Production and characterization of anti-porcine CXCL10 monoclonal antibodies.

Research on C-X-C motif chemokine ligand 10 (CXCL10) has been widely reported for humans and select animal species, yet immune reagents are limited for pig chemokines. Our goal is to provide veterinary immunologists and the biomedical community with new commercial immune reagents and standardized assays. Recombinant porcine CXCL10 (rPoCXCL10) protein was produced by yeast expression and used to generate a panel of α CXCL10 monoclonal antibodies (mAbs). All mAbs were assessed for cross-inhibition and reactivity to orthologous yeast expressed CXCL10 proteins. Characterization of a panel of nine α PoCXCL10 mAbs identified six distinct antigenic determinants. A sensitive quantitative sandwich ELISA was developed with anti-PoCXCL10-1.6 and -1.9 mAb; reactivity was verified with both rPoCXCL10 and native PoCXCL10, detected in supernatants of peripheral blood mononuclear cells stimulated with rPoIFNγ or PMA/Ionomycin. Immunostaining of in vitro rPoIFNγ stimulated pig spleen and blood cells verified CXCL10 + cells as CD3-CD4-CD172+, with occasional CD3-CD4 + CD172 + subsets. Comparison studies determined that α PoCXCL10-1.4 mAb was the ideal mAb clone for intracellular staining, whereas with α PoCXCL10-1.1 and -1.2 mAbs were best for immunohistochemistry analyses. These techniques and tools will be useful for evaluating swine immune development, responses to infectious diseases and vaccines, as well as for improving utility of pigs as an important biomedical model.

Inhibition of elastase enhances the adjuvanticity of alum and promotes anti-SARS-CoV-2 systemic and mucosal immunity.

Alum, used as an adjuvant in injected vaccines, promotes T helper 2 (Th2) and serum antibody (Ab) responses. However, it fails to induce secretory immunoglobulin (Ig) A (SIgA) in mucosal tissues and is poor in inducing Th1 and cell-mediated immunity. Alum stimulates interleukin 1 (IL-1) and the recruitment of myeloid cells, including neutrophils. We investigated whether neutrophil elastase regulates the adjuvanticity of alum, and whether a strategy targeting neutrophil elastase could improve responses to injected vaccines. Mice coadministered a pharmacological inhibitor of elastase, or lacking elastase, developed high-affinity serum IgG and IgA antibodies after immunization with alum-adsorbed protein vaccines, including the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2). These mice also developed broader antigen-specific CD4+ T cell responses, including high Th1 and T follicular helper (Tfh) responses. Interestingly, in the absence of elastase activity, mucosal SIgA responses were induced after systemic immunization with alum as adjuvant. Importantly, lack or suppression of elastase activity enhanced the magnitude of anti-SARS-CoV-2 spike subunit 1 (S1) antibodies, and these antibodies reacted with the same epitopes of spike 1 protein as sera from COVID-19 patients. Therefore, suppression of neutrophil elastase could represent an attractive strategy for improving the efficacy of alum-based injected vaccines for the induction of broad immunity, including mucosal immunity.

Enhanced mucosal immune responses and reduced viral load in the respiratory tract of ferrets to intranasal lipid nanoparticle-based SARS-CoV-2 proteins and mRNA vaccines.

