SARS-CoV-2 DNA Vaccine INO-4800 Induces Durable Immune Responses Capable of Being Boosted in a Phase 1 Open-Label Trial.

BACKGROUND: Additional severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines that are safe and effective as primary vaccines and boosters remain urgently needed to combat the coronavirus disease 2019 (COVID-19) pandemic. We describe safety and durability of immune responses following 2 primary doses and a homologous booster dose of an investigational DNA vaccine (INO-4800) targeting full-length spike antigen. METHODS: Three dosage strengths of INO-4800 (0.5 mg, 1.0 mg, and 2.0 mg) were evaluated in 120 age-stratified healthy adults. Intradermal injection of INO-4800 followed by electroporation at 0 and 4 weeks preceded an optional booster 6-10.5 months after the second dose. RESULTS: INO-4800 appeared well tolerated with no treatment-related serious adverse events. Most adverse events were mild and did not increase in frequency with age and subsequent dosing. A durable antibody response was observed 6 months following the second dose; a homologous booster dose significantly increased immune responses. Cytokine-producing T cells and activated CD8+ T cells with lytic potential were significantly increased in the 2.0-mg dose group. CONCLUSIONS: INO-4800 was well tolerated in a 2-dose primary series and homologous booster in all adults, including elderly participants. These results support further development of INO-4800 for use as primary vaccine and booster. CLINICAL TRIALS REGISTRATION: NCT04336410.

Development of Novel DNA-Encoded PCSK9 Monoclonal Antibodies as Lipid-Lowering Therapeutics.

Elevated low-density lipoprotein cholesterol (LDL-C) is one of the major contributors to cardiovascular heart disease (CHD), the leading cause of death worldwide. Due to severe side effects of statins, alternative treatment strategies are required for statin-intolerant patients. Monoclonal antibodies (mAbs) targeting proprotein convertase subtilisin/kexin type 9 (PCSK9) have shown great efficacy in LDL-C reduction. Limitations for this approach include the need for multiple injections as well as increased costs associated with patient management. Here, we engineered a DNA-encoded mAb (DMAb) targeting PCSK9 (daPCSK9), as an alternative approach to protein-based lipid-lowering therapeutics, and we characterized its expression and activity. A single intramuscular administration of mouse daPCSK9 generated expression in vivo for over 42 days that corresponded with a substantial decrease of 28.6% in non-high-density lipoprotein cholesterol (non-HDL-C) and 10.3% in total cholesterol by day 7 in wild-type mice. Repeated administrations of the DMAb plasmid led to increasing expression, with DMAb levels of 7.5 µg/mL at day 62. daPCSK9 therapeutics may provide a novel, simple, less frequent, cost-effective approach to reducing LDL-C, either as a stand-alone therapy or in combination with other LDL-lowering therapeutics for synergistic effect.

In Vivo Delivery of a DNA-Encoded Monoclonal Antibody Protects Non-human Primates against Zika Virus.

Zika virus (ZIKV) infection is endemic to several world regions, and many others are at high risk for seasonal outbreaks. Synthetic DNA-encoded monoclonal antibody (DMAb) is an approach that enables in vivo delivery of highly potent mAbs to control infections. We engineered DMAb-ZK190, encoding the mAb ZK190 neutralizing antibody, which targets the ZIKV E protein DIII domain. In vivo-delivered DMAb-ZK190 achieved expression levels persisting >10 weeks in mice and >3 weeks in non-human primate (NHPs), which is protective against ZIKV infectious challenge. This study is the first demonstration of infectious disease control in NHPs following in vivo delivery of a nucleic acid-encoded antibody, supporting the importance of this new platform.

Synergy of Immune Checkpoint Blockade with a Novel Synthetic Consensus DNA Vaccine Targeting TERT.

