Abstract
Background
In early 2020, developing vaccines was an urgent need for preventing COVID-19 from a contingency perspective.
Methods
S-268019-a is a recombinant protein-based vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), comprising a modified recombinant spike protein antigen adjuvanted with agatolimod sodium, a Toll-like receptor-9 agonist. In the preclinical phase, it was administered intramuscularly twice at a 2-week interval in 7-week-old mice. Immunogenicity was assessed, and the mice were challenged intranasally with mouse-adapted SARS-CoV-2 at 2 and 8 weeks, respectively, after the second immunization. After confirming the preclinical effect, a Phase 1/2, randomized, parallel-group clinical study was conducted in healthy adults (aged 20–64 years). All participants received 2 intramuscular injections at various combinations of the antigen and the adjuvant (S-910823/agatolimod sodium, in μg: 12.5/250, 25/250, 50/250, 25/500, 50/500, 100/500, 10/500, 100/100, 200/1000) or placebo (saline) in an equivalent volume at a 3-week interval and were followed up until Day 50 in this interim analysis.
Results
In the preclinical studies, S-268019-a was safe and elicited robust immunoglobulin G (IgG) and neutralizing antibody responses in mice. When challenged with SARS-CoV-2, all S-268019-a-treated mice survived and maintained weight until 10 days, whereas all placebo- or adjuvant-treated (without antigen) mice died within 6 days. In the Phase 1/2 trial, although S-268019-a was well tolerated in adult participants, was safe up to Day 50, and elicited robust anti-spike protein IgG antibodies, it did not elicit sufficient neutralizing antibody levels.
Conclusions
The S-268019-a vaccine was not sufficiently immunogenic in Japanese adults despite robust immunogenicity and efficacy in mice. Our results exemplify the innate challenges in translating preclinical data in animals to clinical trials, and highlight the need for continued research to overcome such barriers. (jRCT2051200092)
Keywords: COVID-19 vaccine, Preclinical study, Clinical trial, Immunogenicity, Recombinant spike protein, Safety
1. Introduction
Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), became a global healthcare crisis [1]. Despite the declining number of severe and fatal diseases, the pandemic is yet not fully controlled. Preventing future infections is thus a global public health priority. Increasing prophylactic vaccination outreach to promote herd immunity remains the most effective strategy to control the pandemic [2] in addition to social distancing and appropriate use of fitted face masks. However, emergence of new mutant strains places persistent burden on the healthcare system. Also, shortage of vaccine supply has been a problem in low- and middle-income countries. Furthermore, considering the waning immunity against SARS-CoV-2 following either natural infection or vaccination [3], [4], booster application of vaccines has been initiated worldwide. This places substantial, steady demand for COVID-19 vaccines that are safe and effective against new variants.
As part of a large vaccine research program, we have been developing COVID-19 vaccine candidates, comprising a modified recombinant spike protein of SARS-CoV-2, S-910823 (hereafter referred to as an S-protein antigen), produced using the baculovirus expression system in rhabdovirus-free insect cells and adjuvants [5], [6], [7]. Agatolimod sodium is a CpG-based oligonucleotide that induces immune response by activating Toll-like receptor (TLR) 9, thereby enhancing T-helper 1 cell (Th1) production [8]. Given a possibility that a skewed Th2 immune response and an insufficient neutralizing antibody response may cause eosinophilic immunopathology in the lungs, possibly due to poor TLR stimulation [9], [10], [11], [12], [13], we hypothesized that induction of a Th1-skewed immune response by agatolimod sodium would avoid vaccine-induced disease enhancement (VDE) risk. Herein, we report the preclinical data of S-268019-a in mice, followed by the interim findings of the Phase 1 component of the Phase 1/2 clinical study to assess the safety and immunogenicity of S-268019-a in Japanese adults, with safety and tolerability data collected until 4 weeks following the second S-268019-a dose.
2. Methods
2.1. Preclinical study
2.1.1. Cells and viruses
TMPRSS-2-expressing VeroE6 (VeroE6/TMPRSS2) cells [14] were obtained from the Japanese Collection of Research Bioresources Cell Bank. The cells were cultured in low glucose Dulbecco’s Modified Eagle Medium (DMEM) with 10 % fetal bovine serum and 1 mg/mL of geneticin G418, 100 IU/mL of penicillin, and 100 µg/mL of streptomycin. SARS-CoV-2 strain of the ancestral Pango lineage A (2019-nCoV/Japan/TY/WK-521/2020, hereafter referred to as WK-521) and a mouse-adapted QHmusX strain, originated from the SARS-CoV-2 isolate of Pango lineage B.1 (hCoV-19/Japan/QH-329–037/2020) [15], were developed at the National Institute of Infectious Diseases in Japan. The spike protein of QHmusX has Q498H and D614G mutations compared to that of WK-521 [15]. Stock viruses were propagated in VeroE6/TMPRSS2 cells as reported previously [14]. Viral titers were determined by 50 % of tissue culture infectious dose (TCID50) assay with VeroE6/TMPRSS2 cells. In brief, the cells were infected with a serial dilution of SARS-CoV-2, followed by analysis of cytopathic effect under a microscope at 5 days post-infection, and virus stocks were cryopreserved at −80 °C.
2.1.2. Preparation of antigens and adjuvants
A modified recombinant spike protein from the Pango lineage A of SARS-CoV-2 was produced using the Baculovirus expression system in the rhabdovirus-free insect cells [5]. Agatolimod sodium was synthesized at YMC Co., Ltd., Ishikawa, Japan. The lyophilized agatolimod sodium was dissolved in saline and stored at −20 °C prior to use. Alum (ImjectTM, Alum Adjuvant, Thermo Fisher Scientific) was used as a positive control for the Th2-skewed response. S-protein antigen plus agatolimod sodium or alum solution was formulated with saline.
