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. 2023 May 13;8(1):68.
doi: 10.1038/s41541-023-00665-3.

Intranasal COVID-19 vaccine induces respiratory memory T cells and protects K18-hACE mice against SARS-CoV-2 infection

Béré K Diallo #  1 , Caitlín Ní Chasaide #  1 , Ting Y Wong #  2 , Pauline Schmitt  1 , Katherine S Lee  2 , Kelly Weaver  2 , Olivia Miller  2 , Melissa Cooper  2 , Seyed D Jazayeri  1 , F Heath Damron  2 , Kingston H G Mills  3
Affiliations

Intranasal COVID-19 vaccine induces respiratory memory T cells and protects K18-hACE mice against SARS-CoV-2 infection

Béré K Diallo et al. NPJ Vaccines. .

Abstract

Current COVID-19 vaccines prevent severe disease, but do not induce mucosal immunity or prevent infection with SARS-CoV-2, especially with recent variants. Furthermore, serum antibody responses wane soon after immunization. We assessed the immunogenicity and protective efficacy of an experimental COVID-19 vaccine based on the SARS-CoV-2 Spike trimer formulated with a novel adjuvant LP-GMP, comprising TLR2 and STING agonists. We demonstrated that immunization of mice twice by the intranasal (i.n.) route or by heterologous intramuscular (i.m.) prime and i.n. boost with the Spike-LP-GMP vaccine generated potent Spike-specific IgG, IgA and tissue-resident memory (TRM) T cells in the lungs and nasal mucosa that persisted for at least 3 months. Furthermore, Spike-LP-GMP vaccine delivered by i.n./i.n., i.m./i.n., or i.m./i.m. routes protected human ACE-2 transgenic mice against respiratory infection and COVID-19-like disease following lethal challenge with ancestral or Delta strains of SARS-CoV-2. Our findings underscore the potential for nasal vaccines in preventing infection with SARS-CoV-2 and other respiratory pathogen.

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Conflict of interest statement

Kingston Mills is an inventor on a patent application around a novel vaccine adjuvant and has collaborative research funding from and acts as consultant to Pharmaceutical and Biotech companies. Béré K. Diallo, Caitlín Ní Chasaide, Pauline Schmitt are inventors on a patent application around a novel vaccine adjuvant. The other authors have no competing interests.

