Hau et al. BMC Veterinary Research (2020) 16:167 https://doi.org/10.1186/s12917-020-02377-5 RESEARCH ARTICLE Open Access Evaluation of the recombinant proteins RlpB and VacJ as a vaccine for protection against Glaesserella parasuis in pigs Samantha J. Hau1, Shi-Lu Luan2, Crystal L. Loving1, Tracy L. Nicholson1, Jinhong Wang2, Sarah E. Peters2, David Seilly2, Lucy A. Weinert2, Paul R. Langford3, Andrew N. Rycroft4, Brendan W. Wren5, Duncan J. Maskell2,6, Alexander W. Tucker2†, Susan L. Brockmeier1*† and on behalf of the BRaDP1T Consortium Abstract Background: Glaesserella parasuis, the causative agent of Glӓsser’s disease, is widespread in swine globally resulting in significant economic losses to the swine industry. Prevention of Glӓsser’s disease in pigs has been plagued with an inability to design broadly protective vaccines, as many bacterin based platforms generate serovar or strain specific immunity. Subunit vaccines are of interest to provide protective immunity to multiple strains of G. parasuis. Selected proteins for subunit vaccination should be widespread, highly conserved, and surface exposed. Results: Two candidate proteins for subunit vaccination (RlpB and VacJ) against G. parasuis were identified using random mutagenesis and an in vitro organ culture system. Pigs were vaccinated with recombinant RlpB and VacJ, outer membrane proteins with important contributions to cellular function and viability. Though high antibody titers to the recombinant proteins and increased interferon-γ producing cells were found in subunit vaccinated animals, the pigs were not protected from developing systemic disease. Conclusions: It appears there may be insufficient RlpB and VacJ exposed on the bacterial surface for antibody to bind, preventing high RlpB and VacJ specific antibody titers from protecting animals from G. parasuis. Additionally, this work confirms the importance of utilizing the natural host species when assessing the efficacy of vaccine candidates. Keywords: Subunit vaccine, Glässer’s disease, Glaesserella parasuis Background There are 15 identified serovars of G. parasuis; however, Glaesserella parasuis is a Gram-negative bacterial mem- many isolates are untypable [2]. Multiple serovars can ber of the Pasteurellaceae family and the causative agent circulate within a herd, although it appears some sero- of Glässer’s disease, which is characterized by a fibrinous vars are more capable of causing systemic disease [3, 4]. polyserositis, meningitis, and arthritis. G. parasuis can To prevent G. parasuis disease in the swine industry, cause high morbidity and mortality in herds resulting in efforts have focused on developing broadly protective significant losses to the swine industry annually [1]. vaccines. Commercially available G. parasuis vaccines are predominantly based on a bacterin platform. Bac- * Correspondence: [email protected] terins have been shown to provide good homologous † Alexander W. Tucker and Susan L. Brockmeier contributed equally to this protection [5–7]; however, this protection can be serovar work. – 1USDA, ARS, National Animal Disease Center, 1920 Dayton Ave, Ames, IA or strain specific [7 10], leaving swine susceptible to dis- 50010, USA ease with other serovars or strains in the field. Currently, Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Hau et al. BMC Veterinary Research (2020) 16:167 Page 2 of 9 no available vaccine is able to provide broad cross pro- complete vacJ gene was present in 9 of the 11 strains. tection for G. parasuis. This may be due in part to the The vacJ gene was positioned near the end of a contig in bacterial capsule, which is serovar specific and functions MN-H and was absent from SW140, which may be asso- to mask other antigens on the bacterial surface that may ciated with gaps in the genome of these strains. Amino contribute to the protective immune response [11, 12]. acid identity among the other 9 strains revealed high The importance of a vaccine conferring heterologous pro- conservation, with a 98% or higher identity between tection has led to the pursuit of alternative vaccine plat- isolates. forms that avoid the generation of capsule directed immunity, such as protein and peptide vaccines. Antigens targeted for G. parasuis protein and peptide vaccines Antibody response to vaccination should be highly conserved and widespread amongst iso- Antibody titers (IgG) were determined by ELISA for lates and found on the surface of