Macrophage Polarization Modulated by Porcine Circovirus Type 2 Facilitates Bacterial Coinfection.

Polarization of macrophages to different functional states is important for mounting responses against pathogen infections. Macrophages are the major target cells of porcine circovirus type 2 (PCV2), which is the primary causative agent of porcine circovirus-associated disease (PCVAD) leading to immense economic losses in the global swine industry. Clinically, PCV2 is often found to increase risk of other pathogenic infections yet the underlying mechanisms remain to be elusive. Here we found that PCV2 infection skewed macrophages toward a M1 status through reprogramming expression of a subset of M1-associated genes and M2-associated genes. Mechanistically, induction of M1-associated genes by PCV2 infection is dependent on activation of nuclear factor kappa B (NF-κB) and c-jun N-terminal kinase (JNK) signaling pathways whereas suppression of M2-associated genes by PCV2 is via inhibiting expression of jumonji domain containing-3 (JMJD3), a histone 3 Lys27 (H3K27) demethylase that regulates M2 activation of macrophages. Finally, we identified that PCV2 capsid protein (Cap) directly inhibits JMJD3 transcription to restrain expression of interferon regulatory factor (IRF4) that controls M2 macrophage polarization. Consequently, sustained infection of PCV2 facilitates bacterial infection in vitro. In summary, these findings showed that PCV2 infection functionally modulated M1 macrophage polarization via targeting canonical signals and epigenetic histone modification, which contributes to bacterial coinfection and virial pathogenesis.

IFN-γ-/- Mice Resist Actinobacillus pleuropneumoniae Infection by Promoting Early Lung IL-18 Release and PMN-I Accumulation.

Porcine pleuropneumonia is a common infectious disease of pigs caused by Actinobacillus pleuropneumoniae Interferon gamma (IFN-γ) expression increases in the lung of pigs after A. pleuropneumoniae infection, but the role of IFN-γ during the infection is still obscure. In this study, an IFN-γ-/- mouse infection model was established, and bacterial load, levels of inflammatory cytokines, and types of neutrophils in the lungs were studied at different times post-A. pleuropneumoniae infection. We found that wild-type (WT) mice were more susceptible to A. pleuropneumoniae than IFN-γ-/- mice. At 6 h postinfection (hpi), the expression of interleukin 18 (IL-18) and IL-1ß in the lungs of IFN-γ-/- mice was significantly increased compared to WT mice. The bacterial load and levels of inflammatory cytokines (IL-1ß and IL-6) of IFN-γ-/- mice were significantly reduced at 12 hpi compared to WT mice. After an initial loss, the numbers of lung polymorphonuclear (PMN)-I cells dramatically increased in the lungs of IFN-γ-/- but not WT mice, whereas PMN-II cells continually decreased. Finally, in vivo administration of IL-18 significantly reduced clinical scores and bacterial load in the lungs of A. pleuropneumoniae-infected mice. This study identifies IFN-γ as a target for regulating the inflammatory response in the lung and provides a basis for understanding the course of clinical bacterial pneumonia and for the formulation of treatment protocols.

Development of a non-biased, high-throughput ELISA for the rapid evaluation of immunogenicity and cross-reactivity.

Traditional ELISA-based protein analysis has been predicated on the assumption that proteins bind randomly to the solid surface of the ELISA plate polymer (polystyrene or polyvinyl chloride). Random adherence to the plate ensures equal access to all faces of the protein, an important consideration when evaluating immunogenicity of polyclonal serum samples as well as when examining the cross-reactivity of immune serum against different antigenic variants of a protein. In this study we demonstrate that the soluble form of the surface lipoprotein transferrin binding protein B (TbpB) from three different bacterial pathogens (Neisseria meningitidis, Actinobacillus pleuropneumoniae, and Mannheimia haemolytica) bind the ELISA plate in a manner that consistently obscures the transferrin binding face of the proteins' N-lobe. In order to develop a non-biased ELISA where all faces of the protein are accessible, the strong interaction between biotin and avidin has been exploited by adding a biotin tag to these proteins during Escherichia coli-based cytoplasmic expression and utilizing streptavidin or neutravidin coated ELISA plates for protein capture and display. The use of avidin coated ELISA plates also allows for rapid purification of biotin-tagged proteins from crude E. coli lysates, removing the requirement of prior affinity purification of each protein to be included in the ELISA-based analyses. In proof of concept experiments we demonstrate the utility of this approach for evaluating immunogenicity and cross-reactivity of serum from mice and pigs immunized with TbpBs from human and porcine pathogens.

