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Porcine reproductive and respiratory syndrome (PRRS) is a chronic viral disease of pigs caused by PRRS virus (PRRSV). The PRRSV VR2332 is the prototype North American parental strain commonly used in the preparation of vaccines. Goal of this study was to understand missing information on VR2332 induced immune modulation at the lungs and lymphoid tissues, the sites of PRRSV replication. Pigs were infected intranasally and samples collected at post-infection day (PID) 15, 30, and 60. Microscopically, lungs had moderate interstitial pneumonia, and the virus was detected in all the tested tissues. Peak antibody response and the cytokine IFN-γ secretion were detected at PID 30, with increased TGF-β until PID 60. Population of CD8 + , CD4 + , and CD4 + CD8 + T cells, Natural killer (NK) cells, and γδ T cells in the lungs and lymphoid tissues were significantly modulated favoring PRRSV persistence. The NK cell-mediated cytotoxicity was significantly reduced in infected pigs. In addition, increased population of immunosuppressive T-regulatory cells (Tregs) and associated cytokines were also observed in VR2332 strain infected pigs.

Background Porcine reproductive and respiratory syndrome virus (PRRSV) causes chronic, economically devastating disease in pigs of all ages. Frequent mutations in the viral genome result in viruses with immune escape mutants. Irrespective of regular vaccination, control of PRRSV remains a challenge to swine farmers. In PRRSV-infected pigs, innate cytokine IFN-α is inhibited and the adaptive arm of the immunity is delayed. To elucidate both cellular and innate cytokine responses at very early stages of PRRSV infection, seven weeks old pigs maintained on a commercial pig farm were infected and analyzed. Results One pig in a pen containing 25 pigs was PRRSV infected and responses from this pig and one penmate were assessed two days later. All the infected and a few of the contact neighbor pigs were viremic. At day 2 post-infection, approximately 50% of viremic pigs had greater than 50% reduction in NK cell-mediated cytotoxicity, and nearly a 1-fold increase in IFN-α production was detected in blood of a few pigs. Enhanced secretion of IL-4 (in ~90%), IL-12 (in ~40%), and IL-10 (in ~20%) (but not IFN-γ) in PRRSV infected pigs was observed. In addition, reduced frequency of myeloid cells, CD4-CD8+ T cells, and CD4+CD8+ T cells and upregulated frequency of lymphocytes bearing natural T regulatory cell phenotype were detected in viremic pigs. Interestingly, all viremic contact pigs also had comparable immune cell modulations. Conclusion Replicating PRRSV in both infected and contact pigs was found to be responsible for rapid modulation in NK cell-meditated cytotoxicity and alteration in the production of important immune cytokines. PRRSV-induced immunological changes observed simultaneously at both cellular and cytokine levels early post-infection appear to be responsible for the delay in generation of adaptive immunity. As the study was performed in pigs maintained under commercial environmental conditions, this study has practical implications in design of protective vaccines.

Biodegradable nanoparticle-based vaccine development research is unexplored in large animals and humans. In this study, we illustrated the efficacy of nanoparticle-entrapped UV-killed virus vaccine against an economically important respiratory viral disease of pigs called porcine reproductive and respiratory syndrome virus (PRRSV). We entrapped PLGA [poly (lactide-co-glycolides)] nanoparticles with killed PRRSV antigens (Nano-KAg) and detected its phagocytosis by pig alveolar macrophages. Single doses of Nano-KAg vaccine administered intranasally to pigs upregulated innate and PRRSV specific adaptive responses. In a virulent heterologous PRRSV challenge study, Nano-KAg vaccine significantly reduced the lung pathology and viremia, and the viral load in the lungs. Immunologically, enhanced innate and adaptive immune cell population and associated cytokines with decreased secretion of immunosuppressive mediators were observed at both mucosal sites and blood. In summary, we demonstrated the benefits of intranasal delivery of nanoparticle-based viral vaccine in eliciting cross-protective immune response in pigs, a potential large animal model.

