TbpBY167A-based vaccine is safe in pregnant sows and induces high titers of maternal derived antibodies that reduce Glaesserella parasuis colonization in piglets.

Glässer's disease is one of the main diseases affecting young piglets, particularly during the nursery phase, that can significantly impact pork production. Vaccination of sows has the potential to prevent Glaesserella parasuis infection during the first weeks of life that is to a substantial degree due to the transfer of maternal derived antibodies (MDA) in colostrum. In this study we compare the antibody response to two vaccines administered to pregnant sows. A subunit vaccine containing the mutant transferrin-binding protein, TbpBY167A, and an autogenous vaccine formulated with the LM96/20 strain of G. parasuis (SV4) administered on days 65 and 86 of the gestational period were safe and induced high titers of antibodies in sows. The IgG peak was reached on day 100 of gestation, and the translocation of IgG to the mammary gland was confirmed in colostrum at the time of delivery. Piglets born from vaccinated sows maintained positive IgG titers against TbpBY167A or G. parasuis SV4 for the duration of the experiment (35 days of life). Piglets born from sows vaccinated with the TbpBY167A-based vaccine had a significantly (p = 0.001) lower load of G. parasuis in the respiratory tract compared to those born from sows vaccinated with the autogenous vaccine. Finally, we demonstrate that the LM96/20 (SV4) strain is highly virulent and a primary agent of Glässer's disease.

TbpBY167A-Based Vaccine Can Protect Pigs against Glässer's Disease Triggered by Glaesserella parasuis SV7 Expressing TbpB Cluster I.

Glaesserella parasuis is the etiological agent of Glässer's disease (GD), one of the most important diseases afflicting pigs in the nursery phase. We analyzed the genetic and immunological properties of the TbpB protein naturally expressed by 27 different clinical isolates of G. parasuis that were typed as serovar 7 and isolated from pigs suffering from GD. All the strains were classified as virulent by LS-PCR. The phylogenetic analyses demonstrated high similarity within the amino acid sequence of TbpB from 24 clinical strains all belonging to cluster III of TbpB, as does the protective antigen TbpBY167A. Three G. parasuis isolates expressed cluster I TbpBs, indicating antigenic diversity within the SV7 group of G. parasuis. The antigenic analysis demonstrated the presence of common epitopes on all variants of the TbpB protein, which could be recognized by an in vitro analysis using pig IgG induced by a TbpBY167A-based vaccine. The proof of concept of the complete cross-protection between clusters I and III was performed in SPF pigs immunized with the TbpBY167A-based vaccine (cluster III) and challenged with G. parasuis SV7, strains LM 360.18 (cluster I). Additionally, pigs immunized with a whole-cell inactivated vaccine based on G. parasuis SV5 (Nagasaki strain) did not survive the challenge performed with SV7 (strain 360.18), demonstrating the absence of cross-protection between these two serovars. Based on these results, we propose that a properly formulated TbpBY167A-based vaccine may elicit a protective antibody response against all strains of G. parasuis SV7, despite TbpB antigenic diversity, and this might be extrapolated to other serovars. This result highlights the promising use of the TbpBY167A antigen in a future commercial vaccine for GD prevention.

Proof of Concept for Prevention of Natural Colonization by Oral Needle-Free Administration of a Microparticle Vaccine.

There has been substantial interest in the development of needle-free vaccine administration that has led to a variety of approaches for delivery through the skin for induction of a systemic immune response. The mucosal administration of vaccines has inherently been needle-free, but the simple application of vaccines on the mucosal surface by itself does not lead to mucosal immunity. Since many important bacterial infections develop after initial colonization of the upper respiratory tract of the host, prevention of colonization could not only prevent infection but also eliminate the reservoir of pathogens that reside exclusively in that ecologic niche. This study was designed to provide proof of concept for a needle-free immunization approach that would reduce or eliminate colonization and prevent infection. In order to accomplish this a microparticle vaccine preparation was delivered just below the oral mucosal epithelial cell layer where it would lead to a robust immune response. A vaccine antigen (mutant transferrin binding protein B) shown to be capable of preventing infection in pigs was incorporated into a polyphosphazene microparticle preparation and delivered by a needle-free device to the oral sub-epithelial space of pigs. This vaccination regimen not only provided complete protection from infection after intranasal challenge by Glaesserella parasuis but also eliminated natural colonization by this bacterium. Notably, the complete prevention of natural colonization was dependent upon delivery of the microparticle preparation below the epithelial layer in the oral mucosa as intradermal or intramuscular delivery was not as effective at preventing natural colonization. This study also demonstrated that a primary immunization in the presence of maternal antibody limited the resulting antibody response but a robust antibody response after the second immunization indicated that maternal antibody did not prevent induction of B-cell memory.

New Pathological Lesions Developed in Pigs by a "Non-virulent" Strain of Glaesserella parasuis.