Differences in pig respiratory tract and peripheral blood immune responses to Actinobacillus pleuropneumoniae.

Excessive cytokine production is an important component of the acute respiratory distress syndrome and multiple organ failure. Pneumonia can lead to an overexpression of cytokines, although comparatively little is known about the relevance and differences in cytokines between blood and lung. In this study, piglets were experimentally infected intranasally with Actinobacillus pleuropneumoniae (APP), and transcriptomes of lung tissue and peripheral blood mononuclear cells determined. In addition, the levels of 30 cytokines in broncheoalveolar lavage fluid (BALF) and sera were determined by ELISA. Post infection, there was an early increase in lung monocytes, and a later rise in inflammatory cytokines in BALF. Blood lymphocytes increased early in infection and there was a rise in inflammatory cytokines in the peripheral blood of infected piglets. Genes involved in cytokine production, leukocyte migration and differentiation, lymphocyte activation, and cytokine-mediated signaling pathways in the transcriptomes of lung tissue were significantly down-regulated early in infection. At this early phase of APP infection (0-6 h), the cytokines IL-1ß, MCP-1, and IL-5 in sera increased rapidly and significantly, while many cytokines in BALF decreased. At 48 h post-infection, cytokines in sera were no longer significantly increased, although some were up-regulated in BALF, and there was aggravated pathological damage in the lungs at this time. The data indicate there are substantial differences between immune cells and cytokines in the lung and peripheral blood of APP infected piglets at equivalent time points. The results increase our understanding of pig-APP host interactive biology, and will be important in formulating future therapeutic and preventative strategies to prevent disease caused by APP.

Enhancement of Apx Toxin Production in Actinobacillus pleuropneumoniae Serotypes 1, 2, and 5 by Optimizing Culture Condition.

Actinobacillus pleuropneumoniae (APP) is a causative agent of porcine pleuropneumonia. Therefore, the development of an effective vaccine for APP is necessary. Here, we optimized the culture medium and conditions to enhance the production yields of Apx toxins in APP serotype 1, 2, and 5 cultures. The use of Mycoplasma Broth Base (PPLO) medium improved both the quantity and quality of the harvested Apx toxins compared with Columbia Broth medium. Calcium chloride (CaCl2) was first demonstrated as a stimulation factor for the production of Apx toxins in APP serotype 2 cultures. Cultivation of APP serotype 2 in PPLO medium supplemented with 10 µg/ml of nicotinamide adenine dinucleotide (NAD) and 20 mM CaCl2 yielded the highest levels of Apx toxins. These findings suggest that the optimization of the culture medium and conditions increases the concentration of Apx toxins in the supernatants of APP serotype 1, 2, and 5 cultures and may be applied for the development of vaccines against APP infection.

Genome-wide screening of lipoproteins in Actinobacillus pleuropneumoniae identifies three antigens that confer protection against virulent challenge.

Actinobacillus pleuropneumoniae is an important veterinary pathogen that causes porcine pleuropneumonia. Lipoproteins of bacterial pathogens play pleiotropic roles in the infection process. In addition, many bacterial lipoproteins are antigenic and immunoprotective. Therefore, characterization of lipoproteins is a promising strategy for identification of novel vaccine candidates or diagnostic markers. We cloned 58 lipoproteins from A. pleuropneumoniae JL03 (serovar 3) and expressed them in Escherichia coli. Five proteins with strong positive signals in western blotting analysis were used to immunize mice. These proteins elicited significant antibody responses, and three of them (APJL_0922, APJL_1380 and APJL_1976) generated efficient immunoprotection in mice against lethal heterologous challenge with A. pleuropneumoniae 4074 (serovar 1), both in the active and passive immunization assays. Then immunogenicity of these three lipoproteins (APJL_0922, APJL_1380 and APJL_1976) were further tested in pigs. Results showed that these proteins elicited considerable humoral immune responses and effective protective immunity against virulent A. pleuropneumoniae challenge. Our findings suggest that these three novel lipoproteins could be potential subunit vaccine candidates.