Immune checkpoint blockade antibodies are setting a new standard of care for cancer patients. It is therefore important to assess any new immune-based therapies in the context of immune checkpoint blockade. Here, we evaluate the impact of combining a synthetic consensus TERT DNA vaccine that has improved capacity to break tolerance with immune checkpoint inhibitors. We observed that blockade of CTLA-4 or, to a lesser extent, PD-1 synergized with TERT vaccine, generating more robust anti-tumor activity compared to checkpoint alone or vaccine alone. Despite this anti-tumor synergy, none of these immune checkpoint therapies showed improvement in TERT antigen-specific immune responses in tumor-bearing mice. αCTLA-4 therapy enhanced the frequency of T-bet+/CD44+ effector CD8+ T cells within the tumor and decreased the frequency of regulatory T cells within the tumor, but not in peripheral blood. CTLA-4 blockade synergized more than Treg depletion with TERT DNA vaccine, suggesting that the effect of CTLA-4 blockade is more likely due to the expansion of effector T cells in the tumor rather than a reduction in the frequency of Tregs. These results suggest that immune checkpoint inhibitors function to alter the immune regulatory environment to synergize with DNA vaccines, rather than boosting antigen-specific responses at the site of vaccination.

Antiviral effects of autologous CD4 T cells genetically modified with a conditionally replicating lentiviral vector expressing long antisense to HIV.

We report the safety and tolerability of 87 infusions of lentiviral vector­modified autologous CD4 T cells (VRX496-T; trade name, Lexgenleucel-T) in 17 HIV patients with well-controlled viremia. Antiviral effects were studied during analytic treatment interruption in a subset of 13 patients. VRX496-T was associated with a decrease in viral load set points in 6 of 8 subjects (P = .08). In addition, A → G transitions were enriched in HIV sequences after infusion, which is consistent with a model in which transduced CD4 T cells exert antisense-mediated genetic pressure on HIV during infection. Engraftment of vector-modified CD4 T cells was measured in gut-associated lymphoid tissue and was correlated with engraftment in blood. The engraftment half-life in the blood was approximately 5 weeks, with stable persistence in some patients for up to 5 years. Conditional replication of VRX496 was detected periodically through 1 year after infusion. No evidence of clonal selection of lentiviral vector­transduced T cells or integration enrichment near oncogenes was detected. This is the first demonstration that gene-modified cells can exert genetic pressure on HIV. We conclude that gene-modified T cells have the potential to decrease the fitness of HIV-1 and conditionally replicative lentiviral vectors have a promising safety profile in T cells.

Patient monitoring and follow-up in lentiviral clinical trials.

BACKGROUND: Lentiviral vectors are being used with increasing frequency in human clinical trials. We were the first to use lentiviral vectors in clinical trials in 2003. Our lentiviral vector encoded a long RNA antisense sequence to the HIV-1 envelope and was used in an ex vivo autologous setting to provide viral load control in HIV-1 positive subjects failing anti-HIV therapy. A total of 65 subjects have been treated in Phase 1 and Phase 2 trials in six institutions. METHODS: Good manufacturing practices (GMP) lots of the lentiviral vector used in our clinical trials were assayed for the presence of replication competent lentivirus (RCL). RCL assays were conducted at two stages. The first testing was performed on samples collected immediately following bulk harvest of the GMP product lot and consisted of 1 × 10(8) cells used in production. RCL assays were also performed on aliquots of the final fill of the vector by the inoculation of at least 5% of the GMP final fill volume into C8166 cells, passaged for at least ten passages and tested for RCL by p24 enzyme-linked immunosorbent assay and vesicular stomatitis virus-G envelope DNA. RESULTS: Following 263 infusions of autologous, transduced cells, no adverse events have been detected in these subjects, with some followed for more than 8 years following infusions. More than 4.3 × 10(12) VRX496 proviral copies were administered to these 65 subjects. CONCLUSIONS: Data from this small population suggest that there is no apparent risk for serious adverse events with the use of lentiviral vectors.

Delineation of DNA and mRNA COVID-19 vaccine-induced immune responses in preclinical animal models.

Nucleic acid vaccines are designed based on genetic sequences (DNA or mRNA) of a target antigen to be expressed in vivo to drive a host immune response. In response to the COVID-19 pandemic, mRNA and DNA vaccines based on the SARS-CoV-2 Spike antigen were developed. Surprisingly, head-to-head characterizations of the immune responses elicited by each vaccine type has not been performed to date. Here, we have employed a range of preclinical animal models including the hamster, guinea pig, rabbit, and mouse to compare and delineate the immune response raised by DNA, administered intradermally (ID) with electroporation (EP) and mRNA vaccines (BNT162b2 or mRNA-1273), administered intramuscularly (IM), expressing the SARS-CoV-2 WT spike antigen. The results revealed clear differences in the quality and magnitude of the immune response between the two vaccine platforms. The DNA vaccine immune response was characterized by strong T cell responses, while the mRNA vaccine elicited robust humoral responses. The results may assist in guiding the disease target each vaccine type may be best matched against and suggest mechanisms to further enhance the breadth of each platform's immune response.

