Ipsilateral or contralateral boosting of mice with mRNA vaccines confers equivalent immunity and protection against a SARS-CoV-2 Omicron strain

ABSTRACT

Boosting with mRNA vaccines encoding variant-matched spike proteins has been implemented to mitigate their reduced efficacy against emerging SARS-CoV-2 variants. Nonetheless, in humans, it remains unclear whether boosting in the ipsilateral or contralateral arm with respect to the priming doses impacts immunity and protection. Here, we boosted K18-hACE2 mice with either monovalent mRNA-1273 (Wuhan-1 spike) or bivalent mRNA-1273.214 (Wuhan-1 + BA.1 spike) vaccine in the ipsilateral or contralateral leg after a two-dose priming series with mRNA-1273. Boosting in the ipsilateral or contralateral leg elicited equivalent levels of serum IgG and neutralizing antibody responses against Wuhan-1 and BA.1. While contralateral boosting with mRNA vaccines resulted in the expansion of spike-specific B and T cells beyond the ipsilateral draining lymph node (DLN) to the contralateral DLN, administration of a third mRNA vaccine dose at either site resulted in similar levels of antigen-specific germinal center B cells, plasmablasts/plasma cells, T follicular helper cells, and CD8⁺ T cells in the DLNs and the spleen. Furthermore, ipsilateral and contralateral boosting with mRNA-1273 or mRNA-1273.214 vaccines conferred similar homologous or heterologous immune protection against SARS-CoV-2 BA.1 virus challenge with equivalent reductions in viral RNA and infectious virus in the nasal turbinates and lungs. Collectively, our data show limited differences in B and T cell immune responses after ipsilateral and contralateral site boosting by mRNA vaccines that do not substantively impact protection against an Omicron strain.

IMPORTANCE

Sequential boosting with mRNA vaccines has been an effective strategy to overcome waning immunity and neutralization escape by emerging SARS-CoV-2 variants. However, it remains unclear how the site of boosting relative to the primary vaccination series shapes optimal immune responses or breadth of protection against variants. In K18-hACE2 transgenic mice, we observed that intramuscular boosting with historical monovalent or variant-matched bivalent vaccines in the ipsilateral or contralateral limb elicited comparable levels of serum spike-specific antibody and antigen-specific B and T cell responses. Moreover, boosting on either side conferred equivalent protection against a SARS-CoV-2 Omicron challenge strain. Our data in mice suggest that the site of intramuscular boosting with an mRNA vaccine does not substantially impact immunity or protection against SARS-CoV-2 infection.

INTRODUCTION

The coronavirus disease (COVID-19) pandemic has caused an estimated 776 million infections and 7. 1 million deaths worldwide (https://covid19.who.int/). Multiple vaccines (e.g., mRNA, viral-vectored, inactivated, nanoparticle, and protein subunit-based) targeting the SARS-CoV-2 spike protein were deployed with billions of doses now administered globally. Many of these vaccines are effective against the severe disease, hospitalization, and death caused by SARS-CoV-2, with an estimated 20 million lives saved during the first year of vaccine rollout (1). Despite the success of the COVID-19 vaccines, multiple waves of SARS-CoV-2 variants [e.g., B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 (Delta), BA.1, BA.5, XBB.1.5, and currently circulating KP.3 (Omicron)] continue to emerge, each with different constellations of amino acid substitutions, deletions, and insertions in the spike protein and elsewhere in the genome. As these successive variants often show increased transmissibility and greater antibody escape potential, they pose challenges to the efficacy of existing vaccines. Indeed, the antigen-shifted Omicron variants compromised the protective efficacy of immune responses elicited by vaccination or prior infection with non-Omicron strains (2–5).

Beyond changes in the spike protein of variants, waning immunity poses a separate challenge to vaccine efficacy. Although mRNA vaccines (Moderna mRNA-1273; Pfizer-BioNTech BNT162b2) induce durable germinal center B cells (GCB) responses for at least 6 months (6–9), the serum neutralizing antibody responses wane within 3–6 months and decline further by 8 months, with an estimated half-life of approximately 60 days (10–12). Waning humoral antibodies and the emergence of resistant, highly transmissible variants correlate with an increased frequency of breakthrough infections in vaccinated individuals (13, 14).

To address concerns arising from reduced efficacy of vaccines against current and future variants, third, fourth, fifth, and even sixth doses (herein, boosters) of monovalent (Wuhan-1 or more recently, XBB.1.5) or bivalent mRNA vaccines encoding the historical (Wuhan-1) and variant-matched (BA.1 or BA.5) spike protein have been implemented. Indeed, mRNA booster vaccines have been shown to increase the magnitude and breadth of neutralizing antibodies against SARS-CoV-2 (15–20). One frequent question regarding boosting is which arm should be used and whether the site injected affects immunogenicity and protection with mRNA or other COVID-19 vaccines. A study in mice with alum-adjuvanted influenza protein antigens showed that ipsilateral boosting with a homologous vaccine induced recall germinal center responses with B cells having greater levels of somatic hypermutation and antibodies with higher avidity and cross-reactivity than boosting at a remote site (21). In comparison, data from human studies with Pfizer-BioNTech BNT162b2 mRNA vaccine are less certain, as two studies reached different conclusions. Whereas Ziegler et al. reported that ipsilateral boosting with homologous BNT162b2 mRNA vaccine elicited higher serum neutralizing titers 2 weeks later compared to contralateral boosting (22), Fazli et al. showed that contralateral boosting with a homologous mRNA vaccine was associated with higher binding and neutralizing antibody titers at 8 and 14 months (23). Moreover, a study in mice with a two-dose regimen of BNT162b2 mRNA vaccine reported that ipsilateral compared to contralateral leg boosting produced higher numbers of GCBs that bound the receptor-binding domain (RBD) of an ancestral strain in the draining lymph node (DLN) and higher levels of antibody with enhanced affinity against RBD of ancestral and Omicron strains but only at the early (day 9) but not late (week 19) time points (24). However, the impact of ipsilateral vs contralateral boosting on T cell responses or protective immunity in the context of homologous or heterologous SARS-CoV-2 challenge has not been tested.

Here, we evaluated the impact of ipsilateral and contralateral leg boosting with preclinical versions of historical (mRNA-1273, Wuhan-1 spike) and bivalent (mRNA-1273.214, Wuhan-1 + BA.1 spike) mRNA vaccines on adaptive B and T cell responses and protection against challenge with a BA.1 variant in susceptible K18-hACE2 transgenic mice. We observed that ipsilateral and contralateral boosting elicited comparable levels of serum spike-specific antibody as well as antigen-specific B and T cell responses. The differential impact of boosting site on protective response in the context of virus challenge was limited, as ipsilateral and contralateral boosting with either mRNA-1273 or mRNA-1273.214 vaccines conferred similar levels of virological protection against BA.1 infection.

