In this study, we developed HPV 16/18/58 trivalent L1 VLP vaccines and compared the type specific neutralizing antibody levels induced by the trivalent vaccine with those by corresponding monovalent vaccines. We found that the HPV 58 containing trivalent vaccine could induce high titers of HPV specific antibodies against all component types, and that the type specific neutralizing antibody levels were interfered by co-immunized antigens. HPV 16, 18, 58 L1 genes were codon optimized according to the codon usage bias of Sf9 cells. All modification was made according to Table 1. Optimized genes were synthesized by Sangon Corp. (Shanghai, China) and constructed into

pFastBac I (Invitrogen). Optimized genes were uploaded to Genbank, and the accession numbers are GU556964 (HPV 16 L1), GU556965 (HPV 18 L1) and GU556966 (HPV 58 L1), respectively. HPV 6 and 11 L1 genes were obtained by our lab previously Dabrafenib in vitro [30], [31] and [32]. L1 genes were expressed in baculovirus expression system and purified by CsCl ultracentrifugation

as described previously [33]. The purity of L1 was evaluated by SDS-PAGE with Coomassie blue staining. VLPs were further verified by transmission electron microscopy (TEM) [31]. We formulated pentavalent, trivalent, bivalent and monovalent vaccines with high and low dose of antigens, with or without Aluminium adjuvant according to Table 2. High dose vaccines contained 5 μg VLPs of each type, while low dose vaccines contained 0.1 μg VLPs of each type. IDO inhibitor The adjuvant we used here is Aluminium hydroxide (Sigma–Aldrich). All the vaccines were formulated in a total volume of 100 μl in PBS. The control vaccine

contained 100 μl PBS only. Balb/c mice were purchased from the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences, and kept in the animal facility of the Institute of Basic Medical Science, Chinese Academy of Medical Sciences. All experimental protocols were approved by the Institutional Animal Care and Use Committee. Experiment groups immunized with different vaccine formulations were listed in Table 2. Briefly, for the long-term experiments, mice (n = 4 per group) were immunized intramuscularly with Trivalent-1 vaccine, Mono 16, 18, 58 vaccines or PBS, respectively at week 0, 2, 4, and were given an extra boost at week 52. Serum samples were collected at 2 week’s interval for first 12 first weeks and then at 10 week’s interval until week 52. Samples were also collected 2 weeks after the extra boost. All samples were analyzed by ELISA for type specific antibody responses [30]. Serum samples collected at week 4 and 6 were analyzed for neutralizing antibody level (pseudo-neutralization assay). For dose adjustment experiments, mice (n = 4 per group) were immunized intramuscularly with Trivalent-1, Trivalent-2, Mono 16, Mono 18 and Mono 58 vaccines, respectively at week 0, 2, 4. Serum samples collected at week 4 and 6 were analyzed by pseudo-neutralization assay.