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1.
Virology ; 575: 101-110, 2022 10.
Article in English | MEDLINE | ID: mdl-36096069

ABSTRACT

Respiratory syncytial virus is an important cause of pneumonia in children, the elderly, and immunocompromised individuals. The attachment (G) protein of RSV generates neutralizing antibodies in natural RSV infection which correlate with protection against disease. The immune response to RSV is typically short-lived, which may be related to the heavy glycosylation of RSV-G. In order to improve its immunogenicity, we expressed G protein mutants in a vesicular stomatitis virus (VSV) vector system and tested their ability to protect cotton rats from RSV challenge. We found that the most protective construct was codon-optimized RSV-G, followed by wild-type G and membrane-bound G. Constructs which expressed the G protein with reduced glycosylation or the secreted G protein provided either partial or no protection. Our results demonstrate that modifications to the G protein are not advantageous in a VSV vector system, and that an intact, codon-optimized G is a superior vaccine candidate.


Subject(s)
Respiratory Syncytial Virus Infections , Respiratory Syncytial Virus Vaccines , Respiratory Syncytial Virus, Human , Vesicular Stomatitis , Animals , Antibodies, Neutralizing , Antibodies, Viral , Codon , GTP-Binding Proteins , Immunity , Respiratory Syncytial Virus Vaccines/genetics , Respiratory Syncytial Virus, Human/genetics , Sigmodontinae , Vesicular stomatitis Indiana virus , Vesiculovirus/genetics , Viral Fusion Proteins/genetics
2.
Vaccine ; 39(47): 6817-6828, 2021 11 16.
Article in English | MEDLINE | ID: mdl-34702618

ABSTRACT

Respiratory syncytial virus (RSV) is one of the most important causes of respiratory disease in infants, immunocompromised individuals, and the elderly. Natural infection does not result in long-term immunity, and there is no licensed vaccine. Vesicular stomatitis virus (VSV) is a commonly used vaccine vector platform against infectious diseases, and has been used as a vector for a licensed Ebola vaccine. In this study, we expressed the RSV fusion (F) protein, the RSV F protein stabilized in either a pre-fusion or a post-fusion configuration, the attachment glycoprotein (G), or the G and F proteins of RSV in combination in a VSV vector. Cotton rats were immunized with these recombinants intranasally or subcutaneously to test immunogenicity. RSV F stabilized in either a pre-fusion or a post-fusion configuration proved to be poorly immunogenic and protective when compared to unmodified F. RSV G provided partial protection and moderate levels of neutralizing antibody production, both of which improved with intranasal administration compared to subcutaneous inoculation. The most successful vaccine vector was VSV expressing both the G and F proteins after intranasal inoculation. Immunization with this recombinant induced neutralizing antibodies and provided protection from RSV challenge in the upper and lower respiratory tract for at least 80 days. Our results demonstrate that co-expression of F and G proteins in a VSV vector provides synergistic effects in inducing RSV-specific neutralizing antibodies and protection against RSV infection.


Subject(s)
Ebola Vaccines , Hemorrhagic Fever, Ebola , Respiratory Syncytial Virus Infections , Respiratory Syncytial Virus Vaccines , Respiratory Syncytial Virus, Human , Vesicular Stomatitis , Animals , Antibodies, Neutralizing , Antibodies, Viral , Glycoproteins/genetics , Respiratory Syncytial Virus Infections/prevention & control , Sigmodontinae , Viral Fusion Proteins/genetics
3.
J Virol ; 95(6)2021 02 24.
Article in English | MEDLINE | ID: mdl-33408176