2.1.3. Immunogenicity and efficacy studies
The indicated antigen-adjuvant combinations (50 μL/injection) were administered to 7-week-old female BALB/cAJcl mice (CLEA Japan, Inc.; n = 6) intramuscularly at the thigh twice at a 2-week interval. Two weeks after the second dose, the mice were sacrificed, and their blood and spleen samples were collected. Whole blood was collected from the inferior vena cava, and serum samples were prepared and stored at −80 °C. For the challenge experiments, BALB/cAJcl mice (Japan SLC, n = 6) were administered intramuscularly the indicated dose of S-protein antigen with 10 µg/injection of agatolimod sodium (50 µL/injection). Sera were collected from the mice before immunization and 2 weeks after the first and second doses. Eight weeks after the second dose, the mice were anesthetized and inoculated intranasally with mouse-adapted SARS-CoV-2 virus, QHmusX (2.3 × 104 TCID50/30 µL). The infected mice were daily monitored for survival time and percentage of body weight loss for 10 days. The humane endpoint was defined as the appearance of clinically diagnostic signs of respiratory stress and weight loss of > 25 %. All animal studies were performed according to the Guidelines for Proper Conduct of Animal Experiments of the Science Council of Japan. Animal experiments strictly complied with animal husbandry and welfare regulations and were approved by the Committee on Experimental Animals at the National Institute of Infectious Diseases in Japan (approval No. 521006) and the Institutional Animal Care and Use Committee of Shionogi & Co., Ltd (approval No. S20073C). Measurement of anti-spike IgG titer, neutralizing antibody titer testing, and ELISpot assay in mice are described in the Supplementary information.
2.1.4. Histopathology
After being anesthetized, the mice were sacrificed and the lungs were harvested, fixed, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Eosinophils were identified by Astra Blue/Vital New Red staining, a combined eosinophilmast cell stain (C.E.M. Stain Kit; Diagnostic Biosystems, Pleasanton, California). Using the slides stained with Astra Blue/Vital New Red, peribronchiolar areas in five 147,000-μm2 sections were assessed by light microscopy using the DP71 digital camera and cellSens software (Olympus, Tokyo, Japan), and the numbers of eosinophils counted in the lungs of each mouse were averaged as described previously [15], [16]. Histopathology scores were defined as follows: score 0, ≤5 eosinophils around vessels; score 1: >5 eosinophils around vessels but predominant lymphocytes observed; score 2, eosinophils predominant around vessels; and score 3, eosinophils in the alveolar region. Eosinophil counts results were analyzed using Dunn’s multiple comparison test.
2.2. Clinical study
2.2.1. Design and participants
This Phase 1/2, parallel-group study [jRCT2051200092] was planned to be conducted in 2 phases: (1) Phase 1 consisted of 3 parts (A, B, and C). Part A was a single-center, randomized, double-blind, placebo-controlled study in healthy Japanese adults (aged 20–64 years). In each cohort included in Part A, 8 participants received S-268019-a and 2 received placebo. Notably, placebo-receiving participants from all the cohorts were pooled for analyses. Part B was planned as a single-center, randomized, double-blind, placebo-controlled study in elderly Japanese people (aged ≥ 65 years) after the study intervention had been confirmed to exhibit immunogenicity and no safety and tolerability concerns arose in Part A. Part C was to be conducted as a multicenter, randomized, double-blind, placebo-controlled study in healthy Japanese adults (aged 20–64 years) after confirmation of acceptable safety and tolerability of the study intervention in Part A. (2) Phase 2 was planned to be a multicenter, randomized, double-blind, placebo-controlled study in Japanese adults (aged 20–64 years) after the study intervention had been confirmed to exhibit immunogenicity and acceptable safety and tolerability in Part A. However, since the immunogenicity of S-268019-a was not considered sufficient in Part A of the Phase 1 study, the planned Part B and Phase 2 trial were not conducted.
The study comprised a screening period (Day − 7 to Day − 1 pre-dose), evaluation period (Day 1 post-dose to Day 50), and follow-up period (Day 51 to Day 386) (Fig. 1 ). Participants received one intramuscular dose each of study interventions on Day 1 and Day 22. The study intervention and dose in each of the groups was designated a priori (Table S1). Injectable S-268019-a (S-protein antigen /agatolimod sodium, in μg: 12.5/250, 25/250, 50/250, 25/500, 50/500, 100/500, 10/500, 100/100, 200/1000) or placebo (saline) in an equivalent volume were administered. Japanese healthy adults or elderly participants who provided the informed consent were eligible for inclusion. Those positive for SARS-CoV-2 infection at screening, as determined by a SARS-CoV-2 antigen test, were excluded. The study was conducted in accordance with the protocol, the Declaration of Helsinki [17] and the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) Good Clinical Practice Guidelines [18], and other applicable laws and regulations. The study was approved by Institutional Review Board.
Fig. 1.
Vaccine regimen and key assessments in (A) preclinical and (B) clinical studies. Part B and Phase 2 studies were not conducted. GMT, geometric mean titer; GMFR, geometric mean fold rise; Th, T helper; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.
