2012ZX10004701) from the Ministry of Science and Technology of the Peoples Republic of China, URL: http://www.nmp.gov.cn/. in the immune response of measles vaccine[3]. In addition, single nucleotide polymorphisms of TLRs have been reported to be able to influence the immune effect of vaccines. For example, TLR2 polymorphisms were associated with non-response to hepatitis B vaccine[4]. Furthermore, TLR agonists have been used as vaccine adjuvants for either prophylactic or therapeutic applications: a TLR4 agonist (3-O-desacyl-4-monophosphoryl lipid A/MPL) adsorbed to alum known as AS04 is used in a Chalcone 4 hydrate recombinant hepatitis B vaccine (Fendrix?, GSK)[5]; CPG 7909, a TLR9 agonist, is applied in an influenza vaccine (Fluarix?, GSK)[6]. Dual targeting of TLRs induces synergistic increases in Ag-specific neutralizing antibodies[7]. Vero cells have been used in vaccine production since 1980s[8C15], however, the oncogenic long-fragment residual DNA from vero Dock4 cell substrates pose a potential risk to human health[16C18]. Thus, nucleases, such as Benzonase manufactured by Merck & Co., were used to digest long-fragment DNA into short-fragment DNA (sf-DNA)[19, 20] which was assumed with better safety[21]. Although drug regulatory authorities have made a specification on the oncogenic long-fragment residual DNA for quality control of vero cell derived vaccines[22, 23], the potential effects of inevitable sf-DNA in final products on immune response remain unknown. A vero cell-derived EV71 inactivated vaccine, which showed good protection against EV71-associated HFMD in infants and young children in a Phase III clinical trial, was investigated in this study[24, 25]. TLR4 and TLR9 were assumed as potential receptors to recognize the EV71 inactivated vaccine. Because TLR4 was considered to recognize the structural protein of virus[26] which was the main component in the EV71 inactivated vaccine. Short-fragment DNA residue from host cell seemed more likely to activate TLR9 according to a recent study by Hsiao HB, which indicated the released endogenous DNA from dead cells mediated protection against EV71 infection in mice through TLR9 signaling[27]. In this study, we found sf-DNA residue from vero cell modulated immune response to new inactivated EV71 vaccine by upregulating TLR9 mRNA. The results of this study might provide a proof of the positive immune function of residual sf-DNA in immune response to vaccines. Materials and Methods Chalcone 4 hydrate 1. Inactivated EV71 whole-virus liquid bulk and cell lines Inactivated EV71 whole-virus liquid bulk (subgenotype C4, vero cell), containing 6000U/ml of Ag, less than 5EU/ml bacterial endotoxin, and with residual vero cell DNA below than 10pg per 400U antigen (Ag) measured by the dot blot hybridization assay following Caos[17] protocols, was a gift from Sinovac Biotech. Ltd. (Beijing, China). Vero cells were also obtained from Sinovac Biotech., which were the same as the substrate used for EV71 vaccine production. The cells were maintained in Eagles minimum essential medium containing 2% or 10% fetal bovine serum plus 2mM L-glutamine, 100IU of penicillin, and 100g of streptomycin per Chalcone 4 hydrate ml. 2. Mice immunization and serum collection Normal C57BL/6 mice, TLR4 knockout mice and TLR9 knockout mice on a C57BL/6 background were bred and maintained at the animal facility of National Institutes for Food and Drug Control (NIFDC, Beijing, China). All mice used here were six to eight week-old and specific-pathogen free. The EV71 liquid bulk was diluted to appropriate concentration (25U/ml, 100U/ml and 400U/ml) and then delivered intraperitoneally (i.p.) into female C57BL/6 mice at day 0. PBS was used as negative control and delivered similarly. Blood samples were collected from the female C57BL/6 mice at indicated times (0h, 6h, 12h, 24h, 48h, 72h, 7d and 14d). Blood samples were.