key: cord-0982519-pbjt2thu
authors: Conforti, Antonella; Marra, Emanuele; Palombo, Fabio; Roscilli, Giuseppe; Ravà, Micol; Fumagalli, Valeria; Muzi, Alessia; Maffei, Mariano; Luberto, Laura; Lione, Lucia; Salvatori, Erika; Compagnone, Mirco; Pinto, Eleonora; Pavoni, Emiliano; Bucci, Federica; Vitagliano, Grazia; Stoppoloni, Daniela; Pacello, Maria Lucrezia; Cappelletti, Manuela; Ferrara, Fabiana Fosca; D’Acunto, Emanuela; Chiarini, Valerio; Arriga, Roberto; Nyska, Abraham; Lucia, Pietro Di; Marotta, Davide; Bono, Elisa; Giustini, Leonardo; Sala, Eleonora; Perucchini, Chiara; Paterson, Jemma; Ryan, Kathryn Ann; Challis, Amy-Rose; Matusali, Giulia; Colavita, Francesca; Caselli, Gianfranco; Criscuolo, Elena; Clementi, Nicola; Mancini, Nicasio; Groß, Rüdiger; Seidel, Alina; Wettstein, Lukas; Münch, Jan; Donnici, Lorena; Conti, Matteo; Francesco, Raffaele De; Kuka, Mirela; Ciliberto, Gennaro; Castilletti, Concetta; Capobianchi, Maria Rosaria; Ippolito, Giuseppe; Guidotti, Luca G.; Rovati, Lucio; Iannacone, Matteo; Aurisicchio, Luigi
title: COVID-eVax, an electroporated plasmid DNA vaccine candidate encoding the SARS-CoV-2 Receptor Binding Domain, elicits protective immune responses in animal models of COVID-19
date: 2021-09-01
journal: bioRxiv
DOI: 10.1101/2021.06.14.448343
sha: 7040e5502d62ed22ec1b760c2700231d94e3af7d
doc_id: 982519
cord_uid: pbjt2thu

The COVID-19 pandemic caused by the β-coronavirus SARS-CoV-2 has made the development of safe and effective vaccines a critical global priority. To date, four vaccines have already been approved by European and American authorities for preventing COVID-19 but the development of additional vaccine platforms with improved supply and logistics profiles remains a pressing need. Here we report the preclinical evaluation of a novel COVID-19 vaccine candidate based on the electroporation of engineered, synthetic cDNA encoding a viral antigen in the skeletal muscle, a technology previously utilized for cancer vaccines. We constructed a set of prototype DNA vaccines expressing various forms of the SARS-CoV-2 Spike (S) protein and assessed their immunogenicity in animal models. Among them, COVID-eVax – a DNA plasmid encoding a secreted monomeric form of SARS-CoV-2 S protein RBD – induced the most potent anti-SARS-CoV-2 neutralizing antibody responses (including against the current most common variants of concern) and a robust T cell response. Upon challenge with SARS-CoV-2, immunized K18-hACE2 transgenic mice showed reduced weight loss, improved pulmonary function and significantly lower viral replication in the lungs and brain. COVID-eVax conferred significant protection to ferrets upon SARS-CoV-2 challenge. In summary, this study identifies COVID-eVax as an ideal COVID-19 vaccine candidate suitable for clinical development. Accordingly, a combined phase I-II trial has recently started in Italy.

At the time of writing, SARS-CoV-2 has spread worldwide causing over 200 million confirmed cases and more than 4 million confirmed deaths.

