Supplementary Figure 1 shows the anti-mouse CTLA-4 DMAb design. Supplementary Figure 2 shows the efficacy of the recombinant 9D9 antibody in the Sa1N tumor model. Supplementary Figure 3 shows that the mouse anti-mouse CTLA-4 DMAb induces immune memory and protection from tumor re-challenge. Supplementary Figure 4 shows the efficacy of the mouse anti-mouse CTLA-4 DMAb delivered at an earlier time point. Supplementary Figure 5 shows that the anti-mouse CTLA-4 DMAb induces T cell infiltration into tumors. Supplementary Figure 6 shows the efficiency of CD4 and CD8 depletion antibodies.

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.

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. Two-milligram dose of INO-4800, DNA-based vaccine encoding SARS-CoV-2 spike protein, appears safe and well tolerated and elicits humoral and cell-mediated immunity persisting to 6 months after a second dose. A third dose 6-10.5 months later significantly boosts immune responses.

Abstract Prostate cancer (PCa) represents the most predominant cancer type among males with an estimated 191,930 cases diagnosed in 2020 with 33,330 deaths globally. Notably, different treatment modalities such as surgery, chemo, radiation and hormone therapy exhibited improved outcomes in early stage PCa patients; however, there exist high recurrence rate in the treated patients and leads to castration resistant prostate cancer, the treatment choices for which are highly restricted. This scenario has pressed us to consider synthetic DNA vaccine-based immunotherapy approach, with ability to induce antigen-specific tumor-killing immune cells. Considering the high heterogeneity of prostate cancer cells, we hypothesized that synthetic DNA vaccines targeting multiple prostate specific antigens would elicit improved immune responses in those who are at high risk as well as advanced stages of prostate cancer. Therefore, we generated synthetic enhanced DNA immunogens as DNA constructs (SEV) targeting STEAP1, PAP, PARM1, PSCA, PCTA and PSP94 through utilization of bioinformatics approaches. The SEVs for prostate cancer antigens elicited potent cellular immunity as well as polyfunctionality of antigen-specific T cells in mice. Further, they were capable of generating robust humoral immune responses to each antigen. The anti-tumor activities of the SEVs were studied in prostate tumor which showed that the induced immune responses delayed tumor progression and enhanced infiltration of anti-tumor CD8+ T cells in the tumor microenvironment in a mouse model challenge system. These data highlight further study of these novel immunogens as synthetic DNA immunogens for immunotherapy of PCa, and perhaps other human cancers. Citation Format: Devivasha Bordoloi, Peng Xiao, Hyeree Choi, Michelle Ho, Alfredo Perales-Puchalt, Makan Khoshnejad, J.Joseph Kim, Laurent Humeau, Alagarsamy Srinivasan, David B. Weiner, Kar Muthumani. Novel synthetic DNA vaccines in prostate cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1915.

