage of being a non-invasive procedure suitable for immunization of large populations [26, 27]. Hence, the development of nasal vaccines represents a feasible alternative for a more effective immunization [28] against coronavirus, by providing a first-line of defense in the respiratory tract to prevent both viral infection and transmission. However, mucosal vaccination frequently requires specifically tailored delivery systems to ensure efficient breaching and transit a cross the mucosal barrier. Furthermore, to guarantee antigen uptake, processing and presentation, a danger signal is needed (e.g. adjuvantations) that promotes stimulation and activation of antigen presenting cells and a local microenvironment conductive towards initiation of an adaptive immune responses without toxicity [26]. At the moment, only five different nasal vaccines have been approved by the regulatory authorities for the markets [27].

In this study we evaluated the cyclic di-nucleotide, bis-(3′,5′)-cyclic dimeric adenosine monophosphate (c-di-AMP), as the adjuvant for intranasal vaccination with the RBD and N antigens from SARS-CoV-2. This adjuvant has been recognized as stimulator of IFN genes (STING) pathway, which promote the activation of the immune system, principally to mucosal vaccines against respiratory pathogens [29, 30]. As a result of acting as STING agonist and its subsequent targeting to proteolytic degradation, c-di-AMP promotes a self-restricted local immune activation [31]. Our results show that the nasal vaccine candidate elicits efficient systemic immunity, in terms of neutralizing antibodies and cellular immunity, as well as mucosal immunity against the antigens included in the formulation.

Material and Methods

Cell line, antibody and viral antigens

African monkey kidney (Vero E6) cells were obtained from the ATCC (No. CRL-1586) and were used for isolating, passaging and neutralization tests. Cells were grown at 37°C in Eagle’s MEM supplemented with 10% heat-inactivated of fetal bovine serum (FBS). Monoclonal antibody #8 that recognizes the RBD region was produced by the CIGB-Sancti Spiritus, Cuba. Variant concerns of SARS-CoV-2, Delta (EPI_ISL_7495138) and Omicron BA1.2 (EPI_ISL_12691753) isolates were used for the neutralization experiments.

Antigens of the vaccine formulation

RBD protein was produced as recombinant protein in Pichia pastoris yeast (strain X-33) and obtained as a pyrogen-free product more than 95% pure. The RBD region includes amino acids 331-529 of the Spike protein of SARS-CoV-2 Wuhan-Hu-1 strain (NCBI Acc. No. YP_009724390), it’s the pharmaceutical active ingredient of ABDALA Vaccine developed by the Center for Genetic Engineering and Biotechnology (CIGB, Havana, Cuba). The recombinant full-length N protein of SARS-CoV-2 (Wuhan-Hu-1 strain), 419 amino acids, was obtained as recombinant protein in Escherichia coli bacteria, as a pyrogen-free product with more than 95% purity by the Technology Development Department of the CIGB. The c-di-AMP (BioLog, Bremen, Germany), provided by the Helmholtz Centre for Infection Research (HZI, Germany), was dissolved in water (Ampuwa; Serumwerk, Bernburg, Germany). All components were prepared in buffer Na₂HPO₄ 0.56 mg/mL, NaH₂PO₄ x 2H₂O 0.62 mg/mL, NaCl 8.5 mg/mL diluted in water.

Animals and ethics statement

Female BALB/c (Bc, H-2d) mice (aged 6 – 8 week) were purchased from the Center for the Production of Laboratory Animals (CENPALAB, Cuba) and housed in appropriate animal care facilities during the experimental period. Mouse experiments were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the CIGB. The protocol was approved by the Committee on the Ethics of Animal Experiments (CICUAL/CIGB/22034).

Immunization schedule

Groups of BALB/c mice were injected by intranasal route with volume of 50 μL (25 μL in each nasal cavity) on days 0, 14 and 28 (Figure 1). All immunogens were prepared just before the inoculation (Table 1):

Group 1 (N + c-di-AMP): 10 µg of protein N and 5 µg of c-di-AMP, n = 12.

Group 2 (N): 10 µg of protein N, n = 12.

Group 3 (N + RBD + c-di-AMP): 10 µg of protein N, 50 µg of protein RBD and 5 µg of c-di-AMP, n = 12.

Group 4 (N + RBD): 10 µg of protein N, plus 50 µg of protein RBD, n = 12.

Group 5 (RBD + c-di-AMP): 50 µg of protein RBD and 5 µg of c-di-AMP, n = 12.

Group 6 (Placebo + c-di-AMP): 5 µg of c-di-AMP, n = 8.

Sera samples from each group were collected at day 49 (n=12 or 8) and day 56 (n=6 or 4) after the last dose for evaluating the systemic humoral immune responses. Nasal washes were taken at day 49 (n=6 or 4) and day 56 (n=6 or 4) using PBS buffer. Finally, in both times animals were splenectomized and collected cells to measure the cellular immune responses (Figure 1).

Table 1: Groups of the immunization schedule in BALB/c mice

Figure 1: Representation of the immunization schedule. Immunocompetent BALB/c mice were inoculated with three doses by intranasal route, every fourteen days (day 0, 14, 28). After the third dose, samples (sera, nasal washes and spleen cells) were taken to evaluate the humoral and cellular immune responses on days 49 and 56 of the study.

