Although VLPs of SARS-CoV and SARS-CoV-2 could be readily generated by expression of M and E proteins together in transfected cells16,17, the VLPs to be used as immunogen in SARS-CoV-2 vaccine must contain and display the S protein, a key component inducing protective antibodies that prevents viral attachment to cells (S1 subunit) and/or genome uncoating (S2 subunit)18. The SARS-CoV-2 M protein is a transmembrane protein consisting of 222 amino acid residues that form short N-terminal domain, three transmembrane domains and long C-terminal domain19. M proteins of beta coronaviruses are O-glycosylated with no other post translational modification19,20. Naturally, the M protein functions in virus assembly and defines the shape of the assembled particles18. In the SARS-CoV-2 infected cells, the M protein antagonized the host innate immunity by inhibiting the formation of a functional TRAF3-containing complex of the classical TLR/RLR/TNFR-triggered NF- κB pathway which renders refractoriness of the IRF3/IRF7, hence no innate interferon production19,21. The M proteins of several coronaviruses induce both protective humoral and cytotoxic immune responses22. The N-terminal portion of M protein contains structural and functional cytotoxic T-cell epitope cluster22. Thus, it should be advantageous to include M protein in a SARS-CoV-2 vaccine. From Western blot analysis, the M protein in the VLPs produced in this study appeared as a protein band of about 18–20 kDa, indicating that the protein was intact. The envelope (E) protein is a multifunctional viroporin of coronaviruses that plays role in promoting reproduction and packaging of the progeny viruses. This protein is a major viral factor causing inflammatory response23 which leads to the cytokine storm and ARDS associated with respiratory coronavirus infections24. The E protein has important biological functions in maintaining the virion integrity and pathogenicity; thus, it is one of the attractive targets of drugs/therapeutics against coronaviruses25. SARS-CoV-2 E protein contains 75 amino acids and is a single-spanning membrane protein. The molecular size of the coronavirus E protein ranges from 8.4–12 kDa26. In this study, the E protein in the VLPs appeared as a protein band at about 10–12 kDa, indicating that full-length E protein was produced.
In this study, the versatile and efficient recombinant baculovirus-Sf21 insect cell system of which the recombinant baculovirus served as a vector and the SF21 insect cells as the host, was used for production of the VLPs consisting of SARS-CoV-2 S/S′, M and E proteins. VLPs of several enveloped viruses have been produced successfully by using this binary system27. It is known that the recombinant baculovirus vector can provide not only high levels of the heterologous gene expression, but also it can accommodate multiple and large gene insert; thus, suitable for production of VLPs and other recombinant proteins28. Baculoviruses are non-pathogenic to humans and animals. Usually, the baculovirus-insect cell system yields recombinant proteins with proper folding, glycosylation, phosphorylation, acetylation and acylation.
From both methods of Sf21 cell transfection (co-transfection and separate transfection) with S-bacmids and ME-bacmids, the Western blot analysis of the culture supernatants of the transfected cells containing P1 baculoviruses revealed not only intact S protein with apparent molecular size of approximately 250 kDa, but also the cleaved S products, i.e., the S1 and S2 subunits which the apparent molecular sizes were approximately 100–120 and 70–100 kDa, respectively. Theoretically, the nascent S, S1 and S2 proteins (without post translational modification) of the SARS-CoV-2 wildtype strain are 141.2, 75.3 and 58–60 kDa, respectively29. The molecular sizes of the intact S protein and the S1 and S2 subunits may vary from the theoretical molecular weights due to post translational modifications, post translation cleavages, relative charges, and other experimental factors30,31. The post-translational modification of the recombinant S protein in the insect cells accounted for the apparent larger sizes of the S and its subunits of this study.
The S′-P1 baculovirus derived from Sf21 insect cells separately transfected with S′-bacmids and ME-bacmids showed intact S protein and no S1 and S2 subunits in Western blot analysis. Thus, the S′-P2 baculovirus and S′ME-VLPs derived from the S′-P1 baculovirus should also carry intact S protein. In this study, immunogenicity of the anionic liposome-adjuvanted vaccines consisting of VLPs expressing M, E and S/S′ proteins (with and without furin cleavage site at the S1-S2 junction, respectively) were investigated. They were found to have no difference in immunogenicity in inducing systemic and mucosal immune responses.
