Development of a Multi-Antigenic SARS-CoV-2 Vaccine Using a Synthetic Poxvirus Platform

Modified Vaccinia Ankara (MVA) is a highly attenuated poxvirus vector that is widely used to develop vaccines for infectious diseases and cancer. We developed a novel vaccine platform based on a unique three-plasmid system to efficiently generate recombinant MVA vectors from chemically synthesized DNA. In response to the ongoing global pandemic caused by SARS coronavirus-2 (SARS-CoV-2), we used this novel vaccine platform to rapidly produce fully synthetic MVA (sMVA) vectors co-expressing SARS-CoV-2 spike and nucleocapsid antigens, two immunodominant antigens implicated in protective immunity. Mice immunized with these sMVA vectors developed robust SARS-CoV-2 antigen-specific humoral and cellular immune responses, including potent neutralizing antibodies. These results demonstrate the potential of a novel vaccine platform based on synthetic DNA to efficiently generate recombinant MVA vectors and to rapidly develop a multi-antigenic poxvirus-based SARS-CoV-2 vaccine candidate.


Introduction
Modi ed Vaccinia Ankara (MVA) is a highly attenuated poxvirus vector that is widely used to develop vaccine approaches for infectious diseases and cancer [1][2][3] . As a result of the attenuation process through 570 virus passages on chicken embryo broblast (CEF), MVA has acquired multiple major and minor genome alterations 4,5 , leading to severely restricted host cell tropism 6 . MVA can e ciently propagate on CEF and a baby hamster kidney (BHK) cell line, while in most mammalian cells, including human cells, MVA replication is limited due to a late block in virus assembly 3,6 . Its excellent safety and immunogenicity pro le in addition to its versatile expression system and large capacity to incorporate heterologous DNA make MVA an ideal vector for recombinant vaccine development 1,7 . We developed various MVA vaccine candidates for animal models of cytomegalovirus-associated disease in pregnant women while demonstrating vaccine e cacy in several clinical trials in solid tumor and stem cell transplant patients [8][9][10][11][12][13] .
Since the outbreak of the novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in December 2019 14,15 , the virus has spread to more than 200 countries worldwide, causing a pandemic of global magnitude with over 400,000 deaths. Many vaccine candidates are currently under rapid development to control this global pandemic [16][17][18] , some of which have entered into clinical trials with unprecedented pace 17,19 . Most of these approaches employ antigenic forms of the Spike (S) protein as it is considered the primary target of protective immunity 16,20−22 . The S protein mediates SARS-CoV-2 entry into a host cell through binding to angiotensin-converting enzyme 2 (ACE) and is the major target of neutralizing antibodies (NAb) [23][24][25] . Studies in rhesus macaques show that vaccine strategies based on the S antigen can prevent SARS-CoV-2 infection in this relevant animal model 18 , indicating that the S antigen may be su cient as a vaccine immunogen to elicit SARS-CoV-2 protective immunity. However, a recent study showed that even patients without measurable NAb can recover from SARS-CoV-2 infection, suggesting that protection against SARS-CoV-2 infection is mediated by both humoral and cellular immunity to multiple immunodominant antigens, including S and nucleocapsid (N) antigens 20,26 .
We developed a novel vaccine platform based on a uniquely designed three-plasmid system to e ciently generate recombinant MVA vectors from chemically synthesized DNA. In response to the ongoing global pandemic caused by SARS-CoV-2, we used this novel vaccine platform to rapidly produce synthetic MVA (sMVA) vectors co-expressing full-length S and N antigens. We demonstrate that these sMVA vectors stimulate robust SARS-CoV-2 antigen-speci c humoral and cellular immunity in mice, including potent NAb. These results emphasize the value of a novel vaccine platform based on synthetic DNA to e ciently produce recombinant poxvirus vectors and warrant further pre-clinical and clinical testing of a multiantigenic sMVA vaccine candidate to control the ongoing SARS-CoV-2 pandemic and its devastating consequences.

