Recombinant Lactococcus lactis-based multivalent vaccine targeting Campylobacter jejuni colonisation and modulating cecal microbiota in poultry

Background: Diarrhoeal diseases remain the second leading cause of preventable death globally, particularly among children under the age of five in developing countries, accounting for an estimated 2-3 million deaths annually. Among the bacterial pathogens causing diarrheal illness, Campylobacter jejuni (C. jejuni) remains one of the major contributors, particularly in LMICs. As a common gut pathogen, C. jejuni expresses several secretory or surface-expressed colonisation proteins, namely Hcp, VgrG, CadF, FlpA, and JlpA. Most of these proteins play pivotal roles in bacterial self-survival, host-cell adhesion, and invasion in avian and non-avian hosts. Methods: To minimise the C. jejuni adhesion and subsequent colonisation in the avian gut, we explored the potential of a multivalent mucosal vaccine composition using these putative subunits of C. jejuni. For this purpose, we bioengineered a food-grade Lactic Acid-producing Bacterium, Lactococcus lactis (L. lactis), to express three key immunogenic subunits, Hcp, CadF and JlpA. Utilising this live vector-based multi-component mucosal vaccine platform, we investigated the immunoprotective potential of these antigens in chickens. Since the particular strain of L. lactis is non-colonising, we used chitosan as a natural mucoadhesive, biodegradable polymer to microencapsulate the engineered bacteria to increase their gut retention time for optimal interaction with local immune cells. Results: Our in vivo immunisation study demonstrated that oral administration of this multivalent vaccine formulation elicited a strong local antibody response (sIgA) and upregulated key pro-inflammatory cytokines, leading to robust mucosal immune protection against the cecal colonisation of C. jejuni. Moreover, gut metagenomic analysis of vaccinated birds revealed a marked reduction in the phylum Campylobacterota, accompanied by an increased abundance of the phyla Bacillota and Bacteroidota, as part of a beneficial microbial community. Conclusions: Together, this study underscores the potential of a live vector-based, multivalent mucosal vaccine as a promising, cost-effective strategy to reduce the risk of foodborne transmission of C. jejuni, particularly in poultry production systems.