key: cord-0889913-bh4hfp24 authors: Ahn, J. Y.; Lee, J.; Suh, Y. S.; Song, Y. G.; Choi, Y.-J.; Lee, K. H.; Seo, S. H.; Song, M.; Oh, J.-W.; Kim, M.; Seo, H.-Y.; Kwak, J.-E.; Youn, J. W.; Woo, J. W.; Shin, E. C.; Park, S.-H.; Sung, Y. C.; Choi, J. Y. title: Safety and immunogenicity of a recombinant DNA COVID-19 vaccine containing the coding regions of the spike and nucleocapsid proteins: Preliminary results from an open-label, phase 1 trial in healthy adults aged 19-55 years date: 2021-06-01 journal: nan DOI: 10.1101/2021.05.26.21257700 sha: 2f8d1ddacb5608635316d0db15717d7c4cf0391f doc_id: 889913 cord_uid: bh4hfp24 Background : We investigated the safety and immunogenicity of two recombinant COVID-19 DNA vaccine candidates in first-in-human trials. GX-19 contains plasmid DNA encoding SARS-CoV-2 spike protein, and GX-19N contains plasmid DNA encoding SARS-CoV-2 receptor binding domain (RBD) foldon and nucleocapsid protein (NP) as well as plasmid DNA encoding SARS-CoV-2 spike protein. Methods : Two open-label phase 1 trials of GX-19 and GX-19N safety and immunogenicity were performed in healthy adults aged 19-55 years. GX-19 trial participants received two vaccine injections (1.5 mg or 3.0 mg, 1:1 ratio) four weeks apart. GX-19N trial participants received two 3.0 mg vaccine injections four weeks apart. Findings : Between June 17 and July 30 and December 28 and 31, 2020, 40 and 21 participants were enrolled in the GX-19 and GX-19N trials, respectively. Thirty-two participants (52.5%) reported 80 treatment-emergent adverse events (AE) after vaccination. All solicited AEs were mild except one case of moderate fatigue reported in the 1.5 mg GX-19 group. Binding antibody responses increased after vaccination in all groups. The geometric mean titers (GMTs) of spike-binding antibodies on day 57 were 85.74, 144.20, and 201.59 in the 1.5 mg, 3.0 mg GX-19 groups and the 3.0 mg GX-19N group, respectively. In GX-19N group, neutralizing antibody response (50% neutralizing titer using FRNT) significantly increased after vaccination, but GMT of neutralizing antibody on day 57 (37.26) was lower than those from human convalescent serum (288.78). GX-19N induced stronger T cell responses than GX-19. The magnitude of GX-19N-induced T cell responses was comparable to those observed in the convalescent PBMCs. GX-19N induced both SARS-CoV-2 spike- and NP-specific T cell responses, and the amino acid sequences of 15-mer peptides containing NP-specific T cell epitopes identified in GX-19N-vaccinated participants were identical with those of diverse SARS-CoV-2 variants Interpretation : GX-19N is safe, tolerated and induces humoral and broad SARS-CoV-2-specific T cell response which may enable cross-reactivity to emerging SARS-CoV-2 variants. Funding : This research was supported by Korea Drug Development Fund funded by Ministry of Science and ICT, Ministry of Trade, Industry, and Energy, and Ministry of Health and Welfare (HQ20C0016, Republic of Korea). To overcome COVID-19 outbreak, development of safe and effective vaccines is crucial. Many are in development based on different platforms including mRNA, DNA, viral vectored, protein-based and inactivated virus vaccines. Most vaccines that are currently in development target the spike protein, which binds to the angiotensin converting enzyme 2 receptor to entry into the host cell. over time. 4 Virus-specific T cells can be an alternative to neutralizing antibodies against variants. Given that variants escaping T cell immunity against SARS-CoV-2 also occur 5 , broader T cell immunity would be beneficial and can be achieved by adding well-conserved antigens to induce dominant T cell responses. GX-19 is a candidate recombinant DNA vaccine that contains a plasmid encoding the SARS-CoV-2 S proteins (S1, S2). 