IgG and IgA antibodies specific to the SARS-CoV-2 RBD and N proteins by ELISA

To evaluate systemic humoral immunity, total IgG, IgG1 and IgG2a antibody titers were performed by ELISA on days 49 and 56 against each recombinant antigen. Briefly, 96-well plates were coated with 5 mg/mL of recombinant proteins RBD or N, and incubated overnight at 4°C in coating buffer (0.16% Na₂CO₃, 0.29% NaHCO₃, pH 9.5). The wells were washed three times with phosphate-buffered saline containing 0.05% Tween-20 (PBS-T) after each step of the assay. Subsequently, plates were blocked with 5% (w/v) skim milk, 1 h at 37°C. Then, they were incubated with serum samples diluted in PBS-T and incubated 1 h at 37°C. As previously, three washes with PBS-T were performed after each step of the assay. Finally, anti-mouse IgG (#A0168, Sigma-Aldrich, USA), anti-mouse IgG1 (#A90-105P, ImTec, USA) or anti-mouse IgG2a (#A90-107P, ImTec, USA) antibody-peroxidase conjugated were added and the plates were incubated for 1 h at 37°C. After washing, 0.04% substrate solution (O-phenilendiamine in buffer 2% Na₂HPO₄, 1% citric acid, and 30% H₂O₂, pH 5.0) was added. Plates were kept 10 min at 25°C and the reaction was stopped with 12.5% H₂SO₄. Absorbance at 492 nm was read in a microplate reader (SensIdent Scan; Merck, Germany). For ELISA against antigen RBD, titers were estimated using a standard curve of the monoclonal antibody #8 (CIGB-Sancti Spiritus, Cuba) at know concentrations (0.06 - 4 mg/mL). For ELISA against N antigen, titers were defined as the dilution of serum giving twice the absorbance value of the negative control sera (samples from corresponding Placebo group). For both assays, the positive cut-off values were established as twice the average titer calculates for the placebo group. To assess mucosal IgA antibody titers, an ELISA was performed for each recombinant antigen. The methodology for assessing IgA antibody titers was similar to that for the IgG ELISA, replacing serum samples by nasal washes, which were used directly undiluted. Since the positive signals obtained were usually lower, the results were expressed as absorbance units at 492 nm. Positive cut-off values were set as twice the average values calculated for the placebo group.

Inhibition of interaction RBD-ACE2 receptor by ELISA

The quality of the antibody response generated by immunization; was determined as the capacity of sera to inhibit the binding of RBD to its ACE2 receptor. In this assay, flat bottom 96-well ELISA plates (Costar 3590) were coated in coating buffer (0.16% Na₂CO₃, 0.29% NaHCO₃, pH 9.5) using 5 μg of the recombinant protein ACE2 coupled to the Fc portion of a murine immunoglobulin (ACE2 mFc) and incubated for 16 - 20 h at 4ºC. Subsequently, the plates were washed with 0.1% Tween 20 in H₂O and blocked with 2% skim milk in PBS 1X, 0.05% Tween 20 for 1 h at 37°C. During the same time, sera (dilution 1:500) and RBD protein coupled to the Fc portion of a human immunoglobulin conjugated to peroxidase enzyme (RBD hFc-HRPO) were pre-incubated in U-bottom culture plates (Costar 3799). The serum and RBD hFc-HRPO reagent were diluted in 0.2% skim milk, 0.05% Tween 20 in PBS 1X. ELISA plates were washed three times and 50 mL of the pre-incubation mixture were added to the coated plates. Follow, the plates were incubated during 90 minutes at 37ºC, and washed under the same conditions previously described. Finally, the substrate tetramethyl benzidine was added and the reaction was stopped with 12.5% H₂SO₄, after 10 minutes of incubation. The absorbance at 450 nm was detected in a microplate reader (SensIdent Scan; Merck, Germany). Percentage of inhibition was determined concerning the maximum binding (Umax: absorbance from the binding of RBD hFc-HRPO to the ACE2 in the absence of serum) after subtraction of absorbance values of the background control wells incubated with all the reagents except serum and RBD conjugate. When a negative value arose, it was processed and represented in graphics as zero.

Live virus neutralization assay

Neutralization antibody titers were determined by a traditional virus micro-neutralization assay using SARS-CoV-2 variants under Level 3 Biological Safety Facility (BSL-3) in the Civilian Defense Scientific Research Center, Cuba. Briefly, 96-well culture plates were seeded with 2x10⁴ cells/ well of Vero E6 one day before. Cells were incubated for 24 h at 37^oC and 5% CO₂ to grown until 85-90% confluency. Subsequently, the heat-inactivated sera (by incubation at 56°C for 30 minutes) were serially diluted 1:5 in minimal essential medium (MEM, Gibco, UK) with 2% (v/v) fetal bovine serum (Capricorn, Germany), 25 mM L-glutamine (Sigma, USA), 2 µg/mL sodium bicarbonate (MercK, Germany) and 40 μg/mL of gentamicin (Sigma, USA). Each serum dilution was mix with 100 mean tissue culture infective dose (TCID₅₀) of each SARS-CoV-2 variant, and incubated for 1 h at 37^o C. Serum-virus mixes were added to the semi-confluent cell monolayers and were incubated during 96 h at 37°C, with 5% CO₂. Neutralization was visualized by the colorimetric method, by staining with 0.02 % neutral red and reading the optical density at 540 nm. The virus neutralizing titers (VNT₅₀) were calculated as the highest serum dilution at which 50% of the cells remained intact according to neutral red incorporation in the control wells (no virus added).