ABSTRACT

Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract (LRT) infections, with increased severity in high-risk human populations, such as infants, the immunocompromised, and the elderly. Although the virus was identified more than 60 years ago, there is still no licensed vaccine available. Over the years, several vaccine delivery strategies have been evaluated. In this study, we developed two recombinant vesicular stomatitis virus (rVSV) vector-based vaccine candidates expressing the RSV-G (attachment) protein (rVSV-G) or F (fusion) protein (rVSV-F). All vectors were evaluated in the cotton rat animal model for their in vivo immunogenicity and protective efficacy against an RSV-A2 virus challenge. Intranasal (i.n.) delivery of rVSV-G and rVSV-F together completely protected the lower respiratory tract (lungs) at doses as low as 103 PFU. In contrast, doses greater than 106 PFU were required to protect the upper respiratory tract (URT) completely. Reimmunization of RSV-immune cotton rats was most effective with rVSV-F. In immunized animals, overall antibody responses were sufficient for protection, whereas CD4 and CD8 T cells were not necessary. A prime-boost immunization regimen increased both protection and neutralizing antibody titers. Overall, mucosally delivered rVSV-vector-based RSV vaccine candidates induce protective immunity and therefore represent a promising immunization regimen against RSV infection.IMPORTANCE Even after decades of intensive research efforts, a safe and efficacious RSV vaccine remains elusive. Expression of heterologous antigens from rVSV vectors has demonstrated several practical and safety advantages over other virus vector systems and live attenuated vaccines. In this study, we developed safe and efficacious vaccine candidates by expressing the two major immunogenic RSV surface proteins in rVSV vectors and delivering them mucosally in a prime-boost regimen. The main immune parameter responsible for protection was the antibody response. These vaccine candidates induced complete protection of both the upper and lower respiratory tracts.


Subject(s)
Respiratory Syncytial Virus Infections/prevention & control , Respiratory Syncytial Virus Vaccines/administration & dosage , Respiratory Syncytial Virus, Human/immunology , Vesiculovirus/genetics , Viral Envelope Proteins/immunology , Viral Fusion Proteins/immunology , Administration, Mucosal , Animals , Disease Models, Animal , Genetic Vectors , Immunity, Cellular , Immunity, Humoral , Immunization , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/metabolism , Respiratory Syncytial Virus Infections/immunology , Respiratory Syncytial Virus Vaccines/immunology , Respiratory Syncytial Virus, Human/genetics , Respiratory System/immunology , Respiratory System/virology , Sigmodontinae , Vaccines, Attenuated/administration & dosage , Vaccines, Attenuated/immunology , Vesiculovirus/metabolism , Viral Envelope Proteins/genetics , Viral Envelope Proteins/metabolism , Viral Fusion Proteins/genetics , Viral Fusion Proteins/metabolism
4.
J Control Release ; 247: 194-205, 2017 02 10.
Article in English | MEDLINE | ID: mdl-28057521

ABSTRACT

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.


Subject(s)
Antigens, Viral/administration & dosage , Influenza A Virus, H1N1 Subtype/immunology , Influenza Vaccines/administration & dosage , Lactic Acid/chemistry , Nanoparticles/chemistry , Orthomyxoviridae Infections/prevention & control , Polyglycolic Acid/chemistry , Vaccines, Inactivated/administration & dosage , Administration, Intranasal , Animals , Antigens, Viral/immunology , Antigens, Viral/therapeutic use , Cells, Cultured , Dogs , Immunity, Cellular , Influenza Vaccines/immunology , Influenza Vaccines/therapeutic use , Madin Darby Canine Kidney Cells , Orthomyxoviridae Infections/immunology , Polylactic Acid-Polyglycolic Acid Copolymer , Swine , Vaccines, Inactivated/immunology , Vaccines, Inactivated/therapeutic use
5.
PLoS One ; 11(4): e0151922, 2016.
Article in English | MEDLINE | ID: mdl-27093541

ABSTRACT

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.


