key: cord-1042916-5xnyqrgl
authors: Peng, Lei; Renauer, Paul A.; Ökten, Arya; Fang, Zhenhao; Park, Jonathan J.; Zhou, Xiaoyu; Lin, Qianqian; Dong, Matthew B.; Filler, Renata; Xiong, Qiancheng; Clark, Paul; Lin, Chenxiang; Wilen, Craig B.; Chen, Sidi
title: Variant-specific vaccination induces systems immune responses and potent in vivo protection against SARS-CoV-2
date: 2022-04-26
journal: Cell Rep Med
DOI: 10.1016/j.xcrm.2022.100634
sha: 03b2ac60fb7cd75304496f39364eef071c598ba7
doc_id: 1042916
cord_uid: 5xnyqrgl

Lipid-nanoparticle (LNP)-mRNA vaccines offer protection against COVID-19. However, multiple variant lineages caused widespread breakthrough infections. Here, we generate LNP-mRNAs specifically encoding wildtype (WT), B.1.351 and B.1.617 SARS-CoV-2 spikes, and systematically study their immune responses. All three LNP-mRNAs induced potent antibody and T cell responses in animal models. However, differences in neutralization activity have been observed between variants. All three vaccines offer potent protection against in vivo challenges of authentic viruses of WA-1, Beta and Delta variant. Single cell transcriptomics of WT- and variant-specific LNP-mRNA vaccinated animals reveal a systematic landscape of immune cell populations and global gene expression. Variant-specific vaccination induces a systemic increase of reactive CD8 T cells and altered gene expression programs in B and T lymphocytes. BCR-seq and TCR-seq unveil repertoire diversity and clonal expansions in vaccinated animals. These data provide assessment of efficacy and direct systems immune profiling of variant-specific LNP-mRNA vaccination in vivo.

Severe acute respiratory syndrome coronavirus (SARS-CoV-2), the pathogen of coronavirus disease 2019 2 , has caused the ongoing global pandemic 1 . Although lipid nanoparticle (LNP) -mRNA based 3 vaccines such as BNT162b2 (Pfizer-BioNTech) and mRNA-1273 (Moderna) have demonstrated high efficacy 4 against COVID-19, breakthrough infections have been widely reported in fully vaccinated individuals 2,3 4-8 . 5

Moreover, the virus continues to mutate and multiple dangerous variant lineages have evolved, such as B.1.1.7, 6 B.1.351, and, more recently B.1.617 9,10 . The B.1.1.7 lineage (Alpha variant, or "UK variant") has an increased 7 rate of transmission and higher mortality 11 . The B.1.351 lineage (Beta variant, or "South Africa variant") has 8 an increased rate of transmission, resistance to antibody therapeutics, and reduced vaccine efficacy 12-14 . The 9 lineage B.1.617 ("Indian variant" lineage, including B.1.617.1 "Kappa variant", B.1.617.2 "Delta variant" and 10 B.1.617.3) has recently emerged, spread rapidly, and become dominant in multiple regions in the world 15, 16 . 11

The on-going surge of infections in the US is predominantly caused by the Delta variant, originating from the 12 B.1.617 lineage that has >1,000 fold higher viral load in infected individuals 17, 18 . The B.1.617 lineage has an 13 increased rate of transmission, showing reduced serum antibody reactivity in vaccinated individuals, and 14 exhibits resistance to antibody therapeutics [19] [20] [21] [22] [23] . These variants often spread faster than the ancestral "wildtype" 15 (WT) virus (also noted as Wuhan-1 or WA-1, with identical spike sequences), cause more severe disease, are 16 more likely to escape certain host immune response, cause disproportionally higher numbers of breakthrough 17 infections despite the status of full vaccination 14, [24] [25] [26] [27] , and have been designated by WHO and CDC as "variants 18 of concern" (VoCs) 28 . Regarding their effects on vaccine efficacy, B.1.351, for example, has been known to 19 reduce the efficacy of the Pfizer-BioNTech vaccine from >90% to near 70% 27 . The Delta variant has also 20 resulted in significant reduction of vaccine efficacy especially for individuals who received only a single dose 21 26 , and has caused wide-spread breakthrough infections despite the status of full vaccination 29 . 22 23 It has been widely hypothesized that the next-generation of COVID-19 vaccines can be designed to directly 24 target these variants ("variant-specific vaccines"). However, to date, there has been no literature report on any 25 approved or clinical stage variant-specific vaccine. Moreover, the immune responses, specificity, cross-26 reactivity, and host cell gene expression landscapes upon vaccination have to be rigorously tested for such 27 variant-specific vaccines to be developed. To directly assess the immunogenicity of potential variant-specific 28 SARS-CoV-2 vaccination, we generated LNP-mRNA vaccine candidates that encode the B.1.351 and B.1.617 29 spikes, along with the WT spike. With these variant-specific LNP-mRNAs, we characterized the immune 30 responses they induce in animals against homologous (cognate) and heterologous spike antigens and SARS-31 B). Relatively speaking, higher antibody responses was often observed with ECD antigen, suggesting an 23 immunogenic domain other than RBD contributed to the additional response to spike ECD ( boost response reported in ELISA ( Figure 2C) . The initial level of neutralization was at 10 2 -10 3 level of 30 reciprocal IC50 after priming for most groups ( Figure 2C ). Consistent with findings in ELISA, an 31 approximately two orders of magnitude increase in neutralization titer by boost was observed across all groups 32 (for both vaccine candidates and for all three pseudovirus types) in the low dose (1 µg) setting, and there was 33

