The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in late 2019 in the Hubei province of China led to disastrous human as well as economic losses throughout the world.

The immense effort of the scientific community helped in the implementation of rapid diagnostic tools, immunological monitoring tools, and the development of several vaccines.

SARS-CoV-2 that led to the coronavirus disease 2019 (COVID-19) pandemic belonged to the Sarbecovirus subgenus of Coronaviridae. SARS-CoV-2 like all other zoonotic sarbecovirus uses the human angiotensin-converting enzyme 2 (ACE2) receptor for entry into the cell. SARS-CoV-2 is the third major human infectious disease outbreak that has been caused by zoonotic coronaviruses after SARS-CoV-1 in 2002-2003 and Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012.

Currently, the COVID-19 vaccines are based upon the delivery of SARS-CoV-2 Spike (S) through various vaccine platforms to elicit antibodies against the S protein which also includes the receptor-binding domain (RBD). These vaccines induce Th1 responses that are restricted to the S epitopes but are not very responsive in eliciting CD8+ T-cell responses that are also required for controlling infections.

Furthermore, the control of the pandemic is threatened by the emergence of several SARS-CoV-2 variants of concern (VOCs) such as B.1.1.7 Alpha, B.1.351 Beta, P.1 Gamma, B.1.617.2 Delta, and B.1.1.529 Omicron. These variants are found to consist of several specific or shared mutations in the S sequences that enhance their transmissibility and ability to escape the immune response. Several recent studies indicated decreased efficacy of mRNA vaccines against the VOCs as well as reduced efficacy in immunocompromised and older individuals. Therefore, new and complementary vaccines administered as booster or prophylaxis are required to combat the SARS-CoV-2 variants.

Dendritic cells (DC) are a class of immune system controllers that help to deliver signals to other immune cells with the help of soluble factors and intercellular interactions. An effective strategy to improve subunit-vaccine efficacy while reducing the amount of antigen required can be targeting the vaccine antigens to the DCs with the help of surface receptors. This not only helps in the delivery of specific antigens but also evokes an activation signal that stimulates the immune response without the need for additional immune stimulants. 

Previous studies suggested that vaccines targeting diverse viral antigens to CD40- expressing antigen-presenting cells evoked strong T and B cell responses. Also, studies have reported the efficacy of a new generation of subunit vaccines that target the RBD of the SARS-CoV-2 spike protein to the CD40 receptor.

A new study published in the pre-print server bioRxiv* used in silico approaches for designing a next-generation CD40-targeting vaccine, CD40. CoV2 included new T- and B-cell epitopes from SARS-CoV-2 and was also homologous to 38 sarbecoviruses, including SARS-CoV-2 VOCs. The study reported the antiviral efficacy as well as immunogenicity of this vaccine in a preclinical model.

About the study

The study involved the production of the CD40.CoV2 vaccine using expression plasmids via transfection into mammalian CHO-S cells and followed by Protein A-affinity purification. Infectious stocks of the Wuhan/D614 SARS-CoV-2 virus were grown by inoculating Vero E6 cells and collecting supernatants after cytopathic effects were observed.

Thereafter, 8 to 12 weeks old transgenic mice were injected with CD40.CoV2 vaccine and polyinosinic-polycytidylic acid (Poly-IC; Oncovir) or Poly-IC alone three weeks apart. They were then infected with SARS-CoV-2 on week 4 and monitored daily for mortality and morbidity. Blood was collected from them on day 2 before vaccination, day 28 before infection, and day 40 after vaccination.

The viral load was measured by RT-qPCR along with median tissue-culture infectious dose (TCID50). This was followed by antibody measurement, production of specific SARS-CoV-2 antigens, characterization of SARS-COV-2-specific immune responses in convalescent COVID-19 patients, quantification of culture supernatant analytes from convalescent COVID-19 peripheral blood mononuclear cells (PBMCs) 2 days after CD40.CoV2 vaccine administration, and cytotoxicity assay.

Study findings

The results reported four T-and B-cell epitope-enriched regions in the S, N, and M structural proteins of SARS-CoV-2 that were selected as vaccine regions. The vaccine sequences reported 42 percent and 48 percent CD8+ T-cell epitopes for S and N proteins, respectively while 46 percent and 40 percent were reported for CD4+ 161 T-cell epitopes. Two CD4+ T-cell epitopes and nine CD8+ T cell epitopes were found to be 100 percent homologous across all the sarbecoviruses.

The results also indicated that unvaccinated mice exhibited significant weight loss as well as the development of symptoms post-infection that could lead to death while vaccinated mice showed no symptoms and none of them died. The viral replication and viral infectious particles were found to be lower in the lungs of vaccinated mice as compared to unvaccinated mice. 

Additionally, the CD40.CoV2 vaccine levels were reported to be quite high one week after booster injection in vaccinated mice. The vaccine was also able to elicit cross-neutralizing antibodies responses against RBD from both the original SARS-CoV-2 strain and the VOCs and S from both SARS-CoV-2 and SARS-CoV-1.

Furthermore, the CD40.CoV2 vaccine induced significantly higher proliferation of specific CD4+ and CD8+ T cells and CD19+ B cells. The vaccine also stimulated the production of several chemokines and cytokines. The vaccine elicited a high amount of cross-reactive SARS-CoV-1 CD4+ and CD8+ T cells. The vaccine was found to induce SARS-CoV-1- and SARS-CoV-2-specific T-cell responses that were found to be highly correlated for almost all corresponding antigen sequences. Also, the vaccine responsiveness was not affected by RBD mutations of the VOCs and recognized the SARS-CoV-1 epitopes quite well.

The current study, therefore, demonstrates the urgent need for developing a “pan-sarbecovirus vaccine”. The CD40.CoV2 vaccine involved in the study was found to be quite responsive against the SARS-CoV-2 VOCs and has also shown significant cross-reactivity in both human and mice models. Further research needs to be carried out for the development of protein-based vaccines to combat the emerging SARS-CoV-2 variants as well as any future SARS-like coronaviruses.

Limitations

The study had certain limitations. First, there was no characterization of cross-neutralizing antibodies in vaccinated mice against the B.1.1.529 Omicron variant. Second, various VOC challenges in mice were not evaluated. Finally, the T cell responses were determined using samples obtained from recovered individuals instead of in vivo preclinical models.

*Important notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Written by

Suchandrima Bhowmik

Suchandrima has a Bachelor of Science (B.Sc.) degree in Microbiology and a Master of Science (M.Sc.) degree in Microbiology from the University of Calcutta, India. The study of health and diseases was always very important to her. In addition to Microbiology, she also gained extensive knowledge in Biochemistry, Immunology, Medical Microbiology, Metabolism, and Biotechnology as part of her master's degree.