Subject(s)
Influenza A Virus, H1N1 Subtype/immunology , Influenza Vaccines/chemistry , Lactic Acid/chemistry , Nanoparticles/chemistry , Peptides/chemistry , Polyglycolic Acid/chemistry , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , Animals , Antibodies, Viral/immunology , Cell Line , Dogs , Influenza A Virus, H1N1 Subtype/chemistry , Influenza Vaccines/immunology , Interferon-gamma/immunology , Lactic Acid/immunology , Lung/immunology , Lung/virology , Madin Darby Canine Kidney Cells , Nanoparticles/administration & dosage , Orthomyxoviridae Infections/immunology , Peptides/immunology , Polylactic Acid-Polyglycolic Acid Copolymer , Swine , Swine Diseases/immunology , Vaccines, Inactivated/chemistry , Vaccines, Inactivated/immunology
6.
Vet Res ; 47: 45, 2016 Mar 17.
Article in English | MEDLINE | ID: mdl-26988085

ABSTRACT

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.


Subject(s)
Adjuvants, Immunologic/therapeutic use , Administration, Intranasal/veterinary , Injections, Intramuscular/veterinary , Mycobacterium/immunology , Porcine Reproductive and Respiratory Syndrome/prevention & control , Porcine respiratory and reproductive syndrome virus/immunology , Viral Vaccines/immunology , Adjuvants, Immunologic/administration & dosage , Animals , Antibodies, Viral/blood , Cross Protection , Immunity, Heterologous , Oligodeoxyribonucleotides/administration & dosage , Oligodeoxyribonucleotides/therapeutic use , Porcine Reproductive and Respiratory Syndrome/immunology , Porcine Reproductive and Respiratory Syndrome/virology , Swine , Vaccines, Attenuated/administration & dosage , Vaccines, Attenuated/immunology , Vaccines, Attenuated/therapeutic use , Viral Vaccines/administration & dosage , Viral Vaccines/therapeutic use
7.
Vet Microbiol ; 186: 157-63, 2016 Apr 15.
Article in English | MEDLINE | ID: mdl-27016770

ABSTRACT

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.


Subject(s)
Adaptive Immunity/drug effects , Galactosylceramides/pharmacology , Immunity, Innate/drug effects , Influenza A Virus, H1N1 Subtype/immunology , Influenza Vaccines/immunology , Orthomyxoviridae Infections/veterinary , Swine Diseases/immunology , Adaptive Immunity/immunology , Adjuvants, Immunologic/pharmacology , Animals , Cytokines/immunology , Immunity, Innate/immunology , Immunoglobulin A/blood , Lung/virology , Natural Killer T-Cells/chemistry , Natural Killer T-Cells/immunology , Orthomyxoviridae Infections/immunology , Orthomyxoviridae Infections/physiopathology , Random Allocation , Swine , Swine Diseases/physiopathology , Vaccines, Inactivated/immunology , Viral Load , Weight Gain/drug effects
8.
Arch Virol ; 161(6): 1579-89, 2016 Jun.
Article in English | MEDLINE | ID: mdl-27008569

ABSTRACT

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.


Subject(s)
Porcine Reproductive and Respiratory Syndrome/immunology , Porcine Reproductive and Respiratory Syndrome/prevention & control , Porcine respiratory and reproductive syndrome virus/immunology , Viral Vaccines/immunology , Animals , Antibodies, Viral/blood , Cell Line , Cytokines/metabolism , Genes, Viral , Lung/immunology , Lung/virology , Porcine Reproductive and Respiratory Syndrome/virology , Porcine respiratory and reproductive syndrome virus/genetics , Sus scrofa , Swine , Vaccines, Inactivated/genetics , Vaccines, Inactivated/immunology , Vaccines, Virus-Like Particle/genetics , Vaccines, Virus-Like Particle/immunology , Viral Envelope Proteins/genetics , Viral Envelope Proteins/immunology , Viral Load , Viral Vaccines/genetics
9.
J Virol ; 90(7): 3584-99, 2016 Jan 20.
Article in English | MEDLINE | ID: mdl-26792733

ABSTRACT

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.


