Table of Contents
Abstract
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® 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-ɣ 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-ɣ and TNF-α, 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.
Funding
Coalition for Epidemic Preparedness Innovations (CEPI).
1. Introduction
]. The elderly and those with co-morbid conditions are prone to experience more severe symptoms, including pneumonia and multiorgan disease. Severe and critically-ill COVID-19 patients requiring intensive care and invasive mechanical ventilation can quickly overwhelm hospitals [
]. Despite the increasing number of infections and deaths around the globe, most people remain vulnerable to infection. There is an urgent need for safe and effective vaccines. Many are in development including nucleic acid [
,
], viral vectored [
,
,
], and inactivated virus vaccines [
]. Most of these vaccines target the spike protein, a class I fusion protein of SARS-CoV-2 which binds to the angiotensin converting enzyme 2 (ACE2) receptor to gain entry into the host cell.
]. While natural infection and recovery from SARS-CoV-2 is associated with generation of binding antibodies as well as antibodies that can neutralize virus in recovered individuals [
,
,
], antibody responses are not detectable in all recovered patients [
,
,
], and these antibodies tend to wain within months [
,
]. Studies increasingly underscore the importance of T cell responses in ameliorating the severity of disease, with immunity to the spike Ag as one important immune target [
,
,
,
]. Therefore, eliciting a well-balanced adaptive immune response could be an important hallmark of a promising vaccine candidate.
,
,
] and have demonstrated efficacy against HPV associated cervical dysplasia [
]. This technology has previously been employed in the development of a vaccine candidate (INO-4700) against another betacoronavirus: the Middle Eastern Respiratory virus (MERS), targeting its spike glycoprotein. Preclinical [
] and Phase 1 studies [
] (NCT03721718) demonstrated that INO-4700 was safe and immunogenic, and efficacious in NHP challenge studies (Patel et al., submitted).
], demonstrating protective impact on infection in a nonhuman primate SARS-CoV-2 challenge model (Patel et al., submitted). Here, the initial findings of the first clinical trial evaluating INO-4800 delivered by intradermal injection followed by CELLECTRA® EP, designed to generate a controlled electric field in the injection site to enhance the cellular uptake and expression of the DNA plasmid, are reported.
2. Methods
2.1 Study design and participants
The clinical trial was designed as a Phase 1, open-label, multi-center trial (NCT04336410) to evaluate the safety, tolerability and immunogenicity of INO-4800 administered intradermally (ID) followed by electroporation using the CELLECTRA® 2000 device. The trial was approved by the institutional review board of each clinical site, and all participants provided written informed consent before enrollment. Healthy participants 18 to 50 years of age without a known history of COVID-19 illness received either a 1.0 mg or 2.0 mg dose of INO-4800 in a 2-dose regimen (Weeks 0 and 4). Participants enrolled at two locations in the U.S.: The University of Pennsylvania Clinical trials Unit in Philadelphia and the Alliance for Multispecialty Research in Kansas City (Details regarding inclusion and exclusion criteria and the schedule of events are provided in the protocol and are available with the full text of this article).
2.2 DNA vaccine INO-4800
] at a concentration of 10 mg/ml in a saline sodium citrate buffer. The optimized DNA sequence encoding SARS-CoV-2 insert was created using Inovio's proprietary in silico Gene Optimization Algorithm to enhance expression. The DNA sequence changes do not impact amino acid sequence. INO-4800 is homologous to the Wuhan strain.
2.3 Endpoints
Safety endpoints included systemic and local administration site reactions up to 8 weeks post-dose 1. Immunology endpoints include antigen-specific binding antibody titers, neutralization titers and antigen-specific interferon-gamma (IFN-γ) cellular immune responses after 2 doses of vaccine. For Live Virus Neutralization, a responder is defined as Week 6 PRNT IC50 ≥ 10, or ≥4 if a subject is a responder in ELISA. For S1+S2 ELISA, a responder is defined as a Week 6 value >1. For the ELISpot assay, a responder is defined as a Week 6 or Week 8 value that is ≥12 spot forming units per 106 PBMCs above Week 0.
