Mistake bars represent standard error for each group. conjugate carriers tetanus toxoid (TT) and diphtheria toxoid (DT), but not cross-reacting material HSPB1 197 (CRM197), induced antibody responses that protected mice and guinea pigs against lethal challenge with the respective toxins (7). Although animal models have helped predict how well some vaccines work in humans, careful consideration as to their inherent limitations should allow the identification of an optimal model that more accurately predicts immunogenicity outcomes in clinical trials (8). Here, we describe the development and optimization of a mouse immunogenicity model for the evaluation of candidate meningococcal Eperezolid polysaccharide-protein conjugate vaccines. Methods Ethical Statement Animal studies described here were approved by the Institutional Animal Care Eperezolid and Use Committee (Sanofi Pasteur, Swiftwater, PA) and undertaken in accordance with the recommendations in The Guide for the Care and Use of Laboratory Animals published by the National Research Council (Washington DC). Immunogen polysaccharides specific to serogroups A, C, W or Y were derivatized and conjugated to a carrier protein, either TT or DT (9). These monovalent polysaccharide-protein conjugate vaccine preparations were blended to form bulk tetravalent formulations (MenACYW-TT or MenACYW-DT). MenACYW-TT vaccine formulation was two-fold serially diluted in 0.85% saline from 2 g/dose down to 0.03125?g/dose of each meningococcal serogroup polysaccharide. These serial dilutions were prepared on the day of immunization and used immediately. A non-conjugated (free polysaccharide) MenACYW preparation was also included for comparison Eperezolid at 5?g (per serogroup) dose. MenACYW-DT stored under forced degradation conditions (56C for 1 week) was also evaluated in the optimized mouse model to assess the impact on immune response of the candidate vaccine having undergone a short-term increase of storage temperature outside recommended storage conditions (2C8C). Mouse Strains Female inbred and outbred mouse strains (inbred strains BALB/c [H-2d], C3H/HeN [H-2k], or C57BL/6 [H-2b], and outbred strains ICR [H-2g7] or Swiss Webster [H-2q2]) aged Eperezolid 6C8 weeks were purchased from Hilltop Lab Animals Inc (Scottdale, Pennsylvania) and housed in groups of 10 mice/cage. The animals were rested for a minimum of three days prior to inclusion in the studies. Food and water were available ad libitum. Immunization A number of parameters such as mouse strain, immunization dose, schedule and Eperezolid route were evaluated to develop an optimal immunogenicity model for assessing MenACYW conjugate vaccines. Initially, groups of 10 mice (four mouse strains assessed, excluding the Swiss Webster [H-2q2] strain due to supply issues) were injected subcutaneously with 0.25 mL of each of the candidate conjugate vaccine dose preparation or 0.85% saline as a control on days 0 and 14 (two-dose schedule; Figure?1A ) to identify the optimal mouse strain and immunization dose. An ideal pre-clinical animal model should be sensitive enough to detect a drop in the potency of vaccines either due to alteration of conjugation parameters or deterioration. A three-dose immunization schedule was also assessed, in parallel to the two-dose immunization schedule, using the optimal mouse strain and dose identified, with the MenACYW-TT conjugate vaccine administered as three subcutaneous (SC) or intraperitoneal (IP) injections (on days 0, 14 and 28) ( Figure?1B ). Sera were collected two or three weeks after the last dose for assessment of total IgG and bactericidal antibody responses. Specific details on the immunization regimen of each study are provided in the results section. Open in a separate window Figure?1.