Subject(s)
Host-Pathogen Interactions , Immune Evasion , Immunity, Innate , Porcine respiratory and reproductive syndrome virus/immunology , Viral Nonstructural Proteins/metabolism , Amino Acid Substitution , Animals , Cell Line , DNA Mutational Analysis , Interferon-beta/metabolism , Mutagenesis, Site-Directed , Mutant Proteins/genetics , Mutant Proteins/metabolism , Mutation, Missense , Swine , Viral Nonstructural Proteins/genetics
10.
Vet Res ; 46: 140, 2015 Dec 14.
Article in English | MEDLINE | ID: mdl-26667229

ABSTRACT

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.


Subject(s)
Antibodies, Viral/metabolism , B-Lymphocytes/metabolism , Coronavirus Infections/veterinary , Immunity, Humoral , Immunoglobulin A/metabolism , Immunoglobulin G/metabolism , Porcine epidemic diarrhea virus/physiology , Swine Diseases/immunology , Animals , Antibodies, Viral/blood , Coronavirus Infections/immunology , Coronavirus Infections/virology , Feces/virology , Female , Immunoglobulin A/blood , Immunoglobulin G/blood , Intestines/immunology , Intestines/virology , Lymphoid Tissue/immunology , Lymphoid Tissue/virology , Parity , Swine , Swine Diseases/virology
11.
Int J Nanomedicine ; 9: 1519-35, 2014.
Article in English | MEDLINE | ID: mdl-24711701

ABSTRACT

Porcine reproductive and respiratory syndrome (PRRS) is an economically devastating respiratory disease of pigs. The disease is caused by the PRRS virus (PRRSV), an Arterivirus which is a highly mutating RNA virus. Widely used modified live PRRSV vaccines have failed to prevent PRRS outbreaks and reinfections; moreover, safety of the live virus vaccines is questionable. Though poorly immunogenic, inactivated PRRSV vaccine is safe. The PRRSV infects primarily the lung macrophages. Therefore, we attempted to strengthen the immunogenicity of inactivated/killed PRRSV vaccine antigens (KAg), especially in the pig respiratory system, through: 1) entrapping the KAg in biodegradable poly(lactic-co-glycolic acid) nanoparticles (NP-KAg); 2) coupling the NP-KAg with a potent mucosal adjuvant, whole cell lysate of Mycobacterium tuberculosis (M. tb WCL); and 3) delivering the vaccine formulation twice intranasally to growing pigs. We have previously shown that a single dose of NP-KAg partially cleared the challenged heterologous PRRSV. Recently, we reported that NP-KAg coupled with unentrapped M. tb WCL significantly cleared the viremia of challenged heterologous PRRSV. Since PRRSV is primarily a lung disease, our goal in this study was to investigate lung viral load and various immune correlates of protection at the lung mucosal surfaces and its parenchyma in vaccinated heterologous PRRSV-challenged pigs. Our results indicated that out of five different vaccine-adjuvant formulations, the combination of NP-KAg and unentrapped M. tb WCL significantly cleared detectable replicating infective PRRSV with a tenfold reduction in viral RNA load in the lungs, associated with substantially reduced gross and microscopic lung pathology. Immunologically, strong humoral (enhanced virus neutralization titers by high avidity antibodies) and cell-mediated immune responses (augmented population of interferon-γ secreting CD4(+) and CD8(+) lymphocytes and reduced secretion of immunosuppressive cytokines) in the lungs were observed. In conclusion, combination of NP-KAg and soluble M. tb WCL elicits broadly cross-protective anti-PRRSV immunity in the pig respiratory system.


Subject(s)
Cross Protection/immunology , Lactic Acid/chemistry , Nanocapsules/administration & dosage , Nanocapsules/chemistry , Polyglycolic Acid/chemistry , Porcine Reproductive and Respiratory Syndrome/immunology , Porcine Reproductive and Respiratory Syndrome/prevention & control , Viral Vaccines/administration & dosage , Animals , Lung/immunology , Lung/virology , Polylactic Acid-Polyglycolic Acid Copolymer , Swine , Treatment Outcome , Vaccines, Inactivated/administration & dosage , Vaccines, Inactivated/chemistry , Vaccines, Inactivated/immunology , Viral Vaccines/chemistry
12.
PLoS One ; 9(3): e90066, 2014.
Article in English | MEDLINE | ID: mdl-24594847

ABSTRACT

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.


