Recombinant LAB vector-based multivalent vaccine against Campylobacter jejuni potentially promoting a healthier microbial balance in the poultry gut

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. 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. 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. Beyond targeting C. jejuni, the vaccine may influence the overall gut microbiota, potentially promoting a healthier microbial balance in the poultry gut. Our 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. 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.