Abstract Background Global surveillance has identified emerging SARS-CoV-2 variants of concern (VOC) associated with increased transmissibility, disease severity, and resistance to neutralization by current vaccines under emergency use authorization (EUA). Here we assessed cross-immune responses of INO-4800 vaccinated subjects against SARS-CoV-2 VOCs. Methods We used a SARS-CoV-2 IgG ELISA and a pseudo neutralization assay to assess humoral responses, and an IFNγ ELISpot to measure cellular responses against SARS-CoV-2 VOC in subjects immunized with the DNA vaccine, INO-4800. Results IgG binding titers were not impacted between wild-type (WT) and B.1.1.7 or B.1.351 variants. An average 1.9-fold reduction was observed for the P.1 variant in subjects tested at week 8 after receiving two doses of INO-4800 (Figure 1a). We performed a SARS-CoV-2 pseudovirus neutralization assay using sera collected from 13 subjects two weeks after administration of a third dose of either 0.5 mg, 1 mg, or 2 mg of INO-4800. Neutralization was detected against WT and the emerging variants in all samples tested. The mean ID50 titers for the WT, B.1.1.7, B.1.351 and P.1. were 643 (range: 70-729), 295 (range: 46-886), 105 (range: 25-309), and 664 (range: 25-2087), respectively. Compared to WT, there was a 2.1 and 6.9-fold reduction for B.1.1.7 and B.1.351, respectively, while there was no difference between WT and the P.1 variant (Figure 1b). Next, we compared cellular immune responses to WT and SARS-CoV-2 Spike variants elicited by INO-4800 vaccination. We observed similar cellular responses to WT (median = 82.2 IQR = 58.9-205.3), B.1.1.7 (79.4, IQR = 38.9- 179.7), B.1.351 (80, IQR = 40.0-208.6) and P.1 (78.3, IQR = 53.1-177.8) Spike peptides (Figure 2). Conclusion 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. Disclosures Viviane M. Andrade, PhD, Inovio Pharmaceuticals Inc. (Employee) Aaron Christensen-Quick, PhD, Inovio Pharmaceuticals, Inc (Employee) Joseph Agnes, PhD, Inovio (Employee, Shareholder) Jared Tur, PhD, Inovio (Employee) Charles C. Reed, PhD, Inovio Pharmaceuticals (Employee, Shareholder) Richa Kalia, MS, Inovio Pharmaceuticals (Employee, Other Financial or Material Support, I have stock options with Inovio Pharmaceuticals as an employee.) Idania Marrero, MD, PhD, Inovio Pharmaceuticals (Employee, Shareholder) Dustin Elwood, PhD, Inovio Pharmaceuticals (Employee) Katherine Schultheis, MSc, Inovio Pharmaceuticals (Employee) Emma Reuschel, PhD, Inovio Pharmaceuticals (Employee) Trevor McMullan, MSc, Inovio (Shareholder) Patrick Pezzoli, BS, Inovio (Employee) Kimberly A. Kraynyak, PhD, Inovio Pharmaceuticals (Employee, Other Financial or Material Support, Stock options) Albert Sylvester, MS, Inovio (Employee, Shareholder) Mammen P. Mammen Jr., MD, Inovio Pharmaceuticals (Employee) J Joseph Kim, PhD, Inovio (Employee) David Weiner, PhD, Inovio (Board Member, Grant/Research Support, Shareholder, I serve on the SAB in addition to the above activities) Trevor R. F. Smith, PhD, Inovio (Employee, Shareholder) Stephanie Ramos, PhD, Inovio Pharmaceuticals (Employee) Laurent Humeau, PhD, Inovio Pharmaceuticals (Employee) Jean Boyer, PhD, Inovio (Employee) Kate Broderick, PhD, Inovio (Employee)