Subject(s)
Epithelial Cells/microbiology , Influenza A virus/physiology , Streptococcus pneumoniae/physiology , Virus Replication , Animals , Calibration , Dogs , In Vitro Techniques , Madin Darby Canine Kidney Cells , Microscopy, Fluorescence , Streptococcus pneumoniae/classification
13.
Int J Nanomedicine ; 9: 679-94, 2014.
Article in English | MEDLINE | ID: mdl-24493925

ABSTRACT

Porcine reproductive and respiratory syndrome (PRRS), caused by the PRRS virus (PRRSV), is an economically devastating disease, causing daily losses of approximately $3 million to the US pork industry. Current vaccines have failed to completely prevent PRRS outbreaks. Recently, we have shown that poly(lactic-co-glycolic) acid (PLGA) nanoparticle-entrapped inactivated PRRSV vaccine (NP-KAg) induces a cross-protective immune response in pigs. To further improve its cross-protective efficacy, the NP-KAg vaccine formulation was slightly modified, and pigs were coadministered the vaccine twice intranasally with a potent adjuvant: Mycobacterium tuberculosis whole-cell lysate. In vaccinated virulent heterologous PRRSV-challenged pigs, the immune correlates in the blood were as follows: 1) enhanced PRRSV-specific antibody response with enhanced avidity of both immunoglobulin (Ig)-G and IgA isotypes, associated with augmented virus-neutralizing antibody titers; 2) comparable and increased levels of virus-specific IgG1 and IgG2 antibody subtypes and production of high levels of both T-helper (Th)-1 and Th2 cytokines, indicative of a balanced Th1-Th2 response; 3) suppressed immunosuppressive cytokine response; 4) increased frequency of interferon-γ(+) lymphocyte subsets and expanded population of antigen-presenting cells; and most importantly 5) complete clearance of detectable replicating challenged heterologous PRRSV and close to threefold reduction in viral ribonucleic acid load detected in the blood. In conclusion, intranasal delivery of adjuvanted NP-KAg vaccine formulation to growing pigs elicited a broadly cross-protective immune response, showing the potential of this innovative vaccination strategy to prevent PRRS outbreaks in pigs. A similar approach to control other respiratory diseases in food animals and humans appears to be feasible.


Subject(s)
Cross Protection/immunology , Lactic Acid/chemistry , Nanocapsules/administration & dosage , Nanocapsules/chemistry , Polyglycolic Acid/chemistry , Porcine Reproductive and Respiratory Syndrome/immunology , Porcine Reproductive and Respiratory Syndrome/prevention & control , Viral Vaccines/administration & dosage , Animals , Polylactic Acid-Polyglycolic Acid Copolymer , Swine , Treatment Outcome , Vaccines, Inactivated/administration & dosage , Vaccines, Inactivated/chemistry , Vaccines, Inactivated/immunology , Viral Vaccines/chemistry
14.
Vet J ; 198(1): 158-63, 2013 Oct.
Article in English | MEDLINE | ID: mdl-24011474

ABSTRACT

The effect of sampling material, sample processing, and collection order on the detection of analytes in pig oral fluid specimens was evaluated. Oral fluid samples were collected from 104 pens of commercial wean-to-finish pigs using ropes made of three different materials. Processed (centrifuged and filtered) and unprocessed oral fluid samples were tested using commercial ELISAs for porcine reproductive and respiratory syndrome virus (PRRSV) antibodies and total IgM, IgA, and IgG. Unprocessed samples were tested for PRRSV nucleic acid and processed samples were assayed for PRRSV neutralizing antibodies. Analysis of the data using repeated measures ANOVA and Tukey-Kramer adjusted t tests found statistically significant, non-uniform, and assay-dependent effects of all three factors. Therefore, when testing oral fluid specimens, swine health specialists, veterinarians, and diagnosticians should be aware of the potential impact of these factors on specific analytes. For monitoring health and welfare parameters, oral fluid samples should be collected using cotton-based materials and undergo minimal post-collection processing.