RESULTS

Antibody responses after boosting with mRNA vaccines

To compare serum antibody responses following vaccine boosting on the ipsilateral or contralateral side, cohorts of 7 to 9-week-old female K18-hACE2 transgenic mice were immunized intramuscularly in the left hind leg twice over a 3-week interval with a preclinical version of mRNA-1273 (Fig. 1A) that encodes for the prefusion-stabilized spike protein of SARS-CoV-2 Wuhan-1 strain (25). Animals were boosted 12 weeks later in the corresponding site in the left (ipsilateral) or right (contralateral) leg with 0.25 µg of mRNA-1273 or a preclinical bivalent mRNA-1273.214 vaccine composed of a 1:1 mixture of mRNA-1273 and the BA.1-matched mRNA-1273.529 vaccine (Fig. 1A). Boosting with these two different vaccines enabled analysis of the effects of immunization site on the breadth of the immune response. Serum was collected 1 day before and 4 weeks after boosting, and IgG responses against recombinant Wuhan-1 and BA.1 pre-fusion-stabilized spike (S-2P; Fig. 1B through E) or RBD (Fig. 1F through I) proteins were determined by enzyme-linked immunosorbent assay (ELISA). Prior to boosting, as expected, similar serum IgG titers were detected among all four groups against Wuhan-1 spike (Fig. 1B), and the levels against BA.1 spike were approximately seven- to eightfold lower (P < 0.01; Fig. 1C). Boosting with either mRNA-1273 or mRNA-1273.214 elicited enhanced IgG responses against the spike (Fig. 1B through E) and RBD (Fig. 1F through I) of Wuhan-1 and BA.1. For both mRNA-1273 and mRNA-1273.214 boosts, lower serum IgG titers were observed against the BA.1 spike (4–5-fold, P < 0.01) and RBD (17–20-fold, P < 0.0001) than the corresponding Wuhan-1 proteins. In pre- and post-boost comparisons, boosting with mRNA-1273 increased IgG binding titers against Wuhan-1 spike (8–10-fold, P < 0.01, Fig. 1D) or RBD (six- to eightfold, P < 0.0001, Fig. 1H) with smaller increases against BA.1 RBD. In comparison, mRNA-1273.214 induced larger increases against the BA.1 RBD (11–15-fold, P < 0.01, Fig. 1I). In the context of the booster location, ipsilateral and contralateral injections with mRNA-1273 or mRNA-1273.214 induced similar IgG binding titers against either Wuhan-1 or BA.1 protein.

Fig 1.

Serum IgG responses in K18-hACE2 mice following boosting. Seven- to 9-week-old female K18-hACE2 mice were immunized with a primary two-dose vaccination series spaced 3 weeks apart in the left hind leg with 0.1 μg of mRNA-1273. Animals were boosted 11–12 weeks later in the left (ipsilateral) or right (contralateral) leg with 0.25 μg of mRNA-1273 or bivalent mRNA-1273.214. One day before (pre-boost) or 4 weeks after boost (post-boost), serum was collected and IgG titers were determined by ELISA. (A) Scheme of immunizations and blood collection. (B and C) Serum IgG responses against the spike of Wuhan-1(B) or BA.1 (C). (D and E) Paired analysis of serum IgG titers of pre- and post-boost against WA1/2020 D614G (D) and BA.1 (E). (F and G) Serum IgG responses against RBD domain of Wuhan-1 (F) or BA.1 (G). (H and I) Paired analysis of serum IgG titers of pre- and post-boost against WA1/2020 D614G (H) and BA.1 (I) [n = 15–18, two experiments, column heights indicate geometric mean titers (GMT), and dotted lines show the limit of detection (LOD)]. GMTs or fold-changes are indicated above the corresponding graphs. Statistical analyses: B, C, F, and G. Mann-Whitney test: D, E, H, and I. Wilcoxon matched signed-rank test. *P < 0.05; **P < 0.01, P < 0.001, and P < 0.0001.

We next characterized the inhibitory effects of pre- and post-boost serum on SARS-CoV-2 infectivity using an established focus-reduction neutralization test (FRNT) (26) and authentic SARS-CoV-2 WA1/2020 D614G and BA.1 viruses (Fig. 2A through D; Fig. S1). Because of limited amounts of sera, we started dilutions at 1/60, which is just above the estimated threshold level of neutralization associated with protection in humans (27). As expected, 12 weeks after the primary immunization series with mRNA-1273 and before boosting, the serum neutralizing titers against WA1/2020 D614G were equivalent among all four groups (Fig. 2A). However, as observed in previous studies (16, 17, 28), serum from mice receiving two doses of mRNA-1273 showed poor neutralizing activity against BA.1, with most falling below the limit of detection of the assay (Fig. 2B). One month after boosting with either mRNA-1273 or mRNA-1273.214, serum neutralizing titers against WA1/2020 D614G rose five- to eightfold (P < 0.001; Fig. 2A and C) with minimal differences observed after ipsilateral or contralateral leg boosting. In comparison, ipsilateral or contralateral boosting with mRNA-1273 showed marginally increased neutralizing titers against BA.1, which did not achieve statistical significance (Fig. 2B and D). As expected for a variant-matched vaccine, boosting with mRNA-1273.214 induced three- to fivefold higher neutralizing responses against BA.1 (P < 0.001), with ipsilateral boosters showing slightly higher titers than contralateral boosters, although these differences did not attain statistical significance (Fig. 2B and D). Overall, only small differences in neutralizing activity were observed when comparing the site (ipsilateral vs contralateral) of mRNA vaccine boosting.

Fig 2.

Serum neutralizing antibody responses against WA1/2020 D614G and BA.1. One day before (pre-boost) or 4 weeks after (post-boost) ipsilateral or contralateral hind leg boosting with mRNA-1273 or mRNA-1273.214, serum neutralizing antibody was determined by FRNT against the indicated authentic SARS-CoV-2 strains. (A) WA1/2020 D614G. (B) BA.1 (n = 15–18, two experiments, boxes illustrate GMTs, and dotted lines show the LOD). (C and D) Paired analysis of serum neutralizing titers of pre- and post-boost against WA1/2020 D614G (C) and BA.1 (D) (n = 15–18, two experiments, column heights indicate GMTs, and dotted lines show the LOD). GMTs or fold-changes are indicated above the corresponding graphs. Statistical analyses: (A and B), Mann-Whitney test; (C and D), Wilcoxon matched-pairs signed-rank test. ns, not significant; *P < 0.05; **P < 0.01, P < 0.001, and P < 0.0001.

B cell responses after boosting with mRNA vaccines

To assess further the impact of the site of boosting on immune responses, we interrogated B cells in the DLN and spleen. In this set of experiments, we applied the same vaccination scheme (Fig. 1A) but boosted with a higher 1 µg dose of mRNA-1273 or bivalent mRNA-1273.214 either on ipsilateral (left) or contralateral (right) side; a higher booster dose was used to enhance our ability to detect spike-specific B cell responses by flow cytometry and to overcome the strong imprinting effects of mRNA vaccines (29–31), so we could assess effects on de novo responses. Seven days after boosting, we harvested the left and right inguinal DLNs as well as the spleen to characterize GCBs (CD19⁺IgD^lowGL7⁺Fas⁺) and plasmablast/plasma cell (PB/PC: CD19⁺IgD^lowTACI⁺CD138⁺) responses (Fig. 3 and 4; Fig. S2 to S7). Ipsilateral and contralateral leg boosting with mRNA-1273 or mRNA-1273.214 vaccines induced seven- to eightfold higher frequencies (P < 0.0001) and 100–200-fold higher numbers (P < 0.0001) of GCBs in their respective DLN (ipsilateral, left; contralateral, right) than in naïve mice (Fig. 3A; Fig. S3A). Boosting with either vaccine induced lower GCB responses in the non-draining, contralateral LN with only two- to fivefold higher total numbers of GCB compared to unvaccinated mice (Fig. 3A). These data suggest that mRNA vaccine boosting preferentially induces GC reactions in the DLN, which is consistent with data from human studies (7, 9). Nonetheless, mRNA-1273 and mRNA-1273.214 vaccines generally elicited similar overall GCB responses after ipsilateral and contralateral boosts.

Fig 3.

