Gut Microbiota of Obese Children Influences Inflammatory Mucosal Immune Pathways in the Respiratory Tract to Influenza Virus Infection: Optimization of an Ideal Duration of Microbial Colonization in a Gnotobiotic Pig Model.

The impact of obesity on the human microbiota, immune maturation, and influenza virus infection has not been yet established in natural host animal models of influenza. In this study, gnotobiotic (Gn) pigs were colonized with human fecal microbiota (HFM) of obese (oHFM) or healthy lean (hHFM) children and infected at different periods (2-, 3-, and 5-weeks post-transplantation) using a zoonotic influenza virus strain. The infected oHFM pigs were characterized by lower levels of Firmicutes (Lactococcus, Lactobacillus, Turicibacter, and Streptococcus) and Actinobacteria (Bifidobacterium), which was associated with higher levels of Proteobacteria (Klebsiella), Bacteroidetes, and Verrucomicrobia (Akkermansia) compared with the infected hHFM group (P < 0.01). Furthermore, these genera significantly correlated with the expression of immune effectors, immune regulators, and inflammatory mediators, and displayed opposite trends between oHFM and hHFM groups (P < 0.01). The lymphoid and myeloid immune cell frequencies were differently modulated by the oHFM and hHFM colonization, especially apparent in the 5-weeks HFM colonized piglets. In addition, oHFM group had higher pro-inflammatory cytokines (IL-6, IL-12, TNF-α, and IFNγ) gene expression in the respiratory tract compared with the hHFM colonized pigs was detected. In conclusion, pigs colonized for longer duration, established oHFM increased the immune maturation favoring the activation of inflammatory mediators, however, the influenza virus load remained comparable with the hHFM group. Further, a longer duration of microbial colonization (5 weeks) may be required to reveal the impact of microbiome on the host immune maturation and susceptibility to influenza virus infection in the humanized Gn pig model. IMPORTANCE The diversity of gut microbiome of obese people differs markedly from that of lean healthy individuals which, in turn, influences the severity of inflammatory diseases because of differential maturation of immune system. The mouse model provides crucial insights into the mechanism(s) regulating the immune systems mediated by the gut microbiota but its applicability to humans is questionable because immune cells in mice are poorly activated in microbiota humanized mice. Several important strains of Bifidobacterium, Lactobacillus, and Clostridium fails to colonize the murine gut. Thus, understanding the role of certain important commensal gut bacterial species influences upon health and disease, a suitable large animal model like pig that supports the growth and colonization of most of the important human gut bacteria and possess comparable immunology and physiology to humans is beneficial to improve health.

Circulating integrin α4 ß7+ CD4 T cells are enriched for proliferative transcriptional programs in HIV infection.

HIV preferentially infects α4 ß7+ CD4 T cells, forming latent reservoirs that contribute to HIV persistence during antiretroviral therapy. However, the properties of α4 ß7+ CD4 T cells in blood and mucosal compartments remain understudied. Employing two distinct models of HIV infection, HIV-infected humans and simian-human immunodeficiency virus (SHIV)-infected rhesus macaques, we show that α4 ß7+ CD4 T cells in blood are enriched for genes regulating cell cycle progression and cellular metabolism. Unlike their circulating counterparts, rectal α4 ß7+ CD4 T cells exhibited a core tissue-residency gene expression program. These features were conserved across primate species, indicating that the environment influences memory T-cell transcriptional networks. Our findings provide an important molecular foundation for understanding the role of α4 ß7 in HIV infection.

Chitosan-adjuvanted Salmonella subunit nanoparticle vaccine for poultry delivered through drinking water and feed.

Poor induction of mucosal immunity in the intestines by current Salmonella vaccines is a challenge to the poultry industry. We prepared and tested an oral deliverable Salmonella subunit vaccine containing immunogenic outer membrane proteins (OMPs) and flagellin (F) protein loaded and F-protein surface coated chitosan nanoparticles (CS NPs) (OMPs-F-CS NPs). The OMPs-F-CS NPs had mean particle size distribution of 514 nm, high positive charge and spherical in shape. In vitro and in vivo studies revealed the F-protein surface coated CS NPs were specifically targeted to chicken immune cells. The OMPs-F-CS NPs treatment of chicken immune cells upregulated TLRs, and Th1 and Th2 cytokines mRNA expression. Oral delivery of OMPs-F-CS NPs in birds enhanced the specific systemic IgY and mucosal IgA antibodies responses as well as reduced the challenge Salmonella load in the intestines. Thus, user friendly oral deliverable chitosan-based Salmonella vaccine for poultry is a viable alternative to current vaccines.

Oral Deliverable Mucoadhesive Chitosan-Salmonella Subunit Nanovaccine for Layer Chickens.

