key: cord-0804763-gvoovxuk authors: Thuluva, S.; Paradkar, V.; Turaga, K.; Gunneri, S.; Yerroju, V.; Mogulla, R.; Kyasani, M.; Manoharan, S.; Medigeshi, G. R.; Singh, J.; Shaman, H.; Singh, C.; Rao A, V. title: Selection of optimum formulation of RBD-based protein sub-unit covid19 vaccine (Corbevax) based on safety and immunogenicity in an open-label, randomized Phase-1 and 2 clinical studies date: 2022-03-09 journal: nan DOI: 10.1101/2022.03.08.22271822 sha: 9f06081e4cd040f8a6744f7a75324d145a466e2d doc_id: 804763 cord_uid: gvoovxuk Background- We present the data from an open-label study involved in the selection of optimum formulation of RBD-based protein sub-unit COVID-19 vaccine. Methods- The randomized Phase-1/2 trial followed by a Phase-2 trial was carried out to assess safety and immunogenicity of different formulation of COVID-19 vaccine (Corbevax) and select an optimum formulation for a phase 3 study. Healthy adults without a history of Covid-19 vaccination or SARS-CoV-2 infection, were enrolled. Findings- Low incidence of AEs were reported post vaccination of different Corbevax formulations and majority were mild in nature and no Grade-3 or serious AEs were observed. All formulations in Phase-1/2 study showed similar profile of humoral and cellular immune-response with higher response associated with increasing CpG1018 adjuvant content at same RBD protein content. Hence, high concentration of CpG1018 was tested in phase-2 study, which showed significant improvement in immune-responses in terms of anti-RBD-IgG concentrations, anti-RBD-IgG1 titers, nAb-titers and cellular immune-responses while maintaining the safety profile. Interestingly, binding and neutralizing antibody titers were persisted consistently till 6 months post second vaccine dose. Interpretations- Corbevax was well tolerated with no observed safety concerns. Neutralizing antibody titers were suggestive of high vaccine effectiveness compared with human convalescent plasma or protective thresholds observed during vaccine efficacy trials of other COVID-19 vaccines. The study was prospectively registered with clinical trial registry of India- CTRI/2021/06/034014 and CTRI/2020/11/029032. Funding: Bill and Melinda Gates Foundation, BIRAC- division of Department of Biotechnology, Govt of India, and the Coalition for Epidemic Preparedness Innovations funded the study. Syndrome Coronavirus 2 (SARS-CoV-2) 1 The study describes the clinical studies that led to the optimum formulation of the vaccine (Corbevax). The Phase-1/2 study was conducted to assess the key role played by RBD and the adjuvant CpG1018. The subsequent Phase-2 study was conducted to confirm the safety and immunogenicity of the optimum formulation. In addition, persistence of immune-response till 6-months post completion of primary-vaccination series is also presented for the formulations used in the Phase-1/2 study. 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 March 9, 2022. ; https://doi.org/10.1101/2022.03.08.22271822 doi: medRxiv preprint Overall, 1497 subjects were screened as part of two studies and 460 subjects (n=360 in Phase-1/2 and n=100 in Phase-2 study) were vaccinated with various formulations of Corbevax vaccine (Table 1) . Subject disposition in phase-1/2 and phase-2 study are illustrated in Figure-1 and 2 respectively. The Phase-1/2 and Phase-2 studies were carried out in 5 and 7 centers across India respectively between 11-November-2020 to 20-August-2021. Studies are prospective, open-label, randomized (phase-1/2) to assess best vaccine formulation based on safety, tolerability, reactogenicity and immunogenicity in Covid-19 RT-PCR and sero-negative subjects. Phase-1/2 study had 360 healthy volunteers were randomly assigned into four groups to SARS-CoV-2 antibody concentrations for diagnostic purpose were measured using Diasorin kit 12 . Anti-RBD antibody responses and IgG-subclass responses were measured using validated ELISA-method. SARS-COV-2 neutralizing antibody titers were measured using MicroNeutralizationAssay (MNA) and PseudovirusNeutralizationAssay (PNA) methods and cellular immune-responses were assessed by cytokine secretion using TrueCulture® tubes coated with SARS-COV-2 peptides. Details on assays and methods used for assessing immunological parameters were descried as supplementary information. All data are presented using descriptive statistics. Demographic and primary safety analyses were based on total vaccinated population. Full-analysis-set (FAS) included subjects who provided informed consent for participation. Intent-to-treat (ITT) analysis set included all subjects from FAS who had received both the doses of study vaccine. Per protocol (PP) analysis set included all subjects from ITT set without any major protocol deviations. All subjects entered into the study and who received at least one dose of study vaccine were included in the safety-analysis. All immunological data was log-transformed to obtain a log-normal distribution. All the statistical analyses were conducted using SAS ® 9.4 or higher (SAS Institute, Cary NC). 