Potency testing of most human and veterinary rabies vaccines requires vaccination of mice followed by a challenge test using an intracerebral injection of live rabies virus. NICEATM, ICCVAM, and their international partners organized a workshop to review the availability and validation status of alternative methods that might reduce, refine, or replace the use of animals for rabies vaccine potency testing, and to identify research and development efforts to further advance alternative methods. Workshop participants agreed that general anesthesia should be used for intracerebral virus injections and that humane endpoints should be used routinely as the basis for euthanizing animals when conducting the mouse rabies challenge test. Workshop participants recommended as a near-term priority replacement of the mouse challenge with a test validated to ensure potency, such as the mouse antibody serum neutralization test for adjuvanted veterinary rabies vaccines for which an international collaborative study was recently completed. The workshop recommended that an in vitro antigen quantification test should be a high priority for product-specific validation of human and non-adjuvanted veterinary rabies vaccines. Finally, workshop participants recommended greater international cooperation to expedite development, validation, regulatory acceptance, and implementation of alternative test methods for rabies vaccine potency testing.

The National Institutes of Health (NIH) rabies vaccine potency test is used internationally for evaluating the efficacy of inactivated rabies vaccines, despite concerns about its methods. An alternative test has been developed, using a simplified in vivo method for rabies vaccine testing which has several advantages over the currently recommended method of efficacy testing. The rabies peripheral challenge test more closely models practical vaccine application in target species; decreases the observed effect of vaccine virus strain in testing results and allows sensitive analysis of vaccine and production lot testing.

1429 Several anti-tumor antibody-maytansinoid conjugates, commonly called TAPs (tumor-activated prodrugs) are currently under clinical evaluation. In particular, cantuzumab mertansine (huC242-DM1), has been evaluated in three Phase I clinical trials (A.W. Tolcher et al., J Clin Oncol 2003, 21:211-22; P.R. Helft et al., Clin Cancer Res 2004, 10:4363-4368) using different dosing schedules to treat patients with tumors expressing the CanAg target antigen. The immunoconjugate could be safely administered at pharmacologically meaningful doses and evidence of anti-tumor activity was seen in several patients, which led to a recommendation for broad clinical development of the agent (A.W. Tolcher et al., J Clin Oncol 2003, 21:211-22). During the clinical evaluation, further advancement in the TAP technology led to the development of TAPs with disulfide-containing linkers with increased stability under physiological conditions. One of these new TAPs that demonstrated improved pharmacokinetic behavior and superior anti-tumor activity in murine test models is huC242-DM4, which contains the same antibody as cantuzumab mertansine and targets therefore the same CanAg-expressing tumors. HuC242-DM4 caused complete regressions in several tumor xenograft models even with single i.v. administration at doses far below its maximal tolerated dose (MTD). For example, treatment of SCID mice bearing measurable N87 human gastric tumor xenografts with a single dose of 3.4 mg/kg caused complete regression of the tumors, with significant tumor regression observed at a dose below 2 mg/kg. With a MTD in SCID mice of greater than 40 mg/kg when given as a single i.v. bolus injection, a therapeutic index of over 20 was achieved. HuC42-DM 4 was also curative in mice bearing human colon tumor xenografts of HT29 and COLO 205 cells, at doses that were non-toxic. The disulfide linkage of huC242-DM4 was approximately 2-fold more stable during circulation in mice than that of cantuzumab mertansine. While the huC242-DM4 conjugate demonstrated increased stability and efficacy in vivo compared to cantuzumab mertansine, preliminary acute toxicity studies suggest that the two conjugates have similar tolerability in normal mice. The resulting increased therapeutic window for huC242-DM4 provides a preclinical rationale for its clinical evaluation in patients with CanAg-positive tumors, such as colorectal, pancreatic, gastric, and esophageal carcinomas.

OBJECTIVETo evaluate the effect of various routes of administration and number of doses of 3 commercially produced rabies vaccines on serum antibody responses and protection in mice challenged by intracerebral injection with fixed-strain rabies virus.ANIMALS2,213 mice.PROCEDUREInactivated, adjuvanted rabies vaccines were administered to mice in either 2, 1, or 0 (control) doses via IP, IM, and SC routes, and mice were challenged intracerebrally with fixed-strain rabies virus.RESULTSVaccination route and dose number significantly influenced serum antibody responses and protection from rabies virus challenge, independent of vaccine strain origin and mouse strain, although mouse age significantly affected results. Extended challenge studies revealed that IM vaccination of mice resulted in the highest serum neutralizing antibody responses and protection levels equivalent to IP vaccination. Even multiple doses administered SC (a vaccination route used in dogs) resulted in poor serum anti-rabies neutralizing antibody responses in mice and were far less protective than other routes.CONCLUSIONS AND CLINICAL RELEVANCEFindings suggest possible improvements for the current rabies vaccine potency test in mice by using 1 dose, the IM route, and a delayed time of challenge. These modifications would more closely model vaccination practices in target species and yield more accurate information regarding primary immunogenicity of a vaccine.

OBJECTIVE:To determine effect of route of challenge and strain of rabies virus on efficacy of inactivated rabies vaccines in mice.ANIMALS:3,056 mice.PROCEDURE:Challenge was performed with fixed and street rabies virus strains by use of footpad and intracerebral routes as well as IM injection into the hip, shoulder, neck, and masseter muscles. Intraperitoneal and IM vaccination was performed with 1 or 2 doses of 1 of 3 vaccine-strain inactivated rabies vaccines. For 2 of the vaccine strains, the vaccines were adjuvanted and nonadjuvanted.RESULTS:Incubation periods were dependent on route, dose, and virus strain used for challenge. Use of an intramasseter challenge route with challenge virus-strain rabies virus, which more accurately models natural exposure to rabies virus, resulted in reproducible mortality rates in mice. Use of this route revealed that differences among vaccines and challenge virus strains affected mortality rate less than that observed in the National Institutes of Health potency test, even when street isolates of widely variant origin were used for challenge.CONCLUSIONS AND CLINICAL RELEVANCE:These results, combined with earlier data, support a proposal for a new rabies potency test that more closely models current vaccine administration practices and natural infection routes.

Intraperitoneal vaccination of mice with rabies vaccine results in both dosage-dependent rabies virus neutralizing antibody titres and protection from lethal intracerebral (i.c.) challenge with fixed strain CVS rabies virus. Pre-exposure adoptive intravenous transfer of naive or immune cells did not significantly protect naive Balb/c mice from lethal i.c. CVS challenge, but immune serum and anti-rabies glycoprotein monoclonal antibodies (individually and in combination) did confer significant protection when administered before or up to 24 h after lethal i.c. rabies virus challenge.