Pulmonary inflammatory response to influenza virus infection in pigs is regulated by DAP12 and macrophage M1 and M2 phenotypes.

We delineated the expression of DAP12 (DNAX-Activating Protein) and its associated receptors, TREM-1, TREM-2 and MDL-1 in pig alveolar monocyte/macrophages (AMM) that have attained M1 or M2 phenotypes. Pig AMM stimulated in vitro with IFN-γ and IL-4 induced the expression of M1 (TNFα and iNOS) and M2 (ARG1 and no MMR) phenotypic markers, respectively. In influenza virus infected pigs at seven days post-infection, in addition to substantial modulations in the M1 and M2 markers expression, DAP12, TREM-1 and MDL-1 were downregulated in AMM. Thus, DAP12 signaling promoted the anti-inflammatory pathway in AMM of influenza virus infected pigs.

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.

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.

Chitosan-nanoparticle-based oral Salmonella enteritidis subunit vaccine elicits cross-protection against Salmonella typhimurium in broilers.

Non-typhoidal Salmonella infection is a significant health and economic burden in poultry industry. Developing an oral vaccine to induce robust mucosal immunity in the intestines of birds, especially cross protection against different Salmonella serotypes is challenging. Therefore, a potent oral vaccine platform that can mitigate different serotypes of Salmonella is warranted for the poultry industry. We reported earlier that the Salmonella enteritidis (SE) immunogenic outer membrane proteins (OMPs) and flagellin (FLA) entrapped in mannose chitosan nanoparticles (OMPs-FLA-mCS NPs) administered prime-boost (d-3 and 3-wk later) by oral inoculation elicits mucosal immunity and reduces challenge SE colonization by over 1 log10 CFU in birds. In this study, we sought to evaluate whether the SE antigens containing OMPs-FLA-mCS NPs vaccine induces cross-protection against Salmonella typhimurium (ST) in broilers. Our data indicated that the OMPs-FLA-mCS NPs vaccine induced higher cross-protective antibody responses compared to commercial Poulvac ST vaccine (contains a modified-live ST bacterium). Particularly, OMPs-FLA-mCS-NP vaccine elicited OMPs and FLA antigens specific increased production of secretory IgA and IgY antibodies in samples collected at both post-vaccination and post-challenge timepoints compared to commercial vaccine group. Notably, the vaccine reduced the challenge ST bacterial load by 0.8 log10 CFU in the cecal content, which was comparable to the outcome of Poulvac ST vaccination. In conclusion, our data suggested that orally administered OMPs-FLA-mCS-NP SE vaccine elicited cross protective mucosal immune responses against ST colonization in broilers. Thus, this candidate vaccine could be a viable option replacing the existing both live and killed Salmonella vaccines for birds.

Use of capillary-mediated vitrification to produce thermostable, single-use antibody conjugates as immunoassay reagents.

The performance of enzyme-linked immunoassays is directly dependent on the storage, handling, and long-term stability of the critical reagents used in the assay. Currently, antibody reagents are routinely stored as concentrated, multi-use, frozen aliquots. This practice results in material waste, adds complexity to laboratory workflows, and can compromise reagents via cross-contamination and freeze-thaw damage. While refrigeration or freezing can slow down many degradation processes, the freezing process itself can have damaging effects, including introduction of aggregation and microheterogeneity. To address these challenges, we evaluated the application of capillary-mediated vitrification (CMV) as a tool for storing antibody reagents in a thermostable, single-use format. CMV is a novel biopreservation method that enables vitrification of biological materials without freezing. Using an anti-human IgG-alkaline phosphatase conjugate as a model system, we prepared CMV-stabilized aliquots which were stored in a single-use format at temperatures ranging from 25 to 55 °C for up to 3 months. Each stabilized aliquot contained enough antibody to perform a single assay run. We evaluated the assay performance and functional stability of the CMV-stabilized reagents using a plate-based ELISA. Assays run using the CMV stabilized reagents exhibited good linearity and precision that was comparable to results obtained with a frozen control. Throughout the stability study, the maximum signal and EC50s observed for ELISAs run using CMV-stabilized reagents were generally consistent with those obtained using a frozen control. These results indicate that the CMV process has the potential to improve both reagent stability and long-term assay performance, while also reducing reagent waste and simplifying assay workflows.

