key: cord-0780122-mq6n2w45
authors: Dey, Ayan; Chozhavel Rajanathan, T.M.; Chandra, Harish; Pericherla, Hari P.R.; Kumar, Sanjeev; Choonia, Huzaifa S.; Bajpai, Mayank; Singh, Arun K.; Sinha, Anuradha; Saini, Gurwinder; Dalal, Parth; Vandriwala, Sarosh; Raheem, Mohammed A.; Divate, Rupesh D.; Navlani, Neelam L.; Sharma, Vibhuti; Parikh, Aashini; Prasath, Siva; Rao, Sankar; Maithal, Kapil
title: Immunogenic Potential of DNA Vaccine candidate, ZyCoV-D against SARS-CoV-2 in Animal Models
date: 2021-01-26
journal: bioRxiv
DOI: 10.1101/2021.01.26.428240
sha: 83c7efedafb1bed1a1d729764edb653806e02490
doc_id: 780122
cord_uid: mq6n2w45

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), initially originated in China in year 2019 and spread rapidly across the globe within 5 months, causing over 96 million cases of infection and over 2 million deaths. Huge efforts were undertaken to bring the COVID-19 vaccines in clinical development, so that it can be made available at the earliest, if found to be efficacious in the trials. We developed a candidate vaccine ZyCoV-D comprising of a DNA plasmid vector carrying the gene encoding the spike protein (S) of the SARS-CoV-2 virus. The S protein of the virus includes the receptor binding domain (RBD), responsible for binding to the human angiotensin converting enzyme (ACE-2) receptor. The DNA plasmid construct was transformed into E. coli cells for large scale production. The immunogenicity potential of the plasmid DNA has been evaluated in mice, guinea pig, and rabbit models by intradermal route at 25, 100 and 500μg dose. Based on the animal studies proof-of-concept has been established and preclinical toxicology (PCT) studies were conducted in rat and rabbit model. Preliminary animal study demonstrates that the candidate DNA vaccine induces antibody response including neutralizing antibodies against SARS-CoV-2 and also provided Th-1 response as evidenced by elevated IFN-γ levels.

Several pre-clinical or clinical trials are going on, which include repurposing of already approved drugs but with different indications such as anti-malarial, anti-viral, anti-parasitic drugs, or monoclonal antibodies, etc 6, 7 . However, these drugs may help to prevent worsening of the coronavirus infection only and there is still an unmet need of a vaccine against novel coronavirus SARS-CoV-2.

Huge progresses were made in last one year for bringing effective vaccines against SARS- Three candidates including two mRNA based candidate from Pfizer and Moderna and Chimpanzee adenovirus vector based candidate from AstraZeneca got emergency use approval globally. The emergency use was approved based on Phase-3 efficacy data. The mRNA vaccines from Pfizer reported 95% efficacy 9 , whereas Moderna and AstraZeneca reported 94.5 % and 70.4% efficacy respectively for their vaccine candidate 10 .

The conventional active vaccines are made of a killed or attenuated form of the infectious agent. Vaccination with live attenuated and killed vaccines in most cases results in generation of humoral but not a cell-mediated immune response. What is required in such cases, but not available, are antigens that are safe to use, that can be processed by the endogenous pathway and eventually activating both B and T cell response. The activated lymphocytes generated would destroy the pathogen-infected cell. For these reasons, a new approach of vaccination that involves the injection of a piece of DNA that contains the genes for the antigens of interest are under investigation. DNA vaccines are attractive because they ensure appropriate folding of the polypeptide, produce the antigen over long periods, and do not require adjuvants. These host-synthesized antigens then can become the subject of immune surveillance in the context of both major histocompatibility complex class I (MHC I) and

MHC II proteins of the vaccinated individual 11 . By contrast, standard vaccine antigens are taken up into cells by phagocytosis or endocytosis and are processed through the MHC class II system, which primarily stimulates antibody response. In addition to these properties, the plasmid vector contains immunostimulatory nucleotide sequences-unmethylated cytidine phosphate guanosine (CpG) motifs-that induce strong cellular immunity 12 . Finally, DNA vaccines have been shown to persist and stimulate sustained immune responses. Other advantages are that the technology for producing the vaccine is very simple and rapid, secondly the DNA molecule is stable, has a long shelf life, and does not require a strict cold chain for distribution. DNA vaccines are also safer than certain live-virus vaccines, especially in immunocompromised patients. It also circumvents the numerous problems associated with other vaccines, such as immune responses against the delivery vector and concern about safety related to the use of any viral vector 13 .