Construction of sMVA
To develop the three-plasmid system of the sMVA vaccine platform, we designed three unique synthetic sub-genomic MVA fragments (sMVA F1-F3) based on the MVA genome sequence published by Antoine et al. 4 , which is ~ 178 kbp in length and contains ~ 9.6 kbp inverted terminal repeats (ITRs) (Fig. 1A). The three fragments were designed as follows: sMVA F1 comprises ~ 60 kbp of the left part of the MVA genome, including the left ITR sequences; sMVA F2 contains ~ 60 kbp of the central part of the MVA genome; and sMVA F3 contains ~ 60 kbp of the right part of the MVA genome, including the right ITR sequences (Fig. 1B). sMVA F1 and F2 as well as sMVA F2 and F3 were designed to share ~ 3 kb overlapping homologous sequences to promote recombination of the three sMVA fragments (Fig. 1B). In addition, a duplex copy of the 165-nucleotide long MVA terminal hairpin loop (HL) anked by concatemeric resolution (CR) sequences was added to both ends of each of the three sMVA fragments (Fig. 1C). Such CR/HL/CR sequence arrangements are formed at the genomic junctions in poxvirus DNA replication intermediates and are essential for genome resolution and packaging [27][28][29][30][31] . When circular DNA plasmids containing these CR/HL/CR sequences are transfected into helper virus-infected cells they spontaneously resolve into linear minichromosomes with intact terminal HL sequences 28,29,32 . Based on these ndings, we hypothesized that the three sMVA fragments as shown in Fig. 1B-C, when cotransfected as circular DNA plasmids into helper virus-infected cells, resolve into linear minichromosomes, recombine with each other via the homologous sequences, and are ultimately packaged as full-length genomes into sMVA virus particles. All three sMVA fragments were cloned in E. coli as bacterial arti cial chromosome (BAC) clones.
Using a previously employed procedure to rescue MVA from a BAC 8,9,33 , sMVA virus was reconstituted with Fowl pox (FPV) as a helper virus upon co-transfection of the three DNA plasmids into BHK cells ( Fig. 1D), which are non-permissive for FPV 34 . Two different FPV strains (HP1.441 and TROVAC) 35,36 were used to promote sMVA virus reconstitution ( Fig. 2A). Ultra-puri ed sMVA virus was produced following virus propagation in CEF, which are commonly used for MVA vaccine production. The virus titers achieved with reconstituted sMVA virus were similar to virus titers achieved with "wild-type" MVA (wtMVA) (Table S1).

In vitro characterization of sMVA
To characterize the viral DNA of sMVA, DNA extracts from sMVA and wtMVA-infected CEF were compared for several MVA genome positions by PCR. Similar PCR results were obtained with sMVA and wtMVA for all evaluated genome positions (Fig. 1E), including the F1/F2 and F2/F3 recombination sites, indicating e cient recombination of the three sMVA fragments. Additional PCR analysis indicated the absence of any BAC vector sequences in sMVA viral DNA (Fig. 1E), suggesting spontaneous and e cient removal of bacterial vector elements upon sMVA virus reconstitution. Comparison of viral DNA from ultra-puri ed sMVA and wtMVA virus by restriction enzyme digestion revealed similar genome pattern between sMVA and wtMVA (Fig. 1F). Sequencing analysis of the sMVA viral DNA con rmed the MVA genome sequence at several positions, including the F1/F2 and F2/F3 recombination sites. Furthermore, whole genome sequencing analysis of one of the sMVA virus isolates reconstituted with FPV TROVAC con rmed the assembly of the reference MVA genome sequence and absence of vector-speci c sequences in viral DNA originating from reconstituted sMVA virus.
To characterize the replication properties of sMVA, growth kinetics of sMVA and wtMVA were compared on BHK and CEF cells, two cell types known to support productive MVA replication 6 . This analysis revealed similar growth kinetics of sMVA and wtMVA on both BHK and CEF cells (Fig. 2B). In addition, similar areas of viral foci were determined in BHK and CEF cell monolayers infected with sMVA or wtMVA ( Fig. 2C), suggesting similar capacity of sMVA and wtMVA to spread in MVA permissive cells. Compared to the productive replication of sMVA and wtMVA in BHK and CEF cells 6 , only limited virus production was observed with sMVA or wtMVA following infection of various human cell lines (Fig. 2D). These results are consistent with the severely restricted replication properties of MVA and show that the sMVA virus can e ciently propagate in BHK and CEF cells, while it is unable to propagate in human cells.
In vivo immunogenicity of sMVA To characterize sMVA in vivo, the immunogenicity of sMVA and wtMVA was compared in C57BL/6 mice following two immunizations at high or low dose. MVA-speci c binding antibodies stimulated by sMVA and wtMVA after the rst and second immunization were comparable (Figs. 3A, S1A). While the antibody levels in the high dose vaccine groups exceeded those of the low dose vaccine groups after the rst immunization, similar antibody levels in the high and low dose vaccine groups were observed after the second immunization. In addition, no signi cant differences were detected in the levels of MVA-speci c NAb responses induced by sMVA and wtMVA after the second immunization (Figs. 3B, S1B). MVAspeci c T cell responses determined after the booster immunization by ex vivo antigen stimulation using immunodominant peptides 37 revealed similar MVA-speci c T cell levels in mice receiving sMVA or wtMVA (Figs. 3C-D and S1C-D). These results indicate that the sMVA virus has a similar capacity to wtMVA in inducing MVA-speci c humoral and cellular immunity in mice.

