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Localized and Systemic Immune Responses against SARS-CoV-2 Following Mucosal Immunization - PubMed

  • ️Fri Jan 01 2021

Localized and Systemic Immune Responses against SARS-CoV-2 Following Mucosal Immunization

Shaswath S Chandrasekar et al. Vaccines (Basel). 2021.

Abstract

The rapid transmission of SARS-CoV-2 in the USA and worldwide necessitates the development of multiple vaccines to combat the COVID-19 global pandemic. Previously, we showed that a particulate adjuvant system, quil-A-loaded chitosan (QAC) nanoparticles, can elicit robust immunity combined with plasmid vaccines when used against avian coronavirus. Here, we report on the immune responses elicited by mucosal homologous plasmid and a heterologous immunization strategy using a plasmid vaccine and a Modified Vaccinia Ankara (MVA) expressing SARS-CoV-2 spike (S) and nucleocapsid (N) antigens. Only the heterologous intranasal immunization strategy elicited neutralizing antibodies against SARS-CoV-2 in serum and bronchoalveolar lavage of mice, suggesting a protective vaccine. The same prime/boost strategy led to the induction of type 1 and type 17 T-cell responses and polyfunctional T-cells expressing multiple type 1 cytokines (e.g., IFN-γ, TNFα, IL-2) in the lungs and spleens of vaccinated mice. In contrast, the plasmid homologous vaccine strategy led to the induction of local mono and polyfunctional T-cells secreting IFN-γ. Outcomes of this study support the potential of QAC-nano vaccines to elicit significant mucosal immune responses against respiratory coronaviruses.

Keywords: COVID-19; SARS-CoV-2; heterologous vaccine; intranasal vaccine; nanovaccine.

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Conflict of interest statement

A.M.T. and Y.P. have financial interests in Pan Genome Systems, Inc., a startup company developing animal and human vaccines.

Figures

Figure 1
Figure 1

Design of SARS-CoV-2 vector vaccine constructs expressing S and N proteins. (a) Gene map of pCMV plasmid expressing Truncated S (TrS) protein. (b) Gene map of pCMV plasmid backbone expressing N protein. (c) MVA vaccine construct expressing TrS protein with the addition of C-terminal 6X His tag. (d) The MVA construct expressing N protein. All gene maps were generated using Snapgene software. (e) Western blot analysis with anti 6xHis-HRP antibody (left) and polyclonal mouse anti-SARS-CoV-2 spike sera (right) confirming expression of S protein from vaccine constructs. Lanes are as follows: Supernatant (lanes 2) HEK 293T cells transfected with control plasmid, supernatant (lanes 1 and 7) HEK 293T cells transfected with pCMV-TrS plasmid. Supernatant (lanes 3 and 6) from chicken embryonic fibroblast (CEF) cells infected with MVA-TrS and control non-infected supernatant (lane 4). Purified recombinant SARS-CoV-2 S Glycoprotein (BEI resources-NR-52396). (f) Western blot analysis with anti 6xHis-HRP antibody N from vaccine constructs. Lanes are as follows: Cell pellet (lane 3) HEK 293T cells transfected with control plasmid, cell pellet (lane 1) HEK 293T cells transfected with pCMV-N plasmid. Cell pellet (lane 2) from CEF cells infected with MVA-N.

Figure 2
Figure 2

Generation of humoral immune responses in C57BL/6 mice following immunization with different vaccine constructs. (a) Outline for vaccine construct and immunization protocol using groups of C57BL/6 mice vaccinated with 3 doses of pQAC-CoV (I.N) or pQAC-CoV (I.M) with 3-week interval. Another group of C57BL/5 mice were vaccinated with pQAC-CoV (I.N) at week-0 followed by boost with MVA-CoV (I.N) at week-6. (b) ELISA titers of SARS-CoV-2 S-specific IgG in mice sera, (c) ELISA titers of SARS-CoV-2 spike receptor-binding domain (RBD)-specific IgG in mice serum and (d) ELISA titers of SARS-CoV-2 S-specific IgA in bronchoalveolar lavage (BAL), significance (*, p < 0.05, ****, p < 0.0001) was determined by two-way ANOVA. Data show mean ± SEM.