To date, the regulatory agencies European Medicines Agency (EMA) and Among the latter, DNA-based platforms show the greatest potential in terms of 15 safety and ease of production 3 . Prior work has demonstrated that a DNA-based vaccine approach for SARS-and MERS-CoV induces neutralizing antibody (nAb) responses and provides protection in challenge models 4, 5 . Moreover, in a phase I dose-escalation study subjects immunized with a DNA vaccine encoding the MERS-CoV S protein showed durable nAb and T cell responses and a seroconversion rate of 96% 5 . The 20 SARS-CoV-2 S protein is most similar in sequence and structure to SARS-CoV S and shares a global protein fold architecture with the MERS-CoV S protein 6 . Of note, the 5 receptor-binding site of the S protein is a vulnerable target for antibodies. In fact, anti-MERS antibodies targeting the receptor-binding domain (RBD) of the S protein tend to have greater neutralizing potency than those directed to other epitopes 7 . More recently, a study by Piccoli et al. 8 showed that depletion of anti-RBD antibodies in convalescent patient sera results in the loss of more than 90% neutralizing activity towards SARS-5

CoV-2, suggesting that the SARS-CoV-2 RBD represents a key target for vaccine development.

Here we describe the development of a DNA-based SARS-CoV-2 vaccine.

Synthetic DNA is temperature-stable and cold-chain free, which are important advantages over approved RNA and vector vaccines for delivery to resource-limited 10 settings. Furthermore, synthetic DNA vaccines are amenable to accelerated developmental timelines due to the relative simplicity by which multiple candidates can be designed, preclinically tested, manufactured in large quantities and progressed through established regulatory pathways to the clinic. Injection of DNA plasmid into the skeletal muscle followed by a short electrical stimulation -referred to as electro-gene-15 transfer or electroporation (EP) -enhances DNA uptake and gene expression by several hundred-fold 9-11 , leading to improved antigen expression and a local and transient tissue damage favoring inflammatory cell recruitment and cytokine production at the injection site 12 .

Exploiting our experience in the generation of vaccines based on the 20 electroporation of plasmid DNA in the skeletal muscle 11 , we produced and screened several constructs expressing different portions of the SARS-CoV-2 S protein and 6 identified COVID-eVax -a DNA plasmid encoding a secreted monomeric form of SARS-CoV-2 S protein RBD -as a candidate for further clinical development. COVID-eVax has a favorable safety profile, it induces potent anti-SARS-CoV-2 neutralizing antibody responses also against the current most common variants of concern (VOCs)

as well as T cell responses, and it confers significant protection to hACE2 transgenic 5 mice and ferrets upon SARS-CoV-2 challenge.

We designed five different DNA constructs (Fig. 1A) encoding the following versions of the SARS-CoV-2 (Wuhan Hu-1, GenBank: MN908947) S protein: 1) the fulllength protein (FL); 2) the receptor binding domain (RBD); 3) the highly variable N-5 terminal domain (NTD) and the RBD domain (N/R); 4) the whole S1 subunit (S1); 5) the RBD fused to a human IgG-Fc (RBD-Fc). To promote protein secretion, we introduced a tissue plasminogen activator (tPA) leader sequence in the RBD, N/R and S1 constructs and an IgK leader sequence in the RBD-Fc construct. Western blot analyses confirmed expression of all constructs in cell lysates and of S1, N/R and RBD in the culture 10 supernatants (Fig. 1B) .

Electroporation of these DNA vaccines in the skeletal muscle of BALB/c mice was adopted to evaluate immunogenicity. The vaccination protocol consisted of the injection of 20 µg of DNA into both quadriceps (10 µg of DNA in each muscle) into 6-week-old mice (Fig. 1C) . A DNA plasmid expressing luciferase was used as a control for gene 15 expression whereas a group of mice injected with DNA but not electroporated served as additional controls (Fig. S1 ). Mice received a second vaccination (boost) at day 28 and were sacrificed at day 38 (Fig. 1C) . The humoral response in the sera of vaccinated mice was evaluated by measuring anti-RBD IgG titers by ELISA at day 14 (prime) and at day 38 (boost) (Fig. 1D) . At day 14 all mice showed detectable anti-RBD IgG 20 antibodies, and their levels significantly increased at day 38 (Fig. 1D) . Notably, the most significant increase in antibody response was induced by the secreted RBD construct 8 (Fig. 1D) , with a calculated geometric mean of IgG endpoint titers 13 as high as 1:24,223 after prime and 1:617,648 after boost. Since these preliminary data showed the RBD construct to be the most immunogenic among the five DNA constructs, RBD was chosen as the main vaccine candidate for further development and was directly compared with the FL construct in subsequent experiments. 5