Construction of sMVA SARS-CoV-2 vaccine vectors
Using highly e cient BAC recombination techniques in E. coli, full-length SARS-CoV-2 S and N antigen sequences were inserted into commonly used MVA insertions sites located at different positions within the three sMVA fragments. Combinations of modi ed and unmodi ed sMVA fragments were subsequently co-transfected into FPV-infected BHK cells to reconstitute sMVA SARS-CoV-2 (sMVA-CoV2) vectors expressing the S and N antigen sequences alone or combined ( Figure 4A and 4B). In the single recombinant vectors encoding S or N alone, termed sMVA-S and sMVA-N, the antigen sequences were inserted into the Deletion (Del3) site ( Figures 1B and 4B) 5 . In the double recombinant vectors encoding both S and N, termed sMVA-N/S and sMVA-S/N, the antigen sequences were inserted into Del3 and the Deletion 2 (Del2) site (sMVA-N/S), or they were inserted into Del3 and the intergenic region between 069R and 070L (IGR69/70) (sMVA-S/N) ( Figures 1B and 4B) 5,38 . All antigen sequences were inserted into sMVA together with mH5 promoter to promote antigen expression during early and late phase of MVA replication 39,40 . sMVA-CoV-2 vaccine vectors were reconstituted with FPV HP1.441 or TROVAC. Ultrapuri ed virus of the sMVA-CoV2 vaccine vectors produced using CEF reached titers that were comparable to those achieved with sMVA or wtMVA (Table S1).

In vitro characterization of sMVA-CoV2 vaccine vectors
To characterize S and N antigen expression by the sMVA-CoV2 vectors, BHK cells infected with the sMVA-CoV2 vectors were evaluated by Immunoblot using S and N-speci c antibodies. This analysis con rmed the expression of the S or N antigen alone by the single recombinant vaccine vectors sMVA-S and sMVA-N, while the expression of both the S and the N antigen was con rmed for the double recombinant vectors sMVA-N/S and sMVA-S/N ( Figure 4C).
Further characterization of the antigen expression by the sMVA-CoV2 vectors in HeLa cells using cell surface and intracellular ow cytometry (FC) staining con rmed single and dual S and N antigen expression by the single and double recombinant vaccine vectors. Staining with S-speci c antibodies revealed abundant cell surface and intracellular antigen expression by all vectors encoding the S antigen (sMVA-S, sMVA-N/S, sMVA-S/N) ( Figure 4D). In contrast, staining with anti-N antibody revealed predominantly intracellular antigen expression by all vectors encoding the N antigen (sMVA-N, sMVA-N/S, sMVA-S/N) ( Figure 4D), although cell surface staining was observed to a minor extent. S and N antigen expression by the sMVA-CoV2 vectors was also investigated by immuno uorescence. This analysis con rmed co-expression of the S and N antigens by the double recombinant vaccine vectors and indicated e cient cell surface and intracellular expression of the S antigen, whereas the expression of the N antigen was predominantly observed intracellular ( Figure S2A-C). These results demonstrate e cient antigen expression by the single and double recombinant sMVA-CoV2 vectors.

In vivo immunogenicity of sMVA-CoV2 vectors
To determine the immunogenicity of the sMVA-vectored S and N antigens alone or combined, SARS-CoV-2-speci c humoral and cellular immune responses were evaluated in Balb/c mice by two immunizations with the single or double recombinant vaccine vectors. High-titer antigen-speci c binding antibodies were detected in all vaccine groups after the rst immunization, and an increase in these responses was observed after the booster immunization ( Figure 5A-B and S3A-B). While the single recombinant vectors induced binding antibodies only against the S or N antigen, the double recombinant vectors induced binding antibodies against both the S and N antigens. In addition, all sMVA-CoV2 vectors encoding the S antigen (sMVA-S, sMVA-S/N, sMVA-N/S) stimulated high-titer binding antibodies against the S receptor binding domain (RBD), which is considered the primary target of NAb 22,24 . Antigen-speci c binding antibody titers between the single and double recombinant vaccine groups were comparable. Notably, SARS-CoV-2 antigen-speci c binding antibody responses stimulated by the sMVA-CoV2 vaccine vectors in mice exceeded SARS-CoV-2 S-, RBD-, and N-speci c binding antibody responses measured in human convalescent immune sera ( Figures 5A-B, and Figure S4). Similar binding antibody responses to those induced by sMVA-CoV2 vectors in Balb/c mice were elicited by the vaccine vectors in C57BL/6 mice ( Figure S5). Analysis of the IgG2a/IgG1 isotype ratio of the binding antibodies revealed Th-1-biased immune responses skewed toward IgG2a independently of the investigated vaccine group or antigen ( Figure 5C and S3C).
Potent SARS-CoV-2-speci c NAb responses as assayed using pseudovirus were detected after the rst immunization in all vaccine groups receiving the vectors encoding the S antigen (sMVA-S, sMVA-S/N, sMVA-N/S), and these NAb responses increased after the booster immunization ( Figure 5D-E and S3D-E). Similar potent NAb responses as measured using pseudovirus were also observed in the vaccine groups using infectious SARS-CoV-2 virus ( Figure 5F-G and S3F-G). We also evaluated the immune sera for potential antibody-dependent enhancement of infection (ADE) using THP-1 monocytes. These cells do not express the ACE2 receptor, but express Fcg receptor II, which is considered the predominant mediator of ADE in SARS-CoV infection 41  Stimulation of SARS-CoV-2-speci c immune responses by both the S and N antigen was also evaluated in mice by co-immunization using the single recombinant vectors sMVA-S and sMVA-N at different doses. This study revealed similar SARS-CoV-2 antigen-speci c humoral and cellular immune responses in vaccine groups receiving sMVA-S and sMVA-N alone or in combination ( Figure S9-10). Altogether these results indicate that the sMVA-vectored S and N antigens when expressed alone or combined using a single vector or two separate vectors can stimulate potent SARS-CoV-2-speci c humoral and cellular immune responses in mice.

