
==== Front
NPJ Vaccines
NPJ Vaccines
NPJ Vaccines
2059-0105
Nature Publishing Group UK London

39232020
957
10.1038/s41541-024-00957-2
Article
Broad protection and respiratory immunity of dual mRNA vaccination against SARS-CoV-2 variants
Hajnik Renee L. 12
http://orcid.org/0000-0002-4768-7458
Plante Jessica A. 134
http://orcid.org/0000-0002-2888-2418
Reddy Bonam Srinivasa 1
Rafael Grace H. 1
http://orcid.org/0000-0002-9557-1847
Liang Yuejin 1
Hazell Nicholas C. 12
Walker Jordyn 1
http://orcid.org/0000-0001-8421-8397
Reyna Rachel A. 1
Walker David H. 235
http://orcid.org/0000-0002-5672-6930
Alameh Mohamad-Gabriel 67
Weissman Drew 67
http://orcid.org/0000-0001-8016-8556
Weaver Scott C. 1345
http://orcid.org/0000-0002-7485-3167
Plante Kenneth S. ksplante@UTMB.edu

134
http://orcid.org/0000-0002-6964-2314
Hu Haitao haihu@UTMB.edu

135
1 https://ror.org/016tfm930 grid.176731.5 0000 0001 1547 9964 Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX USA
2 https://ror.org/016tfm930 grid.176731.5 0000 0001 1547 9964 Department of Pathology, University of Texas Medical Branch, Galveston, TX USA
3 https://ror.org/016tfm930 grid.176731.5 0000 0001 1547 9964 Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, TX USA
4 https://ror.org/016tfm930 grid.176731.5 0000 0001 1547 9964 World Reference Center for Emerging Viruses and Arboviruses, University of Texas Medical Branch, Galveston, TX USA
5 https://ror.org/016tfm930 grid.176731.5 0000 0001 1547 9964 Sealy Institute for Vaccine Sciences, University of Texas Medical Branch, Galveston, TX USA
6 grid.25879.31 0000 0004 1936 8972 Department of Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA USA
7 grid.25879.31 0000 0004 1936 8972 Penn Institute for RNA Innovation, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA USA
4 9 2024
4 9 2024
2024
9 16029 3 2024
21 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by/4.0/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
While first-generation, spike (S)-based COVID-19 vaccines were effective against early SARS-CoV-2 strains, the rapid evolution of novel Omicron subvariants have substantially reduced vaccine efficacy. As such, broadly protective vaccines against SARS-CoV-2 are needed to prevent future viral emergence. In addition, it remains less clear whether peripheral immunization, especially with mRNA vaccines, elicits effective respiratory immunity. Our group has developed a nucleoside-modified mRNA vaccine expressing the nucleocapsid (N) protein of the ancestral SARS-CoV-2 virus and has tested its use in combination with the S-based mRNA vaccine (mRNA-S). In this study, we examined efficacy of mRNA-N alone or in combination with mRNA-S (mRNA-S+N) against more immune evasive Omicron variants in hamsters. Our data show that mRNA-N alone induces a modest but significant protection against BA.5 and that dual mRNA-S+N vaccination confers complete protection against both BA.5 and BQ.1, preventing detection of virus in the hamster lungs. Analysis of respiratory immune response in mice shows that intramuscular mRNA-S+N immunization effectively induces respiratory S- and N-specific T cell responses in the lungs and in bronchoalveolar lavage (BAL), as well as antigen-specific binding IgG in BAL. Together, our data further support mRNA-S+N as a potential pan-COVID-19 vaccine for broad protection against current and emerging SARS-CoV-2 variants.

Subject terms

RNA vaccines
Vaccines
https://doi.org/10.13039/100000060 U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID) R24AI120942 R41AI181134 R24AI120942 R24AI120942 Plante Jessica A. Weaver Scott C. Hu Haitao U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)issue-copyright-statement© Springer Nature Limited 2024
==== Body
pmcIntroduction

Since 2020, tremendous efforts have been devoted to the development of vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19. Two vaccines based on the mRNA-lipid nanoparticle (LNP) platform were rapidly developed and clinically approved. These first-generation mRNA vaccines targeted the viral spike (S) protein and showed high clinical efficacy against the early strains1,2. However, a number of immune-escaping SARS-CoV-2 variants have emerged since 2020, including Alpha, Beta, Gamma, Delta, and Omicron variants, largely due to mutations in the viral S protein3,4. A number of new Omicron subvariants have also emerged, including BA.2, BA.3, BA.4, BA.5, BQ.1, and others5. Extensive pre-clinical and clinical studies have shown that the first-generation vaccines had reduced efficacy against SARS-CoV-2 variants, especially the Omicron strains6,7. The emergence of variants has posed continuing challenges to host immunity induced by the monovalent or bivalent S-based vaccines. Thus, development of a pan-SARS-CoV-2 vaccine with broad protection against current and future variants is needed.

A broadly protective SARS-CoV-2 vaccine likely requires targeting the conserved viral epitopes, in addition to the S protein. The viral nucleocapsid (N) protein is relatively conserved across SARS-CoV-2 variants8 and is a main target of host immunity in SARS-CoV-2-infected individuals9,10. In our previous study, we generated a nucleoside-modified mRNA vaccine that expresses the SARS-CoV-2 N protein (mRNA-N) and showed that vaccination with dual mRNA-S+N, both based on the prototypical viral sequence, conferred strong protection against both Delta and Omicron BA.1 variants in animal models11. However, the breadth of protection by this mRNA vaccine approach against newer, more immune evasive SARS-CoV-2 variants12–14 remains to be determined. In addition, clinically approved mRNA vaccines are administered via the intramuscular (IM) route. Our previous study reported induction of strong systemic immunity by IM mRNA vaccination11. Whether peripheral mRNA vaccination elicits effective antigen-specific respiratory immunity is less clear15–18. Addressing these questions will not only facilitate the development of a pan-SARS-CoV-2 vaccine, but also have implications for vaccine development against other respiratory infections.

In this study, we further evaluated efficacy of mRNA-N alone or in combination with mRNA-S (mRNA-S+N) against two newer SARS-CoV-2 Omicron variants (BA.5 and BQ.1) in Syrian hamster models. Our results show that mRNA-N alone induces significant albeit incomplete protection against BA.5 in hamsters. Importantly, dual mRNA-S+N vaccination confers complete protection against both BA.5 and BQ.1, preventing detectable viral loads in the hamster lungs. Serum antibody analysis indicates that the complete protection by mRNA-S+N is independent of neutralizing antibodies against these two variants, further supporting a role of T cell immunity in the vaccine-induced protection. In the C57BL/6 mouse model, we show that IM mRNA-S+N vaccination effectively elicits respiratory S- and N-specific T cell responses in the lungs and bronchoalveolar lavage (BAL). The data provides immunological insights for the robust viral control of immune-escaping SARS-CoV-2 variants by peripheral mRNA-S+N vaccination even in the absence of detectable neutralizing antibodies.

Results

mRNA-N alone induces incomplete but significant protection against SARS-CoV-2 Omicron BA.5 variant in hamsters

The coronavirus N protein exhibits higher sequence conservation when compared to the S protein19,20. Our previous study reported the development of a nucleoside-modified mRNA vaccine expressing ancestral SARS-CoV-2 N (mRNA-N)11. Prior to investigating the extent of protection conferred by mRNA-N against newer variants, we conducted an analysis of the sequence diversity of the N protein among various SARS-CoV-2 strains (BA.1, BA.5, and BQ.1 versus Wuhan-Hu-1 strain), compared to the S protein (Table 1). The analysis reveals that compared to Wuhan-Hu-1 strain, the three Omicron variants demonstrate an overall sequence diversity in 53 (out of 1273) residues for the S protein, and 8 (out of 419) residues for the N protein, supporting that N is more conserved among these SARS-CoV-2 strains relative to S (Table 1).Table 1 Residue Diversity Within the Spike and Nucleocapsid Proteins of SARS-CoV-2 strains

		A	BA.1	BA.5	BQ.1	
		Wuhan-Hu-1	EHC_C19_2811c	COR-22-063113/2022	MDL 5125	
Protein	AA Residue					
Spike	19	T	T	I	I	
24–27	LPPA	LPPA	S	S	
67	A	V	A	A	
69–70	HV	del	del	del	
76	T	T	I	T	
95	T	I	T	T	
142	G	D	D	D	
143–145	VYY	del	VYY	VYY	
211–214	NLVR	IVREPE	NLGR	NLGR	
339	G	D	D	D	
346	R	R	R	T	
371	S	L	F	F	
373	S	P	P	P	
375–376	ST	FT	FA	FA	
405	D	D	N	N	
408	R	R	S	S	
417	K	N	N	N	
440	N	K	K	K	
444	K	K	K	T	
446	G	S	G	G	
452	L	L	R	R	
460	N	N	N	K	
477–478	ST	NK	NK	NK	
484	E	A	A	A	
486	F	F	V	V	
493	Q	R	Q	Q	
496	G	S	G	G	
498	Q	R	R	R	
501	N	Y	Y	Y	
505	Y	H	H	H	
547	T	K	T	T	
614	D	G	G	G	
655	H	Y	Y	Y	
679	N	K	K	K	
681	P	H	H	H	
764	N	K	K	K	
796	D	Y	Y	Y	
856	N	K	N	N	
954	Q	H	H	H	
969	N	K	K	K	
981	L	F	L	L	
Nucleocapsid	13	P	L	L	L	
31–33	ERS	del	del	del	
136	E	E	E	D	
203–204	RG	KR	KR	KR	
413	S	S	R	R	
Amino acid (AA) diversity within the spike and nucleocapsid proteins. AA residue numbering is based on the Wuhan-Hu-1 strain. Protein sequences are assumed on the basis of nucleotide sequences. The Wuhan-Hu-1 sequence reflects NCBI Reference Sequence NC_045512.2. All other strains reflect the next-generation sequencing results of the viral stocks utilized in this manuscript.