Germinal center B cell responses in the lymph nodes following boosting with mRNA-1273 or mRNA-1273.214. Seven- to 9-week-old female K18-hACE2 mice were immunized with a primary two-dose vaccination series spaced 3 weeks apart in the left hind leg with mRNA-1273. Animals then were boosted 11–12 weeks later in the left (ipsilateral) or right (contralateral) leg with 1 µg of mRNA-1273 or mRNA-1273.214. Seven days after boosting, inguinal LNs from the left and right sides were analyzed for GCB responses by flow cytometry. (A) Quantification of total number of CD19⁺IgD^lowGL7⁺Fas⁺ GCBs in respective LNs. Note that the number of GCBs in the LNs on the respective side (L-LN or R-LN) from unvaccinated mice are shown in each graph with the different vaccines (mRNA-1273 or mRNA1273.214) for comparison purposes. (B) Representative flow cytometry scatter plots of Wuhan-1 spike-reactive GCBs. (C) Total numbers of Wuhan-1 spike-reactive GCBs in the respective LNs. (D) Comparison of numbers of Wuhan-1 spike-reactive GCBs in the DLNs. (E) Representative flow cytometry scatter plots of BA.1 spike-reactive GCBs. (F) Total number of BA.1 spike-reactive GCBs in respective LNs. (G) Comparison of numbers of BA.1 spike-reactive GCBs in the DLNs. Data are from two experiments (n = 7–8, each data point represents an individual mouse, column heights indicate geometric mean values, and dotted lines show the LOD). Data in D and G correspond to data in C and F, respectively, and are replotted for direct statistical comparison. Statistical analyses: A; one-way analysis of variance (ANOVA) with Tukey’s post-test: C, D, F, and G; unpaired two-tailed Mann-Whitney test: ns, not significant; *P < 0.05, P < 0.01, P < 0.001, and P < 0.0001.

Fig 4.

Plasmablast and plasma cell responses in the lymph nodes following boosting with mRNA-1273 or mRNA-1273.214. K18-hACE2 mice were immunized and boosted as described in Fig. 3. Seven days after boosting, inguinal LNs from the left and right sides were analyzed for PB/PC responses by flow cytometry. (A) Total number CD19⁺IgD^lowCD138⁺TACI⁺ PBs/PCs. Note that the number of PB/PCs in the LNs on the respective side (L-LN or R-LN) from unvaccinated mice are shown in each graph with the different vaccines (mRNA-1273 or mRNA-1273.214) for comparison purposes. (B) Representative flow cytometry scatter plots of Wuhan-1 spike-reactive PBs/PCs. (C) Total number of Wuhan-1 spike-reactive PBs/PCs in respective LNs. (D) Comparison of numbers of Wuhan-1 spike-reactive PBs/PCs in the DLNs. (E) Representative flow cytometry scatter plots of BA.1 spike reactive PBs/PCs. (F) Total number of BA.1 spike reactive PB/PC cells in respective LNs. (G) Comparison of numbers of BA.1 spike reactive PBs/PCs in the DLNs. Data are from two independent experiments (n = 7–8, each data point represents an individual mouse, column heights indicate geometric mean values, and dotted lines show the LOD). Data in D and G correspond to data in C and F, respectively, and are replotted for direct statistical comparison. Statistical analyses: A; one-way ANOVA with Tukey’s post-test: C, D, F, and G; unpaired two-tailed Mann-Whitney test: ns, not significant; *P < 0.05, P < 0.01, P < 0.001, and P < 0.0001.

We next profiled antigen-specific GCB responses using fluorescently labeled Wuhan-1 and BA.1 spike protein and flow cytometry (Fig. 3B through G; Fig. S3B and C). Ipsilateral and contralateral boosting with mRNA-1273 or mRNA-214 vaccines both elicited robust GCB responses in DLNs against Wuhan-1 (Fig. 3B through D) and BA.1 (Fig. 3E through G) spike proteins. Regardless of the boost location or mRNA vaccine used, higher numbers and frequencies of spike-specific GCBs were detected in the DLN on the side of the boost. When we compared the mRNA-1273 and mRNA-1273.214 response in the DLN where the dominant GC reaction occurred, mRNA-1273 elicited approximately twofold higher numbers (Fig. 3C) and frequency (Fig. S3B) of GCBs that bound Wuhan-1 spike than mRNA-1273.214. In comparison, mRNA-1273.214 elicited approximately two- to threefold higher numbers (Fig. 3F) and frequency (Fig. S3C) of GCBs that bound BA.1 spike than mRNA-1273. Ipsilateral and contralateral leg boosting elicited relatively equivalent overall numbers of Wuhan-1 or BA.1 spike reactive GCBs in the DLNs regardless of vaccines being used (Fig. 3D and G), with substantially reduced responses observed in LNs on the side opposite of boosting (Fig. 3C and F).

We further gated the spike-binding GCB populations (Fig. S4A and B) to determine whether GCBs responses were Wuhan-1 spike-specific (Fig. S4C and D), cross-reactive against Wuhan-1 spike and BA.1 spike (Fig. S4E and F), or BA.1 spike-specific (Fig. S4G and H). We observed two- to threefold higher numbers of Wuhan-1 spike-specific GCBs after mRNA-1273 boosting than with mRNA-1273.214 boosting in the corresponding draining LN (Fig. S4C and D). Whereas the BA.1 spike-binding GCBs in the draining LN of mice boosted with mRNA-1273 were overwhelmingly cross-reactive against Wuhan-1 spike (Fig. S4F through H, left graph), mice boosted with mRNA 1273.214 elicited both cross-reactive and BA.1-specific GCBs (Fig. S4F and H, right graph). Regardless of the vaccines used, ipsilateral and contralateral leg boosting elicited relatively equivalent overall numbers of type-specific and cross-reactive GCBs in the DLNs (Fig. S4D, F and H).

We also assessed total and antigen-specific PB/PC responses in the DLNs (Fig. 4; Fig. S3D through F) and observed a pattern like the GCB response. Ipsilateral and contralateral leg boosting with mRNA-1273 or mRNA-1273.214 vaccines elicited greater total (Fig. 4A; Fig. S3D) and spike-specific (Fig. 4B through G; Fig. S3E and F) PB/PC responses in the DLN on the same side of the boost. mRNA-1273 elicited a higher frequency (Fig. S3E) and number of PB/PCs that bound to Wuhan-1 spike than mRNA-1273.214 (Fig. 4C), and reciprocally, mRNA-1273.214 elicited a greater frequency (Fig. S3F) and number of PB/PCs that bound to BA.1 spike (Fig. 4F). In terms of the effect of the boosting site, ipsilateral and contralateral leg boosting elicited relatively similar numbers of Wuhan-1- or BA.1-spike reactive PBs/PCs in the respective DLNs regardless of the vaccine used (Fig. 4D and G), with lower responses in LNs achieved at the site opposite of boosting (Fig. 4C and F). We performed additional gating (Fig. S5A and B) to determine whether PBs/PCs were Wuhan-1 spike-specific (Fig. S5C and D), Wuhan-1 and BA.1 spike cross-reactive (Fig. S5E and F), or BA.1 spike-specific (Fig. S5G and H). Boosting with mRNA-1273 or mRNA-1273.214 elicited comparable numbers of Wuhan-1 spike-specific (Fig. S5C and D) or cross-reactive PBs/PCs in the corresponding draining LN (Fig. S5E and F). Whereas the BA.1 spike-binding PBs/PCs of mice that received mRNA-1273 boosting were cross-reactive against Wuhan-1 spike (Fig. S5F through H, left graph), mice receiving the mRNA-1273.214 booster had both cross-reactive and BA.1 spike-specific PBs/PCs (Fig. S5F through H, right graph).

