Elicit: Impact of Tozinameran mRNA Design on Antigenicity

Skip to main content

Impact of Tozinameran mRNA Design on Antigenicity

Research reportView only

Create alertChat

May 5, 2026

How does Tozinameran's mRNA design (e.g., prefusion-stabilized spike) affect antigenicity?

Tozinameran's prefusion-stabilized spike design (K986P/V987P mutations) and N1-methylpseudouridine modifications enhance antigenicity by concentrating immune responses on neutralizing epitopes and prolonging antigen expression, producing neutralizing antibody titers 10-18 times higher than natural infection despite recognizing fewer total epitopes.

Abstract

Tozinameran’s mRNA design achieves high antigenicity through multiple synergistic elements centered on prefusion-stabilized spike protein presentation. The K986P and V987P mutations lock the spike in a conformation where approximately 20% of trimers adopt the one-RBD ‘up’ state, authentically presenting the ACE2 binding site and neutralizing epitopes. This design elicits neutralizing antibody titers 10-18 times higher than convalescent sera in non-human primates and preserves neutralization against some variants, supported by clinical efficacy exceeding 95%. N1-methylpseudouridine modification enhances mRNA stability and translation while reducing innate immune sensing, contributing to the superiority of nucleoside-modified vaccines (>94% efficacy) over unmodified designs (48% efficacy). Additional elements including optimized UTRs and TENT5A-mediated poly(A) tail extension prolong antigen production and enhance immunogenicity.

Critically, the prefusion-stabilized design produces a distinct immune profile compared to natural infection: vaccine-elicited sera recognize fewer total linear epitopes (11 vs 37) but achieve higher neutralizing potency by focusing responses on functionally critical determinants like K417, E484, and N501 residues. The 26 epitopes recognized exclusively by convalescent sera map predominantly to post-fusion conformational states masked by prefusion stabilization, representing non-neutralizing targets. This epitope focusing represents an intentional design feature prioritizing neutralizing antibody quality over total antibody diversity. The design also generates strong TH1-type CD4+ and IFNγ+ CD8+ T-cell responses with broader epitope coverage from the full-length spike compared to RBD-only constructs. Advanced stabilization approaches like the S6P mutation further enhance durability and cross-variant neutralization, while self-amplifying platforms demonstrate sustained immune activation through prolonged antigen expression.

Methods

We analyzed 10 sources from an initial pool of 200, using 9 screening criteria. Each paper was reviewed for 6 key aspects that mattered most to the research question. More on methods

Records from Elicit search

n = 200

Papers screened using: Tozinameran Focus, mRNA Design Features, Antigenicity Outcomes, Study Design, Research Context, Comparative Analysis, Beyond Efficacy/Safety Only, mRNA Vaccine Relevance, Adequate Study Type

n = 200

Papers screened out

n = 190

Papers included for extraction

n = 10

Press enter or space to select a node.You can then use the arrow keys to move the node around. Press delete to remove it and escape to cancel.

Press enter or space to select an edge. You can then press delete to remove it or escape to cancel.

Paper search

We performed a semantic search across over 138 million academic papers from the Elicit search engine, which includes all of Semantic Scholar and OpenAlex.

We ran this query: “How does Tozinameran’s mRNA design (e.g., prefusion-stabilized spike) affect antigenicity?”

The search returned 200 total results from Elicit.

We retrieved 200 papers most relevant to the query for screening.

Screening

We screened in sources based on their abstracts that met these criteria:

We considered all screening questions together and made a holistic judgement about whether to screen in each paper.

Data extraction

We asked a large language model to extract each data column below from each paper. We gave the model the extraction instructions shown below for each column.

Extract all mRNA design characteristics relevant to Tozinameran/BNT162b2 antigenicity, including:

Extract all measures of antigenicity and immune response specifically for Tozinameran-type mRNA vaccines, including:

Extract the specific relationships between mRNA design features and antigenicity outcomes for Tozinameran, including:

Extract comparative data between different mRNA vaccine designs or between vaccine-induced and natural immunity, focusing on:

Extract mechanistic explanations for how Tozinameran’s mRNA design affects antigenicity, including:

Extract the experimental model and population studied, including:

Results

Characteristics of Included Studies

Study

Full text retrieved?

Study type

Species

Primary focus

Sample size/characteristics

Annette B. Vogel et al., 2020

Yes

Preclinical

Mice, rhesus macaques

BNT162b2 design and immunogenicity

Male rhesus macaques, 2-4 years old

Annette B. Vogel et al., 2021

Yes

Preclinical

Mice, rhesus macaques

BNT162b1 vs BNT162b2 comparison

Male rhesus macaques, 2-4 years old

Lizhou Zhang et al., 2023

Yes

Preclinical

Mice

Component comparison (Pfizer vs Moderna)

7-week-old female BALB/c mice

Y. Bong et al., 2025

Yes

Preclinical

Mice

S6P mutation effects

Balb/c and K18-hACE2 transgenic mice, 6-8 weeks old

Sascha Hein et al., 2021

Yes

Observational (human sera)

Human

BNT162b2 vs CVnCoV comparison

Healthcare workers (BNT162b2), phase I participants (CVnCoV)

Annette B. Vogel et al., 2020a

Yes

Preclinical

Mice, rhesus macaques

BNT162b vaccine candidates

Male rhesus macaques, 2-4 years old

Sascha Hein et al., 2021a

Yes

Observational (human sera)

Human

Epitope recognition patterns

Vaccinated and convalescent individuals

P. Krawczyk et al., 2025

Yes

Preclinical

Mice

Re-adenylation mechanisms

6-14 week old mice

Kate S. Levine et al., 2025

No (abstract only)

Clinical trial (Phase III)

Human

ARCT-154 vs BNT162b2

Participants with 3 prior mRNA vaccine doses

Rein Verbeke et al., 2021

Yes

Review

Multiple (review)

mRNA vaccine design and performance

Phase 3 clinical trial data

toof

Pageof

The included studies spanned preclinical models (mice and non-human primates), human observational studies, clinical trials, and systematic reviews. Six studies used full text, while one was available as abstract only. Studies employed diverse vaccination schedules, with preclinical studies typically using prime-boost regimens at days 0 and 21, while human studies examined booster responses in previously vaccinated individuals.

mRNA Design Features of Tozinameran

Tozinameran (BNT162b2) incorporates several molecular design elements intended to enhance antigenicity. The vaccine encodes a full-length spike protein stabilized in its prefusion conformation through K986P and V987P substitutions, also referred to as P2 mutations. Structural analysis revealed that approximately 20% of expressed spike trimers adopt the one-RBD ‘up’, two-RBD ‘down’ conformation, which authentically presents the ACE2 binding site and other neutralizing epitopes.

The mRNA contains nucleoside modifications, specifically N1-methylpseudouridine (m1Ψ), which reduces innate immune sensing and enhances RNA translation. The construct includes a 100-nucleotide poly(A) tail, though one study noted BNT162b2 has a composite poly(A) tail structure. The 5’ untranslated region (UTR) derives from human hemoglobin α-globin (HBA1), while the 3’ UTR originates from AES-mtRNR1. Additional optimization includes non-coding sequence elements and a linker (A30LA70) to improve RNA stability and translational efficiency.

Comparative design studies revealed differences between Tozinameran and other mRNA vaccines. BNT162b2 employs the ionizable lipid ALC-0315, enzymatically incorporated Cap 1 for mRNA capping, and a prefusion-stabilized spike configuration. An alternative design, BNT162b1, encodes a soluble, secreted trimerized receptor-binding domain (RBD-foldon) rather than the full-length spike. Advanced stabilization approaches, such as the S6P mutation (incorporating KV986PP plus four additional proline substitutions at F817P, A892P, A899P, and A942P), have been explored in next-generation candidates.

Effects on Antigenicity

Antibody Responses

Tozinameran elicited robust antibody responses across preclinical and clinical studies. In rhesus macaques, prime-boost vaccination produced neutralizing geometric mean titers 10.2 to 18.0 times higher than those in convalescent human serum panels. The vaccine induced dose-dependent increases in pseudovirus neutralization titers in mice, with neutralizing antibodies remaining detectable at 3 months post-vaccination in humans.

Comparative analyses demonstrated superiority over some platforms. BNT162b2-elicited sera showed higher titers and better neutralizing capacity compared to CVnCoV-elicited sera. The vaccine preserved neutralizing antibody activity against the B.1.1.7 variant, though neutralizing capacity was significantly impacted against the B.1.351 variant. Anti-RBD titers from vaccine-elicited sera exceeded those from convalescent sera.