6 A plasmid vector (pGX27), which was used for the HPV DNA vaccine undergoing clinical trials, was used. 7, 8 We produced the following four prototype DNA vaccines expressing the SARS-CoV-2 S protein: 1) S1S2 , 2) S1 , 3) S1S2 with the CD40 ligand , and 4) S1 with the CD40 ligand . SRBD/NP) in a ratio of 1:2 ( Figure 1 ). Preclinical studies in mouse and monkey models showed that higher neutralizing antibody responses and cellular immune responses were expected following vaccination with . Details regarding the results of preclinical studies are provided in the Supplementary Appendix 1. Here, we report the preliminary safety and immunogenicity assessments of two recombinant DNA vaccine candidates for COVID-19, in first-in-human trials. We performed two open-label phase 1 trials to evaluate the safety, tolerability, and immunogenicity of and GX-19N (NCT04715997) after intramuscular vaccination of healthy adults at two hospitals in South Korea (Severance Hospital and Gangnam Severance Hospital). The GX-19 trial was a dose-escalation study conducted prior to the GX-19N trial. GX-19 trial participants received two injections of the trial vaccine four weeks apart at a dose of 1·5 mg or 3·0 mg. The GX-19N trial was subsequently conducted based on the results obtained in the GX-19 trial. Healthy adult participants aged 19-55 years were enrolled through local advertisements. Full details of the eligibility criteria are described in the trial protocol provided in the Supplementary Appendix 2. Written informed consent was obtained from all participants. The trials were conducted in accordance with the principles of the Declaration of Helsinki and Good Clinical Practice and registered with ClinicalTrials.gov (NCT04445389 and NCT04715997). This study was approved in South Korea by the Korea Ministry of Food and Drug Safety (reference 20200261849) and the Institutional Review Board of Severance Hospital (IRB No. 4-2020- uptake by generating a high-voltage pulse after drug injection. In the GX-19 trial, participants were first enrolled in the 1·5 mg group. After safety and tolerability data from this group were reviewed by the independent Data Safety Monitoring Committee (DSMC), participants were enrolled in the 3·0 mg group. Participants were administered 1·5 mg (0·38 ml) or 3·0 mg (0·75 ml) GX-19 intramuscularly in the deltoid muscle on days 1 and 29 and followed for 52 weeks post first vaccination. In the trial, participants were enrolled in a single dose group to receive 3·0 mg (0·75 ml) intramuscularly in the deltoid muscle on days 1 and 29 and followed for 52 weeks post first vaccination. To assure participant safety, all participants were followed-up with daily by telephone for AEs for up to seven days from the first vaccination and had to visit the site on the eighth day to undergo a safety assessment. Follow-up visits were scheduled on days 8, 29, 43, 57, and weeks 24 and 52 after the first vaccination, and safety-related data were collected. A diary was distributed to participants at the time of the first and second vaccination for them to personally list any AEs experienced over approximately four weeks. To assess immunogenicity for this interim analysis report, blood samplings were performed at baseline, 29, 43, and 57 days after the first vaccination. Additional details regarding these procedures are provided in the Supplementary Appendix 3. Local and systemic reactions after each vaccination were monitored for 28 days. Serious AEs were recorded throughout the follow-up period. The solicited AEs appearing in the study participants were graded according to the "Guidance for Adverse Event Grading Scale for Volunteers in Preventive Vaccine Clinical Trials (2011, 12)" issued by the Ministry of Food and Drug Safety. 9 The severity of unsolicited AEs was assessed according to Common Terminology Criteria for Adverse Events (CTCAE V5.0) 10 and Medical Dictionary for Regulatory Activities (MedDRA) classification and relatedness to the vaccine. AEs of special interest (AESIs), including acute respiratory distress syndrome, pneumonitis, enhanced disease following immunization, acute cardiac injury, arrhythmia, septic shock-like syndrome, and acute kidney injury, were also assessed. Laboratory AEs were graded using site-specific toxicity tables, which were adapted from the guideline for assessing the severity of AEs in vaccine clinical trials by the Korea Ministry of Food and Drug Safety. 