Abstract Background First-generation COVID-19 vaccines are matched to spike protein of the Wuhan-H1 (WT) strain. Convalescent and vaccinee samples show reduced neutralization of SARS-CoV-2 variants of concern (VOC). Next generation DNA vaccines could be matched to single variants or synthetically designed for broader coverage of multiple VOCs. Methods The synthetic consensus (SynCon®) sequence for INO-4802 SARS-CoV-2 spike with focused RBD changes and dual proline mutations was codon-optimized (Figure 1). Sequences for wild-type (pWT) and B.1.351 (pB.1.351) were similarly optimized. Immunogenicity was evaluated in BALB/c mice. Pre-clinical efficacy was assessed in the Syrian Hamster model. Figure 1. Design Strategy for INO-4802 Results INO-4802 induced potent neutralizing antibody responses against WT, B.1.1.7, P.1, and B.1.351 VOC in a murine model. pWT vaccinated animals showed a 3-fold reduction in mean neutralizing ID50 for the B.1.351 pseudotyped virus. INO-4802 immunized animals had significantly higher (p = 0.0408) neutralizing capacity (mean ID50 816.16). ID50 of pB.1.351 serum was reduced 7-fold for B.1.1.7 and significantly lower (p = 0.0068) than INO-4802 (317.44). INO-4802 neutralized WT (548.28) comparable to pWT. INO-4802 also neutralized P.1 (1026.6) (Figure 2). pWT, pB.1.351 or INO-4802 induced similar T-cell responses against all variants. INO-4802 skewed towards a TH1-response. All hamsters vaccinated with INO-4802 or pB.1.351 were protected from weight loss after B.1.351 live virus challenge. 4/6 pWT immunized hamsters were completely protected. pWT immunized hamsters neutralized WT (1090) but not B.1.351 (39.16). INO-4802 neutralized both WT (672.2) and B.1.351 (1121) (Figure 3). We observed higher increase of binding titers following heterologous boost with INO-4802 (3.6 – 4.4 log2-fold change) than homologous boost with pWT (2.0 – 2.4 log2 fold change) (Figure 4). Figure 2. INO-4802 Induces Functional Humoral Immune Response Against SARS-CoV-2 Variants of Concern Figure 3. INO-4802 Protects Hamsters Against Challenge With B.1.351 Live Virus Figure 4. Heterologous Boost with INO-4802 Induces Humoral Immune Response Against SARS-CoV-2 Variants Conclusion Vaccines matching single VOCs, like pB.1.351 and pWT, elicit responses against the matched antigen but have reduced cross-reactivity. Presenting a pan-SARS-CoV-2 approach, INO-4802 may offer substantial advantages in terms of cross-strain protection, reduced susceptibility to escape mutants and non-restricted geographical use. Disclosures Katherine Schultheis, MSc, Inovio Pharmaceuticals (Employee) Charles C. Reed, PhD, Inovio Pharmaceuticals (Employee, Shareholder) Viviane M. Andrade, PhD, Inovio Pharmaceuticals Inc. (Employee) Richa Kalia, MS, Inovio Pharmaceuticals (Employee, Other Financial or Material Support, I have stock options with Inovio Pharmaceuticals as an employee.) Jared Tur, PhD, Inovio (Employee) Blake Schouest, PhD, Inovio Pharmaceuticals (Employee) Dustin Elwood, PhD, Inovio Pharmaceuticals (Employee) Arthur Doan, n/a, Inovio (Employee) Patrick Pezzoli, BS, Inovio (Employee) Dinah Amante, BS, Inovio (Employee) David Weiner, PhD, Inovio (Board Member, Grant/Research Support, Shareholder, I serve on the SAB in addition to the above activities) J Joseph Kim, PhD, Inovio (Employee) Laurent Humeau, PhD, Inovio Pharmaceuticals (Employee) Stephanie Ramos, PhD, Inovio Pharmaceuticals (Employee) Trevor R. F. Smith, PhD, Inovio (Employee, Shareholder) Kate Broderick, PhD, Inovio (Employee).

