key: cord-0825723-rdgl6ksj authors: Kraynyak, K. A.; Blackwood, E.; Agnes, J.; Tebas, P.; Giffear, M.; Amante, D.; Reuschel, E. L.; Purwar, M.; Christensen-Quick, A.; Liu, N.; Andrade, V.; Diehl, M.; Wani, S.; Lupicka, M.; Sylvester, A.; Morrow, M. P.; Pezzoli, P.; McMullan, T.; Kulkarni, A. J.; Zaidi, F. I.; Frase, D.; Liaw, K.; Smith, T. R. F.; Ramos, S. J.; Ervin, J.; Adams, M.; Lee, J.; Dallas, M.; Shah Brown, A.; Shea, J. E.; Kim, J. J.; Weiner, D. B.; Broderick, K. E.; Humeau, L. M.; Boyer, J. D.; Mammen, M. P. title: SARS-CoV-2 DNA Vaccine INO-4800 Induces Durable Immune Responses Capable of Being Boosted in a Phase 1 Open-Label Trial date: 2021-10-10 journal: nan DOI: 10.1101/2021.10.06.21264584 sha: 1c994bb04d64d8d561d675d60820cd15a2944cff doc_id: 825723 cord_uid: rdgl6ksj Background: Additional SARS-CoV-2 vaccines that are safe and effective as both primary series and booster remain urgently needed to combat the COVID-19 pandemic. Here we describe the safety and durability of the immune response from two doses of a DNA vaccine (INO-4800) targeting the full-length Spike antigen and a subsequent homologous booster dose. Methods: INO-4800 was evaluated in 120 healthy participants across three dose groups (0.5 mg, 1.0 mg and 2.0 mg), each stratified by age. INO-4800 was injected intradermally followed by electroporation at 0 and 4 weeks followed by an optional booster dose 6-10.5 months following the second dose. Results: INO-4800 was well-tolerated, with no treatment-related serious adverse events reported. Most adverse events were mild in severity 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 detected in the 2.0 mg dose group. Conclusion: INO-4800 was well-tolerated as a 2-dose series and as a homologous booster dose in all adults, including the elderly. These results support further development of INO-4800 as a primary series and as a booster. Keywords: SARS-CoV-2; Clinical trial; DNA Vaccine; COVID-19; Immunogenicity; Booster Despite aggressive vaccination campaigns, most of the world's population remains unvaccinated 2 and susceptible to COVID-19, the disease caused by SARS-CoV-2 [1] . To date, over 3.8 million 3 people have succumbed to COVID-19 [2] . The urgent need remains for additional safe and 4 effective vaccines against SARS-CoV-2 that are affordable, scalable and can be distributed to 5 countries where the infrastructure may not be supportive of ultra-cold chain transport and storage. 6 The preliminary safety and immunogenicity of the COVID-19 DNA vaccine, INO-4800, in both 7 Phase 1 and Phase 2 clinical studies were previously reported [3, 4] . INO-4800 targets the SARS-8 CoV-2 spike protein which exists as a trimer on the virus surface. Attachment to host cells is 9 mediated by binding of the spike protein's receptor binding domain (RBD) onto angiotensin 10 converting enzyme 2 (ACE2) receptors of host cells [5] . Once attached, the viral transport through 11 the host cell membrane is facilitated by the S2 region of spike protein [5] . Eliciting humoral and 12 cellular responses against the spike protein can prevent the virus from gaining access to host 13 cells, and this strategy has led to several impactful vaccine developments. 14 INO-4800 consists of an optimized plasmid DNA that is injected into the skin with enhanced 15 delivery into the cells by in vivo electroporation [6] . This approach potentially offers several 16 advantages, including induction of humoral and cellular immunity, favorable tolerability and 17 thermal stability profiles and its ease of manufacture [7, 8] . 18 The previous preliminary analysis [3] demonstrated that two 1.0 mg or 2.0 mg doses of INO-4800 19 administered one month apart were well-tolerated in 38 healthy participants 18-50 years of age 20 and induced the production of neutralizing antibodies and/or T-cells targeting SARS-CoV-2 spike 21 protein using a two-dose regimen. Here we describe the durability of that response at 6 months 22 following the second dose, as well as the safety and immunogenicity of the 2-dose regimen in 23 older and elderly participants including following a subsequent homologous booster dose. 24 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021. 10 The DNA vaccine INO-4800 was previously described [3, 9] . Briefly, the vaccine expresses the 40 full-length sequence of the SARS-CoV-2 spike glycoprotein derived from the original Wuhan strain 41 based on an optimized synthetic sequence that was created using a proprietary algorithm. The 42 final vaccine drug product, manufactured under Good Manufacturing Practices, was formulated 43 at 10 mg/mL in saline sodium citrate buffer. 