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 March 9, 2022. ; The selection of lab for immunogenicity analysis was based on the recommendations of CEPI. Funding sources were not involved in the study conduct, data analysis/interpretation or writing the manuscript. Subjects' demographics and baseline characteristics are described in Table 2 and subjects' disposition is shown in Figure- In Phase-1/2 study, AEs were reported in 42 subjects (11.67%), in the range of 8.89-15.56% of subjects in different formulations groups. Least number of subjects with AEs reported in "B" formulation and highest was reported in "D" formulation (Table 3) In the Phase-2 study, a total of 27 (27.00%) subjects reported AEs post-vaccination. No AEs were reported in any subject within 120-minutes' post-vaccination. None of the AEs were serious, or of Grade-3 severity. Details of solicited local and systemic AEs and unsolicited AEs were listed in Table- 3 (Phase-1/2) and Table-5 (Phase-2). All the reported AEs were mild to moderate in severity and most of the events were considered related to the study vaccine. MAAEs were reported in 10 subjects (2.78%) and 6 subjects (6%) in Phase-1/2 and phase-2 studies respectively, of which none were reported as serious (Table 4) . No abnormal laboratory values, vital signs or physical examination were reported as clinically significant. There was no SAE reported during the reported study period. 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 March 9, 2022. ; https://doi.org/10.1101/2022.03.08.22271822 doi: medRxiv preprint Data is presented for the four subject cohorts that received the four different vaccine formulations, designated as A, B, C and D respectively. RBD-IgG The anti-RBD-IgG concentrations increased moderately after the first dose and then significantly after the second-dose and the values plateaued between Day42 and 56 ( Figure-3) . Percent seroconversion was highest (90%) for "B" formulation (Table-6 ). IgG1 (Th1-skewed) and IgG4 (Th2-skewed) titers were also measured from all four formulation cohorts. IgG1 titers were significant increased in all four cohorts with the highest GMT (2940 at day56) and GMFR (39.7) observed for B-formulation. The Day0 titers were very low for both isotypes in all the cohorts. In comparison, minor increase was observed in IgG4 titers for the four cohorts at day56 (Table 7) (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 March 9, 2022. ; for these convalescent sera panel was 522.. Formulation-B induced high NT50 (60 at day0 Vs 537 at day56) compared to other formulations tested ( (Table 8) . The subjects in Phase-2 study received the modified vaccine formulation containing higher CpG1018 adjuvant (Formulation-E, Table 1 ) to increase robustness and magnitude of the immune-response. Immunogenicity parameters were measured in 100 subjects that were selected based on anti-SARS-COV-2 IgG seronegative status. As the major aim of this immunogenicity analysis was to assess the increase in immuneresponse due to change in vaccine formulation and hence data is presented for C & B cohorts from Phase-1/2 along with E cohort from Phase-2 trial. Formulation-E induced strong anti-RBD-IgG response (GMC: E-26448 Vs B-17301 and C-11497) compared to C and B, whereas seroconversion rate was comparable in all three formulations (; 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 March 9, 2022. ; seroconversion: E-89%; B-88% and C-82%) at day42 (Figure-7 and Table 9 ). Anti-RBD-IgG1/IgG4 ratio was significantly high in formulation-B (75.4) group compared to formulation-C (38.0) or B (37.1) groups at day42 (Table-10 (Table 11) . The subjects from Phase-1/2 study are planned to be followed for a duration of one year to assess vaccine safety and immune-response. The persistence of immune-response was assessed at 6-months post second-dose i.e.Day208 time-point. All subject sera samples were tested for anti-RBD-IgG concentration and nAb-titers by PNA method at the same laboratories by the same methods. Overall, nAb-titer GMT's for all the formulations at 6- 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 March 9, 2022. ; This was a prospective, open-label, Phase-1/2 (randomized) and Phase-2 studies to assess the safety, tolerability, reactogenicity and immunogenicity of four vaccine formulations (Phase-1/2) and optimal formulation (Phase-2) that contained the same antigen i.e. Receptor Binding Domain (RBD) protein, an important target for vaccine development 14 . The vaccine was safe and well tolerated in all formulation cohorts (phase-1/2 and phase-2). Very similar instances of AE's (the number & percentages of subjects and total number of AE's) reported after two-dose administrations in phase-1/2 cohorts, whereas slightly higher numbers of AEs reported in the Phase-2 study. Most of the AE's were mild and no Grade-3 AE's or Serious AE's and very low number of MAAE's were reported in the study. In the ongoing long-term monitoring, other than two cases of mild COVID-19, no additional AE's were reported in all the cohorts for 10-12 months of phase-1/2 and 5-months of phase-2 study respectively, of monitoring period. Thus, the optimized Corbevax formulation (E) was considered to be safe with minimal reactogenicity to advance into pivotal Phase-3 studies. In Phase-1/2 study, both humoral immune-response and cellular immune-response were analyzed at key time-points in the study to determine the impact of various compositions on the overall immune-response. 