9 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 June 1, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 Immunogenicity Binding antibody responses against the SARS-CoV-2 S protein were assessed by using an inhouse-developed ELISA and antibodies against the SARS-CoV-2 RBD protein were assessed using an ELISA kit (Bionote Inc, Korea). Binding antibodies were assessed before the vaccination, and at days 43 and 57 after the first vaccination. Vaccine-induced neutralizing activity was assessed using a live wild-type SARS-CoV-2 focus reduction neutralization testing (FRNT) assay and a SARS-CoV-2 Spike (S)-pseudotyped murine leukemia virus (MLV) retrovirus-based single-round-of-infection neutralization assay (PsVNA). The 50% neutralization titers were reported as the interpolated reciprocal of the dilutions yielding 50% reductions in viral foci. The FRNT assay was performed on specimens collected before the vaccination and on days 43 and 57 after the first vaccination. For this interim analysis report, PsVNA results were available only before and on day 43 after the first vaccination. T cell responses in participants vaccinated with GX-19 or GX-19N were evaluated before and on day 43 after the first vaccination using a direct ex vivo IFN-γ enzyme-linked immunosorbent spot (ELISpot) assay with peripheral blood mononuclear cells (PBMCs). In these assays, PBMCs were stimulated overnight with overlapping peptides (OLPs) encoding the full-length sequence of S (for GX-19) and S/NP (for GX-19N), respectively. To selectively evaluate vaccine-induced SARS-CoV-2-specific T cell responses, the T-SPOT assay (Oxford Immunotec, UK) was performed. For this assay, PBMCs obtained from participants were stimulated with OLPs spanning S and NP that specific sequences with high homology to endemic (non-SARS-CoV-2) coronaviruses removed. The details of all methods are described in the Supplementary Appendix 4. To compare vaccine-induced immune responses with those induced by natural SARS-CoV-2 infection, we analyzed convalescent blood specimens collected within three months of polymerase chain reaction-based diagnosis of COVID-19. FRNT/PsVNA and ELISpot assays were performed to evaluate neutralizing antibody responses and T cell responses, respectively. The primary outcome was the safety and tolerability of GX-19 and GX-19N based on observation of the frequency, characteristics, and severity of AEs experienced within 28 days of each vaccination. The secondary outcome was the immunogenicity of Immunogenicity endpoints in this interim analysis included changes in SARS-CoV-2 S and RBD antigen-specific binding antibody levels compared to baseline levels within day 57 in all participants (on days 1, 43, and 57 after first vaccination) and changes in SARS-CoV-2 neutralizing antibody levels within day 57 in the participants of the 3·0 mg GX-19N group (on days 1, 43 and 57 for the FRNT assay and day 1 and 43 after first vaccination for PsVNA). Immunogenicity endpoints also included determination of the SARS-CoV-2specific T cell responses of GX-19 and GX-19N measured on days 1 and 43 after the first vaccination. Continuous variables are presented using descriptive statistics (sample size, mean, standard deviation, median, and range), and categorical variables are presented as the frequency and percentage, as well as a 95% two-sided confidence interval (CI), as appropriate. When theoretical hypothesis testing was required, parametric methods were used for continuous values with normal distribution and non-parametric methods were used for continuous values with non-normal distribution. Chi-square and Fisher's exact tests were used for categorical values. For safety assessment, AEs are presented as the number and percentage of subjects who experience AEs and the number of actual cases with a 95% CI. AEs were classified by type, severity, and causality for each group, and the frequency and percentage of each was calculated. For immunological analysis, the GMTs of binding and neutralizing antibodies before and after the vaccination were assessed, and a 95% CI was presented. The proportions of participants with a ≥ 4-fold increase in the titer of binding and neutralizing antibodies compared to baseline levels were also presented. T cell responses were expressed as the number of spot-forming-units per 10 6 cells measured using IFN-γ ELIspot and T-SPOT assays before and after the vaccination, and a 95% CI was presented. The subjects with The funder of the study had no role in data collection, analysis, interpretation or writing of the preliminary report, but was involved in the study design. All authors had full access to all preliminary data in the study and final responsibility for the decision to submit the manuscript for publication. In the GX-19 trial, 96 healthy individuals were screened and 40 were enrolled between June 17 and July 30, 2020, to receive two doses of 1·5 mg or 3·0 mg GX-19 in an 1:1 ratio. In the GX-19N trial, 23 healthy individuals were screened and 21 were enrolled between December 28 and December 31, 2020 and received two