Amish (Rural) vs. non-Amish (Urban) Infant Fecal Microbiotas Are Highly Diverse and Their Transplantation Lead to Differences in Mucosal Immune Maturation in a Humanized Germfree Piglet Model.

The gut microbiome plays an important role in the immune system development, maintenance of normal health status, and in disease progression. In this study, we comparatively examined the fecal microbiomes of Amish (rural) and non-Amish (urban) infants and investigated how they could affect the mucosal immune maturation in germ-free piglets that were inoculated with the two types of infant fecal microbiota (IFM). Differences in microbiome diversity and structure were noted between the two types of fecal microbiotas. The fecal microbiota of the non-Amish (urban) infants had a greater relative abundance of Actinobacteria and Bacteroidetes phyla, while that of the Amish (rural) counterparts was dominated by Firmicutes. Amish infants had greater species richness compared with the non-Amish infants' microbiota. The fecal microbiotas of the Amish and the non-Amish infants were successfully transplanted into germ-free piglets, and the diversity and structure of the microbiota in the transplanted piglets remained similar at phylum level but not at the genus level. Principal coordinates analysis (PCoA) based on Weighted-UniFrac distance revealed distinct microbiota structure in the intestines of the transplanted piglets. Shotgun metagenomic analysis also revealed clear differences in functional diversity of fecal microbiome between Amish and non-Amish donors as well as microbiota transplanted piglets. Specific functional features were enriched in either of the microbiota transplanted piglet groups directly corresponding to the predominance of certain bacterial populations in their gut environment. Some of the colonized bacterial genera were correlated with the frequency of important lymphoid and myeloid immune cells in the ileal submucosa and mesenteric lymph nodes (MLN), both important for mucosal immune maturation. Overall, this study demonstrated that transplantation of diverse IFM into germ-free piglets largely recapitulates the differences in gut microbiota structure between rural (Amish) and urban (non-Amish) infants. Thus, fecal microbiota transplantation to germ-free piglets could be a useful large animal model system for elucidating the impact of gut microbiota on the mucosal immune system development. Future studies can focus on determining the additional advantages of the pig model over the rodent model.

Corn-derived alpha-D-glucan nanoparticles as adjuvant for intramuscular and intranasal immunization in pigs.

Adjuvant potential of positively charged corn-derived nanoparticles (Nano-11) was earlier revealed in mice. We evaluated its adjuvant role to electrostatically adsorbed inactivated/killed swine influenza virus antigen (KAg) (Nano-11 + KAg) in pigs. Nano-11 facilitated the uptake of KAg by antigen presenting cells and induced secretion of proinflammatory cytokines. In pigs vaccinated by an intranasal mist containing Nano-11 + KAg, expression of T-helper 1 and T-helper 2 transcription factors and secretion of cross-reactive influenza antigen-specific mucosal IgA in the nasal cavity were observed. The enhanced frequencies of IFN-γ positive T-helper and cytotoxic T-cells in Nano-11 + KAg-vaccinates after heterologous virus challenge were also observed. Clinically, slightly reduced influenza signs and pneumonic lesions, with mild reduction in virus load in the respiratory tract of vaccinates were observed. In pigs immunized with Nano-11 adsorbed ovalbumin administered by intramuscular (IM) route, enhanced IgG1 and IgG2 antibodies were detected in serum. Thus, Nano-11 vaccine delivery system confers adjuvant effect in pigs.

Liposomal nanoparticle-based conserved peptide influenza vaccine and monosodium urate crystal adjuvant elicit protective immune response in pigs.

