Animal disease surveillance: Its importance & present status in India.

Animal disease surveillance encompasses systematic collection of long-term data on disease events, risk factors and other relevant parameters followed by analyzing the same with reference to temporal and spatial characteristics to arrive at a conclusion so that necessary preventive measures can be taken. In India, the animal disease surveillance is done through National Animal Disease Reporting System, which is a web-based information technology system for disease reporting from States and Union Territories with the aim to record, monitor livestock disease situation and to initiate the preventive and curative action in a swift manner during disease emergencies. National Animal Disease Referral Expert System is a dynamic geographic information system and remote sensing-enabled expert system that captures an incidence of 13 economically important livestock diseases from all over the country and also provides livestock disease forecasting. The laboratories under State and Central governments, several research institutes under the Indian Council of Agricultural Research and veterinary colleges are involved in livestock disease diagnosis including zoonotic diseases. An integrated surveillance system is necessary for early detection of emerging/zoonotic diseases in humans. This review provides information on disease reporting and surveillance systems in animal health sector and the need for One Health approach to improve and strengthen the zoonotic disease surveillance system in India.

Spatial seroprevalence of classical swine fever in India.

Classical swine fever (CSF) is a highly contagious dreadful disease of pigs leading to 100% mortality in acute form in susceptible population thereby causing huge economic loss to pig farmers. This study was undertaken to assess the seroprevalence of CSF at national level. A two-stage random sampling methodology was adopted that included 271 villages from 115 districts of India. A total of 5848 pig serum samples from twenty-five states and one Union Territory of India were collected during 2018-2019. A percent positivity of 38.52 was found at national level. Puducherry and Sikkim showed the highest and lowest percent positivity respectively. Pigs from the west zone showed the highest seroprevalence of 55.83% and those from the south zone showed the lowest of 30.25%. Adult pigs in the north and east zones showed highest percent positivity of 81.8, whereas pigs of more than 3 years of age showed highest percent positivity of 54.9, 75 and 62.5 in the north east, west and central zones respectively. Young ones showed percent positivity of 41.5 in the south zone. Higher rainfall (> 3 mm/day) and lower temperature (< 26 °C) favoured the existence of disease in the north east region combined with high density of pig population. Amidst no fool proof alert system, seroprevalence is the best method to assess the status of CSF in herd/population that provides the policymakers to plan for control of disease.

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.

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.

Epidemiological investigation of ASF outbreaks in Kerala (India): detection, source tracing and economic implications.

This study investigates suspected African swine fever (ASF) outbreaks in two villages of Kannur district in Kerala, India, with the aim of identifying the causative agent and its genotype, the source of infection, and estimating the economic losses due to the outbreaks. Clinically, the disease was acute with high mortality, while gross pathology was characterized by widespread haemorrhages in various organs, especially the spleen, which was dark, enlarged and had friable cut surfaces with diffuse haemorrhages. Notably, histopathological examination revealed multifocal, diffuse haemorrhages in the splenic parenchyma and lymphoid depletion accompanied by lymphoid cell necrosis. The clinico-pathological observations were suggestive of ASF, which was confirmed by PCR. The source of outbreak was identified as swill and it was a likely point source infection as revealed by epidemic curve analysis. The phylogenetic analysis of p72 gene identified the ASFV in the current outbreak as genotype-II and IGR II variant consistent with ASFVs detected in India thus far. However, the sequence analysis of the Central Variable Region (CVR) of the B602L gene showed that the ASFVs circulating in Kerala (South India) formed a separate clade along with those found in Mizoram (North East India), while ASFVs circulating in Arunachal Pradesh and Assam states of India grouped in to different clade. This study represents the first investigation of ASF outbreak in South India, establishing the genetic relatedness of the ASFV circulating in this region with that in other parts of the country. The study also underscores the utility of the CVR of the B602L gene in genetically characterizing highly similar Genotype II ASFVs to understand the spread of ASF within the country.

Characterization of humoral and cellular immune responses elicited by reduced doses of Brucella abortus S19 (calfhood) vaccine in cattle calves of India.

