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

Abstract

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.

Fig. 1. Human antibody and IFNγ responses to N and RBD polypeptides.

a Needle plot indicating the number of amino acid divergence points in the protein sequence of Nucleocapsid (N) and Spike (S) of five variants in relation to the N and S sequences from the SARS-CoV-2 lineage B (Wuhan). The peaks with circles indicate the position of the most frequent amino acid changes. The height of the peaks indicates the frequency of the changes in each divergent point. The blue and purple circles indicate common mutations and those observed on variants of concern, respectively. The sum of the amino acid changes for each segment (S1, RBD, and S2) of the S and N proteins considering the 6 SARS-CoV2 lineages is also shown. The vertical black and red lines below the bars illustrating the N and S polypeptides indicate, respectively, the position of each putative CD4⁺ T and CD8⁺ T cell epitopes identified by in silico epitope prediction. b-g UMAP projection of FACS data showing IFN-γ production by different CD4⁺ (b) or CD8⁺ (c) T cell compartments, determined by the surface markers CD45RO, CD27, and CD69. Data are also represented by the percentage of each subpopulation in total CD4⁺ (d, f) or CD8⁺ (e, g) IFN-γ⁺ T cells. The number of individuals used in these experiments was 5 controls, 8 vaccinated, and 8 convalescents. Statistical analysis of IFN-γ production (f, g) was performed using two-sided Wilcoxon-matched pairs signed rank; “ns” indicate that difference is not statistically significant and NS = non-stimulated PBMCs.

Fig. 2. Evaluation of immune response and protection elicited by vaccination with RBD, N or SpiN proteins associated with Poly ICLC.

a Immunization protocol used in the experiments shown in Figs. 2–8 to analyze the immune response and protection against SARS-CoV-2. Antigen-specific IgG antibodies were measured in the bronchoalveolar lavage (BALF) at 1:1 (b) and serially diluted sera from immunized mice (c). Levels of IFNγ (d) and IL-10 (e) were measured on culture supernatant of splenocytes stimulated with RBD or N antigens. b-e, n = 3/group. f, g Body weight and survival of K18-hACE2 mice immunized with RBD, N or SpiN associated with Poly ICLC and challenged with the Wuhan strain of SARS-CoV-2. h, i Viral load, measured by RT-PCR, in the lung and brain tissues from unvaccinated and mice vaccinated with either RBD, N, or SpiN associated with Poly ICLC. f–i n = 4/group. j, k Levels of circulating total IgG specific for either N, RBD and S proteins or inactivated SARS-CoV-2 in mice immunized with SpiN (n = 4/group). l, m Total IgG response of convalescent individuals to either N or RBD and inactivated SARS-CoV-2 or S (n = 5/group), respectively. n Levels of anti-N (left panel) and anti-RBD (right panel) antibodies were measured in BALF from mice that received Poly ICLC alone or SpiN plus Poly ICLC (n = 4/group). j–n n = 3-5/group. Levels of anti-inactivated SARS-CoV-2 (o) and neutralizing antibodies (p) in pooled sera from mice immunized with PBS, N, RBD or SpiN and from COVID-19 convalescent individuals. q Immunofluorescence of SARS-CoV-2 infected cells stained with sera from mice that received adjuvant alone (left panel) or were immunized with N (left middle panel), RBD (right middle panel) or SpiN (right panel) associated with Poly ICLC. Arrows are pointing to small vesicles containing the S protein that reacted with anti-RBD and anti-SpiN polyclonal antibodies. Statistical analysis of IgG measured in BALF (b, n) was performed using two-tailed unpaired t-test. Cytokine measurements (d, e) were analyzed through Two-way ANOVA followed by Tukey’s multiple tests. Data are representative of two independent experiments. b, d–e, h–i, n, data are presented as mean values ± SEM. * P < 0.05 and *** P < 0.001.

Fig. 3. Levels of cytokines and anti-SARS-CoV-2 IgG isotypes from mice immunized with SpiN.