Cell culture and viral in vitro stimulation

Spleen cells were obtained under aseptic conditions and erythrocytes were lysed by adding 0.83% NH₄Cl solution. Cells were washed twice with PBS + 2% FBS (PAA Laboratories, Ontario, Canada) and resuspended at 2×10⁶ cells/mL in RPMI-1640 medium (Sigma Aldrich) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin (Gibco, UK), 2 mM glutamine (Gibco, UK), 5×10⁻⁵ M 2-mercaptoethanol (Sigma St. Louis, MO) and 5% FBS. Finally, 2×10⁵ cells per well were cultured in 96-well round bottomed plates with the antigens (recombinant protein RBD or N, or mock). Concanavalin A (Sigma) was used as a positive control (5 µg/mL). In all experiments, three wells were plated for each antigen. After 96 h of incubation, culture supernatants were collected and stored at -80°C.

IFNg  detection in culture supernatants by ELISA

The culture supernatants of splenocytes previously stimulated with each antigen were analyzed in duplicate for IFNγ concentrations by ELISA using monoclonal antibody pairs. The ELISA protocol recommended by the manufacturers was used with slight modifications (#3321-1H-6, Mabtech, Sweden). The lowest limit of detection of the cytokine was 4 pg/mL.

Statistical analysis

Direct or transformed (Log10) data normality was verified with the Kolmogorov-Smirnov (n = 6 or 4) or D'Agostino & Pearson (n = 12 or 8) normality test, as well as the homogeneity of variance using the Bartlett´s test, and after were analyzed by ANOVA parametric tests (One-way analysis of variance with Tukey’s post-test). Data that do not fulfill both conditions, even after transformations, were analyzed by a nonparametric test (Kruskal–Wallis non-parametric test with Dunn's multiple comparison post-test). In all cases, the GraphPad Prism version 9.0 for Windows, GraphPad Software, San Diego California USA, www.graphpad.com was employed.

Results

Immunological evaluation in mice of a nasal formulation based on RBD and N antigens combined with c-di-AMP as immune-modulator

The immunogenicity of a nasal formulation comprising RBD and N antigens of SARS-CoV-2 combined with c-di-AMP was tested in BALB/c mice. As control groups were included animals immunized with Placebo (Placebo + c-di-AMP) or immunized with formulations contains each independent antigen (N, N + c-di-AMP, N + RBD, RBD + c-di-AMP). Animals received three doses of the formulations by intranasal route, every fourteen days, containing 50 µg of protein RBD and/or 10 µg of protein N as viral antigens (Table 1). After the third dose, the humoral immune and cellular immune responses were evaluated on day 49 and day 56 of the study (Figure 1).

Systemic and mucosal humoral immune response

Systemic humoral immune responses elicited by the formulations were firstly evaluated by ELISA systems, in terms of total IgG and IgG subclasses (IgG1 and IgG2a) in sera against the RBD or N recombinant antigens (Figure 2A - 2H). As shown in Figure 2, after immunizations with the nasal formulations containing RBD and/or N recombinant proteins, all animals seroconverted and showed high IgG antibody titers against each viral antigen contained in the formulation. Furthermore, formulations including viral antigens combined with the immune-modulator c-di-AMP showed higher antibody titers compared with their mono or bivalent equivalents without adjuvant at both time points studied (P<0.05), (Figure 2).

To broad characterization of the humoral immune response induced, sera from animals immunized were tested by antibody subclasses IgG1 and IgG2a at day 56. As shown in Figure 2C – 2D, animals inoculated with N + RBD + c-di-AMP and RBD + c-di-AMP formulations exhibited higher antibody titers against the RBD recombinant antigen, compared with response observed in animals inoculated with the formulation that included alone both viral antigens (N + RBD), for both subclasses (P<0.05). A similar behavior was observed in the recognition of the N-specific antibody titers. Vaccinated animals with formulations including the immune-modulator c-di-AMP (N + c-di-AMP and N + RBD + c-di-AMP) generated the highest IgG1 and IgG2a antibody titers (P<0.05), indicating the induction of a mixed Th1/Th2 pattern (Figure 2G – 2H).

Figure 2: Systemic humoral response measured by ELISA in serum from immunized mice. (A) anti-RBD IgG antibody titers at day 49 (n = 12 or 8). (B) anti-RBD IgG antibody titers at day 56 (n = 6 or 4). (C) anti-RBD IgG1 antibody titers at day 56 (n = 6). (D) anti-RBD IgG2a antibody titers at day 56 (n = 6). (E) anti-N IgG antibody titers at day 49 (n = 10). (F) anti-N IgG antibody titers at day 56 (n = 6 or 4). (G) anti-N IgG1 antibody titers at day 56 (n = 6). (H) anti-N IgG2a antibody titers at day 56 (n = 6). In all cases, data represent the mean ± SD. The analysis of data was performed using a Kruskal–Wallis non-parametric test with Dunn's multiple comparison tests, (^*: P<0.05; ^**: P<0.01; ^***: P<0.001; ^****: P<0.0001). The dashed lines represent the cut-off value defined as twice the average titer measured in the placebo group.