Subject(s)
Enzyme-Linked Immunosorbent Assay/methods , Porcine Reproductive and Respiratory Syndrome/immunology , Porcine respiratory and reproductive syndrome virus/isolation & purification , Real-Time Polymerase Chain Reaction/methods , Saliva/chemistry , Specimen Handling/methods , Swine Diseases/immunology , Animals , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/metabolism , Antibodies, Viral/blood , Antibodies, Viral/metabolism , Enzyme-Linked Immunosorbent Assay/veterinary , Immunoglobulin Isotypes/blood , Immunoglobulin Isotypes/metabolism , Porcine Reproductive and Respiratory Syndrome/virology , Real-Time Polymerase Chain Reaction/veterinary , Reverse Transcriptase Polymerase Chain Reaction/veterinary , Specimen Handling/veterinary , Swine , Swine Diseases/virology
15.
Clin Vaccine Immunol ; 20(8): 1305-13, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23784856

ABSTRACT

Porcine reproductive and respiratory syndrome virus (PRRSV)-specific neutralizing antibodies (NA) are important for clearing the virus. Pen-based pig oral fluid samples for disease surveillance are gaining in importance due to the ease of collection and low cost. The aim of this study was to develop a PRRSV-specific NA assay to determine NA titers in pig oral fluid samples. At first, we standardized the PRRSV NA assay using pen-based pig oral fluid samples collected over a period of 3 months from a herd of swine that received a PRRSV modified live vaccine (PRRS-MLV), and we also used oral fluid and serum samples collected from individual boars that were vaccinated with PRRS-MLV or infected with a virulent PRRSV strain. Our results suggest that a PRRSV NA titer of >8 in oral fluid samples is virus specific and can be detected beginning at 28 days after vaccination or infection. To validate the assay, we used 104 pen-based pig oral fluid and five representative serum samples from each pen of unknown history, as well as 100 serum samples from repeatedly vaccinated sows and oral fluid samples of their respective litters belonging to four different swine-breeding farms. Our results demonstrated that PRRSV NA titers in oral fluid samples are correlated with serum sample titers, and maternally derived PRRSV-specific NA titers could be detected in litters at the time of weaning. In conclusion, we have standardized and validated the pig oral fluid-based PRRSV NA assay, which has 94.3% specificity and 90.5% repeatability. The assay can be used to monitor herd immunity against PRRSV in vaccinated and infected herds of swine.


Subject(s)
Antibodies, Neutralizing/analysis , Antibodies, Viral/analysis , Mouth/immunology , Neutralization Tests/methods , Porcine Reproductive and Respiratory Syndrome/immunology , Porcine respiratory and reproductive syndrome virus/immunology , Saliva/immunology , Animals , Reproducibility of Results , Sensitivity and Specificity , Swine , Time Factors , Veterinary Medicine/methods
16.
Vet Microbiol ; 166(1-2): 47-58, 2013 Sep 27.
Article in English | MEDLINE | ID: mdl-23764272