Design elements differentially affected antibody production. The ionizable lipid component influenced neutralizing antibody generation, with SM-102 (used in Moderna’s vaccine) producing higher neutralizing activity at days 14 and 35 post-vaccination compared to ALC-0315. UTR sequences from Pfizer-BioNTech’s 5’ region and Moderna’s 3’ region demonstrated superior performance in supporting mRNA translation and antibody production.

T-Cell Responses

The vaccine generated strong cellular immunity characterized by TH1-type CD4+ and IFNγ+ CD8+ T-cell responses in both mice and rhesus macaques. This TH1-biased response profile is favorable for vaccine safety and efficacy. CD4+ T-cell responses were identified in all COVID-19 patients and correlated with IgG and IgA antibody titers. The vaccine facilitated efficient presentation of peptide fragments to CD8+ T cells through intracellular processing.

The full-length prefusion-stabilized spike design (BNT162b2) provided a broader T-cell epitope range compared to the RBD-only construct (BNT162b1), contributing to the selection of BNT162b2 for clinical development. The balanced immune response included secretion of both TH1 cytokines (IFN-γ, IL-2, TNF-α) and TH2 cytokines (IL-4, IL-5, IL-13) in certain contexts.

Epitope Recognition and Structural Considerations

A critical finding emerged regarding epitope recognition patterns. Peptide array analyses identified 37 linear epitopes across the spike protein, with 26 epitopes almost exclusively recognized by convalescent sera but not vaccine-elicited sera. These 26 epitopes predominantly mapped to regions masked in the prefusion structure but exposed in the post-fusion conformation. Specifically, three epitopes in the conserved central helix were only exposed post-fusion.

The prefusion stabilization achieved through K986P and V987P mutations reduced structural flexibility and conferred resistance to proteolytic processing. While this stabilization enhanced presentation of the ACE2 binding site and other key neutralizing epitopes, it simultaneously masked epitopes accessible during natural infection. Vaccine-elicited sera strongly recognized linear epitopes harboring K417, E484, and N501 residues, which are critical for ACE2 binding.

The structural design resulted in approximately 20% of spike trimers adopting the one-RBD ‘up’ conformation, creating a dynamic equilibrium between RBD positions. This equilibrium allowed exposure of epitopes that would otherwise remain buried, enhancing immune recognition while maintaining the prefusion-stabilized state.

mRNA Stability and Processing

The cellular fate of vaccine mRNA significantly influenced antigenicity. Studies revealed that mRNA-1273 (Moderna) underwent more potent poly(A) tail re-adenylation by TENT5A compared to BNT162b2, correlating with higher membrane association and antigen production. Re-adenylation extended poly(A) tails from approximately 100 to 200 nucleotides, stabilizing mRNA and enhancing translation. TENT5A deficiency reduced specific immunoglobulin production in mice immunized with mRNA vaccines.

The capping strategy influenced re-adenylation efficiency, with enzymatically incorporated Cap 1 producing several-fold more antigen than CleanCap-equipped mRNA. The m1Ψ modification enhanced mRNA stability and translation while reducing innate immune signaling, improving overall antigen presentation. However, varying N1-methylpseudouridine content at wobble positions showed little effect on neutralizing antibody production.

Comparative Design Effects

Direct comparisons between BNT162b1 and BNT162b2 revealed that while both candidates elicited strong immune responses, BNT162b2 provided superior protection of the lower respiratory tract from viral RNA. BNT162b2 was selected for clinical advancement based on greater tolerability and broader T-cell epitope coverage, despite comparable immunogenicity.

Comparison with CVnCoV, an unmodified mRNA vaccine encoding a stabilized spike, highlighted the importance of nucleoside modification. BNT162b2 and Moderna (both using modified nucleosides) achieved over 94% efficacy, while CVnCoV demonstrated 48% efficacy. The modified RNA in BNT162b2 (30 μg dose) showed higher efficacy and immune responses compared to unmodified RNA in CVnCoV (12 μg dose). Reactogenicity of unmodified RNA limited achievable dosing in CVnCoV, potentially impacting antigenicity.

Component-level comparisons demonstrated that UTR selection affected translation efficiency. Pfizer-BioNTech’s 5’ UTR outperformed Moderna’s counterpart, while Moderna’s 3’ UTR exceeded Pfizer-BioNTech’s version. These UTR differences contributed to overall antigen expression levels and subsequent immune responses.

Advanced stabilization strategies, exemplified by the S6P mutation, enhanced immunogenicity beyond the standard P2 design. The S6P mutation (incorporating six proline substitutions) elicited significantly stronger B and T cell responses with more durable neutralizing antibodies compared to S2P-based vaccines. This enhanced stability preserved the prefusion conformation more effectively, leading to improved immune recognition and broader cross-variant neutralization.

Synthesis: Reconciling Epitope Diversity with Neutralizing Potency

The studies revealed an apparent paradox: prefusion-stabilized spike designs like BNT162b2 produce fewer recognized linear epitopes than natural infection yet elicit higher neutralizing antibody titers than convalescent sera. This divergence reflects fundamental differences in antigen presentation rather than a deficiency in either response.

Mechanism of Differential Epitope Exposure

The prefusion stabilization through K986P and V987P mutations constrains spike protein flexibility, preventing conformational transitions that would expose post-fusion epitopes. During natural infection, spike proteins undergo fusion-mediated conformational changes, exposing epitopes in the central helix and other regions that become accessible only in post-fusion states. The 26 epitopes preferentially recognized by convalescent sera map predominantly to these conformational-dependent regions. In contrast, the vaccine-stabilized prefusion conformation maintains epitopes critical for ACE2 binding and neutralization in an accessible state, while masking conformational epitopes.

Functional Consequences: Quality Over Quantity

The reduced epitope diversity in vaccine-elicited responses does not indicate inferior immunity; rather, it reflects focused recognition of neutralizing determinants. Vaccine-elicited antibodies target the RBD and S1 domain with high affinity, particularly epitopes containing K417, E484, and N501—residues essential for ACE2 binding. This focused response produces neutralizing titers 10-18 times higher than convalescent sera, demonstrating that epitope quality (neutralizing capacity) outweighs quantity (total epitopes recognized).

Natural infection presents both prefusion and post-fusion spike conformations, generating antibodies to a broader epitope repertoire that includes non-neutralizing targets. While this broader response may provide advantages for variant recognition, it dilutes the neutralizing antibody fraction. The vaccine’s prefusion-only presentation concentrates immune responses on functionally critical epitopes.

Design Element Contributions to Antigenicity

Multiple design elements synergistically enhance antigenicity through distinct mechanisms:

The m1Ψ modification operates primarily through enhanced mRNA stability and reduced innate immune interference, increasing antigen expression duration and quantity. This nucleoside modification proved critical; unmodified mRNA vaccines showed substantially lower efficacy (48% vs >94%) despite encoding similar antigens, likely due to rapid degradation and inflammatory responses that limited dosing.

UTR sequences regulate translation initiation and mRNA stability, with optimal combinations (Pfizer’s 5’ UTR and Moderna’s 3’ UTR) providing superior translation efficiency. The poly(A) tail undergoes enzymatic extension by TENT5A in macrophages, extending from 100 to 200 nucleotides and stabilizing membrane-associated mRNA to prolong antigen production. However, BNT162b2 shows less potent re-adenylation than mRNA-1273, potentially due to differences in membrane association efficiency or capping strategies.

The prefusion stabilization itself provides structural advantages beyond epitope presentation. The K986P and V987P mutations lock the spike in a conformation that authentically presents neutralizing epitopes while maintaining the dynamic RBD equilibrium (~20% ‘up’) necessary for immune recognition. More extensive stabilization (S6P mutation with six proline substitutions) further enhances this effect, producing stronger and more durable responses by improving thermal stability and expression levels.

Implications for Variant Protection

The prefusion-focused epitope recognition has mixed implications for variant responses. While preserved neutralization against B.1.1.7 demonstrates some cross-variant protection, significantly reduced neutralization of B.1.351 reveals limitations. Variants with mutations in conserved prefusion epitopes (particularly K417, E484, N501) can escape vaccine-elicited immunity more readily than they escape convalescent immunity, which targets additional epitopes in the central helix and other regions.

However, advanced designs incorporating broader stabilization (S6P) or self-amplifying platforms (ARCT-154) demonstrate enhanced cross-variant neutralization. The sustained antigen production from self-amplifying mRNA moves immune profiles toward activating phenotypes with broad antigenic coverage, suggesting that prolonged antigen exposure may partially compensate for reduced epitope diversity by allowing affinity maturation against variant epitopes.