Next, we assessed the efficacy of mRNA-N against Omicron BA.5 in hamsters, which was a dominant circulating variant in human population and showed substantial neutralization escape21,22 when this study was conducted. Two groups of hamsters (n = 10 per group) were IM vaccinated with empty LNP (mock) or mRNA-N (2 μg) at weeks 0 and 3, followed by intranasal (IN) challenge with BA.5 (2 × 104 pfu) at week 5 (Fig. 1A). The mRNA vaccine dose was selected based on our recent study11. Two (n = 5) and four (n = 5) days post infection (DPI), hamsters were euthanized and lung tissues were harvested for viral RNA quantification by RT-qPCR (Fig. 1A). Our primary goal was to evaluate the vaccine efficacy based on reductions in viral RNA copies. Since viral replication kinetics for different SARS-CoV-2 strains in animal models varies, lungs are usually harvested at multiple time points after challenge (e.g., 2 and 4 DPI) for quantification of viral loads11. Hamster body weights were also monitored daily until the terminal harvest (4 PDI) as a secondary measure of vaccine-induced protection. At 2 DPI, lung viral RNA copies were reduced in the mRNA-N-vaccinated group compared to the mock-vaccinated group (60-fold reduction in mean viral RNA copies; mock versus mRNA-N; p < 0.05) (Fig. 1B). At 4 DPI, mRNA-N also induced a significant reduction of lung viral RNA copies (8.3-fold reduction in median copies compared to the mock group; p < 0.01; Fig. 1B). Body weight analysis showed that BA.5 infection caused weight reduction in the mock-vaccinated group by ~3% on 4 DPI (Fig. 1C). However, mRNA-N-vaccinated hamsters were protected from weight loss (4 DPI mRNA-N vs. LNP: p < 0.05) (Fig. 1C). Together, these data indicated that mRNA-N induced a modest but significant protection against SARS-CoV-2 BA.5. This level of protection is consistent with its efficacy against the Delta strain reported in our previous study11.Fig. 1 mRNA-N vaccine is efficacious against Omicron variant BA.5 in hamsters.

A Experimental design and timeline (Created with BioRender.com). Two groups of hamsters (n = 10/group) were vaccinated intramuscularly with either empty LNP (mock) or mRNA-N vaccine (2 µg/dose) at weeks 0 and 3. At week 5, hamsters were intranasally challenged with Omicron BA.5 (2 × 104 pfu). B Lungs were harvested at 2 and 4 DPI (days post infection; n = 5 at each time point) for quantification of viral RNA copies by RT-qPCR. C Hamster body weight was monitored from 0 to 4 DPI. In (B) symbols represent individual animals, midlines represent the median, error bars represent the interquartile range (IQR), and the dashed lines represent the lower limit of detection (LOD). The number of animals with viral loads above the LOD is noted. Log10 normalized data was compared by Mann-Whitney test. In (C) symbols represent the mean, error bars represent the standard deviation, and the dashed line highlights 0% weight change. Weight change at individual time point between the two groups was compared by unpaired t-test. *p < 0.05, **p < 0.01.

Intramuscular immunization with mRNA-N alone induces modest respiratory T cell responses in the lung and bronchoalveolar lavage of mice

Prior results are conflicting regarding whether peripheral mRNA immunization (e.g., IM) elicits effective immunity in the airway, in addition to systemic immunity15–18. Our previous study showed induction of systemic T cell responses and circulating antibodies by mRNA-N in BALB/c mice11. Here, we investigated respiratory immune responses following IM mRNA-N vaccination in C57BL/6 mice. Use of C57BL/6 mice was in part because SARS-CoV-2 N-epitope specific MHC-I tetramer (H2-Db; N219-227: LALLLLDRL)23 is available for the identification of N-specific CD8 T cells in the mouse BAL and lung samples. The N219-227 is one of the dominant CD8 T cell epitopes within the N protein that was bioinformatically predicted8 and then confirmed in humans and mice24,25.

Two groups of C57BL/6 mice (n = 5 per group) were IM vaccinated with either empty LNP (mock) or mRNA-N (1 μg) at week 0 (prime) and week 3 (boost). Two weeks after boost vaccination (week 5), lung and BAL samples were collected for analysis of respiratory immune response. Serum and spleen samples were also collected for analysis of systemic immune response for comparison (Fig. 2A). Single cell suspensions prepared from lung and BAL were stained for CD3, CD4, CD8, CD44, CXCR6, and SARS-CoV-2 N-tetramer, to identify activated T cells (based on CD44)26, T cells with tissue-homing potential (CXCR6)27, and vaccine-elicited, N-specific CD8 T cells (tetramer+). Vaccine-induced binding antibodies (IgG and IgA) were also examined in the BAL.Fig. 2 Respiratory T cell response induced by mRNA-N in mice.

A Mouse experimental design and timeline (Created with BioRender.com). Two groups of C57BL/6 mice (n = 5/group) were intramuscularly vaccinated with either empty LNP (mock) or mRNA-N vaccine (1 μg) at weeks 0 and 3. Two weeks after booster dose (week 5), immune responses were analyzed. B, C Analysis of tetramer positive, N-epitope specific T cells in the lungs by flow cytometry. B Representative flow cytometry plots for N-tetramer staining of CD8 T cells from LNP and mRNA-N vaccinated mice. C Frequencies of N epitope-specific CD8 T cells in the lungs of LNP and mRNA-N vaccine group. Analysis of total and N epitope-specific T cells in the BAL. Frequencies of activated (CD44+) CD4 and CD8 T cells (D), CXCR6+ CD4 and CD8 T cells (E), and N epitope-specific CD8 T cells (F) in BAL were examined. Data were presented as median and interquartile range, and were compared by Mann-Whiteney test between the two groups. *p < 0.05, **p < 0.01, ***p < 0.001.

Gating strategy to identify T cell populations and N-tetramer+ CD8 T cells in the lungs is shown in Supplementary Fig. 1. Representative flow cytometric plots for N-tetramer+ CD8 T cells are shown in Fig. 2B. Compared to mock control, mRNA-N vaccination elicited comparable levels of total CD4 and CD8 T cells in the lung (Supplementary Fig. 2A), and slightly higher levels of activated CD44+ (Supplementary Fig. 2B) or CXCR6+ (Supplementary Fig. 2C) CD4 and CD8 T cells in the lung. Analysis of N-tetramer straining (Fig. 2B) revealed that compared to mock control, mRNA-N vaccination elicited significant levels of N epitope-specific CD8 T cells in the lungs (p < 0.001) (Fig. 2C). Of interest, this N epitope-specific CD8 T cell response was not evident in the spleens after mRNA-N vaccination (Supplementary Fig. 3).

We next evaluated T cell response in the BAL after mRNA-N vaccination. Gating strategy for flow cytometric analysis of BAL cells is shown in Supplementary Fig. 4. Compared to mock control, mRNA-N vaccination elicited higher levels of CD44+ CD4 T cells and CD44+ CD8 T cells in the BAL, although statistical significance was only achieved for CD4 T cells (p < 0.05 for CD4) (Fig. 2D). A similar trend was observed for CXCR6+ T cells, with mRNA-N eliciting significantly higher levels of CXCR6+ CD4 T cells in the BAL compared to mock control (p = 0.01 for CD4) (Fig. 2E). Analysis of N-tetramer+ cells revealed that mRNA-N vaccination elicited a trend towards higher levels of N epitope-specific CD8 T cells in the BAL compared to mock control (p = 0.055) (Fig. 2F). Analysis of binding antibodies showed that mRNA-N vaccination induced readily detectable N-specific binding IgG in the BAL (Supplementary Fig. 5A). No binding IgA was detected following IM mRNA-N vaccination (Supplementary Fig. 5B), consistent with the previous report15. Together, the data indicate that mRNA-N vaccine elicits a modest respiratory immune response including N-specific CD8 T cells in the mouse lungs and BAL.

mRNA-S+N vaccination induces strong protection against SARS-CoV-2 Omicron BA.5 in hamsters

After demonstrating that mRNA-N alone elicited modest protection against BA.5, we next explored whether dual mRNA-S+N vaccination induces stronger protection against this variant than mRNA-S alone. Three groups of hamsters were vaccinated with empty LNP (mock), mRNA-S (2 μg), or mRNA-S+N (2 μg for each mRNA) at weeks 0 and 3, followed by intranasal challenge at week 5. On 2 (n = 5) and 4 (n = 5) DPI, vaccine-induced protection was analyzed based on viral loads (Fig. 3A). Analysis of viral RNA in the lungs revealed that, compared to mock vaccination, mRNA-S alone induced modest but significant protection, reducing the lung viral RNA copies by 22 and 4 folds at 2 and 4 DPI, respectively (Fig. 3B–C). Relative to mRNA-S alone, mRNA-S+N induced a more robust effect, leading to complete viral control with no detection of the viral RNAs on 4 DPI (mRNA-S vs. mRNA-S+N: p < 0.0001 on 4 DPI) (Fig. 3C). We previously showed that the stronger protection against an Omicron variant (BA.1) by mRNA-S+N relative to mRNA-S alone was not due to the difference in total mRNA or LNP doses11. Lastly, vaccination with mRNA-S alone and mRNA-S+N both protected the hamsters from weight loss, with no significant difference detected between the two vaccine groups (Fig. 3D).Fig. 3 Dual mRNA-S+N vaccination protects hamsters from Omicron BA.5.