Immunoproteomic characterization of outer membrane vesicles from hyper-vesiculating Actinobacillus pleuropneumoniae.

Outer membrane vesicles (OMVs) are produced and secreted virtually by every known Gram-negative bacterium. Despite their non-live nature, they share antigenic characteristics with the bacteria they originate from. This, together with their relative ease of purification, casts the OMVs as a very promising and flexible tool in both human and veterinary vaccinology. The aim of the current work was to get an insight into the antigenic pattern of OMVs from the pig pathogen Actinobacillus pleuropneumoniae in the context of vaccine development. Accordingly, we designed a protocol combining 2D Western Blotting and mass spectrometric identification to robustly characterize the antigenic protein pattern of the vesicles. Our analysis revealed that A. pleuropneumoniae OMVs carry several immunoreactive virulence factors. Some of these proteins, LpoA, OsmY and MIDG2331_02184, have never previously been documented as antigenic in A. pleuropneumoniae or other pathogenic bacteria. Additionally, we showed that despite their relative abundance, proteins such as FrpB and DegQ do not contribute to the antigenic profile of A. pleuropneumoniae OMVs.

The Effect of Inactivated Mycobacterium Paratuberculosis Vaccine on the Response to a Heterologous Bacterial Challenge in Pigs.

Background: Vaccines may have non-specific effects, affecting resistance to heterologous pathogens. Veterinary vaccines have seldom been investigated for their non-specific effects. However, recent observational studies suggest that an inactivated paratuberculosis vaccine reduced all-cause mortality in goats and cattle. Aim: We tested if vaccination with a killed mycobacterial vaccine may have heterologous effects in swine (Sus domesticus), specifically on the pathogenic and clinical effects of a heterologous challenge with Actinobacillus pleuropneumoniae in young pigs. Methods: Newborn piglets were randomized to vaccination s.c. with the inactivated paratuberculosis vaccine Gudair (Zoetis Inc.) (n = 17) or no vaccine (n = 16). At 4-5 weeks after vaccination, all piglets were challenged intra-nasally with a high (Gudair: n = 8; control: n = 8) or a low (Gudair: n = 9; control: n = 8) dose of the gram-negative bacterium A. pleuropneumoniae causing acute porcine pleuropneumonia. The effect and severity of pathogen challenge was evaluated by measuring acute phase proteins C-reactive protein, haptoglobin and Porcine α1-acid glycoprotein, and by gross pathology 1 day post challenge. Specific and non-specific in vitro cytokine responses to vaccination were evaluated in whole blood before bacterial challenge. Results: The vaccine was immunogenic in the pigs as evidenced by increased IFN-γ responses to purified protein derivative of Mycobacterium paratuberculosis. However, Gudair vaccine did not affect IL-6 responses. The gross pathology of the lungs as well as the acute phase protein responses after the high A. pleuropneumoniae dose challenge was slightly increased in the vaccinated animals compared with controls, whereas this was not seen in the animals receiving the low-dose bacterial challenge. Conclusion: The inactivated paratuberculosis vaccine exacerbated the pathological and inflammatory effects of an experimental A. pleuropneumoniae infection in young pigs.

Development of Actinobacillus pleuropneumoniae ApxI, ApxII, and ApxIII-specific ELISA methods for evaluation of vaccine efficiency.

Among various vaccines against Actinobacillus pleuropneumoniae, subunit vaccines using recombinant proteins of ApxI, ApxII, and ApxIII as vaccine antigens have shown good efficacy in terms of safety and protection. Therefore, subunit vaccines are being applied worldwide and the development of new subunit vaccines is actively being conducted. To evaluate the efficacy of the subunit vaccines, it is important to measure immune responses to each Apx toxin separately. However, the cross-reactivity of antibodies makes it difficult to measure specific immune reactivity to each toxin. In the present study, specific antigen regions among the toxins were identified and cloned to solve this problem. The antigenicity of each recombinant protein was demonstrated by Western blot. Using the recombinant proteins, we developed enzyme-linked immunosorbent assay (ELISA) methods that can detect specific immune responses to each Apx toxin in laboratory guinea pigs. We suggest that the ELISA method developed in this study can be an important tool in the evaluation of vaccine efficiency and vaccine development.

