key: cord-0908498-6reg4jji
authors: Tscherne, Alina; Schwarz, Jan Hendrik; Rohde, Cornelius; Kupke, Alexandra; Kalodimou, Georgia; Limpinsel, Leonard; Okba, Nisreen M.A.; Bošnjak, Berislav; Sandrock, Inga; Halwe, Sandro; Sauerhering, Lucie; Brosinski, Katrin; Liangliang, Nan; Duell, Elke; Jany, Sylvia; Freudenstein, Astrid; Schmidt, Jörg; Werner, Anke; Sera, Michelle Gellhorn; Klüver, Michael; Guggemos, Wolfgang; Seilmaier, Michael; Wendtner, Clemens-Martin; Förster, Reinhold; Haagmans, Bart L.; Becker, Stephan; Sutter, Gerd; Volz, Asisa
title: Immunogenicity and efficacy of the COVID-19 candidate vector vaccine MVA SARS 2 S in preclinical vaccination
date: 2021-01-11
journal: bioRxiv
DOI: 10.1101/2021.01.09.426032
sha: f3fa4fc56341ac5e138dfa1d6032cbc5a6df7dbf
doc_id: 908498
cord_uid: 6reg4jji

The severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) has emerged as the infectious agent causing the pandemic coronavirus disease 2019 (COVID-19) with dramatic consequences for global human health and economics. Previously, we reached clinical evaluation with our vector vaccine based on vaccinia virus MVA against the Middle East respiratory syndrome coronavirus (MERS-CoV), which causes an infection in humans similar to SARS and COVID-19. Here, we describe the construction and preclinical characterization of a recombinant MVA expressing full-length SARS-CoV-2 spike (S) protein (MVA-SARS-2-S). Genetic stability and growth characteristics of MVA-SARS-2-S, plus its robust synthesis of S antigen, make it a suitable candidate vaccine for industrial scale production. Vaccinated mice produced S antigen-specific CD8+ T cells and serum antibodies binding to S glycoprotein that neutralized SARS-CoV-2. Prime-boost vaccination with MVA-SARS-2-S protected mice sensitized with a human ACE2-expressing adenovirus from SARS-CoV-2 infection. MVA-SARS-2-S is currently being investigated in a phase I clinical trial as aspirant for developing a safe and efficacious vaccine against COVID-19. Significance Statement The highly attenuated vaccinia virus MVA is licensed as smallpox vaccine, and as vector it is a component of the approved Adenovirus-MVA-based prime-boost vaccine against Ebola virus disease. Here we provide results from testing the COVID-19 candidate vaccine MVA-SARS-2-S, a poxvirus-based vector vaccine that proceeded to clinical evaluation. When administered by intramuscular inoculation, MVA-SARS-2-S expresses and safely delivers the full-length SARS-CoV-2 spike (S) protein, inducing balanced SARS-CoV-2-specific cellular and humoral immunity, and protective efficacy in vaccinated mice. Substantial clinical experience has already been gained with MVA vectors using homologous and heterologous prime-boost applications, including the immunization of children and immunocompromised individuals. Thus, MVA-SARS-2-S represents an important resource for developing further optimized COVID-19 vaccines.

immunogenicity profiles, and by now first data from large phase 3 clinical trials show promising 97 levels of protective efficacy (4, 11-13). This is good news because efficacious vaccines will provide 98 a strategy to change SARS-CoV-2 transmission dynamics. In addition, multiple vaccine types will 99 be advantageous to meet specific demands across different target populations. This includes the 

The recombinant viruses replicated efficiently in the chicken embryo fibroblast cell line DF-1, but 138 not in the human cell lines HeLa, A549 or HaCat (Fig. 1C ).