Brucella abortus S19 vaccine is a stable attenuated smooth strain, globally used as calfhood vaccine for the prevention of bovine brucellosis. Various agencies demonstrated different doses for vaccinating cattle and buffalo calves leading to ambiguity in selecting a suitable immune vaccine dose. The current study aimed at evaluating four graded doses of S19 vaccine to arrive at the dose which could produce comparable effectiveness as that of full dose prescribed by Indian Pharmacopeia among the Indian calves. Four vaccine doses of which the first dose consisted of full dose (40 × 109 CFU/dose) and the other three were 1/10th, 1/20th, 1/100th reduced doses along with control were tested. Each vaccine dose was administered to 13 cattle calves of 4-5 months of age maintained in separate groups. The blood samples were collected on 0 to 240 days post-vaccination (DPV) at the intervals of 0, 14, 28, 45, 60, 90, 150, 180 and 240 for assessment of vaccine-induced innate, humoral and cell-mediated immune responses. The sero-conversion of all vaccinated animals on DPV 45 and persistence of antibody till DPV 240 were noticed. No significant differences were observed in antibody response between animal groups that received full and 1/10th reduced doses. Innate and cell-mediated response by IL-6, TNF-α¸ IFN-γ, CD4+ and CD8+ cell counts showed dose-dependent responses with no significant difference between full dose and 1/10th reduced doses. The results suggest a possible one log reduction of full dose without compromising immune responses to aid larger vaccination coverage for creating herd immunity.

Seroprevalence of Infectious bovine rhinotracheitis (IBR) in India: A 5-year study.

Infectious bovine rhinotracheitis (IBR) is a highly contagious disease of bovines causing respiratory symptoms, abortions, and reduced milk yield, leading to huge economic losses. Reports on seroprevalence in bovines in India are available and restricted to districts/states. In the present study, a nationwide seroprevalence of IBR in bovines was conducted to provide a national IBR seroprevalence to the Chief Veterinarian who in turn can design the control strategies. A total of 15,592 cattle and buffalo serum samples from 25 states and 3 Union Territories viz., Jammu and Kashmir, Puducherry, and Andaman and Nicobar Islands were tested for IBR antibodies using Avidin­Biotin (AB) ELISA. Cumulative seropositivity was found to be 31.37%. Maharashtra and Rajasthan states, part of the west zone of the country, showed the highest and lowest seroprevalence, respectively. A total of 11,423 cattle and 4,169 buffalo serum samples were tested, which showed 33.91% and 24.39% seropositivity, respectively. India has the highest buffalo population. Presently, India no IBR vaccination programs are implemented in India. Considering the high seroprevalence, the authorities should plan control strategies for vaccinating dairy cows and buffaloes in India.

Evaluation of the immune responses against reduced doses of Brucella abortus S19 (calfhood) vaccine in water buffaloes (Bubalus bubalis), India.

BACKGROUND: Brucella abortus S19 is the most widely used vaccine for the prevention of bovine brucellosis which remains the reference vaccine to which many other vaccine/s are compared. Considering the larger vaccination coverage by reduced dose of vaccine, the study aimed to compare reduced graded doses (1/10th, 1/20th and 1/100th) with standard dose of S19 vaccine (40 × 109CFU /dose) to determine the effective immunizing dose in water buffaloes. METHODS: A total of 25 female buffalo calves (Bubalus bubalis) in the age group of 4-5 months were equally grouped into five animals each in four test and one control groups and given with specified vaccine dose. The blood samples were collected on post vaccination days 14, 28, 45, 60, 90 and 120 for assessing innate (TNF-α and IL-12), humoral (IgG antibodies against Brucella LPS) and cell mediated immune responses (IFN-γ, CD4 + and CD8 + counts). RESULTS: The full dose, 1/10th and 1/20th reduced doses of S19 vaccine was capable of eliciting pathogen-specific antibody response, vaccine induced secretion of IL-12, TNF-α and IFN-γ with CD4 + and CD8 + effector T cell responses. Persistence of antibody and magnitude of immune responses were found dose dependent. CONCLUSION: Comparable immune responses were noticed with 1/10th reduced dose similar to standard dose. With this observation, decline of antibody titre will reduce the number of false positives and reduced dose of vaccine will facilitate larger vaccination coverage in the country.

Development of recombinant nucleocapsid protein-based indirect enzyme-linked immunosorbent assay for sero-survey of porcine reproductive and respiratory syndrome.