A Hamster experimental design and timeline (Created with BioRender.com). Three groups of hamsters (n = 10/group) were vaccinated intramuscularly with empty LNP (mock), mRNA-S (2 μg), or mRNA-S+N (2 μg for each mRNA) at weeks 0 and 3, followed by intranasal challenge with SARS-CoV-2 Omicron BA.5 (2 × 104 pfu) at week 5. Lungs were harvested at 2 (B) and 4 (C) DPI (n = 5 at each time) for quantification of viral RNA copies by RT-qPCR. (D) Hamster body weights were monitored from 0 to 4 DPI. In (B, C), symbols represent individual animals, midlines represent the median, error bars represent the interquartile range, and the dashed line represents the lower limit of detection (LOD). The number of animals with viral loads above the LOD is noted. Data were compared among the three groups by Kruskal-Wallis test. In (D), symbols represent the mean, error bars represent the standard deviation, and the dashed line highlights 0% weight change. Weight change was compared by 2-way ANOVA followed by Tukey’s multiple comparisons test (two factors; hamsters’ weight and time). *p < 0.05, **p < 0.01, ****p < 0.0001.

mRNA-S+N vaccination induces strong protection against SARS-CoV-2 Omicron BQ.1 in hamsters

To further determine the breadth of protection by mRNA-S+N, we next examined efficacy of mRNA-S and mRNA-S+N against BQ.1 in the hamster model. BQ.1, derived from BA.5, emerged with additional spike mutations that contributed to its strong immune evasion and efficient transmission13,28. Hamsters were vaccinated at weeks 0 and 3 before intranasal challenge with BQ.1 (2 × 104 pfu) at week 5 (Fig. 4A). Lungs were harvested at 2 (n = 5) and 4 (n = 5) DPI with hamster body weights monitored throughout the course of the experiment (Fig. 4A). Compared to BA.5 (Fig. 3) and earlier variants (BA.1 and Delta)11, BQ.1 appears to replicate to lower levels in hamsters and with faster clearance by 4 DPI (Fig. 4B, C). Similarly, body weight data indicated that nonvaccinated (mock) hamsters showed marginal weight loss through 4 DPI (Fig. 4D). This is consistent with the observations that BQ.1 is moderately pathogenic compared to BA.513,29. Vaccination with mRNA-S+N led to complete viral control with no detection of viral RNA in four out of five hamsters at 2 DPI (Fig. 4B) and five out of five hamsters at 4 DPI (Fig. 4C). Both mRNA-S and mRNA-S+N protected hamsters from the mild weight loss resulting from BQ.1 infection with no significant difference detected between the two vaccine groups (Fig. 4D).Fig. 4 Dual mRNA-S+N vaccination protects hamsters from Omicron BQ.1.

A Experimental design and timeline (Created with BioRender.com). Three groups of hamsters (n = 10/group) were vaccinated intramuscularly with empty LNP (mock), mRNA-S (2 μg), or mRNA-S+N (2 μg for each mRNA) at weeks 0 and 3, followed by intranasal challenge with SARS-CoV-2 Omicron BQ.1 (2 × 104 pfu) at week 5. Lungs were harvested at 2 (B) and 4 (C) DPI (n = 5 at each time point) for quantification of viral RNA copies by RT-qPCR. D Hamster body weights were monitored from 0 to 4 DPI. In (B, C), symbols represent individual animals, midlines represent the median, error bars represent the interquartile range, and the dashed line represents the lower limit of detection (LOD). The number of animals with viral loads above the LOD is noted. Log10 normalized data was compared among the three groups by Kruskal-Wallis test. In (D), symbols represent the mean, error bars represent the standard deviation, and the dashed line highlights 0% body weight change. Weight change was compared by 2-way ANOVA followed by Tukey’s multiple comparisons test (two factors; hamsters’ weight and time). *p < 0.01, **p < 0.01, ****p < 0.0001.

mRNA-S+N induces binding antibodies that manifest no detectable neutralizing activities against BA.5 and BQ.1 variants

In the above hamster experiments (Fig. 3, 4), serum samples were collected prior to viral challenge (two weeks after booster) for analysis of vaccine-induced antibody responses (Fig. 5). Binding antibody endpoint titers (EPTs) were determined by ELISA. Compared to the mock control, both mRNA-S alone and mRNA-S+N induced significant levels of S-specific binding antibodies in sera (Fig. 5A–C). Median IgG EPTs for both mRNA-S and mRNA-S+N groups were 104.9 (Fig. 5A). Other than IgG, serum IgA and IgM were also detectable. Median IgA EPTs for mRNA-S and mRNA-S + N were 103.95 and 104.43, respectively (Fig. 5B), and median IgM EPTs for mRNA-S and mRNA-S+N were 102.76 and 103, respectively (Fig. 5C). These serum samples were also examined for neutralization against SARS-CoV-2 WA.1, BA.1, BA.5, and BQ.1 using the plaque-reduction neutralization test (PRNT) with corresponding live virus. While the sera of hamsters immunized with mRNA-S and mRNA-S+N showed strong neutralizing activity against the ancestral WA.1 strain (Fig. 5D), their neutralizing activities against BA.1 were substantially reduced: 4/10 samples had weakly detectable neutralization in the mRNA-S alone group and 5/10 weakly detectable neutralization in the mRNA-S+N group. Notably, neutralizing activities of these sera against BA.5 and BQ.1 variants were completely lost, with all samples showing undetectable neutralization activity for these variants (Fig. 5D). These data are consistent with the observations that BA.5 and BQ.1 manifest strong immune escape from the ancestral S-based vaccine induced neutralization. The data also show that the strong viral control by mRNA-S+N in the hamster lungs is occurring even in the absence of detectable neutralizing antibodies, indicating a role of cellular immunity in mRNA-S+N induced protection against variants as described previously11.Fig. 5 Serum antibody response in hamsters following mRNA vaccination.

S-specific binding IgG (A), IgA (B), and IgM (C) endpoint titers (EPTs) in the hamster sera at week 5 after immunization (n = 4 for LNP; n = 10 for mRNA-S or mRNA-S+N). Log10 normalized EPTs for each group is indicated. D Neutralization of the hamster sera (n = 10 for all three groups) against SARS-CoV-2 WA.1, BA.1, BA.5, and BQ.1 as measured by PRNT50. Data are presented as median with interquartile range. Dotted lines in each plot indicates LOD for each assay. Number of animals in each group with neutralizing titers above the LOD is noted in (D). Data were compared among the three groups by Kruskal-Wallis test. *p < 0.05, **p < 0.01, ****p < 0.0001.

mRNA-S+N vaccination induces strong respiratory T cell responses in the lungs and bronchoalveolar lavage

After showing that mRNA-S+N vaccine induced complete viral control in the hamster lungs in the absence of detectable neutralizing antibodies, we next examined respiratory immune responses induced by mRNA-S+N as compared to mRNA-S alone. For this, we utilized the C57BL/6 mouse model and the available S- (S539–546: VNFNFNGL) and N- (N219–227: LLLDRLNQL) specific MHC-I tetramers. Similar to N219–227 (Fig. 2), S539-546 (VNFNFNGL) is a highly dominant CD8 T cell epitope within the S protein that was predicted based on bioinformatic analysis and later validated in the context of both infection and vaccination30–32. Multiple vaccine studies have confirmed a protective role of the CD8 T cell response induced against this particular S epitope33,34.

Three groups of C57BL/6 mice (n = 5 per group) were vaccinated (IM) with empty LNP (mock), mRNA-S alone (1 μg), or mRNA-S+N (1 μg per mRNA) at week 0 and 3, followed by analysis of vaccine-induced immune responses in the lungs and BAL (Fig. 6A). T cell responses in the spleen were also examined for comparison. The gating strategies to identify T cell populations and tetramer+ CD8 T cells are shown in Supplementary Fig. 1, Supplementary Fig. 4, and the main figures.Fig. 6 Respiratory T cell response following different mRNA vaccination.

A Mouse experimental design and timeline (Created with BioRender.com). Three groups of C57BL/6 mice (n = 5/group) were intramuscularly vaccinated with either empty LNP (mock), mRNA-S vaccine (1 μg), or mRNA-S+N vaccine (1 μg for each mRNA) at week 0 and 3. Two weeks after final dose (week 5), mice were euthanized and immune analyses were performed. Analysis of activated T cells in the lungs (B), BAL (C), and spleens (D) of mice. Expression of CD44 on CD4 and CD8 T cells was examined by flow cytometry and shown as percent CD44+ of the parental population. Expression of CXCR6 on CD4 and CD8 T cells in the lungs (E), BAL (F), and spleens (G) was examined by flow cytometry and shown as percent CXCR6+ of the parental population. H Representative flow cytometry plots for S- and N-tetramer staining of CD8 T cells from LNP, mRNA-S, and mRNA-S+N vaccinated mice. Tetramer+ (S- and N-epitope specific CD8 T cells) in the lungs (I), BAL (J), and spleens (K) of mice. Data are presented as median and interquartile range. Kruskal-Wallis test was used for statistical comparison among the three groups. *p < 0.05, **p < 0.01.