BACKGROUND: Unlike the injectable vaccines, intranasal lipid nanoparticle (NP)-based adjuvanted vaccine is promising to protect against local infection and viral transmission. Infection of ferrets with SARS-CoV-2 results in typical respiratory disease and pathology akin to in humans, suggesting that the ferret model may be ideal for intranasal vaccine studies. RESULTS: We developed SARS-CoV-2 subunit vaccine containing both Spike receptor binding domain (S-RBD) and Nucleocapsid (N) proteins (NP-COVID-Proteins) or their mRNA (NP-COVID-mRNA) and NP-monosodium urate adjuvant. Both the candidate vaccines in intranasal vaccinated aged ferrets substantially reduced the replicating virus in the entire respiratory tract. Specifically, the NP-COVID-Proteins vaccine did relatively better in clearing the virus from the nasal passage early post challenge infection. The immune gene expression in NP-COVID-Proteins vaccinates indicated increased levels of mRNA of IFNα, MCP1 and IL-4 in lungs and nasal turbinates, and IFNγ and IL-2 in lungs; while proinflammatory mediators IL-1ß and IL-8 mRNA levels in lungs were downregulated. In NP-COVID-Proteins vaccinated ferrets S-RBD and N protein specific IgG antibodies in the serum were substantially increased at both day post challenge (DPC) 7 and DPC 14, while the virus neutralizing antibody titers were relatively better induced by mRNA versus the proteins-based vaccine. In conclusion, intranasal NP-COVID-Proteins vaccine induced balanced Th1 and Th2 immune responses in the respiratory tract, while NP-COVID-mRNA vaccine primarily elicited antibody responses. CONCLUSIONS: Intranasal NP-COVID-Proteins vaccine may be an ideal candidate to elicit increased breadth of immunity against SARS-CoV-2 variants.

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.

Nanoparticle-based vaccine development and evaluation against viral infections in pigs.

Virus infections possess persistent health challenges in swine industry leading to severe economic losses worldwide. The economic burden caused by virus infections such as Porcine Reproductive and Respiratory Syndrome Virus, Swine influenza virus, Porcine Epidemic Diarrhea Virus, Porcine Circovirus 2, Foot and Mouth Disease Virus and many others are associated with severe morbidity, mortality, loss of production, trade restrictions and investments in control and prevention practices. Pigs can also have a role in zoonotic transmission of some viral infections to humans. Inactivated and modified-live virus vaccines are available against porcine viral infections with variable efficacy under field conditions. Thus, improvements over existing vaccines are necessary to: (1) Increase the breadth of protection against evolving viral strains and subtypes; (2) Control of emerging and re-emerging viruses; (3) Eradicate viruses localized in different geographic areas; and (4) Differentiate infected from vaccinated animals to improve disease control programs. Nanoparticles (NPs) generated from virus-like particles, biodegradable and biocompatible polymers and liposomes offer many advantages as vaccine delivery platform due to their unique physicochemical properties. NPs help in efficient antigen internalization and processing by antigen presenting cells and activate them to elicit innate and adaptive immunity. Some of the NPs-based vaccines could be delivered through both parenteral and mucosal routes to trigger efficient mucosal and systemic immune responses and could be used to target specific immune cells such as mucosal microfold (M) cells and dendritic cells (DCs). In conclusion, NPs-based vaccines can serve as novel candidate vaccines against several porcine viral infections with the potential to enhance the broader protective efficacy under field conditions. This review highlights the recent developments in NPs-based vaccines against porcine viral pathogens and how the NPs-based vaccine delivery system induces innate and adaptive immune responses resulting in varied level of protective efficacy.

Analysis of the efficacy of an adjuvant-based inactivated pandemic H5N1 influenza virus vaccine.

This paper describes a preclinical study analyzing the immunogenicity and protective efficacy of Kazfluvac®, an adjuvant-based inactivated pandemic influenza A/H5N1 virus vaccine. In this study, laboratory animals (ferrets and mice) were vaccinated by the intramuscular or intraperitoneal route at an interval of 14 days with two doses of the vaccine containing different concentrations of influenza virus hemagglutinin (HA) protein. HA protein without adjuvant (aluminum hydroxide and Merthiolate) was used as a control. As a negative control, we utilized PBS. We assessed the protective efficacy of the candidate vaccine by analyzing the response to challenge with the influenza virus strain A/chicken/Astana/6/05 (H5N1). Our experimental results revealed substantially reduced clinical disease and an increased antibody response, as determined by hemagglutination-inhibition (HAI) test and microneutralization assay (MNA). This study showed that the candidate vaccine is safe and elicits an antigen-dose-dependent serum antibody response. In summary, we determined the optimum antigen dose in a Kazfluvac® adjuvant formulation required for induction of heightened immunogenicity and protective efficacy to mitigate H5N1 disease in experimental animals, suggesting its readiness for clinical studies in humans.