An intranasal vaccine comprising SARS-CoV-2 spike receptor-binding domain protein entrapped in mannose-conjugated chitosan nanoparticle provides protection in hamsters.

We developed a novel intranasal SARS-CoV-2 subunit vaccine called NARUVAX-C19/Nano based on the spike protein receptor-binding domain (RBD) entrapped in mannose-conjugated chitosan nanoparticles (NP). A toll-like receptor 9 agonist, CpG55.2, was also added as an adjuvant to see if this would potentiate the cellular immune response to the NP vaccine. The NP vaccine was assessed for immunogenicity, protective efficacy, and ability to prevent virus transmission from vaccinated animals to naive cage-mates. The results were compared with a RBD protein vaccine mixed with alum adjuvant and administered intramuscularly. BALB/c mice vaccinated twice intranasally with the NP vaccines exhibited secretory IgA and a pronounced Th1-cell response, not seen with the intramuscular alum-adjuvanted RBD vaccine. NP vaccines protected Syrian hamsters against a wild-type SARS-CoV-2 infection challenge as indicated by significant reductions in weight loss, lung viral load and lung pathology. However, despite significantly reduced viral load in the nasal turbinates and oropharyngeal swabs from NP-vaccinated hamsters, virus transmission was not prevented to naïve cage-mates. In conclusion, intranasal RBD-based NP formulations induced mucosal and Th1-cell mediated immune responses in mice and protected Syrian hamsters against SARS-CoV-2 infection but not against viral transmission.

Capillary-Mediated Vitrification: Preservation of mRNA at Elevated Temperatures.

RNA is a fundamental tool for molecular and cellular biology research. The recent COVID-19 pandemic has proved it is also invaluable in vaccine development. However, the need for cold storage to maintain RNA integrity and the practical and economic burden associated with cold chain logistics highlight the need for new and improved preservation methods. We recently showed the use of capillary-mediated vitrification (CMV), as a tool for stabilizing temperature-sensitive enzymes. Here, we demonstrate the use of CMV as a method to preserve mRNA. The CMV process was performed by formulating a green fluorescent protein (GFP)-encoding mRNA with common excipients, applying the solution to a porous support, referred to as the scaffold, and drying the samples under vacuum for 30 min. The CMV preserved samples were stored at 55 °C for up to 100 days or 25 °C for 60 days and analyzed by electrophoresis and for transfection efficiency in a cell-based assay. The 55 °C-stressed mRNA exhibited comparable electrophoresis banding patterns and band intensity when compared to a frozen, liquid control. Additionally, the CMV stabilized mRNA maintained 97.5 ± 8.7% transfection efficiency after 77 days and 78.4 ± 3.9% after 100 days when stored 55 °C and analyzed using a cell-based assay in the CHO-K1 cell line. In contrast, a liquid control exhibited no bands on the electrophoresis gel and lost all transfection activity after being stored overnight at 55 °C. Likewise, after 60 days at 25 °C, the CMV-processed samples had full transfection activity while the activity of the liquid control was reduced to 40.1 ± 4.6%. In conclusion, CMV is a simple formulation method that significantly enhances the thermal stability of mRNA, requires minimal processing time, and could enable formulation of mRNA that can tolerate exposure to temperatures well above 25 °C during shipment and deployment in extreme environments.

Capillary-Mediated Vitrification: A Novel Approach for Improving Thermal Stability of Enzymes and Proteins.