Abstract Background DNA vaccines are safe, tolerable, elicit humoral and cellular responses, allow for repeated dosing over time, are thermostable at room temperature, and are easy to manufacture. We present a compilation of Phase 1 and Phase 2 data of Inovio’s US COVID-19 DNA Vaccine (INO-4800) targeting the full-length Spike antigen of SARS-CoV-2. A South Korean Phase 2 study is ongoing. Methods Participants in the open-label Phase 1 trial received 0.5 mg, 1.0 mg or 2.0 mg intradermally (ID) followed by electroporation (EP) at Days 0 and 28. An optional booster dose was administered >6 months post-dose 2. The Phase 2 further compared the 1.0 mg and 2.0 mg doses against placebo in a total of 401 participants randomized at a 3:3:1:1 ratio. ClinicalTrials.gov identifiers: NCT04336410 and NCT04642638 Results The majority of adverse events (AEs) related to INO-4800 across both trials were mild in severity and did not increase in frequency with age and subsequent doses. In Phase 1, 78% (14/18) and 84% (16/19) of subjects generated neutralizing antibody responses with geometric mean titers (GMTs) of 17.4 (95%CI 8.3, 36.5) and 62.3 (95% CI 36.4, 106.7) in the 1.0 and 2.0 groups, respectively (Figure 1). By week 8, 74% (14/19) and 100% (19/19) subjects generated T cell responses by Th1- associated IFNγ ELISPOT assay . Following a booster dose, neutralizing GMTs rose to 82.2 (95% CI 38.2, 176.9) and 124.7 (95% CI 62.8, 247.7) in the 1.0 mg and 2.0 mg groups, respectively, demonstrating the ability of INO-4800 to boost (Figure 2). In Phase 2, neutralizing antibody responses demonstrated GMTs of 93.6 (95%CI 77.3, 113.4) in the 1.0 mg dose group and 150.6 (95%CI 123.8, 183.1) in the 2.0 mg dose group (Figure 3). Conclusion INO-4800 appears safe and tolerable as a primary series and as a booster with the induction of both humoral and cellular immune responses. In addition to eliciting neutralizing antibodies, INO-4800 also induced T cell immune responses as demonstrated by IFNγ ELISpot. Finally, as a homologous booster, INO-4800, when administered 6-10.5 months following the primary series, resulted in an increased immune response without increase in reactogenicity. The 2.0 mg dose was selected for Phase 3 evaluation. Disclosures Joseph Agnes, PhD, Inovio (Employee, Shareholder) Mary Giffear, BS, Inovio Pharmaceuticals, Inc. (Employee) Kimberly A. Kraynyak, PhD, Inovio Pharmaceuticals (Employee, Other Financial or Material Support, Stock options) Dinah Amante, BS, Inovio (Employee) Emma Reuschel, PhD, Inovio Pharmaceuticals (Employee) Aaron Christensen-Quick, PhD, Inovio Pharmaceuticals, Inc (Employee) Viviane M. Andrade, PhD, Inovio Pharmaceuticals Inc. (Employee) Gabriella Garufi, PhD, Inovio Pharmaceuticals, Inc. (Employee) Albert Sylvester, MS, Inovio (Employee, Shareholder) Matthew P. Morrow, PhD, Inovio Pharmaceuticals (Employee) Patrick P. Pezzoli, BS, Inovio Pharmaceuticals, Inc. (Employee) Jan Pawlicki, PhD, Inovio Pharmaceuticals (Employee) Elisabeth Gillespie, PhD, Inovio Pharmaceuticals, Inc. (Employee) Katherine Schultheis, MSc, Inovio Pharmaceuticals (Employee) Hedieh Badie, PhD, INOVIO Pharmaceuticals (Employee) Timothy A. Herring, MPH, Inovio Pharmaceuticals, Inc. (Employee, Other Financial or Material Support, Own stock in the company) Keiko O. Simon, PhD, Inovio Pharmaceuticals (Employee) Trevor R. F. Smith, PhD, Inovio (Employee, Shareholder) Stephanie Ramos, PhD, Inovio Pharmaceuticals (Employee) Jessica Lee, MPH, Inovio Pharmaceuticals (Employee) Michael Dallas, PhD, Inovio Pharmaceuticals, Inc. (Employee, Shareholder) Ami Shah Brown, PhD, Abbot Laboratories (Shareholder)IBB Biotech ETF (Shareholder)Inovio Pharmaceuticals (Employee)J & J (Shareholder)Moderna (Shareholder) Jacqueline E. Shea, PhD, Inovio Pharmaceuticals (Employee, Shareholder) J Joseph Kim, PhD, Inovio (Employee) David Weiner, PhD, Inovio (Board Member, Grant/Research Support, Shareholder, I serve on the SAB in addition to the above activities) Kate Broderick, PhD, Inovio (Employee) Trevor McMullan, MSc, Inovio (Shareholder) Jean Boyer, PhD, Inovio (Employee) Laurent Humeau, PhD, Inovio Pharmaceuticals (Employee) Mammen P. Mammen Jr., MD, Inovio Pharmaceuticals (Employee)

