Mucosal vaccination triggers superior T cell response against SARS-CoV-2 variants

In a recent study posted to the bioRxiv* server, researchers in Singapore and the United States demonstrated that intranasal (I.N.) delivery of a mucosal severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination triggered a superior T cell response compared to an equivalent dose of antigen delivered by the subcutaneous (S.C.) route. In addition, the I.N. vaccine-induced T cell phenotypes had polyfunctional interferon-gamma (IFN-γ) and tumor necrosis factor (TNF) expression and encompassed abundant T central memory (TCM) cells.

Study: Mucosal vaccination for SARS-CoV-2 elicits superior systemic T central memory function and cross-neutralizing antibodies against variants of concern. Image Credit: Design_Cells / ShutterstockStudy: Mucosal vaccination for SARS-CoV-2 elicits superior systemic T central memory function and cross-neutralizing antibodies against variants of concern. Image Credit: Design_Cells / Shutterstock


The ongoing coronavirus disease 2019 (COVID-19) pandemic has necessitated unprecedented advances in vaccination technologies. However, the continuous emergence of new SARS-CoV-2 variants of concern (VOCs) poses a risk of frequent breakthrough infections and severe disease outcomes in multiple age groups, even in populations with high levels of vaccine coverage.

T cells are highly cross-reactive to multiple SARS-CoV-2 VOCs, SARS-CoV, and seasonal coronaviruses. Studies in non-human primates have pointed to a protective role of T cells in vaccine-induced protection. It is also noteworthy that SARS-CoV-2 infection begins at the mucosal surface of the nasal passages and lung airways. Therefore, there is an urgent need for alternative vaccine approaches for SARS-CoV-2, including subunit vaccines, which use adjuvants to promote immune activation.

Currently, there are no mucosal adjuvants approved for use in humans. Mastoparan-7 (M7), an analog of mastoparan, appears to work in vivo and has shown efficacy in enhancing the titer of antigen-specific antibodies in animal models when delivered via the S.C. route as well as application to the nasal mucosae. However, it is unknown if M7  influences T cell phenotypes and functions that are particularly important for combatting certain types of viral pathogens.

About the study

In the present study, researchers tested whether I.N. delivery of an adjuvanted subunit vaccine induced adequate SARS-CoV-2-specific T cell immune responses. They used eight to 10 weeks old C57BL/6 mice for all the vaccination experiments. The vaccine formulation comprised one μg of recombinant S-receptor-binding domain (RBD) protein with or without 20μg of M7.

They harvested spleens, popliteal LNs, or the nasal-associated lymphoid tissue (NALT), a rodent structure analogous to Waldeyer’s ring in humans, of all mice at necropsy on day 35 post-vaccination to prepare single-cell suspensions. They isolated red blood cells (RBCs) from the single-cell suspensions of the spleen and determined total cell numbers using a hemocytometer.

The team measured T cell responses five days post-vaccination by flow cytometry in the draining lymphoid organs for the respective tissues for S.C. or I.N. routes, respectively. They also assessed systemic T cell responses in the spleen following vaccination via both routes. Finally, the researchers visualized populations of T cells in the lymphoid organs using the UMAP algorithm, which they identified using the gating strategy.

Study findings

The currently used COVID-19 vaccines do not appear to induce robust airway-resident antigen-specific T cells, thus, necessitating the potential of next-generation COVID-19 vaccines to improve mucosal and systemic immune responses through modulation of T cells. Mucosal vaccination against the S-RBD antigen of SARS-CoV-2 promoted a T cell-intrinsic phenotype associated with superior systemic immune responses and antibody responses that improved antibody persistence in vivo and cross-protection against SARS-CoV-2 VOCs compared to S.C. vaccination with the same formulation.

The heightened systemic T cell responses in multiple T cell subsets in the spleen also persisted in the TMEM cell compartment for several weeks following the SARS-CoV-2 challenge. These TMEM cells exhibited an improved polyfunctional phenotype, characterized by dual expression of TNF and IFN-γ upon ex vivo stimulation and in vivo memory recall to antigen. An enhanced interleukin-17 (IL-17) production characterized the in vivo splenic T cell responses. Overall, the study results illustrated the polyfunctional nature of TMEM cells, independent of their peripheral tissue homing abilities. Indeed, T cells could help establish systemic mucosal vaccine-induced memory, of which TCM cells are a central component.

The increased numbers of L-selectin (CD62L)-expressing TCM cells within the TMEM compartment following mucosal vaccination is key for the homing of these cells to the spleen, which typified increased systemic immunity. During memory recall, TCM also likely served as a pool of T cells that replenished the TEM population.

The study results also pointed out that site-specific immune responses influence the long-term balance of T cell subpopulations. Therefore, during in vivo antigen challenge, the authors noted a marked increase in the numbers of CD8 TEM in the lung-draining brachial lymph nodes of recipients of I.N. route. Conversely, the ex vivo antigen restimulation of spleen T cells before adoptive transfer resulted in improved activation for CD8 TEM in S.C. vaccinated group.

Systemic T-cell responses also impact B cell-dependent antibody responses. Compared to S.C. vaccination, I.N. vaccination induced a relatively small but substantially higher level of neutralizing antibodies (nAbs) five weeks after the final vaccine boost and more nAbs with greater breadth against multiple SARS-CoV-2 VOCs. Most likely mucosal vaccination strategy preserved antibodies against more diverse epitopes within the polyclonal pool.


The current study is unique in using an M7 adjuvant, which could be injected into the skin and administered at mucosal surfaces, to directly compare the immune responses induced by the same dose of antigen administered via the two routes. The COVID-19 vaccines relying on high titer-specific nAbs with limited induction of mucosal responses need improvements. The mucosal vaccination strategy could prove helpful by helping improve the systemic immune responses and cross-reactive nAbs; thus, this strategy could potentially be used in the next-generation SARS-CoV-2 vaccines.

*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.

Journal reference:
Neha Mathur

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.


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