We next sought to characterize the humoral response to the RBD vaccine in depth, focusing on the specificity, duration and neutralization capacity of the elicited 10 antibodies. As per specificity, we carried out a B cell epitope mapping of the response elicited by the FL and RBD vaccines. To this end, a B cell ELISpot assay was performed by stimulating splenocytes collected from vaccinated BALB/c mice with 338 peptides covering the whole SARS-CoV-2 Spike protein. Sequences of positive hits (Table S1 ) were then mapped on the three-dimensional structure of the S protein 14 , 15 hence outlining the epitope domains ( Fig. 2A) . Mice immunized with the RBD vaccine showed responses mapping mainly on conserved regions of the RBD, and not on regions most commonly affected by mutations in the current circulating VOCs (e.g., N501K, K417N, S477, E484K and L452, Fig. 2A) . Despite the caveat that the abovementioned analysis detects linear and not conformational epitopes, this suggests 20 that antibodies elicited by the RBD vaccine might be functional against the current most commonly circulating SARS-CoV-2 variants.

were present not only in the sera but also in the lungs of vaccinated mice, as shown by analysis of bronchoalveolar lavages (BALs) of mice 38 days after vaccination (Fig. 2B) .

We next sought to analyze whether the RBD-specific antibodies induced by the RBD vaccine were able to neutralize SARS-CoV-2. The neutralization capacity of 5 antibodies induced by the RBD vaccine was comparable with FL after prime and superior after boost (Fig. 2C) , with NT50 at day 38 of 894 ± 249. A dose-response experiment indicated that the neutralizing antibody titers plateaued at an RBD vaccine dose of 10 µg (injected in a single quadricep muscle) in a prime-boost regimen serum ( Fig. 2D) . Finally, total anti-RBD IgG antibodies persisted at high levels up to 6 months 10 after vaccinations (Fig. 2E) .

Next we sought to evaluate the T cell response elicited by the RBD vaccine. To this end, we used peptide pools covering the S1 and S2 portions of the Spike protein 15 (pools S1 and S2, respectively) to stimulate splenocytes collected from BALB/c mice at day 38 after vaccination (see Fig. 1C for experimental setup). IFN-g released by T cells upon peptide re-stimulation was evaluated by ELISpot assay. As expected, in the group vaccinated with the RBD vaccine, we measured only T cell responses against pool S1

(that spans the RBD), whereas in the group vaccinated with the FL construct, we 20 measured T cell responses against both pools S1 and S2 (data not shown). In order to reveal immunodominant epitopes eliciting the T cell response, we performed epitope 10 mapping using thirty-seven matrix mapping pools, covering the entire sequence of the S protein ( Fig. S2A) , or twenty-four matrix pools, covering the RBD alone (Table S2 and Fig. S2B) . Most of the H2-K d -restricted immunodominant epitopes were clustering in the RBD (Fig. S2C) . Cytokine production by antigen-specific T cells was also evaluated by intracellular staining of splenocytes collected from vaccinated BALB/C mice and 5 restimulated with pool S1 (Fig. 2F) . Compared to FL, the RBD vaccine induced the highest frequency of CD8 + T cells producing either IFN-g or TNF-a (Fig. 2F) .