BACKGROUND: Influenza (flu) is a constant threat to humans and animals, and vaccination is one of the most effective ways to mitigate the disease. Due to incomplete protection induced by current flu vaccines, development of novel flu vaccine candidates is warranted to achieve greater efficacy against constantly evolving flu viruses. METHODS: In the present study, we used liposome nanoparticle (<200 nm diameter)-based subunit flu vaccine containing ten encapsulated highly conserved B and T cell epitope peptides to induce protective immune response against a zoonotic swine influenza A virus (SwIAV) H1N1 challenge infection in a pig model. Furthermore, we used monosodium urate (MSU) crystals as an adjuvant and co-administered the vaccine formulation as an intranasal mist to flu-free nursery pigs, twice at 3-week intervals. RESULTS: Liposome peptides flu vaccine delivered with MSU adjuvant improved the hemagglutination inhibition antibody titer and mucosal IgA response against the SwIAV challenge and also against two other highly genetically variant IAVs. Liposomal vaccines also enhanced the frequency of peptides and virus-specific T-helper/memory cells and IFN-γ response. The improved specific cellular and mucosal humoral immune responses in adjuvanted liposomal peptides flu vaccine partially protected pigs from flu-induced fever and pneumonic lesions, and reduced the nasal virus shedding and viral load in the lungs. CONCLUSION: Overall, our study shows great promise for using liposome and MSU adjuvant- based subunit flu vaccine through the intranasal route, and provides scope for future, pre-clinical investigations in a pig model for developing potent human intranasal subunit flu vaccines.

Mucosal Immunity and Protective Efficacy of Intranasal Inactivated Influenza Vaccine Is Improved by Chitosan Nanoparticle Delivery in Pigs.

Annually, swine influenza A virus (SwIAV) causes severe economic loss to swine industry. Currently used inactivated SwIAV vaccines administered by intramuscular injection provide homologous protection, but limited heterologous protection against constantly evolving field viruses, attributable to the induction of inadequate levels of mucosal IgA and cellular immune responses in the respiratory tract. A novel vaccine delivery platform using mucoadhesive chitosan nanoparticles (CNPs) administered through intranasal (IN) route has the potential to elicit strong mucosal and systemic immune responses in pigs. In this study, we evaluated the immune responses and cross-protective efficacy of IN chitosan encapsulated inactivated SwIAV vaccine in pigs. Killed SwIAV H1N2 (δ-lineage) antigens (KAg) were encapsulated in chitosan polymer-based nanoparticles (CNPs-KAg). The candidate vaccine was administered twice IN as mist to nursery pigs. Vaccinates and controls were then challenged with a zoonotic and virulent heterologous SwIAV H1N1 (γ-lineage). Pigs vaccinated with CNPs-KAg exhibited an enhanced IgG serum antibody and mucosal secretory IgA antibody responses in nasal swabs, bronchoalveolar lavage (BAL) fluids, and lung lysates that were reactive against homologous (H1N2), heterologous (H1N1), and heterosubtypic (H3N2) influenza A virus strains. Prior to challenge, an increased frequency of cytotoxic T lymphocytes, antigen-specific lymphocyte proliferation, and recall IFN-γ secretion by restimulated peripheral blood mononuclear cells in CNPs-KAg compared to control KAg vaccinates were observed. In CNPs-KAg vaccinated pigs challenged with heterologous virus reduced severity of macroscopic and microscopic influenza-associated pulmonary lesions were observed. Importantly, the infectious SwIAV titers in nasal swabs [days post-challenge (DPC) 4] and BAL fluid (DPC 6) were significantly (p < 0.05) reduced in CNPs-KAg vaccinates but not in KAg vaccinates when compared to the unvaccinated challenge controls. As well, an increased frequency of T helper memory cells and increased levels of recall IFNγ secretion by tracheobronchial lymph nodes cells were observed. In summary, chitosan SwIAV nanovaccine delivered by IN route elicited strong cross-reactive mucosal IgA and cellular immune responses in the respiratory tract that resulted in a reduced nasal viral shedding and lung virus titers in pigs. Thus, chitosan-based influenza nanovaccine may be an ideal candidate vaccine for use in pigs, and pig is a useful animal model for preclinical testing of particulate IN human influenza vaccines.