To further validate the observations, we also performed bulk BCR-seq and bulk TCR-seq for all these mice on 24 additional tissue samples, including spleen, peripheral blood cells and lymph node (LN). The bulk BCR-seq 25 and TCR-seq data revealed systematic clonality maps of IGH, IGK, IGL, TRA, and TRB repertoires from the 26 spleen, blood, and LN samples of the variant-specific LNP-mRNA vaccinated along with PBS treated animals 27 ( Figure 7F ; Figures S11A and S12A). Analyses of IGK, IGL, TRA, and TRB repertoires showed trends of 28 decreasing unique clonotype numbers in B.1.351-LNP-mRNA and B.1.617-LNP-mRNA vs PBS peripheral 29 blood samples (Figures S11B and S12B), concomitant with an increased proportion of hyperexpanded 30 clonotypes (> 1% total clones) (Figures S11C and S12C ). There is also an increased percent of the repertoire 31 occupied by the top 10 and top 50 most abundant IGK, TRA, and TRB clonotypes in the blood, significantly in 32 TRA chains of 10 µg variant vaccinated and 1 µg B.1.351 vaccinated samples ( Figure 7G) . Lastly, true 33 diversity estimates of TRA and TRB chains were significantly decreased in the blood samples of all 1µg 1 B.1.351 and both 10µg variant vaccinated samples, relative to PBS controls ( Figure 7H ). these combined data 2 unveiled BCR and TCR repertoire clonality, diversity and respective shifts in variant-specific LNP-mRNA 3 vaccinated animals as compared to placebo-treated. In addition, these results are consistent with the 4 observation of decreased clonal diversity from single cell VDJ profiling, which together suggest a clonal 5 expansion of B cells and more notably, T cells. 6 7 Discussion 8

Although efficacious COVID-19 mRNA vaccines have been deployed globally, the rapid spread of SARS-9

CoV-2 VoCs with higher contagiousness as well as resistance to therapies and vaccines demands evaluation 10 of next-generation COVID-19 vaccines specifically targeting these evolving VoCs. Mounting evidence has 11 suggested that the B.1.351 and B.1.617 lineage variants of SARS-CoV-2 possesses much stronger immune 12 escape capability than the original wildtype virus 13, 23 . The lower neutralizing titers in fully vaccinated patients 13 were found associated with breakthrough infections 4 . It has been speculated that the waning immunity from 14 early vaccination and emergence of more virulent SARS-CoV-2 variants may lead to reduction in vaccine 15 protection and increase of breakthrough infections 6, 39 . It has been reported that mRNA vaccines' efficacy 16 against B.1.351 and B.1.617.2 dropped significantly 27, 40 . Moreover, for individuals receiving only a single 17 dose of vaccine, the protective efficacy can be dramatically lower 41 . It is worth noting that efficacy value and 18 definition may vary from study to study 42, 43 , which were conducted in different regions and populations. All 19 these factors prompted us to evaluate the next-generation mRNA vaccine candidates encoding the B.1.351 and 20 B.1.617 spike as antigens. 21

While the findings of differential antibody responses of vaccination against cognate vs. heterologous antigens 23 is in line with the effect of dampening immunity by variants for WT vaccines in human study, however, prior 24 studies were done using WT vaccines against VoCs, not variant-specific vaccines, which are entirely different 25 drug compositions. Currently there is limited published work or immunological data on variant-specific 26 vaccines. Our study directly produced, characterized, and systematically profiled the immunity of variant-27 specific vaccines. It's critical to learn their potential protective benefits against wildtype or variants of SARS-28

CoV-2. In fact, this becomes increasingly important due to the continuous rise of new variants of concern. In 29 reaction to the VoCs, major vaccine producers are actively developing variant-specific vaccines and test their 30 effect in clinical trials (e.g. Pfizer/BioNTech and Moderna), highlighting the clinical relevance. 31

Our study characterized the titers and cross-reactivity of sera from mice vaccinated with WT-/ WA-1-, 1 B.1.351-or B.1.617-LNP-mRNAs to all three WT, B.1.351 and B.1.617 spike antigens, pseudoviruses and 2 authentic viruses. In agreement with findings in patients' sera, we found that the neutralizing titers of WT 3 vaccine sera were several folds lower against the two variants of concerns than against WT pseudovirus. 4