Trade-offs in Design Optimization

Design choices involve inherent trade-offs between different immune response characteristics. Maximum neutralizing potency (achieved through strict prefusion stabilization) comes at the cost of epitope breadth. Higher mRNA doses and enhanced stability (through modifications, optimal UTRs, and re-adenylation) increase antigen expression but may alter the balance between humoral and cellular responses. More extensive proline stabilization (S6P vs S2P) improves durability but requires validation for potential effects on protein processing and presentation.

The selection of BNT162b2 over BNT162b1 illustrates these trade-offs: the full-length spike provides broader T-cell epitope coverage and better respiratory tract protection, while the RBD-only construct offers high-avidity ACE2 binding through flexible tethering. The choice prioritized epitope breadth and T-cell responses over maximizing RBD-focused neutralization.

Conclusion

Tozinameran’s mRNA design achieves high antigenicity through prefusion stabilization that optimally presents neutralizing epitopes, nucleoside modifications that enhance expression, and structural elements that support mRNA stability and translation. The reduced linear epitope recognition compared to natural infection represents a feature rather than limitation—focusing immune responses on functionally critical neutralizing determinants while sacrificing recognition of conformational epitopes with limited protective value. This design philosophy prioritizes neutralizing antibody quality and T-cell breadth over total antibody diversity, an approach validated by clinical efficacy exceeding 95% despite narrower epitope targeting than natural infection.

References

Annette B. Vogel, I. Kanevsky, Y. Che, K. Swanson, A. Muik, and 56 more\ (2020).A prefusion SARS-CoV-2 spike RNA vaccine is highly immunogenic and prevents lung infection in non-human primates. bioRxiv

Rein Verbeke, I. Lentacker, S. D. De Smedt, H. Dewitte\ (2021).The dawn of mRNA vaccines: The COVID-19 case. Journal of Controlled Release

Annette B. Vogel, I. Kanevsky, Y. Che, K. Swanson, A. Muik, and 69 more\ (2021).BNT162b vaccines protect rhesus macaques from SARS-CoV-2. Nature

Lizhou Zhang, Kunal R. More, Amrita Ojha, C. B. Jackson, Brian D. Quinlan, and 5 more\ (2023).Effect of mRNA-LNP components of two globally-marketed COVID-19 vaccines on efficacy and stability. npj Vaccines

Y. Bong, David Brown, Ezra Chung, N. Ananthaswamy, Renxiang Chen, and 10 more\ (2025).S6P mutation in Delta and Omicron variant spike protein significantly enhances the efficacy of mRNA COVID-19 vaccines. Frontiers in Immunology

Sascha Hein, Marie-Luise Herrlein, Ines Mhedhbi, Daniela Bender, Vanessa Haberger, and 11 more\ (2021).Analysis of BNT162b2‐ and CVnCoV‐elicited sera and of convalescent sera toward SARS‐CoV‐2 viruses. Allergy. European Journal of Allergy and Clinical Immunology

Annette B. Vogel, I. Kanevsky, Y. Che, K. Swanson, A. Muik, and 69 more\ (2020).BNT162b vaccines are immunogenic and protect non-human primates against SARS-CoV-2. bioRxiv

Sascha Hein, N. Benz, Jonathan Eisert, Marie-Luise Herrlein, D. Oberle, and 4 more\ (2021).Comirnaty-Elicited and Convalescent Sera Recognize Different Spike Epitopes. Vaccines

P. Krawczyk, M. Mazur, Wiktoria Orzeł, Olga Gewartowska, Sebastian Jeleń, and 20 more\ (2025).Re-adenylation by TENT5A enhances efficacy of SARS-CoV-2 mRNA vaccines. Nature

Kate S. Levine, Ross Blanc, Qixin Wang, Hadar Malca, R. Sekulovich, and 6 more\ (2025).Sustained Humoral Activation through self-amplifying mRNA Vaccination Enhances Longitudinal Antibody Function in a Phase III Trial. bioRxiv

Download BIBDownload RISDownload TXT

Report

Status

Gather sources

200 sources found

Details

Screen sources

10 sources included

Details

Extract data

60 data points extracted

Details

Generate report

Save PDF

BIBLaTeX, ZoteroRISZotero, MendeleyTXTAPA BibliographyPDFPDFDOCXMicrosoft Word

Chat

Got some follow-up questions?

Sign up or sign in to chat with this report.

Back

A prefusion SARS-CoV-2 spike RNA vaccine is highly immunogenic and prevents lung infection in non-human primates

Annette B. Vogel, I. Kanevsky, Y. Che, K. Swanson, A. Muik, M. Vormehr, L. Kranz, Kerstin C. Walzer, Stephanie Hein, Alptekin Güler, Jakob Loschko, M. Maddur, Kristin Tompkins, J. Cole, B. G. Lui, Thomas Ziegenhals, Arianne Plaschke, D. Eisel, Sarah C. Dany, Stephanie Fesser, Stephanie Erbar, F. Bates, D. Schneider, B. Jesionek, Bianca Sänger, A. Wallisch, Yvonne Feuchter, H. Junginger, S. A. Krumm, André P. Heinen, P. Adams-Quack, J. Schlereth, Christoph Kröner, Shannan Hall-Ursone, K. Brasky, M. Griffor, Seungil Han, J. Lees, E. Mashalidis, P. Sahasrabudhe, Charles Y. Tan, D. Pavliakova, Guy Singh, Camila R. Fontes-Garfias, M. Pride, Ingrid L Scully, Tara Ciolino, Jennifer Obregon, M. Gazi, R. Carrion, Kendra J Alfson, W. Kalina, D. Kaushal, P. Shi, T. Klamp, Corinna Rosenbaum, A. Kuhn, Ö. Türeci, P. Dormitzer, K. Jansen, U. Şahin

bioRxiv·

2020·

125 citations

SourceDOI

Plain textPDF

Searching for PDF

Unable to find PDF from source

Back

mRNA Design Features

- Spike protein configuration: Prefusion-stabilized full-length spike protein - Specific stabilization mutations: K986P/V987P (P2 mutations) - Nucleoside modifications: m1Ψ - Poly(A) tail characteristics and length: 100 nucleotides with a linker - Codon optimization details: General optimization for RNA stability and translational efficiency

Antigenicity Outcomes

- Antibody responses: Dose-dependent increases in pseudovirus neutralization titers in mice; neutralizing titers 10.2 to 18.0 times higher than human convalescent sera in rhesus macaques. - Epitope recognition patterns and specificity: Encodes spike glycoprotein in prefusion conformation; S1 and RBD binding antibodies. - T-cell responses: Strong TH1 type CD4+ and IFNγ+ CD8+ T-cell responses in mice and rhesus macaques; TH1-biased response. - Cross-reactivity against variants: Not mentioned. - Duration of immune response: Neutralizing titers remained high five weeks after last immunization. - Cellular uptake and processing measures: Not mentioned. - Measures of immune breadth or diversity: Not mentioned. - Assays and measurement methods: Pseudovirus neutralization assay; ELISpot for T-cell responses; intracellular cytokine staining.

Design-Antigenicity Effects

- Design feature: Prefusion-stabilized spike protein encoded by mRNA. - Direct comparison: BNT162b2 vs. BNT162b1; prefusion-stabilized spike enhances antigenicity. - Quantitative effects: High neutralizing titers; strong TH1 and TFH type T-cell responses. - Enhancing design elements: m1Ψ modification increases RNA translation and dampens innate immune sensing. - Dose-response relationship: Higher doses increase neutralizing GMTs. - Time-dependent effects: Persistent high neutralizing GMTs five weeks post-immunization. - Trade-offs: Greater tolerability of BNT162b2 with comparable immunogenicity compared to BNT162b1.

Vaccine Comparisons

- Direct comparison between BNT162b1 and BNT162b2: Both have similar immunogenicity and protective effects, but BNT162b2 is more tolerable. - Comparison with convalescent immunity: BNT162b2 elicits higher neutralizing titers than convalescent human sera. - No direct comparison with other mRNA vaccines (Moderna, CureVac) or cross-platform comparisons.

Mechanistic Insights

- Structural biology insights: The mRNA design encodes the spike glycoprotein in its prefusion conformation, with approximately 20% of spike molecules in the one-RBD 'up', two-RBD 'down' state. This conformation authentically presents the ACE2 binding site and other epitopes targeted by SARS-CoV-2 neutralising antibodies. - Cellular processing mechanisms: The m1Ψ modification reduces innate immune sensing and enhances RNA translation in vivo, increasing the expression of the spike protein. - Epitope exposure patterns: The prefusion conformation allows for a dynamic equilibrium between RBD 'up' and 'down' states, exposing neutralizing epitopes. - Role of specific design elements: The prefusion-stabilized spike and m1Ψ modification are key for immune recognition by exposing epitopes and enhancing translation. - Molecular explanations for observed antigenicity differences: The dynamic equilibrium between RBD states and the authentic presentation of epitopes contribute to the high immunogenicity of BNT162b2. - Mechanistic models proposed by authors: The design elements work together to enhance antigenicity by exposing neutralizing epitopes and increasing protein expression.

