Vaccine Pre-clinical Toxicology Testing

Pre-clinical Safety Testing of Candidate Vaccines

It may be helpful for the reader to consider the perspective from which FDA reviewers consider pre-clinical toxicology testing of candidate vaccines. It can be summed up in three words: safety, safety, safety. What is (are) a safe starting dose(s) for a Phase 1 study? What is a safe route of administration or safe schedule of administration? Are there any physiological parameters affected by the vaccine candidate that should be examined particularly closely in the Phase 1 clinical trial? In addition, a vaccine manufacturer may wish to consider such factors as suitability of the animal model and time for completion of the studies (so they can plan ahead).

Qualification of Testing Facility: GLP-capable facility

Protocol design:
The pre-clinical protocols should be based on the design of the proposed clinical protocol and the planned dose(s) and dosing regimen. Therefore, a generic protocol is not provided, but general recommendations are described below:

1. General recommendations for local and systemic toxicity/repeat dose⁶ GLP studies in animals
1. The choice of animal model should be appropriate for the product and clinical indication. Often rabbits are used for parenteral vaccine toxicity because their muscle mass can receive a volume equivalent to a full human clinical dose (e.g., 0.5 or 1 mL).
2. High dose should be 1 - 10 times the actual highest planned clinical dose not scaled on weight or body area.
3. To determine if the effects observed are dose-related (and to potentially identify something equivalent to a NOAEL), 2 or 3 concentrations, to cover the range of proposed clinical doses, in addition to a vehicle and/or adjuvant⁷ control, should be used. At a minimum, the highest proposed human dose should be tested.
4. Number of proposed clinical inoculations plus one
5. Period of study varies, depending on the frequency of dose administration (episodic, not daily), which may be abbreviated compared to the proposed clinical dosing schedule. The duration of the GLP safety studies is dependent on the study design. Tissue samples should be processed and data analyzed after intermediate and terminal sacrifice.
6. Timepoints for sacrifice: 1-3 days post-last inoculation; 2-4 weeks post-last inoculation (recovery)
7. Minimum of 5 animals per gender per dose for each time point of sacrifice - this number may vary depending on animal model chosen
8. Same route of administration as the proposed clinical route (with same delivery device, whenever possible)
9. Minimal endpoints examined should include:
1. Clinical observations (daily),
2. Physical examinations (weekly),
3. Evaluation of injection site(s) for irritation and histopathology,
4. body weights (weekly),
5. Food and water consumption, body temperatures (daily in week following inoculations),
6. Ophthalmologic observations,
7. Clinical pathology at regular intervals for hematology, serum chemistry, serology, urinalysis measurements,⁸
8. Gross observations and organ weights at necropsy,
9. Histopathology evaluation to include a select tissue list, especially the immune function organs (e.g., lymph nodes), other highly perfused organs, and the genital organs in the control and high-dose animals and target tissues in the remaining groups. Depending on the route of inoculation, additional organs may need to be examined. (Full tissue collection and preservation should be performed even when only a select list are examined histopathologically),
10. Relevant immunogenicity (Humoral and CMI) studies.

Additional endpoints may be included to address therapeutic-specific concerns.

2. For nucleic acid & some virus-vector based vaccines, specialized studies to examine genetic toxicology (e.g., biodistribution) are so strongly recommended that you should consider them required. These are studies that may incorporate the use of assays which do not (yet) meet GLP standards⁹. General recommendations include:
1. Tissue Distribution studies:
1. 1 - 2 doses, paralleling the doses in the systemic toxicity studies, in addition to the vehicle and/or adjuvant control
2. Same route of injection as the proposed clinical route
3. Time points: from 1 day up to 8 weeks after injection
4. Minimum of 5 animals per gender per dose per time point
5. Endpoint: Using the most sensitive molecular technique(s), detect presence of vaccine in a variety of tissues (which should not be pooled, but rather assayed individually) in the animal (i.e., single tissues from single animals¹⁰), including at a minimum: injection site muscle and skin, lymphatic tissues, other highly perfused organs, body fluids, reproductive tissues to address possible heritable concerns, and other major tissues specific to the vaccine.
2. Integration studies in tissues where vaccine signals persist
1. Single high dose
2. Same route as the studies above
3. Duration: from 10 weeks up to 6 months post injection.
4. Target tissues: injection site skin and muscle and other positive tissues at the late distribution time points.

Additional studies investigating multiple coding regions within the construct using multiplex PCR, confirming the integrity of extracted genomic DNA using housekeeping or constitutively expressed genes, carcinogenesis or tumorigenesis studies may be required if the vaccine is demonstrated to be integrated in tissues.

A detailed summary of the designed protocol is included in the pre-IND document and will frequently be modified based upon the recommendations of CBER at the pre-IND meeting.

After the pre-IND meeting and before the initiation of the pre-clinical safety studies, the following information is necessary to begin:

1. Principal Investigator's and Sponsor's approval of the final study protocol
2. Stability of the vaccine under the conditions of use
3. Homogeneity and uniformity of the formulated vaccine
4. Certificate of Analysis of the lot of vaccine prepared for the pre-clinical studies
5. Standard Operating Procedure (SOP) for dispensing vaccine for dosing. A Material Safety Data Sheet (MSDS) or equivalent is useful.

Items 2 - 5 are generally obtained from the vaccine manufacturer.

All study results should be checked by scientific staff to control quality (QC) and assure integrity of the data. The Quality Assurance (QA) department audits the data and reports. A Draft report is generated and circulated among the sponsor and client(s) for comments and final report(s) are generated, incorporating client(s) comments and findings (if any) by the QA department. The final report(s) are included in the IND document. Alternatively, FDA will review draft unaudited reports, with the expectation that no major differences will be noted during the auditing process and that the final audited reports will be submitted when they become available.