Discussion
We developed a novel vaccine platform based on a fully synthetic form of the highly attenuated and widely used MVA vector. In response to the ongoing global SARS-CoV-2 pandemic, we used this novel vaccine platform to rapidly produce sMVA vectors co-expressing SARS-CoV-2 S and N antigens and show that these vectors can induce potent SARS-CoV-2 antigen-speci c humoral and cellular immune responses in mice, including potent NAb. These results highlight the feasibility to e ciently produce recombinant MVA vectors from chemically synthesized DNA and to rapidly develop a synthetic poxvirusbased vaccine candidate to prevent SARS-CoV-2 infection. We envision that this novel vaccine platform based on synthetic DNA will facilitate the development and clinical use of poxvirus vaccine vectors for infectious diseases and cancer.
Our strategy to produce a synthetic form of MVA using chemically synthesized DNA differs from the recently described approach to produce a synthetic horsepox virus vaccine vector 42 . While our strategy to generate sMVA involves the use of three large circular DNA fragments (~ 60 kbp) with intrinsic HL and CR sequences ( Fig. 1), the approach by Noyce et al. to produce a synthetic horsepox vaccine involves the use of multiple smaller linear DNA fragments (~ 10-30 kbp) and the addition of terminal HL sequences 42 . Because the three sMVA fragments can be used in a circular form for the sMVA reconstitution process they are easily maintained in E. coli as BACs and transferred to BHK cells for sMVA virus reconstitution without the need for additional puri cation steps or modi cations. This feature greatly facilitates the insertion of heterologous antigen sequences into the sMVA DNA by highly e cient bacterial recombination techniques and to produce recombinant sMVA vaccine vectors. Additionally, the threeplasmid system provides the exibility for rapid production of recombinant MVA harboring multiple antigens inserted into different MVA insertion sites, which can be particularly laborious when generating recombinant MVA by the conventional transfection/infection procedure 3,43 . Although the precise mechanism and order of events of the sMVA virus reconstitution using circular plasmids was not investigated, we demonstrate that the sMVA fragments e ciently recombine with one another and produce a synthetic form of MVA that is virtually identical to wtMVA in genome content, replication properties, host cell range, and immunogenicity.
In contrast to most other currently employed SARS-CoV-2 vaccine approaches that solely rely on the S antigen, our SARS-CoV-2 vaccine approach using sMVA employs immune stimulation by S and N antigens, which both are implicated in protective immunity 20,26 . The observation that the sMVA-CoV2 vectors co-expressing S and N antigens can stimulate potent NAb against SARS-CoV-2 pseudovirus and infectious virions suggests that they can elicit antibodies that are considered effective in preventing SARS-CoV-2 infection and disease 16,18,20,21 . We show that the vaccine vectors stimulate a Th1-biased antibody and cellular immune response, which is considered the preferred antiviral adaptive immune response to avoid vaccine-associated enhanced respiratory disease 44,45 . We did not nd any evidence for Fc-mediated ADE promoted by the vaccine-induced immune sera, suggesting that antibody responses induced by the vaccine vectors bear minimal risk for ADE-mediated immunopathology, a general concern in SARS-CoV-2 vaccine development 44,45 . In addition, based on ndings with other viruses associated with ADE, the stimulation of Th1 immunity with a strong T cell response component appears to be the way forward to develop an effective SARS-CoV-2 vaccine candidate 46 .
Other immune responses besides NAb targeting the S antigen may contribute to protection against SARS-CoV-2 infection, which is highlighted by the nding that even patients without measurable NAb can recover from SARS-CoV-2 infection 20 . While antibodies could be particular important to prevent initial SARS-CoV-2 acquisition, T cell responses may impose an additional countermeasure to control sporadic virus spread at local sites of viral infection, thereby limiting virus transmission. Our dual recombinant vaccine approach based on sMVA to induce robust humoral and cellular immune responses to S and N antigens may provide protection against SARS-CoV-2 infection beyond other vaccine approaches that solely employ the S antigen. Our results warrant further preclinical testing of a sMVA vaccine candidate for protective e cacy in animal models towards rapid advancement into phase 1 clinical testing.  4 . The sMVA fragments were produced and assembled by Genscript using chemical synthesis, combined with a yeast recombination system. All sMVA fragments were cloned into a yeast shuttle vector, termed pCCI-Brick, which contains a mini-F replicon for stable propagation of large DNA fragments as low copy BACs in E. coli. sMVA F1 and F3 were cloned and maintained in EPI300 E. coli (Epicentre), while sMVA F1 was cloned and maintained in DH10B E. coli (Invitrogen).

