Investigating in vivo utilization of VLP vaccines adjuvanted with single, dual, or triple pathogen-associated molecular patterns

COVID-19 was first identified in Wuhan, a Chinese city, and it has been a part of our lives since late December 2019. The WHO declared this disease, caused by SARS-CoV-2, a pandemic due to its high transmission rate, a status that remained in effect until May 2023. The escalating death toll and virus spread expedited vaccine development efforts. Various platforms for vaccine development have been utilized, including inactivated virus, mRNA, and subunit vaccines. Given the virus's propensity for mutations due to its high transmission rate, the chosen platform must be easy to produce, effective, and safe for immunocompromised individuals. One such platform is virus-like particles (VLPs). VLP vaccines consist of viral proteins without genetic material and are known for their ease of production. A VLP vaccine, adjuvanted with Alum and K-type CpG ODN against SARS-CoV-2, is tested in Phase II clinical trials. In addition to CpG ODN, this study aimed to evaluate the efficacy of other adjuvants and their combinations to explore potential clinical advantages. The additional adjuvants included poly(I:C) and a member of cyclic dinucleotides, 2,3-cGAMP, which are Toll-like receptor 3 (TLR3) and STING agonists, respectively. Nine groups of male mice (C57BL/6, 6–8 weeks old) received intraperitoneal injections three times with a mixture of VLP vaccine using different combinations of adjuvants, including CpG ODN, CDN, and poly(I:C). Blood samples were collected from the tail vein two weeks after each injection. Antibody ELISA was employed to assess humoral immunity against each combination of vaccines, with plates coated using in-house recombinant 6P-Spike and commercial recombinant RBD. ELISA results indicated that VLPs combined with CpG plus poly(I:C) and VLPs combined with all three adjuvants induced high levels of total IgG against RecS. Two weeks after the third injection, the animals' spleens were isolated, and cytokine profiles were assessed through an antigen recall assay. As a result, only one group exhibited the expected cytokine profile: VLPs combined with the triple adjuvant combination. This profile demonstrated the requisite Th1-biased cytokine response necessary for vaccine development. This study suggests that the triple adjuvant combination holds promise as a vaccine adjuvant for addressing future pandemics.