Prior studies have demonstrated that a DNA vaccine approach for SARS and MERS can induce immune response including neutralizing antibody (nAb) responses in clinical trials and provide protection in challenge models. Previous studies indicated immunization in animal models with DNA vaccines encoding MERS-CoV spike (S) protein provided protection against disease challenge with the wild type virus. In subjects immunized with MERS-CoV DNA vaccine durable neutralizing antibodies (nAbs) and T cell immune responses were measured, and a seroconversion rate of 96% was observed and immunity was followed for 60 weeks in most study volunteers 14 . Similarly, NIH completed Phase 1 clinical trial for SARS-DNA vaccine. Dose of 4.0 mg was tested in healthy adults who were vaccinated on days 0, 28, and 56. The vaccine was found to be well-tolerated and induced antibody responses against the SARS-CoV in 80% of subjects after 3 doses 15 . More recently, Inovio pharmaceuticals developed DNA vaccine INO-4800 against SARS-CoV-2, which was found to be safe and immunogenic in Phase-I trial, eliciting either or both humoral or cellular immune responses 16 .

The spike proteins of SARS-CoV-2 and SARS-CoV were reported to have identical 3-D structures in the receptor-binding domain. SARS-CoV spike protein has a strong binding affinity to human Angiotensin-converting enzyme 2 (ACE-2) receptor, based on biochemical interaction studies and crystal structure analysis. SARS-CoV-2 and SARS-CoV spike proteins have high degree of homology and they share more than 70% identity in amino acid sequences 17 . Further, Wan et al., reported that glutamine residue at position 394 (E394) in the SARS-CoV-2 receptor-binding domain (RBD), corresponding to E479 in SARS-CoV, which is recognized by the critical lysine residue at pos-31 (K31) on the human ACE-2 receptor.

Further analysis suggests that SARS-CoV-2 recognizes human ACE-2 receptor more efficiently than SARS-CoV increasing the ability of SARS-CoV-2 to transmit from person to person 18 . Thus, the SARS-CoV-2 spike protein was predicted to also have a strong binding affinity to human ACE-2 receptor.

ACE-2 is demonstrated as a functional SARS-CoV-2 spike (S) protein receptor in-vitro and in-vivo. It is required for host cell entry and subsequent viral replication. Zhou et al., 19 demonstrated that overexpressing ACE-2 receptor from different species in HeLa cells with human ACE-2, pig ACE-2, and civet ACE-2 receptor allowed SARS-CoV-2 infection and replication, thereby establishing that SARS-CoV-2 uses ACE-2 as a cellular entry receptor. In transgenic mice model with overexpression of human ACE-2 receptor, SARS-CoV infection enhanced disease severity and lung injury, demonstrating that viral entry into cells through ACE-2 receptor is a critical step 19, 20 . Thus for SARS-CoV-2 pathogenesis, spike (S) protein play a critical role by mediating entry of virus into the cell through human ACE-2 receptor and is an important target for vaccine development.

Here we report, design, production and pre-clinical testing of our DNA vaccine candidate. Culture from each clone was used for plasmid isolation using miniprep plasmid isolation kit.

Restriction digestion was carried out with BamH1, Nhe1 and Apa1 for all constructs to check expected band releases of inserts to select the positive clones. Positive clones were selected for preparation of glycerol stocks and stored at -70 0 C.

In-vitro expression of DNA vaccine candidate was checked by transfection of the same in vero cell line. For transfection experiments, vero cells were seeded at density of 3× 10 5 cells/ml in 6 well plates and kept in CO 2 incubator to attain 80-90% confluency. After 24Hrs, once the cells reached the desired confluency, transfection was carried out in OptiMEM serum free medium with Lipofectamine 2000 reagent (Thermo Fisher). Two different concentrations (4µg and 8µg) of DNA construct was used for transfection experiments. After transfection, media was replenished with fresh DMEM media (Biowest) containing FBS.

After 72Hrs, plates were fixed with 1:1 acetone and methanol Anti-S1 rabbit polyclonal antibody (Novus) was added to each well and incubated for 1Hr followed by incubation with FITC labelled anti-rabbit antibody (Merck). Fluorescence images were captured using an inverted microscope (ZeissAX10).

The immunogenicity study for the ZyCoV-D vaccine was carried out in inbred BALB/C mouse, guinea pig, and New Zealand white rabbit model after having ethical approval from Institutional Animal Ethics Committee, CPCSEA Reg. No.: 335/PO/RcBi/S/01/CPCSEA, with IAEC approved application numbers: VAC/010/2020 and VAC/013/2020. BALB/c mouse (five to seven-week-old), guinea pigs (five to seven-week-old) and New Zealand White rabbits (six to twelve-week-old) were used in this study. For mouse intradermal immunization, on day 0; 25 and 100 μg of DNA vaccine was administered to the skin by using 31 gauge needle. Animals injected with empty plasmid served as vehicle control. Two weeks after immunization, animals were given first booster dose. Similarly all mice were given second booster dose two weeks after first booster dose. For guinea pig study, intradermal immunization was carried out using same dosing and schedule. In rabbits, DNA vaccine was administered to the skin by using needle free injection system (NFIS) at 500 μg dose at same 3 dose regimen and schedule. Blood was collected from animals on day 0 (before immunization) & 28 (after 2 dose) and on day 42 (after 3 dose) for immunological assessments from sera samples. In mouse model long term immunogenicity of the vaccine was assessed for up to day 126. Further, IFN-γ response from splenocytes at day 0, 28, and 42 were assessed.