44 INO-4800 is injected ID immediately followed by EP using the CELLECTRA® 2000 device that 45 generates a controlled electric field at the injection site to enhance the cellular uptake and 46 expression of the DNA plasmid as previously described [10, 11] . The device delivers a total of four 47 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Primary safety endpoints included incidence of adverse events (AEs) using the "Toxicity Grading The initial forty participants 18-50 years were enrolled sequentially into 1.0 mg and 2.0 mg dose 64 groups with a safety run-in period [3] . The trial design was expanded to include older participants 65 in all dosing groups (including a 0.5 mg dose level). Upon favorable safety assessment review by 66 an independent Data Safety Monitoring Board (DSMB) of Week 1 data for 0.5 mg dose group 67 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Samples were collected at timepoints described above with screening and pre-dose 1 samples 91 considered baseline. Peripheral blood mononuclear cells (PBMCs) were collected as previously 92 described [3] . After isolation, PBMCs were stored in the vapor phase of a liquid nitrogen freezer 93 until analysis, while serum samples were stored at -80°C. Eight participants were excluded from 94 the immunogenicity analyses due to a seropositive response, as determined by a positive ELISA 95 titer to the SARS-CoV-2 nucleoprotein, indicating SARS-CoV-2 infection 96 SARS-CoV-2 Pseudovirus Neutralization Assay: Serum samples were measured using a 97 pseudovirus neutralization assay as described previously [4] . Data was reported as ID50, which is 98 the reciprocal serum dilution resulting in 50% inhibition of infectivity by comparison to control wells 99 with no serum samples added. Please see supplementary methods for additional information. 100 CoV-2 spike protein were measured by ELISA as described previously [4] . SARS-CoV-2 spike 102 antibody concentrations were determined by interpolation from a dilution curve of SARS-CoV-2 103 convalescent plasma with an assigned concentration of 20,000 Units per mL. Further details are 104 described in the supplementary methods. 105 The SARS-CoV-2 spike antigen-specific IFN-γ 106 T-cell response was measured as described previously [3] . Values were reported as the mean performed as previously described [3] and included cytokines IFNγ, TNFα, and IL-2. The LGL 113 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.10.06.21264584 doi: medRxiv preprint 9 -Confidential-assay was also performed as reported previously [12] following stimulation with overlapping 114 peptides to the full-length spike protein to measure CD8+T cell activation (CD38, CD69, CD137, 115 Ki67) and capacity to produce lytic proteins (granzymes A and B, perforin and granulysin). 116 No formal power analysis was applicable to this trial. Descriptive statistics were used to 118 summarize the safety endpoints based on the safety population: proportions of participants with 119 AEs, through 6 months following dose 2 (non-boosted participants) or 2 weeks following booster signed-rank tests), and flow assay responses (with Wilcoxon signed-rank tests) were performed. 125 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Trial Population Demographics 127 Between 06 April 2020 and 07 July 2020, 154 participants were screened and 120 enrolled into 128 the trial (Figure 1) . The median age was 50.5 years (range 18 to 86 years). Participants were 129 57.5% female (69/120) and 42.5% male (51/120) ( Table 1) (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted October 10, 2021. doses when compared to the first dose (Figure 2) . A decrease in frequency of treatment-related 153 AEs in the older and elderly age cohorts was observed when compared to the younger age group 154 (Supplementary Figure 3) . (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. following the booster dose (P=0.018 and P=0.008, respectively) ( Figure 4A, right panel) . The 191 2.0 mg dose group had a difference in medians of 10 following the booster, resulting in the highest 192 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted October 10, 2021. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.10.06.21264584 doi: medRxiv preprint 14 -Confidential-( Figure 5B and C, respectively) . The frequency of SARS-CoV-2 specific CD38+CD8+T cells 217 statistically significantly increased following 2.0 mg of INO-4800 (P=0.016), with a difference in 218 medians of 1.45 (Figure 5B, left panel) . CD38+CD8+T cells with lytic potential (Figure 5B (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted October 10, 2021. tested, and the increase in antibody levels were statistically significant above baseline out to 6 249 months following dose 2. Importantly, increases in both humoral and cellular immune responses 250 were statistically significant following the booster dose. 251 The contribution of the CD8+T cell response to vaccine efficacy has become increasingly 252 recognized as they have been detected early after vaccination [13] and due to their role in 253 controlling infection [14, 15] . Specifically, it has been established that CD8+T cells expressing 254 cytokines such as IFNγ and TNFα as well as markers involved in activation status and proliferation 255 such as CD38 and Ki67 contribute to limiting disease severity during SARS-CoV-2 infection [14] . 