Levels of cytokines in the supernatant of splenocytes from control and vaccinated mice stimulated (stim) or not (NS) with the RBD (a) and N proteins (b), as measured by the cytometric bead array (CBA). The levels of IgG1 and IgG2c specific for RBD (c), N (d), SARS-CoV-2 (e) and S (f) proteins in sera from mice that received adjuvant alone (black symbols in the bottom) or were immunized with SpiN protein associated with Poly ICLC. Data are representative of two independent experiments, n = 3–4 mice/group. Statistical analysis of CBA was performed with multiple t tests, and antibody measurements were analyzed using Two-way ANOVA followed by Sidak’s multiple comparisons test. **** P < 0.0001.

Fig. 4. T lymphocytes response in the spleen and lungs of SpiN-immunized mice.

The frequency of activated CD4⁺ T (a) and CD8⁺ T (b) lymphocytes was evaluated by measuring the expression of cell surface CD44, data are presented as mea ± SEM. c UMAP projection of FACS data, in which splenocytes were gated on either CD4⁺ T or CD8⁺ T cell populations. Staining with anti-CD62L, anti-CD44 and anti-IFN-γ was used to define IFNγ-producing naïve, central memory (Tcm), and effector/effector memory (Teff/em) T cells. The frequency of naïve, memory and effector CD4⁺ T and CD8⁺ T populations producing IFN-γ with or without stimulation with SpiN are shown in panels d and e respectively. f, g Imunophenotyping of the lungs from mice immunized or not. The cells were cultured with SpiN protein and characterized by flow cytometry as CD4⁺ CD69⁺ CD103⁺ (f, left panel), CD4⁺ CD69⁺ CD103⁺ IFN-γ⁺ (f, middle panel), CD4⁺ CD69⁺ CD103⁺ TNF⁺ (f, right panel), CD8⁺ CD69⁺ CD103⁺ (g, left panel), CD8⁺ CD69⁺ CD103⁺ IFN-γ⁺ (g, middle panel) and CD8⁺ CD69⁺ CD103⁺ TNF⁺ (g, right panel). Data are representative of two independent experiments, n = 3–4 mice/group. Statistical analysis was performed using two-sided Mann–Whitney. * P < 0.05 and ** P < 0.01.

Fig. 5. Protective immunity in hamsters immunized with SpiN and challenged with SARS-CoV-2.

a The levels of total IgG anti-N and anti-RBD in sera of hamsters vaccinated with SpiN associated to Poly ICLC. b Levels of neutralizing antibodies in the sera from hamsters vaccinated with SpiN + Poly ICLC, in comparison with sera from convalescent individuals. c Viral load measured by RT-PCR in the lungs of control and hamsters immunized with SpiN plus Poly ICLC at 4 days post-infection (dpi) with SARS-CoV-2. Data are presented as mean + /- SEM (b, c). d Histopathological analysis at 2,5x, 5x, and 20x magnification of hamsters at 4 dpi with the Wuhan strain, shows that the lungs in the PBS group display an accentuated diffuse alveolar wall thickening, with moderate multifocal collapse (asterisk), associated with accentuated diffuse congestion (black arrow) and mixed inflammatory infiltrate (mononuclear and polymorphonuclear cells). In the bronchial space, inflammatory cells are noted with a predominance of neutrophils associated with cellular debris (arrowhead) (d, right panels). In comparison, hamsters immunized with SpiN adjuvanted with Poly ICLC had mild focal congestion (black arrow) with alveolar space preservation (d, left panels). a–d Data are representative of two independent experiments. a n = 5 hamsters/group. d PBS n = 3, SpiN + Poly ICLC n = 4, Convalescent n = 3. c Pooled data from two independent experiments, n = 7–8 hamsters/group. Data of viral load quantification was analyzed through two-sided Mann–Whitney test.

Fig. 6. Protective immunity in mice immunized with the SpiN chimeric protein and challenged SARS-CoV-2.