Nasal washes from animals vaccinated were used to evaluate by ELISA the local mucosal immune response induced, in terms of IgA antibody titers that recognize the RBD or N antigens in both times (Figure 3). As results, only mice from groups immunized with formulations including c-di-AMP were positives for IgA (N + c-di-AMP, N + RBD + c-di-AMP, and RBD + c-di-AMP) (P<0.05) (Figure 3A – 3D).

Figure 3: Mucosal humoral response measured by ELISA from immunized mice. (A) anti-RBD IgA in nasal washes (Day 49, n = 6 or 4). (B) anti-RBD IgA in nasal washes (Day 56, n = 6 or 4). (C) anti-N IgA in nasal washes (Day 49, n = 6 or 4). (D) anti-N IgA in nasal washes (Day 56, n = 6 or 4). In all cases, data were represented as absorbance units at 492 nm. Graphics represent the mean ± SD. The analysis of data was performed using a Kruskal–Wallis non-parametric test with Dunn's multiple comparison tests, (^*: P<0.05; ^**: P<0.01). The dashed lines represent the cut-off value defined as twice the absorbance unit average measured in the placebo group.

To evaluate the functionality of antibodies two methods were exploited. Firstly, the inhibition capacities to block the interaction between the RBD with ACE2 receptor were tested using sera collected at day 49 and 56. For these assays, only were used sera from animals inoculated with formulations including RBD antigen (N + RBD + c-di-AMP, N + RBD and RBD + c-di-AMP groups). Figure 4 shows that in almost all mice receiving N + RBD + c-di-AMP or RBD + c-di-AMP formulations, more than 50% inhibition was observed at both time points of the study, but only the group N + RBD + c-di-AMP was statistical different (P<0.01) (Figure 4A - B).

In addition, sera collected from vaccinated animals were used to evaluate the neutralizing activity against two viral variants of concern by VNT₅₀. In Figure 4 were represented neutralizing antibody titers to Delta and Omicron BA1.2 isolates. Only neutralizing titers were detected in sera from mice vaccinated with the nasal formulations N + RBD + c-di-AMP and RBD + c-di-AMP, (P<0.05) (Figure 4C - D). However, a discrete increase in the titers were detected in animals immunized with the formulation including N and RBD combined with c-di-AMP, with titer means of 220.8 and 126.2 to Delta and Omicron BA1.2 variants, respectively (P<0.05); compared with the titer means from animals that received RBD + c-di-AMP formulation (68.5 and 62.0 to Delta and Omicron BA1.2 variants) (Figure 4C - D). All sera from the rest of groups did not neutralize.

Figure 4: Functionality of antibodies in serum from vaccinated mice. (A) Percentage of inhibition to the interaction of RBD-ACE2 receptor at day 49 (n = 12). (B) Percentage of inhibition to the interaction of RBD-ACE2 receptor at day 56 (n = 6). (C) Neutralizing antibody titers against Delta measured strain at day 49 (n = 12 or 8). (D) Neutralizing antibody titers against Omicron strain at day 49 (n = 12 or 8). In all cases, data represent the mean ± SD. The analysis of data was performed using a Kruskal–Wallis non-parametric test with Dunn's multiple comparison tests, (^*: P<0.05; ^**: P<0.01; ^***: P<0.001). The dashed lines represent the cut-off value defined as 50% of inhibition to the interaction of RBD-ACE2 receptor.

Cellular immune response

To measure the cell-mediated immunity, IFNg concentration was detected by ELISA in the supernatant collected from spleen cells harvested on day 49 and day 56, after in vitro stimulation with the recombinant antigens (Figure 5A - 5B). Figure 5, shows the results of the two cell-mediated immunity assays performed. In the first experiment, done at day 49, only after stimulation with the recombinant antigen N was detected concentration of IFNg in two groups, N + c-di-AMP and N + RBD + c-di-AMP, with statistical significant differences compared to the rest of groups (P<0.05) (Figure 5A). A second experiment was achieved at day 56, 28 days after the last dose; once again the spleen cells were in vitro stimulated with the recombinant proteins RBD and N. As results, in the supernatants stimulated with N antigen, IFNγ concentrations were detected in animals that received N + c-di-AMP and N + RBD + c-di-AMP intranasal formulations (P<0.05) (Figure 5B). In addition, in this assay a tendency to promote the secretion of this antiviral cytokine was observed in animals from N + RBD + c-di-AMP and RBD + c-di-AMP groups, upon in vitro stimulation with the RBD antigen (Figure 5B). As expected, in both assays, spleen cells from animals immunized with placebo formulation did not produce the antiviral cytokine.

Figure 5: Cell-mediated immune responses induced in BALB/c mice immunized with nasal formulations of RBD and N antigens from SARS-CoV-2. IFNγ secretion measured by ELISA in the culture supernatants of splenocytes after in vitro stimulation with mock, protein N or protein RBD. (A) IFNγ concentration in the culture supernatants of splenocytes at day 49. (B) IFNγ concentration in the culture supernatants of splenocytes at day 56. The graph represents mean ± SEM for each group. The analysis of data was performed using a Kruskal–Wallis non-parametric test with Dunn's multiple comparison tests (n = 4 or 6 per group), (^*: P<0.05; ^**: P<0.01). The dashed lines represent the cut-off values defined as twice the average of IFNγ concentrations measured from Placebo groups after in vitro stimulation with the specific antigen.