ABSTRACT

Porcine reproductive and respiratory syndrome (PRRS) is a chronic viral disease of pigs, has been posing a huge economic concern to pig industry worldwide. In this study, we developed biodegradable PLGA [poly(d,l-lactide-co-glycolide)] nanoparticle-entrapped killed PRRSV vaccine (Nano-KAg), and administered intranasally to pigs once and evaluated the immune correlates. In Nano-KAg vaccinated homologous virus challenged pigs, complete clearance of viremia was observed in 2 weeks, associated with a significant increase in virus neutralizing titers only in the lungs, compared to both unvaccinated and killed vaccine vaccinated pigs. The lung homogenate and sera of Nano-KAg vaccinated pigs had higher levels of IFN-γ and lower levels of TGF-ß than control groups. Restimulation of mononuclear cells isolated from the lungs, blood, BAL, and TBLN of Nano-KAg vaccinated pigs' secreted significantly increased levels of Th1 cytokines, IFN-γ and IL-12. In addition, higher frequencies of CD3(+)CD8(+), CD4(+)CD8(+), and γδ T cells, and reduced frequency of Foxp3(+) T-regulatory cells were observed in Nano-KAg vaccinated pigs. Thus, intranasal delivery of Nano-KAg vaccine may be a suitable strategy to elicit anti-PRRSV immune response required to better clear viremia in pigs.


Subject(s)
Porcine Reproductive and Respiratory Syndrome/prevention & control , Porcine respiratory and reproductive syndrome virus/immunology , Viral Vaccines/immunology , Administration, Intranasal , Animals , Antibodies, Viral/blood , CD8-Positive T-Lymphocytes/immunology , Cytokines/genetics , Cytokines/immunology , Lactic Acid/chemistry , Lung/immunology , Lung/virology , Nanoparticles/chemistry , Polyglycolic Acid/chemistry , Polylactic Acid-Polyglycolic Acid Copolymer , Porcine Reproductive and Respiratory Syndrome/genetics , Porcine Reproductive and Respiratory Syndrome/immunology , Porcine Reproductive and Respiratory Syndrome/virology , Porcine respiratory and reproductive syndrome virus/genetics , Porcine respiratory and reproductive syndrome virus/physiology , Sus scrofa , Swine , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/immunology , Vaccines, Inactivated/administration & dosage , Vaccines, Inactivated/chemistry , Vaccines, Inactivated/immunology , Viral Vaccines/administration & dosage , Viral Vaccines/chemistry , Viral Vaccines/genetics
17.
PLoS One ; 7(12): e51794, 2012.
Article in English | MEDLINE | ID: mdl-23240064

ABSTRACT

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.


Subject(s)
Nanoparticles , Porcine Reproductive and Respiratory Syndrome , Porcine respiratory and reproductive syndrome virus , Respiratory Tract Infections , Viral Vaccines , Administration, Intranasal , Animals , Drug Delivery Systems , Lung/immunology , Lung/virology , Nanoparticles/administration & dosage , Nanoparticles/chemistry , Porcine Reproductive and Respiratory Syndrome/drug therapy , Porcine Reproductive and Respiratory Syndrome/immunology , Porcine Reproductive and Respiratory Syndrome/prevention & control , Porcine respiratory and reproductive syndrome virus/immunology , Porcine respiratory and reproductive syndrome virus/pathogenicity , Respiratory Tract Infections/drug therapy , Respiratory Tract Infections/immunology , Respiratory Tract Infections/prevention & control , Respiratory Tract Infections/veterinary , Respiratory Tract Infections/virology , Sus scrofa/immunology , Swine , Viral Vaccines/administration & dosage , Viral Vaccines/chemistry , Viral Vaccines/immunology
18.
Anim Health Res Rev ; 13(1): 21-37, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22717576

ABSTRACT

Porcine reproductive and respiratory syndrome (PRRS) is an economically important infectious disease of swine. Constant emergence of variant strains of PRRS virus (PPRSV) and virus-mediated immune evasion followed by viral persistence result in increased incidence and recurrence of PRRS in swine herds. Current live and killed PRRSV vaccines administered by a parenteral route are ineffective in inducing complete protection. Thus, new approaches in design and delivery of PRRSV vaccines are needed to reduce the disease burden of the swine industry. Induction of an effective mucosal immunity to several respiratory pathogens by direct delivery of a vaccine to mucosal sites has proven to be effective in a mouse model. However, there are challenges in eliciting mucosal immunity to PRRS due to our limited understanding of safe and potent mucosal adjuvants, which could potentiate the mucosal immune response to PRRSV. The purpose of this review is to discuss methods for induction of protective mucosal immune responses in the respiratory tract of pigs. The manuscript also discusses how PRRSV modulates innate, adaptive and immunoregulatory responses at both mucosal and systemic sites of infected and/or vaccinated pigs. This information may help in the design of innovative mucosal vaccines to elicit superior cross-protective immunity against divergent field strains of PRRSV.


