From Bovine Immune Milk Profiling to Multi-Antigen Vaccine Design: Enhanced Humoral Responses Against H. pylori with
Hongru Li1, Enhao Zhang1, Jingyuan Ning1
1Key Laboratory of Immune Mechanism and Intervention on Serious Disease in Hebei Province, Department of Immunology, Hebei Medical University, Shijiazhuang 050017, China.
Objective:
The aim of this study was to develop and evaluate non-antibiotic strategies against Helicobacter pylori by establishing a bovine immune milk platform and designing a synergistic multi-antigen immunogen to enhance humoral immune responses.
Methods:
Inactivated Helicobacter pylori (H. pylori) was used to immunize dairy cows, and the resulting immune milk was characterized for antibody specificity, acid stability, and target antigens via ELISA, Western blot, agglutination assays, and mass spectrometry. Key identified antigens (UreA, UreB, UreE, UreG, HypA, FlaA, and FlaB) were produced as recombinant proteins. Their immunogenicity was evaluated in a murine model, comparing single antigens with various protein combinations. Immune responses were assessed by antigen-specific IgG ELISA, bacterial agglutination titers, flow cytometry for T-cell activation, and histopathology for safety.
Results:
Immune milk contained high-titer, acid-stable IgG antibodies targeting multiple H. pylori virulence factors. In mice, while single proteins induced specific IgG, a multi-antigen cocktail (FlaA + FlaB + HypA + UreA + UreB + UreE + UreG) elicited significantly higher serum agglutination titers (~7 × 103) than single antigens or inactivated whole-cell vaccine, alongside robust CD4+ T-cell activation. No formulations showed any hepatorenal or splenic toxicity.
Conclusion:
Bovine immune milk is a viable platform for acid-stable antibody delivery. A rationally designed multi-antigen cocktail synergistically enhances functional humoral immunity in vivo, providing a promising foundation for developing antibody-based or subunit vaccine strategies against H. pylori.
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