Engineered DNA-encoded monoclonal antibodies targeting Plasmodium falciparum circumsporozoite protein confer single dose protection in a murine malaria challenge model.

Novel approaches for malaria prophylaxis remain important. Synthetic DNA-encoded monoclonal antibodies (DMAbs) are a promising approach to generate rapid, direct in vivo host-generated mAbs with potential benefits in production simplicity and distribution coupled with genetic engineering. Here, we explore this approach in a malaria challenge model. We engineered germline-reverted DMAbs based on human mAb clones CIS43, 317, and L9 which target a junctional epitope, major repeat, and minor repeat of the Plasmodium falciparum circumsporozoite protein (CSP) respectively. DMAb variants were encoded into a plasmid vector backbone and their expression and binding profiles were characterized. We demonstrate long-term serological expression of DMAb constructs resulting in in vivo efficacy of CIS43 GL and 317 GL in a rigorous mosquito bite mouse challenge model. Additionally, we engineered an Fc modified variant of CIS43 and L9-based DMAbs to ablate binding to C1q to test the impact of complement-dependent Fc function on challenge outcomes. Complement knockout variant DMAbs demonstrated similar protection to that of WT Fc DMAbs supporting the notion that direct binding to the parasite is sufficient for the protection observed. Further investigation of DMAbs for malaria prophylaxis appears of importance.

Comparison of Wild Type DNA Sequence of Spike Protein from SARS-CoV-2 with Optimized Sequence on The Induction of Protective Responses Against SARS-Cov-2 Challenge in Mouse Model.

Genetic optimization of Nucleic Acid immunogens is important for potentially improving their immune potency. A COVID-19 DNA vaccine is in phase III clinical trial which is based on a promising highly developable technology platform. Here, we show optimization in mice generating a pGX-9501 DNA vaccine encoding full-length spike protein, which results in induction of potent humoral and cellular immune responses, including neutralizing antibodies, that block hACE2-RBD binding of live CoV2 virus in vitro. Optimization resulted in improved induction of cellular immunity by pGX-9501 as demonstrated by increased IFN-γ expression in both CD8+ and CD4 + T cells and this was associated with more robust antiviral CTL responses compared to unoptimized constructs. Vaccination with pGX-9501 induced subsequent protection against virus challenge in a rigorous hACE2 transgenic mouse model. Overall, pGX-9501 is a promising optimized COVID-19 DNA vaccine candidate inducing humoral and cellular immunity contributing to the vaccine's protective effects.

Mucosal chemokine adjuvant enhances synDNA vaccine-mediated responses to SARS-CoV-2 and provides heterologous protection in vivo.

The global coronavirus disease 2019 (COVID-19) pandemic has claimed more than 5 million lives. Emerging variants of concern (VOCs) continually challenge viral control. Directing vaccine-induced humoral and cell-mediated responses to mucosal surfaces may enhance vaccine efficacy. Here we investigate the immunogenicity and protective efficacy of optimized synthetic DNA plasmids encoding wild-type severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein (pS) co-formulated with the plasmid-encoded mucosal chemokine cutaneous T cell-attracting chemokine (pCTACK; CCL27). pCTACK-co-immunized animals exhibit increased spike-specific antibodies at the mucosal surface and increased frequencies of interferon gamma (IFNγ)+ CD8+ T cells in the respiratory mucosa. pCTACK co-immunization confers 100% protection from heterologous Delta VOC challenge. This study shows that mucosal chemokine adjuvants can direct vaccine-induced responses to specific immunological sites and have significant effects on heterologous challenge. Further study of this unique chemokine-adjuvanted vaccine approach in the context of SARS-CoV-2 vaccines is likely important.

Induction of tier-2 neutralizing antibodies in mice with a DNA-encoded HIV envelope native like trimer.