To measure the potential recruitment of RBD-specific T-cells to the lungs, 20 µg of the RBD protein was injected intranasally in a group of vaccinated BALB/c mice two weeks after the second immunization and IFN-g production from lymphocytes recovered from 10 bronchoalveolar lavages (BAL) was measured by ELISpot assay one day later (Fig.   2G ). Mice vaccinated with RBD showed a higher recruitment of RBD-specific T cells than mice vaccinated with the FL vaccine (Fig. 2H) . A dose-response experiment conducted in C57BL/6 mice showed an even stronger specific T cell response than in BALB/c mice (data not shown) and a clear dose-dependency (Fig. S3A) . A nonlinear 15 fitting analysis of the curve (after pool S1 stimulation) revealed an ED50 of 2.06 ± 0.86 µg (Fig. S3B) . Cytokine analyses in vaccinated C57BL/6 mice revealed a predominant IFN-g-and TNF-a-producing CD8 + T cell response, independently of the sex and age of the mice (Fig. S3C and data not shown).

Safety and immunogenicity of the RBD vaccine candidate in rats. 11 We next evaluated the safety and immunogenicity of the RBD vaccine candidate in rats, an animal model highly suited for toxicological studies. Seven-week-old female Sprague-Dawley rats were injected intramuscularly with PBS or 100, 200 or 400 µg of the RBD vaccine (divided equally in the two quadriceps) followed by EP at day 0 and day 14 (Fig. 3A) . The immunizations were well tolerated, with only mild to moderate 5 lesions 15 at the injection site that were almost fully recovered within four weeks (Fig.   S4) , and an increased cellularity in the draining lymph nodes (data not shown).

Quantification of the RBD-specific antibody titers showed a robust and dosedependent antibody production, with ELISA endpoint titers up to 152,991 for the highest dose ( Fig. 3B, C) . Immunization with the RBD vaccine induced high neutralizing 10 antibody titers (Fig. 3D) which correlated with the total IgG endpoint titers (Fig. 3E) .

Finally, sera from Sprague-Dawley rats that were immunized with 400 µg of the RBD vaccine at day 0 and 14, or that received a third dose at day 28, were assessed for neutralizing activity against three major SARS-CoV-2 variants (i.e., B.1.1.7, B.1.351 and P.1) utilizing a lentiviral pseudotyped assay ( Fig. 3F-H) . 15

The RBD vaccine candidate elicits protective immune responses in K18-hACE2 transgenic mice and in ferrets.

To explore the in vivo protection efficacy of RBD vaccine against SARS-CoV-2 challenge, K18-hACE2 transgenic mice 16 received two intramuscular immunizations (at 20 day -39 and at day -18) of 10 μg of the RBD vaccine (n = 7) or PBS (n = 6) followed by EP (Fig. 4A) . Pre-challenge sera collected 1 day prior to SARS-CoV-2 infection showed that the RBD vaccine induced robust RBD-specific IgG antibodies (average concentration of ~ 50 µg/ml, Fig. 4B ). Eighteen days after the boost immunization, all mice were infected intranasally with 1 × 10 5 TCID50 of SARS-CoV-2 (hCoV-19/Italy/LOM-UniSR-1/2020; GISAID Accession ID: EPI_ISL_413489) (Fig. 4A) . As expected 17 , beginning 3-4 days post infection (p.i.) PBS-treated K18-hACE2 transgenic mice 5 infected with SARS-CoV-2 exhibited a weight loss close to 20% of their body weight and a lethargic behavior ( Fig. 4C and data not shown). By contrast, K18-hACE2 transgenic mice immunized with RBD vaccine maintained stable body weight upon SARS-CoV-2 challenge and appeared more active ( Fig. 4C and data not shown). We used wholebody plethysmography to evaluate several complementary metrics of pulmonary 10 function, obstruction, and bronchoconstriction, including frequency, enhanced pause (PenH), and the fraction of expiration time at which the peak occurs (Rpef) 18, 19 . PBStreated mice infected with SARS-CoV-2 exhibited a decreased respiratory rate (Fig.   4D ), an increased PenH (Fig. 4E ) and a decreased Rpef (Fig. 4F) , indicative of pronounced loss of pulmonary function. By contrast, K18-hACE2 transgenic mice 15 immunized with the RBD vaccine prior to infection maintained a relatively stable respiratory rate (Fig. 4D) , had a much lower PenH (Fig. 4E ) and a higher Rpef (Fig.   4F) , indicative of better pulmonary function. Much higher amounts of viral RNA, infectious SARS-CoV-2 and viral N protein were detected in the lungs and brain of PBStreated mice compared to mice immunized with the RBD vaccine ( Fig. 4G-L) . The lower 20 viral titers in the lungs of immunized mice were associated with the detection of RBD-13 specific CD4 + T cells producing IFN-g, TNF-a or both as well as RBD-specific IFN-gproducing CD8 + T cells (Fig. 4M) .