BACKGROUND AND AIM: Porcine reproductive and respiratory syndrome (PRRS) is a disease endemic in many countries and is of economic importance. India was free from PRRS until the first outbreak was reported from a North-East Indian state in 2013. Since then, disease outbreaks have been reported from North-East India and the pilot study conducted earlier showed that it is gradually spreading to the rest of India. Considering there are no locally developed population screening tests available for PRRS and imported diagnostic/screening tests are expensive, the present study was aimed at developing recombinant nucleocapsid (rN) protein-based indirect enzyme-linked immunosorbent assay (iELISA). MATERIALS AND METHODS: The rN protein of PRRS virus (PRRSV) was produced following standard cloning, expression, and purification procedures. Using this antigen, iELISA was optimized for the detection of serum antibodies to PRRSV. The sensitivity and specificity of the test were assessed by comparing it with a commercial PRRSV antibody detection kit. RESULT: A total of 745 serum samples from ten different states of India were screened using the developed iELISA. The iELISA had a relative specificity of 76.18% and sensitivity of 82.61% compared to the commercial ELISA (Priocheck PRRSV ELISA kit, Thermo Fisher Scientific, USA). CONCLUSION: The iELISA, which deployed rN protein from Indian PRRSV, was found to be suitable in the serological survey and may be a useful tool in future disease surveillance programs.

Detection of torque teno sus virus infection in Indian pigs.

BACKGROUND AND AIM: Torque teno viruses (TTVs) are circular, single-stranded DNA viruses, which infect a wide range of animals including livestock and companion animals. Swine TTVs (torque teno sus viruses [TTSuVs]) are thought to act as a primary or coinfecting pathogen in pathological conditions such as porcine dermatitis and nephropathy syndrome and post-weaning multisystemic wasting syndrome. So far, the presence of the virus has not been reported in India. Considering that TTSuVs have the potential to cross the species barrier into humans and that pork consumption is common in North-Eastern states of India, the current study aims to investigate the presence of TTSuV in the Indian pig population. MATERIALS AND METHODS: A total of 416 samples were collected during 2014-2018, from both apparently healthy pigs and also from pigs suspected of having died from classical swine fever and/or porcine reproductive and respiratory syndrome. These samples were screened for TTSuV infection by polymerase chain reaction (PCR) and DNA sequencing techniques. RESULTS: The presence of the virus was confirmed in 110 samples from 12 different states of India. Phylogenetic analysis of the nucleotide sequences obtained from the PCR products indicated the presence of viruses of both Iotatorquevirus and Kappatorquevirus genera in India. CONCLUSION: The study is the first report on the presence of TTSuVs in India and highlights the circulation of both genera of the virus in the country.

Serological and molecular analysis for brucellosis in selected swine herds from Southern India.

BACKGROUND: Swine brucellosis is a zoonotic disease caused by Brucella suis. The study describes the occurrence of brucellosis in two organized piggeries in Southern India. METHODS: A total of 585 serum samples comprising 575 from pigs and 10 from animal handlers were collected and tested by serological tests and PCR. Tissue samples were collected for isolation of the pathogen. RESULTS: Out of 575 serum samples screened, 236 (41.04%) were positive for brucellosis by both Rose Bengal plate test (RBPT) and indirect ELISA (iELISA) and 47 (8.17) samples showed Brucella DNA amplification by genus specific PCR. The sows those aborted and 19 boars with orchitis were seropositive for brucellosis indicating association of clinical symptoms with brucellosis seropositivity. Two of 10 pig handlers were positive by RBPT and showed significant serum agglutination test (SAT) titres of >1:160 and 1:320. B. suis bvI was isolated and identified by biochemical tests and confirmed by amplification Brucella genus and Bruce ladder PCRs from vaginal and testicular samples. CONCLUSIONS: The introduction of untested breeding boars in the farms might have resulted in the disease transmission and spread. The present study highlighted the diagnosis of B. suis bvI as a cause of abortions in the pigs and occupational exposure to farm personnel.

Supplementation of inactivated influenza vaccine with norovirus P particle-M2e chimeric vaccine enhances protection against heterologous virus challenge in chickens.