Discussion

After the COVID-19 pandemic, an unprecedented global effort was triggered to develop safe and effective vaccines against the variants of concern of SARS-CoV-2. COVID-19 vaccines mostly contain the Spike antigen or fragments thereof, and are designed to generate robust neutralizing antibodies to prevent not only SARS-CoV-2 infection, but also severe outcomes of the disease, hospitalizations and deaths [5, 6]. To date, the WHO has approved several vaccines against COVID-19, which are based on nucleic acid, protein subunits, viral vectors and inactivated viruses platforms [4]. However, the effectiveness of these vaccines is significantly reduced in relation to newer strains [32, 33]. Data shows that current COVID-19 vaccines confer around 70% protection against intensive care unit admission some months after immunization, despite marked reductions in protection from symptomatic infection [34]. These observations suggest that vaccines continue to provide protection against severe disease despite the emergence of progressively more antibody-evasive and increasingly transmissible variants [34].

In this pipeline, a new generation of vaccines is emerging, aimed to solve the limitations of actual approved vaccines. In this regard, the goal is to induce broader immune responses, stimulate longer-lasting immunity, and activate the mucosal immune response [35]. To achieve these outcomes, various approaches have been pursued, including the incorporation of more viral antigens, the use of adjuvants, alternatives administration routes, and more effective delivery systems, among others [36]. Recombinant subunit proteins provide advantages regarding safety, costs, and speed of vaccine production; making those very attractive platforms for the development of vaccines for emerging viruses [37]. In correspondence, until last year near to 32% of vaccine candidates to COVID-19 diseases were based on protein subunits [4]. In our center was develop the ABDALA Vaccine, based on RBD region of the surface antigen of SARS-CoV-2 obtained as a recombinant protein in the yeast Pichia pastoris [38]. The ABDALA Vaccine has demonstrated that is safe, well tolerated, and protect humans against the COVID-19 disease [39]. The ABDALA Vaccine has successfully delivered as part of the initial vaccination or booster scheme of the population in several countries [40, 41]. On the other hand, the N antigen from SARS-CoV-2 have been used in several vaccine approaches [11, 17-19, 42, 43], as this protein serves as a representative antigen for eliciting T-cell response in a vaccine setting. It induces SARS-specific T-cell proliferation and cytotoxic activity [15] and cross-reactive responses to other relevant coronavirus [17]. Various studies have evaluated in preclinical models the ability to induce the immune response of vaccine candidates that combine the RBD and N antigens as subunit proteins [13, 43-48].

In the present study, we explore the immunogenicity in BALB/c mice of a nasal vaccine candidate based on recombinant proteins RBD and N in combination with c-di-AMP as immune-modulator. In our strategy was used the nasal vaccination to promote mucosal and systemic immunity. To this path has an important role to prevent seeding of the initial reservoir and control viral transmission [49]. This immunization path could be an advantage as non-invasive procedure suitable for immunization of large populations [26]. Furthermore, the inclusion of c-di-AMP as an adjuvant seeks to increase the mucosal immunogenicity generated by both viral antigens. Prior studies have been evaluated the adjuvant properties of cyclic di-nucleotides in respiratory diseases [29, 30, 50, 51], these molecules are bacterial second messengers which are consider as PAMP, rapidly sense by the immune system as a danger signal, allowing their utilization as activators of the immune response [52]. Cyclic di-nucleotides are able to boost antibody titers, increase Th1/Th2-associated responses, and expanded germinal center B cells and memory T cells [51, 53, 54].

First, our results demonstrated that RBD and N recombinant proteins combined with c-di-AMP, in a nasal formulation, induced systemic and mucosal humoral immune responses. High IgG and IgA antibody titers against both recombinant antigens were detected, which were significantly boosted when c-di-AMP was incorporated in the nasal formulation. Neutralizing antibodies have been well established as a key correlate of protection against COVID-19 [55, 56]. In the study was demonstrated that these humoral responses elicited against ancestral strain were functional in terms of induction neutralizing antibodies against two relevant SARS-CoV-2 isolates (Delta and Omicron BA1.2), primarily due to the presence of the recombinant RBD subunit. In this sense, clinical data show that current COVID-19 vaccines continue to provide good protection against severe disease, despite the generation of low neutralizing antibody titers against new variants of concern [34]. Nevertheless, the addition of N antigen to nasal formulation N + RBD + c-di-AMP suggests a modulatory effect of this component, since a slight increase in neutralizing titers was observed. N antigen might stimulate high-affinity antibody clones to the ACE2 cellular receptor. Further studies should be performed to confirm this statement. Although, in the present study was not evaluated the functional role of antibodies generated to N subunit, several reports have shown that N-specific IgG antibodies could mediate antibody-dependent cell-mediated cytotoxicity (ADCC) [57-59].