In the lungs, compared to mock control, both mRNA-S and mRNA-S+N elicited slightly higher levels of total activated (CD44+) CD4 T cells, although only the mRNA-S group reached statistical significance (p < 0.05 for mock versus mRNA-S) (Fig. 6B, left). A significant increase in the frequency of total activated CD8 T cells in the lungs of mRNA-S+N-vaccinated mice (median: 15%) was observed when compared to the mock group (median: 6.53%) (p < 0.01) (Fig. 6B, right). In the BAL, a stronger effect was observed. Compared to mock control, mRNA-S+N vaccine elicited substantially higher levels of activated CD4 (Mock versus mRNA-S+N: 20.1%, 88.5%; p < 0.01) and CD8 (Mock versus mRNA-S+N: 14.6%, 92.9%; p < 0.01) T cells (Fig. 6C). Compared to mRNA-S+N, mRNA-S alone induced weaker CD4 and CD8 T cell activation in the BAL and no statistical significance was detected between the two vaccine groups (Fig. 6C). In contrast, in the spleen, we only noted a modest increase in CD4 and CD8 T cell activation in the mRNA-S+N group compared to mock control (Fig. 6D). In all these compartments, no significant difference in the frequency of total CD4 and CD8 T cells was observed among the groups (Fig. S6).

We next examined frequencies of T cells positive for CXCR6 in the lungs, BAL, and spleen (Fig. 6E–G). A pattern similar to the activated T cells was revealed. Compared to the mock control, mRNA-S+N elicited significantly higher levels of CXCR6+ CD8 T cells in the lungs (1.47% versus 6.99%; p < 0.01) (Fig. 6E) as well as in the BAL (6.08% versus 51.6%, p < 0.01) (Fig. 6F). In the BAL, mRNA-S+N also induced a markedly higher level of CXCR6+ CD4 T cells when compared to the mock control (7.87% versus 66.9%, p < 0.01) (Fig. 6F). These data indicate lung-homing potential of the induced T cells by mRNA-S+N. In contrast, mRNA-S alone only elicited a trend towards increase in CXCR6+ CD4 and CXCR6+ CD8 T cells in the BAL when compared to the mock control (Fig. 6F). As anticipated, lower levels of CXCR6+ T cells were observed in the spleens when compared to those in the BAL and lung following mRNA-S or mRNA-S+N vaccination (Fig. 6G).

Antigen-specific CD8 T cells in these compartments were measured (Fig. 6H–K). Representative flow cytometry plots for S- and N-tetramer staining showed that mRNA-S alone elicited S-specific, but not N-specific, CD8 T cells, while mRNA-S+N vaccine elicited both S- and N-specific CD8 T cells, supporting the specificity of the tetramer staining (Fig. 6H). We observed that, compared to mRNA-S alone, mRNA-S+N vaccine elicited higher levels of S-specific CD8 T cells in the lungs (p < 0.05) (Fig. 6I). A more profound effect was observed in the BAL, where mRNA-S+N induced much higher levels of S-specific CD8 T cells (~36%) than mRNA-S (~14%) (Fig. 6J). Of note, while mRNA-S+N also elicited detectable N-specific CD8 T cells in the lungs (Fig. 6I), their levels in the BAL were low (Fig. 6J). Lastly, only low levels of S-epitope specific CD8 T cells were detected in the spleen after mRNA-S+N vaccination (Fig. 6K). Together, these data indicate that mRNA-S+N elicits strong respiratory T cell responses, especially S-specific CD8 T cells, after IM mRNA immunization. The data also reveal that the presence of N for co-immunization has some synergistic effect and augments S-specific T cell response in the respiratory tract, a finding also observed for systemic immunity in our previous study11.

Vaccine-induced binding antibodies in the BAL were also examined. Compared to mock control, both vaccines induced significant levels of S-specific binding IgG (Fig. S7). As expected, only mRNA-S+N vaccination resulted in the production of both S- and N-specific binding IgG in the BAL (Fig. S7), whereas mRNA-S alone only elicited S-specific binding IgG. Unlike IgG, there was a lack of detectable antigen-specific IgA in the BAL, consistent with the result of mRNA-N vaccination (Supplementary Fig. 5) as well as with a previous study reporting limited mucosal IgA production following IM mRNA immunization15.

Discussion

The emergence of novel SARS-CoV-2 variants has greatly reduced effectiveness of the first-generation COVID-19 vaccines35. Our previous study reported the efficacy of the dual mRNA-S+N vaccine against Delta and Omicron BA.1 strain in animal models11. The present study demonstrates that mRNA-S+N, based on the ancestral viral sequence, induces robust protection against the more immune evasive Omicron subvariants, BA.5 and BQ.1, in the absence of detectable neutralizing antibodies. Additionally, in contrast to our previous study which examined vaccine-induced systemic immunity11, this study investigates mRNA vaccine-induced respiratory immunity and shows that peripheral immunization with mRNA-S+N effectively elicits respiratory S- and N-specific CD8 T cell responses in the animal lungs and BAL at the time examination (2 weeks after booster). The data together support mRNA-S+N as a promising pan-SARS-CoV-2 vaccine approach and provide additional immunological insights in mechanisms of protection conferred by mRNA-S+N against immune-escaping variants.

There are ongoing efforts to develop a next-generation, broadly protective SARS-CoV-2 vaccine against current and emerging variants. Targeting conserved regions of the virus, e.g., N protein, in addition to the S protein, is considered an attractive strategy for pan-COVID-19 vaccine development36,37. In support, our analysis reveals that N protein has higher sequence conservation among the different SARS-CoV-2 variants (BA.1, BA.5, BQ.1., and Wuhan-Hu-1) as compared to the S protein (Table 1). N is a structural protein of SARS-CoV-2 with abundant expression in infected cells and plays a role in the coronavirus life cycle, including viral genome packaging and immune regulation38. Notably, the N-induced memory T cell response is cross-reactive39. In this study, we further demonstrate that mRNA-N alone induces incomplete but significant control of Omicron BA.5 infection in hamster lungs. The data are consistent with our previous report showing a modest protective effect of this vaccine alone against mouse-adapted SARS-CoV-2 and Delta variant11.

Our study also determines the efficacy of dual mRNA-S+N vaccine against newly emerged immune-escaping Omicron subvariants BA.5 and BQ.1. Compared to the early variants, BA.5 possesses additional changes (69-70del, L452R, and F486V) and a reversion (R493Q) in the S protein (Table 1), which contribute to its enhanced fusogenicity and infectivity22. Studies in rodents showed that BA.5 has superior viral fitness to that BA.240. In addition, when the present study was conducted, the BQ.1 variant which bears additional mutations in its S protein (i.e., N460K, R346T, and K444T) (Table 1) had emerged as a dominant circulating strain with enhanced transmissibility and immune evasion35,41,42. In line with these observations, our data showed that while the sera of mRNA-S or mRNA-S+N-immunized hamsters manifested strong neutralization of the prototypical WA.1 strain, and even the Delta strain11, their neutralizing activities were greatly reduced against Omicron BA.1 (only a few animals in each vaccine group showed weak neutralization) and were completely lost against BA.5 and BQ.1 (Fig. 5D)43,44. However, even in the absence of detectable neutralizing antibodies, we still observed complete viral control in hamster lungs by mRNA-S+N against both BA.5 and BQ.1. Indeed, our previous study using in vivo CD8 cell depletion did support a role of T cell immunity in protection11. Despite these data, immune mechanisms other than T cell immunity, such as other effector functions of antibodies induced by S and N23,45,46, should not be excluded and warrant further investigation. Nevertheless, our data confirmed the breadth of protection conferred by mRNA-S+N, irrespective of S-based mutations, and support the idea that vaccine approaches inducing robust, cross-protective cellular immunity should be pursued47,48.

Respiratory immunity is important for protection against SARS-CoV-2 infection. Studies on induction of respiratory immunity by peripheral mRNA vaccination have reported inconsistent results. Some studies reported minimal T cell and antibody responses in the airway mucosa after peripheral vaccination15,16, while others showed detectable mucosal IgA along with CD4 and CD8 T cells in vaccinated individuals17,18. In our study, we analyzed total and antigen-specific T cells in the lungs and BAL of the immunized mice. We demonstrated that both S and N epitope-specific CD8 T cells were detectable following IM mRNA-S+N vaccination in the lungs and/or BAL. This induction of a robust respiratory T cell response correlates with the strong control of BA.5 and BQ.1 by mRNA-S+N in the absence of neutralizing antibodies. Thus, our data favor that peripheral mRNA vaccination could induce T cell responses in the airway, although it remains unclear if these T cell responses are durable, since we only examined the response at the peak immunogenicity following vaccination (2 weeks after booster). Our analysis of antibodies showed that only binding IgG was detectable in the BAL. Lack of detection of mucosal IgA following IM mRNA vaccination in our study is consistent with previous reports15,16 and supports the need for a mucosal booster to further improve respiratory immunity after peripheral mRNA vaccination.