PURPOSE: Salmonellosis in poultry is a serious economic burden. A major concern is the public health hazard caused by consumption of Salmonella-contaminated poultry products. Currently used Salmonella vaccines are ineffective in combating poultry Salmonellosis warranting the need of a potent vaccine, especially an oral vaccine that can elicit robust local intestinal immunity. MATERIALS AND METHODS: A Salmonella subunit chitosan nanoparticles (NPs)-based vaccine was prepared that contained immunogenic outer membrane proteins (OMPs) and -flagellin (F) protein (OMPs-F-CS NPs). OMPs-F-CS NPs were administered as an oral vaccine in layer chickens and the resultant humoral and cell-mediated immune responses and localization of NPs were examined using standard detection methods. RESULTS: We demonstrated targeting of surface F-protein coated chitosan NPs to immune cells when delivered orally to layer chickens, the particles were localized in ileal Peyer's patches. The OMPs-F-CS NPs vaccinated layer chickens had significantly higher OMPs-specific mucosal IgA production and lymphocyte proliferation response. The candidate vaccine increased the expression of toll-like receptor (TLR)-2, TLR-4, IFN-γ, TGF-ß and IL-4 mRNA expression in chicken cecal tonsils. CONCLUSION: Our study demonstrated that the chitosan-based oral Salmonella nanovaccine targets immune cells of chickens and induced antigen-specific B and T cell responses. This candidate oral Salmonella nanovaccine has the potential to mitigate Salmonellosis in poultry.

Surface engineered polyanhydride-based oral Salmonella subunit nanovaccine for poultry.

PURPOSE: Salmonellosis is a severe economic threat in poultry and a public health concern. Currently available vaccines are ineffective, and thus, developing effective oral Salmonella vaccine is warranted. Especially, a potent oral vaccine such as the mucoadhesive polyanhydride nanoparticle (PNP) protects the vaccine cargo and delivers to intestinal immune sites to elicit robust mucosal immunity and mitigate Salmonella colonization and shedding. MATERIALS AND METHODS: We designed a Salmonella subunit vaccine using PNP containing immunogenic Salmonella outer membrane proteins (OMPs) and flagellar (F) protein-entrapped and surface F-protein-coated PNPs (OMPs-F-PNPs) using a solvent displacement method. Using high-throughput techniques, we characterized the OMPs-F-PNPs physicochemical properties and analyzed its efficacy in layer birds vaccinated orally. RESULTS: The candidate vaccine was resistant in acidic microenvironment and had ideal physicochemical properties for oral delivery in terms of particle size, charge, morphology, biocompatibility, and pH stability. In vitro, in vivo, and ex vivo studies showed that F-protein surface-anchored nanoparticles were better targeted to chicken immune cells in peripheral blood and splenocytes and intestinal Peyer's patch sites. In layer chickens inoculated orally with OMPs-F-PNPs, substantially higher OMPs-specific IgG response and secretion of Th1 cytokine IFN-γ in the serum, enhanced CD8+/CD4+ cell ratio in spleen, and increased OMPs-specific lymphocyte proliferation were observed. OMPs-F-PNPs vaccination also upregulated the expression of toll-like receptor (TLR)-2 and -4, TGF-ß, and IL-4 cytokines' genes in chicken cecal tonsils (lymphoid tissues). Importantly, OMPs-F-PNPs vaccine cleared Salmonella cecal colonization in 33% of vaccinated birds. CONCLUSION: This pilot in vivo study demonstrated the targeted delivery of OMPs-F-PNPs to ileum mucosal immune sites of chickens and induced specific immune response to mitigate Salmonella colonization in intestines.

Intranasal delivery of influenza antigen by nanoparticles, but not NKT-cell adjuvant differentially induces the expression of B-cell activation factors in mice and swine.

Intranasal vaccination of pigs with poly lactic-co-glycolic acid and polyanhydride nanoparticles delivered inactivated influenza virus provides cross-reactive T-cell response, but not antibody response, resulting in incomplete protection and no reduction in nasal virus shedding. Expression of BAFF and Th2 transcription factor GATA-3 were downregulated in lungs of pigs vaccinated with influenza nanovaccine, but in mice it upregulated the expression of BAFF and cytokine TGFß in cervical lymph nodes. However, the intranasal iNKT cell adjuvant, α-Galctosylceramide upregulates the expression of BAFF in pig lungs. In conclusion, expression of BAFF is differentially regulated by intranasal nanovaccine and α-Galctosylceramide in pig respiratory tract.

Polyanhydride nanovaccine against swine influenza virus in pigs.

