Prime-pull immunization of mice with novel subunit vaccine elicits mucosal immunity to prevent severe SARS CoV-2 infection

In a recent study posted to the bioRxiv* pre-print server, researchers tested whether a subunit vaccine could generate mucosal immunity in mice and prevent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and related pathology.

The vaccine contained SARS-CoV-2 spike (S) adjuvanted with alum or Bordetella colonization factor A (BcfA).

While systemic immunity prevents SARS-CoV-2 infection and dissemination, mucosal immunity clears the virus and infected cells from the respiratory tract. Hence, there is an urgent need for novel vaccines and immunization strategies that generate both types of immunity to combat SARS-CoV-2 effectively.

About the study

In the present study, researchers immunized C57 black 6 (C57BL/6) mice intramuscularly (i.m.) with one µg of S adsorbed to alum (S/A) or S protein with alum and 10 µg BcfA (S/A/B) on day 0. On day 28, they boosted mice intranasally (i.n.) with S alone, S/A, or S/B and evaluated them 14 days later. The prime-pull immunization generated both systemic and mucosal immunity in the test animals.

The team infected anesthetized mice i.n. with 10⁵ plaque-forming units (PFU) of SARS-CoV-2 diluted in phosphate buffer saline (PBS). Importantly, they randomized test mice and assigned them to particular harvest days before beginning the experiments, and monitored clinical signs of weight loss in mice daily.

Post-euthanizing the mice, the researchers collected samples for evaluating viral titers from the caudal right lung lobe and nasal septum and the left lung lobe for histopathological analyses. They used plaque assays to estimate lung viral titers.

Study findings

The BcfA-adjuvanted vaccine-induced T helper 17 (Th17) polarized CD4⁺ tissue-resident memory T cell responses (TRM) in C57BL/6 mice, whereas alum-adjuvanted vaccines generated T helper 2 (Th2) polarized systemic and mucosal CD4⁺ T cell responses. Additionally, the former efficiently protected the respiratory tract against infection-associated lung damage, while the latter did not.

Contrastingly, interleukin 17 (IL-17) knockout mice immunized with the same vaccine formulation and immunization dosages had high viral titers in the lower and upper respiratory tracts and showed respiratory pathologies. Thus, suggesting IL-17⁺ T cell responses were critical for protection conferred by the BcfA-adjuvanted vaccine.

Overall, the Th17 polarized mucosal and systemic T cell response and neutralizing antibodies (nAbs), generated by systemic priming with an alum BcfA-adjuvanted vaccine and a BcfA-adjuvanted vaccine booster, prevented SARS-CoV-2 related severe illness and respiratory pathology.

Alum-adjuvanted vaccines are delivered i.m. only, whereas prime-pull immunization with S/A produced immunoglobulin A (IgA) in the serum and lungs, demonstrating that the vaccine delivery route altered the immune response in mice.

The authors observed minimal CD8⁺ T cell responses to the S protein, suggesting that the T cell responses induced by the same adjuvant vary with the antigenic composition of the vaccine, further suggesting that CD8⁺ T cells are not needed for SARS-CoV-2 clearance from the respiratory tracts.

The percentage of the cluster of differentiation (CD45) to CD3⁺, CD4⁺, CD44⁺, and CD62L to CD69⁺ TRM cells increased in all the groups of vaccinated mice compared to naïve mice. However, the only group which showed a statistically significant increase in the total number of CD4⁺ TRM in the lungs was the one primed with S/A/B and boosted with S/B.

Mice primed and boosted with S/A produced interferon-gamma (IFNγ) and interleukin-5 (IL-5), while mice primed and boosted with S/A/B and S/B, respectively, primarily produced IL-17, indicating the generation of a Th1/2 polarized immune response.

Notably, the BcfA adjuvant in the vaccine attenuated the proportion and number of IL-5 producing CD4⁺ TRM. Another finding suggesting that BcfA primarily elicited CD4⁺ T cells was that the percentage and number of IFNy-producing CD8⁺ TRM increased in S/A immunized mice but not in mice primed with S/A/B and boosted with S/B.

Interestingly, while the proportion and number of SARS-CoV-2-specific circulating memory T cells (CD45⁺) increased, changes in the percentage and number of cytokine-producing cells did not reach statistical significance. This indicates that antigen-specific cells are largely tissue-resident, and this vaccine did not elicit SARS-CoV-2-specific CD8⁺ T cells.

Mice immunized with alum or alum/BcfA containing vaccines had low viral titers in the lungs and nose, indicating that both the vaccines provided similar protection against severe coronavirus disease 2019 (COVID-19). However, histopathology and immunohistochemistry (IHC) analysis revealed that S/A immunized mice had pneumonitis, epithelial damage, and prolonged expression of nucleoprotein antigen, despite the production of mucosal IgA. Contrastingly, the lungs of mice immunized with BcfA-adjuvanted vaccines had minimal damage and showed no nucleoprotein expression.

Conclusions

The study identified a prime-pull immunization strategy and vaccine formulation that induced both mucosal and systemic immunity against SARS-CoV-2 infection. Together, the study data showed that alum generated strong S-specific systemic responses, and BcfA attenuated Th2 responses when combined with alum. Overall, i.m. priming and i.n. boosting with S/A, generated systemic and mucosal T cells and nAbs were highly effective in precluding severe SARS-CoV-2 infection and respiratory damage.

However, as the need for booster vaccines that extend the protection provided by currently approved messenger ribonucleic acid (mRNA) vaccines will rise, it will be important to validate whether i.n. booster with S/B could generate mucosal immunity in individuals previously immunized with mRNA vaccines to increase the durability of protection.

*Important notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Written by

Neha Mathur

Neha is a digital marketing professional based in Gurugram, India. She has a Master’s degree from the University of Rajasthan with a specialization in Biotechnology in 2008. She has experience in pre-clinical research as part of her research project in The Department of Toxicology at the prestigious Central Drug Research Institute (CDRI), Lucknow, India. She also holds a certification in C++ programming.