We also profiled the total antigen-specific B cell response in the LN by gating on activated B cells (CD19⁺IgD^low; Fig. S6A and D). Ipsilateral or contralateral leg boosting elicited relatively equivalent numbers of Wuhan-1 or BA.1 spike-reactive B cells in the DLNs (Fig. S6C and F), with reduced responses observed in LNs on the side opposite of boosting (Fig. S6B and E). Overall, total antigen-specific B cell responses against homologous or heterologous spike antigens in the DLN were comparable regardless of whether the mRNA vaccine booster occurred ipsilateral or contralateral to the site of the primary vaccination series.

We also measured the total and spike-specific PB/PC response in the spleen 7 days after boosting (Fig. S7). Ipsilateral or contralateral boosting with mRNA-1273 or mRNA-1273.214 vaccines induced 5–10-fold higher numbers (P < 0.0001) of total PB/PC in the spleen than in unvaccinated mice (Fig. S7A). Mice boosted with either mRNA-1273 or mRNA-1273.214 vaccine had six- to ninefold higher numbers (P < 0.05) of antigen-specific PB/PCs that bound spike of Wuhan-1 compared to BA.1 (Fig. S7B and C). However, as we observed in the DLN, ipsilateral or contralateral boosting with mRNA-1273 or mRNA-1273.214 induced equivalent levels of PB/PCs in the spleen that bound Wuhan-1 (Fig. S7B) and BA.1 (Fig. S7C) spike proteins.

T cell responses after boosting with mRNA vaccines

Since T cell immunity also contributes to protection against SARS-CoV-2 infection and disease (8, 32–35), we interrogated their responses after mRNA vaccine boosting (Fig. 5; Fig. S8 and S9). At 7 days after ipsilateral or contralateral leg boosting with mRNA-1273 or mRNA-1273.214 vaccines, similar or slightly higher numbers of total CD3⁺ T cells were present in the respective DLN (ipsilateral, left; contralateral, right) compared to non-boosted mice (Fig. 5A). We next evaluated spike-specific CD8⁺ T responses using class I MHC tetramers displaying a conserved immunodominant peptide (S_539-546; VNFNFNGL). Ipsilateral and contralateral boosting with mRNA-1273 or mRNA-1273.214 vaccines elicited higher frequencies and numbers of tetramer⁺ CD8⁺ T cells in the DLN, with reduced responses observed on the side opposite of boosting (Fig. 5B through D). Ipsilateral and contralateral mRNA vaccine boosting elicited comparable numbers of tetramer⁺ CD8⁺ T cells in the DLNs regardless of the vaccine used (Fig. 5E). Ipsilateral or contralateral boosting with mRNA-1273 or mRNA-1273.214 mRNA also elicited comparable frequencies and numbers of tetramer⁺ CD8⁺ T cells in the spleen (Fig. 5F).

Fig 5.

CD8⁺ T cell responses in the lymph nodes and spleen following boosting with mRNA-1273 or mRNA-1273.214. Seven- to 9-week-old female K18-hACE2 mice were immunized with a primary two-dose vaccination series spaced 3 weeks apart in the left hind leg with mRNA-1273. Animals then were boosted 11–12 weeks later in the left (ipsilateral) or right (contralateral) leg with 1 µg of mRNA-1273 or mRNA-1273.214. Seven days after boosting, inguinal LNs from the left and right side as well as spleen were analyzed for CD8⁺ T cell responses by flow cytometry. (A) Quantification of total number of CD3⁺ T cells in respective LNs. Note that the number of CD3⁺ T cells in the LNs on the respective side (L-LN or R-LN) from unvaccinated mice are shown in each graph with the different vaccines (mRNA-1273 or mRNA-1273.214) for comparison purposes. (B) Representative flow cytometry scatter plots of spike-specific tetramer⁺CD44⁺CD8⁺ T cells in respective LNs. (C and D) Spike-specific tetramer⁺CD44⁺CD8⁺ T cells in respective LNs: C, frequency, D, total cell number. (E) Comparison of numbers of spike-specific tetramer⁺CD44⁺CD8⁺ T cells in the DLNs. (F) Frequency and total cell number of spike-specific tetramer⁺CD44⁺CD8⁺ T cells in the spleen. Data are from two experiments (n = 7–8, each data point represents an individual mouse, and column heights indicate geometric mean values). Data in E correspond to data in D and is replotted for direct statistical comparison. Statistical analyses: A; one-way ANOVA with Tukey’s post-test: C, D, E, and F; unpaired two-tailed Mann-Whitney test: ns, not significant; *P < 0.05, P < 0.01, and P < 0.001.

Follicular T helper cells (T_FH: CD4⁺CD45⁺CXCR5⁺PD-1⁺) shape B cell responses by instructing GC reactions including the process of somatic hypermutation and affinity selection (36). We assessed the impact of ipsilateral and contralateral boosting on T_FH cell responses in the LNs at 7 days post boosting (Fig. S9A through D). Like that observed with CD8⁺ T cell responses, ipsilateral and contralateral leg boosting with mRNA-1273 or mRNA-1273.214 vaccines elicited higher frequencies (Fig. S9A and B) and numbers (Fig. S9C, 3–10-fold, P < 0.05) of T_FH cells in the respective DLN, with reduced responses observed in the LN on the side opposite of boosting (Fig. S9B and C). Ipsilateral and contralateral boosting elicited equivalent numbers of T_FH cells in the DLNs regardless of the vaccine used (Fig. S9D). Likewise, ipsilateral and contralateral boosting with mRNA-1273 or mRNA-1273.214 mRNA elicited comparable frequencies and numbers of T_FH cells in the spleen (Fig. S9E).

Effect of ipsilateral or contralateral boosting on protection against BA.1 challenge

To evaluate the protective activity of the mRNA vaccines following ipsilateral or contralateral boosting, the cohorts of K18-hACE2 mice were challenged 9 weeks later via an intranasal route with 10⁴ focus-forming units (FFUs) of BA.1 (Fig. 6A). One limitation of these studies is that BA.1 is inherently less pathogenic in rodents (37–40) and replicates to lower levels in both the upper and lower respiratory tracts of K18-hACE2 mice without the development of clinical signs of disease compared to earlier variants (16, 37); as such, we focused our analysis on virological endpoints. Nasal turbinates and lungs were harvested on day 4 after BA.1 infection and assayed for viral RNA levels by quantitative reverse transcription-PCR (qRT-PCR) (Fig. 6B and C). In the nasal turbinates of control mRNA vaccine immunized mice, although some variability was observed, moderate amounts of BA.1 viral RNA were detected (approximately 5 × 10⁴ copies of N transcript per milligram; Fig. 6B). Ipsilateral and contralateral boosting with mRNA-1273 or mRNA-1273.214 vaccines conferred protection in the nasal turbinates, with 100–1,000-fold reductions (P < 0.05) in viral RNA compared to the control vaccinated mice. In the lungs of control vaccinated K18-hACE2 mice, 10⁵–10⁷ copies of viral RNA per milligram of tissue were detected (Fig. 6C). Ipsilateral and contralateral boosting with either mRNA-1273 or mRNA-1273.214 substantially reduced (mRNA-1273, ipsilateral or contralateral, 90–190-fold; mRNA-1273.214, ipsilateral or contralateral 370–1,000-fold, P < 0.0001) levels of viral RNA in the lung compared to the control vaccinated mice with no statistically significant differences conferred by the site of boosting (P > 0.05). Consistent with these data, no differences in infectious BA.1 were detected by plaque assay after ipsilateral or contralateral boosting with mRNA-1273 or mRNA-1273.214, with each vaccine and immunization site resulting in significant reductions (14–26-fold, P < 0.0001) compared to control mRNA vaccinated and challenged controls (Fig. 6D).