Besides inducing potent adaptive immune responses, the protection induced by the RBD vaccine might lie in the competitive inhibition of SARS-CoV-2 binding to ACE2 by the secreted RBD. Indeed, RBD is detectable in the sera and in the BAL of 5

immunized BALB/c mice as early as 2 days after immunization ( Fig. S5A-C) , time point in which anti-RBD antibodies are not yet detectable (Fig. S5D) . To test whether this secreted RBD would compete with SARS-CoV-2 for ACE2 binding, we immunized K18-hACE2 transgenic mice with the RBD vaccine 2 days prior to intranasal inoculation with a luciferase-encoding lentiviral vector pseudotyped with the SARS-CoV-2 Spike protein. Two days later the lungs of treated mice were assessed for bioluminescence using an in vivo imaging system (IVIS). As shown in Fig. S5E , compared to mice injected with PBS, mice immunized with the RBD vaccine exhibited a reduced bioluminescence, indicative of a significantly lower in vivo transduction. Further experiments should determine the extent to which the abovementioned mechanism confers protection by the RBD vaccine. 15

To confirm the immunogenicity and protective efficacy of the RBD vaccine against SARS-CoV-2 infection in a different and larger animal model, sixteen female ferrets weighing over 750 g were either left untreated (control) or injected with 400 µg of the RBD vaccine followed by electroporation 42 and 14 days prior to intranasal infection with 5 x 10 6 pfu of SARS-CoV-2 isolate Victoria/1/2020 (Fig. 5A) . Compared to control 20 animals, viral subgenomic RNA detected in nasal washes and throat swabs at day 7 post challenge in immunized ferrets were significantly reduced (Fig. 5B, C) . 14 Together, the results obtained in two distinct animal models of SARS-CoV-2 infection indicate that the RBD vaccine induces protective immune responses. We believe that this vaccine will pave the way for the approval of other DNA vaccines, such as COVID-eVax.

DNA-based vaccines are engineered for maximal gene expression and immunogenicity, they can be quickly designed from new genetic viral sequences, and they allow for fast and scalable manufacturing as well as long-term stability at room 5 temperature. Moreover, DNA vaccines do not require complex formulations such as those based on nanoparticles (necessary for peptide-or RNA-based vaccines). An efficient DNA uptake can be obtained with different methods 20 . Among others, electroporation (EP) increases the initial uptake of DNA plasmid by local cells by approximately 500-fold [20] [21] [22] . Here, we adopted an EP technology manufactured by the 10 Italian company IGEA, a leader in tissue EP, and extensively tested both in mice and other animal species 11, [23] [24] [25] [26] . This platform technology has been referred to as X-eVax, where X represents the antigen (or the disease). to 65 years of age. In the Phase I Dose Escalation part, COVID-eVax is administered at 3 escalating doses (20 subjects/cohort), in a prime-boost setting (4 weeks apart), from 20 0.5 to 2 mg/dose. In addition, a cohort in a single 2 mg dose schedule will also be tested. The vaccine is administered by the intramuscular route followed by EP using the 19 IGEA new ElectroPoration System (EPSGun), and the EGT technology for pulse generation (Cliniporator®) commercially available in the EU. In both phases, subjects will be followed up for a total duration of 6 months after the first vaccination.

