The global pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to efforts in developing effective vaccine approaches. Currently, approved coronavirus disease 2019 (COVID-19) vaccines are administered through an intramuscular (I.M.) route. Here, we show that the SARS-CoV-2 spike (S) glycoprotein receptor-binding domain (RBD), when displayed on immunogenic liposomes, can be intranasally (I.N.) administered, resulting in the production of antigen-specific IgA and antigen-specific cellular responses in the lungs. Following I.N. immunization, antigen-presenting cells of the lungs took up liposomes displaying the RBD. K18 human ACE2-transgenic mice that were immunized I.M or I.N with sub-microgram doses of RBD liposomes and that were then challenged with SARS-CoV-2 had a reduced viral load in the early course of infection, with I.M. achieving complete viral clearance. Nevertheless, both vaccine administration routes led to full protection against lethal viral infection, demonstrating the potential for the further exploration and optimization of I.N immunization with liposome-displayed antigen vaccines.

Cobalt porphyrin phospholipid (CoPoP) can incorporate within bilayers to enable non-covalent surface-display of antigens on liposomes by mixing with proteins bearing a polyhistidine tag (his-tag); however, the mechanisms for how this occurs are poorly understood. These were investigated using the his-tagged model antigen Pfs25, a protein antigen candidate for malaria transmission-blocking vaccines. Pfs25 was found to associate with the small molecule aquocobalamin, a form of vitamin B12 and a cobalt-containing corrin macrocycle, but without particle formation, enabling comparative assessment. Relative to CoPoP liposomes, binding and serum stability studies indicated a weaker association of Pfs25 to aquocobalamin or cobalt nitrilotriacetic acid (Co-NTA) liposomes, which have cobalt displayed in the aqueous phase on lipid headgroups. Antigen internalization by macrophages was enhanced with Pfs25 bound to CoPoP liposomes. Immunization in mice with Pfs25 bound to CoPoP liposomes elicited antibodies that recognized ookinetes and showed transmission-reducing activity. To explore the physical mechanisms involved, we employed molecular dynamics (MD) simulations of bilayers containing phospholipid, cholesterol, as well as either CoPoP or NTA-functionalized lipids. The results show that the CoPoP-containing bilayer creates nanodomains that allow access for a limited but sufficient amount of water molecules that could be replaced by his-tags due to their favorable free energy properties allowing for stabilization. The position of the metal center within the NTA liposomes was much more exposed to the aqueous environment, which could explain its limited capacity for stabilizing Pfs25. This study illustrates the impact of CoPoP-induced antigen particleization in enhancing vaccine efficacy, and provides molecular insights into the CoPoP bilayer properties that enable this.

Background: Intranasal (I.N.) vaccination holds promise to elicit mucosal immunity that counters respiratory pathogens at the site of infection. For subunit protein vaccines, immunostimulatory adjuvants are typically required. Methods: We screened a panel of 22 lipid-phase adjuvants to identify which ones elicited antigen-specific IgA with I.N. immunization of liposome-displayed SARS-CoV-2 receptor-binding domain (RBD). Results: Initial screening showed the TLR-4 agonist Kdo2-Lipid A (KLA) effectively elicited RBD-specific IgA. A second round of screening identified further inclusion of the invariant NKT cell ligands α-Galactosylceramide (α-GalCer) and its synthetic analog 7DW8-5 as complementary adjuvants for I.N. immunization, resulting in orders-of-magnitude-greater mucosal IgA response relative to intramuscular (I.M.) immunization. The inclusion of cationic lipids conferred capacity for mucosal adhesion and maintained immune responses. In K18 hACE2 transgenic mice, vaccination significantly reduced viral replication and prevented mortality from SARS-CoV-2 challenge. Conclusions: These results point towards the potential for the use of KLA and α-GalCer for I.N. subunit vaccines.

Background: Effective vaccines are essential to overcome the limitations of livestock immunisation, particularly in low- and middle-income countries (LMICs), where scalable, thermostable, and easy-to-administer solutions are needed. Nanoparticle-based delivery systems, such as the Spontaneous Nanoliposome Antigen Particle (SNAP) technology using CoPoP liposomes, offer a promising alternative for subunit vaccine development, although their performance in large animal species remains poorly characterised. CoPoP enables the rapid non-covalent multimeric display of His-tagged protein antigens combined with immunomodulators on liposomes incorporating cobalt porphyrin–phospholipid (CoPoP). Objective: To evaluate the immunogenicity of CoPoP-based liposomes delivering the Theileria parva p67C antigen in cattle and compare their performance in murine models. Methods: Cattle and mice were immunised with p67C formulated in CoPoP liposomes incorporating QS-21 and/or PHAD immunomodulators. Humoral and cellular responses were assessed. Parallel in vitro stimulation of bovine PBMC with Quil-A was used to investigate the mechanistic effects of saponins on bovine cells. Results: CoPoP liposome formulations did not improve p67C immunogenicity in cattle, with antibody responses at least two-fold lower than previously reported results and no detectable cellular responses. In contrast, the same platform induced up to 2000-fold higher antibody titres in mice. This disparity is likely driven by differences in antigen dose relative to body mass, tissue architecture, lymphatic accessibility, and innate immune signalling differences. PHAD-mediated TLR4 activation appeared less effective in cattle, whereas QS-21 induced a broader immune activation, likely through conserved inflammasome pathways. Despite limited immunogenicity, antigen presentation by CoPoP liposomes was preserved. Conclusions: SNAP-based CoPoP liposomes showed strong immunogenicity in mice but limited efficacy in cattle, highlighting the challenges of cross-species translation. Optimisation of antigen dose and adjuvant selection for the targeted species is required, with QS-21 representing a more promising candidate than the TLR4 agonist. The scalability and versatility of SNAP technology support its continued development for multivalent livestock vaccines.