doses of 3·0 mg GX-19N, (Figure 2 ). All 61 participants received their second vaccine dose, and all continued to attend the scheduled trial visits until day 57 and were included in both safety and immunogenicity analyses. The demographic characteristics of the participants at enrollment are presented in Table 1 . The mean age was 34·9 years (range, 20-52 years) and 52·4% were men. All participants were Korean. Similar demographic characteristics were observed in all groups. Thirty-two participants (52·5%), of whom 12 (60%) were in the 1·5 mg GX-19 group, eight (40%) in the 3·0 mg GX-19 group, and 12 (57·1%) in the 3·0 mg GX-19N group, reported 80 treatment-emergent adverse events (TEAEs) after vaccination. (Supplementary Appendix 5) The majority of AEs were mild, and none of the participants experienced serious drug-related AEs or AESIs. (Supplementary Appendix 6) No participants discontinued the trial due to AEs. After the first vaccination, solicited local AEs were reported by six participants (30%) in the 1·5 mg GX-19 group, two (10%) in the 3·0 mg GX-19 group, and five (23·8%) in the 3·0 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 June 1, 2021. ; https://doi.org/10.1101/2021.05.26.21257700 doi: medRxiv preprint GX-19N group. After both doses, solicited local AEs were reported by six (30%) participants in the 1·5 mg GX-19 group, three (15%) in the 3·0 mg GX-19 group, and five (23·8%) in the 3·0 mg GX-19N group. Solicited systemic AEs were reported by one (5%) participant in the 1·5 mg GX-19 group and two (9·5%) in the 3·0 mg GX-19N group after the first vaccination and three (15%) in the 1·5 mg GX-19 group and two (9·5%) in the 3·0 mg GX-19N group after both doses. There were no reported solicited systemic AEs in the 3·0 mg GX-19 group. The most common solicited AEs were injection site pain, itching, tenderness, and fatigue. The GMTs of binding antibodies to S protein increased after the first vaccination by day 57 in both the 1·5 mg GX-19 and 3·0 mg GX-19 groups. In the GX-19N group, antibody responses to S and RBD proteins increased after vaccination by day 43, but the GMTs on day 57 slightly decreased compared to those on day 43 (Table 2, Figure 4A ). In each group, there was a statistically significant increase in GMTs by day 43 and day 57 over the baseline. The GMTs of S-binding antibody on day 57 were 85·74 (95% CI 49·67-148·00) in the 1·5 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 June 1, 2021. ; https://doi.org/10.1101/2021.05.26.21257700 doi: medRxiv preprint GX-19 group, 144·20 (95% CI 80·81-257·32) in the 3·0 mg GX-19 group, and 201·59 (105·32-385·83) in the 3·0 mg GX-19N group. The proportion of subjects with ≥ 4-fold rises in the S or RBD protein antibody levels after the vaccination was 55% in the 1·5 mg GX-19 group, 65% in the 3·0 mg GX-19 group, and 80·95% in the 3·0 mg GX-19N group. in PsVNA were 1·00 (95% CI 1·00-1·00) before the first vaccination and 21·87 (95% CI 15·30-31·07) on day 43. Higher than 4-fold rises in the 50% neutralization titer were observed in all participants using PsVNA. However, the GMT of ID50 values on day 43 was lower than that of convalescent sera from 20 patients (GMT = 222·20) ( Figure 4B ). Using FRNT, a statistically significant increase in the neutralizing antibody GMT after vaccination was observed in the 3·0 mg GX-19N group by days 43 and 57. Neutralizing antibody GMTs were 32·47 (95% CI 22·71-46·41) on day 43 and 37·26 (95% CI 25·29-54·90) on day 57. The proportion of subjects with ≥ 4-fold rises in antibody levels was 23·81% by day 57. Neutralizing GMTs by day 57 were also much lower than those in 22 human convalescent serum samples (GMT = 288·78) ( Figure 4C ). In the GX-19 trial, both the 1·5 mg and 3·0 mg GX-19 groups showed significantly enhanced S-specific T cell responses following vaccination. Specifically, 10 (50%) participants in the 1·5 mg GX-19 group and 10 (50%) in the 3·0 mg GX-19 group mounted augmented Sspecific T cell responses on day 43, respectively (Table 3and Figure 5A ). In the GX-19N trial, GX-19N-immunized participants exhibited significantly enhanced S-and NP-specific T cell responses on day 43 after vaccination (Table 3, Figure 5B and 5C). The results revealed that 15 (75%) participants generated vaccine-induced T cell responses after vaccination 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 June 1, 2021. ; https://doi.org/10.1101/2021.05.26.21257700 doi: medRxiv preprint (Supplementary Appendix 7), and the magnitude of GX-19N-induced T cell responses was comparable to those observed in the convalescent PBMCs from six COVID-19 patients ( Figure 5B ). Furthermore, we tried to evaluate whether five participants (19Na, 19Nd, 19Nf, 19Nq, and 19Nt ), who did not show enhanced T cell responses based on the results of the IFN-γ ELIspot assay using OLPs spanning the full length of S and NP, induced vaccineinduced SARS-CoV-2-specific T cell responses. To this end, we used the T-SPOT kit with PBMCs from four participants (19Na, 19Nd, 19Nf, and 19Nq) using OLPs that did not contain specific sequences with high homology to endemic (non-SARS-CoV-2) coronaviruses. The results revealed that three out of four participants mounted increased GX- We report the interim findings from a phase 1 clinical trial of two recombinant DNA vaccines administered to healthy adults. GX-19 and GX-19N were well tolerated and showed an 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 June 1, 2021. ; https://doi.org/10.1101/2021.05.26.21257700 doi: medRxiv preprint acceptable safety profile without serious vaccine-related AEs. The overall incidence of solicited AEs in this study is lower than the frequencies observed in other platforms of licensed SARS-CoV-2 vaccines. 11-13 The favorable safety profile in this study was comparable with the trial results of another DNA vaccine candidate for The GX-19N group showed stronger cellular responses than those of the GX-19 groups, which were similar to the T cell responses observed after natural COVID-19 infection. Recent studies have suggested the critical roles of SARS-CoV-2-specific T cells in the clearance of SARS-CoV-2 and protection from developing severe COVID-19. It has been demonstrated that among several immune parameters, the frequency of SARS-CoV-2-specific IFN-γ-producing CD8 + T cells showed the strongest inverse correlation with the severity of COVID-19. 15 One study reported that SARS-CoV-2-specific polyfunctional T cells in the circulation of antibody negative convalescent patients presented asymptomatic and mild COVID-19 16 , suggesting in the absence of antibodies, a robust T cell response might provide immune protection against SARS-CoV-2. 17 In another study, it has been shown that T cells contributed to protection of hosts with hematologic malignancy even when B cells were depleted by anti-CD20 therapy. 18 Moreover, a study using a SARS-CoV-2 infection model of rhesus macaques showed that the depletion of CD8 + T cells in convalescent animals partially abolished the protective immunity against SARS-CoV-2 re-challenge. 19 It was also reported that a T cell vaccination in the absence of neutralizing antibodies protected mice from SARS-CoV-2 infection. 20 Furthermore, GX-19N not only induced S-specific T cell responses but also induced broad NP-specific T cell responses, which were also specific to SARS-CoV-2 variants. A key feature of GX-19N is that it contains a plasmid encoding NP, which is highly conserved (with >90% amino acid homology and fewer mutations over time) in diverse SARS-CoV-2 variants. To our knowledge, this is the first study reporting a phase 1 clinical trial of a SARS-CoV-2 DNA vaccine candidate that includes a non-spike protein-encoding DNA sequence. While escaping neutralizing antibody-based immunity is easily achieved via a few RBD mutations, escaping T cell-based immunity is far more complex because T cell epitopes are more abundant and widespread throughout the whole antigens. 21 Therefore, considering the growing evidence suggesting that a T cell response is important for rapid virological control as well as long-term protection against SARS-CoV-2 infection, especially when neutralizing antibody titers decline, the fact that GX-19N induces broad T cell responses to highly 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 June 1, 2021. ; https://doi.org/10.1101/2021.05.26.21257700 doi: medRxiv preprint conserved regions among SARS-CoV-2 variants indicates that this vaccine would be beneficial in inducing protective immunity against future SARS-CoV-2 variants. Based on the binding and neutralizing antibody response assay results, GX-19N-induced antibody responses to SARS-CoV2 antigens are likely to be weaker than those induced by natural COVID-19 infection or commercialized mRNA-based or adenoviral vector-based vaccines 11-13 , even though immune responses are not directly compared in the same setting. Previous studies on DNA vaccines have determined that the number of doses required for effective vaccination is dependent on the antigen and virus types and how they interact with the immune system. 