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.

Mutations in a Highly Conserved Motif of nsp1ß Protein Attenuate the Innate Immune Suppression Function of Porcine Reproductive and Respiratory Syndrome Virus.

UNLABELLED: Porcine reproductive and respiratory syndrome virus (PRRSV) nonstructural protein 1ß (nsp1ß) is a multifunctional viral protein, which is involved in suppressing the host innate immune response and activating a unique -2/-1 programmed ribosomal frameshifting (PRF) signal for the expression of frameshifting products. In this study, site-directed mutagenesis analysis showed that the R128A or R129A mutation introduced into a highly conserved motif ((123)GKYLQRRLQ(131)) reduced the ability of nsp1ß to suppress interferon beta (IFN-ß) activation and also impaired nsp1ß's function as a PRF transactivator. Three recombinant viruses, vR128A, vR129A, and vRR129AA, carrying single or double mutations in the GKYLQRRLQ motif were characterized. In comparison to the wild-type (WT) virus, vR128A and vR129A showed slightly reduced growth abilities, while the vRR129AA mutant had a significantly reduced growth ability in infected cells. Consistent with the attenuated growth phenotype in vitro, pigs infected with nsp1ß mutants had lower levels of viremia than did WT virus-infected pigs. Compared to the WT virus in infected cells, all three mutated viruses stimulated high levels of IFN-α expression and exhibited a reduced ability to suppress the mRNA expression of selected interferon-stimulated genes (ISGs). In pigs infected with nsp1ß mutants, IFN-α production was increased in the lungs at early time points postinfection, which was correlated with increased innate NK cell function. Furthermore, the augmented innate response was consistent with the increased production of IFN-γ in pigs infected with mutated viruses. These data demonstrate that residues R128 and R129 are critical for nsp1ß function and that modifying these key residues in the GKYLQRRLQ motif attenuates virus growth ability and improves the innate and adaptive immune responses in infected animals. IMPORTANCE: PRRSV infection induces poor antiviral innate IFN and cytokine responses, which results in weak adaptive immunity. One of the strategies in next-generation vaccine construction is to manipulate viral proteins/genetic elements involved in antagonizing the host immune response. PRRSV nsp1ß was identified to be a strong innate immune antagonist. In this study, two basic amino acids, R128 and R129, in a highly conserved GKYLQRRLQ motif were determined to be critical for nsp1ß function. Mutations introduced into these two residues attenuated virus growth and improved the innate and adaptive immune responses of infected animals. Technologies developed in this study could be broadly applied to current commercial PRRSV modified live-virus (MLV) vaccines and other candidate vaccines.

Comparative analysis of routes of immunization of a live porcine reproductive and respiratory syndrome virus (PRRSV) vaccine in a heterologous virus challenge study.