2.2.2. Outcomes
The primary objective of the Phase 1 study was to assess the safety and tolerability of S-268019-a in healthy Japanese adults through endpoints such as incidence of treatment-emergent adverse events (TEAEs, AEs reported after the initial administration of the study intervention), treatment-related AEs (TRAEs, AEs that could be reasonably explained as having been caused by the study intervention), serious AEs (SAEs), and solicited AEs, including systemic AEs such as fever, nausea/vomiting, diarrhea, headache, fatigue, and myalgia, and local AEs such as pain, erythema/redness, induration, and swelling. Secondary endpoints were geometric mean titer (GMT) and geometric mean of fold rise (GMFR) for neutralizing antibodies, anti-spike protein IgG antibodies, and seroconversion rate (defined as a ≥ 4-fold change from baseline, where titer values reported as below the lower limit of quantification [LLOQ] are imputed to 0.5 × LLOQ for neutralizing antibody titers). Exploratory endpoints included evaluation of immunological indices (cytokine-producing cell counts, T-cell cytokines, and Th1/Th2 balance). The methodology for quantification of anti-spike protein IgG titer, neutralizing antibody titer, intracellular cytokine staining by flow cytometry, and interferon gamma (IFN-γ) ELISpot assay has been previously described [6]. While this manuscript only describes interim analysis results, the long-term follow-up is ongoing.
2.2.3. Immunogenicity in recovered COVID-19 patients
Twenty-nine samples were collected from recovered patients aged 19–88 years at 31–91 days after COVID-19 onset (4 severe, 9 moderate, and 16 mild) and other 30 samples collected from patients aged 36–78 years at 29–52 days after COVID-19 onset (5 severe, 25 moderate) from Summit Pharmaceuticals International. Neutralizing antibody titer and anti-spike protein IgG titer were measured and used as reference data as described previously [6].
2.2.4. Statistical analyses
All analyses were descriptive and were performed using SAS® Version 9.4 (SAS Institute, Cary, North Carolina); no confirmatory hypothesis testing was performed.
3. Results
3.1. Preclinical study
3.1.1. Immunogenicity of S-268019-a in mice
In the preliminary study, the neutralizing antibody titer was at a detectable level 2 weeks after the mice were administered 2 doses of 1 μg of S-protein antigen plus 10 μg of agatolimod sodium. Therefore, we examined IgG and neutralizing antibody titers when mice were given 1 and 10 μg of S-protein antigen along with 1, 10, or 50 μg of agatolimod sodium. The specific IgG antibody was detected 2 weeks after the second dose in all tested conditions (Fig. 2 A). When administered in combination with S-protein antigen, agatolimod sodium increased the IgG titers in a dose-dependent manner. Immunization of 1 and 10 μg of S-protein antigen with agatolimod sodium also elicited neutralizing antibodies against ancestral SARS-CoV-2 WK-521 strain, and the neutralizing antibody titer was elevated in an S-protein antigen dose-dependent manner (Fig. 2 B). These data indicate that S-protein antigen plus agatolimod sodium combination elicits IgG antibody with neutralizing activity.
Fig. 2.
Immunogenicity of S-910823 adjuvanted with agatolimod sodium or alum in mice after intramuscular dosing. BALB/cAJcl mice (n = 6/group) were immunized twice at a 2-week interval with indicated doses of S-910823 as antigen and agatolimod sodium or alum as adjuvant. Two weeks after the second dose, sera were collected and the levels of (A) anti-SARS-CoV-2 S-protein total IgG, (B) neutralizing antibody against ancestral SARS-CoV-2 WK-521 strain, and (C) anti-S protein IgG2a, and (D) IgG1were analyzed. Open circles represent titers of individual animals. Dotted lines represent the detection limit. The bars and error bars in the IgG2a and IgG1 graphs are mean and SD, respectively. GMT, geometric mean titer; IgG, immunoglobulin G; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; SD, standard deviation.
Based on the segregation of IgG2a and IgG1 immunoglobulin isotypes as markers for Th1 and Th2 lymphocytes, respectively, we next analyzed induction of IgG subclasses to assess Th1/Th2 polarity. Compared to the mice given S-protein antigen alone, anti-spike specific IgG2a was increased when mice were given 1 and 10 μg of S-protein antigen adjuvanted with 10 or 50 μg agatolimod sodium (Fig. 2 C). Of note, agatolimod sodium elevated IgG2a levels in a dose-dependent fashion. As expected, IgG2a levels in S-protein antigen plus alum groups showed almost undetectable levels, and a high level of anti-spike IgG1 was detected in S-protein antigen plus alum-adjuvanted groups (Fig. 2 C and 2D). In this setting, the IgG1 levels in the 1 μg S-protein antigen plus the agatolimod sodium-adjuvanted groups were comparable to the levels in the S-protein antigen alone group. When mice were given 10 μg of S-protein antigen, relatively high levels of IgG1 were detected, but this elevation was reduced by combining agatolimod sodium in a dose-dependent manner (Fig. 2 D). To further confirm the Th1 and Th2 immune responses, we isolated splenocytes from the immunized mice and re-stimulated with spike-derived overlapping peptides for ELISpot assay. The spot-forming units (SFUs) for IFN-γ-secreting cells in the splenocytes immunized with 1 and 10 µg of S-protein antigen were higher than those without immunization (Fig. 3 A). The SFUs for interleukin 4 (IL-4)-secreting cells immunized with S-protein antigen adjuvanted with agatolimod sodium were lower than those with S-protein antigen only and S-protein antigen with alum (Fig. 3 B). The SFUs for IL-5-secreting cells mostly were not detected in the splenocytes immunized with any dose of S-protein antigen with agatolimod sodium. In contrast, the SFUs for IL-5-secreting cells were detected only in splenocytes immunized with S-protein antigen alone and S-protein antigen with alum (Fig. 3 C). These data suggest that S-protein antigen adjuvanted with agatolimod sodium elicited a Th1 cellular response, whereas Th2 response was conversely decreased by a combination of agatolimod sodium.