Characterization of SARS-CoV-2 S expressed by recombinant MVA. To determine the 140 expression pattern of the recombinant SARS-CoV-2 S protein, we stained MVA-SARS-2-S infected To examine the MVA-produced recombinant S glycoprotein in more detail, we prepared total 150 lysates from MVA-SARS-2-S infected CEF or Vero cells for separation by SDS-PAGE and 151 subsequent immunoblot analysis (Fig. 2) . The mouse monoclonal antibody directed against the 152 HAtag at the C-terminus of the recombinant SARS-2-S protein revealed two prominent protein 153 bands that migrated with molecular masses of approximately 190 kDa and 90-100 kDa (Fig 2B) .

As in the SDS-PAGE the detected protein bands migrated at molecular masses significantly higher 155 than the 145 kDa predicted for full-length SARS-CoV-2 S protein based on its amino acid sequence, 

Western blot analysis using serum antibodies from a COVID-19 patient hospitalized with 170 pneumonia also revealed protein bands corresponding to the molecular masses of full-length S 171 protein or the S2 polypeptide (Fig. 2D ). intramuscular (i.m.) administration and prime-boost immunization schedules with a 3-week interval 176 (Fig. 3, SI Appendix, Fig. S2 ). At day 18 after the prime inoculation, we detected serum IgG 177 antibodies binding to whole recombinant SARS-CoV-2 S protein in the sera from 3/8 LD-vaccinated 178 and 4/6 HD-vaccinated animals by ELISA (Fig. 3A) . Following the booster immunization on day 21, 179 all vaccinated animals mounted high levels of S-binding serum IgG antibodies with mean titers of 180 1:900 for the LD vaccination group and 1:1257 for the HD group (Fig. 3A ). More importantly, sera 181 from vaccinated mice also contained antibodies binding to the S protein receptor-binding domain 182 (RBD). Already at day 18 post priming, the RBD-binding antibodies were detected in 33% of the 183 mice in the LD dose group (2/6 mice, mean OD value 0.35) and 50% of the mice receiving the HD 184 immunization (3/6, mean OD 0.63). The boost vaccinations increased the levels of RBD specific 185 antibodies with 87.5% seropositive mice in the 10 7 dose group (7/8, mean OD 1.81) and 100% of 186 the animals vaccinated with 10 8 PFU MVA-SARS2-S (8/8, mean OD 2.92) ( Figure 3B ). Since live 187 virus neutralization is the gold standard for coronavirus serologic analysis, we next assessed the 188 mouse sera in two different assays for SARS-CoV-2 neutralization, a plaque reduction 189 neutralization test 50 (PRNT50) (24) and a complete virus neutralization test (VNT100) (9) ( 

In addition, using SARS-2-S derived peptides with predicted capacity for MHC II binding we also 238 monitored the presence of activated CD4+ T cells. Using three different peptide pools (SI Appendix, were generated, all peptides with an IC50 score of 500nM or less were selected for inclusion in the top 5% list. All the peptides in this list were further analyzed using the MHC-I Processing Prediction 519 tool 'Proteasomal cleavage/TAP transport/MHC class I combined predictor'. All peptides with an 520 IC50 score of 500nM or less and a high total score were chosen and subsequently included in the 521 top peptides list. To confirm that these peptides were potential binders of MHC class I alleles H2-522 K d , H2-D d and H2-L d , they were further screened for MHC I binding using the RankPep server(44).

Peptides that were found to bind to any of the above alleles were selected for synthesis and testing. About two weeks after the last immunization the mice were sensitized with an adenovirus expressing hACE2 and mCherry and infected with SARS-CoV-2 five days after transduction. Four days post challenge the animals were sacrificed and samples were taken for further analysis. (A) Lung tissues were harvested to determine SARS-CoV-2 RNA loads by viral genome copies, (B) the expression of mCherry by mRNA copies, or (C) the amounts of infectious SARS-CoV-2 by TCID 50 /ml. (D) Sera were tested for SARS-CoV-2 neutralizing antibodies by virus neutralization (VNT100). Statistical evaluation was performed with GraphPad Prism for Windows. Statistical significance of differences between groups is indicated as follows: *, p < 0.05; ***, p < 0.001. 