The present study utilized MHC-I tetramer staining to identify antigen-specific CD8 T cells in the mouse lungs and BAL. Compared to intracellular cytokine staining (ICS), tetramer staining requires fewer cells and no peptide restimulation, thus making it less challenging to measure antigen-specific T cells in samples of low cell numbers (e.g., BAL), although tetramer staining only identifies T cells specific to single epitope. In our study, two MHC-I epitopes (N219-227: LLLDRLNQL; S539-546: VNFNFNGL) were used to detect N- and S-specific CD8 T cells in mouse BAL/lungs. Both are dominant CD8 T cell epitopes within the SARS-CoV-2 N and S protein, respectively. The two epitopes were initially predicted by bioinformatical analysis8 and subsequently validated in humans and mice24,25,30–32. In particular, the protective role of CD8 T cell responses against the single S539-546 epitope was confirmed in multiple SARS-CoV-2 vaccine studies33,34. Consistent with these reports, our study shows that IM mRNA-S+N vaccination elicits both S539-546 and N219-227 epitope-specific CD8 T cell populations in the mouse airway (Fig. 6). It is interesting to note that the magnitude of S539-546-specific CD8 T cell response is markedly higher than N219-227-specific CD8 T cell response in the mouse BAL/lungs, although the dose of mRNA-S and mRNA-N used in the dual immunization regimen (mRNA-S+N) is identical. This is likely due to multiple reasons. First, the co-immunization of N augments S-specific immunity, but not vice versa. This pattern is particularly evident in the CD8 T cell responses, as seen here and in our previous study11. The underlying mechanisms are not yet clear. Second, the S539-546 epitope may be more dominant within the S protein than the N219-227 epitope within the N protein following mRNA vaccination. Also, it is possible that CD8 T cells specific to other N epitopes are also induced by mRNA vaccines but not detectable using the N219-227 tetramer staining.

Other than CD8 epitopes, a number of CD4 T cell epitopes within the SARS-CoV-2 S and N proteins were also identified8,49, despite their protective roles in vaccination are less clear. Our study examined respiratory CD4 T cells with activation (CD44) or lung-tissue residency (CXCR6) phenotype and showed that IM mRNA-S+N vaccination elicits high levels of activated and CXCR6+ CD4 T cells, especially in the BAL (Fig. 6C, F). The present study did not measure respiratory antigen-specific CD4 T cells, largely due to the current lack of tetramers for these CD4 epitopes. However, our previous study used ICS assay and showed the induction of robust systemic antigen-specific CD4 T cells by mRNA-S+N vaccine in BALB/c mice11. We thus speculate that peripheral mRNA vaccination should also elicit antigen-specific CD4 T cells in BAL/lungs. Nevertheless, a more comprehensive characterization of the respiratory CD4 and CD8 T cell responses specific to the full S and N protein in the BAL/lungs following mRNA vaccination is needed in future studies.

The present study has several limitations. First, in the respiratory immune analysis, single cell suspensions were prepared from the whole lung tissue without discriminating immune cells from circulating blood, raising the concern that the total and antigen-specific T cells identified in the lungs could also contain contaminated cells from the blood. However, our analysis of T cells in the BAL supports that mRNA vaccination induces respiratory T cell responses in the airway. Second, while we show the induction of antigen-specific T cells in the lung and BAL at peak immunogenicity, whether they are long-lasting, tissue-resident T cells and can confer durable protection remain unclear. Lastly, with the vast majority of the human population having received the S-based vaccines or being naturally infected with SARS-CoV-2, mRNA-N as a booster component for inducing a broad protection in the context of pre-existing immunity should be investigated, which could expand the potential utility of this vaccine candidate.

Materials and methods

Study design

The aim of the present study was to investigate efficacy of mRNA-N and mRNA-S+N against newer SARS-CoV-2 Omicron variants and the respiratory immunity induced by these mRNA vaccines in animal models. Evaluation of vaccine efficacy against Omicron BA.5 and BQ.1 challenges were conducted in Syrian hamsters, and analysis of vaccine-induced respiratory immunity was conducted in C57BL/6 mice. The animal study protocols were approved by the Institutional Animal Care and Use Committee at the University of Texas Medical Branch (Protocol numbers: 1703020, 2009087). All animal experiments were carried out following the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. During the studies, all animals were monitored by animal resources center or laboratory staff daily. The number of animals utilized per group was determined by a power analysis for a medium effect size prior to the onset of the study and all data points were utilized for analysis. All animals were allowed a minimum of 3 days to acclimate to their environment before study onset and anesthetized with 1–5% isoflurane via vaporizer prior to each procedure apart from weights. Mice were humanely euthanized with CO2 asphyxiation followed by cervical dislocation. Hamsters were humanely euthanized with CO2 asphyxiation followed by bilateral thoracotomy. The study design was not blinded to researchers or staff at the animal facility, and animals were randomly assigned to each group.

mRNA synthesis and LNP formulation

mRNA synthesis and LNP formulations were prepared as published previously11. Briefly, mRNA-N (full-length) and mRNA-S (prefusion-stabilized S protein with two proline mutations; mRNA-S-2P) were synthesized by using the T7 RNA polymerase (MegaScript, Thermo Fisher Scientific) in vitro transcription kit. The sequences are based on the ancestral SARS-CoV-2 Wuhan-Hu-1 strain (GenBank MN908947.3). Uridine triphosphate was replaced with one-methylpseudouridine (m1)-5′-triphosphate. For improved protein expression, polyadenylated tails were added to the ends of modified mRNAs. ScriptCap m7G capping system and ScriptCap 2′-O-methyltransferase kit were used to cap in vitro transcribed mRNAs, which were then underwent cellulose-based purification. As previously reported, the mRNAs were formulated into LNPs using an ethanolic lipid mixture of ionizable cationic lipid and an aqueous buffer system. mRNA-LNPs were prepared in accordance with RNA concentrations (1 µg/µl) and stored at −80 °C prior to animal immunizations. Protein expression in cells after mRNA-N and mRNA-S LNP treatment was confirmed in our previous publication11.

Animal immunizations and SARS-CoV-2 challenge

mRNA vaccine-induced immune response was evaluated in mice. 6-week-old female C57BL/6 mice purchased from the Jackson Laboratory (strain no. 000664) were randomly assigned into four groups (n = 5 per group) that were respectively immunized IM (thigh muscles of the hind limb) with 50 µl of either empty LNP (mock), mRNA-S (1 μg), mRNA-N (1 μg), or mRNA-S+N (1 μg per mRNA) at week 0 (prime) and week 3 (boost). 2 weeks after booster vaccination (week 5), all mice were euthanized (CO2 and physical dislocation) for terminal blood collection and tissue harvests. Lung and BAL samples were collected for analysis of vaccine-induced respiratory immune responses. In some experiments, sera and spleen samples were also collected to analyze vaccine-induced systemic immune response for comparison.

mRNA vaccine efficacy was evaluated in hamsters. 3-week-old male golden Syrian hamsters purchased from Envigo (strain HsdHan: AURA, catalog no. 8901M) were randomly assigned into four groups (n = 10 per group). They were respectively vaccinated IM (thigh muscles of the hind limb) with 100 µL of either LNP (mock), mRNA-S (2 µg), mRNA-N (2 µg), or combined mRNA-S+N (2 µg per mRNA) at weeks 0 and 3. Two weeks after booster (week 5) and prior to viral challenge, all hamsters were subject to serum collection under anesthesia. The sera were used to measure vaccine-induced antibodies. Hamsters were then transferred to ABSL-3 facility and intranasally challenged with either SARS-CoV-2 Omicron BA.5 (2 × 104 pfu in 100 µl/hamster) or BQ.1 (2 × 104 pfu in 100 µl/hamster) under anesthesia. At 2- and 4 DPI, five hamsters of each group (for each time point) were euthanized and the lung tissues were collected for evaluating vaccine-induced protection. Hamster body weights were also monitored daily from the day of viral challenge to 4 DPI.

Spike-specific binding antibodies in hamster sera

Vaccine-induced S-specific binding antibody titers (IgG, IgA, and IgM) in the hamster sera were determined by ELISA. Plates (Greiner Bio-One) were coated with recombinant S protein (1 µg/ml; 40589-V08B1, Sino Biological) overnight at 4°C. Plates were washed three times and then blocked with blocking buffer [8% fetal bovine serum (FBS) in Dulbecco’s PBS (DPBS)] for 1 h at 37 °C, followed by washing and incubation at 37 °C for 2 h with serially diluted serum samples (initial dilution 1:100, followed by 3-fold serial dilution) in blocking buffer at 50 µl per well. Plates were washed again and incubated with HRP-conjugated anti-hamster IgG (1:1000; Southern Biotech; 6060-05), anti-hamster IgA (1:1000; Brookwood Biomedical; sab3003a), or anti-hamster IgM (1:500; Brookwood Biomedical; sab3003m) secondary antibodies for 1 h (IgG) or overnight (IgA and IgM) at 37 °C. After incubation, plates were washed and developed using TMB 1-Component Peroxidase Substrate (Thermo Fisher Scientific), followed by termination of reaction using the 2 N HCl solution. Plates were read at 450 nm wavelength within 15 min by using a Microplate Reader (BioTek). Binding antibody EPTs were calculated.