Fig 6.

Protection against BA.1 infection in K18-hACE2 mice following boosting with mRNA-1273 or mRNA-1273.214. (A) Scheme of immunizations and virus challenge. Seven- to 9-week-old female K18-hACE2 mice were immunized with a primary two-dose vaccination series spaced 3 weeks apart in the left hind leg with 0.1 μg of mRNA-1273. Animals were boosted 11–12 weeks later in the left (ipsilateral) or right (contralateral) leg with 0.25 μg of mRNA-1273 or bivalent mRNA-1273.214. Mice immunized with mRNA-control (left hind leg) were used as a negative control group. Nine weeks after boosting, mice were challenged via an intranasal route with 10⁴ FFUs of SARS-CoV-2 BA.1. Viral RNA levels were determined at 4 dpi in the nasal turbinates (B) and lungs (C). (D) Infectious virus in the lungs. Data are from two independent experiments (n = 15–18, each data point represents an individual mouse, and column heights indicate median values). Note that the viral yield in the nasal turbinates or lungs (B–D) after immunization with mRNA-control is shown in each graph with the different vaccines (mRNA-1273 or mRNA1273.214) for comparison purposes. Statistical analyses: one-way ANOVA with Tukey’s post-test: ns, not significant; *P < 0.05, P < 0.01, P < 0.001, and P < 0.0001.

DISCUSSION

One of the key immunization strategies to mitigate waning immunity and reduced efficacy against SARS-CoV-2 variants that escape neutralization is boosting with vaccines that encode variant spike proteins. A frequent question regarding the clinical practice of boosting is which arm should be used, and whether boosting in the alternating arm impacts immunogenicity and protection against SARS-CoV-2. In this study, we evaluated in mice the impact of ipsilateral or contralateral site boosting of mRNA vaccines on the adaptive immune responses and protection against SARS-CoV-2 challenge. We observed that ipsilateral and contralateral boosting elicited comparable overall levels of serum spike-specific antibody as well as antigen-specific B and T cell responses. The differential impact of boosting site on protective response in the context of virus challenge was limited, as ipsilateral and contralateral boosting with mRNA-1273 or mRNA-1273.214 vaccines both conferred similar levels of virological protection against BA.1 infection in the respiratory tract.

Our data showed that ipsilateral and contralateral boosting elicited comparable levels of anti-spike IgG in serum at 4 weeks after immunization. Functional analysis of antibody responses revealed similar neutralizing activities against SARS-CoV-2 WA1/2020 D614G and BA.1 viruses with small differences that did not achieve statistical significance between booster site regimens. Our results in K18-hACE2 mice with COVID-19 mRNA vaccines generally are consistent with a two-dose vaccination study in conventional C57BL/6 mice using homologous influenza hemagglutinin (H1-HA) antigen, which showed comparable levels of serum anti-HA IgG at 8 days post boost, although that study did not examine neutralizing antibody activities (21). Our results contrast with this study when analyzing the effects of a heterologous antigen boost. In the influenza vaccine study, ipsilateral, but not contralateral, boosting with heterologous H3-HA enhanced H1-binding serum IgG levels, whereas contralateral boosting with H3-HA did not. While we did not observe such effects when comparing ipsilateral and contralateral administration of monovalent (mRNA-1273) and bivalent (mRNA-1273.214) mRNA vaccine boosters, the bivalent vaccine contains the original mRNA-1273, which likely affects recall responses against the original Wuhan-1 spike antigen. Future studies with a fully heterologous monovalent boost [e.g., mRNA-1273.529 (BA.1) or mRNA-1273.815 (XBB.1.5)] may allow for more direct comparisons to the influenza immunization experiments.

Our serum antibody data showed some similarities and differences with a recent study in C57BL/6 mice that used a two-dose homologous BNT162b2 (Pfizer-BioNTech) mRNA vaccination regimen (24). That study showed that ipsilateral boosting with BNT162b2 mRNA vaccine produced higher titers of serum antibodies with enhanced affinity against RBD of Wuhan-1 and Omicron strains at early (day 9) but not the late (week 19) timepoints post boost. However, like our results, no difference was observed in overall serum IgG levels after ipsilateral or contralateral boosting when the analysis was performed with the spike proteins at either the early or later timepoints. Beyond pre-clinical studies in small animals, the data on the impact of ipsilateral vs contralateral boosting on antibody responses in humans remain inconclusive. Two observational studies conducted among SARS-CoV-2-uninfected individuals who received the BNT162b2 mRNA vaccine reached different conclusions. Ziegler et al. reported that spike-specific IgG levels did not differ between two groups that received a second dose of BNT162b2 on the ipsilateral (n = 147) or contralateral side (n = 156), although inhibitory activity was higher in the group boosted on the ipsilateral side at 2 weeks after the second dose based on an assay that measured inhibition of RBD binding to hACE2 (22). In contrast, Fazli et al. reported that contralateral boosting (n = 440) with BNT162b2 mRNA vaccine was associated with slightly higher spike- and RBD-specific serum IgG titers compared to ipsilateral boosting (n = 507), and this effect increased over time from a 1.1-fold to a 1.4-fold effect by 14 months. Contralateral boosting also resulted in increases (1.3-fold to 4.0-fold) in neutralizing antibody titers against B.1.1.529 at 8 and 14 months, with lesser effects against the WA1/2020 D614G strain (no difference at 8 months and twofold difference at 14 months) (23).

To investigate the mechanisms by which boosting site selection might influence humoral immunity, we interrogated B cells in the LNs and spleen. Ipsilateral and contralateral leg boosting with mRNA-1273 or mRNA-1273.214 vaccines elicited robust GCB responses in the DLNs against Wuhan-1 and BA.1 spike proteins at 7 days post boost, with reduced responses observed in the LNs on the site opposite of boosting. As expected, bivalent mRNA-1273.214 elicited higher numbers of GCBs that specifically bound BA.1 spike than mRNA-1273. Whereas contralateral boosting extended the GCB response to the contralateral DLN, ipsilateral or contralateral boosting with mRNA-1273 or mRNA-1273.214 vaccines overall induced similar levels of GCBs that bound Wuhan-1 or BA.1 spike in the DLNs. Our results are consistent with fate-mapping studies in mice, which showed that recruitment of antigen-experienced memory B cells to secondary GCs is inefficient (41, 42), as the progeny of primed GC B cells account for only a subset of total GCBs. Our analysis of PBs/PCs also revealed that boosting with the bivalent mRNA-1273.214 mRNA vaccine elicited higher numbers of PBs/PCs that bound specifically to BA.1 spike than the mRNA-1273 vaccine. Ipsilateral and contralateral boosts elicited equivalent PBs/PCs responses in the respective DLNs and spleen, which is consistent with the similar spike- and RBD-specific IgG responses in serum by the two booster regimens. However, our results both agree and contrast with the two-dose influenza immunization study in mice (21), which showed that ipsilateral and contralateral boosting with homologous influenza hemagglutinin H1 antigen elicited comparable total GCB but better quality of H1-reactive GCBs in the DLN; in that study, ipsilateral boosting with the heterologous H3-HA antigen increased the breadth of GCBs that bound to H1-HA. Our GCB results also differ from the study with two homologous doses of BNT162b2 mRNA in mice (24), which showed that ipsilateral boosting induced a higher number of GCBs that bound RBD of the Wuhan-1 strain in the ipsilateral DLN than that contralateral boosting elicited in the contralateral DLN.