Figure 3
Figure 3

Heterologous vaccine strategy elicits spike-binding antibody responses. Groups of C57BL/5 mice were either unvaccinated (PBS) or immunized with pQAC/MVA-CoV (I.N). Serum and BAL samples were collected 3 weeks post boost. (a) SARS-CoV-2 spike-specific serum IgG binding endpoint titers measured by ELISA. (b) Serial serum dilutions of IgG binding to SARS-CoV-2 spike protein. (c) SARS-CoV-2 spike-specific BAL IgA binding endpoint titers measured by ELISA. (d) Serial BAL dilutions of IgA binding to SARS-CoV-2 spike protein. Significance (*, p < 0.05) was determined by one-way ANOVA. Data show mean ± SEM.

Figure 4
Figure 4

Heterologous vaccine strategy elicits SARS-CoV-2 neutralizing antibody responses. Groups of C57BL/5 mice were either unvaccinated (PBS) or immunized with pQAC/MVA-CoV (I.N). Serum and BAL samples were collected 3 weeks post boost. (a) 50% serum neutralization titer of pseudo-virus expressing SARS-CoV-2 spike, (b) serum neutralization titer of wild-type SARS-CoV-2, isolate USA-WA1/2020, (c) 50% BAL neutralization titer of pseudo-virus expressing SARS-CoV-2 spike, and (d) BAL neutralization titer of wild-type SARS-CoV-2, isolate USA-WA1/2020. Significance (*, p < 0.05; **, p < 0.01) was determined by one-way ANOVA. Data show mean ± SEM. The dotted line indicates the lower limit of detection.

Figure 5
Figure 5

SARS-CoV-2 spike specific T cell responses in lungs of vaccinated C57BL/6 mice. Intracellular cytokine staining was performed on lungs harvested 3 weeks after final boost to assess T-cell responses. (a) Type 1 helper (Th1) responses (IFN-γ or TNFα or IL-2+), (b) type 2 helper (Th2) responses (IL-13+), (c) type 17 helper (Th17) responses (IL-17+), (d) type 1 cytotoxic (Tc1) responses (IFN-γ or TNFα or IL-2+), (e) type 2 cytotoxic (Tc2) responses (IL-13+), (f) type 17 cytotoxic (Tc17) responses (IL-17+) intracellular cytokine staining assays for lung T-cells in response to recombinant SARS-CoV-2 spike stimulation. Samples with lower live cells (<10,000) were excluded from analysis. Significance (**, p < 0.01) was determined by ANOVA compared to PBS controls. Data show mean ± SEM.

Figure 6
Figure 6

SARS-CoV-2 spike specific T cell responses in spleen of vaccinated C57BL/6 mice. Intracellular cytokine staining was performed on spleens harvested 3 weeks after final boost to assess T-cell responses. (a) Type 1 helper (Th1) responses (IFN-γ or TNFα or IL-2+), (b) type 2 helper (Th2) responses (IL-13+), (c) type 17 helper (Th17) responses (IL-17+), (d) type 1 cytotoxic (Tc1) responses (IFN-γ or TNFα or IL-2+), (e) type 2 cytotoxic (Tc2) responses (IL-13+), (f) type 17 cytotoxic (Tc17) responses (IL-17+) intracellular cytokine staining assays for spleen T-cells in response to recombinant SARS-CoV-2 spike stimulation. Significance (*, p < 0.05; **, p < 0.01) was determined by ANOVA compared to PBS controls. Data show mean ± SEM.

Figure 7
Figure 7

Immunization with SARS-CoV-2 vaccine constructs induces polyfunctional T cells. (a) CD4+ IFN-γ, TNFα, IL-2, and IL-17, (b) CD 4+ IL-2 and IL-17 (c) CD8+ IFN-γ, TNFα, IL-2, and IL-17 polyfunctional T-cells in the spleen of vaccinated mice. (d) CD4+ IFN-γ, TNFα, IL-2, and IL-17, (e) CD8+ IFN-γ and TNFα, (f) CD8+ IFN-γ, TNFα, and IL-17 polyfunctional T-cells in the lungs of vaccinated mice following intracellular cytokine staining assays for after recombinant SARS-CoV-2 spike stimulation. Significance (*, p < 0.05; **, p < 0.01) was determined by ANOVA compared to PBS controls. Data show mean ± SEM.

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