Capillary-mediated vitrification (CMV) is a novel method for stabilizing biological molecules and complexes. CMV leverages capillary evaporation to enable rapid desiccation of aqueous solutions while avoiding both freezing and boiling. In the CMV process, an aqueous solution containing the biological material of interest and common excipients is applied to a solid, porous support, referred to as the scaffold, and desiccated under vacuum. The pores within the scaffold accelerate drying by increasing surface area while preventing boiling through the interaction of the vapor pressure, capillary forces, and viscous forces. The process, which can be completed in under an hour, produces an amorphous dried product with enhanced thermal stability. In this study, CMV is demonstrated using luciferase as a model system. Using a 30-minute drying time, residual moisture levels of <4% were achieved. CMV-stabilized luciferase maintained full activity when stored for up to 6 weeks at 25 °C and >70% activity after 6 weeks at temperatures up to 45 °C. The liquid formulated enzyme lost all activity after 1 day at 37 °C or 4 h at temperatures above 37 °C. The data presented in this report demonstrate that CMV is a promising alternative to traditional biopreservation methods.

Evaluation of a Novel Adjuvanted Vaccine for Ultrashort Regimen Therapy of Artemisia Pollen-Induced Allergic Bronchial Asthma in a Mouse Model.

Wormwood (Artemisia) pollen is among the top 10 aeroallergens globally that cause allergic rhinitis and bronchial asthma. Allergen-specific immunotherapy (ASIT) is the gold standard for treating patients with allergic rhinitis, conjunctivitis, and asthma. A significant disadvantage of today's ASIT methods is the long duration of therapy and multiplicity of allergen administrations. The goal of this study was to undertake a pilot study in mice of a novel ultrashort vaccine immunotherapy regimen incorporating various adjuvants to assess its ability to treat allergic bronchial asthma caused by wormwood pollen. We evaluated in a mouse model of wormwood pollen allergy candidates comprising recombinant Art v 1 wormwood pollen protein formulated with either newer (Advax, Advax-CpG, ISA-51) or more traditional [aluminum hydroxide, squalene water emulsion (SWE)] adjuvants administered by the intramuscular or subcutaneous route vs. intranasal administration of a mucosal vaccine formulation using chitosan-mannose nanoparticle entrapped with Art v 1 protein. The vaccine formulations were administered to previously wormwood pollen-sensitized animals, four times at weekly intervals. Desensitization was determined by measuring decreases in immunoglobulin E (IgE), cellular immunity, ear swelling test, and pathological changes in the lungs of animals after aeroallergen challenge. Art v 1 protein formulation with Advax, Advax-CpG, SWE, or ISA-51 adjuvants induced a significant decrease in both total and Art v 1-specific IgE with a concurrent increase in Art v 1-specific IgG compared to the positive control group. There was a shift in T-cell cytokine secretion toward a Th1 (Advax-CpG, ISA-51, and Advax) or a balanced Th1/Th2 (SWE) pattern. Protection against lung inflammatory reaction after challenge was seen with ISA-51, Advax, and SWE Art v 1 formulations. Overall, the ISA-51-adjuvanted vaccine group induced the largest reduction of allergic ear swelling and protection against type 2 and non-type 2 lung inflammation in challenged animals. This pilot study shows the potential to develop an ultrashort ASIT regimen for wormwood pollen-induced bronchial asthma using appropriately adjuvanted recombinant Art v 1 protein. The data support further preclinical studies with the ultimate goal of advancing this therapy to human clinical trials.

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.

Efficacy of a nanoparticle vaccine administered in-ovo against Salmonella in broilers.