Porcine reproductive and respiratory syndrome (PRRS) is caused by PRRS virus (PRRSV), which infects primarily the respiratory tract of pigs. Thus intranasal (IN) delivery of a potent vaccine-adjuvant formulation is promising. In this study, PRRS-MLV (VR2332) was coadministered ± an adjuvant Mycobacterium vaccae whole cell lysate or CpG ODN through intramuscular (IM) or IN route as a mist, and challenged with a heterologous PRRSV 1-4-4 IN at 42 days post-vaccination (dpv). At 14 and 26 dpv, vaccine viral RNA copies were one log greater in the plasma of PRRS-MLV IM compared to IN vaccinated pigs, and the infectious replicating vaccine virus was detected only in the IM group. In PRRS-MLV ± adjuvant IM vaccinated pigs, reduced viral RNA load and absence of the replicating challenged virus was observed at 7, 10 and 14 days post-challenge (dpc). At 14 dpc, in BAL fluid ≥ 5 log viral RNA copies were detected in all the pig groups, but the replicating challenged virus was undetectable only in IM groups. Immunologically, virus neutralizing antibody titers in the plasma of IM (but not IN) vaccine groups was ≥ 8 against the vaccine and challenged viruses. At 26 dpv, PRRS-MLV IM (without adjuvant) received pigs had significantly increased population of CD4 and CD8 T cells in PBMC. At 14 dpc, relatively increased population of IFN-γ(+) total lymphocytes, NK, CD4, CD8 and γδ T cells were observed in the MLV-IM group. In conclusion, PRRS-MLV IM vaccination induced the virus specific T cell response in pigs, but still it is required to improve its cross-protective efficacy.

Development of a porcine reproductive and respiratory syndrome virus-like-particle-based vaccine and evaluation of its immunogenicity in pigs.

Porcine reproductive and respiratory syndrome (PRRS) is a leading cause of economic burden to the pork industry worldwide. The routinely used modified live PRRS virus vaccine (PRRS-MLV) induces clinical protection, but it has safety concerns. Therefore, in an attempt to develop a safe and protective inactivated PRRSV vaccine, we generated PRRS-virus-like-particles (PRRS-VLPs) containing the viral surface proteins GP5-GP4-GP3-GP2a-M or GP5-M using a novel baculovirus expression system. Our in vitro results indicated that the desired PRRSV proteins were incorporated in both the VLPs preparations based on their reactivity in immunogold electron microscopy and ELISA. To boost their immunogenicity in pigs, we entrapped the PRRS-VLPs in PLGA nanoparticles and coadministered them intranasally with a potent adjuvant. We then evaluated their efficacy in pigs against a viral challenge using a virulent heterologous field isolate. Our results indicated that PRRS-VLPs induced an anamnestic immune response, since we observed boosted IgG and IFN-γ production in vaccinated and virus-challenged animals, but not during the pre-challenge period. Importantly, a two-log reduction in the lung viral load was detected in PRRS-VLP-vaccinated animals. In conclusion, we generated PRRS-VLPs containing up to five viral surface proteins and demonstrated their immunogenicity in pigs, but further studies are required to improve its immunogenicity and efficacy as a vaccine candidate.

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.

Inhibition of CD1d-mediated antigen presentation by the transforming growth factor-ß/Smad signalling pathway.

CD1d-mediated lipid antigen presentation activates a subset of innate immune lymphocytes called invariant natural killer T (NKT) cells that, by virtue of their potent cytokine production, bridge the innate and adaptive immune systems. Transforming growth factor (TGF-ß) is a known immune modulator that can activate the mitogen-activated protein kinase p38; we have previously shown that p38 is a negative regulator of CD1d-mediated antigen presentation. Several studies implicate a role for TGF-ß in the activation of p38. Therefore, we hypothesized that TGF-ß would impair antigen presentation by CD1d. Indeed, a dose-dependent decrease in CD1d-mediated antigen presentation and impairment of lipid antigen processing was observed in response to TGF-ß treatment. However, it was found that this inhibition was not through p38 activation. Instead, Smads 2, 3 and 4, downstream elements of the TGF-ß canonical signalling pathway, contributed to the observed effects. In marked contrast to that observed with CD1d, TGF-ß was found to enhance MHC class II-mediated antigen presentation. Overall, these results suggest that the canonical TGF-ß/Smad pathway negatively regulates an important arm of the host's innate immune responses - CD1d-mediated lipid antigen presentation to NKT cells.

CD1d expression on and regulation of murine hematopoietic stem and progenitor cells.