the bacterium. rRlpB and rVacJ. Minimal reactivity was seen in animals Several mechanisms have been employed to identify prior to vaccination. Modest increases in IgG titer to subunit vaccine candidates, including the use of hyper- rRlpB and rVacJ were seen in the control and bacterin immune or post-challenge serum from pigs to identify vaccinated groups prior to challenge, while significant proteins separated by gel electrophoresis and in silico increases in titer with a memory response were seen to prediction methods [13–15]. In this report, we utilized a both rRlpB and rVacJ for the subunit vaccinated pigs previously reported functional genomic screen to iden- (Fig. 1a and b). Additionally, animals were screened for tify subunit vaccine candidates [16]. This screen identi- antibody response to G. parasuis HS069. There was fies proteins associated with bacterial fitness and an increase in titer for bacterin vaccinated animals, resulted in the selection of RlpB and VacJ as vaccine but no change in titer for subunit vaccinated or con- candidates. The rlpB gene (lptE) is best studied in trol animals (Fig. 1c). Titers for bacterin vaccinated Escherichia coli. RlpB is a low abundance outer mem- animals were significantly higher at day 21 (p = 0.03) brane lipoprotein that functions in outer membrane as- and day 42 (p < 0.01) than that of subunit vaccinated sembly, specifically in mobilizing lipopolysaccharide to and control animals. the outer membrane’s outer surface, and plays an essen- Western blotting was utilized to evaluate the specifi- tial role in cellular viability [17–19]. The vacJ gene has city of the antibody response. Reactivity to G. parasuis been assessed in G. parasuis previously [20]. VacJ is an HS069 whole cell sonicate was not seen at 25 kDa or 35 outer membrane lipoprotein that contributes to outer kDa, which would correlate to intact RlpB and VacJ re- membrane integrity [20]. It has also been associated with spectively (Fig. 2a); however, some reactivity was noted stress tolerance, serum resistance, and host cell inter- at lower molecular weights. Probing with serum from action in G. parasuis and other Gram negative patho- the bacterin vaccinated animals revealed no reactivity to gens [20–23]. Additionally, the vacJ gene was previously the recombinant proteins (Fig. 2b). assessed for potential as a subunit vaccine against G. para- suis in a guinea pig model of disease [15]. In order to as- sess antigenicity and the potential of recombinant RlpB Cell mediated immune response and VacJ (rRlpB and rVacJ) to stimulate a protective im- Peripheral blood mononuclear cells (PBMCs) were col- mune response in swine, we vaccinated and boosted naïve lected at the time of boost (day 21), 1 week after boost pigs with rRlpB and rVacJ 3 weeks apart. Their antibody (day 28), and at challenge (day 42) to evaluate the preva- response was quantified and protection was evaluated lence of interferon-γ (IFN-γ) secreting cells. Animals im- through challenge with the G. parasuis strain HS069. munized with the subunit vaccine were found to have more IFN-γ producing cells showing reactivity to pooled Results rRlpB and rVacJ than control animals and bacterin vac- Comparison of RlpB and VacJ sequence identity cinated animals on day 21 (p = 0.014 and 0.006, respect- RlpB and VacJ amino acid sequences were compared to ively), but differences did not reach the statistical evaluate protein sequence diversity among G. parasuis threshold on day 28 and 42 (Fig. 3). Additionally, more isolates. The genome sequence was obtained for 11 G. IFN-γ producing PBMCs were noted in the subunit vac- parasuis strains representing 9 different serovars and cine group on day 21 than day 28 or 42, although this amino acid sequences of RlpB and VacJ were generated. was not statistically significant. Minimal reactivity The rlpB gene was obtained for 9 of the 11 strains, the was seen in both the control animals and subunit SW114 and 174 genomes are both draft sequences that vaccinated animals to stimulation with heat killed G. contain gaps and no rlpB was identified. The RlpB parasuis HS069, significantly less than that seen in amino acid sequence for the remaining 9 strains showed HS069 bacterin vaccinated animals on day 21 and 28 an identity greater than 96% among all strains. A (p <0.01)(Fig. 3). Hau et al. BMC Veterinary Research (2020) 16:167 Page 3 of 9 a c b Fig. 1 ELISA titers against rRlpB (a), rVacJ (b), and HS069 (c).