Fig. 3.
Cytokine production of splenocytes of mice immunized with S-910823 adjuvanted with agatolimod sodium or alum after intramuscular dosing. BALB/cAJcl mice (n = 6/group) were immunized twice at a 2-week interval with indicated doses of S-910823 as antigen and agatolimod sodium or alum as adjuvant. Two weeks after the second dose, splenocytes were collected and the number of cytokine-secreting cells were evaluated using ELISpot assay. (A) IFN-γ, (B) IL-4, and (C) IL-5 were measured after stimulation of splenocytes with S-overall peptide mixture. Each bar represents mean spot-forming units (SFUs; error bars indicate standard error of mean). The open and solid bars indicate splenocytes stimulated with distilled water (open circles) or S-overall peptide mixture (filled circles). Symbols represent individual animals. IFN-γ, interferon gamma; IL, interleukin.
3.1.2. Protective efficacy of S-268019-a against SARS-CoV-2 challenge in mice
A previous study showed that mice models with mouse-adapted QHmusX strain may be useful in evaluating protection from SARS-CoV-2 infection and estimation of VDE risks [15]. Therefore, we investigated the protective efficacy of S-268019-a against QHmusX infection. Since 10 µg of S-protein antigen plus 10 µg of agatolimod sodium elicited the highest level of neutralizing antibody titer, we tested protective efficacy against SARS-CoV-2 infection at doses of 0.1, 1, or 10 µg of S-protein antigen plus 10 µg of agatolimod sodium for identifying the adequate dose of S-protein antigen. Mice were vaccinated twice with S-910823 alone or in combination of S-protein antigen with agatolimod sodium or alum at a 2-week interval, and serum IgG and neutralizing antibody titer levels were positively confirmed, as expected (Fig. S1 A and S1 B). All mice administrated saline or adjuvant (1000 µg of alum or 10 µg of agatolimod sodium alone) had lost weight 2 days after the infection (Fig. 4 A), and died within 6 days (Fig. 4 B). While all mice immunized with 0.1, 1, and 10 µg of S-protein antigen either maintained or gained weight after 4 or 5 days of infection, and survived (Fig. 4 B). There were no differences in survival rates and weight changes between the mice treated with and without agatolimod sodium.
Fig. 4.
Protective efficacy against SARS-CoV-2 infection of mice vaccinated with S-910823 adjuvanted with agatolimod sodium or alum. BALB/cAJcl mice (n = 6/group) were immunized twice at a 2-week interval with indicated doses of S-910823 as antigen and agatolimod sodium or alum as adjuvant. Eight weeks after the second dose, mice were intranasally challenged with mouse-adapted SARS-CoV-2 virus QHmusX strain, and the (A) body weight and (B) survival were monitored daily for 10 days; (C) Histopathological analysis of the lungs from mice that survived the SARS-CoV-2 infection challenge was conducted. Representative histopathological observations from the mice with the highest eosinophilic infiltration were detected by HE staining and eosinophilic staining using the combined eosinophilmast cell staining CEM kit. Yellow arrows indicate representative eosinophils. (D) The numbers of eosinophils counted in the lungs of each mouse were averaged. Histopathology scores were defined as follows: 0, ≤5 eosinophils around vessels; 1: >5 eosinophils around vessels but predominant lymphocytes observed; 2, eosinophils predominant around vessels; and 3, eosinophils in the alveolar region. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001 by Dunn’s multiple comparison test. CEM, combined eosinophil-mast cell stain; HE, hematoxylin and eosin stain; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
We next conducted histopathological analysis of lung from the survived mice to investigate vaccine-related eosinophilic immunopathology after QHmusX infection. Eosinophilic infiltrations surrounding the bronchioles and blood vessels of the lungs were observed in mice in the groups immunized with S-protein antigen alone, similar to results from ectodomain of Spike protein in previous study [15]. In the groups immunized with S-protein antigen 3 plus alum, reduced number of the eosinophilic infiltrations was observed compared to the groups of S-protein antigen alone, while this was almost undetectable in the groups immunized with S-protein antigen plus agatolimod sodium (Fig. 4 C). The number of eosinophils was significantly lower in the groups immunized with 0.1 or 10 µg of S-protein antigen plus agatolimod sodium than the cognate groups immunized with S-protein antigen alone, and a similar trend was observed in the group of 1 µg of S-protein antigen plus agatolimod sodium (Fig. 4 D). These results suggest that insufficient neutralizing antibody levels and Th2-skewed immune response induce eosinophilic infiltration, which may cause VDE, after SARS-CoV-2 infection, but adjuvanting with agatolimod sodium reduces risks of VDE.
3.2. Clinical study
3.2.1. Participant characteristics
Sixty-one participants were randomized in Part A, and 30 in Part C (Fig. 5 ). Table S1 shows the baseline characteristics of the patients. All patients were Japanese and had no evidence of previous SARS-CoV-2 infection. Their mean age ranged between 29.4 and 48.0 years in the S-268019-a groups and 42.3 years in the placebo group (Table S1).
Fig. 5.