In summary, COVID-eVax is a highly efficient vaccination platform capable of inducing robust, protective neutralizing antibody and T cell responses in a variety of 5 animal models. COVID-eVax can be administered multiple times, without the risk of inducing antibody responses to the vaccine itself, which may happen in the case of virus-based vector vaccines. We believe that the DNA vaccination platform described here offers unique advantages over other candidate vaccines, such as rapid manufacturing in response to sequence mutations (compared to protein-or viral vector-10 based vaccines), and greater stability at room temperature (compared to RNA-based platforms). With an increasing number of people having been immunized against SARS-CoV-2 with an RNA-, adenovirus-, or protein-based vaccine, COVID-eVax might be also considered as an additional platform for booster immunizations to extend the duration of protective immunity. 15

We thank the entire Rottapharm Biotech and IGEA Teams for useful scientific and regulatory discussions and for setting up the EPSgun technology in a short time.

We also thank M. Mainetti, M. 

All data are available in the main text or the supplementary material. 

The synthesis and codon optimization analysis of a cDNA encoding the SARS-CoV-2 protein S has been performed at Genscript (China). All constructs were completely synthetic and optimized for codon usage. Codon-optimized variants took into 5 account codon usage bias, GC content, CpG dinucleotides content, mRNA secondary structure, cryptic splicing sites, premature PolyA sites, internal chi sites and ribosomal binding sites, negative CpG islands, RNA instability motif (ARE), repeat sequences (direct repeat, reverse repeat, and Dyad repeat) and restriction sites that may interfere with cloning. In addition, to improve translational initiation and performance, Kozak and Rats. After a suitable quarantine period, animals were divided in three different experimental groups (4 females/group) and immunized by intramuscular electroporation, alternating quadriceps at each vaccine administration. 15

Ferrets. Eight ferrets were immunized twice at day -42 and -14 with 400µg RBD intra-muscularly in the quadriceps muscle of the right leg using followed by electroporation. An additional eight ferrets remained unvaccinated.

The hCoV-19/Italy/LOM-UniSR-1/2020 (GISAID Accession ID: EPI_ISL_413489) isolate of SARS-CoV-2 was used in this study. Virus isolation studies were carried out in BSL-3 workspace and performed in Vero E6 cells, which were cultured at 37°C, 5% 37 CO2 in complete medium (DMEM supplemented with 10% FBS, 1% penicillin plus streptomycin, 1% L-glutamine). Virus stocks were titrated using both Plaque Reduction Assayurn (PRA, PFU/ml) and Endpoint Dilutions Assay (EDA, TCID50/ml). In PRA, confluent monolayers of Vero E6 cells were infected with eight 10-fold dilutions of virus stock. After 1 h of adsorption at 37°C, the cell-free virus was removed. Cells were then 5 incubated for 48 h in DMEM containing 2% FBS and 0.5% agarose. Cells were fixed and stained, and viral plaques were counted. In EDA, Vero E6 cells were seeded into 96 wells plates and infected at 95% of confluency with base 10 dilutions of virus stock.

After 1 h of adsorption at 37°C, the cell-free virus was removed, cells were washed with PBS 1X, and complete medium was added to cells. After 48 h, cells were observed to 10 evaluate the presence of a cytopathic effect (CPE). TCID50/ml of viral stocks were then determined by applying the Reed-Muench formula.

K18-hACE2 mice were immunized with 10 ug of COVID-eVax or saline solution twice 21 days apart intra-muscularly followed by electroporation as described above.

Virus infection was performed via intranasal administration of 1 x 10 5 TCID50 per mouse 15 under Isoflurane 2% (# IsoVet250) anesthesia. Mice were monitored to record body weight, clinical and respiratory parameters.

Eight ferrets were immunized twice at day -42 and -14 with 400µg RBD intra-muscularly in the quadriceps muscle of the right leg using followed by electroporation as described above. An additional eight ferrets remained unvaccinated. All ferrets were challenged Elmer) at 10 µl/g of body weight.