Glaesserella parasuis is a Gram-negative bacterium that causes Glässer's disease, a common pathology found in young pigs characterized by polyarthritis, polyserositis, and meningitis. The bacterium has 15 known serovars that have been classified by virulence. Serovars 1, 4, 5, and 12 are considered highly virulent and used in most studies. Serovars 3, 6, 7, 9, and 11 are considered avirulent. Recent reports that serovar 7 is an emerging problem in the pig industry indicate that the association of virulence and serovar may not always be reliable. This led us to infect colostrum-deprived piglets with the reference serovar 7 strain (SV7 strain 174) that had been passaged through pigs and characterize the clinical and pathological signs. We observed that SV7 strain 174 caused clinical signs consistent with Glässer's disease in all infected piglets that succumbed to infection for up to day 5 post-infection. Macroscopic and microscopic lesions were consistent with those found in piglets infected with conventional virulent serovars. In addition, we describe novel microscopic lesions associated with Glässer's disease such as endophthalmitis and thymic depletion. Thus, our findings indicate that SV7 strain 174 causes classical signs of Glässer's disease in colostrum-deprived piglets and some caution should be used in employing vaccine strategies based on association between capsular serovar and virulence.

Molecular serotyping of clinical strains of Haemophilus (Glaesserella) parasuis brings new insights regarding Glässer's disease outbreaks in Brazil.

Glässer's disease (GD) is an important infectious disease of swine caused by Haemophilus (Glaesserella) parasuis. Vaccination with inactivated whole cell vaccines is the major approach for prevention of H. parasuis infection worldwide, but the immunity induced is predominantly against the specific polysaccharide capsule. As a consequence, the available vaccines may not induce adequate protection against the field strains, when the capsules present in the vaccine strains are different from those in strains isolated from the farms. Therefore, it is crucial to map H. parasuis serovars associated with regional outbreaks so that appropriate bacterin vaccines can be developed and distributed for prevention of infection. In this study, 459 H. parasuis field strains isolated from different Glässer's disease outbreaks that occurred in 10 different Brazilian States were analyzed for serotype using PCR-based approaches. Surprisingly, non-typeable (NT) strains were the second most prevalent group of field strains and along with serovars 4, 5 and 1 comprised more than 70% of the isolates. A PCR-based approach designed to amplify the entire polysaccharide capsule locus revealed 9 different band patterns in the NT strains, and 75% of the NT strains belonged to three clusters, suggesting that a number of new serovars are responsible for a substantial proportion of disease. These results indicate that commercially available vaccines in Brazil do not cover the most prevalent H. parasuis serovars associated with GD.

The amino acid selected for generating mutant TbpB antigens defective in binding transferrin can compromise the in vivo protective capacity.

Haemophilus parasuis is the causative agent of the Glässer's disease (GD), one of the most important bacterial diseases that affect young pigs worldwide. GD prevention based on vaccination is a major concern due to the limited cross-protection conferred by the inactivated whole cell vaccines used currently. In this study, vaccines based on two mutant recombinant proteins derived from transferrin binding protein B of H. parasuis (Y167A-TbpB and W176A-TbpB) were formulated and evaluated in terms of protection against lethal challenge using a serovar 7 (SV7) H. parasuis in a high susceptibility pig model. Our results showed that H. parasuis strain 174 (SV7) is highly virulent in conventional and colostrum-deprived pigs. The Y167A-TbpB and W176A-TbpB antigens were immunogenic in pigs, however, differences in terms of antigenicity and functional immune response were observed. In regard to protection, animals immunized with Y167A-TbpB antigen displayed 80% survival whereas the W176A-TbpB protein was not protective. In conjunction with previous studies, our results demonstrate, (a) the importance of testing engineered antigens in an in vivo pig challenge model, and, (b) that the Y167A-TbpB antigen is a promising antigen for developing a broad-spectrum vaccine against H. parasuis infection.

New insights about functional and cross-reactive properties of antibodies generated against recombinant TbpBs of Haemophilus parasuis.

Vaccines have become fundamental in the control and elimination of Glässer Disease, a systemic disease of pigs caused by Haemophilus parasuis. The classic vaccines available for prevention of this infection were developed without a robust knowledge about host immunological mechanisms. In this study, we demonstrated the presence of cross-reactive epitopes on both the N-lobe and C-lobe of variants of transferrin binding protein B (TbpBs) expressed on the surface of 6 virulent serovars of H. parasuis. Antibodies against TbpB-derived antigens were capable of increasing the phagocytic capacity of neutrophils and were also capable of blocking porcine transferrin from binding to TbpB. Surprisingly, none of the pig or mice antisera from animals immunized with TbpB-derived antigens mixed with Montanide IMS 2215 VG PR adjuvant were able to activate the classical complement pathway (CCP). In contrast, antisera from mice immunized with TbpB-derived antigens adjuvanted with Freund's adjuvants or Montanide Gel 01 were able to activate the CCP and kill H. parasuis. Our results demonstrate that the type of adjuvant can modulate the functional response induced by TbpB-derived antigens. Based on these results, we propose that a properly formulated TbpB-based vaccine may elicit a functional protective antibody response with broad cross-reactivity against heterologous strains of H. parasuis.