In the present study, surface CD1d, which is involved in immune cell interactions, was assessed for effects on hematopoiesis. Mouse BM hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) express CD1d. The numbers and cycling status of HPCs in the BM and spleen of different strains of cd1d(-/-) mice were enhanced significantly, suggesting that CD1d is a negative regulator of HPCs. In support of this, CD1d was required for the SCF and Flt3 ligand synergistic enhancement of CSF induction of HPC colony formation and for HPC response to myelosuppressive chemokines. Colony formation by immature subsets of HPCs was greatly enhanced when normal, but not cd1d(-/-), BM cells were pretreated with CD1d Abs in vitro. These effects required the full CD1d cytoplasmic tail. In contrast, long-term, but not short-term, repopulating HSC engraftment was impaired significantly, an effect that was minimally influenced by the presence of a truncated CD1d cytoplasmic tail. Pretreatment of normal BM cells with CD1d Abs greatly enhanced their engraftment of HSCs. The results of the present study implicate CD1d in a previously unrecognized regulatory role of normal and stressed hematopoiesis.

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.

Evaluation of Efficacy of Surface Coated versus Encapsulated Influenza Antigens in Mannose-Chitosan Nanoparticle-Based Intranasal Vaccine in Swine.

This study focuses on the development and characterization of an intranasal vaccine platform using adjuvanted nanoparticulate delivery of swine influenza A virus (SwIAV). The vaccine employed whole inactivated H1N2 SwIAV as an antigen and STING-agonist ADU-S100 as an adjuvant, with both surface adsorbed or encapsulated in mannose-chitosan nanoparticles (mChit-NPs). Optimization of mChit-NPs included evaluating size, zeta potential, and cytotoxicity, with a 1:9 mass ratio of antigen to NP demonstrating high loading efficacy and non-cytotoxic properties suitable for intranasal vaccination. In a heterologous H1N1 pig challenge trial, the mChit-NP intranasal vaccine induced cross-reactive sIgA antibodies in the respiratory tract, surpassing those of a commercial SwIAV vaccine. The encapsulated mChit-NP vaccine induced high virus-specific neutralizing antibody and robust cellular immune responses, while the adsorbed vaccine elicited specific high IgG and hemagglutinin inhibition antibodies. Importantly, both the mChit-NP vaccines reduced challenge heterologous viral replication in the nasal cavity higher than commercial swine influenza vaccine. In summary, a novel intranasal mChit-NP vaccine platform activated both the arms of the immune system and is a significant advancement in swine influenza vaccine design, demonstrating its potential effectiveness for pig immunization.

An Orf-Virus (ORFV)-Based Vector Expressing a Consensus H1 Hemagglutinin Provides Protection against Diverse Swine Influenza Viruses.

Influenza A viruses (IAV-S) belonging to the H1 subtype are endemic in swine worldwide. Antigenic drift and antigenic shift lead to a substantial antigenic diversity in circulating IAV-S strains. As a result, the most commonly used vaccines based on whole inactivated viruses (WIVs) provide low protection against divergent H1 strains due to the mismatch between the vaccine virus strain and the circulating one. Here, a consensus coding sequence of the full-length of HA from H1 subtype was generated in silico after alignment of the sequences from IAV-S isolates obtained from public databases and was delivered to pigs using the Orf virus (ORFV) vector platform. The immunogenicity and protective efficacy of the resulting ORFVΔ121conH1 recombinant virus were evaluated against divergent IAV-S strains in piglets. Virus shedding after intranasal/intratracheal challenge with two IAV-S strains was assessed by real-time RT-PCR and virus titration. Viral genome copies and infectious virus load were reduced in nasal secretions of immunized animals. Flow cytometry analysis showed that the frequency of T helper/memory cells, as well as cytotoxic T lymphocytes (CTLs), were significantly higher in the peripheral blood mononuclear cells (PBMCs) of the vaccinated groups compared to unvaccinated animals when they were challenged with a pandemic strain of IAV H1N1 (CA/09). Interestingly, the percentage of T cells was higher in the bronchoalveolar lavage of vaccinated animals in relation to unvaccinated animals in the groups challenged with a H1N1 from the gamma clade (OH/07). In summary, delivery of the consensus HA from the H1 IAV-S subtype by the parapoxvirus ORFV vector decreased shedding of infectious virus and viral load of IAV-S in nasal secretions and induced cellular protective immunity against divergent influenza viruses in swine.