Coronaviridae Study Group of the International Committee on Taxonomy of

The species Severe acute respiratory syndrome-related coronavirus: 600 classifying 2019-nCoV and naming it SARS-CoV-2

SARS-CoV-2 immunity: review 603 and applications to phase 3 vaccine candidates

What defines an efficacious COVID-19 vaccine? A review 605 of the challenges assessing the clinical efficacy of vaccines against SARS-CoV-606 2

Safety and immunogenicity of

COV002): a single-blind, randomised, controlled, phase 2/3 trial

Antigen-Specific Adaptive Immunity to SARS

CoV-2 in Acute COVID-19 and Associations with Age and Disease Severity

Cryo-EM structure of the 2019-nCoV spike in the prefusion 615 conformation

SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2

Potently neutralizing human antibodies that block SARS-CoV-2 620 12

Phase I/II study of COVID-19 RNA vaccine BNT162b1 in 629 adults

Safety and Immunogenicity of Two RNA-Based Covid-19

Nonreplicating vaccinia vector efficiently expresses 633 recombinant genes

Chapter Five -Modified Vaccinia Virus Ankara: History, Value 635 in Basic Research, and Current Perspectives for Vaccine Development

Development of a multi-antigenic SARS-CoV-2 vaccine 639 candidate using a synthetic poxvirus platform

Middle East respiratory syndrome coronavirus spike protein 641 delivered by modified vaccinia virus Ankara efficiently induces virus-neutralizing 642 antibodies

Protective Efficacy of Recombinant Modified Vaccinia Virus Ankara 644

Delivering Middle East Respiratory Syndrome Coronavirus Spike Glycoprotein

An orthopoxvirus-based vaccine reduces virus excretion 647 after MERS-CoV infection in dromedary camels

Severe Acute Respiratory Syndrome Coronavirus 660 2−Specific Antibody Responses in Coronavirus Disease Patients. Emerging 661

Low serum neutralizing anti-SARS-CoV-2 S antibody levels in 663 mildly affected COVID-19 convalescent patients revealed by two different 664 detection methods

Poxvirus CD8+ T-Cell Determinants and Cross-Reactivity 666 in BALB/c Mice

Generation of a Broadly Useful Model for COVID-19 Pathogenesis

Sensitization of Non-permissive Laboratory Mice to SARS

CoV-2 with a Replication-Deficient Adenovirus Expressing Human ACE2

Proteolytic Cleavage of the SARS

CoV-2 Spike Protein and the Role of the Novel S1/S2 Site

A comparison of four serological assays for detecting anti

SARS-CoV-2 antibodies in human serum samples from different populations

Syncytia formation by SARS-CoV-2-infected cells

SARS-CoV-2 vaccines in development

The structure of an antigenic determinant in a protein

Safety and immunogenicity of a modified-vaccinia-virus

Ankara-based influenza A H5N1 vaccine: a randomised, double-blind phase 1/2a 698 clinical trial

Detection of 2019 novel coronavirus (2019-nCoV) by real-700 time RT-PCR

A Soluble Version of Nipah Virus Glycoprotein G Delivered 702 by Vaccinia Virus MVA Activates Specific CD8 and CD4 T Cells in Mice

The Immune Epitope Database and Analysis Resource in Epitope 705 Discovery and Synthetic Vaccine Design

IEDB-AR: immune epitope database-analysis resource in 707 2019

Enhancement to the 709 RANKPEP resource for the prediction of peptide binding to MHC molecules using 710 profiles

) at a MOI of 10 and collected after the indicated time points. Deglycosylation with PNGase F (MVA-Sd) was performed with the sample collected after 24 hpi. Polypeptides in cell lysates were separated by SDS-PAGE and analyzed with a monoclonal antibody against the HA-tag (1:8000) (a, b) or with human serum