Likewise, these results are in agreement with previous studies in mice using the molecule c-di-AMP as adjuvant in a subunit SARS-CoV-2 vaccine based on the RBD, but administered by intramuscularly, which showed that c-di-AMP improves the specific immune response to SARS-CoV-2 [29]. However, the clinical applicability of mucosal vaccine strategies remains promising, it is evident that the induction of upper respiratory tract immune responses by these vaccines could significantly enhance protection from transmission and improve durability against emerging variants [24]. Afterward, we demonstrate that incorporation of c-di-AMP to the formulations including N antigen, stimulated functional cell-mediated immune responses that secreted IFNγ after the in vitro stimulation with this recombinant protein. Thus, an immune-modulator effect in the cell-mediated immune response was detected in mice immunized with the nasal bivalent formulation that included the two recombinant antigens, RBD and N, plus c-di-AMP. These results corroborate the capacity of N antigen as a distinctive antigen for the T-cell response induction [15, 17]. In addition, these results are relevant in the context of infection with antibody-evasive variants; where T-cells with antigen specificity for conserved SARS-CoV-2 epitopes could control viral replication to prevent progression to more severe disease [34, 60]. A prior study showed that recombinant N protein mixed with a specific oligonucleotide (ODN-39M), promoted the aggregate formation of the nucleocapsid [18]. Although in the present study this effect not was assessed, we do not discard that the cyclic di-nucleotide can also induce the same behavior in the nasal formulation RBD + N + c-di-AMP. In this regards, next experiments could be focused on confirming the induction of self-assembling particles based on RBD and N proteins after combination with the c-di-AMP, which offer advantages over other antigen presentation forms [61]. In summary, this study establishes that an intranasal vaccine formulation based on RBD and N recombinant subunits plus c-di-AMP as adjuvant elicits comprehensive specific immunity against SARS-CoV-2.

Statements and Declarations:

Acknowledgments:

The authors would like to thank the staff of the animal experimentation facilities at Center for Genetic Engineering and Biotechnology (CIGB, Cuba) for their assistance in the handling and maintenance of the animals.

Competing Interests and Funding:

This investigation received financial support from the Cuban Program for Vaccine Development and the Management Office to Funds and International Projects from Ministry of Science, Technology and Environment (CITMA), (#PN385LH007-048). The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Clinical trial number: not applicable

CRediT authorship contribution statement:

Iris Valdes: Writing–review & editing, Writing–original draft, Validation, Supervision, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Laura Lazo: Methodology, Investigation, Formal analysis, Conceptualization. Edith Suzarte: Methodology, Investigation, Formal analysis, Conceptualization. Karem Cobas: Methodology, Investigation. Yusleidi Pérez: Methodology. Rocío Garateix: Methodology. Enrique Noa: Methodology. Rubén Amaya: Methodology. Dionne Casillas: Methodology. Elias Nelson: Methodology. Yinet Cartaya: Methodology. Monica Bequet: Methodology, Formal analysis. Julio C. Aguilar: Methodology, Conceptualization. Carlos A. Guzmán: Conceptualization, Writing–review & editing. Gerardo Guillen: Writing–review & editing, Conceptualization, Resources, Funding acquisition, Project administration.