Spike- and nucleocapsid-specific binding antibodies in mouse bronchoalveolar lavage

S- and N-specific binding IgG and IgA in mouse BAL were measured by ELISA as described above except the BAL samples underwent 1:1 dilution.

Flow cytometry analysis

Single cell suspensions prepared from lung, BAL, and spleen were directly stained for viability (65-0866-14, Invitrogen; Fixable Viability Dye eFluor 506) and surface markers including CD3- BUV805 (741982, BD Biosciences; clone 17A2), CD4-APC/Fire 570 (100460, BioLegend; clone GK1.5), CD8-BV570 (100740, Biolegend; clone 53-6.7), CD44-PerCP (103036, Biolegend; clone IM7), CXCR6-BV711 (151111, Biolegend; clone SA051D1). To detect S- and N-specific CD8 T cells, samples were incubated with S539–546 MHC-I tetramer-Alexa488 (H-2Kb, 1:500 dilution) and N219–227 MHC-I tetramer-BV421 (H2-Db, 1:500 dilution) (NIH Tetramer Core) for 30 min at room temperature. Cells were washed and then acquired on a flow cytometer FACSymphony A5 (BD Biosciences). Data were analyzed using FlowJo (Flowjo LLC).

Quantification of viral RNA by RT-qPCR

Hamster lungs were collected in maintenance medium (2% FBS in DMEM with 1% pen-strep) and subjected to homogenization for 1 min. Debris was pelleted by centrifugation for 5 min at 16,000 g. Following homogenization, the supernatants of tissue homogenates were combined with a fivefold volume of TRIzol LS Reagent (Thermo Fisher Scientific). Extraction of viral RNAs was performed in accordance with the instructions provided by the manufacturer. The final RNA solutions were stored at −80 °C until use for RT-qPCR. Viral RNA copies were determined in the lungs by using the one-step RT-qPCR kit (Bio-Rad, 1725151) on CFX Connect Real-Time PCR Detection System (Bio-Rad). SARS-CoV-2 E gene primers (forward, 5′-GGAAGAGACAGGTACGTTAATA-3′; reverse, 5′-AGCAGTACGCACACAATCGAA-3′) were used. The PCR reaction was composed of primers (10 μM), RNA sample (2 μl), iTaq universal SYBR Green 1-step reaction mix (2×; 10 μl), iScript reverse transcriptase (0.25 μl), and molecular grade water, for a total volume of 20 μl. The PCR cycling conditions were as follows: 95 °C for a duration of 3 min, followed by 45 cycles of 95 °C for 5 s and 60 °C for 30 s. A standard curve was included in each RT-qPCR analysis, utilizing an RNA standard that was synthesized in vitro. This RNA standard consisted of 3839 base pairs and encompassed genomic nucleotide locations 26,044 to 29,883 of the SARS-CoV-2 genome. By using the standard curve we determined the absolute number of viral RNA copies present in the lung tissue11.

Virus neutralization analysis

Serum neutralizing activity was examined using PRNT assay11. The experiments were carried out on Vero E6 cells (ATCC, CRL-1586) with the SARS-CoV-2 wild-type or Omicron variants. Briefly, serum samples were heat-inactivated (at 56 °C for 30 min) and serially diluted twice (1:10 initial dilution and then twofold serial dilutions), followed by incubation for 1 h at 37 °C with wild-type SARS-CoV-2 (USA-WA1/2020), BA.5, or BQ.1, respectively. The above mixtures were added to monolayers of Vero E6 cells in 6-well plates for incubation at 37 °C for 1 h. After incubation, a 2 ml of semisolid overlay medium (minimum essential medium containing 1.6% agarose, 2% FBS, and 1% penicillin-streptomycin) was added to the cells, and then incubated for 48 h at 37 °C. After incubation, monolayer cells were stained with 0.03% liquid neutral red for 3–6 h. A manual counting method was used to count the number of plaques and PRTN50 was calculated.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 10.1.1 software. Data were presented as median ± IQR or mean ± standard deviation, as denoted in the figure legends. Statistical comparison among groups was performed using Mann-Whitney test, Kruskal-Wallis test, or two-way ANOVA with Tukey’s multiple comparison test where appropriate as denoted in the figure legends. Two-tailed p values were denoted, and p < 0.05 were considered significant. All analyses were conducted assuming a 95% confidence interval.

Supplementary information

Supplementary Information

Supplementary information

The online version contains supplementary material available at 10.1038/s41541-024-00957-2.

Acknowledgements

Virus stocks were obtained from the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA). We thank Meredith Weglarz and the flow cytometry and cell sorting (FCCS) core at UTMB for assistance in flow cytometry. The research was supported by a UTMB COVID-19 pilot grant (to H.H.) and NIH grant R24AI120942 (to S.C.W.). H.H. was supported by NIH grants AI157852 and AI181134. D.W. was supported by NIH grant P01-AI158571. R.L.H. was supported by UTMB Mclaughlin Fellowship. The funders played no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author contributions

S.R.B., H.H., R.L.H., and K.S.P., designed experiments; R.L.H. and S.R.B. performed animal immunization experiments with assistance from Y.L. and N.C.H.; S.R.B., J.A.P., K.S.P., R.A.R. and J.H.W., performed animal challenge experiments. R.L.H. and S.R.B. performed ELISA, flow cytometry, RT-qPCR, and data analysis. J.A.P., J.H.W., and G.H.R. performed viral neutralizing experiments. D.H.W. performed data analysis; M-G.A., D.W. provided experimental materials; H.H., K.S.P., S.C.W. supervised experiments and the study. S.R.B., H.H., and R.L.H. wrote manuscript draft with editing from all authors.

Data availability

The datasets generated and analyzed in this study are presented in the manuscript and the supplementary files. Original raw data are also available from the corresponding authors upon request.

Competing interests

H.H. and D.W. are inventors on a patent (PCT) titled COVID-19 mRNA vaccine (WO2023056045A1). All other authors declare no competing interests where relevant.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

These authors contributed equally: Renee L. Hajnik, Jessica A. Plante, Srinivasa Reddy Bonam.
==== Refs
References