Study Model

- Species: Mouse, Rhesus macaque - Study design: Preclinical - Sample characteristics: Male rhesus macaques, 2-4 years old - Vaccination schedule and dosing: Two doses of BNT162b2 (30 or 100 µg) on Days 0 and 21 - Time points of immune assessment: Day 21 after first dose, Day 28 after second dose - Relevant study limitations: Preclinical model, specific age group of rhesus macaques

To contain the coronavirus disease 2019 (COVID-19) pandemic, a safe and effective vaccine against the new severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is urgently needed in quantities sufficient to immunise large populations. In this study, we report the design, preclinical development, immunogenicity and anti-viral protective effect in rhesus macaques of the BNT162b2 vaccine candidate. BNT162b2 contains an LNP-formulated nucleoside-modified mRNA that encodes the spike glycoprotein captured in its prefusion conformation. After expression of the BNT162b2 coding sequence in cells, approximately 20% of the spike molecules are in the one-RBD ‘up’, two-RBD ‘down’ state. Immunisation of mice with a single dose of BNT162b2 induced dose level-dependent increases in pseudovirus neutralisation titers. Prime-boost vaccination of rhesus macaques elicited authentic SARS-CoV-2 neutralising geometric mean titers 10.2 to 18.0 times that of a SARS-CoV-2 convalescent human serum panel. BNT162b2 generated strong TH1 type CD4+ and IFNγ+ CD8+ T-cell responses in mice and rhesus macaques. The BNT162b2 vaccine candidate fully protected the lungs of immunised rhesus macaques from infectious SARS-CoV-2 challenge. BNT162b2 is currently being evaluated in a global, pivotal Phase 2/3 trial (NCT04368728).

Main Introduction

Due to the shattering impact of the coronavirus disease 2019 (COVID-19) pandemic on human health and society, multiple collaborative research programs have been launched, leading to new insights and progress towards vaccine development. Soon after it emerged in December 2019, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was identified as a βcoronavirus with high sequence similarity to bat-derived SARS-like coronaviruses 1,2 . The globalised response is mirrored by the upload of over 92,000 viral genome sequences as of August 29, 2020, to GISAID (Global Initiative on Sharing All Influenza Data).

The trimeric spike glycoprotein (S) of SARS-CoV-2 binds its cellular receptor, angiotensin converting enzyme 2 (ACE2), through a receptor-binding domain (RBD), which is part of its N-terminal furin cleavage fragment (S1) 3,4 . S rearranges to translocate the virus into cells by membrane fusion 5,6 . The C-terminal furin cleavage fragment (S2) contains the fusion machinery 7 . Membrane fusion can be blocked by mutating S residues 986 and 987 to prolines, producing an S antigen stabilised in the prefusion conformation (P2 S) [8][9][10] . The RBD is a key target for virus neutralising antibodies, with an 'up' conformation, in which more neutralising epitopes are exposed, and a 'down' conformation in which many epitopes are buried 5,[10][11][12] . In addition, some neutralising antibodies bind S epitopes outside the RBD.

During this pandemic, fast vaccine availability is critical. COVID-19 vaccine candidates based on different platforms are already in clinical trials, with the most advanced based on viral vector and nucleic acid technologies [13][14][15][16] . We report the preclinical development of BNT162b2, a lipidnanoparticle (LNP) formulated N 1 -methyl-pseudouridine (m1Ψ) nucleoside-modified mRNA (modRNA) vaccine candidate that encodes P2 S with a native furin cleavage site resulting in the S1 and S2 cleavage fragments (Fig. 1a ). The m1Ψ-modification dampens innate immune sensing, and, together with optimised non-coding sequence elements, increases RNA translation in vivo [17][18][19] . ModRNA vaccines have already proven immunogenic for several viral targets 20,21 . BNT162b2 has been evaluated in phase 1 clinical trials in the US (NCT04368728) and Germany (NCT04380701, EudraCT: 2020-001038-36), and is now being evaluated in a pivotal, global, phase 2/3 safety and efficacy study 15 .

preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Results

BNT162b2 RNA in vitro transcribed by T7 polymerase from a plasmid DNA template has a single, sharp-peak microfluidic capillary electrophoresis profile, consistent with its calculated length of 4,283 nucleotides, indicating purity and integrity (Fig. 1b ). When HEK293T/17 cells were incubated with BNT162b2 (which is LNP-formulated) or with BNT162b2 RNA mixed with a transfection reagent, robust expression of P2 S was detectable by flow cytometry (Extended Data Fig. 1a ).

For structural characterisation, P2 S was expressed in Expi293F cells from DNA that encodes the same amino acid sequence as BNT162b2 RNA, with the addition of a C-terminal TwinStrep tag for affinity purification. The trimeric P2 S bound the human ACE2 peptidase domain (PD), and an anti-RBD human neutralising antibody B38 with high affinity (KD 1 nM, Extended Data Fig. 1b, c ) 22 . Structural analysis by cryo-electron microscopy (cyro-EM) produced a 3.29 Å nominal resolution mass density map, into which a previously published atomic model 10 was fitted and rebuilt (Fig. 1c, d ; Extended Data Fig. 2 , Extended Data Table 1 ). The rebuilt model shows good agreement with reported structures of prefusion full-length wild type S and its ectodomain with P2 mutations 5,10 . Three-dimensional classification of the dataset showed a class of particles that was in the one RBD 'up' (accessible for receptor binding), two RBD 'down' (closed) conformation and represented 20.4% of the trimeric molecules. The remainder were in the all RBD 'down' conformation (Fig. 1e , Extended Data Fig. 2c ). The RBD in the 'up' conformation was less well resolved than other parts of the structure, suggesting conformational flexibility and a dynamic equilibrium between RBD 'up' and RBD 'down' states as also suggested by others 5,23 . Nevertheless, the binding and structural analyses indicate that the BNT162b2 RNA sequence encodes a recombinant P2 S that authentically presents the ACE2 binding site and other epitopes targeted by SARS-CoV-2 neutralising antibodies.

To characterise BNT162b2-elicited B-and T-cell responses, BALB/c mice were immunized intramuscularly (IM) once with 0.2, 1, or 5 µg BNT162b2 or received a buffer control. S1-and RBD-binding serum IgG developed rapidly at all dose levels in a dose-dependent manner. For S1-binding antibodies, the geometric mean concentration (GMC) in the 5 µg group was 386 µg/mL at Day 28 (Fig. 2a , Extended Data Fig. 3a ). At Day 28 after immunisation, vaccineelicited IgG had a strong binding affinity for S1 (geometric mean KD 12 nM) and the RBD (geometric mean KD 0.99 nM), with both having a low off-rate (Extended Data Fig. 3b ). SARS-CoV-2 neutralising activity in mouse serum was measured by a vesicular stomatitis virus (VSV)-based SARS-CoV-2 pseudovirus neutralisation assay. Fifty percent pseudovirus neutralisation geometric mean titers (pVNT50 GMTs) increased steadily after immunisation to 26, 176, and 296 on Day 28 for the 0.2, 1, and 5 µg dose levels, respectively (Fig. 2b , Extended Data Fig. 3c ).

A high fraction of splenocytes of CD4 + and CD8 + T-cell phenotype isolated from mice on Days 12 and 28 after BNT162b2-immunisation had a strong antigen-specific IFNγ and IL-2 response in ELISpot and intracellular cytokine staining flow cytometry analysis when re-stimulated ex vivo with a full-length S peptide pool (Fig. 2c-e ). Total splenocytes harvested on Day 28 and ex vivo re-stimulated with the full-length S peptide pool secreted high levels of the TH1 cytokines IL-2 or IFNγ, but minute amounts of the TH2 cytokines IL-4, IL-5 and IL-13 as measured in multiplex immunoassays (Fig. 2f ).