Porcine reproductive and respiratory syndrome (PRRS) is an immunosuppressive chronic respiratory viral disease of pigs that is responsible for major economic losses to the swine industry worldwide. The efficacy of parenteral administration of widely used modified live virus PRRS vaccine (PRRS-MLV) against genetically divergent PRRSV strains remains questionable. Therefore, we evaluated an alternate and proven mucosal immunization approach by intranasal delivery of PRRS-MLV (strain VR2332) with a potent adjuvant to elicit cross-protective immunity against a heterologous PRRSV (strain MN184). Mycobacterium tuberculosis whole cell lysate (Mtb WCL) was chosen as a potent mucosal adjuvant due to its Th1 biased immune response to PRRS-MLV. Unvaccinated pigs challenged with MN184 had clinical PRRS with severe lung pathology; however, vaccinated (PRRS-MLV+ Mtb WCL) pigs challenged with MN184 were apparently healthy. There was a significant increase in the body weight gain in vaccinated compared to unvaccinated PRRSV challenged pigs. Vaccinated compared to unvaccinated, virus-challenged pigs had reduced lung pathology associated with enhanced PRRSV neutralizing antibody titers and reduced viremia. Immunologically, an increased frequency of Th cells, Th/memory cells, γδ T cells, dendritic cells, and activated Th cells and a reduced frequency of T-regulatory cells were detected at both mucosal and systemic sites. Further, reduced secretion of immunosuppressive cytokines (IL-10 and TGF-β) and upregulation of the Th1 cytokine IFN-γ in blood and lungs were detected in mucosally vaccinated, PRRSV-challenged pigs. In conclusion, intranasal immunization of pigs with PRRS-MLV administered with Mtb WCL generated effective cross-protective immunity against PRRSV.

Important roles played by invariant natural killer T (iNKT) cells in asthma pathogenesis have been demonstrated. We identified functional iNKT cells and CD1d molecules in pig lungs. Pig iNKT cells cultured in the presence of α-GalCer proliferated and secreted Th1 and Th2 cytokines. Like in other animal models, direct activation of pig lung iNKT cells using α-GalCer resulted in acute airway hyperreactivity (AHR). Clinically, acute AHR-induced pigs had increased respiratory rate, enhanced mucus secretion in the airways, fever, etc. In addition, we observed petechial hemorrhages, infiltration of CD4(+) cells, and increased Th2 cytokines in AHR-induced pig lungs. Ex vivo proliferated iNKT cells of asthma induced pigs in the presence of C-glycoside analogs of α-GalCer had predominant Th2 phenotype and secreted more of Th2 cytokine, IL-4. Thus, baby pigs may serve as a useful animal model to study iNKT cell-mediated AHR caused by various environmental and microbial CD1d-specific glycolipid antigens.

Histamine (HA) is one of the most versatile biogenic amines with multiple physiological functions in the central nervous system (CNS), the respiratory and the intestinal tract due to its ability to induce severe inflammatory reactions. More recently, a number of studies have established that besides its most obvious contribution in allergic reactions, HA also exerts more subtle regulatory functions influencing the orientation of the immune response, thus rekindling interest in this field of investigation. It can influence numerous functions of the cells involved in the regulation of immune responses and hematopoiesis of macrophages, dendritic cells, T lymphocytes, B lymphocytes and endothelial cells. All these cells express histamine receptors and also secrete histamine, which can selectively recruit major effector cells into tissue sites and affect their maturation, activation, polarization, and effector functions leading to chronic inflammation. Histamine regulates antigen-specific T-helper 1 (Th1) and T-helper 2 (Th2) cells, as well as related isotype specific antibody responses. Histamine acts through its receptor called as histamine receptor (H1-H4) subtypes, which positively interferes with the peripheral antigen tolerance induced by T-regulatory cells (Tregs) through several pathways. Natural killer T (NKT) cells are the heterogeneous population of innate immune T cells that have been attracted the attention of many researchers due to their potential to regulate immune responses to a variety of pathogens, tumors, autoimmune diseases etc. A majority of NKT cells in mice are invariant NKT (iNKT) cells and are considered to be immunoregulatory in nature, due to their ability to promptly produce both Th1 and Th2 cytokines rapidly upon activation. In this chapter, we have tried to focus on HA participation in NKT cell activation by functional tuning to ensure optimal cytokine production, which leads to the recruitment and activation of other immune cells involved in inflammatory responses mediated through eosinophils, mast cells, neutrophils, conventional T lymphocytes, dendritic cells etc.