Salmonella is a zoonotic pathogen that persists in poultry. Salmonella vaccines that can be delivered in-ovo can be cost-effective and can decrease Salmonella load in poultry. This study evaluates the efficacy of a Salmonella chitosan-nanoparticle (CNP) vaccine, administered in-ovo, in broilers. CNP vaccine was synthesized with Salmonella Enteritidis (SE) outer-membrane-proteins (OMPs) and flagellin proteins. At embryonic-d18, one-hundred-thirty-six eggs were injected with 200µl PBS or 1000µg CNP into the amniotic cavity. At d1-of-age, 132 chicks were allocated in 6 pens/treatment with 11 chicks/pen. At d7, birds were orally challenged with 1×109 CFU/bird SE. At d1, 8h-post-challenge, d14, and d21, serum anti-SE-OMPs IgY were analyzed. At d14 and d21, cloacal swabs and bile anti-SE-OMPs IgA, CD4+/CD8+-T-cell ratios, and ceca SE loads were analyzed. At d21, cecal tonsil IL-1ß, IL-10, and iNOS mRNA were analyzed. Body-weight-gain (BWG) and feed-conversion-ratio (FCR) were recorded weekly. Data were analyzed by Student's t-test at P<0.05. There were no significant differences in BWG or FCR between vaccinated birds compared to control. At d1, CNP-vaccinated birds had 5.62% greater levels (P<0.05) of anti-SE-OMPs IgY, compared to control. At 8h-post-challenge, CNP-vaccinated birds had 6.39% greater levels (P<0.05) of anti-SE-OMPs IgY, compared to control. At 2wk-post-challenge, CNP-vaccinated birds had 7.34% lower levels (P<0.05) of anti-SE-OMPs IgY, compared to control. At 1wk-post-challenge, CNP-vaccinated birds had 15.30% greater levels (P<0.05) of bile anti-SE-OMPs IgA, compared to control. At d14 and d21, CNP-vaccinated birds had 0.62 and 0.85 Log10 CFU/g, decreased SE ceca load (P<0.05), respectively, compared to control. There were no significant differences in CD4+/CD8+-T-cell ratios between vaccinated birds compared to control. There were no significant differences in IL-1ß, IL-10, iNOS mRNA between vaccinated birds compared to control. Findings demonstrate that the in-ovo administration of CNP vaccine can induce an antigen-specific immune response against SE and can decrease SE cecal load in broilers.

Immunity and Protective Efficacy of Mannose Conjugated Chitosan-Based Influenza Nanovaccine in Maternal Antibody Positive Pigs.

Parenteral administration of killed/inactivated swine influenza A virus (SwIAV) vaccine in weaned piglets provides variable levels of immunity due to the presence of preexisting virus specific maternal derived antibodies (MDA). To overcome the effect of MDA on SwIAV vaccine in piglets, we developed an intranasal deliverable killed SwIAV antigen (KAg) encapsulated chitosan nanoparticles called chitosan-based NPs encapsulating KAg (CS NPs-KAg) vaccine. Further, to target the candidate vaccine to dendritic cells and macrophages which express mannose receptor, we conjugated mannose to chitosan (mCS) and formulated KAg encapsulated mCS nanoparticles called mannosylated chitosan-based NPs encapsulating KAg (mCS NPs-KAg) vaccine. In MDA-positive piglets, prime-boost intranasal inoculation of mCS NPs-KAg vaccine elicited enhanced homologous (H1N2-OH10), heterologous (H1N1-OH7), and heterosubtypic (H3N2-OH4) influenza virus-specific secretory IgA (sIgA) antibody response in nasal passage compared to CS NPs-KAg vaccinates. In vaccinated upon challenged with a heterologous SwIAV H1N1, both mCS NPs-KAg and CS NPs-KAg vaccinates augmented H1N2-OH10, H1N1-OH7, and H3N2-OH4 virus-specific sIgA antibody responses in nasal swab, lung lysate, and bronchoalveolar lavage (BAL) fluid; and IgG antibody levels in lung lysate and BAL fluid samples. Whereas, the multivalent commercial inactivated SwIAV vaccine delivered intramuscularly increased serum IgG antibody response. In mCS NPs-KAg and CS NPs-KAg vaccinates increased H1N2-OH10 but not H1N1-OH7 and H3N2-OH4-specific serum hemagglutination inhibition titers were observed. Additionally, mCS NPs-KAg vaccine increased specific recall lymphocyte proliferation and cytokines IL-4, IL-10, and IFNγ gene expression compared to CS NPs-KAg and commercial SwIAV vaccinates in tracheobronchial lymph nodes. Consistent with the immune response both mCS NPs-KAg and CS NPs-KAg vaccinates cleared the challenge H1N1-OH7 virus load in upper and lower respiratory tract more efficiently when compared to commercial vaccine. The virus clearance was associated with reduced gross lung lesions. Overall, mCS NP-KAg vaccine intranasal immunization in MDA-positive pigs induced a robust cross-reactive immunity and offered protection against influenza virus.