ELISA was performed to determine antibody titres in different animal sera samples. In brief, Maxisorp ELISA plates (Nunc) were coated with 50ng/well of recombinant S1 spike protein of SARS-CoV-2 (Acro, USA) in phosphate-buffered saline (PBS) overnight at 4 °C. Plates were washed three times with PBS then blocked with 5% skimmed milk (BD Difco) in PBS for 1 Hr at 37 °C. After blocking plates were then washed thrice with PBS and incubated with serial dilutions of mouse, guinea pig and rabbit sera and incubated for 2 Hrs at 37 °C. After that, plates were again washed thrice followed by incubation with 1:5,000 dilution of horse radish peroxidase (HRP) conjugated anti-guinea pig IgG secondary antibody (Sigma-Aldrich) or 1:2,000 dilution of HRP conjugated anti-mouse IgG secondary antibody (Sigma-Aldrich) or 1:5,000 dilution of HRP conjugated anti rabbit IgG secondary antibody (Sigma-Aldrich) for 1 Hr at 37 °C. After that again plates were washed thrice with PBS and then developed 

In-silico analysis confirmed more than 99% homology of the spike protein amino acid sequence from Wuhan strain with other circulating strains world including India. Humoral immune response to DNA vaccine candidate Immunization with DNA vaccine candidate by intradermal route elicited significant serum IgG responses against the S protein in doses-dependent manner in BALB/c mice, guinea pigs and rabbits with mean end point titres reaching ~28000 in BALB/C mice, ~140000 in guinea pigs and ~17000 in rabbits respectively on day 42 after 3 doses (Figure 3, 4 and Figure 5 ).

Long term antibody response was studied in mice almost 3.5 months after the last dose and a mean end point IgG titres of ~18000 was detected (Figure 3 ) suggesting sustainable immune response was generated by DNA vaccine candidate.

Neutralizing antibody titres were evaluated in BALB/C mice, guinea pigs and rabbits following immunization by using micro-neutralization assay and Genscript neutralizing antibody detection kit. Neutralizing antibodies were elicited by DNA vaccine candidate in mice, guinea pigs and rabbits. Sera from DNA vaccine candidate immunized BALB/c mice could neutralize wild SARS-CoV-2 virus strains with average MNT titres of 40 and 160 at day 42 with 25 and 100 μg dose regimens respectively (Table 1 ). Using Genscript neutralizing antibody detection kit average IC 50 titres of 82 and 168 were obtained at day 42 with 25 and 100 μg dose regimens respectively. Further, neutralizing antibodies were also detected in long term immunogenicity studies in BALB/c mice. Significant rise in neutralizing antibodies levels were also observed in guinea pigs and rabbits (Table 1) .

Cellular immune response to DNA vaccine candidate T cell response against SARS-CoV-2 spike antigen was studied by IFN-γ ELISpot assay. samples including the site of injection, brain, blood, lungs, intestine, kidney, heart and spleen at different time points post injection as described above. RT-PCR was performed by plasmid specific primers to detect copy numbers. Following injection of maximum ~ 10 14 plasmid DNA copies in Wistar Rats, maximum local concentration of 10 3 -10 7 plasmid copies at the site of injection were detected two hours post injection. (Figures 7A and 7B) . We also observed bio-distribution of plasmid molecule in blood, lungs, intestine, kidney, heart, spleen, skin post 24 Hrs of injection which got cleared by 336 Hrs (day 14) in most of the organs except skin (site of injection) where only 10 2 -10 3 copies were detected. Although by 672 Hrs (day 28) post injection, no plasmid copies were detected at site of injection as well.