The present study was envisaged to evaluate potential of combination therapy comprising of immunomodulator picroliv and antimalarial chloroquine against drug resistant Plasmodium yoelii (P. yoelii) infection in BALB/c mice.The immunomodulatory potential of picroliv was established by immunizing animals with model antigen along with picroliv. Immune response was assessed using T-cell proliferation assay and also by determining the antibody isotype-profile induced in the immunized mice. In the next set of experiment, prophylactic potential of picroliv to strengthen antimalarial properties of chloroquine against P. yoelii (MDR) infection in BALB/c mice was assessed.T-cell proliferation as well as antibody production study reveals that picroliv helps in evoking strong immuno-potentiating response against model antigen in the immunized mice. Co-administration of picroliv enhances efficacy of CHQ against experimental murine malaria.The activation of host immune system can increase the efficacy of chloroquine for suppression of drug resistant malaria infection in BALB/c mice.

In our previous studies we established fusogenic properties of lipids isolated from edible yeast Saccharomyces cerevisiae ( S. cerevisiae ). We demonstrated that liposomes prepared from S. cerevisiae membrane lipid (saccharosome) can deliver encapsulated antigen into cytosol of the antigen presenting cells and elicit antigen specific cell mediated as well as humoral immune responses. In this study, we evaluated immunological behavior of saccharosome encapsulated cytosolic proteins (sAg) of Plasmodium yoelii nigeriensis in BALB/c mice . Immunization with antigen (sAg) encapsulated in saccharosome resulted in enhancement of CD4 + and CD8 + T cell populations and also up-regulated the expression of CD80 and CD86 molecules on the surface of antigen presenting cells. Further, immunization with saccharosome-encapsulated sAg-induced elevated levels of both IFN-γ and IL-4 cytokines in the immunized mice when compared to egg PC liposome encapsulated sAg or its IFA emulsified form. Saccharosome-mediated immunization resulted in induction of high level of total antibody response with preponderance of IgG2a isotype as well. The data of this study suggest that saccharosome-based vehicle can emerge as an effective vaccine in imparting protection against various intracellular pathogens including Plasmodium yoelii nigeriensis.

Triton X-114 phase separated integral membrane proteins (IMPs) of a multidrug resistant strain of Plasmodium yoelii nigeriensis (P. yoelii) were screened for their potential to impart protection against malaria infection in BALB/c mice. As revealed by immunoblotting, antibodies present in parasite specific sera from convalescent (protected) as well as immunized (partially protected) animals recognized different membrane proteins. A thorough investigation reveals that P. yoelii specific convalescent sera recognized IMPs with molecular masses ranging from 21 to 81 kDa. Among various membrane proteins, the IMPs corresponding to 81 and 66 kDa molecular weight were highly prominent in the immunoblots probed with the sera from convalescent animals, whereas sera from immunized animals failed to produce impressive band pattern. Immunofluorescence assay revealed that the 66-kDa IMP specific antibodies reacted with fixed smears of mature schizonts and merozoites. Further immunization with 66 kDa IMP (PyIMP) purified through polyclonal IgG sepharose 4B affinity did not impart effective immune response (in its free form) and could provided partial protection only. On the other hand, animals immunized with 66 kDa PyIMP entrapped in phosphatidyl-choline/cholesterol (PC/chol) liposomes protected BALB/c mice against lethal P. yoelii challenge.