Interestingly, the B.1.617-LNP-mRNA vaccinated sera also showed particularly strong neutralization activity 5 against its cognate B.1.617 pseudovirus, while the B.1.351-LNP-mRNA showed similar neutralization activity 6 against all three pseudoviruses. It is worth noting that all three forms of vaccine candidates can induce potent 7 B and T cell responses to WT as well as the two VoCs' spikes. The in vivo challenge experiments showed that 8 all three vaccine candidates, i.e. WT/WA-1-, B.1.351-and B.1.617-LNP-mRNAs offer strong protection 9 against all three authentic viruses (WA-1, Beta and Delta) in mice. 10 11

The T cell-biased immune response is important for antiviral immunity and thereby the efficacy and safety of 12 viral vaccines 44 . To evaluate the Th1 and Th2 immune response by the variant vaccines, we performed 13 intracellular staining of Th1 and Th2 cytokines in splenocytes. After stimulation with peptide pools covering 14 the entire S protein, the splenocytes from three mRNA vaccine groups produced more hallmark Th1 cytokine 15 IFN-γ in both CD4 + and CD8 + T cells than those from PBS group. Our flow cytometry data suggested that the 16 two variant vaccine candidates induced strong Th1-biased immune responses, just like the WT vaccine, of 17 which Th1 response had been observed by previous studies in animal models 31,35 . 18 19 Single cell sequencing is a powerful technology for immune and gene expression profiling, which has been 20 utilized for mapping immune responses to COVID-19 infection 45, 46 . In order to gain insights on the 21 transcriptional landscape of the immune cells, and clonal repertoire changes specifically in B and T cells, we 22 performed single cell transcriptomics, as well as BCR and TCR repertoire sequencing. The single cell 23 transcriptomics data revealed a systematic landscape of immune cell populations in B.1.351-LNP-mRNA and 24 B.1.617-LNP-mRNA vaccinated animals. We mapped out the repertoires and associated global gene 25 expression status of the immune populations including B cells, T cells, and innate immune cells. From the 26 overall splenocyte population, we observed a distinct and significant increase in the CD8 T cell, activated B 27 cell, and macrophage cell populations in vaccinated animals. Interestingly, differential expression between 28 vaccinated and placebo-treated animals showed a strong signature of increased expression of transcriptional 29 and translational machinery in both B and T cells. While the actual mechanism awaits future studies, these 30 phenomena are potentially reflective of the active proliferation and immune responses in these lymphocytes. 31 BCR and TCR sequencing are efficient tools for mapping of clonal repertoire diversity, which has been rapidly 1 utilized for sequencing COVID-19 patients 46, 47 . BCR-seq and TCR-seq unveiled the diversity and clonality 2 and respective shifts in variant-specific LNP-mRNA inoculated animals as compared to placebo-treated. The 3 decrease in VDJ clonal diversity, along with clonal expansion of a small number of clones, are observed in 4 vaccinated animals as compared to placebo group. Vaccinated animals from both B.1.351-LNP-mRNA and 5 B.1.617-LNP-mRNA groups have clonal TCR expansion, especially pronounced in peripheral blood samples. 6

The induction of diverse and expanding clones is a signature of vaccine induced protective immunity 38 . The 7 goal of this experiment is to profile the global repertoire of BCR and TCR, rather than just the antigen-specific 8 cells. Alternatively, antigen-specific sorting will zoom into the picture of those clones that are reactive to the 9 spike antigen, but may miss other antigenic or bystander clones. The population of B or plasma cells contain 10 antigen-specific clones in the vaccinated animals, as suggested by positive ELISA, neutralization and 11 protection data. While outside the scope of this study, it is of future interest to further dissect the clonal 12 expanded populations of B cells or plasma cell for antigen-specific responses, for example, whether they are 13 monoclonal, oligoclonal, polyclonal and whether there are increased mutations for GC selection. In addition, 14 the T cell clonal evolution is complex as it involves responses to both structural proteins (S, M, N, E) and non-15 structural proteins (NSPs). We performed the unsorted single cell and bulk BCR/TCR-seq analysis using the 16 entire populations from the samples to charter a comprehensive landscape of the BCR/TCR repertoires in the 17 WT-and variant-specific vaccinated animals. 18 19 In summary, our study provided direct assessment of in vivo immune responses to vaccination using LNP-20 mRNAs encoding specific SARS-CoV-2 variant spikes in pre-clinical animal models. The single cell and bulk 21 VDJ repertoire mapping also provided unbiased datasets and robust systems immunology of SARS-CoV-2 22 vaccination by LNP-mRNA specifically encoding B.1.351 and B.1.617 spikes. Last but not least, all three 23 vaccine candidates, including WT-LNP-mRNA, B.1.351-LNP-mRNA, and B.1.617-LNP-mRNA showed full 24 protective potency for mice against the challenge of authentic SARS-CoV-2 viruses, not only the ancestral 25 WA-01, but also two variants of concern, Beta and Delta. These original data may offer valuable insights for 26 the development of the next-generation COVID-19 vaccines against the SARS-CoV-2 pathogen and especially 27 its emerging variants of concern 21 . 28 29