HIV Envelope (Env) is the main vaccine target for induction of neutralizing antibodies. Stabilizing Env into native-like trimer (NLT) conformations is required for recombinant protein immunogens to induce autologous neutralizing antibodies(nAbs) against difficult to neutralize HIV strains (tier-2) in rabbits and non-human primates. Immunizations of mice with NLTs have generally failed to induce tier-2 nAbs. Here, we show that DNA-encoded NLTs fold properly in vivo and induce autologous tier-2 nAbs in mice. DNA-encoded NLTs also uniquely induce both CD4 + and CD8 + T-cell responses as compared to corresponding protein immunizations. Murine neutralizing antibodies are identified with an advanced sequencing technology. The structure of an Env-Ab (C05) complex, as determined by cryo-EM, identifies a previously undescribed neutralizing Env C3/V5 epitope. Beyond potential functional immunity gains, DNA vaccines permit in vivo folding of structured antigens and provide significant cost and speed advantages for enabling rapid evaluation of new HIV vaccines.

Prime-boost vaccination regimens with INO-4800 and INO-4802 augment and broaden immune responses against SARS-CoV-2 in nonhuman primates.

The enhanced transmissibility and immune evasion associated with emerging SARS-CoV-2 variants demands the development of next-generation vaccines capable of inducing superior protection amid a shifting pandemic landscape. Since a portion of the global population harbors some level of immunity from vaccines based on the original Wuhan-Hu-1 SARS-CoV-2 sequence or natural infection, an important question going forward is whether this immunity can be boosted by next-generation vaccines that target emerging variants while simultaneously maintaining long-term protection against existing strains. Here, we evaluated the immunogenicity of INO-4800, our synthetic DNA vaccine candidate for COVID-19 currently in clinical evaluation, and INO-4802, a next-generation DNA vaccine designed to broadly target emerging SARS-CoV-2 variants, as booster vaccines in nonhuman primates. Rhesus macaques primed over one year prior with the first-generation INO-4800 vaccine were boosted with either INO-4800 or INO-4802 in homologous or heterologous prime-boost regimens. Both boosting schedules led to an expansion of T cells and antibody responses which were characterized by improved neutralizing and ACE2 blocking activity across wild-type SARS-CoV-2 as well as multiple variants of concern. These data illustrate the durability of immunity following vaccination with INO-4800 and additionally support the use of either INO-4800 or INO-4802 in prime-boost regimens.

Nucleic acid delivery of immune-focused SARS-CoV-2 nanoparticles drives rapid and potent immunogenicity capable of single-dose protection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines may target epitopes that reduce durability or increase the potential for escape from vaccine-induced immunity. Using synthetic vaccinology, we have developed rationally immune-focused SARS-CoV-2 Spike-based vaccines. Glycans can be employed to alter antibody responses to infection and vaccines. Utilizing computational modeling and in vitro screening, we have incorporated glycans into the receptor-binding domain (RBD) and assessed antigenic profiles. We demonstrate that glycan-coated RBD immunogens elicit stronger neutralizing antibodies and have engineered seven multivalent configurations. Advanced DNA delivery of engineered nanoparticle vaccines rapidly elicits potent neutralizing antibodies in guinea pigs, hamsters, and multiple mouse models, including human ACE2 and human antibody repertoire transgenics. RBD nanoparticles induce high levels of cross-neutralizing antibodies against variants of concern with durable titers beyond 6 months. Single, low-dose immunization protects against a lethal SARS-CoV-2 challenge. Single-dose coronavirus vaccines via DNA-launched nanoparticles provide a platform for rapid clinical translation of potent and durable coronavirus vaccines.

Immunotherapy of prostate cancer using novel synthetic DNA vaccines targeting multiple tumor antigens.

Prostate cancer is a prevalent cancer in men and consists of both indolent and aggressive phenotypes. While active surveillance is recommended for the former, current treatments for the latter include surgery, radiation, chemo and hormonal therapy. It has been observed that the recurrence in the treated patients is high and results in castration resistant prostate cancer for which treatment options are limited. This scenario has prompted us to consider immunotherapy with synthetic DNA vaccines, as this approach can generate antigen-specific tumor-killing immune cells. Given the multifocal and heterogeneous nature of prostate cancer, we hypothesized that synthetic DNA vaccines targeting different prostate specific antigens are likely to induce broader and improved immunity who are at high risk as well as advanced clinical stage of prostate cancer, compared to a single antigen approach. Utilizing a bioinformatics approach, synthetic enhanced DNA vaccine (SEV) constructs were generated against STEAP1, PAP, PARM1, PSCA, PCTA and PSP94. Synthetic enhanced vaccines for prostate cancer antigens were shown to elicit antigen-specific immune responses in mice and the anti-tumor activity was evident in a prostate tumor challenge mouse model. These studies support further evaluation of the DNA tools for immunotherapy of prostate cancer and perhaps other cancers.