1. Baden LR Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine N. Engl. J. Med. 2020 384 403 416 10.1056/NEJMoa2035389 33378609
Baden, L. R. et al. Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine. N. Engl. J. Med. 384, 403–416 (2020).33378609 10.1056/NEJMoa2035389
2. Polack FP Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine N. Engl. J. Med. 2020 383 2603 2615 10.1056/NEJMoa2034577 33301246
Polack, F. P. et al. Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine. N. Engl. J. Med. 383, 2603–2615 (2020).33301246 10.1056/NEJMoa2034577
3. Harvey WT SARS-CoV-2 variants, spike mutations and immune escape Nat. Rev. Microbiol. 2021 19 409 424 10.1038/s41579-021-00573-0 34075212
Harvey, W. T. et al. SARS-CoV-2 variants, spike mutations and immune escape. Nat. Rev. Microbiol. 19, 409–424 (2021).34075212 10.1038/s41579-021-00573-0
4. Viana R Rapid epidemic expansion of the SARS-CoV-2 Omicron variant in southern Africa Nature 2022 603 679 686 10.1038/s41586-022-04411-y 35042229
Viana, R. et al. Rapid epidemic expansion of the SARS-CoV-2 Omicron variant in southern Africa. Nature 603, 679–686 (2022).35042229 10.1038/s41586-022-04411-y
5. Chatterjee S Bhattacharya M Nag S Dhama K Chakraborty C A detailed overview of SARS-CoV-2 omicron: its sub-variants, mutations and pathophysiology, clinical characteristics, immunological landscape, immune escape, and therapies Viruses 2023 15 167 10.3390/v15010167 36680207
Chatterjee, S., Bhattacharya, M., Nag, S., Dhama, K. & Chakraborty, C. A detailed overview of SARS-CoV-2 omicron: its sub-variants, mutations and pathophysiology, clinical characteristics, immunological landscape, immune escape, and therapies. Viruses 15, 167 (2023).36680207 10.3390/v15010167
6. Garcia-Beltran WF Multiple SARS-CoV-2 variants escape neutralization by vaccine-induced humoral immunity Cell 2021 184 2372 83.e9 10.1016/j.cell.2021.03.013 33743213
Garcia-Beltran, W. F. et al. Multiple SARS-CoV-2 variants escape neutralization by vaccine-induced humoral immunity. Cell 184, 2372–83.e9 (2021).33743213 10.1016/j.cell.2021.03.013
7. Lau JJ Real-world COVID-19 vaccine effectiveness against the Omicron BA.2 variant in a SARS-CoV-2 infection-naive population Nat. Med 2023 29 348 357 10.1038/s41591-023-02219-5 36652990
Lau, J. J. et al. Real-world COVID-19 vaccine effectiveness against the Omicron BA.2 variant in a SARS-CoV-2 infection-naive population. Nat. Med 29, 348–357 (2023).36652990 10.1038/s41591-023-02219-5
8. Grifoni A A sequence homology and bioinformatic approach can predict candidate targets for immune responses to SARS-CoV-2 Cell Host Microbe 2020 27 671 680.e2 10.1016/j.chom.2020.03.002 32183941
Grifoni, A. et al. A sequence homology and bioinformatic approach can predict candidate targets for immune responses to SARS-CoV-2. Cell Host Microbe 27, 671–680.e2 (2020).32183941 10.1016/j.chom.2020.03.002
9. Grifoni A Targets of T cell responses to SARS-CoV-2 coronavirus in humans with COVID-19 disease and unexposed individuals Cell 2020 181 1489 501.e15 10.1016/j.cell.2020.05.015 32473127
Grifoni, A. et al. Targets of T cell responses to SARS-CoV-2 coronavirus in humans with COVID-19 disease and unexposed individuals. Cell 181, 1489–501.e15 (2020).32473127 10.1016/j.cell.2020.05.015
10. Peng Y Broad and strong memory CD4(.) and CD8(.) T cells induced by SARS-CoV-2 in UK convalescent individuals following COVID-19 Nat. Immunol. 2020 21 1336 1345 10.1038/s41590-020-0782-6 32887977
Peng, Y. et al. Broad and strong memory CD4(.) and CD8(.) T cells induced by SARS-CoV-2 in UK convalescent individuals following COVID-19. Nat. Immunol. 21, 1336–1345 (2020).32887977 10.1038/s41590-020-0782-6
11. Hajnik RL Dual spike and nucleocapsid mRNA vaccination confer protection against SARS-CoV-2 Omicron and Delta variants in preclinical models Sci. Transl. Med. 2022 14 eabq1945 10.1126/scitranslmed.abq1945 36103514
Hajnik, R. L. et al. Dual spike and nucleocapsid mRNA vaccination confer protection against SARS-CoV-2 Omicron and Delta variants in preclinical models. Sci. Transl. Med. 14, eabq1945 (2022).36103514 10.1126/scitranslmed.abq1945
12. Saksena NK SARS-CoV-2 variants, its recombinants and epigenomic exploitation of host defenses Biochim. Biophys. Acta Mol. Basis Dis. 2023 1869 166836 10.1016/j.bbadis.2023.166836 37549720
Saksena, N. K. et al. SARS-CoV-2 variants, its recombinants and epigenomic exploitation of host defenses. Biochim. Biophys. Acta Mol. Basis Dis. 1869, 166836 (2023).37549720 10.1016/j.bbadis.2023.166836
13. Ito J Convergent evolution of SARS-CoV-2 Omicron subvariants leading to the emergence of BQ.1.1 variant Nat. Commun. 2023 14 2671 10.1038/s41467-023-38188-z 37169744
Ito, J. et al. Convergent evolution of SARS-CoV-2 Omicron subvariants leading to the emergence of BQ.1.1 variant. Nat. Commun. 14, 2671 (2023).37169744 10.1038/s41467-023-38188-z
14. Case JB Characterization of the SARS-CoV-2 BA.5.5 and BQ.1.1 Omicron variants in mice and hamsters J. Virol. 2023 97 e0062823 10.1128/jvi.00628-23 37676002
Case, J. B. et al. Characterization of the SARS-CoV-2 BA.5.5 and BQ.1.1 Omicron variants in mice and hamsters. J. Virol. 97, e0062823 (2023).37676002 10.1128/jvi.00628-23
15. Tang J Respiratory mucosal immunity against SARS-CoV-2 following mRNA vaccination Sci. Immunol. 2022 7 eadd4853 10.1126/sciimmunol.add4853 35857583
Tang, J. et al. Respiratory mucosal immunity against SARS-CoV-2 following mRNA vaccination. Sci. Immunol. 7, eadd4853 (2022).35857583 10.1126/sciimmunol.add4853
16. Lim JME SARS-CoV-2 breakthrough infection in vaccinees induces virus-specific nasal-resident CD8+ and CD4+ T cells of broad specificity J. Exp. Med. 2022 219 e20220780 10.1084/jem.20220780 35972472
Lim, J. M. E. et al. SARS-CoV-2 breakthrough infection in vaccinees induces virus-specific nasal-resident CD8+ and CD4+ T cells of broad specificity. J. Exp. Med. 219, e20220780 (2022).35972472 10.1084/jem.20220780
17. S Guerrieri S Lazzarin C Zanetta A Nozzolillo M Filippi L Moiola Serological response to SARS-CoV-2 vaccination in multiple sclerosis patients treated with fingolimod or ocrelizumab: an initial real-life experience J. Neurol. 2022 269 39 43 10.1007/s00415-021-10663-x 34189719
S, Guerrieri, S, Lazzarin, C, Zanetta, A, Nozzolillo, M, Filippi & L, Moiola Serological response to SARS-CoV-2 vaccination in multiple sclerosis patients treated with fingolimod or ocrelizumab: an initial real-life experience. J. Neurol. 269, 39–43 (2022).34189719 10.1007/s00415-021-10663-x
18. Ssemaganda A Expansion of cytotoxic tissue-resident CD8(+) T cells and CCR6(+)CD161(+) CD4(+) T cells in the nasal mucosa following mRNA COVID-19 vaccination Nat. Commun. 2022 13 3357 10.1038/s41467-022-30913-4 35688805
Ssemaganda, A. et al. Expansion of cytotoxic tissue-resident CD8(+) T cells and CCR6(+)CD161(+) CD4(+) T cells in the nasal mucosa following mRNA COVID-19 vaccination. Nat. Commun. 13, 3357 (2022).35688805 10.1038/s41467-022-30913-4
19. Yoshimoto FK The proteins of severe acute respiratory syndrome coronavirus-2 (SARS CoV-2 or n-COV19), the cause of COVID-19 Protein J. 2020 39 198 216 10.1007/s10930-020-09901-4 32447571
Yoshimoto, F. K. The proteins of severe acute respiratory syndrome coronavirus-2 (SARS CoV-2 or n-COV19), the cause of COVID-19. Protein J. 39, 198–216 (2020).32447571 10.1007/s10930-020-09901-4
20. Wu A Genome composition and divergence of the novel coronavirus (2019-nCoV) originating in China Cell Host Microbe 2020 27 325 328 10.1016/j.chom.2020.02.001 32035028
Wu, A. et al. Genome composition and divergence of the novel coronavirus (2019-nCoV) originating in China. Cell Host Microbe 27, 325–328 (2020).32035028 10.1016/j.chom.2020.02.001
21. Miller Substantial neutralization escape by SARS-CoV-2 Omicron variants BQ.1.1 and XBB.1 N. Engl. J. Med. 2023 388 662 664 10.1056/NEJMc2214314 36652339
Miller et al. Substantial neutralization escape by SARS-CoV-2 Omicron variants BQ.1.1 and XBB.1. N. Engl. J. Med. 388, 662–664 (2023).36652339 10.1056/NEJMc2214314
22. Tegally H Emergence of SARS-CoV-2 Omicron lineages BA.4 and BA.5 in South Africa Nat. Med. 2022 28 1785 1790 10.1038/s41591-022-01911-2 35760080
Tegally, H. et al. Emergence of SARS-CoV-2 Omicron lineages BA.4 and BA.5 in South Africa. Nat. Med. 28, 1785–1790 (2022).35760080 10.1038/s41591-022-01911-2
23. Dangi T Improved control of SARS-CoV-2 by treatment with a nucleocapsid-specific monoclonal antibody J. Clin. Investig. 2022 132 e162282 10.1172/JCI162282 36219482
Dangi, T. Improved control of SARS-CoV-2 by treatment with a nucleocapsid-specific monoclonal antibody. J. Clin. Investig. 132, e162282 (2022).36219482 10.1172/JCI162282
24. Saini SK SARS-CoV-2 genome-wide T cell epitope mapping reveals immunodominance and substantial CD8(+) T cell activation in COVID-19 patients Sci. Immunol. 2021 6 eabf7550 10.1126/sciimmunol.abf7550 33853928
