Comparative Analysis of the Immunogenicity and Protective Effects of Inactivated EV71 Vaccines in Mice

et al. (2012) Comparative Analysis of the Immunogenicity and Protective Effects of Inactivated EV71 Vaccines in Mice. PLoS ONE 7(9): e46043. doi:10.1371/journal.pone.0046043 Comparative Analysis of the Immunogenicity and Protective Effects of Inactivated EV71 Vaccines in Mice Qunying Mao 0 Chenghong Dong 0 Xiuling Li 0 Qiang Gao 0 Zengbing Guo 0 Xin Yao 0 Yiping Wang 0 Fan Gao 0 Fengxiang Li 0 Miao Xu 0 Weidong Yin 0 Qihan Li 0 Xinliang Shen 0 Zhenglun Liang 0 Junzhi Wang 0 Lijun Rong, University of Illinois at Chicago, United States of America 0 1 National Institutes for Food and Drug Control , Beijing , China , 2 Institute of Medical Biology, Chinese Academy of Medical Sciences , Kunming , China , 3 National Vaccine and Serum Institute , Beijing, China, 4 Sinovac Biotech Co. , Ltd. , Beijing , China , 5 Hualan Biological Engineering Inc , Henan , China Background: Enterovirus 71 (EV71) is the major causative agent of hand, foot, and mouth disease (HFMD). Three inactivated EV71 whole-virus vaccines of different strains developed by different manufacturers in mainland China have recently entered clinical trials. Although several studies on these vaccines have been published, a study directly comparing the immunogenicity and protective effects among them has not been carried out, which makes evaluating their relative effectiveness difficult. Thus, properly comparing newly developed vaccines has become a priority, especially in China. Methods and Findings: This comparative immunogenicity study was carried out on vaccine strains (both live and inactivated), final container products (FCPs) without adjuvant, and corresponding FCPs containing adjuvant (FCP-As) produced by three manufacturers. These vaccines were evaluated by neutralizing antibody (NAb) responses induced by the same or different dosages at one or multiple time points post-immunization. The protective efficacy of the three vaccines was also determined in one-day-old ICR mice born to immunized female mice. Survival rates were observed in these suckling mice after challenge with 20 LD50 of EV71/048M3C2. Three FCP-As, in a dose of 200 U, generated nearly 100% NAb positivity rates and similar geometric mean titers (GMTs), especially at 14-21 days post-inoculation. However, the dynamic NAb responses were different among three vaccine strains or three FCPs. The FCP-As at the lowest dose used in clinical trials (162 U) showed good protective effects in suckling mice against lethal challenge (90-100% survival), while the ED50 of NAb responses and protective effects varied among three FCP-As. Conclusions: These studies establish a standard method for measuring the immunogenicity of EV71 vaccines in mice. The data generated from our mouse model study indicated a clear dose-response relationship, which is important for vaccine quality control and assessment, especially for predicting protective efficacy in humans when combined with future clinical trial results. - Funding: The current study was sponsored by the National Science Project (No. 2008BAI69B01) and the National 11th Five Major Special Projects Funding Program (No. 2009ZX10004-804) from the Ministry of Science and Technology of the Peoples Republic of China. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Q Gao and WD Yin are affiliated to Sinovac Biotech Co., Ltd. ZB Guo is affiliated to Hualan Biological Engineering Inc.; XL Li and XL Shen are affiliated to National Vaccine and Serum Institute; CH Dong and QH Li are affiliated to Institute of Medical Biology, Chinese Academy of Medical Sciences. As a Board member, WD Yin holds shares of Sinovac Biotech Co., Ltd. There are EV71 vaccines in development to declare. There are no consultancies, patents, or marketed products to declare. This does not alter the authors adherence to all the PLOS ONE policies concerning sharing data and materials as detailed online in the guide for authors. Enterovirus 71 (EV71) is a small RNA virus belonging to the Enterovirus genus. It is a spherical particle with icosahedral (cubic) symmetry and contains a positive-sense single-stranded RNA approximately 7.4 kb long. Each subunit of the viral capsid contains a copy of the four structural viral proteins (VP1VP4); VP1, VP2, and VP3 are external, while VP4 is completely within the interior of the viral particle and is not, therefore, exposed to the host antibody response [1]. VP1 displays the predominant neutralizing epitope [2]. EV71 infection mainly leads to hand, foot, and mouth disease (HFMD) and EV71-associated neurological diseases, including aseptic meningitis, brainstem encephalitis, and acute flaccid paralysis indistinguishable from poliomyelitis [3]. In recent years EV71 has caused epidemics and is a growing public health concern due to a high incidence of severe symptoms and high fatality rates in Asia-Pacific regions [413]. Because of the lack of preventative and therapeutic measures, the development of safe and effective EV71 vaccines has become an urgent matter, especially in China. Currently, there are several commercial manufacturers and research institutes developing different types of EV71 vaccines, including inactivated virus vaccines, attenuated live vaccines, engineered virus-like particle (VLP) vaccines, and polypeptide vaccines [1421]. Benefiting from the research communitys extensive experience in developing other enterovirus vaccines, such as the polio and hepatitis A vaccines, development of inactivated virus vaccines has proceeded faster than the others and exhibits the highest apparent immunogenicity [18,20,22]. In mainland China [23], Taiwan [24] and Singapore [25], these inactivated virus vaccines have been tested in clinical trials and are expected to be the first class of vaccines to be employed to prevent EV71-associated diseases worldwide [23]. In mainland China, three inactivated EV71 vaccines have been developed by different manufacturers. Although the three vaccines are all inactivated virus vaccines, differences in their manufacturing processes exist, including the strains (though all three are the C4 genotype), cell substrate (Vero or diploid cells), cell culture system (roller bottles, cell factories or microcarrier bioreactor system), production process, and vaccine dose (Table 1). All these factors may lead to differences in immunogenicity [15,26]. Although good immunogenicity and protective effects have been reported at particular time points after immunization, the antigen content of these vaccines was reported in different units (mg/ml, KU/ml, EU/ml), and different animal models were empolyed by the different manufacturers to test these vaccines [27,28]. These differences make it difficult to compare the immunogenicity and protective effects among the different EV71 vaccines, which will be important for testing in clinical trials. A prior collaborative effort was carried out to standa (...truncated)