A Novel Approach against Salmonella: A Review of Polymeric Nanoparticle Vaccines for Broilers and Layers.

This work discusses the present-day limitations of current commercial Salmonella vaccines for broilers and layers and explores a novel approach towards poultry vaccination using biodegradable nanoparticle vaccines against Salmonella. With the increasing global population and poultry production and consumption, Salmonella is a potential health risk for humans. The oral administration of killed or inactivated vaccines would provide a better alternative to the currently commercially available Salmonella vaccines for poultry. However, there are currently no commercial oral killed-vaccines against Salmonella for use in broilers or layers. There is a need for novel and effective interventions in the poultry industry. Polymeric nanoparticles could give way to an effective mass-administered mucosal vaccination method for Salmonella. The scope of this work is limited to polymeric nanoparticles against Salmonella for use in broilers and layers. This review is based on the information available at the time of the investigation.

Salmonella chitosan nanoparticle vaccine administration is protective against Salmonella Enteritidis in broiler birds.

Salmonella control strategies include vaccines that help reduce the spread of Salmonella in poultry flocks. In this study we evaluated the efficacy of administering a live Salmonella vaccine followed by a killed Salmonella chitosan nanoparticle (CNP) vaccine booster on the cellular and humoral immunity of broilers. The CNP vaccine was synthesized with Salmonella Enteritidis (S. Enteritidis) outer-membrane-proteins (OMPs) and flagellin-proteins. At d1-of-age, one-hundred-sixty-eight chicks were allocated into treatments: 1) No vaccine, 2) Live vaccine (Poulvac®ST), 3) CNP vaccine, 4) Live+CNP vaccine. At d1-of-age, birds were orally vaccinated with PBS, Live vaccine, or CNP. At d7-of-age, the No vaccine, Live vaccine and CNP vaccine groups were boosted with PBS and the Live+CNP vaccine group was boosted with CNP. At d14-of-age, birds were challenged with 1×109 CFU/bird S. Enteritidis. There were no significant differences in body-weight-gain (BWG) or feed-conversion-ratio (FCR). At 8h-post-challenge, CNP and Live+CNP-vaccinated birds had 17% and 24% greater levels (P<0.05) of anti-Salmonella OMPs IgA in bile, respectively, compared to control. At d28-of-age, CNP, Live, and Live+CNP-vaccinated birds had 33%, 18%, and 24% greater levels (P<0.05) of anti-Salmonella OMPs IgA in bile, respectively, compared to control. At d14-of-age, Live+CNP-vaccinated birds had 46% greater levels (P<0.05) of anti-Salmonella OMPs IgY in serum, compared to control. At d21-of-age, splenocytes from CNP and Live-vaccinated birds had increased (P<0.05) T-lymphocyte proliferation at 0.02 mg/mL OMPs stimulation compared to the control. At d28-of-age, CNP and Live+CNP-vaccinated birds had 0.9 Log10 CFU/g and 1 Log10 CFU/g decreased S. Enteritidis cecal loads (P<0.05), respectively, compared to control. The CNP vaccine does not have adverse effects on bird's BWG and FCR or IL-1ß, IL-10, IFN-γ, or iNOS mRNA expression levels. It can be concluded that the CNP vaccine, as a first dose or as a booster vaccination, is an alternative vaccine candidate against S. Enteritidis in broilers.

Chitosan Nanoparticle Based Mucosal Vaccines Delivered Against Infectious Diseases of Poultry and Pigs.