Participant flow in the Phase 1/2 clinical study. FAS, full analysis set; PPS, per protocol set; SAS, safety analysis set. The SAS population included randomized participants who received at least one dose of study intervention. The FAS population included all randomized participants who received at least one dose of the study intervention and who had at least one post-vaccination immunogenicity data. The PPS population included all randomized participants who were included in FAS but either violated the enrollment criteria, met any of the exclusion criteria in the protocol, or deviated from the protocol. *Since the immunogenicity of S-268019-a was not considered sufficient in Part A of Phase 1, the planned Part B and Phase 2 trials were not conducted.
3.2.2. Safety outcomes
Most AEs were considered treatment-related (Table S2 A and S2B). The incidence of solicited systemic TRAEs was similar between the S-268019-a and the placebo groups after the first and second interventions in Part A and Part C, except after the second intervention in Part C (25.0 % to 50.0 % across the S-268019-a groups; 0 % in the placebo group). The main AEs were headache and fatigue. The incidence of solicited local TRAEs was higher in the S-268019-a groups than in the placebo group after the first and second interventions in Part A and after the second intervention in Part C. Pain was the most common local TRAE (Table 1 ). All of the reported TRAEs in Part A and Part C were mild or moderate, and most of them recovered/resolved within 7 days from TRAE onset. No notable findings were noted in clinical laboratory evaluation, vital signs, and 12-lead ECGs during the study. Finally, no deaths, SAEs, or AEs leading to discontinuation of study intervention were reported in this study.
Table 1.
Solicited systemic/local TRAE after first and second vaccine injections.
| A) Phase 1 Part A | |||||||
|---|---|---|---|---|---|---|---|
|
S-268019-a (S-910823/agatolimod sodium, in μg) |
Placebo n = 12 |
||||||
|
12.5 μg/250 μg n = 8 |
25 μg/250 μg n = 8 |
50 μg/250 μg n = 8 |
25 μg/500 μg n = 8 |
50 μg/500 μg n = 8 |
100 μg/500 μg n = 8 |
||
| After first vaccination | |||||||
| Participants with any solicited systemic TRAEs | 1 (12.5) | 3 (37.5) | 0 | 1 (12.5) | 1 (12.5) | 3 (37.5) | 4 (33.3) |
| Nausea/vomiting | 1 (12.5) | 1 (12.5) | 0 | 0 | 0 | 0 | 0 |
| Diarrhea | 0 | 0 | 0 | 0 | 0 | 2 (25.0) | 0 |
| Headache | 1 (12.5) | 2 (25.0) | 0 | 1 (12.5) | 0 | 0 | 3 (25.0) |
| Fatigue | 1 (12.5) | 2 (25.0) | 0 | 1 (12.5) | 1 (12.5) | 1 (12.5) | 3 (25.0) |
| Myalgia | 0 | 0 | 0 | 1 (12.5) | 0 | 0 | 1 (8.3) |
| Participants with any solicited local TRAEs | 3 (37.5) | 4 (50.0) | 2 (25.0) | 4 (50.0) | 5 (62.5) | 4 (50.0) | 0 |
| Pain | 3 (37.5) | 4 (50.0) | 2 (25.0) | 4 (50.0) | 5 (62.5) | 4 (50.0) | 0 |
| After second vaccination | |||||||
| Participants with any solicited systemic TRAEs | 0 | 2 (28.6) | 1 (12.5) | 3 (37.5) | 2 (25.0) | 1 (12.5) | 2 (16.7) |
| Fever | 0 | 0 | 0 | 1 (12.5) | 0 | 0 | 0 |
| Diarrhea | 0 | 1 (14.3) | 0 | 0 | 0 | 0 | 0 |
| Headache | 0 | 2 (28.6) | 1 (12.5) | 1 (12.5) | 2 (25.0) | 0 | 2 (16.7) |
| Fatigue | 0 | 1 (14.3) | 0 | 3 (37.5) | 0 | 0 | 1 (8.3) |
| Myalgia | 0 | 0 | 0 | 2 (25.0) | 0 | 1 (12.5) | 1 (8.3) |
| Participants with any solicited local TRAEs | 4 (50.0) | 7 (100.0) | 4 (50.0) | 4 (50.0) | 5 (62.5) | 4 (50.0) | 1 (8.3) |
| Pain | 4 (50.0) | 7 (100.0) | 4 (50.0) | 4 (50.0) | 5 (62.5) | 3 (37.5) | 1 (8.3) |
| Erythema/redness | 0 | 0 | 0 | 0 | 0 | 1 (12.5) | 0 |
| Induration | 0 | 0 | 0 | 0 | 0 | 1 (12.5) | 0 |
| B) Phase 1 Part C | |||||
|---|---|---|---|---|---|
|
S-268019-a (S-910823/agatolimod sodium, in μg) |
Placebo |
||||
|
10 μg/500 μg n=8 |
100 μg/100 μg n=8 |
200 μg/1000 μg n=8 |
n=6 | ||
| TRAEs; % (95% CI) | 87.5 (47.3, 99.7) | 75.0 (34.9, 96.8) | 87.5 (47.3, 99.7) | 66.7 (22.3, 95.7) | |
| After first vaccination | |||||
| Participants with any solicited systemic TRAEs | 1 (12.5) | 4 (50.0) | 3 (37.5) | 2 (33.3) | |
| Fever | 0 | 0 | 1 (12.5) | 0 | |
| Diarrhea | 0 | 0 | 0 | 1 (16.7) | |
| Headache | 0 | 3 (37.5) | 2 (25.0) | 1 (16.7) | |
| Fatigue | 1 (12.5) | 2 (25.0) | 1 (12.5) | 1 (16.7) | |
| Myalgia | 0 | 1 (12.5) | 0 | 0 | |
| Participants with any solicited local TRAEs | 2 (25.0) | 2 (25.0) | 1 (12.5) | 2 (33.3) | |
| Pain | 2 (25.0) | 2 (25.0) | 1 (12.5) | 2 (33.3) | |
| After second vaccination | |||||
| Participants with any solicited systemic TRAEs | 4 (50.0) | 2 (25.0) | 4 (50.0) | 0 | |
| Fever | 1 (12.5) | 0 | 3 (37.5) | 0 | |
| Nausea/vomiting | 0 | 0 | 1 (12.5) | 0 | |
| Headache | 2 (25.0) | 1 (12.5) | 4 (50.0) | 0 | |
| Fatigue | 1 (12.5) | 2 (25.0) | 3 (37.5) | 0 | |
| Myalgia | 2 (25.0) | 0 | 2 (25.0) | 0 | |
| Participants with any solicited local TRAEs | 4 (50.0) | 3 (37.5) | 5 (62.5) | 1 (16.7) | |
| Pain | 4 (50.0) | 3 (37.5) | 5 (62.5) | 1 (16.7) | |
| Swelling | 1 (12.5) | 0 | 0 | 0 | |
AE, adverse event, TRAE, treatment-related AE.