References

1. COVID-19 Coronavirus Pandemic. 2024. https://www.worldometers.info/coronavirus/
2. Almadhi MA, Abdulrahman A, Sharaf SA, et al. The high prevalence of asymptomatic SARS-CoV-2 infection reveals the silent spread of COVID-19. Int J Infect Dis. 105 (2021): 656-61.
3. Ma Q, Liu J, Liu Q, et al. Global percentage of asymptomatic SARS-CoV-2 infections among the tested population and individuals with confirmed COVID-19 diagnosis: a systematic review and meta-analysis. JAMA Netw Open. 4 (2021): e2137257.
4. COVID-19 vaccination, World data. 2023. https://data.who.int/dashboards/covid19/vaccines
5. Pang J, Wang MX, Ang IYH, T, et al. Potential rapid diagnostics, vaccine and therapeutics for 2019 novel coronavirus (2019-nCoV): a systematic review. J Clin Med. 9 (2020): 623.
6. Chen WH, Strych U, Hotez PJ, Bottazzi ME. The SARS-CoV-2 vaccine pipeline: an overview. Curr Trop Med Rep. 7 (2020): 61-4.
7. Sunagar R, Singh A, Kumar S. SARS-CoV-2: immunity, challenges with current vaccines, and a novel perspective on mucosal vaccines. Vaccines (Basel). 11 (2023): 849.
8. Dutta A. COVID-19 waves: variant dynamics and control. Sci Rep. 12 (2022): 9332.
9. Abebe EC, Dejenie TA. Protective roles and protective mechanisms of neutralizing antibodies against SARS-CoV-2 infection and their potential clinical implications. Front Immunol. 14 (2023): 1055457.
10. Moss P. The T cell immune response against SARS-CoV-2. Nat Immunol. 23 (2022): 186-93.
11. Matchett WE, Joag V, Stolley JM, et al. Cutting edge: nucleocapsid vaccine elicits spike-independent SARS-CoV-2 protective immunity. J Immunol. 207 (2021): 376-9.
12. Hong SH, Oh H, Park YW, et al. Immunization with RBD-P2 and N protects against SARS-CoV-2 in nonhuman primates. Sci Adv. 7 (2021): eabg7156.
13. Dangi T, Class J, Palacio N, et al. Combining spike- and nucleocapsid-based vaccines improves distal control of SARS-CoV-2. Cell Rep. 36 (2021): 109664.
14. Mendoza-Ramírez NJ, García-Cordero J, Shrivastava G, Cedillo-Barrón L. The key to increase immunogenicity of next-generation COVID-19 vaccines lies in the inclusion of the SARS-CoV-2 nucleocapsid protein. J Immunol Res. 2024 (2024): 9313267.
15. Dutta NK, Mazumdar K, Gordy JT. The nucleocapsid protein of SARS-CoV-2: a target for vaccine development. J Virol. 94 (2020): e00647-20.
16. López-Muñoz AD, Kosik I, Holly J, Yewdell JW. Cell surface SARS-CoV-2 nucleocapsid protein modulates innate and adaptive immunity. Sci Adv. 8 (2022): eabp9770.
17. Ahlén G, Frelin L, Nikouyan N, et al. The SARS-CoV-2 N protein is a good component in a vaccine. J Virol. 94 (2020): e01279-20.
18. Lobaina Y, Chen R, Suzarte E, et al. The nucleocapsid protein of SARS-CoV-2, combined with ODN-39M, is a potential component for an intranasal bivalent vaccine with broader functionality. Viruses. 16 (2024): 418.
19. Rabdano SO, Ruzanova EA, Pletyukhina IV, et al. Immunogenicity and in vivo protective effects of recombinant nucleocapsid-based SARS-CoV-2 vaccine Convacell®. Vaccines (Basel). 11 (2023): 874.
20. Phatarphekar A, Vidyadhar Reddy GEC, Gokhale A, et al. RelCoVax®, a two antigen subunit protein vaccine candidate against SARS-CoV-2 induces strong immune responses in mice. Vaccine. 40 (2022): 4522-30.
21. Sathe N, Shaikh S, Bhavsar M, et al. Safety, immunogenicity and efficacy of RelCoVax®, a dual receptor binding domain (RBD) and nucleocapsid (N) subunit protein vaccine candidate against SARS-CoV-2 virus. Vaccine. 42 (2024): 1051-64.
22. Castro JT, Azevedo P, Fumagalli MJ, et al. Promotion of neutralizing antibody-independent immunity to wild-type and SARS-CoV-2 variants of concern using an RBD-Nucleocapsid fusion protein. Nat Commun. 13 (2022): 4831.
23. Almeida GG, Pinto JA, Pinto PM, et al. Safety and immunogenicity of SpiN-Tec, a T-cell based RBD-Nucleocapsid chimeric vaccine for COVID-19. Vaccine. 64 (2025): 127756.
24. Tobias J, Steinberger P, Wilkinson J, et al. SARS-CoV-2 Vaccines: the advantage of mucosal vaccine delivery and local immunity. Vaccines (Basel). 12 (2024): 795.
25. Yusuf H, Kett V. Current prospects and future challenges for nasal vaccine delivery. Hum Vaccin Immunother. 13 (2017): 34-45.
26. An X, Martinez-Paniagua M, Rezvan A, et al. Single-dose intranasal vaccination elicits systemic and mucosal immunity against SARS-CoV-2. iScience. 24 (2021): 103037.
27. Kehagia E, Papakyriakopoulou P, Valsami G. Advances in intranasal vaccine delivery: a promising non-invasive route of immunization. Vaccine. 41 (2023): 3589-603.
28. Tiboni M, Casettari L, Illum L. Nasal vaccination against SARS-CoV-2: Synergistic or alternative to intramuscular vaccines? Int J Pharm. 603 (2021): 120686.
29. Germanó MJ, Giai C, Cargnelutti DE, et al. Receptor-binding domain-based SARS-CoV-2 vaccine adjuvanted with cyclic di-adenosine monophosphate enhances humoral and cellular immunity in mice. J Med Virol. 95 (2023): e28584.
30. Sanchez MV, Ebensen T, Schulze K, et al. Intranasal delivery of influenza rNP adjuvanted with c-di-AMP induces strong humoral and cellular immune responses and provides protection against virus challenge. PLoS One. 9 (2014): e104824.
31. Rueckert C, Rand U, Roy U, et al. Cyclic dinucleotides modulate induced type I IFN responses in innate immune cells by degradation of STING. FASEB J. 31 (2017): 3107-15.
32. Feikin DR, Higdon MM, Andrews N, et al. Assessing COVID-19 vaccine effectiveness against Omicron subvariants: Report from a meeting of the World Health Organization. Vaccine. 41 (2023): 2329-38.