Earlier we have demonstrated that novel fusogenic liposomes made up of lipid from Escherichia coli (escheriosomes) have strong tendency to fuse with the plasma membrane of target cells and thereby delivering the entrapped contents into their cytosol. The delivery of entrapped antigen in cytosol of the target cells ensues its processing and presentation along with MHC class I pathway that eventually elicit antigen specific cytotoxic T cells. The result of the present study revealed that immunization of BALB/c mice with escheriosome-encapsulated Salmonella typhimurium (S. typhimurium) cytosolic antigens resulted in the augmentation of antigen specific cytotoxic T cell lymphocyte as well as IgG responses. In contrast, free or conventional liposome (PC liposome) encapsulated antigen failed to induce CD8+ CTLs in the immunized animals. Further, immunization with escheriosome-encapsulated antigen resulted in significant enhancement in the release of IFN-gamma and IgG2a in the experimental animals. Interestingly, the immunization with escheriosome-encapsulated antigen resulted in upregulation of CD80 and CD86 on the surface of antigen presenting cells (APCs) as well. Finally, the results of the present study reveal that immunization of animals with escheriosomes encapsulated antigen protected them against virulent S. typhimurium infection. This was evident by increased survival, and reduced bacterial burden in vital organs of the immunized animals. The data of the present study suggest that escheriosomes can emerge as an effective vehicle for intracellular delivery of antigen and thus hold promise in development of liposome based vaccine against Salmonella and other intracellular pathogens.

Lung immunopathology is the major cause of influenza induced morbidity and mortality. DAP12 (DNAX-Activating Protein of 12kDa) is a membrane adaptor protein associated with varied surface receptors, and it is known to regulate influenza induced lung immunopathology. DAP12 associated receptor expression and their association with phenotype of bronchoalveolar lavage fluid (BAL) cells during swine influenza virus (SIV) infection in pigs is unknown. Since pig is a suitable large animal model for influenza research, our aim was to understand the effects of zoonotic SIV H1N1 infection on expression of DAP12 associated MDL-1, TREM-1 and TREM-2 receptors in pig BAL cells through qRT-PCR profiling. Results indicated that DAP12, MDL-1 and TREM-1 were constitutively expressed, but TREM-2 was upregulated. Stimulation of uninfected pig BAL cells in vitro using the cytokines IFNgamma and IL4 revealed that upregulation of TREM-2 was associated with enhanced expression of ARG1, but not with high levels of expression of TNFalpha and iNOS. ARG1 is a phenotype of alternatively activated alveolar macrophages that are known to protect lung tissue; hence TREM2 upregulation in pig BAL cells with similar phenotype suggests the possible beneficial role of these molecules in preventing the lung immunopathology. In conclusion, DAP12 and associated receptors are differentially expressed in pig BAL cells and TREM2 appears to have a beneficial role in regulating the lung immunopathology in SIV infection.

Porcine reproductive and respiratory syndrome (PRRS) is caused by a highly mutagenic RNA virus, PRRS virus (PRRSV). Currently used vaccines have failed to prevent PRRS outbreaks induced losses of estimated one billion dollar annually to the US pig farmers. The PRRSV infects primarily the lung macrophages. Because of safety advantage, we attempted to strengthen the immunogenicity of killed PRRSV vaccine antigens (KAg) in the pig respiratory system. We entrapped the KAg in biodegradable PLGA nanoparticles (NP-KAg) and coadministered the NP-KAg with a potent mucosal adjuvant, whole cell lysate of Mycobacterium tuberculosis (M. tb WCL), twice intranasally to growing pigs and challenged with a heterologous virus. Our results indicated that formulation of NP-KAg and unentrapped soluble M. tb WCL significantly cleared detectable replicating infective PRRSV with a 10-fold reduction in viral RNA load in the lungs, associated with substantially reduced microscopic lung pathology. Immunologically, enhanced virus neutralizing antibody titers by high avidity antibodies and augmented populations of IFN-γ secreting CD4+ and CD8+ lymphocytes, and reduced secretion of TGFbeta was detected in the lungs. Currently, we are in the process of identifying PRRSV epitope-specific T-cell response and frequency of apoptotic/necrotic immune cells in the lungs of vaccinated pigs. In conclusion, our vaccine formulation elicited broadly cross-protective anti-PRRSV immunity in the pig respiratory system.