Cytoplasmic glycoengineering of Apx toxin fragments in the development of Actinobacillus pleuropneumoniae glycoconjugate vaccines.

BACKGROUND: Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia and represents a major burden to the livestock industry. Virulence can largely be attributed to the secretion of a series of haemolytic toxins, which are highly immunogenic. A. pleuropneumoniae also encodes a cytoplasmic N-glycosylation system, which involves the modification of high molecular weight adhesins with glucose residues. Central to this process is the soluble N-glycosyl transferase, ngt, which is encoded in an operon with a subsequent glycosyl transferase, agt. Plasmid-borne recombinant expression of these genes in E. coli results in the production of a glucose polymer on peptides containing the appropriate acceptor sequon, NX(S/T). However to date, there is little evidence to suggest that such a glucose polymer is formed on its target peptides in A. pleuropneumoniae. Both the toxins and glycosylation system represent potential targets for the basis of a vaccine against A. pleuropneumoniae infection. RESULTS: In this study, we developed cytoplasmic glycoengineering to construct glycoconjugate vaccine candidates composed of soluble toxin fragments modified by glucose. We transferred ngt and agt to the chromosome of Escherichia coli in order to generate a native-like operon for glycoengineering. A single chromosomal copy of ngt and agt resulted in the glucosylation of toxin fragments by a short glycan, rather than a polymer. CONCLUSIONS: A vaccine candidate that combines toxin fragment with a conserved glycan offers a novel approach to generating epitopes important for both colonisation and disease progression.

Escherichia coli-Derived Outer Membrane Vesicles Deliver Galactose-1-Phosphate Uridyltransferase and Yield Partial Protection against Actinobacillus pleuropneumoniae in Mice.

In our previous studies, we have identified several in vivo-induced antigens and evaluated their potential as subunit vaccine candidates in a murine model, in which the recombinant protein GalT showed the most potent immunogenicity and immunoprotective efficacy against Actinobacillus pleuropneumoniae. To exploit a more efficient way of delivering GalT proteins, in this study, we employed the widely studied E. coli outer membrane vesicles (OMVs) as a platform to deliver GalT protein and performed the vaccine trial using the recombinant GalT-OMVs in the murine model. Results revealed that GalT-OMVs could elicit a highly-specific, IgG antibody titer that was comparable with the adjuvant GalT group. Significantly higher lymphocyte proliferation and cytokines secretion levels were observed in the GalT-OMVs group. 87.5% and 50% of mice were protected from a lethal dose challenge using A. pleuropneumoniae in active or passive immunization, respectively. Histopathologic and immunohistochemical analyses showed remarkably reduced pathological changes and infiltration of neutrophils in the lungs of mice immunized with GalT-OMVs after the challenge. Taken together, these findings confirm that OMVs can be used as a platform to deliver GalT protein and enhance its immunogenicity to induce both humoral and cellular immune responses in mice.

Effective Pro-Inflammatory Induced Activity of GALT, a Conserved Antigen in A. Pleuropneumoniae, Improves the Cytokines Secretion of Macrophage via p38, ERK1/2 and JNK MAPKs Signal Pathway.

GALT is a highly conserved antigen in gram-negative bacteria, and has been shown to play a crucial role in the pathogenesis of many zoonoses. Actinobacillus pleuropneumoniae (APP) is a widespread respiratory system pathogen belonging to the Pasteuriaceae family. The functional mechanisms of GALT in the process of infection remain unclear. The aim of this study is to analyze roles of GALT in the pathogenesis of APP infection. Recombinant GALT was expressed in E. coli, purified, and was used to treat a Raw 264.7 macrophage line. Stimulation of Raw 264.7 macrophages with recombinant GALT protein induced the expression of pro-inflammatory cytokines (TNF-α, IL-1ß, and IL-6). Compared with negative control, GALT led to increased production of pro-inflammatory cytokines in treated cells. Furthermore, specific inhibitors of the extracellular signal-regulated P38 and JNK MAPKs pathways significantly decreased GALT-induced pro-inflammatory cytokine production, and a western blot assay showed that GALT stimulation induced the activation of the MAPKs pathway. This process included cell-signaling pathways like P38, ERK1/2 and JNK MAPKs, and NF-κB. Both TLR2 and TLR4 were receptors of GALT antigens, whereas they played negative and positive roles (respectively) in the process of induction and expression of pro-inflammatory cytokines. Taken together, our data indicate that GALT is a novel pro-inflammatory mediator and induces TLR2 and TLR4-dependent pro-inflammatory activity in Raw 264.7 macrophages through P38, ERK1/2, and JNK MAPKs pathways.

