# From Bovine Immune Milk Profiling to Multi-Antigen Vaccine Design: Enhanced Humoral Responses Against H. pylori with a Flagellin and Urease Subunit Cocktail

**Authors:** Hongru Li, Enhao Zhang, Jingyuan Ning, Yushan Lin, Guanyuan Wang, Hong Zhang, Cuixia Ma, Jiachao Wang, Miao Li, Xue Gao, Chenhui Li, Lin Wei, Xian Wang, Cuiqing Ma

PMC · DOI: 10.3390/vaccines14020110 · Vaccines · 2026-01-23

## TL;DR

This study explores using bovine immune milk and a multi-antigen vaccine to boost immunity against Helicobacter pylori, a harmful stomach bacterium.

## Contribution

The study introduces a novel multi-antigen cocktail vaccine and a bovine immune milk platform for acid-stable antibody delivery against H. pylori.

## Key findings

- Bovine immune milk produced high-titer, acid-stable IgG antibodies targeting multiple H. pylori virulence factors.
- A multi-antigen cocktail of FlaA, FlaB, HypA, UreA, UreB, UreE, and UreG significantly enhanced serum agglutination titers and CD4+ T-cell activation in mice.
- No hepatorenal or splenic toxicity was observed with any tested formulations.

## Abstract

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.

## Linked entities

- **Proteins:** ureA (urease subunit gamma), ureB (urease subunit beta), ureE (urease accessory protein UreE), UREG (urease accessory protein G), PRPF40A (pre-mRNA processing factor 40A), flaA (flagellin A), flaB (flagellin B)
- **Species:** Helicobacter pylori (taxon 210)

## Full-text entities

- **Genes:** Cd4 (CD4 antigen) [NCBI Gene 12504] {aka L3T4, Ly-4}, CD4 (CD4 molecule) [NCBI Gene 920] {aka CD4mut, IMD79, Leu-3, OKT4D, T4}, CD79A (CD79a molecule) [NCBI Gene 973] {aka IGA, IGAlpha, MB-1, MB1}, VacA [NCBI Gene 48201093], CD8A (CD8 subunit alpha) [NCBI Gene 925] {aka CD8, CD8alpha, IMD116, Leu2, p32}, Cd3e (CD3 antigen, epsilon polypeptide) [NCBI Gene 12501] {aka CD3, CD3epsilon, T3e}, CagA [NCBI Gene 48200769], PRPF40A (pre-mRNA processing factor 40A) [NCBI Gene 55660] {aka FBP-11, FBP11, FLAF1, FNBP3, HIP-10, HIP10}
- **Diseases:** chronic dyspepsia (MESH:D004415), chronic gastritis (MESH:D005756), necrosis (MESH:D009336), splenic toxicity (MESH:D013158), hepatic, renal, or splenic toxicity (MESH:D056486), enteric (MESH:D004751), gastric adenocarcinoma (MESH:D013274), Toxicity (MESH:D064420), Hepatorenal (MESH:D006530), ulcer (MESH:D014456), infection (MESH:D007239), peptic ulcer disease (MESH:D010437), cancer (MESH:D009369), upper gastrointestinal disorders (MESH:D005767), deaths (MESH:D003643), H. pylori infection (MESH:D016481), bacterial colonization (MESH:D015179), gastric inflammation (MESH:D007249), injury to (MESH:D014947)
- **Chemicals:** H2SO4 (MESH:C033158), Tween 20 (MESH:D011136), PBS (MESH:D007854), SDS (MESH:D012967), Freund's incomplete adjuvant (MESH:C114843), linolenic acid (MESH:D017962), agar (MESH:D000362), 3,3,5,5-tetramethylbenzidine (MESH:C021758), nickel (MESH:D009532), UreA (MESH:D014508), FlaA (-), H&amp;E (MESH:D006371)
- **Species:** Streptococcus pyogenes (species) [taxon 1314], Rotavirus (genus) [taxon 10912], Bos taurus (bovine, species) [taxon 9913], Homo sapiens (human, species) [taxon 9606], Helicobacter pylori (species) [taxon 210], Mus musculus (house mouse, species) [taxon 10090], Bacteria Latreille et al. 1825 (Bacteria stick insect, genus) [taxon 629395], Escherichia coli BL21(DE3) (strain) [taxon 469008], Escherichia coli (E. coli, species) [taxon 562]
- **Mutations:** D1013S

## Full text

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## Figures

5 figures with captions in the complete paper: https://tomesphere.com/paper/PMC12945149/full.md

## References

30 references — full list in the complete paper: https://tomesphere.com/paper/PMC12945149/full.md

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Source: https://tomesphere.com/paper/PMC12945149