BNT162b2-induced effects on proliferation and dynamics of immune cell populations were assessed in injection site draining lymph nodes (dLNs), which are the principal immuneeducated compartments for proficient T-and B-cell priming, and in blood and spleen for evaluation of its systemic effects. Higher numbers of plasma cells, class switched IgG1-and IgG2a-positive B cells, and germinal center B cells were observed in dLNs and spleens of mice 12 days after immunisation with 5 µg BNT162b2 than after immunisation with buffer (Extended Data Fig. 4a, b ). In Day 7 post-immunisation blood, there were significantly fewer circulating B cells than in blood from buffer-immunised mice (Extended Data Fig. 4c ), which may imply that B-cell homing to lymphoid compartments augments B cell counts in dLN and spleen. The dLNs from BNT162b2-immunised mice also have significantly elevated counts of CD8 + and CD4 + T cells, which was most pronounced for T follicular helper (TFH) cells, including ICOS + subsets essential for germinal center formation (Extended Data Fig. 4a ) 24 .

BNT162b2 immunisation increased CD8 + T cell counts in the blood and TFH cell counts in the spleen and blood (Extended Data Fig. 4b, c ). These data indicate that BNT162b2 concurrently elicits strong SARS-CoV-2 pseudovirus neutralising titers and systemic TH1-driven CD4 + and CD8 + T-cell responses.

To assess BNT162b2-mediated protection in non-human primates, groups of six male, 2-4 year old rhesus macaques were immunised IM with 30 or 100 µg of BNT162b2 or saline control on Days 0 and 21. S1-binding IgG was readily detectable by Day 21 after Dose 1, and levels increased further after Dose 2 through Day 28 (Fig. 3a ). Seven days after Dose 2 (Day 28), the GMCs of S1-binding IgG were 30,339 units (U)/mL (30 µg dose level) and 34,668 U/mL preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

(100 µg dose level). For comparison, the S1-binding IgG GMC of a panel of 38 SARS-CoV-2 convalescent human sera was 631 U/mL, substantially lower than the GMCs of the immunised rhesus macaques after one or two doses.

Fifty percent virus neutralisation GMTs, measured by an authentic SARS-CoV-2 neutralisation assay 25 , were detectable in rhesus macaque sera by Day 21 after Dose 1 and peaked at a GMT of 962 (Day 35, 14 days after Dose 2 of 30 µg) or 1,689 (Day 28, 7 days after Dose 2 of 100 µg; Fig. 3b ). Robust GMTs of 285 for 30 µg and 310 for 100 µg dose levels persisted to at least Day 56 (most recent time point tested). For comparison, the neutralisation GMT of the human convalescent serum panel was 94.

S-specific T-cell responses were analysed in BNT162b2-immunised rhesus macaques and saline-immunised controls by ELISpot and intracellular cytokine staining (ICS). Peripheral blood mononuclear cells (PBMCs) were collected before immunisation and at the times indicated after Doses 1 and 2. Strong IFNγ but minimal IL-4 responses were detected by ELISpot after Dose 2 (Fig. 3c, d , Extended Data Fig. 5 ). ICS confirmed that BNT162b2 elicited strong S-specific IFNγ producing T-cell responses, including a high frequency of CD4 + T cells that produced IFNγ, IL-2, and TNF but a low frequency of CD4 + T cells that produced IL-4, indicating a TH1-biased response (Fig. 3e, f ). BNT162b2 also elicited S-specific IFNγ + producing CD8 + T cells (Fig. 3g ).

Six rhesus macaques that had received two immunisations with 100 µg BNT162b2 and three age-matched macaques that had received saline were challenged 55 days after Dose 2 with 1.05 × 10 6 plaque forming units of SARS-CoV-2 (strain USA-WA1/2020), split equally between intranasal and intratracheal routes, as previously described 26 . Three additional nonimmunised, age-matched rhesus macaques (sentinels) were mock-challenged with cell culture medium. Nasal and oropharyngeal (OP) swabs were collected and bronchoalveolar lavage (BAL) was performed at the times indicated, and samples were tested for SARS-CoV-2 RNA (genomic RNA or subgenomic transcripts) by reverse-transcription quantitative polymerase chain reaction (RT-qPCR; Fig. 4 ). All personnel performing clinical, radiological, histopathological, or RT-qPCR evaluations were blinded to the group assignments of the macaques.

Viral RNA was detected in BAL fluid from 2 of the 3 control-immunised macaques on Day 3 after challenge and from 1 of 3 on Day 6 (Fig. 4a ). At no time point sampled was viral RNA detected in BAL fluid from the BNT162b2-immunised and SARS-CoV-2 challenged macaques (Fig. 4a ). The difference in viral RNA detection in BAL fluid between BNT162b2-immunised and control-immunised rhesus macaques after challenge is highly statistically significant (by a nonparametric test, p=0.0014).

From control-immunised macaques, viral RNA was detected in nasal swabs obtained on Days 1, 3, and 6 after SARS-CoV-2 challenge; from BNT162b2-immunised macaques, viral RNA was detected only in nasal swabs obtained on Day 1 after challenge and not in swabs obtained on Day 3 or subsequently (Fig. 4b ). The pattern of viral RNA detection from OP swabs was similar to that for nasal swabs (Fig. 4c ).

In general, virus-challenged animals showed no clinical signs of significant disease. We conclude that the 2-4 year old male rhesus macaque challenge model is primarily a SARS-CoV-2 infection model and not a COVID-19 disease model.

Discussion

We demonstrate that BNT162b2, an LNP-formulated, m1Ψ nucleoside-modified mRNA encoding SARS-CoV-2 S captured in a prefusion conformation is highly immunogenic in mice and rhesus macaques. Expression from DNA of protein with the BNT162b2-encoded amino acid sequence confirmed the P2 S prefusion conformation by cryo-EM. This analysis confirmed that the antigenically important RBD can assume the 'up' conformation, with the receptor binding site, rich in neutralising epitopes, accessible in a proportion of the molecules 27 . The alternative states observed likely reflect a dynamic equilibrium between RBD 'up' and 'down' positions 10,23 . Binding of expressed and purified P2 S to ACE2 and a neutralising monoclonal antibody further demonstrates its conformational and antigenic integrity.

In mice, a single injection of BNT162b2 elicited high neutralizing titers and strong TH1 and TFH type CD4 + and IFNγ + IL-2 + CD8 + T-cell responses. Both BNT162b2 induced CD4 + T-cell types may support antigen-specific antibody generation and maturation, and potentially protection from infectious challenge. Limitation and clearance of virus infection is promoted by the interplay of neutralising antibodies with CD8 + T cells that eliminate intracellular virus reservoirs. CD8 + T cells may also reduce the influx of monocytes into infected lung tissue, which can be associated with undesirable IL-6 and TNF production and impaired antigen presentation 28,29 . The contributions of the immune effector systems to human protection from preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

SARS-CoV-2

is not yet understood. Therefore, it appears prudent to develop COVID-19 vaccines that enlist concomitant cognate B cell, CD4 + T cell, and CD8 + T-cell responses.

The immunogenicity of BNT162b2 in rhesus macaques paralleled its immunogenicity in mice.

Seven days after Dose 2 of 100 µg, the neutralising GMT reached 18-times that of a human SARS-CoV-2 convalescent serum panel and remained 3.3-times higher than this benchmark five weeks after the last immunisation. The strongly TH1-biased CD4 + T-cell response and IFNγ + CD8 + T-cell response to BNT162b2 is a pattern favoured for vaccine safety and efficacy, providing added reassurance for clinical translation 30 . BNT162b2 protected 2-4 year old rhesus macaques from infectious SARS-CoV-2 challenge, with reduced detection of viral RNA in immunised animals compared to those that received saline and with no evidence of clinical exacerbation. Strong RT-qPCR evidence for lower respiratory tract protection was demonstrated by the absence of detectable SARS-CoV-2 RNA in serial BAL samples obtained starting 3 days after challenge of BNT162b2-immunised rhesus macaques.

We recently presented data from immunisation with BNT162b1, a vaccine candidate that has the same LNP-formulated m1Ψ nucleoside-modified RNA platform but expresses a trimerised, secreted RBD (Vogel et al., manuscript in preparation). The pattern, magnitude and durability of humoral and cellular responses to BNT162b1 in mice and macaques were in the range of those elicited by BNT162b2, as was protection of macaques from virus challenge, indicating that these features are largely class-intrinsic for this particular vaccine platform. BNT162b1 elicits high SARS-CoV-2 neutralizing titers and strong TH1-biased CD4 + and IFNγ + and IL-2 + CD8 + T cell responses in humans, consistent with the preclinical findings 15,31,32 .

The selection of BNT162b2 over BNT162b1 for further clinical testing was largely driven by greater tolerability of BNT162b2 with comparable immunogenicity in clinical trials 15

Ethics statement.