Galactose-1-phosphate uridyltransferase (GalT), an in vivo-induced antigen of Actinobacillus pleuropneumoniae serovar 5b strain L20, provided immunoprotection against serovar 1 strain MS71.

GALT is an important antigen of Actinobacillus pleuropneumoniae (APP), which was shown to provide partial protection against APP infection in a previous study in our lab. The main purpose of the present study is to investigate GALT induced cross-protection between different APP serotypes and elucidate key mechanisms of the immune response to GALT antigenic stimulation. Bioinformatic analysis demonstrated that galT is a highly conserved gene in APP, widely distributed across multiple pathogenic strains. Homologies between any two strains ranges from 78.9% to 100% regarding the galT locus. Indirect enzyme-linked immunosorbent assay (ELISA) confirmed that GALT specific antibodies could not be induced by inactivated APP L20 or MS71 whole cell bacterin preparations. A recombinant fusion GALT protein derived from APP L20, however has proven to be an effective cross-protective antigen against APP sevorar 1 MS71 (50%, 4/8) and APP sevorar 5b L20 (75%, 6/8). Histopathological examinations have confirmed that recombinant GALT vaccinated animals showed less severe pathological signs in lung tissues than negative controls after APP challenge. Immunohistochemical (IHC) analysis indicated that the infiltration of neutrophils in the negative group is significantly increased compared with that in the normal control (P<0.001) and that in surviving animals is decreased compared to the negative group. Anti-GALT antibodies were shown to mediate phagocytosis of neutrophils. After interaction with anti-GALT antibodies, survival rate of APP challenged vaccinated animals was significantly reduced (P<0.001). This study demonstrated that GALT is an effective cross-protective antigen, which could be used as a potential vaccine candidate against multiple APP serotypes.

Proposal of serovars 17 and 18 of Actinobacillus pleuropneumoniae based on serological and genotypic analysis.

The aim of this study was to investigate isolates of Actinobacillus pleuropneumoniae previously designated serologically either as non-typable (NT) or as 'K2:07', which did not produce serovar-specific amplicons in PCR assays. We used whole genome sequencing to identify the capsule (CPS) loci of six previously designated biovar 1 NT and two biovar 1 'K2:O7' isolates of A. pleuropneumoniae from Denmark, as well as a recent biovar 2 NT isolate from Canada. All of the NT isolates have the same six-gene type I CPS locus, sharing common cpsABC genes with serovars 2, 3, 6, 7, 8, 9, 11 and 13. The two 'K2:O7' isolates contain a unique three-gene type II CPS locus, having a cpsA gene similar to that of serovars 1, 4, 12, 14 and 15. The previously NT isolates share the same O-antigen genes, found between erpA and rpsU, as serovars 3, 6, 8, and 15. Whereas the 'K2:O7' isolates, have the same O-antigen genes as serovar 7, which likely contributed to their previous mis-identification. All of the NT and 'K2:O7' isolates have only the genes required for production of ApxII (apxIICA structural genes, and apxIBD export genes). Rabbit polyclonal antisera raised against representative isolates with these new CPS loci demonstrated distinct reactivity compared to the 16 known serovars. The serological and genomic results indicate that the isolates constitute new serovars 17 (previously NT) and 18 (previously 'K2:O7'). Primers designed for amplification of specific serovar 17 and 18 sequences for molecular diagnostics will facilitate epidemiological tracking of these two new serovars of A. pleuropneumoniae.