For the B cell Elispot assay, pools of sera collected from mice vaccinated with RBD or FL constructs were tested against each of the 338 peptides covering the entire 20

Spike protein, pre-coated on 96 well plate, in order to identify the linear epitopes.

Sequences of positive hits were then mapped on three-dimensional structure of Spike protein, hence outlining the epitope domains. The T cell ELISPOT for mouse IFNg was 39 performed as previously described 36 . RBD peptides are 132 out of the 338 peptides covering the whole Spike protein (from peptide nr.4 to peptide nr.136). In order to identify immunodominant RBD epitopes (here highlighted in yellow), Elispot assay was performed by stimulating splenocytes from RBD vaccinated Balb/c mice for 20h with RBD peptide pools. Pools (from 1 to 24) were distributed as a matrix (the intersection of 5 two pools identifies one RBD peptide), with each pool comprising up to 12 RBD peptides. Immunodominant RBD peptides were identified at the intersection of pools showing >50 SFCs.

Antibody titration was performed both on sera, obtained by retro-orbital bleeding, and on bronchoalveolar lavages (BALs), obtained by flushing 1ml PBS in the lungs. The In experiments performed with SARS-CoV-2-infected mice, lung was perfused through the right ventricle with PBS at the time of autopsy and after the brain was removed from the skull. Lung tissue was digested in RPMI 1640 containing 3.2 mg/ml Collagenase IV (Sigma) and 25 U/ml DNAse I (Sigma) for 30 minutes at 37°C. Brain was digested in 15 RPMI 1640 containing 1 mg/ml Collagenase D (Sigma) and 6,3 µg/ml DNAse I (Sigma) for 30 minutes at 37°C. Homogenized lung and brain were passed through 70 μm nylon mesh to obtain a single cell suspension. Cells were resuspended in 36% percoll solution (Sigma) and centrifuged for 20 minutes at 2000 rpm (light acceleration and low brake).

The remaining red blood cells were removed with ACK lysis. 20 

Whole-body plethysmography (WBP) was performed using WBP chamber (DSI Buxco respiratory solutions, DSI). First mice were allowed to acclimate inside the chamber for 10 minutes, then respiratory parameters were acquired for 15 minutes using FinePointe software. 15

Lungs of infected mice were collected and fixed in 4% paraformaldehyde (PFA).

Samples were then dehydrated in 30% sucrose prior to embedding in OCT freezing 20 media (Bio-Optica). Twenty micrometer sections were cut on a CM1520 cryostat (Leica) and adhered to Superfrost Plus slides (Thermo Scientific). Sections were then 47 permeabilized and blocked in PBS containing 0.3% Triton X-100 (Sigma-Aldrich) and 5% FBS followed by staining in PBS containing 0.3% Triton X-100 and 1% FBS. Slides were stained for SARS-CoV-2 nucleocapsid (GeneTex) for 1h RT. Then, slides were stained with Alexa Fluor 568 Goat Anti-Rabbit antibody for 2h RT. ) the whole S1 subunit (S1); 5) the RBD fused to a human IgG-Fc (RBD-Fc). The RBD, N/R and S1 constructs include a tPA leader sequence at the N-terminus, whereas the RBD-Fc construct contains a IgK leader sequence. (B) Western blot analysis of SARS-CoV-2 DNA vaccine constructs after transfection in HEK293 cells. Forty-eight hours after transfection, both cell lysates and supernatants were resolved on a gel and blotted with a polyclonal SARS-CoV Spike S1 Subunit antibody. Cells transfected with empty plasmid vector were used as negative control (control). Non-specific bands were detected both in cell lysates and in supernatants, likely due to non-specific binding of primary antibody. (C) Schematic representation of the experimental setup. Each DNA construct was injected intramuscularly (20 μg total, 10 μg each quadriceps) into BALB/c mice (n = 5) at day 0 (prime) and day 28 (boost). Intramuscular injection was followed by electroporation 

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