Data are presented as n (%) unless stated otherwise.
3.2.3. Immunogenicity outcomes
The GMTs of anti-spike protein IgG in the S-268019-a groups were about 8- to 38-fold higher than those in the placebo group on Day 36 in both Part A and Part C (Fig. 6 A and 6B). In the S-268019-a groups, the GMFRs of anti-spike protein IgG were approximately 8 to 38 on Day 36 and somewhat reduced to approximately 3 to 25 on Day 50 (Table S3). The seroconversion rates for the anti-spike protein IgG on Day 36 were 75.0 % to 100.0 % in the S-268019-a groups while 0 % in the placebo group in both Part A and Part C (Table S4). The rates were 37.5 % to 100.0 % on Day 50. In Part A, the GMT and seroconversion rates for the anti-spike protein IgG gradually decreased on Day 113.
Fig. 6.
GMT of anti-spike protein IgG antibody titers. (A) GMT of anti-spike protein IgG (Phase 1 Part A) (B) GMT of anti-S protein IgG (Phase 1 Part C). GMT, geometric mean titer; IgG, immunoglobulin G.
In both Part A and Part C, the GMTs of SARS-CoV-2 neutralizing antibody on Day 36 and Day 50 in the S-268019-a groups were under 5, which were almost similar to those in the placebo group (Fig. 7 A and 7B). The GMFRs of SARS-CoV-2 neutralizing antibody titer were below 2 in both Part A and Part C (Table S3). Only 2 participants of the S-268019-a groups showed seroconversion with respect to SARS-CoV-2 neutralizing antibody in both Part A and Part C (data not shown). The immunogenicity after the second dose of S-268019-a (Day 36 and/or 50) in Part A was considered insufficient to start administration of study intervention in Part B and Phase 2. In terms of cellular immunity, the vaccine induced predominantly Th1 response with a substantial increase in CD4 T-cell-mediated IFN-γ and IL-2 production on Day 36 in participants from Part C of Phase 1 (Fig. 8 ). Notably, the results were comparable across antigen and adjuvant concentrations used. In the ELISpot assay, the increase in IFN-γ spots was observed in all cohorts in Part C at every visit (Fig. 9 ).
Fig. 7.
GMT of neutralizing antibody titers. (A) GMT of neutralizing antibody titer (Phase 1 Part A). (B) GMT of neutralizing antibody titer (Phase 1 Part C). GMT, geometric mean titer; IgG, immunoglobulin G; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.
Fig. 8.
Th1- and Th2-type cytokines in patients from Part C of the Phase 1 study measured by flow cytometry. The groups represent doses of antigen/antibody (in μg). CD, cluster of differentiation cells; IFN-γ, interferon gamma; IL, interleukin.
Fig. 9.
IFN-γ-producing spots in ELISpot assay in patients from Part C of the Phase 1 study. X axis represents study days. CD; cluster of differentiation cells; IFN-γ, interferon gamma; IL, interleukin; Th, T-helper.
3.2.4. Immunogenicity in the recovered COVID-19 patients
The GMT [95 % CI] of anti-spike protein IgG was 1279 [822.9, 1990.83; Figure S2]. The GMT [95 % CI] of SARS-CoV-2 neutralizing antibodies was 28.5 [21.1, 38.4], as previously noted by Iwata et al [6].
4. Discussion
The S-268019-a vaccine, comprising a modified recombinant spike protein of SARS-CoV-2 and agatolimod sodium adjuvant, was safe and well tolerated in both these preclinical and clinical studies. Notably, the S-268019-a formulation also elicited satisfactory immunogenic response in terms of IgG and neutralizing antibodies in mice. However, these findings did not translate in human participants. Considering that neutralizing antibody levels are a proxy marker for the conferred immune protection from symptomatic SARS-CoV-2 infection [19], [20], S-268019-a was deemed to be insufficiently immunogenic and/or protective to pursue subsequent clinical trials with the formulation.