Infectious disease of poultry and pig are major threat to health and cause severe economic loss to the food industry and a global food safety issue. Poultry and pig act as a mixing vessel of zoonotic transmission of disease to humans. Effective mucosal vaccines used in animals could reduce the impact of diseases in food animals. Chitosan is a biocompatible polymer, and its positive charge makes it a natural mucoadhesive agent. Therefore, since last one-decade chitosan derived nanoparticles (CS NPs) have been in use widely to deliver vaccine antigens in animals through mucosal route. Primary route of entry of most infectious disease pathogen is through oral and nasal routes, and the CS NPs based vaccines delivered through that routes enhance the immunogenicity of encapsulated vaccine antigens by targeting the cargo to mucosal microfold cells, dendritic cells and macrophages. Resulting in induction of robust secretory and systemic antibodies and/or cell mediated immune response which provides protection against infections. To date, CS NPs is being widely used for mucosal vaccine delivery in poultry and pigs to control bacterial and viral infections, and tested in several preclinical trials for vaccine delivery in humans. In this review, we highlighted the progress so far made in using CS NPs as a vehicle for mucosal vaccine delivery against infectious and zoonotic diseases of poultry and pigs. Discussed about the need of CS NPs modifications, CS NPs based vaccines induced immune responses and its role in protection, and challenges in vaccination and future directions.

Incorporated plant extract fabricated silver/poly-D,l-lactide-co-glycolide nanocomposites for antimicrobial based wound healing.

Polymeric nanocomposites have gained extensive attention in modern nanotechnology by reason of its design, flexibility, sole applications and lower life cycle costs. Preparation of composites using spreading of inorganic metal nanoparticles in organic polymeric matrices has plenty of scope and applications in the biomedical field. Poly-D,l-lactide-co-glycolide (PLGA) is an appreciated polymer for composites preparation because of its non-toxic and promising biodistribution. The consideration of metal nanoparticles has extended rapidly with the presence of new nanocomposites into a range of products and technologies. Compared to bulk materials the synthesized metal nanoparticles have unique character and biomedical uses due to its shape, size, and huge surface to volume ratio. Among different inorganic metal nanoparticles, silver nanoparticles (Ag NPs) have dominated in the biomedical field owing to its diverse potential applications including imaging, sensor, diagnosis and disease treatment. Further, medicinal plant extract mediated Ag NPs shown superior advantages and its antimicrobial based wound healing prospective has been established. However, not much information on plant extract mediated Ag NPs integrated PLGA nanocomposites wound healing applications. In the present review, we discussed necessity, preparation, characterization and antimicrobial based wound healing mechanism of incorporated plant extract mediated silver/PLGA nanocomposites.

In vitro characterization and immunogenicity of chitosan nanoparticles loaded with native and inactivated extracellular proteins from a field strain of Clostridium perfringens associated with necrotic enteritis.

There are currently no licensed vaccines against Clostridium perfringens which causes necrotic enteritis in poultry. Chitosan nanoparticles were formulated with native (CN) or toxoids (CT) of extracellular proteins (ECP) of C. perfringens, both surface-tagged with Salmonella flagellar proteins. In a pH stability assay, CN and CT nanoparticles released 6% and 0% of their protein at 8.0 pH. In a protein release assay, CN and CT nanoparticles released 16% and 10% of their protein respectively at 7.4 pH after 24 h. CN and CT nanoparticles incubated at 100 µg/mL PBS with Chicken RBCs released 1% and 0% hemoglobin respectively. Ninety broilers were randomly assigned to treatments; sham-vaccinated (Control), CN-vaccinated (CN), and CT-vaccinated (CT). Each bird was orally gavaged with 50 µg vaccine in 0.5 mL PBS or 0.5 mL PBS only on d 0, 3, 7 and 14 of age. At 21 d of age, the CN group had higher anti-ECP IgA than control (P < 0.05). At 21 d of age, the CN and CT group had higher anti-ECP IgA than control (P < 0.05). At 17 d of age, the CN group had higher anti-flagellar IgG than control (P < 0.05). At 10 d of age, the CN group had higher anti-flagellar IgA than control (P < 0.05). Splenic T cells from chickens in the CN and CT group ex-vivo stimulated with 0.05 mg/mL ECP, had higher proliferation control (P < 0.05, P < 0.01 respectively). Splenic T cells from chickens in the CN and CT groups ex-vivo stimulated with 0.1 mg/mL ECP had proliferation than control (P < 0.05). Pooled serum from 17 d of age CN and CT-vaccinated birds partially neutralized toxins in 50 µg of ECP (P < 0.05). Pooled serum from 28 d of age CN-vaccinated birds also partially neutralized toxins in 50 µg of ECP. The result from this study indicates the potential for chitosan loaded with Clostridium perfringens extracellular proteins to be applied to necrotic enteritis challenge studies.

