Rapid development of a synthetic DNA vaccine for COVID-19

The coronavirus family member, SARS-CoV-2 has been identified as the causal agent for the outbreak of viral pneumonia disease, COVID-19 which first emerged in mid-December 2019 in the city of Wuhan in central China. As of February 25, 2020 there are 80,994 people infected and 2,760 deaths, and documented human-to-human transmission across multiple continents. At this time, no vaccine is available to control further dissemination of the disease. We have previously developed a synthetic DNA vaccine targeting the MERS coronavirus Spike (S) protein that was deployed in response to the MERS outbreak in South Korea. This vaccine induced potent antibody and CTL responses, and provided protection in a NHP challenge model. In the clinic, the vaccine generated humoral immunity including neutralizing antibody responses, as well as T cell immunity. Here we build on this prior work and report on the rapid development of a synthetic DNA-based vaccine targeting the major surface antigen Spike protein of SARS-CoV-2. The engineered construct, INO-4800 induced robust expression of the Spike protein in vitro, and generated antibody and T cell responses following a single immunization in mice and guinea pigs. This preliminary dataset identifies INO-4800 as a potential COVID-19 vaccine candidate, supporting further study for mobilization against this emerging disease threat. All in vitro and in vivo studies described in the current manuscript were executed within 6 weeks of the SARS-CoV–2 genome sequence becoming available. Our data support the expression and immunogenicity of the INO–4800 synthetic DNA vaccine candidate in multiple animal models. Robust humoral and T cells responses were observed in mice after a single dose. In guinea pigs we employed clinical delivery parameters, and observed robust antibody titers after a single dose.

8 vaccine candidates. We show the expression of the SARS-CoV-2 S antigen RNA and protein after in vitro transfection of COS and 293T cells, respectively with the vaccine candidates.
We followed the induction of immunity by the selected immunogens in mice and guinea pigs. The data demonstrate that the synthetic DNA SARS-CoV-2 S antigen vaccine induces robust cellular and humoral host immune responses that can be observed within days following a single immunization. T cell and B cell responses are highly potent for the SARS-CoV-2 S protein and responses cross react at a lower level against SARS-CoV S protein.

Results
Design and synthesis COVID-19 synthetic DNA vaccine constructs Four spike protein sequences were retrieved from the first four available SARS-CoV-2 full genome sequences published on GISAID (Global Initiative on Sharing All Influenza Data).
Three Spike sequences were 100% matched and one was considered an outlier (98.6% sequence identity with the other sequences). After performing a sequence alignment, the SARS-CoV-2 spike glycoprotein sequence was generated and an N-terminal IgE leader sequence was added. The highly optimized DNA sequence encoding SARS-CoV-2 IgE-spike was created using Inovio's proprietary in silico Gene Optimization Algorithm to enhance expression and immunogenicity. The optimized DNA sequence was synthesized, digested with BamHI and XhoI, and cloned into the expression vector pGX0001 under the control of the human cytomegalovirus immediate-early promoter and a bovine growth hormone polyadenylation signal. The resulting plasmids were designated as pGX9501 and pGX9503, designed to encode the SARS-CoV-2 S protein from the 3 matched sequences and the outlier sequence, respectively (Figure 2a)..

In vitro characterization of COVID-19 synthetic DNA vaccine constructs
We measured the expression of the encoded SARS-CoV-2 spike transgene at the RNA level in COS-7 cells transfected with pGX9501 and pGX9503. Using the total RNA extracted from the transfected COS-7 cells we confirmed expression of the spike transgene by RT-PCR ( Figure 2b). In vitro spike protein expression in 293T cells was measured by Western blot analysis using a cross-reactive antibody against SARS-CoV S protein on cell lysates. HEK-293T cells transfected with pGX9501 or pGX9503 constructs expressed the S protein at the predicted molecular weight, 140-142 kDa (Figure 2c).. In immunofluorescent studies the S protein was detected in 293T cells transfected with pGX9501 or pGX9503 ( Figure   2d).. In summary, in vitro studies revealed the expression of the Spike protein at both the RNA and protein level after transfection of cell lines with the candidate vaccine constructs.
Robust humoral immune responses to SARS-CoV-2 S protein antigens measured in mice immunized with INO-4800 Since candidate design, it has been observed that newly published SARS-CoV-2 Spike protein sequences match pGX9501 with >99.7% amino acid sequence identity. pGX9501 was therefore selected as the vaccine construct to advance to immunogenicity studies, due to the broader coverage it would likely provide compared to the outlier, pGX9503.
pGX9501 was subsequently termed INO-4800. The immunogenicity of INO-4800 was evaluated in BALB/c mice, post-administration to the TA muscle followed with CELLECTRA® delivery device 16 . The reactivity of the sera from a group of mice immunized with INO-4800 was measured against a panel of SARS-CoV-2 and SARS-CoV antigens ( Figure 3). Analysis revealed robust IgG binding against SARS-CoV-2 S protein antigens, with limited cross-reactivity to SARS-CoV S protein antigens, in the serum of INO-4800 immunized mice. We proceeded to measure the serum IgG binding endpoint titers in mice immunized with pDNA against recombinant SARS-CoV-2 spike protein S1+S2 regions In summary, rapid and robust T cell responses against SARS-CoV-2 S protein epitopes were detected in mice immunized with INO-4800.