Multivalent DNA Vaccines as A Strategy to Combat Multiple Concurrent Epidemics: Mosquito-Borne and Hemorrhagic Fever Viruses.

The emergence of multiple concurrent infectious diseases localized in the world creates a complex burden on global public health systems. Outbreaks of Ebola, Lassa, and Marburg viruses in overlapping regions of central and West Africa and the co-circulation of Zika, Dengue, and Chikungunya viruses in areas with A. aegypti mosquitos highlight the need for a rapidly deployable, safe, and versatile vaccine platform readily available to respond. The DNA vaccine platform stands out as such an application. Here, we present proof-of-concept studies from mice, guinea pigs, and nonhuman primates for two multivalent DNA vaccines delivered using in vivo electroporation (EP) targeting mosquito-borne (MMBV) and hemorrhagic fever (MHFV) viruses. Immunization with MMBV or MHFV vaccines via intradermal EP delivery generated robust cellular and humoral immune responses against all target viral antigens in all species. MMBV vaccine generated antigen-specific binding antibodies and IFNγ-secreting lymphocytes detected in NHPs up to six months post final immunization, suggesting induction of long-term immune memory. Serum from MHFV vaccinated NHPs demonstrated neutralizing activity in Ebola, Lassa, and Marburg pseudovirus assays indicating the potential to offer protection. Together, these data strongly support and demonstrate the versatility of DNA vaccines as a multivalent vaccine development platform for emerging infectious diseases.

Phase 1 study of safety, tolerability and immunogenicity of the human telomerase (hTERT)-encoded DNA plasmids INO-1400 and INO-1401 with or without IL-12 DNA plasmid INO-9012 in adult patients with solid tumors.

BACKGROUND: Human telomerase reverse transcriptase (hTERT) is frequently classified as a 'universal' tumor associated antigen due to its expression in a vast number of cancers. We evaluated plasmid DNA-encoded hTERT as an immunotherapy across nine cancer types. METHODS: A phase 1 clinical trial was conducted in adult patients with no evidence of disease following definitive surgery and standard therapy, who were at high risk of relapse. Plasmid DNA encoding one of two hTERT variants (INO-1400 or INO-1401) with or without plasmid DNA encoding interleukin 12 (IL-12) (INO-9012) was delivered intramuscularly concurrent with the application of the CELLECTRA constant-current electroporation device 4 times across 12 weeks. Safety assessments and immune monitoring against native (germline, non-mutated, non-plasmid matched) hTERT antigen were performed. The largest cohort of patients enrolled had pancreatic cancer, allowing for additional targeted assessments for this tumor type. RESULTS: Of the 93 enrolled patients who received at least one dose, 88 had at least one adverse event; the majority were grade 1 or 2, related to injection site. At 18 months, 54.8% (51/93) patients were disease-free, with median disease-free survival (DFS) not reached by end of study. For patients with pancreatic cancer, the median DFS was 9 months, with 41.4% of these patients remaining disease-free at 18 months. hTERT immunotherapy induced a de novo cellular immune response or enhanced pre-existing cellular responses to native hTERT in 96% (88/92) of patients with various cancer types. Treatment with INO-1400/INO-1401±INO-9012 drove hTERT-specific IFN-γ production, generated hTERT-specific CD4+ and CD8+ T cells expressing the activation marker CD38, and induced hTERT-specific activated CD8 +CTLs as defined by cells expressing perforin and granzymes. The addition of plasmid IL-12 adjuvant elicited higher magnitudes of cellular responses including IFN-γ production, activated CD4+ and CD8+ T cells, and activated CD8+CTLs. In a subset analysis of pancreatic cancer patients, the presence of immunotherapy-induced activated CD8+ T cells expressing PD-1, granzymes and perforin correlated with survival. CONCLUSIONS: Plasmid DNA-encoded hTERT/IL-12 DNA immunotherapy was well-tolerated, immune responses were noted across all tumor types, and a specific CD8+ phenotype increased by the immunotherapy was significantly correlated with survival in patients with pancreatic cancer.