Despite increasing data on antibody and B cell responses induced after immunization at different boosting sites in mice and humans, the results remain inconsistent and challenging to develop paradigms. Multiple factors in study design likely contribute to discrepant results among studies. (i) Vaccine platforms could impact responses. Whereas the influenza study used HA protein-based vaccines to interrogate the impact of ipsilateral or contralateral boosting, we used monovalent and bivalent mRNA vaccines. Vaccine platforms can affect the extent of GC responses in the DLN, as mRNA vaccines can induce persistent GC responses that last months compared to traditional protein-based platforms (9, 43–46). (ii) The frequency of boosters and interval between primary immunization could change the dynamics of B cell and antibody responses generated from ipsilateral and contralateral boosting. Whereas the previous studies in mice boosted after one-dose prime immunization, our study evaluated the impact of boost location following a two-dose primary vaccination series. Indeed, a third COVID-19 mRNA vaccine dose in humans has been associated with an increase in RBD-specific memory B cells both from expanded clones present after the second dose and the emergence of new clones (47, 48). The boosting interval also differs between our study (12 weeks) and the prior BNT162b2 mRNA vaccine study (3 weeks) in mice (24), which has been shown to independently impact antibody responses (49, 50). (iii) The relative dosing of the booster could impact responses. Higher doses could result in prolonged antigen retention in the follicular dendritic cells in the DLN that differentially shape B cell and antibody responses (51, 52). (iv) The timing of assessment. The two human studies on ipsilateral and contralateral boosting may have reached different conclusions because they evaluated antibody responses at different time points, ranging from 2 weeks to 14 months (22, 23). Analogously, the study in C57BL/6 mice with two homologous doses of BNT162b2 mRNA vaccine showed different affinity measurements at early and late time points after ipsilateral and contralateral boosting (24). Together, these data suggest that at the early phase after boosting, memory B cells in the DLN on the ipsilateral side with respect to priming likely differentiate into PB/PC rapidly after ipsilateral boosting, resulting in earlier serum antibody production than contralateral boosting. In comparison, after contralateral boosting, in the contralateral DLN, newly recruited antigen-specific B cells from the naive pool expand, affinity mature, and differentiate into plasmablasts to produce antibodies. (v) Antigenic distance between primary and boost vaccines could impact the responses. As shown in the influenza study (21), ipsilateral boosting with a heterologous antigen increased cross-reactive antibody responses more than contralateral boosting, which might be better at overcoming immune imprinting. The bivalent vaccine in our study contains the original mRNA-1273, which likely affects recall responses against the original Wuhan-1 spike antigen. For mRNA vaccines, more study of the effects of ipsilateral and contralateral boosting is likely needed for evaluating fully heterologous monovalent vaccines.

T cell immune responses contribute to protection against SARS-CoV-2 infection and disease, especially in the setting of poor neutralizing antibody responses against variants (8, 32–35). We interrogated spike-specific CD8 and total T_FH cell responses locally and systemically. Ipsilateral and contralateral boosting with mRNA-1273 or mRNA-1273.214 vaccines both elicited enhanced spike-specific CD8⁺ T cell and overall T_FH responses in the spleen and DLN at 7 days post boosting, with reduced responses observed in LN at the site opposite of boosting. Notwithstanding these data, ipsilateral and contralateral boosting with mRNA-1273 or mRNA-1273.214 vaccines elicited comparable spike-specific CD8⁺ T cells and overall T_FH responses in the spleen and respective DLN. These results are consistent with a study in C57BL/6 mice that used self-amplifying mRNA vaccine encoding the influenza A virus nucleoprotein (53) and showed comparable antigen-specific CD4⁺ and CD8⁺ T cells in the spleen 28 days after ipsilateral and contralateral boosting. Our results with T_FH cells differ from this study, as we characterized these cells in the spleen and respective DLNs at 7 days after ipsilateral and contralateral boosting. Our CD8⁺ T cell results also differ from a study in humans, which showed that frequencies of spike-specific CD8⁺ T cell in the blood were higher after ipsilateral than contralateral boosting (22). The reasons for the differences are not clear beyond the different anatomic compartments (spleen vs blood) sampled.

We uniquely assessed the impact of ipsilateral and contralateral boosting on vaccine-mediated protection against virus challenge. Boosting with either mRNA-1273 or mRNA-1273.214 efficiently reduced BA.1 viral RNA levels in the upper and lower respiratory tracts with a trend toward a higher level of protection in animals boosted with mRNA-1273.214, as observed previously (16). Notably, our data showed that the site of boosting with mRNA-1273 or mRNA-1273.214 vaccines did not affect protection against BA.1 as judged by measurement of viral RNA and infectious virus in the nasal turbinates and lungs. Boosting at either site with mRNA-1273 or mRNA-1273.214 reduced the levels of viral infection, with each vaccine and immunization site resulting in significant reductions compared to unvaccinated, challenged controls. Overall, these data in mice show that the limited differences in overall immune response induced by ipsilateral or contralateral boosting with historical (mRNA-1273) or variant-matched bivalent (mRNA-1273.214) vaccines do not substantively impact virological protection against challenge by a SARS-CoV-2 Omicron strain.

We acknowledge several limitations in our study. (i) We evaluated humoral or cellular immune responses at only one time point following boosting. Longitudinal studies after ipsilateral and contralateral boosting are needed to assess whether the dynamics of immune responses change over time. (ii) A higher 1 mg booster dose was used in our analysis of B and T cell responses. In pilot experiments, the response after a lower 0.25 mg dose was detectable in most but not all mice. We selected the higher booster dose to discern statistical differences in immune cell populations and also because it enables mice to overcome the strong imprinting effects of mRNA vaccines (29–31), so de novo BA.1 spike-specific responses could be measured. (iii) Our study evaluated immune responses after one ipsilateral or contralateral boost after a primary vaccination series. How additional boosting on the contralateral side shifts the recall responses remains to be determined. (iv) We used monovalent and bivalent mRNA vaccine formulations in our boosting schemes in part because of when the studies were initiated. Experiments that test monovalent heterologous boosting with more contemporary vaccines (encoding spikes from BA.1, BA.5, XBB.1.5, or emerging KP.2/KP.3 strains) are needed to determine how the boosting site affects or potentially overcomes immune imprinting. (v) We performed studies only in uninfected mice receiving vaccines. Future studies on boost location are needed in the setting of antecedent SARS-CoV-2 infection to extrapolate our findings to conditions of hybrid immunity. (vi) We did not analyze the impact of boost location on bone marrow-derived long-lived plasma cells (LLPCs). LLPCs constitutively secrete high levels of antibodies for extended periods of time and dominantly contribute to serum antibody levels at times remote from vaccination or infection. (vii) Finally, studies with different vaccine platforms, as well as other animal models and ultimately humans, are required for corroboration.

Our studies in K18-hACE2 mice provide evidence that ipsilateral and contralateral boosting with mRNA vaccines elicit comparable immune responses, and this was associated with equivalent control of infection by a SARS-CoV-2 Omicron challenge strain. Thus, at least in mice, the boost site location for mRNA vaccines appears to have limited impacts on vaccine immunogenicity and protection against SARS-CoV-2.