We note a few limitations of our study. 1) The characterized VoC-specific vaccine candidates target the WT 31 and the two variants dominant in 2020-2021, while new VoCs continue to emerge and evolve. Investigation 32 of vaccine candidates targeting emerging variants are warranted. 2) Although commonly used, the animal 33 J o u r n a l P r e -p r o o f SARS-CoV-2 Variant Vaccine 13 model in this study is mouse, which has certain species specific immune response different from human. Further information and requests for resources and reagents should be directed to and will be fulfilled by the 9 Lead Contact, Sidi Chen (sidi.chen@yale.edu). 10 11 Materials Availability Statement 12

All unique/stable reagents generated in this study are available from the lead contact. Certain materials such 13 as vaccine candidates will be shared with a completed Materials Transfer Agreement. 14 15 Data and Code Availability 16

All data generated or analyzed during this study are included in this article, supplementary information, and 17 source data files. Specifically, source data and statistics for non-high-throughput experiments are provided in 18 a supplementary table excel file. Processed data and statistics for NGS experiments are provided in Data S1. 19

The raw NGS data have been deposited at SRA and are publicly available. Additional Supplemental flow 20 cytometry raw data are available from Mendeley Data at http://dx.doi.org/ 10.17632/2m6hvhhmr4.1. 21

The original codes of data analysis are available from the lead contact upon reasonable request. 22

Any additional information required to reanalyze the data reported in this paper is available from the lead 23 contact upon request. This study has received institutional regulatory approval. All recombinant DNA (rDNA) and biosafety work 30

were performed under the guidelines of Yale Environment, Health and Safety (EHS) Committee with approved 31 protocols (20) (21) (22) (23) (24) (25) (26) M. musculus (mice), 6-8 weeks old females of C57BL/6Ncr were purchased from Charles River. M. musculus 3 (mice), 6-8 weeks old females of K18-hACE2 mice (B6.Cg-Tg(K18-ACE2)2Prlmn/J) were purchased from 4 Jackson Laboratory and used for immunogenicity study. Animals were housed in individually ventilated cages 5 in a dedicated vivarium with clean food, water, and bedding. Animals are housed with a maximum of 5 mice 6 per cage, at regular ambient room temperature (65-75°F, or 18-23°C), 40-60% humidity, and a 14 h:10 h light 7 cycle. All experiments utilize randomized littermate controls. 8 9 Cell Lines 10 HEK293T (ATCC) and 293T-hACE2 (gifted from Dr Bieniasz' lab) cell lines were cultured in complete 11 growth medium, Dulbecco's modified Eagle's medium (DMEM; Thermo fisher) supplemented with 10% Fetal 12 bovine serum (FBS, Hyclone),1% penicillin-streptomycin (Gibco) (D10 media for short). Cells were typically 13 passaged every 1-2 days at a split ratio of 1:2 or 1:4 when the confluency reached at 80%. 14 15

The protective efficacy of SARS-CoV-2 WT and variant mRNA-LNP against replication-competent SARS- One week after boost, the LNP-mRNA vaccinated, and control mice were subdivided into three groups 26 randomly, then sedated with isoflurane. SARS-CoV-2 isolated USA-WA1/2020, Beta variant, or Delta variant 27 was inoculated intranasally at a dose of 10^3 PFU/mouse (determined using WT Vero E6) in 50 ul of DPBS. 28

Survival, body conditions, and weights of mice were monitored daily for 10 consecutive days. 29 30 31 METHOD DETAILS 32

The DNA sequences of B.1.351 and B.1.617 SARS-CoV-2 spikes for the mRNA transcription and pseudovirus 2 assay were synthesized as gBlocks (IDT) and cloned by Gibson Assembly (NEB) into pcDNA3.1 plasmids. 3

To improve expression and retain prefusion conformation, six prolines (HexaPro variant, 6P) were introduced 4 to the SARS-CoV-2 spike sequence in the mRNA transcription plasmids. The plasmids for the pseudotyped 5 virus assay including pHIVNLGagPol and pCCNanoLuc2AEGFP are gifts from Dr. Bieniasz' lab 48 . The C-6 terminal 19 amino acids were deleted in the SARS-CoV-2 spike sequence for the pseudovirus assay. 7 8 Cell culture 9 HEK293T (ATCC) and 293T-hACE2 (gifted from Dr Bieniasz' lab) cell lines were cultured in complete 10 growth medium, Dulbecco's modified Eagle's medium (DMEM; Thermo fisher) supplemented with 10% Fetal 11 bovine serum (FBS, Hyclone),1% penicillin-streptomycin (Gibco) (D10 media for short). Cells were typically 12 passaged every 1-2 days at a split ratio of 1:2 or 1:4 when the confluency reached at 80%. by an encoded polyA tail was used as template. The above DNA templates were obtained from circulated 20 plasmids pVP22b (B.1351 variant (6P)) and pVP29b (B.1.617 variant (6P)). pVP22b and pVP29b plasmids 21 were linearized with BbsI restriction enzyme digestion and cleaned up with gel purification. 22

