Use of Praziquantel as an Adjuvant Enhances Protection and Tc-17 Responses to Killed H5N1 Virus Vaccine in Mice

et al. (2012) Use of Praziquantel as an Adjuvant Enhances Protection and Tc-17 Responses to Killed H5N1 Virus Vaccine in Mice. PLoS ONE 7(4): e34865. doi:10.1371/journal.pone.0034865 Use of Praziquantel as an Adjuvant Enhances Protection and Tc-17 Responses to Killed H5N1 Virus Vaccine in Mice Qiang Zou 0 Yanxin Hu 0 Jia Xue 0 Xiaoxu Fan 0 Yi Jin 0 Xianghua Shi 0 Di Meng 0 Xianzheng Wang 0 Congcong Feng 0 Xiaoping Xie 0 Yizhi Zhang 0 Youmin Kang 0 Xiaoxuan Liang 0 Bing Wu 0 Ming Wang 0 Bin Wang 0 Yi Guan, The University of Hong Kong, China 0 1 Key Laboratory of Medical Molecular Virology of MOH and MOE, Fudan University Shanghai Medical College , Shanghai , China , 2 State Key Laboratory for Agro- Biotechnology, College of Biological Science, China Agricultural University , Beijing , China , 3 College of Veterinary Medicine, China Agricultural University , Beijing , China Background: H5N1 is a highly pathogenic influenza A virus, which can cause severe illness or even death in humans. Although the widely used killed vaccines are able to provide some protection against infection via neutralizing antibodies, cytotoxic T-lymphocyte responses that are thought to eradicate viral infections are lacking. Methodology/Principal Findings: Aiming to promote cytotoxic responses against H5N1 infection, we extended our previous finding that praziquantel (PZQ) can act as an adjuvant to induce IL-17-producing CD8+ T cells (Tc17). We found that a single immunization of 57BL/6 mice with killed viral vaccine plus PZQ induced antigen-specific Tc17 cells, some of which also secreted IFN-c. The induced Tc17 had cytolytic activities. Induction of these cells was impaired in CD8 knockout (KO) or IFN-c KO mice, and was even lower in IL-17 KO mice. Importantly, the inoculation of killed vaccine with PZQ significantly reduced virus loads in the lung tissues and prolonged survival. Protection against H5N1 virus infection was obtained by adoptively transferring PZQ-primed wild type CD8+ T cells and this was more effective than transfer of activated IFN-c KO or IL-17 KO CD8+ T cells. Conclusions/Significance: Our results demonstrated that adding PZQ to killed H5N1 vaccine could promote broad Tc17mediated cytotoxic T lymphocyte activity, resulting in improved control of highly pathogenic avian influenza virus infection. - Funding: This work was partly supported by the National High-Tech 863 Project of China (Development of Novel Adjuvants for Influenza Vaccines) and Scientific Technology Development Foundation of Shanghai (09DZ1908602). No additional external funding received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Avian A/H5N1 Influenza A virus, emerged as a cause of human disease in recent years that is associated with high mortality and it poses a major pandemic threat [13]. Vaccination has its advantages over other approaches for limiting potential pandemic influenza outbreaks. Although, the widely used killed H5N1 vaccine is helpful by eliciting a neutralizing antibody response in chickens [4], protection against H5N1 infection in humans is likely to require eliciting both humoral and cellular virus-specific responses. Eliciting cytolytic T lymphocyte responses may be particularly important since this has been demonstrated to eradicate viral infections [58]. The induction of broad cytotoxic T lymphocyte responses to killed H5N1 vaccine is an urgent and challenging issue. Adjuvant could be added into the killed vaccine to elicit a robust and broad cellular immune response [9,10]. Till now, alum and MF59 are the only two adjuvants approved by the U.S. Food and Drug Administration (FDA) for use in influenza vaccines [9,11], but both are limited by minimal induction of cell-mediated immunity [1216]. Novel adjuvants are therefore needed to enhance cellular immunity for these killed viral vaccines. Praziquantel (PZQ) has an excellent record of safety in treating Schistosomiasis infections [17], and recently we demonstrated that PZQ could enhance cellular responses to HBsAg DNA vaccination [18,19]. Therefore we investigate here whether PZQ could enhance cellular immune responses to the killed H5N1 vaccination. In this study, we demonstrated that by using PZQ with a killed H5N1 vaccine mice could be better protected against a lethal-dose challenge with H5N1 virus. This protection was strongly associated with elicitation of more IL-17-producing cytotoxic CD8+ T cells (Tc17) when PZQ was used with the killed H5N1 vaccination. This is the first report to show that PZQ has great potential as an adjuvant to induce strong CD8+ T cell-immunity with killed H5N1 vaccine. PZQ as adjuvant induced a high level of cytolytic response Since virus specific cytotoxic T lymphocyte activity is indispensable for controlling viral infections [7,8], we first examined whether adding PZQ to the killed H5N1 vaccine (0.5 mg PZQ/ 0.1 mg antigen) could induce higher levels of nucleoprotein (NP)specific cytolytic responses. Mice were immunized a single time and in vivo cytotoxic activity was assayed 7 days later. Compared to the groups that were immunized with the killed vaccine alone or the killed vaccine with vehicle, the highest level of antigen-specific lysis was observed in the mice immunized with the killed vaccine plus PZQ (Figure 1AB). Since antibody production is also an important arm of immune response, we also tested antibody titers of serum samples taken 14 days after the vaccination. There were no differences between the groups (Figure 1C), suggesting that PZQ did not affect the humoral response. PZQ promoted Tc17 cell activation Because both IFN-c-producing CD8+ T cells (Tc1) and IL-17producing CD8+ T cells (Tc17) could contribute to the cytolytic response [2022], we sought to determine which subset of T cells were the main effectors. Splenocytes were isolated 7 days after immunization and stimulated with NP peptide (ASNENMETM) in vitro before they were stained for intracellular IFN-c, IL-17 or both and assayed by flow cytometry. As shown in Figure 2AB, the expression of antigen-induced IL-17, but not IFN-c in CD8+ T cells was significantly higher in the mice immunized with killed vaccine plus PZQ compared to other groups. This was confirmed by the result of double staining shown in Fig. 2.CD, which Figure 1. In vivo cytotoxic responses and antibody responses in immunized mice. C57BL/6 mice were immunized with 0.1 mg killed H5N1 vaccine in 100 ul of delivery vehicle, with vehicle alone, with vehicle containing PZQ (0.5 mg PZQ/0.1 mg antigen) or with vaccine in vehicle containing PZQ. (A) Analysis of in vivo cytotoxic lysis on day 7 after primary immunization. (B) The percentage of specific lysis summarized as the means of the three independent experiments. (C) Antibody levels in serum collected on day 14 after immunization (...truncated)