Low-cost, refrigerator-stable COVID-19 vaccines will facilitate global access and improve vaccine coverage in low- and middle-income countries. To this end, subunit-based approaches targeting the receptor-binding domain (RBD) of SARS-CoV-2 Spike protein remain attractive. Antibodies against RBD neutralize SARS-CoV-2 by blocking viral attachment to the host cell receptor, ACE2. Here, a yeast-produced recombinant RBD antigen (RBD-L452K-F490W or RBD-J) was formulated with various combinations of aluminum-salt (Alhydrogel®, AH; AdjuPhos®, AP) and CpG 1018 adjuvants. We assessed the effect of antigen-adjuvant interactions on the stability and mouse immunogenicity of various RBD-J preparations. While RBD-J was 50% adsorbed to AH and <15% to AP, addition of CpG resulted in complete AH binding, yet no improvement in AP adsorption. ACE2 competition ELISA analyses of formulated RBD-J stored at varying temperatures (4, 25, 37°C) revealed that RBD-J was destabilized by AH, an effect exacerbated by CpG. DSC studies demonstrated that aluminum-salt and CpG adjuvants decrease the conformational stability of RBD-J and suggest a direct CpG-RBD-J interaction. Although AH+CpG-adjuvanted RBD-J was the least stable in vitro , the formulation was most potent at eliciting SARS-CoV-2 pseudovirus neutralizing antibodies in mice. In contrast, RBD-J formulated with AP+CpG showed minimal antigen-adjuvant interactions, a better stability profile, but suboptimal immune responses. Interestingly, the loss of in vivo potency associated with heat-stressed RBD-J formulated with AH+CpG after one dose was abrogated by a booster. Our findings highlight the importance of elucidating the key interrelationships between antigen-adjuvant interactions, storage stability, and in vivo performance to enable successful formulation development of stable and efficacious subunit vaccines.

Authorized vaccines against SARS-CoV-2 remain less available in low- and middle-income countries due to insufficient supply, high costs, and storage requirements. Global immunity could still benefit from new vaccines using widely available, safe adjuvants, such as alum and protein subunits, suited to low-cost production in existing manufacturing facilities. Here, a clinical-stage vaccine candidate comprising a SARS-CoV-2 receptor binding domain–hepatitis B surface antigen virus–like particle elicited protective immunity in cynomolgus macaques. Titers of neutralizing antibodies (>10 4 ) induced by this candidate were above the range of protection for other licensed vaccines in nonhuman primates. Including CpG 1018 did not significantly improve the immunological responses. Vaccinated animals challenged with SARS-CoV-2 showed reduced median viral loads in bronchoalveolar lavage (~3.4 log 10 ) and nasal mucosa (~2.9 log 10 ) versus sham controls. These data support the potential benefit of this design for a low-cost modular vaccine platform for SARS-CoV-2 and other variants of concern or betacoronaviruses.

Vaccines against SARS-CoV-2 have been distributed at massive scale in developed countries, and have been effective at preventing COVID-19. Access to vaccines is limited, however, in low- and middle-income countries (LMICs) due to insufficient supply, high costs, and cold storage requirements. New vaccines that can be produced in existing manufacturing facilities in LMICs, can be manufactured at low cost, and use widely available, proven, safe adjuvants like alum, would improve global immunity against SARS-CoV-2. One such protein subunit vaccine is produced by the Serum Institute of India Pvt. Ltd. and is currently in clinical testing. Two protein components, the SARS-CoV-2 receptor binding domain (RBD) and hepatitis B surface antigen virus-like particles (VLPs), are each produced in yeast, which would enable a low-cost, high-volume manufacturing process. Here, we describe the design and preclinical testing of the RBD-VLP vaccine in cynomolgus macaques. We observed titers of neutralizing antibodies (>104) above the range of protection for other licensed vaccines in non-human primates. Interestingly, addition of a second adjuvant (CpG1018) appeared to improve the cellular response while reducing the humoral response. We challenged animals with SARS-CoV-2, and observed a ~3.4 and ~2.9 log10 reduction in median viral loads in bronchoalveolar lavage and nasal mucosa, respectively, compared to sham controls. These results inform the design and formulation of current clinical COVID-19 vaccine candidates like the one described here, and future designs of RBD-based vaccines against variants of SARS-CoV-2 or other betacoronaviruses.

Campylobacter infection is one of the most common causes of bacterial gastroenteritis worldwide and a major global health threat due to the rapid development of antibiotic resistance. Currently, there are no vaccines approved to prevent campylobacteriosis, and rehydration is the main form of therapy. Secretory immunoglobulin A (SIgA) is the main antibody class found in mucous secretions, including human milk, and serves as the first line of defense for the gastrointestinal epithelium against enteric pathogens. In this study, we describe the prophylactic activity of orally delivered recombinant SIgA generated from two human monoclonal antibodies (CAA1 and CCG4) isolated for their reactivity against the flagellar-capping protein FliD, which is essential for bacteria motility and highly conserved across Campylobacter species associated with severe enteritis. In an immunocompetent weaned mouse model, a single oral administration of FliD-reactive SIgA CAA1 or CCG4 at 2 h before infection significantly enhances Campylobacter clearance at early stages post-infection, reducing the levels of inflammation markers associated with epithelial damage and polymorphonuclear (PMN) cells infiltration in the cecum lamina propria. Our data indicate that the prophylactic activity of CAA1 and CCG4 is not only dependent on the specificity to FliD but also on the use of the SIgA format, as the immunoglobulin G (IgG) versions of the same antibodies did not confer a comparable protective effect. Our work emphasizes the potential of FliD as a target for the development of vaccines and supports the concept that orally administered FliD-reactive SIgA can be developed to prevent or mitigate the severity of Campylobacter infections as well as the development of post-infection syndromes.

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