MATERIALS AND METHODS

Cells

African green monkey Vero-TMPRSS2 (54) and Vero-hACE2-TMPRRS2 (55) cells were cultured at 37°C in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES pH 7.3, 1 mM sodium pyruvate, 1× non-essential amino acids, and 100 U/mL of penicillin-streptomycin. Vero-TMPRSS2 cells were cultured in a medium supplemented with 5 µg/mL of blasticidin. Vero-hACE2-TMPRSS2 cells were propagated in a medium supplemented with 5 µg/mL of blasticidin and 10 µg/mL of puromycin. All cells were routinely tested negative for mycoplasma using a PCR-based assay.

Viruses

The WA1/2020 strain with a D614G substitution was described previously (56). The BA.1 (B.1.1.529) isolate (hCoV-19/USA/WI-WSLH-221686/2021) was passaged once on Vero-TMPRSS2 cells and described previously (57). All viruses were subjected to next-generation sequencing to confirm substitutions.

Mice

Heterozygous K18-hACE2 C57BL/6J mice (strain: 2B6.Cg-Tg(K18-ACE2)2Prlmn/J, Cat # 034860) were obtained from The Jackson Laboratory. Virus inoculations were performed under anesthesia that was induced and maintained with ketamine hydrochloride and xylazine, and all efforts were made to minimize animal suffering. Experiments were neither randomized nor blinded.

mRNA vaccine and lipid nanoparticle production process

Sequence-optimized mRNA encoding prefusion-stabilized Wuhan-1 (mRNA-1273) and BA.1 (mRNA-1273.529, the Omicron gene component in mRNA-1273.214) SARS-CoV-2 S-2P proteins were synthesized in vitro using an optimized T7 RNA polymerase-mediated transcription reaction with complete replacement of uridine by N1m-pseudouridine (58). Bivalent mRNA-1273.214 vaccine is a 1:1 bench side mix of separately formulated mRNA-1273 and mRNA-1273.529 vaccines. A non-translating control mRNA was synthesized and formulated into lipid nanoparticles as previously described (59). The transcription reaction included a DNA template containing the immunogen open-reading frame flanked by 5' untranslated region (UTR) and 3' UTR sequences and was terminated by an encoded poly A tail. After RNA transcription, the cap-1 structure was added using the vaccinia virus capping enzyme and 2ʹ-O-methyltransferase (New England Biolabs). The mRNA was purified, sterile filtered, and kept frozen at –20°C until further use.

The mRNA was encapsulated in a lipid nanoparticle as described previously (60). Vials were filled with formulated lipid nanoparticle and stored frozen at –20°C until further use. The vaccine product underwent analytical characterization, which included the determination of particle size and polydispersity, encapsulation, mRNA purity, double-stranded RNA content, osmolality, pH, endotoxin, and bioburden, and the material was deemed acceptable for in vivo study.

Viral antigens

Recombinant soluble spike and RBD proteins from Wuhan-1 and BA.1 SARS-CoV-2 strains were expressed as described (61, 62). Recombinant proteins were produced in Expi293F cells (Thermo Fisher) by transfection of DNA using the ExpiFectamine 293 Transfection Kit (ThermoFisher). Supernatants were harvested 3 days post-transfection, and recombinant proteins were purified using Ni-NTA agarose (Thermo Fisher). The proteins were then buffer exchanged into phosphate-buffered saline (PBS) and concentrated using Amicon Ultracel centrifugal filters (EMD Millipore).

ELISA

Purified recombinant Wuhan-1 or BA.1 spike or RBD proteins were coated onto 96-well Maxisorp clear plates at 2 mg/mL (spike) or 4 µg/mL (RBD) in 50 mM Na₂CO₃ pH 9.6 (50 μL) overnight at 4°C. Coating buffers were aspirated, and wells were blocked with 200 µL of 1× PBS + 0.05% Tween-20 (PBST) +2% bovine serum albumin (BSA) +0.02% NaN₃ (Blocking buffer, PBSTBA) overnight at 4°C. Sera were serially diluted in blocking buffer and added to the plates. Plates were incubated for 1 h at room temperature and then washed three times with PBST, followed by the addition of 50 µL of 1:2,000 dilution of horeseradish peroxidase (HRP)-conjugated anti-mouse IgG (Southern Biotech Cat. #1030–05) in PBST. Following a 1 h incubation at room temperature, plates were washed three times with PBST, and 50 µL of 1-Step Ultra TMB-ELISA was added (Thermo Fisher Cat. #34028). Following a 2–5 min incubation, reactions were stopped with 50 µL of 2 M H₂SO₄. The absorbance of each well at 450 nm was determined using a microplate reader (BioTek) within 5 min of the addition of sulfuric acid. The endpoint serum dilution was calculated with curve fit analysis of optical density values for serially diluted sera with a cut-off value set to six times the mean of the background signal.

Focus reduction neutralization test

Serial dilutions of sera were incubated with 10² FFUs of WA1/2020 D614G or BA.1 for 1 h at 37°C. Antibody-virus complexes were added to Vero-TMPRSS2 cell monolayers in 96-well plates and incubated at 37°C for 1 h. Subsequently, cells were overlaid with 1% (wt/vol) methylcellulose in modified Eagle medium (MEM). Plates were harvested 30 h (WA1/2020 D614G) or 70 h (BA.1) later by removing overlays and fixed with 4% paraformaldehyde (PFA) in PBS for 20 min at room temperature. Plates were washed and sequentially incubated with an oligoclonal pool (SARS2-02, -08, -09, -10, -11, -13, -14, -17, -20, -26, -27, -28, -31, -38, -41, -42, -44, -49, -57, -62, -64, -65, -67, and -71) (63) of anti-spike murine antibodies (including cross-reactive mAbs to SARS-CoV) and HRP-conjugated goat anti-mouse IgG (Sigma Cat # A8924, RRID: AB_258426) in PBS supplemented with 0.1% saponin and 0.1% bovine serum albumin. SARS-CoV-2-infected cell foci were visualized using KPL TrueBlue peroxidase substrate (SeraCare) and quantitated on an ImmunoSpot microanalyzer (Cellular Technologies).

Mouse experiments

Seven- to 9-week-old female K18-hACE2 C57BL/6 mice were immunized 3 weeks apart with 0.1 μg of mRNA-1273 vaccine in 50 µL of PBS via intramuscular injection in the left hind leg. Animals were boosted 12 weeks later in the left (ipsilateral) or right (contralateral) leg with 0.25 µg of mRNA-1273 or bivalent mRNA-1273.214 vaccine (1:1 mixture of mRNA-1273 and the BA.1-matched mRNA-1273.529). Animals were bled 1 day before and 4 weeks after boosting for antibody analysis.

To evaluate the protective activity of the mRNA vaccines 9 weeks after boosting, the cohorts of K18-hACE2 mice were challenged via an intranasal route with 10⁴ FFUs of BA.1. Animals were euthanized at 4 dpi. Nasal turbinates and lungs were harvested for virological and immunological analyses. For B and T cells analysis, the same vaccination scheme was applied, but animals were boosted with 1 µg of mRNA-1273 or bivalent mRNA-1273.214 in the ipsilateral (left) or contralateral (right) leg. Seven days after boosting, the left and right inguinal LN and spleen were harvested for immune cells analysis.

