parasuis. == Physique 1. involved in the protective effects of HPS_06257. Our findings extend our understanding of how antibody-dependent phagocytosis may contribute to the immune protection afforded by other outer membrane proteins. Thus, our study provides insight into the protective antigens ofGlaesserella parasuisand useful information for the development of novel vaccines to preventGlaesserella parasuisinfection. == Abstract == HPS_06257 has been identified as an important protective antigen againstGlaesserella parasuisinfection. However, little is known about the role of HPS_06257 in the protective immune response. A whole-genome data analysis showed that among 18 isolates ofGlaesserella parasuis, 11 were positive for theHPS_06257gene, suggesting that not every strain contains this gene. We used PCR to investigate the presence of theHPS_06257gene among 13 reference strains and exhibited that 5 strains contained the gene. A polyclonal antibody against HPS_06257 was generated with a recombinant protein to study the expression of HPS_06257 in those 13 strains. Consistent with the PCR data, five strains expressed HPS_06257, whereas eight strains were HPS_06257 null. We also compared the protective effects of HPS_06257 against an HPS_06257-expressing strain (HPS5) and an HPS_06257-null strain (HPS11). Immunization with HPS_06257 only guarded against HPS5 and not HPS11. Moreover, phagocytosis of antibody-opsonized bacteria demonstrates that this antibody against HPS_06257 increased the phagocytosis of the HPS5 strain by macrophages but not the phagocytosis of the HPS11 strain, suggesting that antibody-dependent phagocytosis is responsible for the protective role exerted by HPS_06257 in the immune response to HPS5. Our data also show that this antibody against HPS_06257 increased the phagocytosis of the other HPS_06257-expressing strains by macrophages but not that of HPS_06257-null strains. In summary, our findings demonstrate that antibody-dependent phagocytosis contributes to Anguizole the protective immune response induced by immunization with HPS_06257 against HPS_06257-expressing strains. Keywords:Glaesserella parasuis, HPS_06257, immunization, antibody-dependent phagocytosis == 1. Introduction == Glaesserella parasuis(G. parasuis), formerly known asHaemophilus parasuis(HPS), is one of the opportunistic pathogens causing Glssers disease in piglets, which is usually characterized by fibrinous polyserositis, polyarthritis, and meningitis [1]. With increasing reports of this disease,G. parasuishas become a significant concern for pig farmers. Vaccination has been shown to be effective in preventingG. parasuisinfection by inducing the protective immune response. Notably, a humoral immune response plays an important role in protection ofG. parasuisinfection. For example, passive immunization of pigs with anti-serum againstG. parasuisprotected against lethal challenge [2]. The mechanism of protection by antibodies has been shown to be associated with antibody-opsonized phagocytosis, which facilitates uptake ofG. parasuisby phagocytes such as macrophages [3]. Regarding vaccination, as Anguizole a promising approach to prevention and control of Glssers disease, different kinds of vaccines have been developed. While inactiveG. parasuisvaccines have some limitations, including the different levels of cross-protection betweenG. parasuisserovars, a short time of protection, and so on, they are widely used throughout the world. In order to overcome the limitations of inactivated vaccines, several other methods have been used to develop novel vaccines. Among them, subunit vaccines have been extensively investigated for this purpose [4]. Several different kinds of proteins have been used to develop subunit Anguizole vaccines [5,6,7,8,9,10]. Notably, outer membrane proteins are often chosen and confer effective protection againstG. parasuisinfection, mainly of homologous strains [5,11,12]. HPS_06257 is one of the outer membrane proteins that has been shown to confer protection againstG. parasuisinfection [5]. It has a molecular weight of 28 kDa and contains a lipoprotein, GNA1870, domain name at its C-terminus. Although data have shown that anti-HPS_06257 serum may safeguard againstG. parasuisinfection, the specific mechanism of Anguizole this protective effect remains unclear. Antibody-dependent phagocytosis is an important mechanism underlying the Anguizole protective roles of some immune antigens [9]. However, little is known about the role of antibody-dependent phagocytosis in HPS_06257-mediated protection againstG. parasuisinfection. PreviousG. parasuisgenome analyses have identified similarities and divergence in the genomes of differentG. parasuisstrains [13,14,15]. Whether HPS_06257 is present in all strainsG. parasuisremains unknown. In this study, we identified the presence Tm6sf1 and absence of HPS_06257 in differentG. parasuisstains and clarified the role of antibody-dependent phagocytosis in the protection it affords against HPS_06257-expressing strains. == 2. Materials and Methods == == 2.1. Glaesserella parasuis == Glaesserella parasuiswas recovered from a stock frozen in 20% glycerol and stored at 80 C, and was grown on tryptic soy agar (TSA; Difco, Detroit, MI, USA) supplemented with 20 g/mL nicotinamide adenine dinucleotide.