Intranasal Delivery of Inactivated Influenza Virus and Poly(I:C) Adsorbed Corn-Based Nanoparticle Vaccine Elicited Robust Antigen-Specific Cell-Mediated Immune Responses in Maternal Antibody Positive Nursery Pigs.

We designed the killed swine influenza A virus (SwIAV) H1N2 antigen (KAg) with polyriboinosinic:polyribocytidylic acid [(Poly(I:C)] adsorbed corn-derived Nano-11 particle based nanovaccine called Nano-11-KAg+Poly(I:C), and evaluated its immune correlates in maternally derived antibody (MDA)-positive pigs against a heterologous H1N1 SwIAV infection. Immunologically, in tracheobronchial lymph nodes (TBLN) detected enhanced H1N2-specific cytotoxic T-lymphocytes (CTLs) in Nano-11-KAg+Poly(I:C) vaccinates, and in commercial vaccinates detected CTLs with mainly IL-17A+ and early effector phenotypes specific to both H1N2 and H1N1 SwAIV. In commercial vaccinates, activated H1N2- and H1N1-specific IFNγ+&TNFα+, IL-17A+ and central memory T-helper/Memory cells, and in Nano-11-KAg+Poly(I:C) vaccinates H1N2-specific central memory, IFNγ+ and IFNγ+&TNFα+, and H1N1-specific IL-17A+ T-helper/Memory cells were observed. Systemically, Nano-11-KAg+Poly(I:C) vaccine augmented H1N2-specific IFNγ+ CTLs and H1N1-specific IFNγ+ T-helper/Memory cells, and commercial vaccine boosted H1N2- specific early effector CTLs and H1N1-specific IFNγ+&TNFα+ CTLs, as well as H1N2- and H1N1-specific T-helper/Memory cells with central memory, IFNγ+&TNFα+, and IL-17A+ phenotypes. Remarkably, commercial vaccine induced an increase in H1N1-specific T-helper cells in TBLN and naive T-helper cells in both TBLN and peripheral blood mononuclear cells (PBMCs), while H1N1- and H1N2-specific only T-helper cells were augmented in Nano-11-KAg+Poly(I:C) vaccinates in both TBLN and PBMCs. Furthermore, the Nano-11-KAg+Poly(I:C) vaccine stimulated robust cross-reactive IgG and secretory IgA (SIgA) responses in lungs, while the commercial vaccine elicited high levels of serum and lung IgG and serum hemagglutination inhibition (HI) titers. In conclusion, despite vast genetic difference (77% in HA gene identity) between the vaccine H1N2 and H1N1 challenge viruses in Nano-11-KAg+Poly(I:C) vaccinates, compared to over 95% identity between H1N1 of commercial vaccine and challenge viruses, the virus load and macroscopic lesions in the lungs of both types of vaccinates were comparable, but the Nano-11-KAg+Poly(I:C) vaccine cleared the virus from the nasal passage better. These data suggested the important role played by Nano-11 and Poly(I:C) in the induction of polyfunctional, cross-protective cell-mediated immunity against SwIAV in MDA-positive pigs.