Discussion
The novel coronavirus, SARS-CoV-2, has spread across China, and as of this writing, a rapidly increasing number of infections and associated COVID-19 disease are being reported across the globe. Currently, there are no COVID-19 vaccines available, and global dissemination of SARS-CoV-2 may continue until there is a high level of herd immunity within the human population. Here, we have described accelerated preclinical development of a synthetic DNA-based COVID-19 vaccine, INO-4800 to combat this emerging infectious disease. Synthetic DNA vaccine design and synthesis was immediately initiated upon public release of the SARS-CoV-2 genome sequences on January 11, 2020.
All in vitro and in vivo studies described in the current manuscript were executed within 6 weeks of the SARS-CoV-2 genome sequence becoming available. Our data support the expression and immunogenicity of the INO-4800 synthetic DNA vaccine candidate in multiple animal models. Robust humoral and T cells responses were observed in mice after a single dose. In guinea pigs we employed clinical delivery parameters, and observed robust antibody titers after a single dose.
Halting a rapidly emerging infectious disease requires an orchestrated response from the global health community and requires improved strategies to accelerate vaccine development. In response to the 2019/2020 coronavirus outbreak we immediately employed our highly adaptable synthetic DNA medicine platform. The design and manufacture of synthetic DNA vaccines for novel antigens is a plug and play process in which we insert the target antigen sequence into a highly characterized and clinicallytested plasmid vector backbone (pGX0001). The construct design and engineering parameters have been optimized for in vivo gene expression, and previously applied to MERS, EBOV, Zika and Lassa DNA vaccine constructs which are all undergoing clinical testing 7,9,11,19,20 .
Based upon our previous experience developing a vaccine against MERS coronavirus, and previous published studies of SARS vaccines, SARS-CoV-2 S protein was chosen as the antigen target. The SARS-CoV-2 S protein is a class I membrane fusion protein, which the major envelope protein on the surface of coronaviruses. Initial studies have already been performed which indicate SARS-CoV-2 interaction with its host receptor (ACE-2) can be blocked by antibodies 21 . In vivo immunogenicity studies in both mouse and guinea pig models revealed robust levels of S protein-reactive IgG in the serum of INO-4800 immunized animals. In addition to full-length S1+S2 and S1, INO-4800 immunization induced potent RBD binding antibodies (Figures 3&4),, a domain known to be a target for neutralizing antibodies from SARS-CoV convalescent patients 22,23  In addition to the ability of INO-4800 to rapidly elicit humoral and cellular responses following a single immunization, our synthetic DNA medicine platform has several synergistic characteristics which position it well to respond to disease outbreaks, such as COVID-19. As mentioned previously, the ability to design and synthesize candidate vaccine constructs means that in vitro and in vivo testing can potentially begin within days of receiving the viral sequence, allowing for an accelerated response to vaccine development. The well-defined and established production processes for DNA plasmid manufacture result in a rapid and scalable manufacture process which has the potential to circumvent the complexities of conventional vaccine production in eggs or cell culture.
The cost of goods related to DNA manufacture is also significantly lower than currently seen for mRNA-based technologies. We have recently published on the stability profile afforded to our products through the use of our optimized DNA formulation 7 . The stability characteristics mean that our DNA drug product is non-frozen and can be stored for 4.5+ years at 2-8°C, room temperature for 1 year and 1 week at 37°C, while maintaining potency at temperatures upwards of 60°C. In the context of a pandemic outbreak, the stability profile of a vaccine plays directly to its ability to be deployed and stockpiled in an efficient and executable manner.
Additional preclinical studies are ongoing to further characterize INO-4800 in small and larger animals. Availability of reagents is a major challenge for development of vaccines against newly emerging infectious diseases. For initial vaccine expression studies, we successfully used a cross-reactive SARS-CoV polyclonal antibody for detection. Once the tools become available, studies will begin to determine the functionality of the antibodies raised in animals immunized with INO-4800 in terms of virus neutralization. The ability of INO-4800 immunization to mediate protection against viral challenge will be assessed in multiple animal models. Even though we are still in the early days of this outbreak the scientific community has been rapidly mobilized, and we believe the tools to do these studies will be available soon.
In summary, the initial result describing the immunogenicity of COVID-19 vaccine