Figures

Fig. 1
Fig. 1. The Spike-LP-GMP vaccine delivered by i.n. and/or i.m. routes induces potent antibodies and peripheral T cell responses.
a Schematic of experimental design; C57BL/6 mice (n = 7/group) were immunized twice (0 and 21 days) i.n./i.n., i.m./i.n. or i.m./i.m. with the Spike-LP-GMP vaccine or PBS and euthanized on day 35 for samples collection. Spike-specific IgG b, IgG2c c, and IgA d, and hACE-2-Spike neutralizing antibodies e in the serum, lung homogenate and nasal wash. f Antigen-specific IFN-γ, IL-17 and GrB production by cervical and inguinal LN cells stimulated for 72 h with medium alone or Spike trimer (2.5 µg/ml), quantified by ELISA. Data were analyzed by one-way ANOVAs followed by post hoc Tukey’s test for multiple comparisons (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001), error bars, SEM. Results are representative of three independent experiments.
Fig. 2
Fig. 2. Immunization with Spike-LP-GMP by i.n. route elicits CD4+ TRM and Tfh cells in the respiratory mucosa.
Mice were immunized as described in Fig. 1, lung and nasal tissue samples were collected on day 35. a Number of CD4+ TRM cells (CD44+CD62L-CD45-CD69+) in the lung and nasal tissue. b Representative flow cytometry plot of lung CD4+ TRM cells. c-f Number of Spike-specific CD4+ TRM cells secreting IFN-γ c, TNF d, IL-17 e, or GrB f in the lung and nasal tissue following 18 h stimulation with Spike trimer (2.5 µg/ml). g Representative flow cytometry plot of IL-17 and TNF secreting lung CD4+ TRM cells. h Number of CD4+ Tfh cells in the lung and nasal tissue. Data were analyzed by one-way ANOVAs followed by post hoc Tukey’s test for multiple comparisons (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001), error bars, SEM. Results are representative of three independent experiments.
Fig. 3
Fig. 3. Immunization with Spike-LP-GMP by i.n. route elicits CD8+ TRM in the respiratory mucosa.
Mice were immunized as described in Fig. 1, lung and nasal tissue samples were collected on day 35 and stimulated for 18 h with Spike trimer (2.5 µg/ml). Supernatants were collected for cytokine quantification by ELISA and cells analyzed by flow cytometry a Number of CD8+ TRM cells (CD44+CD62L-CD45-CD69+) in the lung and nasal tissue. b Representative flow cytometry plot of lung CD8+ TRM cells. c–e Number of Spike-specific CD8+ TRM cells secreting TNF c, GrB d, or IFN-γ e in the lung and nasal tissue. f Representative flow cytometry plot of TNF and GrB secreting CD8+ TRM cells (lung). Antigen-specific IFN-γ, IL-17 and GrB production quantified by ELISA on supernatants of lung g and nasal tissue h cells stimulated for 18 h with Spike trimer (2.5 µg/ml). Data were analyzed by one-way ANOVAs followed by post hoc Tukey’s test for multiple comparisons (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001), error bars, SEM. Results are representative of three independent experiments.
Fig. 4
Fig. 4. The Spike-LP-GMP vaccine induces persistent systemic and mucosal immune response in mice.
a Schematic of experimental design; C57BL/6 mice (n = 7/group) were immunized twice (0 and 21 days) i.n./i.n., i.m./i.n. or i.m./i.m. with the Spike-LP-GMP vaccine or PBS and euthanized on day 111 for samples collection. Spike-specific IgG b, IgA c in the serum, lung homogenate and nasal wash. d Antigen-specific IFN-γ, IL-17 and GrB production by cervical and inguinal LN cells stimulated for 72 h with medium alone or Spike trimer (2.5 µg), quantified by ELISA. Data were analyzed by one-way ANOVAs followed by post hoc Tukey’s test for multiple comparisons (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001), error bars, SEM. Results are representative of three independent experiments.
Fig. 5
Fig. 5. Spike-specific TRM cells persist in the lungs and nasal tissue for at least 3 months post immunization.
Mice were immunized as described in Fig. 4, lung and nasal tissue samples were collected on day 111. a Number of CD4+ TRM cells (CD44+CD62L-CD45-CD69+) in the lung and nasal tissue. b Representative flow cytometry plot of CD4+ TRM cells from lung. Number of Spike-specific CD4+ TRM cells secreting TNF c, IL-17 d, GrB e, or IFN-γ f in the lung and nasal tissue following 18 h stimulation with Spike trimer (2.5 µg/ml), g Representative flow cytometry plot of IL-17 and TNF secreting CD4+ TRM cells from lung. Data were analyzed by one-way ANOVAs followed by post hoc Tukey’s test for multiple comparisons (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001), error bars, SEM.
Fig. 6
Fig. 6. The Spike-LP-GMP vaccine protects K18-hACE2 transgenic mice against challenge with SARS-CoV-2 variants.
a Experimental design. In study 1, K18-hACE2 (n = 8/group) were immunized twice (0 and 21 days) i.n./i.n., i.m./i.n. or i.m./i.m. with the Spike-LP-GMP vaccine (WT virus strain) or PBS and challenged with SARS-CoV-2 WA-1 on day 35. Disease score (based on weight loss, appearance, activity, eye closure, respiration, and hypothermia) b and survival c and lung acute inflammatory score d. Virus RNA copies in the lung e brain f and nasal wash g 12 days after SARS-CoV-2 WA-1 challenge. Dashed line indicates limit of detection by qPCR. In study 2, K18-hACE2 mice were immunized twice (0 and 21 days) i.n./i.n. or i.m./i.n. with the Spike-LP-GMP vaccine (WT or Beta virus strain) or PBS and challenged with SARS-CoV-2 Delta on day 35. Disease score h and survival i and lung acute inflammatory score j, with representative images of H&E stained lung sections (PBS (1), LP-GMP (2), Spike(WT)-LP-GMP i.m./i.n. (3), Spike(WT)-LP-GMP i.n./i.n. (4) and Spike(β)-LP-GMP i.n./i.n. (5) k. Virus RNA copies in the lung l, brain m, and nasal wash n 10 days after SARS-CoV-2 Delta challenge. Dashed line indicates limit of detection by qPCR. Data were analyzed by one-way ANOVAs followed by post hoc Tukey’s test for multiple comparisons (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001), error bars, SEM. Log-rank Mantel-Cox test was used to determine statistical significance for survival analyzes (***P < 0.001).
Fig. 7
Fig. 7. Antibodies induced by nasal Spike-LP-GMP vaccine neutralize multiple variants of SARS-CoV-2 but have reduced efficacy against Omicron.
Serum neutralizing antibody responses, based on inhibition of binding of SARS-CoV-2 RBD Spike from WT, Alpha, Beta, Delta, Gamma and Omicron to hACE-2 (MSD assay) were assessed on day 35 (before SARS-CoV-2 challenge), 2 weeks after i.n./i.n. or i.m./i.n. immunization with the Spike-LP-GMP vaccine (WT or Beta virus strains) or PBS. Data are represented as percentage of neutralization. Data were analyzed by one-way ANOVAs followed by post hoc Tukey’s test for multiple comparisons (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001), error bars, SEM.
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