Mice were challenged with the Wuhan strain of SARS-CoV-2. Body weight (a), survival (b) and viral load in lungs (c) and brain (d) were evaluated in K18-hACE-2 mice immunized with SpiN plus Poly ICLC and controls that received Poly ICLC alone. e Titers of nAbs in the sera from control and immunized mice at 5 dpi. Convalescent patients were used as positive control. c–e Data are presented as mean ± SEM. f Histopathological analysis at 2,5x, 5x, and 20x magnification of the lungs from Poly ICLC or SpiN + Poly ICLC groups, at 5 dpi. Black arrows: congestion; white arrows: intra-alveolar exudate; white star: hemorrhagic foci; asterisks: alveolar collapse; red arrows: inflammatory infiltrate. g, h mRNA expression of cytokines (g) and chemokines (h) quantified by qRT-PCR in mice lungs at 5 dpi. i–o, Frequency of myeloid (i) and lymphoid (j) as well as total numbers of neutrophils (k), monocytes (l), monocyte derived dendritic cells (m), resident CD8⁺ T cells (n) and conventional dendritic cells (o) in the lungs of control, vaccinated, challenged (infected) or not (NI) with SARS-CoV2 at 5 dpi. g–o Data are presented as mean ± SEM. a, b Individual values of pooled data from two independent experiments, Poly ICLC n = 4 and SpiN + Poly ICLC n = 7. c–o Data are representative of two independent experiments, n = 3 mice/group (c–h). i–o, n = 4 PBS N.I. and infected, n = 3 SpiN + Poly ICLC N.I., n = 6 SpiN + Poly ICLC infected. Statistical analysis of weight measurements (a) was performed using Two-way ANOVA. Survival analysis (b) was performed with Log–rank test. Data of viral quantification (c, d) was analyzed using Two-way ANOVA followed by Sidak’s multiple comparisons test. qRT-PCR data (g, h) was analyzed with unpaired two-tailed t tests. Flow cytometry of the lungs (i–o) was analyzed by Kruskal–Wallis followed by Dunn’s multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001.

Fig. 7. The importance of T lymphocytes and antibodies in the protective immunity elicited by SpiN.

K18-hACE2 mice were immunized with SpiN adjuvanted with Poly ICLC (a, b) or Covishield (e, f) and treated with anti-CD4⁺, anti-CD8⁺ or both on days −3, −2, and −1 before infectious challenge. Naïve K18-hACE2 mice were administered, at day −1 before infection, with pooled sera from control or SpiN- (c, d) or Covishield- (g, h) immunized mice. Body weight (a, c, e, g) and survival (b, d, f, h) were monitored for 11 days after infection with the SARS-CoV-2 Wuhan strain. a–h 4 mice/group.

Fig. 8. SpiN-induced protective immunity to the Delta and Omicron variants of SARS-CoV-2.

K18-hACE2 mice were vaccinated with SpiN associated to Poly ICLC and challenged 30 days after the second dose with 5 × 10⁴ PFU of Delta (a, b) or 2.5 × 10⁴ PFU of Omicron (c, d) variants. Body weight (a, c) and survival (b) were measured for 11 days. d Viral load was measured by RT-PCR at 6 dpi with Omicron, data are presented as mean + /- SEM. a–e 6 mice/group. e Photomicrographs of lung tissue from mice infected with SARS-CoV-2 Omicron strain. Furthermore, in control mice that received Poly ICLC only, the lungs showed a diffuse interstitial pneumonia characterized by a mixed inflammatory infiltration (mononuclear and polymorphonuclear cells), accompanied by intense congestion (black arrows), intra-alveolar exudate (white arrows with black outline), hemorrhagic foci (white star) and areas of alveolar collapse (asterisks) (e, top panels). In the immunized group it is noted the preservation of the pulmonary architecture, with the presence of predominantly mononuclear inflammatory infiltrate (e, bottom panels). a–e Data are representative of two independent experiments. Statistical analysis of viral load (d) was performed with Two-way ANOVA followed by Sidak’s multiple comparisons test. **** P < 0.0001.