2.4 Study procedures
,
]. The device delivers total four electrical pulses, each 52 ms in duration at strengths of 0.2 A current and voltage of 40–200 V per pulse.
The dose groups were enrolled sequentially with a safety run-in for each. The 1.0 mg dose group enrolled a single participant per day for 3 days. An independent Data Safety Monitoring Board (DSMB) reviewed the Week 1 safety data and based on a favorable safety assessment, made a recommendation to complete enrollment of the additional 17 participants into that dose group. In a similar fashion, the 2.0 mg dose group was subsequently enrolled.
Participants were assessed for safety and concomitant medications at all time points, including screening, Week 0 (Dose 1), post dose next day phone call, Week 1, 4 (dose 2), 6, 8, 12, 28, 40 and 52 post-dose 1. Local and systemic AEs, regardless of relationship to the vaccine, were recorded and graded by the investigator. Safety laboratory testing (complete blood count, comprehensive metabolic panel and urinalysis) were and will continue to be conducted at screening, Week 1, 6, 8, 12, 28 and 52 post-dose 1. Immunology specimens were obtained at all time points post-dose 1 except at Day 1 and Week 1. AEs were graded according to the Toxicity Grading Scale for Healthy Adult and Adolescent Volunteers Enrolled in Preventive Vaccine Clinical Trials guidelines that were issued by the Food and Drug Administration in September 2007. The DSMB reviewed laboratory and AE data for the participants up to 8 weeks included in this report. There were protocol-specified safety stopping rules and adverse events of special interest (AESIs). For the purpose of this report, clinical and laboratory safety assessments up to 8 weeks post the first dose are presented.
2.5 Protocol eligibility
Eligible participants must have met the following criteria: healthy adults aged between 18 and 50 years; able and willing to comply with all study procedures; Body Mass Index of 18–30 kg/m2 at screening; negative serological tests for Hepatitis B surface antigen, Hepatitis C antibody and Human Immunodeficiency Virus antibody; screening electrocardiogram (ECG) deemed by the Investigator as having no clinically significant findings; use of medically effective contraception with a failure rate of < 1% per year when used consistently be post-menopausal, or surgically sterile or have a partner who is sterile. Key exclusion criteria included the following: individuals in a current occupation with high risk of exposure to SARS-CoV-2; previous known exposure to SARS-CoV-2 or receipt of an investigational product for the prevention or treatment of COVID-19; autoimmune or immunosuppression as a result of underlying illness or treatment; hypersensitivity or severe allergic reactions to vaccines or drugs; medical conditions that increased risk for severe COVID-19; reported smoking, vaping, or active drug, alcohol or substance abuse or dependence; and fewer than two acceptable sites available for intradermal injection and electroporation.
2.6 Immunogenicity assessment methods
For this report, samples collected at screening, Week 0 (prior to dose) and at Weeks 6 and 8 were analyzed. Peripheral Blood Mononuclear Cells (PBMCs) were isolated from blood samples by a standard overlay on ficoll hypaque followed by centrifugation. Isolated cells were frozen in 10% DMSO and 90% fetal calf serum. The frozen PBMCs were stored in liquid nitrogen for subsequent analyses. Serum samples were stored at −80 C until used to measure binding and neutralizing antibody titers.
2.6.1 SARS-CoV-2 wildtype virus neutralization assays
]. Neutralizing virus titers were measured in serum samples that had been heat-inactivated at 56 °C for 30 min. SARS-CoV-2 (Australia/VIC01/2020 isolate44) was diluted to a concentration of 933 pfu/ml and mixed 50:50 in 1% FCS/MEM containing 25 mM HEPES buffer with doubling serum dilutions. After a 1 h incubation at 37 °C, the virus-antibody mixture was transferred to confluent monolayers of Vero E6 cells (ECACC 85020206; PHE, UK). Virus was allowed to adsorb onto cells at 37 °C for a further hour in an incubator, and the cell monolayer was overlaid with MEM/4% FBS/1.5% CMC. After 5 days incubation at 37 °C, the plates were fixed, stained, with 0.2% crystal violet solution (Sigma) in 25% methanol (v/v). Plaques were counted.