Overall safety and immunogenicity outcomes in mice in our study were in line with previously published preclinical studies on SARS-CoV-2 vaccines [21], [22], [23]. S-268019-a elicited Th1-skewed IgG and neutralizing antibodies 2 weeks following the second injection. Notably, the induction of neutralizing antibodies was dose-dependent, a feature considered critical in preclinical therapeutic development [24]. The neutralizing antibody titers with S-268019-a were considerably higher than those with the S-protein antigen alone, highlighting the potential contribution of agatolimod sodium adjuvant to at least partial enhancement of neutralizing antibody response in mice. Additionally, agatolimod sodium could have also contributed to minimizing the eosinophil infiltration and overall inflammation, as S-268019-a resulted in lower eosinophil infiltrations compared with non-adjuvanted formulations in histopathological evaluation. Some preclinical trials of vaccines against SARS-CoV indicated VDE in mice, presumably through Th2-type responses [25], [26]. However, no evidence of Th2-type response and VDE were noted in vaccinated mice in our study. Notably, S-268019-a administration resulted primarily in Th1 response evident through induction of anti-spike IgG2a antibody in serum and IFN- γ in splenocytes. Interestingly, all mice in the S-268019-a-treated groups, either with or without adjuvants, maintained their weight and survived for at least 10 days after infection, whereas all mice treated with saline or adjuvants alone (all without antigen) died within 6 days of infection. These findings imply the therapeutic potential of the antigen and its ability to promote survival on its own without any dependence on adjuvants. While protection from SARS-CoV-2 challenge was shown with S-protein antigen alone, humoral immunogenicity was insufficient without adjuvants. S-protein antigen adjuvanted with alum elicited a level of IgG and neutralizing antibodies similar to S-protein antigen adjuvanted with agatolimod sodium, but, in terms of the Th1/Th2 balance, the latter demonstrated a more Th1-skewed response, which was considered to be important for SARS-CoV-2 vaccines to reduce the risk of VDE. Therefore, we decided to employ agatolimod sodium as the adjuvant for S-protein antigen when we planned the clinical study.
When transitioned to a Phase 1/2 clinical study, no major AEs occurred with S-268019-a from the time of study interventions until the data cutoff date. Most of the reported AEs were vaccination site pain, headache, and fatigue, which were treatment-related and resolved within 7 days from AE onset. These TRAEs were in alignment with reactogenicity expected from vaccines with various mechanisms [27], [28], [29], [30]. Therefore, S-268019-a was considered to have no substantial safety issues.
GMTs of anti-spike protein IgG on Day 36 and Day 50 were higher than those on Day 1 and Day 22 across all S-268019-a groups. The IgG levels in the placebo group were often below the detection limit, implying the immunogenicity of the S-268019-a formulation in eliciting IgG response. Moreover, the IgG titers in our study were generally comparable with those seen in sera samples of patients recovered from COVID-19. However, GMTs of SARS-CoV-2 neutralizing antibody on Day 36 and Day 50 in the S-268019-a groups were almost similar to those in the placebo group, implying insufficient immunogenicity after the second dose of S-268019-a and the need to enhance immune response. Many vaccines, containing inactivated whole viruses or subunit viral protein, have inherent immune-potentiating activity. While many modern vaccines use rationally designed recombinant antigens with excellent safety profiles, the immunogenicity of some recombinant vaccines may be low with little to no T-cell response compared with vaccines containing live attenuated or inactivated virus antigens. Inclusion of adjuvants in vaccine formulations can induce appropriate immune responses not sufficiently induced in the absence of adjuvant, enhance the efficacy of weak antigens, or both. Adjuvants also enable the use of lower vaccine doses, thereby helping fast production of large batches and mitigate the demand–supply crisis [31].
TLRs play an important role in modulating humoral and cellular immune response. Thus, agonists for TLR4, TLR7, TLR8, and TLR9 have been tested as adjuvants for infectious diseases and in certain cancers [8]. Notably, TLR9 activation with synthetic CpG oligodeoxynucleotides, such as agatolimod (also known as CpG 7909, ODN 2006, PF-3512676, VaxImmune, and ProMuneT) induces predominantly Th1 innate and adaptive immune responses and activates dendritic and B cells against antigens. Over the years, safety of TLR9 activation with CpG oligodeoxynucleotides has been established as acceptable as an adjuvant for vaccines, exemplified by Bacillus anthracis and hepatitis B vaccines [32]. However, literature on its efficacy as an adjuvant is limited. A recombinant vaccine based on a prefusion-stabilized spike trimer of SARS-CoV-2 and formulated with aluminum hydroxide and agatolimod adjuvants elicited robust neutralizing antibody responses and substantial CD4 + T-cell responses in both mice and non-human primates [33]. Ongoing Phase 1 and 2 trials [34], [35] will shed further light on the clinical efficacy of CpG oligonucleotides adjuvants in the SARS-CoV-2 vaccine.
Animal research plays a crucial role in identifying successful vaccine candidates. In fact, regulatory authorities require all candidates to be tested for safety and efficacy in animals before potential evaluation in humans. Mouse models have provided vital information about the COVID-19 disease course and continue to be used by medical researchers to understand the COVID-19 disease research because of their rapid reproduction, a well-characterized immune system, and a defined genome [36]. However, animal models may not recapitulate an entire disorder or disease [37]. Furthermore, challenges related to difficulty in modeling heterogeneity of the patient population in more homogenously bred animals often result in suboptimal predictive utility of animal models [38]. Animal models used in preclinical studies often have different TLR expression patterns compared to humans [39]. Moreover, the TLR specificity of adjuvant molecules could vary in different species. For instance, human and rodent TLR9 receptors interact with ligands slightly differently. Selection of appropriate preclinical animal models is therefore essential for the efficient development of new vaccines. Heterogeneity in responses of B cells to TLR9 agonists may be explained by different TLR9 expression levels, genetic alterations, and signaling pathway activation [40]. Other than translational issues, many adjuvants fail during preclinical/clinical development phases because of factors such as stability, lack of innate efficacy, and unacceptable levels of tolerability or safety concerns [31]. In the context of our study, agatolimod sodium was probably not the most suitable adjuvant for the S-protein antigen in humans from the immunogenicity viewpoint. The development of safe and potent immunologic adjuvants that can increase and direct vaccine-specific immunity is therefore required [41]. Regardless of the limited translation to clinical studies, preclinical studies in animals will continue to provide vital information to identify new vaccine candidates as new SARS-CoV-2 variants continue to emerge. We believe that building on valuable lessons provided by our study will further aid vaccine development in SARS-CoV-2 and other infectious diseases.
