Characterization and immunogenicity assessment of MERS-CoV pre-fusion spike trimeric oligomers as vaccine immunogen

ABSTRACT

Middle East respiratory syndrome coronavirus (MERS-CoV) is a lethal beta-coronavirus that emerged in 2012. The virus is part of the WHO blueprint priority list with a concerning fatality rate of 35%. Scientific efforts are ongoing for the development of vaccines, anti-viral and biotherapeutics, which are majorly directed toward the structural spike protein. However, the ongoing effort is challenging due to conformational instability of the spike protein and the evasion strategy posed by the MERS-CoV. In this study, we have expressed and purified the MERS-CoV pre-fusion spike protein in the Expi293F mammalian expression system. The purified protein was extensively characterized for its biochemical and biophysical properties. Thermal stability analysis showed a melting temperature of 58°C and the protein resisted major structural changes at elevated temperature as revealed by fluorescence spectroscopy and circular dichroism. Immunological assessment of the MERS-CoV spike immunogen in BALB/c mice with AddaVax^TM and Imject alum adjuvants showed elicitation of high titer antibody responses but a more balanced Th1/Th2 response with AddaVax^TM squalene like adjuvant. Together, our results suggest the formation of higher-order trimeric pre-fusion MERS-CoV spike proteins, which were able to induce robust immune responses. The comprehensive characterization of MERS-CoV spike protein warrants a better understanding of MERS spike protein and future vaccine development efforts.

KEYWORDS:

• MERS-CoV vaccine
• prefusion spike
• immunogenicity
• thermostability, antibody response

Acknowledgments

We sincerely thank Prof Pramod Kumar Garg former Director, THSTI, and Dr. Jayanta Bhattacharya, Dean for full support and valuable input and guidance. We thank our industry partner Panacea Biotec ltd, India and The Coalition for Epidemic Preparedness Innovations (CEPI) scientific coordinators for continuous support and technical guidance. We thank Vikas Maithil, Anup Singh and Amit Kumar from our lab for helping in biochemical and animal experimentation. We thank Prof. Amit Awasthi for timely suggestions and critical inputs. The following reagent was deposited by the Centres for Disease Control and Prevention and obtained through BEI Resources, NIAID, NIH: SARS Related Coronavirus 2, Isolate USA-WA1/2020, NR-52281. We thank Dr. B Graham (VRC/NIAID/NIH) for providing us the spike construct (SARS-2-CoV S 2P) to raise mouse polyclonal sera. We thank the THSTI EAF facility and staffs for providing support and timely help for immunization experiments. We thank Mr. Vijay Kumar of Regional Centre for Biotechnology (RCB), for assisting with DSC and CD experiments. Part of the work was carried out at the Advanced Technology Platform Centre (ATPC) of RCB.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Author contribution

S.S and S.A. designed the immunogen; S.R. expressed and purified the immunogen under the guidance of S.A. P.V, R.A, V.K carried out the biochemical and biophysical characterization. P.V. and Sh.S. (Shikha Saxena) carried out in vivo study, A.K. assisted in in vivo experiments for animal handling during immunization and blood collection at different time points. R.K. and B.L conducted the CoV-1 and CoV-2 in vitro studies, Sh.S carried out in vitro experiment with Influenza. Sh.A. (Shailendra Asthana) and G.S. made the bioinformatics analysis and molecular modelling. S.S. and S.A conceived the study and designed experiments. S.S. S.A. S.R., P.V., R.A, V.K, Sh.A, Sh.S and M.S. analyzed the data. S.S. wrote the original manuscript. S.A. S.R., P.V., R.A, V.K, Sh.A, Sh.S and M.S edited the manuscript. All authors have read and agreed to the published version of the manuscript.

Supplementary material

Supplemental data for this article can be accessed on the publisher’s website at https://doi.org/10.1080/21645515.2024.2351664.

Data availability

All is available from corresponding author upon request.

Abbreviations

SARS-CoV: severe acute respiratory syndrome coronavirus, MERS-CoV: Middle East Respiratory Syndrome, M-CoV-S: Middle East Respiratory Syndrome Coronavirus Spike, DPP4: dipeptidyl peptidase-4, TLR: Toll-like Receptor, RBD: Receptor binding domain, DNA: deoxynucleic acid

Additional information

Funding

In this study the MERS antigen design and characterization was partially supported by the grant from the Coalition for Epidemic Preparedness Innovations (“CEPI”) towards a Consortium involving THSTI and Panacea Biotec ltd., to develop a Multi-epitope, Nanoparticle based broadly protective Beta coronavirus candidate vaccine (“Project”). The other works were supported by Translational Health Science and Technology Institute (THSTI) core funding and INDCEPI project, an institute functioning under the Biotechnology Research and Innovation Council (BRIC) of the Department of Biotechnology, Ministry of Science and Technology, Government of India.