INO-4800 DNA vaccine induces neutralizing antibodies and T cell activity against global SARS-CoV-2 variants.

Global surveillance has identified emerging SARS-CoV-2 variants of concern (VOC) associated with broadened host specificity, pathogenicity, and immune evasion to vaccine-induced immunity. Here we compared humoral and cellular responses against SARS-CoV-2 VOC in subjects immunized with the DNA vaccine, INO-4800. INO-4800 vaccination induced neutralizing antibodies against all variants tested, with reduced levels detected against B.1.351. IFNγ T cell responses were fully maintained against all variants tested.

Identification of Novel Neutralizing Monoclonal Antibodies against SARS-CoV-2 Spike Glycoprotein.

Coronavirus disease 2019 (COVID-19) is caused by the newly emerged human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Due to the highly contagious nature of SARS-CoV-2, it has infected more than 137 million individuals and caused more than 2.9 million deaths globally as of April 13, 2021. There is an urgent need to develop effective novel therapeutic strategies to treat or prevent this infection. Toward this goal, we focused on the development of monoclonal antibodies (mAbs) directed against the SARS-CoV-2 spike glycoprotein (SARS-CoV-2 Spike) present on the surface of virus particles as well as virus-infected cells. We isolated anti-SARS-CoV-2 Spike mAbs from animals immunized with a DNA vaccine. We then selected a highly potent set of mAbs against SARS-CoV-2 Spike protein and evaluated each candidate for their expression, target binding affinity, and neutralization potential using complementary ACE2-blocking and pseudovirus neutralization assays. We identified a total of 10 antibodies, which specifically and strongly bound to SARS-CoV-2 Spike, blocked the receptor binding domain (RBD) and angiotensin-converting enzyme 2 (ACE2) interaction, and neutralized SARS-CoV-2. Furthermore, the glycomic profile of the antibodies suggested that they have high Fc-mediated effector functions. These antibodies should be further investigated for elucidating the neutralizing epitopes on Spike for the design of next-generation vaccines and for their potential in diagnostic as well as therapeutic utilities against SARS-CoV-2.

Intradermal delivery of a synthetic DNA vaccine protects macaques from Middle East respiratory syndrome coronavirus.

Emerging coronaviruses from zoonotic reservoirs, including severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), have been associated with human-to-human transmission and significant morbidity and mortality. Here, we study both intradermal and intramuscular 2-dose delivery regimens of an advanced synthetic DNA vaccine candidate encoding a full-length MERS-CoV spike (S) protein, which induced potent binding and neutralizing antibodies as well as cellular immune responses in rhesus macaques. In a MERS-CoV challenge, all immunized rhesus macaques exhibited reduced clinical symptoms, lowered viral lung load, and decreased severity of pathological signs of disease compared with controls. Intradermal vaccination was dose sparing and more effective in this model at protecting animals from disease. The data support the further study of this vaccine for preventing MERS-CoV infection and transmission, including investigation of such vaccines and simplified delivery routes against emerging coronaviruses.

One or two dose regimen of the SARS-CoV-2 synthetic DNA vaccine INO-4800 protects against respiratory tract disease burden in nonhuman primate challenge model.

Safe and effective vaccines will provide essential medical countermeasures to tackle the COVID-19 pandemic. Here, we assessed the safety, immunogenicity and efficacy of the intradermal delivery of INO-4800, a synthetic DNA vaccine candidate encoding the SARS-CoV-2 spike protein in the rhesus macaque model. Single and 2 dose vaccination regimens were evaluated. Vaccination induced both binding and neutralizing antibodies, along with IFN-γ-producing T cells against SARS-CoV-2. Upon administration of a high viral dose (5 × 106 pfu) via the intranasal and intratracheal routes we observed significantly reduced virus load in the lung and throat, in the vaccinated animals compared to controls. 2 doses of INO-4800 was associated with more robust vaccine-induced immune responses and improved viral protection. Importantly, histopathological examination of lung tissue provided no indication of vaccine-enhanced disease following SARS-CoV-2 challenge in INO-4800 immunized animals. This vaccine candidate is currently under clinical evaluation as a 2 dose regimen.