Tthe mRNA was synthesized and purified by following the manufacturer's instructions and kept frozen at -24 80 °C until further use. The mRNA was encapsulated in a lipid nanoparticle (Genvoy-ILM TM , Precision 25 Nanosystem) using the NanoAssemblr ® Ignite™ machine (Precision Nanosystems). All procedures are 26 following the guidance of manufacturers. In brief, Genvoy-ILM TM , containing 50% PNI ionizable lipid,10% 27 DSPC, 37.5% cholesterol, 2.5% PNI stabilizer, were mixed with mRNA in acetate buffer, pH 5.0, at a ratio of 28 6:1 (Genvoy-ILM TM : mRNA). The mixture was neutralized with Tris-Cl pH 7.5, sucrose was added as a 29 cryoprotectant. The final solution was sterile filtered and stored frozen at -80 °C until further use. Negative-stain TEM 2 5 μl of the sample was deposited on a glow-discharged formvar/carbon-coated copper grid (Electron 3 Microscopy Sciences, catalog number FCF400-Cu-50), incubated for 1 min and blotted away. The grid was 4 washed briefly with 2% (w/v) uranyl formate (Electron Microscopy Sciences, catalog number 22450) and 5 stained for 1 min with the same uranyl formate buffer. Images were acquired using a JEOL JEM-1400 Plus 6 microscope with an acceleration voltage of 80 kV and a bottom-mount 4k × 3k charge-coupled device camera 7 (Advanced Microscopy Technologies, AMT). To detect surface-protein expression, the cells were stained with 10 μg/mL ACE2-Fc chimera (Genescript, 14 Z03484) in MACS buffer for 30 min on ice. Thereafter, cells were washed twice in MACS buffer and incubated 15 with PE-anti-human FC antibody (Biolegend, M1310G05) in MACS buffer for 30 min on ice. Live/Dead aqua 16 fixable stain (Invitrogen) were used to assess viability. Data acquisition was performed on BD FACSAria II 17

Cell Sorter (BD). Analysis was performed using FlowJo software. 18 19

A standard two-dose schedule given 21 days apart was adopted 49 . 1 g or 10 g LNP-mRNA were diluted in 21 1X PBS and inoculated into mice intramuscularly for prime and boost. Control mice received PBS. Two weeks 22 post-prime (day14) and two weeks post-boost (day 35), sera were collected from experimental mice and 23 utilized for following ELISA and neutralization assay of pseudovirus. Forty days (day 40) after prime, mice 24 were euthanized for endpoint data collection. Splenocytes were collected for T cell stimulation and cytokine 25 analysis, and single cell profiling. Lymphocytes were separately collected from mouse blood, spleen and 26 draining lymph nodes and applied for Bulk BCR and TCR profiling. 27 28

For every mouse treated with either LNP-mRNA or PBS. Blood, spleens and draining lymph nodes were 30 separately collected. Spleen and lymph node were homogenized gently and filtered with a 100 μm cell strainer 31 (BD Falcon, Heidelberg, Germany). The cell suspension was centrifuged for 5 min with 400 g at 4 °C. 32

Erythrocytes were lysed briefly using ACK lysis buffer (Lonza) with 1mL per spleen for 1~2 mins before 33 adding 10 mL PBS containing 2% FBS to restore iso-osmolarity. The single-cell suspensions were filtered 1 through a 40 μm cell strainer (BD Falcon, Heidelberg, Germany). 2 3

Spleens from three mice in LNP mRNA vaccine groups and four mice in PBS group were collected five days 5 post boost. Mononuclear single-cell suspensions from whole mouse spleens were generated using the above 6 method. 0.5 million splenocytes were resuspended with 200μl into RPMI1640 supplemented with 10% FBS, 7 1% penicillin-streptomycin antibiotic, Glutamax and 2mM 2-mercaptoethonal, anti-mouse CD28 antibody 8 (Biolegend, Clone 37.51) and seed into 96-well plate for overnight. The splenocytes were incubated for 6 hr 9