All mouse studies were performed at BioNTech SE, and protocols were approved by the local authorities (local welfare committee), conducted according to FELASA recommendations and in compliance with the German Animal Welfare Act and Directive 2010/63/EU. Only animals with an unobjectionable health status were selected for testing procedures.

Protein and peptide reagents.

Purified recombinant SARS-CoV-2 S1 subunit including a histidine tag and the RBD tagged with the Fc region of human IgG1 (both Sino Biological) were used in ELISA to detect SARS-CoV-2 S-specific IgG in mice. Purified recombinant SARS-CoV-2 S1 and RBD with a histidine tag (both Sino Biological) were used for surface plasmon resonance (SPR) spectroscopy. An overlapping 15-mer peptide pool of the S protein was used for ELISpot, cytokine profiling and intracellular cytokine staining. An irrelevant peptide control (SPSYVYHQF, derived from gp70 AH-1 33 ) or a CMV peptide pool was used as control for ELISpot assays. All peptides were obtained from JPT Peptide Technologies.

Human convalescent sera.

Human COVID-19 convalescent sera (n=38) were drawn from donors 18-83 years of age at least 14 days after PCR-confirmed diagnosis and at a time when the participants were asymptomatic. Serum donors had symptomatic infections (35/38), or had had been hospitalised

(1/38). Sera were obtained from Sanguine Biosciences (Sherman Oaks, CA), the MT group (Van Nuys, CA) and Pfizer Occupational Health and Wellness (Pearl River, NY) and used across different studies as reference benchmark.

Cell culture.

Human embryonic kidney (HEK)293T/17 and Vero 76 cells (both ATCC) were cultured in Dulbecco's modified Eagle's medium (DMEM) with GlutaMAX™ (Gibco) supplemented with 10% fetal bovine serum (FBS [Sigma-Aldrich]). Cell lines were tested for mycoplasma contamination after receipt, before expansion and cryopreservation. For studies including NHP samples, Vero 76 and Vero CCL81 (both ATCC) cells were cultured in DMEM (Gibco) containing 2% HyClone fetal bovine and 100 U/mL penicillium/streptomycin (Gibco).

Expi293F™ cells were grown in Expi293™ media and transiently transfected using ExpiFectamine™293 (all from Thermo Fisher Scientific).

In vitro transcription and purification of RNA.

To generate the template for RNA synthesis, a DNA fragment encoding the SARS-CoV-2 P2 S protein (based on GenBank: MN908947), including the amino acid exchanges K986P and V987P, was cloned into a starting plasmid vector with backbone sequence elements for improved RNA stability and translational efficiency 19,34 . Non-coding backbone elements included the regions from the T7 promoter to the 5′ and 3′ UTR plus a poly(A) tail (100 nucleotides) interrupted by a linker (A30LA70, 10 nucleotides). The DNA was purified, spectrophotometrically quantified, and in vitro transcribed by T7 RNA polymerase in the presence of a trinucleotide cap1 analogue ((m2 7,3'-O )Gppp(m 2'-O )ApG; TriLink) and of N 1methylpseudouridine-5'-triphosphate (m1ΨTP; Thermo Fisher Scientific) instead of uridine-5'-triphosphate (UTP) 35 . RNA was purified using magnetic particles 36 , integrity assessed by microfluidic capillary electrophoresis (Agilent Fragment Analyser), and concentration, pH, osmolality, endotoxin level and bioburden determined.

Lipid-nanoparticle formulation of the RNA.

Purified RNA was formulated into LNPs using an ethanolic lipid mixture of ionisable cationic lipid and transferred into an aqueous buffer system via diafiltration to yield an LNP composition similar to one previously described 37 . BNT162b2 was stored at -70 °C at a concentration of 0.5 mg/mL.

P2 S expression and purification.

To express P2 S for structural characterisation, a gene encoding the full length of SARS-CoV-2 (GenBank: MN908947) with two prolines substituted at residues 986 and 987 followed with a C-terminal HRV3C protease site and a TwinStrep tag was cloned into a modified pcDNA3.1(+) vector with the CAG promoter. The TwinStrep-tagged P2 S was expressed in Expi293 cells. Purification of the recombinant protein was based on a procedure described previously, with minor modifications 5 . Upon cell lysis, P2 S was solubilized in 1% NP-40 detergent. The TwinStrep-tagged protein was then captured with StrepTactin Sepharose HP resin in 0.5% NP-40. P2 S was further purified by size-exclusion chromatography and eluted as three distinct peaks in 0.02 % NP-40 as previously reported 5 . Peak 1, which consists of intact P2 S migrating at around 150 kDa, as well as dissociated S1 and S2 subunits, which co-migrate at just above 75 kDa, was used in the structural characterisation. Spontaneous dissociation of the S1 and S2 subunits mostly occurs throughout the course of the protein purification, starting at the point of detergent-mediated protein extraction.

Biolayer interferometry.

The binding of detergent NP-40 solubilized, purified P2 S to human ACE2 peptidase domain (ACE2 PD) and human neutralising monoclonal antibody B38 22 was performed on Octet RED384 (FortéBio) at 25 ⁰C in a running buffer (RB) consisting of 25 mM Tris pH7.5, 150 mM NaCl, 1 mM EDTA and 0.02% NP-40. Avi-tagged ACE2-PD was captured on streptavidin coated sensors and B38 antibody was captured on sensors coated with protein G. After initial baseline equilibration of 120 s, the sensors were dipped in 10 µg/mL solution of Avi-tagged ACE2-PD or B38 mAb for 300 s to achieve capture levels of 1 nM using the threshold function.

The sensors were dipped in RB for 120 s for collecting baseline before they were dipped in a concentration series of purified P2 S samples for 300 s (association phase). The sensors were immersed in RB for measuring 600 s (dissociation phase). Data were reference subtracted and fit to a 1:1 binding model with R 2 value greater than 0.95, to determine kinetics and affinity of binding, using Octet Data Analysis Software v10.0 (FortéBio).

Cryo-electron microscopy sample preparation, data collection and data processing.

For TwinStrep-tagged P2 S, 4 μL purified protein at 0.5 mg/mL were applied to gold Quantifoil R1.2/1.3 300 mesh grids freshly overlaid with graphene oxide. Sample was blotted using a Vitrobot Mark IV for 4 s with a force of -2 before being plunged into liquid ethane cooled by liquid nitrogen. 27,701 micrographs were collected from a two identically prepared grids on a Titan Krios operating at 300 keV equipped with a Gatan K2 Summit direct electron detector in super-resolution mode at a magnification of 165,000x, for a magnified pixel size of 0.435 Å at the specimen level. Data were collected from each grid over a defocus range of -1.2 to -3.4 μm with a total electron dose of 50.32 and 50.12 e -/Å 2 , respectively, fractionated into 40 frames over a 6-second exposure for 1.26 and 1.25 e -/Å 2 /frame. On-the-fly motion correction, CTF estimation, and particle picking and extraction with a box size of 450 pixels were performed in Warp 38 , during which super-resolution data were binned to give a pixel size of 0.87 Å. A total of 1,119,906 particles were extracted. All subsequent processing was performed in RELION 3.1-beta 39 1 .

Immunisation.

Mice. Female BALB/c mice (Janvier; 8-12 weeks) and were randomly allocated to groups.

BNT162b2 was diluted in PBS, 300 mM sucrose or saline (0.9% NaCl) and injected IM into the gastrocnemius muscle at a volume of 20 µL under isoflurane anaesthesia.

Rhesus macaques (Macaca mulatta). Male rhesus macaques (2-4 years) were randomly assigned to receive either BNT162b2 or saline placebo control in 0.5 mL volume administered by IM injection in the left quadriceps muscle on Days 0 and 21.

Tissue preparation.

Mice. Peripheral blood was collected from the retro-orbital venous plexus under isoflurane anaesthesia or vena facialis without prior anesthetisation. Blood was centrifuged for 5 minutes at 16.000 x g, and the serum was immediately used for downstream assays or stored at -20 °C.

Spleen single-cell suspensions were prepared in PBS by mashing tissue against the surface of a 70 µm cell strainer (BD Falcon). Erythrocytes were removed by hypotonic lysis. Popliteal, inguinal and iliac lymph nodes were pooled, cut into pieces, digested with collagenase D (1 mg/mL; Roche) and passed through cell strainers.

Rhesus macaques (Macaca mulatta). Serum was obtained before immunisation and on Days 14, 21, 28, 35, 42, and 56. PBMCs were obtained before immunisation and on Days 7, 28, and 42, except that PBMCs were not obtained from the buffer-immunised group on Day 28. Blood for serum and PBMCs was collected in compliance with animal protocol 2017-8725-023 approved by the NIRC Institutional Animal Care and Use Committee. Animals were anesthetised with ketamine HCl (10 mg/kg; IM) during blood collection and immunisation, and monitored for adequate sedation.