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. Studies have shown that an appropriate Th1 immune-response can clear the infection and a poor prognosis was associated with cytokine storm that triggers Th2-cells 15, 16 . In this study, anti-RBD-IgG1 (Th1) and IgG4 (Th2) isotype titers were measured. All four formulations showed significant and consistent high ratio of IgG1 to IgG4 titers at Day56 which is a hallmark of skewed Th1-type immune-response. This is thought to be due to the presence of adjuvant CpG1018 in the formulation which is known to skew the immune-response toward Th1 response. Two key cytokines monitored in the study were IFN-γ (Th1-biased) and Interleukin-4 (Th2-biased). A significant increase was observed in active INF-γ 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 March 9, 2022. ; concentration at Day56 compared to baseline samples for all cohorts with highest average concentration observed for the B-formulation. In contrast, only small increase was 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. Thus, Corbevax vaccination yields the most consistent cross-protection against two most relevant VOC's and this protection potential is significantly superior to other vaccines. 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. infection which corresponds to COVID-19 incidence rate of approximately seven cases per 1000 person-years which corresponds to very high vaccine effectiveness. Based on the analysis conducted during Phase-3, efficacy studies was part of product approval for Spikevax 22 (Moderna Inc;) and Vaxzveria 23 (AstraZeneca Inc;); nAb-titers in sera samples after two-dose vaccine administration were observed to correlate with the protection against symptomatic COVID-19 infection. Both studies also reported this CoP information in terms of nAb-titers expressed in IU/mL by using calibration factors to convert the study specific nAb-assay titers to the WHO-International Standard. This CoP evaluation showed that if nAb GMT's of >100 IU/mL post two-dose 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. (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 March 9, 2022. ; The present study is an open-label study and not a randomized double-blind study. The study population did not include pediatric and elderly age group (65+Years). Immuneresponse data for the optimized formulation is available till two weeks post second-dose and longevity of the immune-response for the optimum formulation will be available in the future during long-term monitoring. The subject cohorts were limited in number in Phase-1/2 and Phase-2 studies and the vaccine performance in larger cohorts in wider age-groups (5-80) will be assessed in the ongoing Phase-3 studies. We are thankful to all the study participants, the principal investigators, and the study staff at all the clinical sites. In addition, we are thankful to the team at Dang's Lab, New (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 March 9, 2022. ; and produced the recombinant Pichia Pastoris strain expressing the RBD protein. (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 March 9, 2022. ; 18. Davis 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 March 9, 2022. ; https://doi.org/10.1101/2022.03.08.22271822 doi: medRxiv preprint Buffer (Tris and NaCl in WFI) q.s to 0.5 mL q.s to 0.5 mL q.s to 0.5 mL q.s to 0.5 mL q.s to 0.5 mL 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 March 9, 2022. ; 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 March 9, 2022. ; https://doi.org/10.1101/2022.03.08.22271822 doi: medRxiv preprint Note: Cytokine measured in the supernatants of whole-blood samples incubated in tubes coated with SARS-COV-2 peptides (Active) and without coating (Null) for a subset of subjects from all four cohorts 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 March 9, 2022. ; https://doi.org/10.1101/2022.03.08.22271822 doi: medRxiv preprint 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 March 9, 2022. ; https://doi.org/10.1101/2022.03.08.22271822 doi: medRxiv preprint (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 March 9, 2022. ; https://doi.org/10.1101/2022.03.08.22271822 doi: medRxiv preprint The cytokine concentrations are measured in the supernatants of whole-blood samples incubated in tubes coated with SARS-COV-2 peptides (Active) and without coating (Null) for a subset of subjects from Formulation C&B cohorts from Phase-1/2 study and Formulation-E cohort from Phase-2 study. 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 March 9, 2022. ; Novel Coronavirus-Infected Pneumonia SARS-CoV-2 Vaccines: Where Are We Now? Neutralizing antibody responses to SARS-CoV-2 in symptomatic COVID-19 is persistent and critical for survival Expansion of SARS-CoV-2-Specific Antibody-Secreting Cells and Generation of Neutralizing Antibodies in Hospitalized COVID-19 Patients Convergent antibody responses to SARS-CoV-2 in convalescent individuals A vaccine targeting the RBD of the S protein of SARS-CoV-2 induces protective immunity Genetic modification to design a stable yeastexpressed recombinant SARS-CoV-2 receptor binding domain as a COVID-19 vaccine Process development and scale-up optimization of the SARS-CoV-2 receptor binding domain-based vaccine candidate Yeast-expressed SARS-CoV recombinant receptor-binding domain (RBD219-N1) formulated with aluminum hydroxide induces protective immunity and reduces immune enhancement Yeast-expressed recombinant SARS-CoV-2 receptor binding domain RBD203-N1 as a COVID-19 protein vaccine candidate CoV-2 S1/S2 IgG. The fully automated serology test for the detection of SARS-CoV-2 IgG Antibodies Interim Results of a Phase 1-2a Trial of S Covid-19 Vaccine Antibodies and Vaccines Target RBD of SARS-CoV-2 SARS-CoV-2-Specific T Cells Exhibit Phenotypic Features of Helper Function, Lack of Terminal Differentiation, and High Proliferation Potential Signals of Th2 immune-response from Systemic Adverse events Pyrexia