at 37°C three times using the 50TS microplate washer (Fisher Scientific, NC0611021) and blocked with 0.5% BSA in 1 PBST at room temperature for one hour. Plasma was serially diluted twofold or fourfold starting at a 1:2000 2 dilution. Samples were added to the coated plates and incubate at room temperature for one hour, followed by 3 washes with PBST five times. Anti-mouse secondary antibody was diluted to 1:2500 in blocking buffer and 4 incubated at room temperature for one hour. Plates were washed five times and developed with 5 tetramethylbenzidine substrate (Biolegend, 421101). The reaction was stopped with 1 M phosphoric acid, and 6 OD at 450 nm was determined by multimode microplate reader (PerkinElmer EnVision 2105). The binding 7 response (OD450) were plotted against the dilution factor in log10 scale to display the dilution-dependent 8 response. The area under curve of the dilution-dependent response (Log10 AUC) was calculated to evaluate 9 the potency of the serum antibody binding to spike antigens. The pseudovirus neutralization assays were performed on 293T-hACE2 cell. One day before, 293T-hACE2 26 cells were plated in a 96 well plate, 0.01 x10 6 cells per well. The following day, serial dilution serum plasma, 27 collected from PBS or LNP-mRNA vaccine immunized mice and started from 1:100 (5-fold serial dilution 28 using complete growth medium), 55 μL aliquots were mixed with the same volume of SARS-CoV-2 WT, 29 B.1.351 variant, and B.1.617 variant pseudovirus. The mixture was incubated for 1 hr at 37 °C incubator, 30 supplied with 5% CO2. Then 100 μL of the mixtures were added into 96-well plates with 293T-hACE2 cells. 31

Plates were incubated at 37°C supplied with 5% CO2. 48 hr later, 293T-hACE2 cells were collected and the 32 GFP+ cells were analyzed with Attune NxT Acoustic Focusing Cytometer (Thermo Fisher). The 50% 33 inhibitory concentration (IC50) was calculated with a four-parameter logistic regression using GraphPad Prism 1 (GraphPad Software Inc.). 2 3 Bulk BCR and TCR sequencing 4

Lymphocytes from blood, draining lymph node, spleen of each mRNA-LNP vaccinated and control mice were 5 collected as described above for mouse immunization and sample collection. mRNA of lymphocytes from 6 three tissues were extracted using a commercial RNeasy® Plus Mini Kit (Qiagen). Following bulk BCR and 7 TCR are prepared using SMARTer Mouse BCR IgG H/K/L Profiling Kit and SMARTer Mouse TCR a/b 8 profiling kit separately (Takara). Based on the extracted mRNA amount of each sample, the input RNA 9 amounts for bulk BCR libraries were as follows: lymphocytes from blood (100 ng), lymphocytes from lymph 10 node (1000 ng), and lymphocytes from spleen (1000 ng). The input RNA amounts for bulk TCR libraries were 11 as follows: lymphocytes from blood (100 ng), lymphocytes from lymph node (500 ng), and lymphocytes from 12 spleen (500 ng). All procedures followed the standard protocol of the manufacture. The pooled library was 13 sequenced using MiSeq (Illumina) with 2*300 read length. 14 Single cell profiling 16

Splenocytes were collected from mRNA-LNP vaccinated and control mice were collected as described above 17

for mouse immunization and sample collection, and normalized to 1000 cells/L. Standard volumes of cell 18 suspension were loaded to achieve targeted cell recovery to 10000 cells. The samples were subjected to 14 19 cycles of cDNA amplification. Following this, gene expression (GEX), TCR-enriched and BCR-enriched 20 libraries were prepared according to the manufacturer's protocol (10x Genomics). All libraries were sequenced 21 using a NovaSeq 6000 (Illumina) with 2*150 read length. Cells were clustered by generating a shared nearest neighbors (SNN) graph (k = 20, first 15 PCA dimensions) 1 and optimizing modularity using the Louvain algorithm with multilevel refinement algorithm and an 2 empirically chosen resolution, based on the best spatial separation of major immune populations cells via Cd3d, 3

Cd19, Ncr1, Itgam, Itgax, and Sdc1 expression on UMAP visualization. The clusters were then labeled using 4 the expression patterns of immune cell markers (Dataset S1), based on (a) the proportion of cells within each 5 cluster that express the markers (>10% of cells with scaled expression > 1) and (b) the cluster-averaged scaled 6 expression > 0 ( Figures 5B, S3-6 ). For better resolution of complex cell types, B cells, T cells and dendritic 7 cells (DCs) (Cd45+Cd19+, Cd45+Cd3d+, and Cd45+Itgax+ clusters, respectively) were separately subset, 8 rescaled, visualized in low dimensional UMAP space, clustered, and populations were identified using the 9 method above. Labeled cell types were tested for homogeneity by performing Wilcoxon rank sum testing of 10 scaled data and assessing discreet hierarchical clustering of populations using the top 10 DEGs in each cell 11 type compared to all others. Downstream analyses were performed using differentially expressed genes (DEGs) with an FDR-adjusted p 24 value < 0.01 and a log fold-change (log-FC) > 0.5 or < -0.5 for upregulated and downregulated genes, 25 respectively. First, DEGs were sorted by significance and analyzed by the gProfiler2 R package with biological 26 process gene ontology (GO) terms for mus musculus, against known genes as the analysis domain 56,57 . 27 Analysis results were filtered to include those with an adjusted p value (gProfiler gSCS method) < 0.01, GO 28 terms <= 600 genes, and terms that included > 4 DEGs. If there were more than 3 filtered terms, results were 29 clustered into "supra-pathways" by constructing an undirected network graph with (1) edges weighted by 30 filtered pathway similarity coefficients (coefficient = Jaccard + Overlap of genes between GO terms; 31 coefficients > 0.375), (2) a layout calculated via Fruchterman-Reingold algorithm, and (3) terms clustered by 32 the Leiden algorithm (modularity function, 1000 iterations, resolution = 0.8), all of which using the iGraph, 33 network, and sna R packages. The clustered pathways were labeled by the most significant pathway from each 1 cluster. 2 3 VDJ sequencing data analysis 4 Bulk VDJ sequencing data had adapters trimmed by Trimmomatic v0.39 in single-end mode, clipping Illumina 5