Another formulation of S-268019-b with a squalene-based adjuvant has been evaluated in a subsequent study. S-268019-b displayed sufficient safety and immunogenicity to a similar extent as that of S-268019-a in a preclinical study in mice [42]. Additionally, S-268019-b yielded favorable results in (a) a randomized, placebo-controlled, Phase 1/2 study as a primary series, (b) a large open-label Phase 2/3 study as a primary series, and (c) a randomized Phase 2/3 study as a booster application in Japanese participants [6], [7]. Based on the favorable results, S-268019-b is currently being evaluated in large Phase 3 trials. These findings are in alignment with results of another vaccine program with SCB-2019, a recombinant protein-based vaccine. In the Phase 1 study, the immunogenicity in participants injected with SCB-2019 adjuvanted with a squalene-based adjuvant was stronger than in those immunized with SCB-2019 adjuvanted with CpG1018/Alum with TLR9 agonism [43]. One of the possible reasons that the change in adjuvant led to a successful clinical trial result could be because agatolimod functions through receptor-mediated pathogen-associated molecular patterns, whereas squalene-based adjuvant follows damage-associated molecular patterns. Both adjuvants share the mechanism of activating NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome, but the mechanism upstream of NLRP3 is different.
We acknowledge that our study has its limitations. Intentionally limited ethnic diversity as a function of the study design could limit the generalizability of results. Moreover, absence of an “adjuvant only” group precludes the possibility of understanding the true contribution of agatolimod sodium in this formulation to the overall immunogenicity in humans. Given the low immunogenicity observed in the present study, the current data do not support an agatolimod sodium-based formulation and warrants evaluation of new formulations for developing SARS-CoV-2 vaccines containing the S-protein antigen. Nonetheless, our findings highlight the inherent challenges in translation of preclinical findings to clinical success, underscore the critical role of adjuvants in vaccine development, and demonstrate how changes in formulation parameters can have an important impact on translation to favorable clinical results.
5. Conclusion
Despite the promising safety and immunogenicity in the rodent model, S-268019-a vaccine comprising the SARS-CoV-2 spike protein adjuvanted with agatolimod sodium did not elicit sufficient immunogenic response in terms of neutralizing antibodies in this Phase 1/2 study in Japanese participants though it was well tolerated.
6. Disclosures/potential conflicts on interests
Takuhiro Sonoyama, Yukio Tada, Tomoyuki Homma, Shinya Omoto, Risa Yokokawa Shibata, Kenji Igarashi, and Mari Ariyasu are employees of Shionogi & Co. Ltd. Satoshi Iwata received payment or honoraria for lectures, presentations, speakers’ bureaus, manuscript writing, and educational events from Astellas Pharma Inc., Japan Vaccine Co., Ltd., Meiji Seika Pharma Co., Ltd., Pfizer Japan Inc., and Taisho Toyama Pharmaceutical Co. Ltd. Masaharu Shinkai, Naoko Iwata-Yoshikawa, Nozomi Shiwa-Sudo, Takuya Hemmi, Akira Ainai, Noriyo Nagata, Nobuaki Mastunaga, Tadaki Suzuki, and Hideki Hasegawa have no conflicts of interest to declare.
Funding
This work was supported by Shionogi & Co., Ltd. and Japan Agency for Medical Research and Development (AMED) under Grant No. JP21nf0101626.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
We acknowledge Drs. Masayuki Hashimoto and Soichi Tofukuji (Shionogi & Co., Ltd.) for conducting immunological analysis. We also thank Ms. Haruna Hayashi and Mr. Satoshi Kojima (Shionogi & Co., Ltd.) for supporting manuscript development. We also thank Vidula Bhole, MD, MHSc, and Ivan D’Souza, MS, of MedPro Clinical Research for providing medical writing support for this manuscript, which was funded by Shionogi & Co., Ltd.
Footnotes
Supplementary data to this article can be found online at https://doi.org/10.1016/j.vaccine.2022.12.025.
Appendix A. Supplementary material
The following are the Supplementary data to this article:
Supplementary figure 1.
Protective efficacy of S-268019-a against SARS-CoV-2 challenge in mice as seen with (A) anti-spike IgG titer at multiple timepoints (before immunization and 14 days after the first and second vaccine injections) and (B) neutralizing antibody titer at 14 days after second injections.
GMT, geometric mean titer; IgG, immunoglobulin G; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.
Supplementary figure 2.
(A) IgG titers in human convalescent sera from recovered COVID-19 patients; (B) correlation between neutralizing antibody and IgG antibody titer.
COVID-19, Coronavirus Disease 2019; GMT, geometric mean titers; ID50, concentration of neutralizing antibodies required to reduce the virus titer by 50%; IgG, immunoglobulin G.
Data availability
Data will be made available on request.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
Data will be made available on request.