Evaluation of the humoral and mucosal immune response of a multiepitope vaccine against COVID-19 in pigs

Abstract

Introduction: This study evaluated the immune response to a multiepitope recombinant chimeric protein (CHIVAX) containing B- and T-cell epitopes of the SARS-CoV-2 spike's receptor binding domain (RBD) in a translational porcine model for pre-clinical studies.

Methods: We generated a multiepitope recombinant protein engineered to include six coding conserved epitopes from the RBD domain of the SARS-CoV-2 S protein. Pigs were divided into groups and immunized with different doses of the protein, with serum samples collected over time to determine antibody responses by indirect ELISA and antibody titration. Peptide recognition was also analyzed by Western blotting. A surrogate neutralization assay with recombinant ACE2 and RBDs was performed. Intranasal doses of the immunogen were also prepared and tested on Vietnamese minipigs.

Results: When the immunogen was administered subcutaneously, it induced specific IgG antibodies in pigs, and higher doses correlated with higher antibody levels. Antibodies from immunized pigs recognized individual peptides in the multiepitope vaccine and inhibited RBD-ACE2 binding for five variants of concern (VOC). Comparative antigen delivery methods showed that both, subcutaneous and combined subcutaneous/intranasal approaches, induced specific IgG and IgA antibodies, with the subcutaneous approach having superior neutralizing activity. CHIVAX elicited systemic immunity, evidenced by specific IgG antibodies in the serum, and local mucosal immunity, indicated by IgA antibodies in saliva, nasal, and bronchoalveolar lavage secretions. Importantly, these antibodies demonstrated neutralizing activity against SARS-CoV-2 in vitro.

Discussion: The elicited antibodies recognized individual epitopes on the chimeric protein and demonstrated the capacity to block RBD-ACE2 binding of the ancestral SARS-CoV-2 strain and four VOCs. The findings provide proof of concept for using multiepitope recombinant antigens and a combined immunization protocol to induce a neutralizing immune response against SARS-CoV-2 in the pig translational model for preclinical studies.

Keywords: COVID-19; SARS-CoV-2; humoral response; mucosal immunity; multiepitopic vaccine; recombinant protein.

Figure 1

(A) Immunization schemes for two independent experiments, the first using subcutaneous (SC) (I) route and the second using SC, IN, and a combined SC/IN protocol (II) (B) Selected peptides that make up the multi-epitope vaccine (CHIVAX). Name, size, sequence, and type of epitope predicted in the designed RBD peptides. (C) A polyacrylamide gel was loaded with the induced culture medium (lane 2), induced E coli soluble protein lysate fraction (lane 3), induced E coli lysate insoluble fraction (lane 4), non-induced culture medium (lane 5), non-induced E coli lysate soluble protein fraction (lane 6) and non-induced E coli lysate insoluble fraction (lane 7). (D) The recombinant protein was loaded onto a polyacrylamide gel, transferred to a nitrocellulose membrane, and subsequently divided into 3 membranes. The serum sample from a COVID-19-positive human was incubated with the first membrane (lane 1), the serum sample from a COVID-19-negative human was incubated with the second membrane (lane 2), and the control monoclonal anti-His tag antibody was incubated with the third membrane (lane 3). These samples were employed to detect the recombinant protein on the membrane. (E) The purified and dialyzed protein fractions were loaded onto a polyacrylamide gel under denaturing conditions for subsequent analysis by SDS-PAGE. The protein fractions ranging from the second to the eightieth were loaded onto the gel individually in lanes 2 to 9. The black arrows indicate the molecular size of target protein (48 KDa).

Figure 2

Serum antibody production in pigs immunized with CHIVAX 17.4 by the subcutaneous route. (A) Effect of the immunization dose on the production of specific IgG antibodies to the chimeric protein (samples diluted 1:2000). The animals were immunized two times (red arrows). (B) Specific IgG antibody titers to the chimeric protein detected in the serum of pigs 31 days after the first immunization with the chimeric protein in the groups immunized with 30, 60 or 100 µg, and the control group. The results are expressed as the mean ± SEM of five independent experiments in quadruplicates. The blue plus symbol represents the statistical difference with the control group, the green asterisk represents differences with 60 µg, and the number violet symbol differences with 30 µg group. Unpaired t-test with Tukey-Kramer´s comparison. The differences were considered significant at: p ≤ 0.01 for two symbols, p ≤ 0.001 for three symbols, and p ≤ 0.0001 for four symbols.

