Optimizing immunogenicity of novel Cryptosporidium vaccine candidates

Cryptosporidiosis is a top ten cause of infant diarrhea in low and middle-income countries (LMICs), as well as malnutrition and impaired neurocognitive development, leading to substantial yearly morbidity, mortality and lost disability-adjusted life years. A vaccine for cryptosporidiosis is not available but would be appealing for prevention of cryptosporidiosis in children in LMICs where the burden of infection is high. In a recent Phase 1 SBIR study, Antigen Discovery, Inc. (ADI) in collaboration with University of Virginia (UVA) successfully developed a pan-proteomic Cryptosporidium protein microarray. This tool was used to characterize the IgA and IgG antibody responses in children from a high transmission area of Bangladesh at one year of life. Cryptosporidial proteins targeted by antibodies and associated with protection from Cryptosporidium reinfection were discovered and published by Gilchrist and Campo et al in the J. Clinical Investigation. This work also established the importance of antibodies and mucosal immunity in protection against Cryptosporidium. The next steps are to formulate novel Cryptosporidium proteins and a potent mucosal immunity-stimulating adjuvant as vaccine candidates that can be tested for immunogenicity and protection in preclinical animal studies. A mouse model of cryptosporidiosis with challenge in BL6 mice has been established at UVA. Outstanding questions are which novel Cryptosporidium proteins, and which route(s) of immunization and which adjuvants stimulate mucosal immunity. In this new Phase 1 study, vaccine candidates will be produced as purified recombinant proteins for animal immunization experiments. These proteins will be mixed with a GLA-3M052 liposome adjuvant that has been demonstrated as safe and immunogenic in non-human primates and mice. Intranasal (IN) delivery of vaccines has been shown to elicit a durable and functional systemic and mucosal immune response. We hypothesize that novel Cryptosporidium vaccine candidates adjuvanted with GLA-3M052 and delivered IN or by heterologous prime + IN boost regimens will stimulate robust mucosal and systemic antibody and cellular immune responses in female and male BL6 mice. To test this, a two-phase adaptive immunization study design will be used with an interim immunogenicity analysis between phases. In the first phase, vaccine candidates Cp17 and Cp23 will be administered to BL6 mice as a bivalent vaccine, and novel antigens CCgd4_4270 and Chro.30111 will be given as monovalent vaccines adjuvanted with GLA-3M052. Blood and stool before and after immunization will be assessed for antigen-specific antibody binding and antibody functional analysis, including mouse complement deposition and opsonophagocytosis. Post-euthanasia lymphocytes from spleen and mesenteric lymph nodes will be tested for antigen-specific IFNγ production and multiplex cytokine/chemokine secretion. If a mucosal immune response to vaccination is observed, a second phase of immunizations will test novel antigens cgd2_1520, cgd8_700 and cgd4_1970 adjuvanted with GLA-3M052. If mucosal responses are not detected, the second phase will repeat the phase 1 antigens using the LTA1 modified enterotoxin as an alternative adjuvant. A final immunogenicity analysis will reveal which antigen(s) and which immunization regimen elicits strong mucosal and systemic immunogenicity. Successful completion of this work will provide proof of concept for a vaccine candidate that should be tested in preclinical studies going forward.