Influenza A virus (IAV) and Streptococcus pneumoniae (pneumococcus) are two major upper respiratory tract pathogens responsible for exacerbated disease in coinfected individuals. Information on the direct effect of S. pneumoniae on IAV replication in humans is unknown. Such an investigation is important because S. pneumoniae is a commensal of the human upper respiratory tract. The aim of this study was to determine whether treatment of epithelial cells with S. pneumoniae affects IAV replication, determined with the help of a standard immunofluorescence assay (IFA). For this study, four IAV permissive epithelial cell lines including two human-derived cell lines, 12 pneumococcal strains including recent human clinical isolates which represent different genetic backgrounds and serotypes, and six IAV strains of varying genetic nature and pathogenic potential including the pandemic 2009 H1N1 virus were used. Our results suggested that pretreatment of MDCK cells with 7.5×10^6 colony-forming unit (CFU) of live S. pneumoniae resulted in gradual cell-death in a time-dependent manner (1/2 hr to 4 hr). However, pretreatment of cells with 7.5×10^5 and lower CFU of S. pneumoniae had no detectable effect on either the morphology of epithelial cells or on the IAV replication. However, the effect of pneumococci on IAV replication may be different during a coinfection in vivo in the human upper respiratory tract.

Influenza A virus (IAV) and Streptococcus pneumoniae (pneumococcus) are two major upper respiratory tract pathogens responsible for exacerbated disease in coinfected individuals. Despite several studies showing increased susceptibility to secondary bacterial infections following IAV infection, information on the direct effect of S. pneumoniae on IAV in vitro is unknown. This is an important area of investigation as S. pneumoniae is a common commensal of the human upper respiratory tract, present as an important coinfecting pathogen with IAV infection. A recent study showed that S. pneumoniae enhances human metapneumovirus infection in polarized bronchial epithelial cells in vitro. The aim of the current study was to determine whether treatment of epithelial cells with S. pneumoniae affects IAV replication using a standard immunofluorescence assay (IFA). For this study we used four IAV permissive epithelial cell lines including two human-derived cell lines, 12 pneumococcal strains including recent human clinical isolates which represent different genetic backgrounds and serotypes, and six IAV strains of varying genetic nature and pathogenic potential including the pandemic 2009 H1N1 virus. Our results suggested that pretreatment of MDCK cells with 7.5×10(6) colony-forming units (CFUs) of live S. pneumoniae resulted in gradual cell-death in a time-dependent manner (0.5 to 4 hr). But, pretreatment of cell lines with 7.5×10(5) and lower CFUs of S. pneumoniae had no detectable effect on either the morphology of cells or on the IAV replication. However, unlike in epithelial cell lines, due to influence of secreted host factors the effect of pneumococci on IAV replication may be different during coinfections in vivo in the human upper respiratory tract, and in vitro with primary human polarized bronchial epithelial cells.