Figure 3

Recognition of individual peptides by the sera of immunized pigs. (A) Individual peptide recognition by the serum of animals immunized with 100 µg of CHIVAX protein. (B) Individual peptide recognition by the serum of animals immunized with 60 µg. (C) Individual peptide recognition by the serum of animals immunized with 30 µg. (D) Detection of individual peptides by the serum of animals in the control group, Dilution of serum in all graphs is 1:80. The results are expressed as the mean ± SEM of five independent experiments in quadruplicate. Small letters express significant differences between all the samples with letters different (p ≤ 0.01) by Unpaired t-test with Sidak´s multiple comparisons.

Figure 4

Sera of immunized pigs block the RBD-ACE2 binding of four VOCs and the ancestral strain of SARS-CoV-2. (A) The percentage inhibition of RBD-ACE2 binding of serum from pigs immunized with 30 µg of recombinant protein. (B) The percentage inhibition of RBD-ACE2 binding of serum from pigs immunized with 60 µg of recombinant protein, and (C) The percentage inhibition of RBD-ACE2 binding of serum from pigs immunized with 100 µg of recombinant protein. All graphs represent inhibition percentage (%) against Wuhan-1 (D614G), Alpha (B.1.1.7), Gamma (B.1.1.248), Beta (B.1.351), and Delta (B.1617.2) variants determined at 450 nm. The results are expressed as the mean ± SEM of five independent experiments in triplicate. Each experiment corresponds to an animal immunized with 30, 60, 100 µg of vaccines for duplicated. Each symbol represents an individual. The threshold was 30% inhibition. Modified from .

Figure 5

Serum and mucosal IgG and IgA antibody responses in vaccinated minipigs. The animals were immunized at days 0, 7, 14 and 42 by the subcutaneous (SC, red triangles), intranasal (IN, green squares), or a combined SC (days 0 and 7)/IN (days 21 and 42) (purple diamonds) route, and their IgG and IgA concentration were determined by a quantitative ELISA. Serum IgG (A), and IgA (B) antibodies. Nasal IgG (C), and IgA (D) antibodies. Saliva IgG (E), and IgA (F) antibodies. Each symbol is the mean value of 4-6 animals ± SEM. Statistical significance was tested by paired multiple t-tests with the Tukey method. The blue plus symbol represents the statistical difference with the control group, the green and red asterisks represent differences between the IN and SC groups respectively, and the number violet symbol differences with the SC/IN group. The level of significance is as follows: One symbol p ≤ 0.05, two symbols p ≤ 0.01, three symbols p ≤ 0.001, and four symbols p ≤ 0.0001. Unpaired t-test with Mann-Whitney comparison. Scales are different in the graphs.

Figure 6

Bronchioalveolar lavage IgG and IgA anti CHIVAX response in immunized pigs, and neutralizing activity evaluation. Pigs were immunized on days 0, 7, 14 and 42 by the subcutaneous (SC), intranasal (IN), or a combined SC (days 0 and 7)/IN (days 21 and 42) route, and their IgG (A) and IgA (B) concentration were determined by a quantitative ELISA in serum samples from day 56 after the first immunization. The horizontal bar is the mean, and each symbol represents one animal. The scales of the graphs are different. Statistical significance: *p≤0.05, **p≤0.01. Unpaired t-test with Mann-Whitney comparison. (C) Anti SARS-CoV-2 neutralizing antibody activity in serum of vaccinated minipigs. Samples were taken at day 56 after four immunizations by the SC, IN, or a combined SC/IN route, and their neutralizing activity was tested using the SARS-CoV-2 Surrogate Virus Neutralization Test (sVNT). Statistical significance was tested by an unpaired t-Student test with Mann-Whitney comparison. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.