New trends in innovative vaccine development against Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, a respiratory disease leading to severe economic losses in the swine industry. The most widely used commercial vaccines are bacterins comprising inactivated whole cells of A. pleuropneumoniae but these have only been partially effective in preventing disease. Innovative immuno-prophylactic preparations of A. pleuropneumoniae based on ApxI, ApxII, ApxIII, ApxIV toxins and outer membrane proteins, among others (i.e. RnhB, GalU, GalT, HflX, ComL, LolB, LppC), have high protective efficacy in mice and pigs. Some vaccine preparations have efficacy against homologous and heterologous A. pleuropneumoniae serovars, which constitute an important advance to control porcine pleuropneumonia. In this arena, subunit vaccines based on toxins are one of the most advanced and promising developments. Many research groups have focussed on the development of live attenuated vaccines comprising strains with inactivated Apx toxins and/or other virulence factors, their protective efficacy being determined in mouse and/or swine models. Other innovative approaches such as bacteria, yeast and plants as production and oral delivery platforms have been explored in animal models and the definitive host with encouraging results. In addition, further research into A. pleuropneumoniae-based DNA and nano-vaccines, as well as bioencapsulation of antigens in plants, is envisaged. Here, the recent findings and future trends in innovative vaccine development against A. pleuropneumoniae are reviewed and placed in perspective.

Generation, safety and immunogenicity of an Actinobacillus pleuropneumoniae quintuple deletion mutant SLW07 (ΔapxICΔapxIICΔorf1ΔcpxARΔarcA).

We inactivated a virulence determinant, ArcA, in an Actinobacillus pleuropneumoniae quadruple deletion mutant SLW06 (ΔapxICΔapxIICΔorf1ΔcpxAR, serovar 1), and a quintuple deletion mutant SLW07 was generated. SLW07 showed decreased adherence to and invasion of host cells, compared to its parent strain SLW06. SLW07 was more sensitive in RAW264.7 macrophage-mediated phagocytosis and clearance. SLW07 was less virulent in mice. An immunization assay indicated that both SLW07 and SLW06 preferentially stimulated T helper cell type 2 response in mice. Live vaccines induced the production of interleukin-6 and tumor necrosis factor-α by splenic lymphocytes. Furthermore, the protective immunity of SLW07 was not affected after ArcA mutation. Immunization with SLW07 could provide a complete protection following virulent A. pleuropneumoniae challenge in mice. Our results suggest that SLW07 is a promising live vaccine candidate, which is further attenuated from and shares similar protective efficacy with its quadruple deletion parent SLW06.

Recombinant ApxIV protein enhances protective efficacy against Actinobacillus pleuropneumoniae in mice and pigs.

AIMS: Available bacterins, commercial or autogenous, for Actinobacillus pleuropneumoniae disease control have, thus far, shown debatable protective efficacy and only in homologous challenges. Our study sought to determine whether the addition of reombinant protein ApxIV to the multicomponent vaccine could enhance protection against homologous and heterologous challenge of A. pleuropneumoniae. METHODS AND RESULTS: The virulence of ApxI, ApxII, ApxIV and OMP were cloned and expressed using a prokaryotic system; these recombinant proteins were combined with inactivated A. pleuropneumoniae serovar 1 to formulate different multicomponent vaccines. Immune response and protective efficacy of the vaccines were evaluated in mice and pigs. A protection rate of 67% was observed against heterologous challenge in mice vaccinated with the rApxIV formulation. Piglets vaccinated with vaccine containing ApxIV produced significantly higher antibody titre and provided complete protection and reduced gross lesions by 67% when compared with the nonimmunized group after homologous challenge. Additionally, flow cytometry analysis showed significant cellular immune response. CONCLUSIONS: The results of our vaccination experiments revealed that a combination of inactivated bacteria and the recombinant antigens rApxI, rApxII, rApxIV and rOMP can provide effective protection against heterologous A. pleuropneumoniae challenge. SIGNIFICANCE AND IMPACT OF THE STUDY: The addition of ApxIV to the multicomponent vaccine could enhance homologous and heterologous protection in mice and pigs, respectively, against challenge by A. pleuropneumoniae.

A trivalent Apx-fusion protein delivered by E. coli outer membrane vesicles induce protection against Actinobacillus pleuropneumoniae of serotype 1 and 7 challenge in a murine model.