TruSeq adapters with default settings and filtering reads with an average quality score < 30 58 . Clonotypes 6 were called using MiXCR v2.1.5 with the recommended settings for 5' RACE (RNA alignment to V gene 7 transcripts with P region) 59 . Single-cell sequencing data was processed using the Cellranger v5.0.1 (10x 8 Genomics) pipeline and aligned to the mm10 VDJ reference. The MiXCR clonotype output or Cell Ranger 9 AIRR-formated output (bulk and single cell VDJ analyses, respectively) were used as inputs to Immunarch 10 v0.6.6 R package for calculating summary statistics, diversity metrics, and repertoire overlaps. 11

12 Standard Statistical analysis 13

The statistical methods are described in figure legends and/or supplementary Excel tables. The statistical 14 significance was labeled as follows: n.s., not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 15 0.0001. Prism (GraphPad Software) and RStudio were used for these analyses. Additional information can be 16 found in the Nature Research Reporting Summary. 17 18

Replicate experiments have been performed for all key data shown in this study. 20

Biological or technical replicate samples were randomized where appropriate. In animal experiments, mice 21 were randomized by littermates. 22

Experiments were not blinded. 23 NGS data processing were blinded using metadata. Subsequent analyses were not blinded. 24

Commercial antibodies were validated by the vendors, and re-validated in house as appropriate. Custom 25 antibodies were validated by specific antibody -antigen interaction assays, such as ELISA. Isotype controls 26 were used for antibody validations. 27 Cell lines were authenticated by original vendors, and re-validated in lab as appropriate. 28

All cell lines tested negative for mycoplasma. 29 with Tukey's multiple comparisons test was used to assess statistical significance. 32 K-L, T cell response of WT-LNP mRNA vaccinated animals (n = 4). CD8 + (k) and CD4 + (l) T cell responses 1 were measured by intracellular cytokine staining 6 hours after addition of BFA. The unpaired parametric t test 2 was used to evaluate the statistical significance. 3

In this figure: 5

Each dot represents data from one mouse. 6

Data are shown as mean ± s.e.m. plus individual data points in dot plots. 7

Statistical significance labels: n.s., not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0. analyses were performed using Wilcoxon rank sum test for each cell type vs all other cells, and the heatmap 20 includes the 10 DEGs from each analysis (absolute log2-FC > 4, q < 0.01). 21 D, Boxplots of overall cell type proportions compared across vaccine groups (n = 6 for each). Comparisons 22 were performed using a 2-way ANOVA test, accounting for vaccine and cell type as covariates, with Dunnet's 23 post-hoc analysis for multiple comparisons against PBS as the control. Data were analyzed together, but 24 displayed separately for clarity. 25 E, Stacked bar chart of cell proportions between different vaccination groups (n = 6 for each). 26

are labeled by cell types, assigned by the expression of cell type-specific markers. 28 G, Boxplots of B and T subset proportions compared across vaccine groups (n = 6 for each). Comparisons 29 were performed using a 2-way ANOVA test, accounting for vaccine and cell type as covariates, with Dunnet's 30 post-hoc analysis for multiple comparisons against PBS as the control. Data were analyzed together, but 31 displayed separately for clarity. 32

In this figure, panels D and G: 1 Each dot represents data from one mouse. 2

The high dose (n = 3 each) and low dose (n = 3 each) groups for each vaccine were merged (n = 6 total) in 3 single cell data analysis, same thereafter. 4

Statistical significance labels: n.s., not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0. Statistics for F and G performed using two-way ANOVA tests with Dunnet's multiple comparison test. 21 Statistical significance labels: n.s., not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 22 See also: Figure Lopez Bernal, J., Andrews, N., Gower, C., Gallagher, E., Simmons, R., Thelwall, S., Stowe, J., Tessier, E., 1 Groves, N., Dabrera, G., et al. (2021) Lopez Bernal, J., Andrews, N., Gower, C., Gallagher, E., Simmons, R., Thelwall, S., Stowe, J., Tessier, E., 1 Groves, N., Dabrera, G., et al. (2021) 

S29. BCR clonotype statistics, including relative clone abundance and top clone proportions across different ranges.