Development of safe and effective vaccine against SARS-CoV-2 is need of hour to curb the global pandemic. DNA vaccine platform has several advantages, which positions it well to respond to disease outbreaks, such as COVID-19. The ability to design and immediately synthesize candidate vaccine constructs allow us to carry out in-vitro and in-vivo testing within days of receiving the viral sequence. The expression and localization of S protein expressed by ZyCoV-D were investigated using an immunofluorescence assay. The immunofluorescence assay with rabbit anti-S1 antibody revealed a strong signal in the vero cells transfected with ZyCoV-D. In contrast, the positive signal was not detected in cells transfected with control vector. This demonstrates the ability of the ZyCoV-D vaccine to express strongly in mammalian cells and that antibodies induced by this construct can bind their target antigen. Further, the DNA plasmid manufacturing process is easily scalable with substantial yields, and has the potential to overcome the challenges of conventional vaccine production in eggs or cell culture. Additionally, we have also studied stability profile of our vaccine candidate (unpublished data). The stability data suggests that our DNA vaccine candidate can be stored at 2-8 °C for long term and further at 25°C for short term (few months). In the context of a pandemic outbreak, the stability profile of a vaccine plays a vital role easy deployment and distribution for mass vaccination. Further, we will like to highlight that ZyCoV-D was developed using a pVAX-1 vector, which has been used in number of other DNA vaccines in past and have been proven to be very safe for human use 14, 21 .

ZyCoV-D was evaluated in-vivo in different animal models and has demonstrated ability to elicit immunogenic response against SARS-CoV-2, S-antigen in animal species. Primary antibody response starts mounting in serum two weeks after two doses and reaches pick two weeks after third immunization. The serum IgG levels against spike antigen in mice were maintained even after three months post last dosing suggesting a long-term immune response generated by the DNA vaccine candidate. This also indicates that ZyCoV-D can possibly induce robust secondary anamnestic immune response upon re-exposure, generated by balanced memory B and helper T cells expression and has been reported for other DNA vaccine candidates 22 .

We reported serum neutralizing (Nab) titres following DNA vaccination, which was tested by micro-neutralization assay and Genscript neutralizing antibody detection kit. The Nab titre values tested by both methods demonstrated that the DNA vaccine candidate generates robust response and neutralizes the SARS CoV-2 virus conferring protective immunity against infection. In future if these Nab titres will be established as correlate of protection across multiple vaccine studies in both animals and humans, then this parameter can be utilized as a benchmark for clinical development of SARS-CoV-2 vaccines.

We also observed that ZyCoV-D vaccine is capable of inducing T-cell response complementary to antibody response in mice model as demonstrated by IFN-γ ELISPOT. This is very important as successful DNA vaccination is known to induce both humoral and cellular responses in both animals and human 14, 15, 16, 23 . The mechanism of action for DNA vaccine candidate includes both class-I antigen-processing pathways (i.e., intracellular processing of viral proteins and subsequent loading onto MHC class-I molecules) and class-II antigen-processing pathways (i.e., endosomal loading of peptides generated from Bio-distribution pattern for ZyCoV-D was also evaluated and level of plasmid DNA was measured at different intervals in various tissues in Wistar rats post intradermal injection.

Post intradermal injection, the plasmid was found to clear off from most of the organs by 14 days post injection except site of injection, which also cleared off by 28 days post injection.

Our outcome was very similar to other DNA vaccine candidate including HIV-1, Ebola, Severe Acute Respiratory Syndrome (SARS), and a West Nile Virus candidate developed 32 .

Biodistribution and plasmid copy number detection studies for the vaccine candidates were done in different animal models 32 . It was observed that animals injected with 2mg (equivalent to 10 14 plasmid copies) by both intramuscular and subcutaneous route have detectable plasmid copies in first one week of vaccination at the site of injection with copies in order of 10 4 -10 6 . Over the period of 2 months, the plasmid clears from the site of injection with only a small percentage of animals in group (generally 10-20%) retaining few copies (around 100 copies) at the injection site. Directly after injection into skin or muscle, low levels of plasmids are transported via the blood stream and detected in various organs at early time points. However, the plasmids are eventually, cleared from the organs and are normally found exclusively at the site of injection at later time points.

In summary, these initial results demonstrate the immunogenicity of our ZyCoV-D DNA vaccine candidate in multiple animal models. These studies strongly support the clinical evaluation as a vaccine candidate for COVID-19 infection. were collected at day 28 (orange), day 42 (blue) and day 126 (green) evaluated for SARS-CoV-2 S1-specific IgG antibodies. Figure 4 -Antibody response after DNA vaccination in Guinea Pigs. Guinea pigs were immunized at week 0, 2 and 4 with DNA vaccine construct or empty control vector as described in the methods. Sera were collected at day 28 (blue) and day 42 (pink) and evaluated for SARS-CoV-2 S1-specific IgG antibodies.

White Rabbits were immunized at week 0, 2 and 4 with DNA vaccine construct or empty control vector as described in the methods. Sera were collected at day 28 (pink) and day 42 (green) and evaluated for SARS-CoV-2 S1-specific IgG antibodies. Figure 7B -Bio-distribution of DNA vaccine after 0.5mg dose. Plasmid copy levels as determined by RT-PCR, in the tissue samples at site of injection, brain, blood, lungs, intestine, kidney, heart and spleen at different time point after injection in animals. Table 1 

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