2.6.2 S1±S2 enzyme-linked immunosorbent assay (ELISA)
ELISA plates were coated with 2.0 µg/mL recombinant SARS-CoV-2 S1+S2 spike protein (Acro Biosystems; SPN-C52H8) and incubated overnight at 2–8 °C. The S1+S2 contains amino acids residues Val 16 – Pro 1213 of the full length spike protein, GenBank # QHD43416.1. It contains two mutations to stabilize the protein to the trimeric prefusion state (R683A, R685A) and also contains a C-terminal 10× His tag. The plates were then washed with PBS with 0.05% Tween-20 (Sigma; P3563) and blocked (Starting Block, Thermo Scientific; 37,538) for 1–3 h at room temperature. Samples were serially diluted using blocking buffer and were added in duplicate, along with prepared controls, to the washed and blocked assay plates. The samples were incubated on the blocked assay plates for one hour at room temperature. Following sample and control incubation, the plates were washed and a 1/1000 preparation of anti-human IgG HRP conjugate (BD Pharmingen; 555,788) in blocking buffer was then added to each well and allowed to incubate for 1 h at room temperature. The plates were washed and TMB substrate (KPL; 5120-0077) was then added and allowed to incubate at room temperature for approximately 10 min. TMB Stop Solution (KPL; 5150-0021) was next added and the plates read at 450 nm and 650 nm on a Synergy HTX Microplate Reader (BioTek). The magnitude of the assay response was expressed as titers which were defined as the greatest reciprocal dilution factor of the greatest dilution serial dilution at which the plate corrected optical density is 3 SD above background a subject's corresponding Week 0.
2.6.3 SARS-CoV-2 spike ELISpot assay description
Peripheral mononuclear cells (PBMCs) pre- and post-vaccination were stimulated in vitro with 15-mer peptides (overlapping by 9 residues) spanning the full-length consensus spike protein sequence. Cells were incubated overnight in an incubator with peptide pools at a concentration of 5 μg per ml in a precoated ELISpot plate, (MabTech, Human IFN-g ELISpot Plus). The next day, cells were washed off, and the plates were developed via a biotinylated anti-IFN-γ detection antibody followed by a streptavidin-enzyme conjugate resulting in visible spots. Each spot corresponds to an individual cytokine-secreting cell. After plates were developed, spots were scanned and quantified using the CTL S6 Micro Analyzer (CTL) with ImmunoCapture and ImmunoSpot software. Values are shown as the background-subtracted average of measured triplicates. The ELISpot assay qualification determined that 12 spot forming units was the lower limit of detection. Thus, anything above this cutoff is considered to be a signal of an antigen specific cellular response.
2.6.4 INO-4800 SARS-CoV-2 spike flow cytometry assay
PBMCs were also used for Intracellular Cytokine Staining (ICS) analysis using flow cytometry. One million PMBCs in 200 µL complete RPMI media were stimulated for six hours (37 °C, 5% CO2) with DMSO (negative control), PMA and Ionomycin (positive control, 100 ng/mL and 2 μg/mL, respectively), or with the indicated peptide pools (225 ug/mL). After one hour of stimulation, Brefeldin A and Monensin (BD GolgiStop and GolgiPlug, 0.001% and 0.0015%, respectively) were added to block secretion of expressed cytokines. After stimulation the cells were moved to 4 °C overnight. Next, cells were washed in PBS for live/dead staining (Life Technologies Live/Dead aqua fixable viability dye), and then resuspended in FACS buffer (0.5% BSA, 2 mM EDTA, 20 mM HEPES). Next, extracellular markers were stained, the cells were fixed and permeablized (eBioscience™ Foxp3 Kit) and then stained for the indicated cytokines (Table S2) using fluorescently-conjugated antibodies. Fig. S1A shows representative gating strategies for CD4+ and CD8+ T cells as well as examples of positive expression of IFNγ, TNFα, IL-2 and IL-4.