Materials And Methods
Antigen insertion SARS-CoV-2 S and N antigen sequences were inserted into the sMVA fragments by En passant mutagenesis in GS1783 E. coli cells 49,50 . Brie y, transfer constructs were generated that consisted of the S or N antigen sequence with upstream mH5 promoter sequence and downstream Vaccinia transcription termination signal (TTTTTAT), and a kanamycin resistance cassette anked by a 50 bp gene duplication was introduced into the antigen sequences. The transfer constructs were ampli ed by PCR with primers providing ~ 50 bp extensions for homologous recombination and the resulting PCR products were used to insert the transfer constructs into the sMVA DNA by a rst Red-recombination reaction 49 BHK cells, and CEF was determined as follows. The cells were seeded in 6-well plate tissue culture format and at 70-90% con uency infected in duplicates with 0.01 MOI of sMVA or wtMVA using MEM2. At 2 hours post infection, the cells were washed twice with PBS and incubated for two days in normal growth medium (as described under cells and viruses). After the incubation period, virus was prepared by conventional freeze/thaw method and the virus titers of each duplicate infection was determined in duplicate on CEF.

Replication kinetics
To compare the replication kinetics of sMVA and wtMVA, CEF or BHK cells were seeded in 6 well-plate tissue culture format and at 70-90% con uency infected in triplicates at 0.02 MOI with sMVA or wtMVA using MEM2. After 2 hours of incubation, the cells were grown in MEM10. At 24 and 48 hours post infection, virus was prepared by freeze/thaw method and the virus titers of each triplicate infection and the inoculum was determined in duplicate on CEF.

Plaque size analysis
To compare the plaque size of sMVA virus and wtMVA, CEF or BHK cells were seeded in 6-well plate tissue culture format and at 70-90% con uency infected with 0.002 MOI with sMVA or wtMVA using MEM2. After 2 hours of incubation, MEM10 was added and the cells were grown for 16-24 hours. The cell monolayers were stained with Vaccinia virus polyclonal antibody and viral plaques were imaged using Leica DMi8 inverted microscope and measured using LAS X software. The size of 25 viral plaques per sMVA or wtMVA was calculated using the formula = × × , where a and b are the major and minor radius of the ellipse, respectively.