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.

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.

Coronavirus disease 2019 (COVID-19), caused by the SARS-CoV-2 virus, has had a dramatic global impact on public health and social and economic infrastructures. Here, we assess the immunogenicity and anamnestic protective efficacy in rhesus macaques of an intradermal (i.d.)-delivered SARS-CoV-2 spike DNA vaccine, INO-4800, currently being evaluated in clinical trials. Vaccination with INO-4800 induced T cell responses and induced spike antigen and RBD binding antibodies with ADCP and ADCD activity. Sera from the animals neutralized both the D614 and G614 SARS-CoV-2 pseudotype viruses. Several months after vaccination, animals were challenged with SARS-CoV-2 resulting in rapid recall of anti-SARS-CoV-2 spike protein T cell and neutralizing antibody responses. These responses were associated with lower viral loads in the lung. These studies support the immune impact of INO-4800 for inducing both humoral and cellular arms of the adaptive immune system, which are likely important for providing durable protection against COVID-19 disease.

Background: A vaccine against SARS-CoV-2 is of high urgency. Here the safety and immunogenicity induced by a DNA vaccine (INO-4800) targeting the full length spike antigen of SARS-CoV-2 are described. Methods: INO-4800 was evaluated in two groups of 20 participants, receiving either 1.0 mg or 2.0 mg of vaccine intradermally followed by CELLECTRA (R) EP at 0 and 4 weeks. Thirty-nine subjects completed both doses; one subject in the 2.0 mg group discontinued trial participation prior to receiving the second dose. ClinicalTrials.gov identifier: NCT04336410. Findings: The median age was 34.5, 55% (22/40) were men and 82.5% (33/40) white. Through week 8, only 6 related Grade 1 adverse events in 5 subjects were observed. None of these increased in frequency with the second administration. No serious adverse events were reported. All 38 subjects evaluable for immunogenicity had cellular and/or humoral immune responses following the second dose of INO-4800. By week 6, 95% (36/38) of the participants seroconverted based on their responses by generating binding (ELISA) and/or neutralizing antibodies (PRNT IC50), with responder geometric mean binding antibody titers of 655.5 [95% CI (255.6, 1681.0)] and 994.2 [95% CI (395.3, 2500.3)] in the 1.0 mg and 2.0 mg groups, respectively. For neutralizing antibody, 78% (14/18) and 84% (16/19) generated a response with corresponding geometric mean titers of 102.3 [95% CI (37.4, 280.3)] and 63.5 [95% CI (39.6, 101.8)], in the respective groups. By week 8, 74% (14/19) and 100% (19/19) of subjects generated T cell responses by IFN-gamma ELISpot assay with the median SFU per 106 PBMC of 46 [95% CI (21.1, 142.2)] and 71 [95% CI (32.2, 194.4)] in the 1.0 mg and 2.0 mg groups, respectively. Flow cytometry demonstrated a T cell response, dominated by CD8(+) T cells co-producing IFN-gamma and TNF-alpha, without increase in IL-4. Interpretation: INO-4800 demonstrated excellent safety and tolerability and was immunogenic in 100% (38/38) of the vaccinated subjects by eliciting either or both humoral or cellular immune responses. Crown Copyright (c) 2020 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Summary Coronavirus disease 2019 (COVID-19), caused by the SARS-CoV-2 virus, has had a dramatic global impact on public health, social, and economic infrastructures. Here, we assess immunogenicity and anamnestic protective efficacy in rhesus macaques of the intradermal (ID)-delivered SARS-CoV-2 spike DNA vaccine, INO-4800. INO-4800 is an ID-delivered DNA vaccine currently being evaluated in clinical trials. Vaccination with INO-4800 induced T cell responses and neutralizing antibody responses against both the D614 and G614 SARS-CoV-2 spike proteins. Several months after vaccination, animals were challenged with SARS-CoV-2 resulting in rapid recall of anti-SARS-CoV-2 spike protein T and B cell responses. These responses were associated with lower viral loads in the lung and with faster nasal clearance of virus. These studies support the immune impact of INO-4800 for inducing both humoral and cellular arms of the adaptive immune system which are likely important for providing durable protection against COVID-19 disease.

Hepatitis B virus (HBV) causes a potentially life-threatening liver infection that frequently results in life-long chronic infection. HBV is responsible for 887,000 deaths each year, most resulting from chronic liver diseases and hepatocellular carcinoma. Presently, there are 250 million chronic HBV carriers worldwide who are at a high risk for developing cirrhosis and hepatocellular carcinoma (HCC). HCC is the most common type of liver cancer with a strong association with HBV infection. HBV transmission through blood transfusions and perinatal transfer from infected mother to child have been common routes of infection. In the present study, we describe the development of a synthetic DNA plasmid encoding an anti-HBV human monoclonal antibody specific for the common "a determinant region" of HBsAg of hepatitis B virus and demonstrate the ability of this platform at directingin vivoantibody expression.In vivodelivery of this DNA encoded monoclonal antibody (DMAb) plasmid in mice resulted in expression of human IgG over a period of one month following a single injection. Serum antibody was found to recognize the relevant conformational epitope from plasma purified native HBsAg as well as bound HBV in HepG2.2.15 cells. The serum DMAb efficiently neutralized HBV and prevented infection of HepaRG cellsin vitro. Additional study of these HBV-DMAb as a possible therapy or immunoprophylaxis for HBV infection is warranted.

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.

The coronavirus family member, SARS-CoV-2 has been identified as the causal agent for the pandemic viral pneumonia disease, COVID-19. At this time, no vaccine is available to control further dissemination of the disease. We have previously engineered a synthetic DNA vaccine targeting the MERS coronavirus Spike (S) protein, the major surface antigen of coronaviruses, which is currently in clinical study. Here we build on this prior experience to generate a synthetic DNA-based vaccine candidate targeting SARS-CoV-2 S protein. The engineered construct, INO-4800, results in robust expression of the S protein in vitro. Following immunization of mice and guinea pigs with INO-4800 we measure antigen-specific T cell responses, functional antibodies which neutralize the SARS-CoV-2 infection and block Spike protein binding to the ACE2 receptor, and biodistribution of SARS-CoV-2 targeting antibodies to the lungs. This preliminary dataset identifies INO-4800 as a potential COVID-19 vaccine candidate, supporting further translational study.