22 One to two doses of the DNA vaccine are enough for eliciting sufficient neutralizing antibody responses to influenza virus, whereas three to four doses are needed to produce protective humoral responses against HIV. 23 In a preclinical study of a plasmid DNA vaccine for SARS-CoV-2, it was reported that the magnitude of binding and neutralizing antibody responses was the highest after three doses. 22 Further studies on humoral immunogenicity after another booster dose of GX-19N should be considered. As this is a phase 1 interim analysis, we could not report persistent safety outcomes or vaccine-induced immune responses and the results do not permit a vaccine efficacy assessment. The interpretation of the results of this trial is also limited by the open-labeled, non-randomized design and small size. The participants in this trial were not ethnically diverse, and participants more susceptible to SARS-CoV-2, such as older individuals or individuals with comorbidities, were not included. However, this study has several strengths. This is the first trial to assess the safety and immunogenicity of a DNA vaccine that encodes both the S protein and the highly conserved NP. Given the concerns of decreased efficacy of the current COVID-19 vaccines against SARS-CoV-2 variants, we also performed an additional detailed assessment of T cell responses and analyzed the reactivity of vaccineinduced T cell responses to SARS-CoV-2 variants. Our preliminary results indicate that the recombinant DNA vaccines for COVID-19, GX-19 and GX-19N are safe and that GX-19N is able to induce both humoral and cellular responses, more focused on T cell responses rather than neutralizing antibody responses. Importantly, GX-19N, which contains a plasmid encoding both the S protein and NP, showed a broad SARS-CoV-2-specific T cell response, which may allow cross-reactivity to emerging SARS-CoV-2 variants. Based on these safety and immunogenicity findings, GX-19N was selected as a vaccine candidate for phase 2 immunogenicity trials (NCT04715997). 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 June 1, 2021. Deidentified individual participant data will be made available when the trial is complete, upon request directed to the corresponding author. Proposals should be sent to the corresponding authors, and data can be shared following proposal approval. a h a n K , Y u J , M e r c a d o N B , e t a l . C o r r e l a t e s o f p r o t e c t i o n a g a i n s t S A R S -C o V -2 i n r h e s u s m a c a q u e s . N a t u r e 2 0 2 0 ; 5 9 0 ( 7 8 4 7 ) : h u a n g Z , L a i X , S u n J , e t a l . M a p p i n g a n d r o l e o f T c e l l r e s p o n s e i n S A R S -C o V -2 -i n f e c t e 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 June 1, 2021. ; https://doi.org/10.1101 https://doi.org/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 June 1, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 Healthy volunteers aged 19-55 were vaccinated at day 1 and 29, with 1·5 mg (n=20), 3·0 mg (n=20) . Sera were collected at day 1, 43, 57 and assessed for SARS-CoV-2 Spike-and RBD-specific IgG antibodies by ELISA Yes 10 (50·00) 6 (30·00) 12 (57·14) No n (%) 14 (70·00) 15 (75·00) 11 (52·38) Ex-smoker n (%) 6 (30·00) 5 (25·00) 10 (47·62) No n (%) 11 (55·00) 11 (55·00) 3 (14·29) Ex-drinker n (%) 9 (45·00) 9 (45·00) 18 (85·71) No n (%) 20 (100·00) 20 (100·00) 21 (100·00) SD = Standard deviation, CI = confidence interval, Min = minimum, Max = maximum, BMI = body mass index. 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. GMTs = geometric mean titers, SD = Standard deviation, CI = confidence interval, Min = minimum, Max = maximum. [1] p-value was calculated in Wilcoxon signed rank test. Denominator of percentage is the number of subjects in each cohort. 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 June 1, 2021. ; https://doi.org/10.1101/2021.05.26.21257700 doi: medRxiv preprint T cell responses are presented as spot-forming-unit (SFU) per 10 6 cells [1] p-value was calculated in Wilcoxon signed rank test. Denominator of percentage is the number of subjects in each cohort. 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 June 1, 2021. GX-19 (1.5 mg) GX-19 (3.0 mg) GX-19N