PCR analysis
To characterize the viral DNA of the sMVA vectors by PCR, CEF were seeded in 6-well plate tissue culture format and at 70-90% con uency infected at 5 MOI with sMVA or wtMVA. DNA was extracted at 16- Restriction pattern analysis BHK cells were seeded in 20 × 150 mm tissue culture dishes, grown to ~ 70-90% con uency, and infected at 0.01 MOI with wtMVA, sMVA tv1, or sMVA tv2. Ultra-puri ed virus was prepared two days post-infection as previously described 48 . Viral DNA (vDNA) was phenol/chloroform extracted, followed by ethanol precipitation as previously described 52 . DNA concentration and A 260 /A 280 ratios were determined using NanoVue (GE Healthcare Bio-sciences Corp). 10 µg of vDNA were digested with 3 units of either KpnI or XhoI, followed by visualization on 0.5% EtBr-stained agarose gel that was run at 2.4v/cm, overnight.
Sequencing of sMVA fragments and genome PacBio Long Read Sequencing analysis con rmed the integrity of the sMVA fragments and sMVA genome, including a single point mutation in a non-coding determining region at 3 base pairs downstream of 021L 4 that was found both in sMVA F1 and in reconstituted sMVA. Brie y, 5 ug of fragmented DNAs were converted to SMRTbell libraries using the SMRTbell Template Prep Kit 1.0 (PacBio). The libraries were size-selected (7-kb size cutoff) with BluePippin (Sage Science). The sizeselected libraries were loaded to SMRT cells using MagBeads loading and sequenced on a PacBio RSII with 10 hour movie. Read demultiplexing, mapping to the reference sequence (Vaccinia virus strain Ankara U94848.1), and variants calling were performed using the SMRT Link (v6.0.0.47841). The identi ed variants were visually inspected in SMRT view Genome Browser for con rmation. De novo assembly was done using either canu v1.7.1 or wtdbg2 v2.5. The 5' start position of the assembled contig was edited by comparing to the U94848.1 reference.
Immunblot analysis BHK cells infected at 5 MOI were harvested 24-post infection. Proteins were solubilized in PBS with 0.1% Triton X-100, supplemented with protease inhibitor, then reduced and denatured in Laemmli buffer containing DTT and boiled at 95 °C for ~ 10 minutes. Proteins were resolved on a 4-20% Mini Protean TGX gradient gel (BioRad), and transferred onto PVDF membrane. S protein was probed with anti-SARS-CoV-1 S1 subunit rabbit polyclonal antibody (40150-T62-COV2, Sino Biological); N protein was probed with anti-SARS-CoV1 NP rabbit polyclonal antibody (40413-T62, Sino Biological). Vaccinia BR5 protein was probed as a loading control. Anti-rabbit polyclonal antibody conjugated with horseradish peroxidase (Sigma-Aldrich) was used as a secondary antibody and protein bands were visualized with chemiluminescent substrate (ThermoFisher).
Immuno uorescence BHK or HeLa cells were grown on glass coverslips and infected with sMVA or recombinant sMVAs encoding S and/or N proteins at an MOI of 5 for 6 hours at 37°C in a humidi ed incubator (5% CO 2 ). After infection, cells were xed for 15 min in 2% PFA and then directly permeabilized by addition of ice cold 1:1 acetone/methanol for 5 min on ice. Cells were blocked for 1 hr with 3% BSA at room temperature, incubated with primary antibody mix (1:500) against the S2 subunit or N for 1 hr at 37°C, and then incubated with Alexa-conjugated secondary antibodies (ThermoFischer) (1:2000) for 1 hr at 37°C, with washing (PBS + 0.1% Tween20) between each step. For detection of cell membranes and nuclei, cells were incubated with Alexa-conjugated wheat germ agglutinin at 5 µg/mL (Thermo Fisher) and DAPI for 10 minutes at room temperature. Coverslips were washed and mounted onto slides with Fluoromount-G (SouthernBiotech). Microscopic analysis was performed using a laser-scanning confocal microscope (Zeiss, LSM700). Images were acquired and processed using Zen software (Zeiss).

Mouse immunization
The

Binding antibodies
Binding antibodies in mice immunized with sMVA, wtMVA, or sMVA-CoV2 vectors were evaluated by ELISA. ELISA plates (3361, Corning) were coated overnight with 1 µg/ml of MVA expressing Venus uorescent marker 9 , S (S1 + S2, 40589-V08B1, Sino Biological), RBD (40592-V08H, Sino Biological) or N (40588-V08B, Sino Biological). Plates were blocked with 3% BSA in PBS for 2 hours. Serial dilutions of the mouse sera were prepared in PBS and added to the plates for two hours. After washing the plate, 1:3,000 dilution of HRP-conjugated anti-mouse IgG secondary antibody (W402B, Promega) was added and incubated for one additional hour. Plates were developed using 1-Step Ultra TMB-ELISA (34028, Thermo Fisher) for one to two minutes after which the reaction was stopped with 1M H 2 SO 4 . Plates were read at 450 nanometers wave length using FilterMax F3 microplate reader (Molecular Devices). Binding antibodies endpoint titers were calculated as the latest serum dilution to have an absorbance higher than 0.1 absorbance units (OD) or higher than the average OD in mock immunized mice plus 5 times the standard deviation of the OD in the same group at the same dilution. For evaluation of the IgG2a/IgG1 ratio, mouse sera were diluted 1:10,000 in PBS. The assay was performed as described above except for the secondary antibodies (1:2,000. goat Anti-Mouse IgG2a cross absorbed HRP antibody, Southern biotech, 1083-05; Goat anti-Mouse IgG1 cross absorbed HRP antibody, Thermo Fisher, A10551). The IgG2a/IgG1 ratio was calculated by dividing the absorbance read in the well incubated with the IgG2a secondary antibody divided the absorbance for the same sample incubated with the IgG1 antibody.
ARPE-19 cells were seeded in 96 well plates (1.5 × 10 4 cells/well). The following day, serial dilutions of mouse sera were incubated for 2 hours with MVA expressing the uorescent marker Venus 9 (1.5 × 10 4 PFU/well). The serum-virus mixture was added to the cells in duplicate wells and incubated for 24 hours. After the 24 hours incubation period, the cells were imaged using Leica DMi8 inverted microscope.
Pictures from each well were processed using Image-Pro Premier (Media Cybernetics) and the uorescent area corresponding to the area covered by MVA-Venus infected cells was calculated.