S30. Diversity statistics for BCR clonotypes, including data for Chao1, true diversity and Gini-Simpson methods.

S31. BCR clonotype counts.

S32. BCR clonotype statistics, including relative clone abundance and top clone proportions across different ranges. S49. TRD clonotype counts.

S50. TRD clonotype statistics, including relative clone abundance and top clone proportions across different ranges.

S51. Diversity statistics for TRD clonotypes, including data for Chao1, true diversity and Gini-Simpson methods. Details of the

617; 1ug; Blood B.1.617; 10ug; Blood PBS; 0ug; Spleen B.1.351; 1ug; Spleen B.1.351; 10ug; Spleen B.1.617; 1ug; Spleen B.1.617; 10ug

351; 10ug; Blood B.1.617; 1ug; Blood B.1.617; 10ug; Blood PBS; 0ug; Spleen B.1.351; 1ug; Spleen B.1.351; 10ug; Spleen B.1.617; 1ug; Spleen B.1.617; 10ug

351; 10ug; Blood B.1.617; 1ug; Blood B.1.617; 10ug; Blood PBS; 0ug; Spleen B.1.351; 1ug; Spleen B.1.351; 10ug; Spleen B.1.617; 1ug; Spleen B.1.617; 10ug

Miltenyi Biotec Cat#130-127-951

SARS-CoV-2 (2019-nCoV) Spike S1+S2 ECD-His Recombinant Protein SINO Cat#40589-V08B1

SARS-CoV-2 (2019-nCoV) Spike RBD Quote UQ7100 Cat#40592-V08B

SARS-CoV-2 Spike RBD (L452R,T478K) SINO Cat#40592-V08H90

SARS-CoV-2 Spike S1+S2 (E154K, L452R, E484Q, D614G, P681R, E1072K, K1073R) Protein (ECD, His Tag) SINO Cat#40589-V08B12

SARS-CoV-2 (2019-nCoV) Spike RBD (L452R, E484Q) Protein (His Tag) SINO Cat#40592-V08H88

SARS-CoV-2 (2019-nCoV) Spike S1+S2

SARS-CoV-2 (2019-nCoV) Spike RBD(N501Y)-His Recombinant Protein SINO Cat#40592-V08H82

SARS-CoV-2 (2019-nCoV) Spike RBD(K417N, E484K, N501Y)-His Recombinant Protein SINO Cat#40592-V08H85

Chromium Next GEM Single Cell 5ʹ Kit v2, 16 rxns PN-1000263 10X Genomics Cat#PN-1000263

Chromium Next GEM Chip K Single Cell Kit, 16 rxns PN-1000287 10X Genomics Cat#PN-1000287

Dual Index Kit TT Set A, 96 rxns PN-1000215 10X Genomics Cat#PN-1000215

SPRIselect -60 mL Beckman Coulter Cat#B23318 Chromium Single Cell Mouse TCR Amplification Kit

Statistics for comparison of cell type proportions between vaccination groups, using a two-way ANOVA test

Dataset 2 | T cell and B cell specific analyses of single cell GEX of PBS, WA-1-LNP-mRNA, B.1.351-LNP-mRNA, and 20 B.1.617-LNP-mRNA treated animals 21 Tabs in this dataset: S16. Pathway network analysis results for B.1.351 DE analysis in B cells

Pathway network analysis results for B.1.351 DE analysis in CD8 T cells

Pathway network analysis results for B.1.351 DE analysis in CD4 T cells

Pathway network analysis results for B.1.617 DE analysis in B cells

Pathway network analysis results for B.1.617 DE analysis in CD8 T cells

Pathway network analysis results for B.1.617 DE analysis in CD4 T cells

Pathway network analysis results for B.1.351 -WA-1 DE analysis in B cells

Pathway network analysis results for B.1.351 -WA-1 DE analysis in CD8 T cells

Pathway network analysis results for B.1.351 -WA-1 DE analysis in CD4 T cells

Pathway network analysis results for B.1.617 -WA-1 DE analysis in B cells

Pathway network analysis results for B.1.617 -WA-1 DE analysis in CD8 T cells

Diversity statistics for IGL clonotypes, including data for Chao1, true diversity and Gini-Simpson methods

WA-1-LNP-mRNA, B.1.351-LNP-mRNA, and B.1.617-LNP-mRNA

TRA clonotype statistics, including relative clone abundance and top clone proportions across different ranges

Diversity statistics for TRA clonotypes, including data for Chao1, true diversity and Gini-Simpson methods. Details of the 17 two-way ANOVA test for true diversity are included

TRB clonotype statistics, including relative clone abundance and top clone proportions across different ranges

Diversity statistics for TRB clonotypes, including data for Chao1, true diversity and Gini-Simpson methods. Details of the 21 two-way ANOVA test for true diversity are included