: Background: Recurrent respiratory papillomatosis (RRP) is a rare disorder characterized by the generation of papillomas of the aerodigestive tract, usually associated with human papilloma virus (HPV) subtypes 6, 11. INO-3106 is a DNA plasmid-based immunotherapy targeting E6 and E7 proteins of HPV6, in order to create a robust immune T cell response. Methods: Testing of INO-3016 in animal models confirmed immunogenicity of the DNA-based therapy. A single-site open-label Phase 1 study was initiated for patients with HPV6-positive RRP. Patients were dosed with INO-3106 with or without INO-9012, a DNA plasmid immunotherapy that encodes IL-12, delivered intramuscularly (IM) in combination with electroporation (EP) with the CELLECTRA® device. Patients received an escalating dose of INO-3106, 3 mg once and then 6 mg for three additional doses, each dose three weeks apart, with the third and fourth doses co-administered with INO-9012. The primary objective of the study was to evaluate the safety and tolerability of INO-3106 with and without INO-9012. The secondary objective was to determine cellular immune responses to INO-3106 with and without INO-9012. Exploratory objectives included preliminary clinical efficacy to the therapy. Results: Three patients were enrolled in this study, of which two had RRP. Study therapy was well-tolerated, with no related serious adverse events and all related adverse events (AEs) were low-grade. Injection site pain was the most common related AE reported. Immunogenicity was evidenced by multiple immune assays showing engagement and expansion of an HPV6-specific cellular response, including cytotoxic T cells. Preliminary efficacy was demonstrated in patients with RRP in the form of reduction in need for surgical intervention for papilloma growth. Prior to intervention, both patients required surgical intervention approximately every 180 days. One patient demonstrated a greater than three-fold increase in surgery avoidance (584 days) and the other patient remains completely surgery-free as of the last contact at 915 days, a greater than 5-fold increase in surgery interval. Conclusion: INO-3106 with and without INO-9012 was well tolerated, immunogenic and demonstrated preliminary efficacy in patients with HPV6-associated RRP aerodigestive lesions. Further clinical study is indicated.

Significant concerns have arisen over the past 3 y from the increased global spread of the mosquito-borne flavivirus, Zika. Accompanying this spread has been an increase in cases of the devastating birth defect microcephaly as well as of Guillain–Barré syndrome in adults in many affected countries. Currently there is no vaccine or therapy for this infection; however, we sought to develop a combination approach that provides more rapid and durable protection than traditional vaccination alone. A novel immune-based prophylaxis/therapy strategy entailing the facilitated delivery of a synthetic DNA consensus prME vaccine along with DNA-encoded anti-ZIKV envelope monoclonal antibodies (dMAb) were developed and evaluated for antiviral efficacy. This immediate and persistent protection strategy confers the ability to overcome shortcomings inherent with conventional active vaccination or passive immunotherapy. A collection of novel dMAbs were developed which were potent against ZIKV and could be expressed in serum within 24–48 h of in vivo administration. The DNA vaccine, from a previous development, was potent after adaptive immunity was developed, protecting against infection, brain and testes pathology in relevant mouse challenge models and in an NHP challenge. Delivery of potent dMAbs protected mice from the same murine viral challenge within days of delivery. Combined injection of dMAb and the DNA vaccine afforded rapid and long-lived protection in this challenge model, providing an important demonstration of the advantage of this synergistic approach to pandemic outbreaks.

Mayaro virus (MAYV) of the genus alphavirus is a mosquito-transmitted emerging infectious disease that causes an acute febrile illness, rash, headaches, and nausea that may turn into incapacitating, persistent arthralgias in some victims. Since its discovery in Trinidad in 1954, cases of MAYV infection have largely been confined there and to the northern countries of South America, but recently, MAYV cases have been reported in some island nations in the Caribbean Sea. Accompanying these reports is evidence that new vectors, including Aedes spp. mosquitos, recently implicated in the global spread of Zika and chikungunya viruses, are competent for MAYV transmission, which, if true, could facilitate the spread of MAYV beyond its current range. Despite its status as an emerging virus, there are no licensed vaccines to prevent MAYV infection nor therapeutics to treat it. Here, we describe the development and testing of a novel DNA vaccine, scMAYV-E, that encodes a synthetically-designed consensus MAYV envelope sequence. In vivo electroporation-enhanced immunization of mice with this vaccine induced potent humoral responses including neutralizing antibodies as well as robust T-cell responses to multiple epitopes in the MAYV envelope. Importantly, these scMAYV-E-induced immune responses protected susceptible mice from morbidity and mortality following a MAYV challenge.