SARS-CoV-2 pseudovirus production
The day before transfection, HEK293T/17 were seeded in a 15 cm dish at a density of 5 × 10 6 cells in DMEM supplemented with 10% heat inactivated FBS, non-essential amino acids, HEPES, and glutamine 53 . Next day, cells were transfected with a mix of packaging vector (pALDI-Lenti System, Aldevron), luciferase reporter vector and a plasmid encoding for the wild type SARS-CoV2 Spike protein (Sino Biological) or vesicular stomatitis virus G (VSV-G, Aldevron), using FuGENE6 (Roche) as a transfection reagent : DNA ratio of 3:1, according to manufacturer's protocol. Sixteen hours posttransfection, the media was replaced and cells were incubated for an additional 24-72 hours. Supernatants were harvested at 24-, 48-and 72 hours, clari ed by centrifugation at 1,500 RPM for 5 minutes and ltered using a sterile 0.22 µm pore size lter. Clari ed lentiviral particles were concentrated by ultracentrifugation at 20,000 RPM for 2 hours at 4 °C. The pellet was resuspended in DMEM containing 2% heat inactivated-FBS and stored overnight at 4 °C to allow the pellet to completely dissolve. Next day, samples were aliquoted, snap frozen and stored at -80 °C for downstream assays.

SARS-CoV-2 pseudotype neutralization and ADE assay
Levels of p24 antigen in the puri ed SARS-CoV-2 pseudotype solution was measured by ELISA (Takara).
Mouse sera were heat inactivated, pooled and diluted at a linear range of 1:100 to 1:50,000 in complete DMEM. For the neutralization assay, diluted serum samples were pre-incubated overnight at 4 °C with SARS-CoV-2-Spike pseudotyped luciferase lentiviral vector, normalized to 100 ng/mL of p24 antigen.
HEK293T cells overexpressing ACE-2 receptor were seeded the day before transduction at a density of 2 × 10 5 cells per well in a 96-well plate in complete DMEM 47 . Before infection, 5 µg/mL of polybrene was added to each well. Neutralized serum samples were then added to the wells and the cells were incubated for an additional 48 hours at 37 °C and 5% CO 2 atmosphere. Following incubation, cells were lysed using 40 µL of Luciferase Cell Culture Lysis 5x Reagent per well (Promega). Luciferase activity was quanti ed using 100 µL of Luciferase Assay Reagent (Promega) as a substrate. Relative luciferase units (RLU) were measured using a microplate reader (SpectraMax L, Molecular Devices) at a 570 nm wave length. The percent neutralization titer for each dilution was calculated as follows: NT = [1-(mean luminescence with immune sera/mean luminescence without immune sera)] x 100. The titers that gave 90% neutralization (NT90) were calculated by determining the linear slope of the graph plotting NT versus serum dilution by using the next higher and lower NT. In all the experiments RLU of uninfected cells was measured and was always between 50 and 90.
For the ADE assay, THP1 cells were seeded at a con uency of 2 × 10 6 cells/mL in a 96 well plate and coincubated for 48 hours with serum samples diluted at 1:5,000 or 1:50,000 in the presence of SARS-CoV-2-Spike pseudotyped or VSV-G luciferase lentiviral vector, normalized to 100 ng/mL of p24 antigen.
Following incubation, cells were lysed using 100 µL of ONE-Glo Luciferase Assay System per well (Promega). RLU were measured as above.

SARS-CoV-2 focus reduction neutralization test (FRNT)
FRNT assay was performed as described recently 54  were obtained from UCSD. Individuals were con rmed to be infected in the previous three to ten weeks by PCR and lateral ow assay. All individuals were symptomatic with mild to moderate-severe symptoms. Serum samples (DS-626-G and DS-626-N, Seracare) purchased before SARS-CoV-2 pandemic were used as a negative control. SARS-CoV-2-speci c binding antibodies in plasma samples were measured as described above. Cross-adsorbed goat antihuman IgG (H + L) secondary antibody (A18811, Invitrogen) was used at a dilution of 1:3,000.