S1-and RBD-binding IgG assay.

For mouse sera, MaxiSorp plates (Thermo Fisher Scientific) were coated with recombinant S1 or RBD (100 ng/100 µL) in sodium carbonate buffer, and bound IgG was detected using an HRP-conjugated secondary antibody and TMB substrate (Biotrend). Data collection was performed using a BioTek Epoch reader and Gen5 software version 3.0.9. For concentration analysis, the signal of the specific samples was correlated to a standard curve of an isotype control. For rhesus macaque and human sera, a recombinant SARS-CoV-2 S1 containing a Cterminal Avitag™ (Acro Biosystems) was bound to streptavidin-coated Luminex microspheres.

Bound rhesus macaque or human anti-S1 antibodies present in the serum were detected with a fluorescently labelled goat anti-human polyclonal secondary antibody (Jackson ImmunoResearch). Data were captured as median fluorescent intensities (MFIs) using a Bioplex200 system (Bio-Rad) and converted to U/mL antibody concentrations using a reference standard consisting of 5 pooled human COVID-19 convalescent serum samples (obtained >14 days PCR diagnosis), diluted in antibody depleted human serum with arbitrary assigned concentrations of 100 U/mL and accounting for the serum dilution factor.

Binding kinetics of antigen-specific IgGs using surface plasmon resonance spectroscopy

Binding kinetics of murine S1-and RBD-specific serum IgGs was determined using a Biacore T200 device (Cytiva) with HBS-EP running buffer (BR100669, Cytiva) at 25 °C. Carboxyl groups on the CM5 sensor chip matrix were activated with a mixture of 1-ethyl-3-(3dimethylaminopropyl) carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) to form active esters for the reaction with amine groups. Anti-mouse-Fc-antibody (Jackson ImmunoResearch) was diluted in 10 mM sodium acetate buffer pH 5 (30 µg/mL) for covalent coupling to immobilisation level of ~10,000 response units (RU). Free N-hydroxysuccinimide esters on the sensor surface were deactivated with ethanolamine.

Mouse serum was diluted 1:50 in HBS-EP buffer and applied at 10 µL/min for 30 seconds to the active flow cell for capture by immobilised antibody, while the reference flow cell was treated with buffer. Binding analysis of captured murine IgG antibodies to S1-His or RBD-His (Sino Biological) was performed using a multi-cycle kinetic method with concentrations ranging from 25 to 400 nM or 1.5625 to 50 nM, respectively. An association period of 180 seconds was followed by a dissociation period of 600 seconds with a constant flow rate of 40 μL/min and a final regeneration step. Binding kinetics were calculated using a global kinetic fit model (1:1 Langmuir, Biacore T200 Evaluation Software Version 3.1, Cytiva).

VSV-SARS-CoV-2 spike variant pseudovirus neutralisation.

A recombinant replication-deficient vesicular stomatitis virus (VSV) vector that encodes GFP instead of VSV-G (VSVΔG-GFP) was pseudotyped with SARS-CoV-2 S protein according to published pseudotyping protocols 42,43 . In brief, HEK293T/17 monolayers transfected to express SARS-CoV-2 S truncated of the C-terminal cytoplasmic 19 amino acids (SARS-CoV-2-S-CΔ19) were inoculated with VSVΔG-GFP vector. After incubation for 1 hour at 37 °C, the inoculum was removed and cells were washed with PBS before medium supplemented with anti-VSV-G antibody (clone 8G5F11, Kerafast Inc.) was added to neutralise residual input virus. VSV/SARS-CoV-2 pseudovirus-containing medium was harvested 20 hours after inoculation, 0.2 µm filtered and stored at -80 °C.

Serial dilutions of mouse serum samples were prepared and pre-incubated for 10 minutes at room temperature with VSV/SARS-CoV-2 pseudovirus suspension (4.8 × 10 3 infectious units [IU]/mL) before transferring the mix to Vero 76 cells. Inoculated Vero-76 cells were incubated for 20 hours at 37 °C. Plates were placed in an IncuCyte Live Cell Analysis system (Sartorius) and incubated for 30 minutes prior to the analysis (IncuCyte 2019B Rev2 software). Whole well scanning for brightfield and GFP fluorescence was performed using a 4× objective. The 50% pseudovirus neutralisation titre (pVNT50) was reported as the reciprocal of the first serum dilution yielding a 50% reduction in GFP-positive infected cell number per well compared to the mean of the no serum pseudovirus positive control. Each serum sample dilution was tested in duplicates.

IFNγ and IL-4 ELISpot.

Murine ELISpot assays were performed with mouse IFNγ ELISpot PLUS kits according to the manufacturer's instructions (Mabtech). A total of 5 × 10 5 splenocytes was ex vivo restimulated with the full-length S peptide mix (0.1 µg/mL final concentration per peptide) or controls (gp70-AH1 [SPSYVYHQF] 33 , 4 µg/mL; Concanavalin A [ConA], 2 µg/mL [Sigma]).

Streptavidin-alkaline phosphatase (ALP) and BCIP/NBT-plus substrate were added, and spots counted using an ELISpot plate reader (ImmunoSpot® S6 Core Analyzer [CTL]). Spot numbers were evaluated using ImmunoCapture Image Aquision Software V7.0 and ImmunoSpot 7.0.17.0 Professional. Spot counts denoted too numerous to count by the software were set to 1,500. For T-cell subtyping, CD8 + T cells and CD4 + T cells were isolated from splenocyte suspensions using MACS MicroBeads (CD8a [Ly-2] and CD4 [L3T4] [Miltenyi Biotec]) according to the manufacturer's instructions. 1 × 10 5 CD8 + or CD4 + T cells were subsequently restimulated with 5 × 10 4 syngeneic bone marrow-derived dendritic cells loaded with fulllength S peptide mix (0.1 µg/mL final concentration per peptide) or cell culture medium as control. Purity of isolated T-cell subsets was determined by flow cytometry.

Rhesus macaque PBMCs were tested with commercially available NHP IFNγ and IL-4 ELISpot assay kits (Mabtech, Sweden). Cryopreserved rhesus macaque PBMCs were thawed in pre-warmed AIM-V media (Thermo Fisher Scientific, US) with Benzonase (EMD Millipore, US).

For IFNγ ELISpot, 1.0 x 10 5 PBMCs and 2.5 x 10 5 PBMCs for IL-4 ELISpot were stimulated ex vivo with 1 μg/mL of the full-length S overlapping peptide mix. Tests were performed in triplicate wells and media-DMSO, a CMV peptide pool and PHA (Sigma) were included as controls. After 24 hours for IFNγ and 48 hours for IL-4, Streptavidin-HRP and AEC substrate (BD Bioscience) were added, and spots counted using a CTL ImmunoSpot S6 Universal Analyzer (CTL, US). Results shown are background (Media-DMSO) subtracted and normalized to SFC/10 6 PBMCs.

Flow cytometry for analysis of cell mediated immunity.

For mouse T-cell analysis in peripheral blood, erythrocytes from 50 µL freshly drawn blood were lysed (ACK lysing buffer [Gibco]), and cells were stained with Fixable Viability Dye (eBioscience) and primary antibodies in the presence of Fc block in flow buffer (DPBS [Gibco] supplemented with 2% FCS, 2 mM EDTA [both Sigma] and 0.01% sodium azide [Morphisto]).

After staining with secondary biotin-coupled antibodies in flow buffer, cells were stained extracellularly against surface markers with directly labelled antibodies and streptavidin in For mouse T-cell analysis in lymphoid tissues, 1.5 × 10 6 lymph node and 4 × 10 6 spleen cells were stained for viability and extracellular antigens with directly labelled antibodies. Fixation, permeabilisation and intracellular staining was performed as described for blood T-cell staining.

For mouse B-cell subtyping in lymphoid tissues, 2.5 × 10 5 lymph node and 1 × 10 6 spleen cells were treated with Fc block, stained for viability and extracellular antigens as described for blood T-cell staining and fixed with 2% RotiHistofix overnight.

For mouse intracellular cytokine staining in T cells, 4 x 10 6 spleen cells were ex vivo restimulated with 0.5 µg/mL final concentration per peptide of full-length S peptide mix or cell culture medium (no peptide) as control in the presence of GolgiStop and GolgiPlug (both BD Bioscience) for 5 hours. Cells were stained for viability and extracellular antigens as described for lymphoid T-cell staining. Cells were fixed with 2% RotiHistofix and permeabilised overnight. Intracellular staining was performed as described for blood T-cell staining.