The VLPs of this study mimic the structural organization and conformation of the authentic native SARS-CoV-2 particles but lacking the viral ribonucleoprotein (RNP)27. The SME-VLPs and the S′ME-VLPs without spikes were approximately 179 and 207 nm in median diameters, respectively, which are conformed to the size of the commercialized SARS-CoV-2 VLPs produced from transfected HEK293 cells32. The median size of the authentic native SARS-CoV-2 particles without spikes was 100 nm33. After being encapsulated by liposome (L), the median sizes of the liposome-encapsulated SME-VLPs (L-SME-VLP vaccine) and S′ME-VLPs (L-S′ME-VLP vaccine) were 316 and 237 nm in the average, respectively, which were not significantly different from the average size of the liposome-entrapped PBS (placebo; L-PBS; 342 nm). The correlation of the particulate adjuvant characteristics (including sizes and surface charges) with the resultant immune responses against the adjuvanted vaccines has been reviewed extensively34. The size of the particulate adjuvants may have different effects on the type of the vaccine-induced immune responses. For targeted-delivery systems, nanoparticles (1-1000 nm) are considered more effective than microparticles (1-1000 µm), as the former is more efficient in diffusing through biological barriers, passing through capillaries and being relatively stable in blood circulation34–36. For vaccines, however, experimental results pertaining to the optimal size ranges of the particulate-based delivery system that will generate strong and sustained immune responses to the co-administered antigen are conflicting34,37. Nevertheless, evidence indicated that immunization with the 200–600 nm particles favored Th1 immune responses, whereas immunization with the 2–8-µm particles favored Th2 response38. For respiratory infections, like respiratory syncytial virus (RSV) and SARS-CoV/SARS-CoV-2, Th1 response to vaccine is preferred to the Th2 response as it was observed in vaccinated animal models as well as in children that the Th2 response may exacerbate lung inflammation upon experiencing new infection due to immunopathology that reminiscent the type 1 hypersensitivity with eosinophil infiltration and immune complex deposition in the lung39,40.
Both SME-VLPs and S′ME-VLPs carried negatively charged surface (-1.96 and − 5.31, respectively). After liposome encapsulation, the negative charges of the vaccine micelles were increased to -5.12 and − 18.63, respectively. For parenteral immunization, e.g., intramuscular or subcutaneous route, vaccines using cationic liposome as adjuvant and delivery vehicle offers benefit by causing tissue damage and a release of damage-associated molecular patterns (DAMPs) at the injection site that act as the endogenous adjuvant to activate inflammatory response via binding with pattern recognition receptors (PRRs) of cells of the innate immune system including antigen-presenting cells (APCs) such as dendritic cells (DCs) and macrophages, and stimulation of both helper and cytotoxic lymphocyte responses41. Besides, the cationic liposome are prone to coalesce with the negatively charged surface of the APCs42 and, in the effect, may cause a release of the liposome-entrapped antigen into the APC cytoplasm which is then processed and presented to the CD8 + T cells via the MHC class I (cytotoxic response) and cross-presented to the CD4 + T cells by the MHC class II pathway to elicit the T helper response, i.e., Th1 or Th2 response, or both, depending upon the cytokine milieu. However, administration of cationic liposome made of lipids with quaternary ammonium head groups intravenously to mice (mimicking natural infection such as hematophagous insect bite) causes cell disruption and hemolysis42,43. In this study, mice were immunized with anionic liposome-adjuvanted VLP vaccines, either intraperitoneally (IP) or intranasally (IN). In the peritoneal cavity, macrophages (that functions in immune surveillance against invader) effectively detect, phagocytose, and process the antigen for T cell presentation and B1 cell stimulation. At the mucosal surface, like nasal cavity and intestine, the mucin glycoproteins in the mucus gel layer carry strongly net-negative surface due to their high sialic acid and sulfate content44. Thus, for the mucosal vaccination, anionic or neutral nanoparticles with encapsulated immunogen will not bind to or trapped in the negatively charged mucosa, allowing them to be easily approachable, endocytosed, and transported (by microfold/M cells) to the inductive site of the respective mucosal lymphoid tissue, such as, mouse organized and diffuse nasal-associated lymphoid tissues (ONALT and DNALT), tonsils, Peyer's patches, draining lymph nodes43. Thus, negatively charged carriers may favor mucosal vaccination such as intranasal, oral, or vaginal vaccination42,44.