33. Lau JJ, Cheng SMS, Leung K, et al. Real-world COVID-19 vaccine effectiveness against the Omicron BA.2 variant in a SARS-CoV-2 infection-naive population. Nat Med. 29 (2023): 348-57.
34. Mahrokhian SH, Tostanoski LH, Vidal SJ, Barouch DH. COVID-19 vaccines: Immune correlates and clinical outcomes. Hum Vaccin Immunother. 20 (2024): 2324549.
35. Miquel-Clopés A, Bentley EG, Stewart JP, Carding SR. Mucosal vaccines and technology. Clin Exp Immunol. 196 (2019): 205-14.
36. Gupta T, Gupta SK. Potential adjuvants for the development of a SARS-CoV-2 vaccine based on experimental results from similar coronaviruses. Int Immunopharmacol. 86 (2020): 106717.
37. Wang M, Jiang S, Wang Y. Recent advances in the production of recombinant subunit vaccines in Pichia pastoris. Bioengineered. 7 (2016): 155-65.
38. Limonta-Fernández M, Chinea-Santiago G, Martín-Dunn AM, et al. An engineered SARS-CoV-2 receptor-binding domain produced in Pichia pastoris as a candidate vaccine antigen. N Biotechnol. 72 (2022): 11-21.
39. Hernández-Bernal F, Ricardo-Cobas MC, Martín-Bauta Y, et al. A phase 3, randomised, double-blind, placebo-controlled clinical trial evaluation of the efficacy and safety of a SARS-CoV-2 recombinant spike RBD protein vaccine in adults (ABDALA-3 study). Lancet Reg Health Am. 21 (2023): 100497.
40. Islas-Vazquez L, Alvarado-Alvarado YC, Cruz-Aguilar M, et al. Evaluation of the Abdala vaccine: antibody and cellular response to the RBD domain of SARS-CoV-2. Vaccines (Basel). 11 (2023): 1787.
41. Thanh TT, Tu NTK, Nguyet LA, et al. Immunogenicity of Abdala COVID-19 vaccine in Vietnamese people after primary and booster vaccinations: A prospective observational study in Vietnam. Int J Infect Dis. 147 (2024): 107173.
42. Rabdano SO, Ruzanova EA, Vertyachikh AE, et al. N-protein vaccine is effective against COVID-19: Phase 3, randomized, double-blind, placebo-controlled clinical trial. J Infect. 89 (2024): 106288.
43. Mendoza-Ramírez NJ, García-Cordero J, Martínez-Frías SP, et al. Combination of recombinant proteins S1/N and RBD/N as potential vaccine candidates. Vaccines (Basel). 11 (2023): 864.
44. Nazarian S, Olad G, Abdolhamidi R, et al. Preclinical study of formulated recombinant nucleocapsid protein, the receptor binding domain of the spike protein, and truncated spike (S1) protein as vaccine candidates against COVID-19 in animal models. Mol Immunol. 149 (2022): 107-18.
45. Mamedov T, Yuksel D, Ilgin M, et al. Production and characterization of Nucleocapsid and RBD cocktail antigens of SARS-CoV-2 in Nicotiana benthamiana plant as a vaccine candidate against COVID-19. Vaccines (Basel). 9 (2021): 1337.
46. Lobaina Y, Chen R, Suzarte E, et al. A nasal vaccine candidate, containing three antigenic regions from SARS-CoV-2, to induce a broader response. Vaccines (Basel). 12 (2024): 588.
47. Ghaemi A, Roshani AP, Zargaran H, et al. Recombinant COVID-19 vaccine based on recombinant RBD/Nucleoprotein and saponin adjuvant induces long-lasting neutralizing antibodies and cellular immunity. Front Immunol. 13 (2022): 974364.
48. Rezaei A, Nazarian S, Samiei AH, et al. Antibodies produced toward recombinant RBD and Nucleocapsid neutralize SARS-COV-2. Avicenna J Med Biotechnol. 14 (2022): 270-7.
49. Dhama K, Dhawan M, Tiwari R, et al. COVID-19 intranasal vaccines: current progress, advantages, prospects, and challenges. Hum Vaccin Immunother. 18 (2022): 2045853.
50. Ning H, Zhang W, Kang J, et al. Subunit vaccine ESAT-6:c-di-AMP delivered by intranasal route elicits immune responses and protects against Mycobacterium tuberculosis infection. Front Cell Infect Microbiol. 11 (2021): 647220.
51. Junkins RD, Gallovic MD, Johnson BM, et al. A robust microparticle platform for a STING-targeted adjuvant that enhances both humoral and cellular immunity during vaccination. J Control Release. 270 (2018): 1-13.
52. Ebensen T, Libanova R, Schulze K, et al. Bis-(3',5')-cyclic dimeric adenosine monophosphate: strong Th1/Th2/Th17 promoting mucosal adjuvant. 29 (2011): 5210-20.
53. Lirussi D, Weissmann SF, Ebensen T, et al. Cyclic di-adenosine monophosphate: a promising adjuvant candidate for the development of neonatal vaccines. Pharmaceutics. 13 (2021): 188.
54. Libanova R, Becker PD, Guzmán CA. Cyclic di-nucleotides: new era for small molecules as adjuvants. Microb Biotechnol. 5 (2012): 168-76.
55. Feng S, Phillips DJ, White T, et al. Correlates of protection against symptomatic and asymptomatic SARS-CoV-2 infection. Nat Med. 27 (2021): 2032-40.
56. Wajnberg A, Amanat F, Firpo A, et al. Robust neutralizing antibodies to SARS-CoV-2 infection persist for months. 370 (2020): 1227-30.
57. Hagemann K, Riecken K, Jung JM, et al. Natural killer cell-mediated ADCC in SARS-CoV-2-infected individuals and vaccine recipients. Eur J Immunol. 52 (2022): 1297-307.
58. Yu Y, Wang M, Zhang X, et al. Antibody-dependent cellular cytotoxicity response to SARS-CoV-2 in COVID-19 patients. Signal Transduct Target Ther. 6 (2021): 346.
59. Dangi T, Sanchez S, Class J, et al. Improved control of SARS-CoV-2 by treatment with a nucleocapsid-specific monoclonal antibody. J Clin Invest. 132 (2022): e162282.
60. Rydyznski MC, Ramirez SI, Dan JM, et al. Antigen-Specific Adaptive Immunity to SARS-CoV-2 in Acute COVID-19 and Associations with Age and Disease Severity. 183 (2020): 996-1012.
61. Okba NMA, Widjaja I, van DB, et al. Particulate multivalent presentation of the receptor binding domain induces protective immune responses against MERS-CoV. Emerg Microbes Infect. 9 (2020): 1080-91.