Mouse cells were acquired on a BD Symphony A3 or BD Celesta (B-cell subtyping) flow cytometer (BD Bioscience) using BD FACSDiva software version 9.1 or 8.0.1.1, respectively, and analysed with FlowJo 10.6 (FlowJo LLC, BD Biosciences).

For rhesus macaques intracellular cytokine staining in T cells, 1.5 x 10 6 PBMCs were stimulated with the full-length S peptide mix at 1 μg/mL, Staphyloccocus enterotoxin B (SEB; 2 μg/mL) as positive control, or 0.2% DMSO as negative control. GolgiStop and GolgiPlug (both BD Bioscience) were added. Following 37 °C incubation for 12 to 16 h, cells were stained for viability and extracellular antigens after blocking Fc binding sites with directly labelled antibodies. Cells were then fixed and permeabilized with BDCytoFix/CytoPerm solution (BD Bioscience), intracellular staining was performed in perm buffer for 30 min at RT. Cells were washed, resuspended in 2% FBS/PBS buffer and acquired on a LSR Fortessa. Data analyzed by FlowJo (10.4.1). Results shown are background (Media-DMSO) subtracted.

Cytokine profiling.

Mouse splenocytes were re-stimulated for 48 hours with full-length S peptide mix (0.1 µg/mL final concentration per peptide) or cell culture medium (no peptide) as control. Concentrations of IFNγ, IL-2, IL-4, IL-5 and IL-13 in supernatants were determined using a bead-based, 11plex TH1/TH2 mouse ProcartaPlex multiplex immunoassay (Thermo Fisher Scientific) according to the manufacturer's instructions. Fluorescence was measured with a Bioplex200 system (Bio-Rad) and analysed with ProcartaPlex Analyst 1.0 software (Thermo Fisher Scientific). Values below the lower limit of quantification (LLOQ) were set to zero.

SARS-CoV-2 neutralisation.

The SARS-CoV-2 neutralisation assay used a previously described strain of SARS-CoV-2 (USA_WA1/2020) that had been rescued by reverse genetics and engineered by the insertion of an mNeonGreen (mNG) gene into open reading frame 7 of the viral genome 25 . This reporter virus generates similar plaque morphologies and indistinguishable growth curves from wildtype virus. Viral master stocks were grown in Vero 76 cells as previously described 44

SARS-CoV-2 challenge of rhesus macaques (Macaca mulatta).

The SARS-CoV-2 inoculum was obtained from a stock of 2.1 × 10 6 PFU/mL previously prepared at Texas Biomedical Research Institute (San Antonio, TX), aliquoted into single use vials, and stored at -70 °C. The working virus stock was generated from two passages of the SARS-CoV-2 USA-WA1/2020 isolate (a 4 th passage seed stock purchased from BEI Resources; NR-52281) in Vero 76 cells. The virus was confirmed to be SARS-CoV-2 by deep sequencing and identical to the published sequence (GenBank accession number MN985325.1).

BNT162b2-immunised (n=6) and age-matched saline control-immunised (n=3) male rhesus macaques (control) were challenged with 1.05 × 10 6 plaque forming units of SARS-CoV-2 USA-WA1/2020 isolate, split equally between the intranasal (IN; 0.25 mL) and intratracheal (IT; 0.25 mL) routes as previously described 26 . The challenge was performed 55 days after the second immunisation. A separate sentinel group of non-immunised age-and sex-matched animals (n=3) was mock challenged with DMEM supplemented with 10% FCS IN (0.25 mL) and IT (0.25 mL). Approximately two weeks prior to challenge, animals were moved to the ABSL-3 facility at Southwest National Primate Research Center (SNPRC; San Antonio, TX). Animals were monitored regularly by a board-certified veterinary clinician for rectal body temperature, weight and physical examination. Specimen collection was performed under tiletamine zolazepam (Telazol) anaesthesia as described 26 . Nasal and oropharyngeal swabs were collected from all macaques at Day 0, 1, 3, and 6 (relative to the day of challenge), from BNT162b2-immunised macaques on Day 7 or 8, and from control and sentinel macaques on Day 10. Bronchoalveolar lavage (BAL) was performed on all macaques the week before challenge and on Days 3 and 6 post-challenge and on BNT162b2-immunised macaques on Day 7 or 8. BAL was performed by instilling four times 20 mL of saline. These washings were pooled, aliquoted and stored frozen at -70 °C. Necropsy was performed on BNT162b2immunised animals on Day 7 or 8. Control and sentinel animals were not necropsied to allow re-challenge (control) or challenge (sentinel) on a subsequent day.

Reverse-transcription quantitative polymerase chain reaction.

To detect and quantify SARS-CoV-2 in NHP, viral RNA was extracted from nasal swabs, OP swabs, and BAL specimens as previously described [45][46][47] and tested by RT-qPCR as previously described 26 . Briefly, 10 µg yeast tRNA and 1 × 10 3 PFU of MS2 phage (Escherichia coli bacteriophage MS2, ATCC) were added to each thawed sample, and RNA extraction performed using the NucleoMag Pathogen kit (Macherey-Nagel). The SARS-CoV-2 RT-qPCR was performed on extracted RNA using a CDC-developed 2019-nCoV_N1 assay on a QuantStudio 3 instrument (Applied Biosystems). The cut-off for positivity (limit of detection, LOD) was established at 10 gene equivalents (GE) per reaction (800 GE/mL). Samples were tested in duplicate. On day 6, one BAL specimen from the control group and one day 1 nasal swab from the BNT162b1-immunised group had, on repeated measurements, viral RNA levels on either side of the LLOD. These specimens were categorised as indeterminate and excluded from the graphs and the analysis.

Statistics and reproducibility.

No statistical methods were used to predetermine group and samples sizes (n). All experiments were performed once. P-values reported for RT-qPCR analysis were determined by nonparametric analysis (Friedman's test) based on the ranking of viral RNA shedding data within each day. PROC RANK and PROC GLM from SAS® 9.4 were used to calculate the pvalues. Samples from post challenge days (Days 3, 6 and end of protocol [EOP] for BAL; Days 1, 3, 6 and 10 [control and sentinel] or EOP [BNT162b2-immunised] for nasal and oropharyngeal swabs) were included in the analysis. Indeterminate results were excluded from this analysis. All remaining analyses were carried out using GraphPad Prism 8.4.

Data availability.

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Extended Data Table 1 . Electron cryomicroscopy data collection, 3D reconstruction and refinement statistics.

Competing interests

The authors declare: U.S. and Ö.T. are management board members and employees at no other relationships or activities that could appear to have influenced the submitted work.

preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Additional Information

Supplementary Information is available for this study.

Correspondence and requests for materials should be addressed to Ugur Sahin.

Acknowledgements

AcknowledgementsWe thank T. Garretson for advice on NHP serology studies, R. F. Sommese and K. F. Fennell for technical assistance for molecular cloning and cell based binding.Valuable support and assistance from R. de la Caridad Güimil Garcia1, A. Su, M. Dvorak, M. Drude, F. Zehner, T. Lapin, B. Ludloff, S. Hinz, F. Bayer, J. Scholz, A.L. Ernst, T. Sticker and S. Wittig resulted in a rapid availability of oligonucleotides and DNA templates.E. Boehm, K. Goebel, R. Frieling, C. Berger, S. Koch, T. Wachtel, J. Leilich, M. Mechler, R. Wysocki, M. Le Gall, A. Czech and S. Klenk carried out RNA production and analysis.Without their commitment during this pandemic situation, this vaccine candidate could not have been transferred to non-clinical studies in light speed.B. Weber, J. Vogt, S. Krapp, K. Zwadlo, J. Mottl, J. Mühl and P. Windecker supported the mouse studies and serological analysis with excellent technical assistance.We thank A. Ota-Setlik for ELISpot testing of NHP samples.Radiologists A. K. Voges, M. R. Gutman and E. Clemmons advised on the radiographic scoring.We thank Polymun Scientific for excellent formulation services as well as Acuitas Therapeutics for fruitful discussions.

Funding

FundingBioNTech is the Sponsor of the study, and Pfizer it its agent.BioNTech and Pfizer are responsible for the design, data collection, data analysis, data interpretation, and writing of the report.The corresponding authors had full access to all the data in the study and had final responsibility for the decision to submit the data for publication.This study was not supported by any external funding at the time of submission.

Failed to load PDF:

Paper sources

Abstract screening pilot

Abstract screening results

Extraction pilot

Extraction results

Research report

Modify setup

StripeM-Inner