The current inactivated influenza vaccines provide satisfactory protection against homologous viruses but limited cross-protection against antigenically divergent strains. Consequently, there is a need to develop more broadly protective vaccines. The highly conserved extracellular domain of the matrix protein 2 (M2e) has shown promising results as one of the components of a universal influenza vaccine in different animal models. As an approach to overcome the limited, strain specific, protective efficacy of inactivated influenza vaccine (IIV), a combination of recombinant M2e expressed on the surface of norovirus P particle (M2eP) and IIV was tested in chickens. Co-immunization of birds with both vaccines did not affect the production of M2e-specific IgG antibodies compared to the group vaccinated with M2eP alone. However, the co-immunized birds developed significantly higher pre-challenge hemagglutination inhibition antibody titers against the homologous IIV antigen and heterologous challenge virus. These combined vaccine groups also had cross reactive antibody responses against different viruses (H5, H6, and H7 subtypes) compared to the IIV alone vaccinated group. Upon intranasal challenge with homologous and heterologous viruses, the combined vaccine groups showed greater reduction in viral shedding in tracheal swabs compared to those groups receiving IIV alone. Moreover, M2eP antisera from vaccinated birds were able to bind to the native M2 expressed on the surface of whole virus particles and infected cells, and inhibit virus replication in vitro. Our results support the potential benefit of supplementing IIV with M2eP, to expand the vaccine cross protective efficacy.

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.

Adjuvant effects of invariant NKT cell ligand potentiates the innate and adaptive immunity to an inactivated H1N1 swine influenza virus vaccine in pigs.

Pigs are considered as the source of some of the emerging human flu viruses. Inactivated swine influenza virus (SwIV) vaccine has been in use in the US swine herds, but it failed to control the flu outbreaks. The main reason has been attributed to lack of induction of strong local mucosal immunity in the respiratory tract. Invariant natural killer T (iNKT) cell is a unique T cell subset, and activation of iNKT cell using its ligand α-Galactosylceramide (α-GalCer) has been shown to potentiate the cross-protective immunity to inactivated influenza virus vaccine candidates in mice. Recently, we discovered iNKT cell in pig and demonstrated its activation using α-GalCer. In this study, we evaluated the efficacy of an inactivated H1N1 SwIV coadministered with α-GalCer intranasally against a homologous viral challenge. Our results demonstrated the potent adjuvant effects of α-GalCer in potentiating both innate and adaptive immune responses to SwIV Ags in the lungs of pigs, which resulted in reduction in the lung viral load by 3 logs compared to without adjuvant. Immunologically, in the lungs of pigs vaccinated with α-GalCer an increased virus specific IgA response, IFN-α secretion and NK cell-cytotoxicity was observed. In addition, iNKT cell-stimulation enhanced the secretion of Th1 cytokines (IFN-γ and IL-12) and reduced the production of immunosuppressive cytokines (IL-10 and TGF-ß) in the lungs of pigs⋅ In conclusion, we demonstrated for the first time iNKT cell adjuvant effects in pigs to SwIV Ags through augmenting the innate and adaptive immune responses in the respiratory tract.

Entrapment of H1N1 Influenza Virus Derived Conserved Peptides in PLGA Nanoparticles Enhances T Cell Response and Vaccine Efficacy in Pigs.

Pigs are believed to be one of the important sources of emerging human and swine influenza viruses (SwIV). Influenza virus conserved peptides have the potential to elicit cross-protective immune response, but without the help of potent adjuvant and delivery system they are poorly immunogenic. Biodegradable polylactic-co-glycolic acid (PLGA) nanoparticle (PLGA-NP) based vaccine delivery system enhances cross-presentation of antigens by the professional antigen presenting cells. In this study, Norovirus P particle containing SwIV M2e (extracellular domain of the matrix protein 2) chimera and highly conserved two each of H1N1 peptides of pandemic 2009 and classical human influenza viruses were entrapped in PLGA-NPs. Influenza antibody-free pigs were vaccinated with PLGA-NPs peptides cocktail vaccine twice with or without an adjuvant, Mycobacterium vaccae whole cell lysate, intranasally as mist. Vaccinated pigs were challenged with a virulent heterologous zoonotic SwIV H1N1, and one week later euthanized and the lung samples were analyzed for the specific immune response and viral load. Clinically, pigs vaccinated with PLGA-NP peptides vaccine had no fever and flu symptoms, and the replicating challenged SwIV was undetectable in the bronchoalveolar lavage fluid. Immunologically, PLGA-NP peptides vaccination (without adjuvant) significantly increased the frequency of antigen-specific IFNγ secreting CD4 and CD8 T cells response in the lung lymphocytes, despite not boosting the antibody response both at pre- and post-challenge. In summary, our data indicated that nanoparticle-mediated delivery of conserved H1N1 influenza peptides induced the virus specific T cell response in the lungs and reduced the challenged heterologous virus load in the airways of pigs.