Background. Zika virus (ZIKV) infection has been associated with prolonged viral excretion in human semen and causes testicular atrophy and infertility in 10-week-old immunodeficient mice. Male IFNAR(/) mice, knockout for type I interferon receptor, were immunized with GLS-5700, a deoxyribonucleic acid-based vaccine, before a subcutaneous ZIKV challenge with 6 10(5) plaque-forming units at 13 weeks of age. On day 28 postinfection, testes and epididymides were collected in some mice for histological and functional analyses, whereas others were mated with naive female wild-type C57BL/6J. Although all mice challenged with ZIKV developed viremia, most of them were asymptomatic, showed no weight loss, and survived infection. On day 28 postinfection, none of the unvaccinated, infected mice (9 of 9) exhibited abnormal spermatozoa counts or motility. However, 33% (3 of 9) and 36% (4 of 11) of mated males from this group were infertile, from 2 independent studies. Contrarily, males from the noninfected and the vaccinated, infected groups were all fertile. On days 75 and 207 postinfection, partial recovery of fertility was observed in 66% (2 of 3) of the previously infertile males. This study reports the effects of ZIKV infection on male fertility in a sublethal, immunodeficient mouse model and the efficacy of GLS-5700 vaccination in preventing male infertility.Male IFNAR(/) mice infected with ZIKV at an older age were shown to be infertile without overt signs of disease. This outcome was prevented by immunization with a DNA vaccine. A partial recovery of fertility was observed later after infection.

Abstract Antibody-based immune therapies targeting the T-cell checkpoint inhibitors CTLA4 and PD1 have made incredible advances in cancer therapy. In particular, the combination of CTLA4 and PD1 blockade (using ipilimumab and nivolumab, respectively) was recently shown to be the most effective immune therapy for improving progression-free survival in advanced melanoma patients. However, this immune therapy combination strategy is extraordinarily expensive due to high manufacturing costs of monoclonal antibodies, with an estimated annual cost of almost one million dollars per patient. In this study, we focused on the development of a DNA-encoded monoclonal antibody (DMAbTM) approach for delivery of anti-CTLA4 monoclonal antibodies in vivo. With this technology, formulated DMAbTM plasmids are injected into the muscle with electroporation, allowing for muscle cells to produce and secrete the DMAbTM for a prolonged period of time without the need for repeated administration. In proof-of-concept studies in mice, we show that delivery of a DMAbTM plasmid for a monoclonal antibody targeting mouse CTLA4 (clone 9D9) elicits high serum expression (peak expression of 7.9µg/mL in immune-competent C57Bl/6 mice with one injection of DNA). This anti-mouse CTLA4 DMAbTM is capable of inducing tumor regression in A/J mice that were implanted with the immunogenic Sa1N tumor cell line without repeated administration. Furthermore, this anti-mouse CTLA4 DMAbTM is capable of synergizing with a DNA vaccine targeting the tumor antigen TERT in slowing tumor growth for the nonimmunogenic TC-1 tumor cell line. We also examined DNA delivery of the anti-human CTLA4 antibodies ipilimumab and tremelimumab. For these anti-human CTLA4 DMAbsTM, we achieved steady-state levels of approximately 75µg/mL and 50ug/mL for ipilimumab and tremelimumab, respectively, in mice, which is greater than the mean trough levels of ipilimumab achieved in patients (21.8µg/mL serum concentration for 3mg/kg dose). These anti-human CTLA4 DMAbsTM produced in vivo bind to human CTLA4 protein and induce T-cell activation in a functional assay ex vivo. These results demonstrate the feasibility of delivering immune checkpoint blockade monoclonal antibodies using DNA for cancer immune therapy. Future studies will explore combination therapies with CTLA4 DMAbsTM and chemotherapy. Citation Format: Elizabeth Duperret, Aspen Trautz, Ami Patel, Joseph Kim, Kar Muthumani, David B. Weiner. Synthetic DNA-encoded monoclonal antibody delivery (DMAbTM) of anti-CTLA4 antibodies induces tumor shrinkage in vivo [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B036.