256 Additional studies have identified the expression of CD69 and CD137 on SARS-CoV-2 specific 257 CD8+T cells being associated with less severe disease [15] . This expanded Phase 1 trial 258 demonstrates that immunization with INO-4800 induces SARS-CoV-2 specific CD8+ T cells 259 exhibiting these specific characteristics, suggesting the induction of a vaccine induced cellular 260 response that has potential to protect against severe COVID-19. As variants of concern continue 261 to emerge, the generation of cross-reactive activated CD8+T cells with lytic potential are likely to 262 play an important role in preventing severe disease. We have previously demonstrated that 263 vaccination with INO-4800 induces T cells and neutralizing antibodies that are active against the 264 parental SARS-CoV-2 strain as well as the Variants of Concern [16] . We acknowledge limitations 265 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. times, with high tolerability, along with its ease of scalability and thermostability, contribute to its 280 potential value in combatting the COVID-19 pandemic and addressing the persistence of SARS-281 CoV-2 as an ongoing endemic threat to global health. 282 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted October 10, 2021. From Vaccine Nationalism to Vaccine Equity -Finding a Path Forward An interactive web-based dashboard to track COVID-19 in real time Safety and immunogenicity of INO-4800 DNA vaccine against SARS-CoV-2: A preliminary report of an open-label, Phase 1 clinical trial Safety and immunogenicity of INO-4800 DNA vaccine against SARS-CoV-2: a preliminary report of a randomized, blinded, placebo-controlled, Phase 2 clinical trial in adults at high risk of viral exposure. medRxiv : the preprint server for health sciences Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein DNA vaccines: prime time is now Addressing the Cold Reality of mRNA Vaccine Stability A Comparison of Plasmid DNA and mRNA as Vaccine Technologies Immunogenicity of a DNA vaccine candidate for COVID-19 Intradermal SynCon(R) Ebola GP DNA Vaccine Is Temperature Stable and Safely Demonstrates Cellular and Humoral Immunogenicity Advantages in Healthy Volunteers No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted Safety and Immunogenicity of an Anti-Zika Virus DNA Vaccine -Preliminary Report Immune Therapy Targeting E6/E7 Oncogenes of Human Paillomavirus Type 6 (HPV-6) Reduces or Eliminates the Need for Surgical Intervention in the Treatment of HPV-6 Associated Recurrent Respiratory Papillomatosis Early T cell and binding antibody responses are associated with COVID-19 RNA vaccine efficacy onset T cell responses in patients with COVID-19 Antigen-Specific Adaptive Immunity to SARS-CoV-2 in Acute COVID-19 and Associations with Age and Disease Severity INO-4800 DNA Vaccine Induces Neutralizing Antibodies and T cell Activity Against Global SARS-CoV-2 Variants The investigators and sponsor express their gratitude for the contribution of all the trial participants All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021. 10 All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. -Confidential-All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.10.06.21264584 doi: medRxiv preprint 24 -Confidential-All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted October 10, 2021. All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.10.06.21264584 doi: medRxiv preprint 26 -Confidential-All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.10.06.21264584 doi: medRxiv preprint 27 -Confidential-the minimum to maximum values. The mean is denoted with a "+" sign. Wilcoxon signed-rank was used to assess significance versus baseline. The dose groups are represented by triangles (0.5 mg), circles (1.0 mg) and squares (2.0 mg). All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.10.06.21264584 doi: medRxiv preprint 28 -Confidential- (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.10.06.21264584 doi: medRxiv preprint 29 -Confidential-is denoted with a "+" sign. Wilcoxon signed-rank was used to assess significance versus baseline.The dose groups are represented by orange triangles (0.5 mg), blue circles (1.0 mg) and green squares (2.0 mg). All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.10.06.21264584 doi: medRxiv preprint