2.7 Statistical analysis
No formal power analysis was applicable to this trial. Descriptive statistics were used to summarize the safety end-points: proportions with AEs, administration site reactions, and AESIs through 8 weeks. Descriptive statistics were also used to summarize the immunogenicity endpoints: median responses (with 95% confidence intervals) and percentage of responders for cellular results, and geometric mean titers (with 95% confidence intervals) and percentage of responders for humoral results. Post-hoc analyses of post-vaccination minus pre-vaccination paired differences in SARS-CoV-2 neutralization responses (on the natural log-scale, with a paired t-test), ELISpot responses (with Wilcoxon signed-rank tests), and Intracellular Flow Assay responses (with Wilcoxon signed-rank tests) were performed.
2.8 Role of funding sources
The COVID19-001 Phase 1 clinical study is in part funded by the Coalition for Epidemic Preparedness Innovations (CEPI). CEPI had not role in the study design, collection, analysis, interpretation of the preliminary study data, writing of the interim report and decision to submit the manuscript for publication to EClinicalMedicine. Furthermore, all authors had full access to all the preliminary data in the study and accept responsibility to submit for publication.
4. Discussion
This report provides initial data from a Phase 1 trial on the safety, tolerability and immunogenicity of INO-4800, a SARS-CoV-2 vaccine encoding the spike protein (S). INO-4800 was well tolerated with a frequency of product-related Grade 1 AEs of 15% (3/20 subjects) and 10% (2/20 subjects) of the participants in 1.0 mg and 2.0 mg dose group, respectively. Only Grade 1 AEs were noted in the study, which compares favorably with existing licensed vaccines. The safety profile of a successful COVID-19 vaccine is important and supports broad development of INO-4800 in at-risk populations who are at more serious risk of complications from SARS-CoV-2 infection, including the elderly and those with comorbidities.
Lee WT, Girardin RC, Dupuis AP, et al. Neutralizing Antibody Responses in COVID-19 Convalescent Sera. medRxiv. J Infect Dis 2020. doi:10.1093/infdis/jiaa673.
,
] as well as the PRNT IC50 titers in NHPs which were protected in a SARS-CoV-2 challenge [
]. Furthermore, there was a statistically significant increase in titers. It is important to note that all but one vaccine recipient that did not develop neutralizing antibody titers responded positively in the T cell ELISpot assay, suggesting that the immune responses generated by the vaccine are registering differentially in these assays. Cellular immune responses were observed in 74% (14/19) and 100% (19/19) of 1.0 mg and 2.0 mg dose groups, respectively. Importantly, INO-4800 generated T cell responses that were more frequent and with higher responder median responses (46 [95% CI (21.1, 142.2)] vs. 71 [95% CI (32.2, 194.4)] SFU 106 PBMC) in the 1.0 mg and 2.0 mg dose groups respectively. These T cell responses in the 2.0 mg dose group were higher in magnitude than convalescent samples tested (Fig. 4A) and were similar or greater responses to those previously reported for other vaccine candidates [
,
,
,
], although the results should be interpreted in the context of variability of the immunological responses after natural infection and among different trials. Furthermore, there was a statistically significant increase in SFU. In the flow cytometric assays, both the 1.0 mg and 2.0 mg Dose Groups showed increases in cytokine production from both the CD4+ and CD8+ T cell compartments, especially in the 2.0 mg group. The 2.0 mg group exhibited a number of statistically significant cytokine outputs, including IFN-ɣ and TNF-α and “any cytokine” from the CD8+ T cell compartment and TNF-α from the CD4+ T cell compartment (Fig. 4D). Of considerable importance is that CD8+ T cell responses in the 2.0 mg dose group were dominated by cells expressing both IFN-ɣ and TNF-α with or without IL-2 (Fig. 4F and Table S4). In total, these cells amounted to nearly half of the total CD8+ T cell response (42.7%, Table S4). The contribution of this set of cytokines in the context of multi-cytokine production from CD8+ T cells appears to exceed those from previously reported vaccine studies [
,
]. The importance of such cells in mediating COVID-19 disease is underscored in a number of clinical studies [
,
,
,
] including a recent study which reported that recovered COVID-19 patients demonstrated a substantial frequency of CD8+ T cells expressing IFN-ɣ that also expressed TNF-α [
]. Additionally, a comprehensive review of currently available clinical data puts forth a model for mild vs severe COVID disease in which the presence of IFN-ɣ and TNF-α producing CD8+ T cells is proposed to be associated with a positive clinical outcome [
].