Although intraperitoneal route of immunization is not in medical practice, however, intraperitoneal immunization of mice has been utilized extensively in research for vaccine development against respiratory viruses, including influenza, respiratory syncytial virus (RSV), and SARS-CoV-2 vaccines, to gain primary data on innocuity and immunogenicity45–47. Intraperitoneal inoculation with live influenza A virus confers protection against intranasal infections in mice and ferrets46. Intraperitoneal immunization induced acute and memory immune responses capable of effector functions and protection at distal nasal mucosa and lung against RSV45. Intraperitoneal immunization was performed in this study for testing innocuousness and immunogenicity of the anionic liposome-adjuvanted VLP vaccines against SARS-CoV-2. The peritoneal cavity is the largest serosal body space that harbors most of the abdominal organs and an important visceral adipose tissue called omentum. The omentum contains milky spots which are clusters of leukocytes that the cells are organized like those in the secondary lymphoid tissues, i.e., a central B cell area surrounded by T cells and myeloid cells that are supported by a fibroblastic stromal cell network48,49. The omentum (and other serous cavities, e.g., pleural cavity) is a site of B1 cell lymphopoiesis and T cell-independent immune responses to multivalent antigens. B1 cells produce cross-reactive antibodies that are mainly IgM but can be IgG3 and IgA isotypes50. Besides, activated B1 cells can migrate to mucosal surface such as intestinal lamina propria and differentiate into plasma cells that secrete IgA antibodies for protection of the mucosal surface51. Activated B1 cells can migrate to spleen where they serve as precursor of splenic IgM producing cells52. Peritoneal B1 cells can switch readily to IgA producing cells in splenic marginal zone53.
After three IP doses of the L-SME-VLP/L-S′ME-VLP vaccines, the titers of the ELISA antibodies to SARS-CoV-2 S1 subunit and VN antibodies were markedly induced. The predominant isotype of the serum anti-S1 antibodies of 5 of 6 immunized mice (83%) were IgG3 which can be either from activated B1 cells that migrated to other lymphoid tissues, i.e., spleen and lymph nodes, or from the activated B2 cells in the peripheral lymphoid tissues in response to the antigen that entered systemic circulation. Mouse IgG3 is highly efficient in complement activation, opsonophagocytic activities as well as antibody-dependent cell-mediated cytotoxicity (ADCC)54 which cause virus clearance upon infection. Nevertheless, complement fixation and immune-complex formation may exacerbate the inflammation and cytokine storm as well as causing infiltration of inflammatory cells (especially eosinophils) into lungs of the virus infecting host which exacerbates the critical morbidity39,55.
The upper respiratory tract is an important prime site of host defense against inhalant pathogens, e.g., respiratory viruses like influenza virus, RSV and SARS-CoV-2. Advantages of intranasal vaccination in induction of the mucosal and systemic immunity have been reviewed56. Intranasal immunization effectively induces protective immunity by triggering both mucosal and systemic responses following antigen administration which contrasts with intramuscular injection that primarily induces systemic immune responses56,57. Intranasal vaccination can confer protection against infections at other mucosal sites, such as the lower respiratory tract and lungs, intestines, and genital tract, and may provide cross-protection against variant strains due to cross-reactive/poly-reactive nature of antibodies produced by activated B1 cells58. Three doses of the L-SME-VLP and L-S′ME-VLP vaccines containing 30 µg VLPs/dose, administered intranasally did not induce significant rise of serum antibodies, indicating that the immunizing dose might be too low, or the time of sample collection was too soon59. Both vaccines were found to induce predominantly Th1 response as shown by IgG2a and/or IgG2b antibody isotypes in the BALF, and less IgG3 production. IgG2a and IgG2b are relatively poor in complement activation and opsonophagocytic activities compared to the IgG360. On contrary, several studies have demonstrated that specific IgG2a exhibits stronger anti-viral effects than other antibody isotypes61,64. Predominant IgG2a among many IgG antibody responses elicited by live viruses could confer the best protection for the infected host65. The intranasal route of vaccination is best suited for pandemic/epidemic control of highly contagious/infectious respiratory viruses following the outbreak, because it is easy to do; thus, less skilled allied health personnels can be recruited for doing the mass vaccinations56. The intranasal route causes minimal discomfort with no intrusive and pain; therefore, it should receive better compliance from children, needle-fear subjects, and patients with morbidities that required multiple/frequent injections60,66.
In conclusion, the anionic liposome (L) encapsulated/adjuvanted VLPs (L-SME-VLP and L-S′ME-VLP vaccines) were innocuous and immunogenic in mice after IP and IN immunization. Both vaccines induced principally serum IgG3 antibody isotype response in the IP immunized mice while mice immunized intranasally with the vaccines had principally Th1-type response as shown by predominant IgG2a and/or IgG2b antibody isotypes in the bronchoalveolar lavage fluids. The intranasal anionic liposome-adjuvanted VLP vaccines should be tested further towards the clinical use as an effective, safe and well-compliant vaccine that induces the first line defense against the inhalant SARS-CoV-2.