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.

Intradermal Vaccination against Influenza with a STING-Targeted Nanoparticle Combination Adjuvant Induces Superior Cross-Protective Humoral Immunity in Swine Compared with Intranasal and Intramuscular Immunization.

The development of cross-protective vaccines against the zoonotic swine influenza A virus (swIAV), a potential pandemic-causing agent, continues to be an urgent global health concern. Commercially available vaccines provide suboptimal cross-protection against circulating subtypes of swIAV, which can lead to worldwide economic losses and poor zoonosis deterrence. The limited efficacy of current swIAV vaccines demands innovative strategies for the development of next-generation vaccines. Considering that intramuscular injection is the standard route of vaccine administration in both human and veterinary medicine, the exploration of alternative strategies, such as intradermal vaccination, presents a promising avenue for vaccinology. This investigation demonstrates the first evaluation of a direct comparison between a commercially available multivalent swIAV vaccine and monovalent whole inactivated H1N2 swine influenza vaccine, delivered by intradermal, intranasal, and intramuscular routes. The monovalent vaccines were adjuvanted with NanoST, a cationic phytoglycogen-based nanoparticle that is combined with the STING agonist ADU-S100. Upon heterologous challenge, intradermal vaccination generated a stronger cross-reactive nasal and serum antibody response in pigs compared with intranasal and intramuscular vaccination. Antibodies induced by intradermal immunization also had higher avidity compared with the other routes of vaccination. Bone marrow from intradermally and intramuscularly immunized pigs had both IgG and IgA virus-specific antibody-secreting cells. These studies reveal that NanoST is a promising adjuvant system for the intradermal administration of STING-targeted influenza vaccines.

Characterization of the Efficacy of a Split Swine Influenza A Virus Nasal Vaccine Formulated with a Nanoparticle/STING Agonist Combination Adjuvant in Conventional Pigs.

Swine influenza A viruses (SwIAVs) are pathogens of both veterinary and medical significance. Intranasal (IN) vaccination has the potential to reduce flu infection. We investigated the efficacy of split SwIAV H1N2 antigens adsorbed with a plant origin nanoparticle adjuvant [Nano11-SwIAV] or in combination with a STING agonist ADU-S100 [NanoS100-SwIAV]. Conventional pigs were vaccinated via IN and challenged with a heterologous SwIAV H1N1-OH7 or 2009 H1N1 pandemic virus. Immunologically, in NanoS100-SwIAV vaccinates, we observed enhanced frequencies of activated monocytes in the blood of the pandemic virus challenged animals and in tracheobronchial lymph nodes (TBLN) of H1N1-OH7 challenged animals. In both groups of the virus challenged pigs, increased frequencies of IL-17A+ and CD49d+IL-17A+ cytotoxic lymphocytes were observed in Nano11-SwIAV vaccinates in the draining TBLN. Enhanced frequency of CD49d+IFNγ+ CTLs in the TBLN and blood of both the Nano11-based SwIAV vaccinates was observed. Animals vaccinated with both Nano11-based vaccines had upregulated cross-reactive secretory IgA in the lungs and serum IgG against heterologous and heterosubtypic viruses. However, in NanoS100-SwIAV vaccinates, a slight early reduction in the H1N1 pandemic virus and a late reduction in the SwIAV H1N1-OH7 load in the nasal passages were detected. Hence, despite vast genetic differences between the vaccine and both the challenge viruses, IN vaccination with NanoS100-SwIAV induced antigen-specific moderate levels of cross-protective immune responses.