Actinobacillus pleuropneumoniae (APP) causes serious economic losses in the swine industry, and is the etiologic agent of porcine pleuropneumonia. In this study we have engineered a trivalent Apx fusion protein enclosed in outer membrane vesicles (Apxr-OMV) and studied its immunoprotective efficacy against APP serotypes 1 and 7 challenge in mice. The results showed that the IgG levels in the Apxr-OMVs immune group were significantly higher than those of the negative control (P < 0.05). Up-regulation of both Th1 (IFN-γ, IL-2) and Th2 (IL-4) cytokines were detected in splenocytes of Apxr-OMVs immune group. The survival rates 87.5% and 62.5% were observed against APP strain 1516 of serotype 7 and APP strain 2701 of serotype 1 in the groups of Apxr-OMVs immune group, respectively. Histopathological lesions of the pulmonary structure alveoli were found to be minimal in APX-OMV group challenged with APP serotypes 1 and 7. These results strongly indicated that engineered OMVs could effectively induce specific humoral or cellular immune responses. Moreover, Apxr-OMVs used as novel vaccine provides cross-protective immunity against different serotype 1 and 7 of APP infection in a mouse model. In contrast, the OMV-empty and PBS as negative controls or inactivated strain of APP-2701 and APP-1516 as positive controls for the animal study cannot provide protection or cross-protection.

In vivo testing of novel vaccine prototypes against Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae (A. pleuropneumoniae) is a Gram-negative bacterium that represents the main cause of porcine pleuropneumonia in pigs, causing significant economic losses to the livestock industry worldwide. A. pleuropneumoniae, as the majority of Gram-negative bacteria, excrete vesicles from its outer membrane (OM), accordingly defined as outer membrane vesicles (OMVs). Thanks to their antigenic similarity to the OM, OMVs have emerged as a promising tool in vaccinology. In this study we describe the in vivo testing of several vaccine prototypes for the prevention of infection by all known A. pleuropneumoniae serotypes. Previously identified vaccine candidates, the recombinant proteins ApfA and VacJ, administered individually or in various combinations with the OMVs, were employed as vaccination strategies. Our data show that the addition of the OMVs in the vaccine formulations significantly increased the specific IgG titer against both ApfA and VacJ in the immunized animals, confirming the previously postulated potential of the OMVs as adjuvant. Unfortunately, the antibody response raised did not translate into an effective protection against A. pleuropneumoniae infection, as none of the immunized groups following challenge showed a significantly lower degree of lesions than the controls. Interestingly, quite the opposite was true, as the animals with the highest IgG titers were also the ones bearing the most extensive lesions in their lungs. These results shed new light on A. pleuropneumoniae pathogenicity, suggesting that antibody-mediated cytotoxicity from the host immune response may play a central role in the development of the lesions typically associated with A. pleuropneumoniae infections.

Activation of Porcine Alveolar Macrophages by Actinobacillus pleuropneumoniae Lipopolysaccharide via the Toll-Like Receptor 4/NF-κB-Mediated Pathway.

Actinobacillus pleuropneumoniae is the causative agent of porcine contagious pleuropneumonia. Overproduction of proinflammatory cytokines, like interleukin-1ß (IL-1ß), IL-6, tumor necrosis factor alpha, and resistin, in the lung is an important feature of A. pleuropneumoniae infection. These proinflammatory cytokines enhance inflammatory and immunological responses. However, the mechanism that leads to cytokine production remains unclear. As a major virulence factor of A. pleuropneumoniae, lipopolysaccharide (LPS) may act as a potent stimulator of Toll-like receptor 4 (TLR4), triggering a number of intracellular signaling pathways that lead to the synthesis of proinflammatory cytokines. Porcine alveolar macrophages (PAMs) are the first line of defense against pathogenic microbes during pathogen invasion. The results of the present study demonstrate that A. pleuropneumoniae LPS induces PAMs to produce inflammatory cytokines in time- and dose-dependent manners. Moreover, PAMs were activated by A. pleuropneumoniae LPS, resulting in upregulation of signaling molecules, including TLR4, MyD88, TRIF-related adaptor molecule, and NF-κB. In contrast, the activation effects of A. pleuropneumoniae LPS on PAMs could be suppressed by specific inhibitors, like small interfering RNA and Bay11-7082. Taken together, our data indicate that A. pleuropneumoniae LPS can induce PAMs to produce proinflammatory cytokines via the TLR4/NF-κB-mediated pathway. These findings partially reveal the mechanism of the overproduction of proinflammatory cytokines in the lungs of swine with A. pleuropneumoniae infection and may provide targets for the prevention of A. pleuropneumoniae-induced pneumonia. All the data could be used as a reference for the pathogenesis of respiratory infection.