Efficacy of chitosan-based nanoparticle vaccine administered to broiler birds challenged with Salmonella.

Two experiments were conducted to evaluate the immune response of broilers vaccinated with Salmonella chitosan-nanoparticle (CNP) vaccine and challenged with Salmonella. The Salmonella CNP vaccine was synthesized with Salmonella enterica outer membrane proteins (OMPs) and flagellin proteins. In Experiment I, birds were orally gavaged with PBS or 500, 1000, or 2000µg of CNP vaccine 1 and 7d-of-age. At 14d-of-age, birds were orally challenged with 1 X 105 CFU/bird of live S. Enteritidis (SE). Macrophage-nitrite production 11d-post-challenge was higher (P<0.05) in the 500µg group when compared to the control. At d14 (8h-post-challenge), broilers vaccinated with 1000µg CNP had higher (P<0.05) serum anti-OMPs IgG and IgA and cloacal anti-OMP IgA amounts. At 11d-post-challenge, birds vaccinated with 1000µg CNP vaccine had greater (P<0.05) bile anti-OMP and anti-flagellin IgA amounts. At 11d-post-challenge, birds administered 1000µg CNP vaccine has increased (P<0.05) IL-1ß and IL-10 mRNA in cecal tonsils. In Experiment II, birds were orally gavaged with PBS or 1000µg CNP or a live commercial vaccine at 1 and 7d-of-age. At 14d-of-age, birds were orally challenged with 1 X 105 CFU/bird of live SE or S. Heidelberg (SH). Birds vaccinated with CNP showed higher (P<0.05) serum anti-OMPs IgG amounts at 8h-post-challenge. At 4d-post-SH challenge, birds vaccinated with CNP had higher (P<0.05) bile anti-flagellin IgA amounts. CNP decreased (P<0.05) anti-OMPs IgG levels in serum at 2d-post-SE challenge and 4d-post-SH or SE challenge. Salmonella Enteritidis loads in cecal content at 2d-post-challenge was decreased (P<0.05) by 65.9% in birds vaccinated with CNP, when compared to the control. Chitosan-nanovaccine had no adverse effects on bird's production performance. In conclusion, 1000µg CNP vaccine can induce a specific immune response against Salmonella and has the potential to mitigate SE cecal colonization in broiler birds.

Poly(I:C) augments inactivated influenza virus-chitosan nanovaccine induced cell mediated immune response in pigs vaccinated intranasally.

To improve the innate and adaptive immune responses elicited by a killed/inactivated swine influenza virus antigen (KAg)-loaded chitosan nanoparticles (CS NPs-KAg), we used the adjuvant, poly(I:C). The formulated CS NPs-KAg and CS NPs-poly(I:C) had a net surface charge of +30.7 mV and +25.1 mV, respectively. The CS NPs-KAg was coadministered with CS NPs-poly(I:C) (chitosan nanovaccine) as intranasal mist. Vaccinations enhanced homologous (H1N2-OH10) and heterologous (H1N1-OH7) hemagglutination inhibition (HI) titers in both vaccinated and virus-challenged animals compared to the control soluble poly(I:C) vaccinated pigs. In addition, the chitosan nanovaccine induced the proliferation of antigen-specific IFNγ secreting T-helper/memory and γδ T cells compared to control poly(I:C) group; and an increased Th1 (IFNγ, IL-6 and IL-2) and Th2 (IL-10 and IL-13) cytokines mRNA expression in the tracheobronchial lymph nodes compared to lymphoid tissues obtained from pigs given commercial influenza vaccine. The virus load in nasal passages and microscopic lung lesions were partially reduced by both chitosan nanovaccine and commercial vaccine. The HA gene homology between the vaccine and challenge viruses indicated that the chitosan nanovaccine induced a cross-protective immune response. In conclusion, coadministration of CS NPs-poly(I:C) with CS NPs-KAg augmented the cross-reactive specific HI titers and the cell-mediated immune responses in pigs.