Statistics
Statistical evaluation was pursued using GraphPad Prism (v8.3.0). For evaluation of differences in sMVA and wtMVA plaque area in BHK-21 and CEF cells and differences in sMVA and wtMVA host cell range, one-way ANOVA followed by Tukey's and Dunnet's multiple comparison tests were used, respectively. For sMVA and wtMVA growth kinetic analysis, mixed-effects model with the Geisser-Greenhouse correction, followed by Tukey's multiple comparisons test were applied. For ELISAs, one-way ANOVA and Tukey's multiple comparison tests were used to calculate differences in endpoint titers and group means between groups. For IgG2a/IgG1 ratio analysis, one-way ANOVA with Dunnett's multiple comparison test was used to compare the IgG2a/IgG1 ratio measured in each group to a ratio of 1. Pearson correlation analysis was performed to calculate the correlation coe cient r and its signi cance. For T cell responses analysis, oneway ANOVA followed by Dunnett's multiple comparisons test with a single pooled variance was used to compare the mean of each group.  inoculum based on 0.01 MOI. Differences between groups in C-D were calculated using one-way ANOVA followed by Tukey's (C) or Dunnett's (D) multiple comparison tests. ns = not signi cant. sMVA in vivo immunogenicity. sMVA derived either with FPV HP1.441 (sMVA hp) or TROVAC from two independent virus reconstitution (sMVA tv1 and sMVA tv2) was compared by in vitro analysis with wtMVA. C57BL/6 mice were immunized twice at three week interval with low (1x107 PFU) or high (5x107 PFU) dose of sMVA or wtMVA. Mock-immunized mice were used as controls A) Binding antibodies. MVAspeci c binding antibodies (IgG titer) stimulated by sMVA or wtMVA were measured after the rst and second immunization by ELISA. B) NAb responses. MVA-speci c NAb titers induced by sMVA or wtMVA were measured after the booster immunization against recombinant wtMVA expressing a GFP marker. C-D) T cell responses. MVA-speci c IFN , TNFα, IL-4, and IL-10-secreting CD8+ (C) and CD4+ (D) T cell responses induced by sMVA or wtMVA after two immunizations were measured by ow cytometry following ex vivo antigen stimulation using B8R immunodominant peptides. Differences between groups were evaluated using one-way ANOVA with Tukey's multiple comparison test. ns = not signi cant.
Construction and characterization of sMVA-CoV2 vectors. A) Schematic representation of vector construction. S and N antigen sequences (red spheres and green triangles) were inserted into sMVA fragments F2 and F3 by bacterial recombination methods in E. coli. The modi ed sMVA fragments of F1 and F2 with inserted antigen sequences and the unmodi ed sMVA fragment F1 were isolated from E. coli and co-transfected into FPV-infected BHK cells to initiate virus reconstitution. B) Schematics of single (sMVA-S, sMVA-N) and double (sMVA-N/S, sMVA-S/N) recombinant sMVA-CoV2 vectors with S and N antigen sequences inserted into commonly used MVA insertion sites (Del2, IGR69/70, Del3). All antigens were expressed via the Vaccinia mH5 promoter. C) Western Blot. BHK cells infected with the single and double recombinant sMVA-CoV2 vectors derived with FPV HP1.441 (sMVA-S/N hp, sMVA-N/S hp) or TROVAC (sMVA-S/N tv, sMVA-N/S tv, sMVA-S tv, sMVA-N tv) were evaluated for antigen expression by Western Blot using anti-S1 and N antibodies (αS1 and αN Ab). Vaccinia B5R protein was veri ed as infection control. Higher and lower molecular weight bands may represent mature and immature protein species. D) Flow cytometry staining. HeLa cells infected with the vaccine vectors were evaluated by cell surface and intracellular ow staining using anti-S1, S2, and N antibodies (αS1, αS2, and αN Ab). Live cells were used to evaluate cell surface antigen expression. Fixed and permeabilized cells were used to evaluate intracellular antigen expression. Anti-Vaccinia virus antibody (αVAC) was used as staining control to verify MVA protein expression. Cells infected with sMVA or wtMVA or uninfected cells were used as controls for experiments in C and D as indicated.
duplicate (D-E) or triplicate (F-G) infection. N/A=failed quality control of the samples. Dotted lines indicate lowest antibody dilution included in the analysis. H) SARS-CoV-2/SARS-CoV-2pv correlation analysis. Correlation analysis of NT90 measured in mouse sera after one and two immunizations using infectious SARS-CoV-2 virus and SARS-CoV-2pv. Pearson correlation coe cient (r) was calculated in H. *p<0.05. ns= not signi cant.