Subject(s)
Porcine Reproductive and Respiratory Syndrome/prevention & control , Porcine respiratory and reproductive syndrome virus/immunology , Respiratory Mucosa/immunology , Viral Vaccines , Adjuvants, Immunologic/administration & dosage , Animals , Antibodies, Viral/biosynthesis , Antibodies, Viral/immunology , Cross Protection , Humans , Immune Evasion , Immunity, Mucosal/immunology , Lymphoid Tissue/immunology , Porcine Reproductive and Respiratory Syndrome/immunology , Swine , Vaccines, Inactivated/immunology , Viral Vaccines/administration & dosage , Viral Vaccines/immunology
19.
Virol J ; 9: 45, 2012 Feb 16.
Article in English | MEDLINE | ID: mdl-22340040

ABSTRACT

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.


Subject(s)
Cytokines/metabolism , Immunity, Cellular , Immunity, Innate , Porcine Reproductive and Respiratory Syndrome/immunology , Porcine respiratory and reproductive syndrome virus/immunology , Animals , CD4 Antigens/analysis , CD8 Antigens/analysis , Killer Cells, Natural/immunology , Porcine Reproductive and Respiratory Syndrome/virology , Porcine respiratory and reproductive syndrome virus/pathogenicity , Swine , T-Lymphocyte Subsets/immunology , Viremia
20.
Viral Immunol ; 24(6): 475-82, 2011 Dec.
Article in English | MEDLINE | ID: mdl-22111597

ABSTRACT

Reactive oxygen species (ROS) are produced predominantly by phagocytic cells in response to microbial infections. When produced at optimal levels ROS have potent antimicrobial properties. However, excessive production of ROS induces apoptosis/necrosis of infected as well as bystander cells, resulting in inflammatory pathology. Previously we showed that vaccination of pigs with a modified live porcine reproductive and respiratory syndrome virus vaccine (PRRS-MLV) administered intranasally with a potent mucosal adjuvant M. tuberculosis whole-cell lysate (Mtb WCL) induces protective immunity against PRRSV challenge. In this study, using bronchoalveolar lavage fluid cells and peripheral blood mononuclear cells harvested from that study were quantified for the levels of ROS produced. Our results indicated that in vaccinated pigs, levels of ROS were lower compared to unvaccinated PRRSV-challenged pigs. In unvaccinated but PRRSV-challenged pigs, the higher ROS production was associated with increased inflammatory lung pathology. In conclusion, our results suggest that intranasal immunization using PRRS-MLV along with a potent mucosal adjuvant protects pigs against both homologous and virulent heterologous PRRSV challenge, which was associated with reduced ROS production and reduced lung pathology compared to control virus-challenged pigs.


Subject(s)
Porcine Reproductive and Respiratory Syndrome/therapy , Porcine respiratory and reproductive syndrome virus/immunology , Reactive Oxygen Species/metabolism , Viral Vaccines/immunology , Adjuvants, Immunologic/metabolism , Adjuvants, Immunologic/pharmacology , Administration, Intranasal , Animals , Flow Cytometry , Immunity , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/metabolism , Lung/immunology , Lung/metabolism , Lung/pathology , Lung/virology , Mycobacterium tuberculosis/immunology , Mycobacterium tuberculosis/metabolism , Porcine Reproductive and Respiratory Syndrome/immunology , Porcine Reproductive and Respiratory Syndrome/virology , Reactive Oxygen Species/immunology , Swine/immunology , Vaccination , Vaccines, Attenuated/administration & dosage , Vaccines, Attenuated/immunology , Viral Vaccines/administration & dosage
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