Swine influenza virus (SIV) causes an acute respiratory disease in pigs, and pigs are infectable by both avian and mammalian influenza viruses. Activation of NKT cell induces heterologous protection against influenza viruses in rodent studies. We discovered CD1d-restricted NKT cell in pigs. Aim of this study was to determine the efficacy of UV-inactivated bivalent SIV vaccine, comprising of triple reassortant zoonotic H1N1 (Sw/OH/24366/07) and H3N2 (Sw/OH/04) viruses; coadministered intranasally with NKT cell adjuvants, phosphatidylinositolmannosides-2 (PIM2) and α-Galctosylceramide (α-GalCer). In vaccinated homologous H1N1 virus challenged pigs, reduced viral load in the lungs associated with increased IFN-γ+ lymphocytes in the lungs and tracheobronchial lymph nodes by ELISPOT, and an increase in IFN-γ+CD8+ and IFN-γ+ γδ T cells was observed. Further, increased virus specific IgA response in the BAL fluid and enhanced lung NK cell-cytotoxicity was detected. However, in vaccinated pandemic 2009 H1N1 virus challenged pigs, enhanced nasal viral shedding and lung viral load was detected. Immunologically, reduced frequency of total lymphocytes and CD8+ T cells, and reduced frequency of total IFN-γ+ T cells was detected in the lungs. In conclusion, NKT cell adjuvanted inactivated SIV vaccine in pigs enhanced the pandemic 2009 H1N1 replication and dampened the anti-viral immune response.

Porcine reproductive and respiratory syndrome (PRRS) is an economically very important endemic chronic respiratory viral disease of pigs. Live PRRS virus (PRRSV) vaccines are not safe in pregnant sows and boars, and currently used killed vaccines are ineffective. In this study, killed PRRSV Ags were entrapped in poly-DL-lactide-co-glycolide (PLGA) nanoparticles (100-500nm by SEM) with an approximate 50% entrapment efficiency. Internalization and localization of nanoparticles-PRRSV Ags inside the endosomes of pig alveolar macrophages was demonstrated by confocal microscopy. Immune correlates of protection were analyzed in nanoparticles-PRRSV Ags immunized and genetically variant PRRSV challenged pigs, inoculated intranasally, at post-challenge day 15. In nanoparticles immunized pig lungs, reduced PRRSV load and reduced gross lung lesions were detected compared to pigs unimmunized or immunized with free killed PRRSV Ags and challenged. Immunologically, increased frequency of lymphocytes, CD4+, CD8+, γδ T cells, myeloid cells, and reduced frequency of Tregs were detected in lungs, blood, and tracheobronchial lymph nodes. In addition, increased secretion of cytokines, IFN-γ, IL-6, and reduced IL-10 and TGF-β by ex vivo restimulated mucosal immune cells was detected. Thus, our results suggested that mucosal delivery of nanoparticles- PRRSV killed Ags has the potential to control PRRS.

Biomedical research in pigs is critical for better human health, as they are immunologically and physiologically more similar to humans, and are an excellent animal model to alleviate human asthma. Natural Killer T (NKT) cells with an invariant TCR are called "invariant NKT" (iNKT). iNKT cells are activated by CD1d-bound glycolipids with a unique α-anomerically linked sugar (e.g. α-GalCer). For the first time in pigs, we detected iNKT cells and CD1d expression. The pig iNKT cells proliferated and secreted both Th1 and Th2 cytokines when cultured in the presence of α-GalCer. In particular, iNKT cells from lung, and lung draining lymph nodes proliferated to higher numbers (30%), with the secretion of more cytokines, compared to iNKT cells from other tissues. Recently, “many pivotal roles” played by iNKT cells in asthma pathogenesis have been reported. Direct activation of pig lung iNKT cells using α-GalCer resulted in airway hyperreactivity (AHR), and clinically those pigs had dyspnea with elevated body temperature. The AHR induced pig lung had petechial hemorrhages with increased mucus secretion in the airway. The α-GalCer received pigs had three-fold increase in cytokines, IL-6, IL-12 and IL-10, and two-fold increase in CD1d expression on the dendritic cells and macrophages at post-24 of inoculation. A moderate increase in iNKT cells in lungs of AHR induced pigs was also detected, compared to controls. This work is supported by USDA and NPB grants to RJG.