Saini, S. K. et al. SARS-CoV-2 genome-wide T cell epitope mapping reveals immunodominance and substantial CD8(+) T cell activation in COVID-19 patients. Sci. Immunol. 6, eabf7550 (2021).33853928 10.1126/sciimmunol.abf7550
25. Joag V Cutting edge: mouse SARS-CoV-2 epitope reveals infection and vaccine-elicited CD8 T cell responses J. Immunol. 2021 206 931 935 10.4049/jimmunol.2001400 33441437
Joag, V. et al. Cutting edge: mouse SARS-CoV-2 epitope reveals infection and vaccine-elicited CD8 T cell responses. J. Immunol. 206, 931–935 (2021).33441437 10.4049/jimmunol.2001400
26. Ponta H Sherman L Herrlich PA CD44: from adhesion molecules to signalling regulators Nat. Rev. Mol. Cell Biol. 2003 4 33 45 10.1038/nrm1004 12511867
Ponta, H., Sherman, L. & Herrlich, P. A. CD44: from adhesion molecules to signalling regulators. Nat. Rev. Mol. Cell Biol. 4, 33–45 (2003).12511867 10.1038/nrm1004
27. Wein AN CXCR6 regulates localization of tissue-resident memory CD8 T cells to the airways J. Exp. Med 2019 216 2748 2762 10.1084/jem.20181308 31558615
Wein, A. N. et al. CXCR6 regulates localization of tissue-resident memory CD8 T cells to the airways. J. Exp. Med 216, 2748–2762 (2019).31558615 10.1084/jem.20181308
28. Scarpa F Genetic and structural data on the SARS-CoV-2 Omicron BQ.1 variant reveal its low potential for epidemiological expansion Int. J. Mol. Sci. 2022 23 15264 10.3390/ijms232315264 36499592
Scarpa, F. et al. Genetic and structural data on the SARS-CoV-2 Omicron BQ.1 variant reveal its low potential for epidemiological expansion. Int. J. Mol. Sci. 23, 15264 (2022).36499592 10.3390/ijms232315264
29. Case J. B., et al. Characterization of the SARS-CoV-2 BA.5.5 and BQ.1.1 Omicron variants in mice and hamsters. J. Virol. 97, e0062823 (2023).
30. Li Mechanisms of innate and adaptive immunity to the Pfizer-BioNTech BNT162b2 vaccine Nat. Immunol. 2022 23 543 555 10.1038/s41590-022-01163-9 35288714
Li et al. Mechanisms of innate and adaptive immunity to the Pfizer-BioNTech BNT162b2 vaccine. Nat. Immunol. 23, 543–555 (2022).35288714 10.1038/s41590-022-01163-9
31. Kingstad-Bakke B Vaccine-induced systemic and mucosal T cell immunity to SARS-CoV-2 viral variants Proc. Natl Acad. Sci. USA 2022 119 e2118312119 10.1073/pnas.2118312119 35561224
Kingstad-Bakke, B. et al. Vaccine-induced systemic and mucosal T cell immunity to SARS-CoV-2 viral variants. Proc. Natl Acad. Sci. USA 119, e2118312119 (2022).35561224 10.1073/pnas.2118312119
32. Poluektov Y George M Daftarian P Delcommenne MC Assessment of SARS-CoV-2 specific CD4(+) and CD8 (+) T cell responses using MHC class I and II tetramers Vaccine 2021 39 2110 2116 10.1016/j.vaccine.2021.03.008 33744048
Poluektov, Y., George, M., Daftarian, P. & Delcommenne, M. C. Assessment of SARS-CoV-2 specific CD4(+) and CD8 (+) T cell responses using MHC class I and II tetramers. Vaccine 39, 2110–2116 (2021).33744048 10.1016/j.vaccine.2021.03.008
33. Pardieck IN A third vaccination with a single T cell epitope confers protection in a murine model of SARS-CoV-2 infection Nat. Commun. 2022 13 3966 10.1038/s41467-022-31721-6 35803932
Pardieck, I. N. et al. A third vaccination with a single T cell epitope confers protection in a murine model of SARS-CoV-2 infection. Nat. Commun. 13, 3966 (2022).35803932 10.1038/s41467-022-31721-6
34. Montoya B., et al. mRNA-LNP vaccine-induced CD8(+) T cells protect mice from lethal SARS-CoV-2 infection in the absence of specific antibodies. Mol. Ther. 32, 1790–1804 (2024).
35. Carabelli AM SARS-CoV-2 variant biology: immune escape, transmission and fitness Nat. Rev. Microbiol. 2023 21 162 177 36653446
Carabelli, A. M. et al. SARS-CoV-2 variant biology: immune escape, transmission and fitness. Nat. Rev. Microbiol. 21, 162–177 (2023).36653446
36. Dutta NK Mazumdar K Gordy JT The nucleocapsid protein of SARS-CoV-2: a target for vaccine development J. Virol. 2020 94 e00647 20 10.1128/JVI.00647-20 32546606
Dutta, N. K., Mazumdar, K. & Gordy, J. T. The nucleocapsid protein of SARS-CoV-2: a target for vaccine development. J. Virol. 94, e00647–20 (2020).32546606 10.1128/JVI.00647-20
37. Wu W Cheng Y Zhou H Sun C Zhang S The SARS-CoV-2 nucleocapsid protein: its role in the viral life cycle, structure and functions, and use as a potential target in the development of vaccines and diagnostics Virol. J. 2023 20 6 10.1186/s12985-023-01968-6 36627683
Wu, W., Cheng, Y., Zhou, H., Sun, C. & Zhang, S. The SARS-CoV-2 nucleocapsid protein: its role in the viral life cycle, structure and functions, and use as a potential target in the development of vaccines and diagnostics. Virol. J. 20, 6 (2023).36627683 10.1186/s12985-023-01968-6
38. Bai Z Cao Y Liu W Li J The SARS-CoV-2 nucleocapsid protein and its role in viral structure, biological functions, and a potential target for drug or vaccine mitigation Viruses 2021 13 1115 10.3390/v13061115 34200602
Bai, Z., Cao, Y., Liu, W. & Li, J. The SARS-CoV-2 nucleocapsid protein and its role in viral structure, biological functions, and a potential target for drug or vaccine mitigation. Viruses 13, 1115 (2021).34200602 10.3390/v13061115
39. Le Bert N SARS-CoV-2-specific T cell immunity in cases of COVID-19 and SARS, and uninfected controls Nature 2020 584 457 462 10.1038/s41586-020-2550-z 32668444
Le Bert, N. et al. SARS-CoV-2-specific T cell immunity in cases of COVID-19 and SARS, and uninfected controls. Nature 584, 457–462 (2020).32668444 10.1038/s41586-020-2550-z
40. Uraki R Characterization of SARS-CoV-2 Omicron BA.4 and BA.5 isolates in rodents Nature 2022 612 540 545 10.1038/s41586-022-05482-7 36323336
Uraki, R. et al. Characterization of SARS-CoV-2 Omicron BA.4 and BA.5 isolates in rodents. Nature 612, 540–545 (2022).36323336 10.1038/s41586-022-05482-7
41. Li D Neutralization of BQ.1, BQ.1.1, and XBB with RBD-Dimer Vaccines N. Engl. J. Med. 2023 388 1142 1145 10.1056/NEJMc2216233 36856580
Li, D. et al. Neutralization of BQ.1, BQ.1.1, and XBB with RBD-Dimer Vaccines. N. Engl. J. Med. 388, 1142–1145 (2023).36856580 10.1056/NEJMc2216233
42. Jiang XL Omicron BQ.1 and BQ.1.1 escape neutralisation by omicron subvariant breakthrough infection Lancet Infect. Dis. 2023 23 28 30 10.1016/S1473-3099(22)00805-2 36543471
Jiang, X. L. et al. Omicron BQ.1 and BQ.1.1 escape neutralisation by omicron subvariant breakthrough infection. Lancet Infect. Dis. 23, 28–30 (2023).36543471 10.1016/S1473-3099(22)00805-2
43. Zou J Neutralization of BA.4-BA.5, BA.4.6, BA.2.75.2, BQ.1.1, and XBB.1 with bivalent vaccine N. Engl. J. Med. 2023 388 854 857 10.1056/NEJMc2214916 36734885
Zou, J. et al. Neutralization of BA.4-BA.5, BA.4.6, BA.2.75.2, BQ.1.1, and XBB.1 with bivalent vaccine. N. Engl. J. Med. 388, 854–857 (2023).36734885 10.1056/NEJMc2214916
44. Hachmann NP Neutralization escape by SARS-CoV-2 Omicron subvariants BA.2.12.1, BA.4, and BA.5 N. Engl. J. Med. 2022 387 86 88 10.1056/NEJMc2206576 35731894
Hachmann, N. P. et al. Neutralization escape by SARS-CoV-2 Omicron subvariants BA.2.12.1, BA.4, and BA.5. N. Engl. J. Med. 387, 86–88 (2022).35731894 10.1056/NEJMc2206576
45. Mackin SR Fc-gammaR-dependent antibody effector functions are required for vaccine-mediated protection against antigen-shifted variants of SARS-CoV-2 Nat. Microbiol. 2023 8 569 580 10.1038/s41564-023-01359-1 37012355
Mackin, S. R. et al. Fc-gammaR-dependent antibody effector functions are required for vaccine-mediated protection against antigen-shifted variants of SARS-CoV-2. Nat. Microbiol. 8, 569–580 (2023).37012355 10.1038/s41564-023-01359-1
46. Zhang A Beyond neutralization: Fc-dependent antibody effector functions in SARS-CoV-2 infection Nat. Rev. Immunol. 2023 23 381 396 10.1038/s41577-022-00813-1 36536068
Zhang, A. et al. Beyond neutralization: Fc-dependent antibody effector functions in SARS-CoV-2 infection. Nat. Rev. Immunol. 23, 381–396 (2023).36536068 10.1038/s41577-022-00813-1
47. Wherry EJ Barouch DH T cell immunity to COVID-19 vaccines Science 2022 377 821 822 10.1126/science.add2897 35981045
Wherry, E. J. & Barouch, D. H. T cell immunity to COVID-19 vaccines. Science 377, 821–822 (2022).35981045 10.1126/science.add2897
48. Moss P The T cell immune response against SARS-CoV-2 Nat. Immunol. 2022 23 186 193 10.1038/s41590-021-01122-w 35105982
Moss, P. The T cell immune response against SARS-CoV-2. Nat. Immunol. 23, 186–193 (2022).35105982 10.1038/s41590-021-01122-w
49. Tye EXC Mutations in SARS-CoV-2 spike protein impair epitope-specific CD4(+) T cell recognition Nat. Immunol. 2022 23 1726 1734 10.1038/s41590-022-01351-7 36456735
Tye, E. X. C. et al. Mutations in SARS-CoV-2 spike protein impair epitope-specific CD4(+) T cell recognition. Nat. Immunol. 23, 1726–1734 (2022).36456735 10.1038/s41590-022-01351-7