SARS-CoV-2 spike probe generation

Recombinant spike proteins were biotinylated with EZ-Link NHS-PEG4-Biotin (Thermo Fisher, Cat. # A39259) for 2 h at 4°C and processed through Zeba spin desalting columns (Thermo Fisher) to remove excess unbound biotin. Purified biotinylated full-length Wuhan-1 and BA.1 spike proteins were subsequently fluorescently conjugated by mixing with fluorophore-labeled streptavidin at 1:1.5 molar ratio for 1 h at 4°C (Wuhan-1 spike: strepavidin-BV421 or -BV786; BA.1-spike: streptavidin-APC or -AF594) prior to antigen-specific B cell staining.

B cell phenotyping

Seven days after boosting, ipsilateral and contralateral inguinal LNs and spleens were collected, and single-cell suspensions were generated after tissue disruption and passage through a 70 µm cell strainer. Splenocytes were pelleted by centrifugation, and erythrocytes were lysed using ACK lysis buffer (Thermo Fisher). Cells were collected in DMEM with 10% FBS on ice. All staining steps were performed at 4°C in PBS with 2% heat inactivated FBS (FACS buffer). Single-cell suspensions were blocked for FcγR binding with anti-CD16/CD32 monoclonal antibody (eBioscience, #14-0161-82) on ice for 15 min prior to staining. Cells were subsequently incubated for 60 min on ice with a pool of biotin-streptavidin conjugated recombinant spike protein (Wuhan-1-spike BV421, Wuhan-1-spike BV786, BA.1-spike APC, BA.1-spike AF594, oval-biotin-streptavidin-BUV737) in FACS buffer (2% FBS and 2 mM EDTA in PBS), washed twice, then stained for 30 min on ice with Fixable Viability dye eFluor506 and a cocktail of labeled mAbs including CD4-AF700 (Biolegend, #100536), CD19-PerCP-Cy5.5. (BD, #551001), IgD-BV711 (BD, #564275), Fas-PE-Cy7 (BD, #557653), GL7-FITC (Biolegend, #144604), CD138-BV605 (Biolegend, #142516), and TACI-PE (Biolegend, #133403). Cells were washed twice with FACS buffer, fixed with 1% PFA for 30 min prior to data acquisition. Data were acquired on an Aurora (Cytek) spectral flow cytometer and analyzed in FlowJo v10 software.

T cell phenotyping

For T cell analysis, single-cell suspensions were incubated with FcγR antibody (clone 93, BioLegend) to block non-specific antibody binding, followed by staining with Fixable Viability dye eFluor506 and a cocktail of labeled mAbs, including CD45 (BUV395; Clone 30-F11; Cat: 564279; BD Biosciences), CD3 (BV711; Clone 145–2C11; Cat: 563123; BD Biosciences), CD8α (PerCP/Cy5.5; Clone 53–6.7; Cat: 100734; BioLegend), CD4 (BV785; Clone GK1.5; Cat: 100453; BioLegend), CD44 (APC/Cy7; Clone IM7; Cat: 103028; BioLegend), PD-1 PE/Cy7 (clone RMP1-30; Biolegend Cat# 109110), CXCR-5 BV421 (clone L138D7; Biolegend, Cat# 145511), and APC-labeled SARS-CoV-2 S-specific tetramer (MHC class I tetramer, residues 539–546, VNFNFNGL, H-2K^b) for 60 min at room temperature. Cells were washed twice with FACS buffer and fixed with 2% paraformaldehyde for 5 min before data acquisition. Data were acquired on an Aurora (Cytek) spectral flow cytometer and analyzed with FlowJo v10 software.

Measurement of viral RNA

Tissues were weighed and homogenized with zirconia beads in a MagNA Lyser instrument (Roche Life Science) in 1 mL of DMEM medium supplemented with 2% heat-inactivated FBS. Tissue homogenates were clarified by centrifugation at 10,000 rpm for 5 min and stored at −80°C. RNA was extracted using the MagMax mirVana. Total RNA isolation kit (Thermo Fisher) on the Kingfisher Flex extraction robot (Thermo Fisher). RNA was reverse transcribed and amplified using the TaqMan RNA-to-CT 1-Step Kit (Thermo Fisher). Reverse transcription was carried out at 48°C for 15 min followed by 2 min at 95°C. Amplification was accomplished over 50 cycles as follows: 95°C for 15 s and 60°C for 1 min. Copies of SARS-CoV-2 N gene RNA in samples were determined using a published assay (64).

Viral plaque assays

Plaque assays for titration of infectious virus were performed on Vero-hACE2-hTRMPSS2 cells in 24-well plates. Lung tissue homogenates were serially diluted 10-fold, starting at 1:10, in cell infection medium (DMEM + 2% FBS + 100 U/mL penicillin-streptomycin). Two hundred microliters of the diluted virus were added to a single well per dilution per sample. After 1 h at 37°C, the cells were overlayed with 1 mL of 1% methylcellulose in MEM supplemented with 2% FBS. Ninety-six hours after virus inoculation, the cells were fixed with 10% formalin, and the monolayer was stained with crystal violet [0.05% (wt/vol) in 25% methanol in water] for 30 min at 20°C. Plaque numbers were counted and used to calculate the PFU per gram.

Statistical analysis

Statistical significance was assigned when P values were <0.05 using GraphPad Prism version 9.3. Tests, number of animals, median, mean or geometric mean values, and statistical comparison groups are indicated in the Figure legends. Changes in viral RNA levels or serum antibody responses were compared to unvaccinated animals and were analyzed by one-way ANOVA with a multiple comparisons correction, Mann-Whitney test, or Wilcoxon signed-rank test depending on the type of results, number of comparisons, and distribution of the data.

DATA AVAILABILITY

All data supporting the findings of this study are available within the paper and from the corresponding author upon request. All requests for resources and reagents should be directed to and will be fulfilled by the corresponding author. All reagents will be made available on request after completion of a Materials Transfer Agreement. Preclinical mRNA vaccines can be obtained under an MTA with Moderna (contact: Darin Edwards, darin.edwards@modernatx.com).

ETHICS APPROVAL

All virus experiments were performed with Institutional Biosafety Committee approval in an approved biosafety level 3 (BSL-3 or A-BSL3) facility at Washington University School of Medicine. Animal studies were carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocols were approved by the Institutional Animal Care and Use Committee at the Washington University School of Medicine (assurance number A3381-01).

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/jvi.00574-24.

Figure S1. jvi.00574-24-s0001.tif.

Serum neutralization of WA1/2020 D614G and BA.1 viruses.

Figure S2. jvi.00574-24-s0002.tif.

Flow cytometry gating strategies for B cells in the LN and spleen.

Figure S3. jvi.00574-24-s0003.tif.

Frequency of germinal center B cells and plasmablast/plasma cells.

Figure S4. jvi.00574-24-s0004.tif.

Virus type-specific and cross-reactive GCB responses in lymph node.

Figure S5. jvi.00574-24-s0005.tif.

Virus type-specific and cross-reactive PB/PC responses in lymph nodes.

Figure S6. jvi.00574-24-s0006.tif.

Spike-specific total B cell responses in lymph nodes.

Figure S7. jvi.00574-24-s0007.tif.

PB/PC responses in the spleen following boosting with mRNA-1273 or mRNA1273.214.

Figure S8. jvi.00574-24-s0008.tif.

Gating strategies for analyzing spike specific CD8+ T and total TFH cells.

Figure S9. jvi.00574-24-s0009.tif.

TFH cell responses in the lymph node and spleen.

Supplemental figure legends. jvi.00574-24-s0010.docx.

Legends for Fig. S1 to S9.

ASM does not own the copyrights to Supplemental Material that may be linked to, or accessed through, an article. The authors have granted ASM a non-exclusive, world-wide license to publish the Supplemental Material files. Please contact the corresponding author directly for reuse.