We have recently demonstrated the effectiveness of an influenza A virus (IAV) subunit vaccine based on biodegradable polyanhydride nanoparticles delivery in mice. In the present study, we evaluated the efficacy of ∼200nm polyanhydride nanoparticles encapsulating inactivated swine influenza A virus (SwIAV) as a vaccine to induce protective immunity against a heterologous IAV challenge in pigs. Nursery pigs were vaccinated intranasally twice with inactivated SwIAV H1N2 (KAg) or polyanhydride nanoparticle-encapsulated KAg (KAg nanovaccine), and efficacy was evaluated against a heterologous zoonotic virulent SwIAV H1N1 challenge. Pigs were monitored for fever daily. Local and systemic antibody responses, antigen-specific proliferation of peripheral blood mononuclear cells, gross and microscopic lung lesions, and virus load in the respiratory tract were compared among the groups of animals. Our pre-challenge results indicated that KAg nanovaccine induced virus-specific lymphocyte proliferation and increased the frequency of CD4+CD8αα+ T helper and CD8+ cytotoxic T cells in peripheral blood mononuclear cells. KAg nanovaccine-immunized pigs were protected from fever following SwIAV challenge. In addition, pigs immunized with the KAg nanovaccine presented with lower viral antigens in lung sections and had 6 to 8-fold reduction in nasal shedding of SwIAV four days post-challenge compared to control animals. Immunologically, increased IFN-γ secreting T lymphocyte populations against both the vaccine and challenge viruses were detected in KAg nanovaccine-immunized pigs compared to the animals immunized with KAg alone. However, in the KAg nanovaccine-immunized pigs, hemagglutination inhibition, IgG and IgA antibody responses, and virus neutralization titers were comparable to that in the animals immunized with KAg alone. Overall, our data indicated that intranasal delivery of polyanhydride-based SwIAV nanovaccine augmented antigen-specific cellular immune response in pigs, with promise to induce cross-protective immunity.

Biodegradable nanoparticle delivery of inactivated swine influenza virus vaccine provides heterologous cell-mediated immune response in pigs.

Swine influenza virus (SwIV) is one of the important zoonotic pathogens. Current flu vaccines have failed to provide cross-protection against evolving viruses in the field. Poly(lactic-co-glycolic acid) (PLGA) is a biodegradable FDA approved polymer and widely used in drug and vaccine delivery. In this study, inactivated SwIV H1N2 antigens (KAg) encapsulated in PLGA nanoparticles (PLGA-KAg) were prepared, which were spherical in shape with 200 to 300nm diameter, and induced maturation of antigen presenting cells in vitro. Pigs vaccinated twice with PLGA-KAg via intranasal route showed increased antigen specific lymphocyte proliferation and enhanced the frequency of T-helper/memory and cytotoxic T cells (CTLs) in peripheral blood mononuclear cells (PBMCs). In PLGA-KAg vaccinated and heterologous SwIV H1N1 challenged pigs, clinical flu symptoms were absent, while the control pigs had fever for four days. Grossly and microscopically, reduced lung pathology and viral antigenic mass in the lung sections with clearance of infectious challenge virus in most of the PLGA-KAg vaccinated pig lung airways were observed. Immunologically, PLGA-KAg vaccine irrespective of not significantly boosting the mucosal antibody response, it augmented the frequency of IFN-γ secreting total T cells, T-helper and CTLs against both H1N2 and H1N1 SwIV. In summary, inactivated influenza virus delivered through PLGA-NPs reduced the clinical disease and induced cross-protective cell-mediated immune response in a pig model. Our data confirmed the utility of a pig model for intranasal particulate flu vaccine delivery platform to control flu in humans.

Evaluation of humoral immune status in porcine epidemic diarrhea virus (PEDV) infected sows under field conditions.

Porcine epidemic diarrhea virus (PEDV) is an economically devastating enteric disease in the swine industry. The virus infects pigs of all ages, but it cause severe clinical disease in neonatal suckling pigs with up to 100% mortality. Currently, available vaccines are not completely effective and feedback methods utilizing PEDV infected material has variable success in preventing reinfection. Comprehensive information on the levels and duration of effector/memory IgA and IgG antibody secreting B cell response in the intestines and lymphoid organs of PEDV-infected sows, and their association with specific antibody levels in clinical samples such as plasma, oral fluid, and feces is important. Therefore, our goal in this study was to quantify PEDV specific IgA and IgG B cell responses in sows at approximately 1 and 6 months post-infection in commercial swine herds, including parity one and higher sows. Our data indicated that evaluation of both PEDV specific IgA and IgG antibody levels in the plasma and oral fluid (but not feces) samples is beneficial in disease diagnosis. PEDV specific B cell response in the intestine and spleen of infected sows decline by 6 months, and this associates with specific antibody levels in the plasma and oral fluid samples; but the virus neutralization titers in plasma remains high beyond 6 months post-infection. In conclusion, in sows infected with PEDV the presence of effector/memory B cell response and strong virus neutralization titers in plasma up to 6 months post-infection, suggests their potential to protect sows from reinfection and provide maternal immunity to neonates, but challenge studies are required to confirm such responses.