,
,
,
]. The INO-4800 Phase 1 safety data further suggest that the vaccine could be a safe booster as there was no increase in frequency of side effects after the second dose compared to the first dose, an important aspect for the safety profiles of SARS-CoV-2 vaccines. Given the uncertainty about the durability of the natural infection or vaccine induced responses against COVID 19 disease, vaccine boosting by a benign approach may be an important way to maintain protection over subsequent epidemic waves of COVID 19. It is also possible that INO-4800 could serve as a useful booster shot for other S protein-targeted vaccine candidates with limitations in boosting ability. In addition, planning is underway to further test if INO-4800 could provide booster immunity for COVID-19 recovered patients whose immunity is reported to wain rapidly. Many such subjects include persons in high risk groups who would especially benefit from longer term immune protection.
,
]. The clinical plan is to follow the current Phase 1 participants for 12 months for long-term safety as well as to measure the durability of immune response. Lastly, it is interesting that one of the volunteers in 1.0 mg dose group was seropositive at baseline, indicating that the person had been previously infected by the virus. This person, who had received both doses of vaccine as scheduled, did not have any AEs. A separate study of INO-4800 in seropositive individuals is planned for the future.
The development of a safe and effective vaccine remains the ultimate goal of preventive efforts against COVID-19. Multiple vaccine candidates and platforms are being tested, and it is unlikely that a single platform will prove suitably safe, effective, and logistically feasible, in terms of cold chain distribution, in all segments of the global population. Our data suggest that INO-4800 demonstrates a pristine safety profile and that immunization induces both humoral and cellular responses, supporting its further development to prevent infection, disease, and death in the global population. The safety profile could potentially make it a preferred vaccine option for high-risk populations, such as the elderly and those living with co-morbid conditions. The study of this vaccine's efficacy is planned for additional trials.
Declaration of Interests
SY, JDB, ACQ, VMA, MPM, KK, JA, AS, JP, EG, IM, PP, KS, TRFS, SR, TMcM, MD, EB, MPM, JL, MD, ASB, JES, JJK, KEB and LMH report grants from Coalition for Epidemic Preparedness Innovations, during the conduct of the study; other from Inovio Pharmaceuticals, outside the submitted work.
PT, ELR, AP, MP, FIZ, KYK, YD, DF, KB, MWC, JE and DBW report grants from Coalition for Epidemic Preparedness Innovations, during the conduct of the study.
Funding
This work is funded by Coalition for Epidemic Preparedness Innovations (CEPI).
Data sharing
The preliminary COVID19-001 Phase 1 clinical study datasets are subject to access restriction to protect subject confidentiality as the clinical study is still ongoing.
Acknowledgments
The investigators express their gratitude for the contribution of all the trial participants and the invaluable advice of the international Data Safety Monitoring Board. We also acknowledge the broader support from the various teams within Inovio: Greta Kcomt Del Rio, BS; Neiman Liu, MS, Alysia Ryan, BS; Dennis Van De Goor, MS; David Valenta, PhD; Snehal Wani, MS; EJ Brandreth, MBA; Dan Jordan, BS; Robert J. Juba Jr, MS, Stephen Kemmerrer, BSME, MBA, PE; Timothy Herring, MPH, Susan Duff, BS, the University of Pennsylvania: Sukyung Kim, RN, PhD; Alan Wanicur, BS; Zuleika Guzman, BS, the Wistar Institute: Dr. Ziyang Xu and Edgar Tello Ruiz; National Infections Service, Public Health England: Naomi Coombes, PhD; Mike Elmore, PhD and the Alliance for Multispecialty Research, Kansas City.
