An adjuvanted subunit SARS-CoV-2 spike protein vaccine provides protection against Covid-19 infection and transmission

Abstract

Recombinant protein approaches offer major promise for safe and effective vaccine prevention of SARS-CoV-2 infection. We developed a recombinant spike protein vaccine (called NARUVAX-C19) and characterized its ability when formulated with a nanoemulsion adjuvant to induce anti-spike antibody and T-cell responses and provide protection including against viral transmission in rodent. In mice, NARUVAX-C19 vaccine administered intramuscularly twice at 21-day interval elicited balanced Th1/Th2 humoral and T-cell responses with high titers of neutralizing antibodies against wild-type (D614G) and delta (B.1.617.2) variants. In Syrian hamsters, NARUVAX-C19 provided complete protection against wild-type (D614G) infection and prevented its transmission to naïve animals (n = 2/group) placed in the same cage as challenged animals (n = 6/group). The results contrasted with only weak protection seen with a monomeric spike receptor-binding domain (RBD) vaccine even when formulated with the same adjuvant. These encouraging results warrant the ongoing development of this COVID-19 vaccine candidate.

Fig. 1. Antigen-specific IgG, IgG1 and IgG2a titers in serum of BALB/c mice vaccinated with rRBD or rSpike protein ECD at 21 days after prime and booster immunization.

a Data post prime vaccination, b data post booster vaccination. Antigens were used at doses of 1.25, 2.5, and 5 µg with SWE adjuvant. For comparison, 5 µg vaccine was given without adjuvant and a negative control vaccine comprised SWE adjuvant with no antigen. Antibody levels are presented as geometric mean titers (GMT) with a 95% confidence interval. Differences in IgG titers were assessed using Tukey’s multiple comparisons test. A P < 0.05 value was considered as a significant difference.

Fig. 2. RBD-ACE2 blocking and virus-neutralizing antibody titers in BALB/c mice at 21 days after prime and booster immunization with SWE-adjuvanted rRBD and rSpike protein vaccines.

a, c Data post prime vaccination, b, d data post booster vaccination. Controls included rRBD or rSpike protein alone (Ag 5.0 µg alone) or SWE adjuvant alone (Control). RBD-ACE2 blocking antibodies were determined according to the level of inhibition: negative (<30%), low (30–59%), medium (60–89), and high (90≤). Virus neutralizing antibody levels are presented as geometric mean titers (GMT) with a 95% confidence interval. Differences in antibody titers between groups were assessed using Tukey’s multiple comparisons test.

Fig. 3. Antigen-stimulated cytokine production and CD4 + and CD8 + T cell proliferation in splenocytes from rRBD or rSpike protein vaccinated BALB/c mice.

Controls included rRBD or rSpike protein alone at 5.0 µg (Ag 5.0 µg alone) and SWE adjuvant-alone group (Control). Cytokine data (a) were presented as the difference (delta) in cytokine concentrations between samples with and without protein stimulation. CD4 + and CD8 + T cell proliferation (b) was calculated as the difference (Δ) in number of proliferating (CFSE + ) lymphocytes between stimulated vs non-stimulated cells. Statistical differences were assessed using Šídák’s multiple comparisons test. A P < 0.05 value was considered as a significant difference.

Fig. 4. Spike-specific IgG, RBD-ACE2 blocking antibody and neutralizing antibody levels in hamsters at 21 days after booster immunization with SWE-adjuvanted rRBD or rSpike protein as compared to convalescent sera.

a Data for spike-specific IgG, b RBD-ACE2 blocking antibody, c, d neutralizing antibody levels. Control refers to a group of animals injected with adjuvant-alone with no antigen. Convalescent refers to post-infection serum samples from four unvaccinated hamsters that had previously been challenged with the wild-type SARS-CoV-2 virus. Viral neutralizing antibodies were assessed against wild-type D614G (c) and delta variant (d) viruses. Differences between groups assessed using Dunnett’s multiple comparisons test.

Fig. 5. Vaccine protection in Syrian hamsters against SARS-CoV-2 infection and virus transmission.

To assess vaccine efficacy animals (n = 6/group) were intranasally infected with WT SARS-CoV-2 and the following parameters then measured: 1) changes in body weight (a; n = 3-6/group); virus excretion from oropharyngeal swabs Day 2 post challenge (e; n = 6/group) 2) viral load in nasal turbinates (n = 3/group) and lungs (n = 3/group) by PCR (represented by Ct; b, c) and culture of infectious virus (expressed as log₁₀ TCID₅₀/0.2 mL; f, g); 3) histopathological changes in the lungs (i, j), on Days 3 (n = 3/group) and 7 (n = 3/group) after challenge. Efficacy against virus transmission in contact sentinel animals (n = 2/group) was evaluated by detection of viral load (d, h), lung pathology scoring (i, j). Control included an adjuvant-alone group. Scale bars are 500 µm. Differences in the studied parameters between animal groups were assessed using Tukey’s (virus titer; Lung Histology), Dunnett’s (postchallenge weight), or Šídák’s (viral load in PCR) multiple comparisons test. A P < 0.05 value was considered as a significant difference. * in comparison with the control group. LOD limit of detection.

Fig. 6. Correlation matrix analysis between immune markers including various antibody measurements and measures of vaccine protection including weight changes, viral loads, and lung pathology.

The color refers to r value scale (−1 to 1) shown on the right. The number in each cell indicates the actual r value and the lower digits represent p-values. The analysis was conducted by multivariable Pearson correlation method.