HIV/AIDS

Pre-clinical Toxicology Testing of Vaccines

By CAPT Rebecca L. Sheets, Ph.D. and Polly Y. Chang, Ph.D.

Introduction

These web pages are intended to provide HIV vaccine researchers with general advice on pre-clinical toxicology testing and pre-clinical product development to aid in translating research concepts into vaccine candidates suitable for human clinical trial testing. The recommendations provided on this webpage do not reflect official U.S. Food and Drug Administration (FDA) policy. Thus, the reader is encouraged to contact the Office of Vaccines Research and Review (OVRR) in the Center for Biologics Evaluation and Research (CBER) at FDA regarding pre-clinical safety testing requirements for particular vaccine candidates to support investigational new drug applications. In addition, specific guidance on regulatory requirements for a particular product should be sought from the FDA (www.fda.gov/cber). These web pages will provide advice on pre-clinical toxicology study designs, in general, and with additional focus on specialized toxicology studies for DNA plasmid or other recombinant vectored vaccines. Many of the resources needed to accomplish this translation of research concepts into actual product vaccine candidates may be requested from the Division of AIDS.

The purpose of this webpage is to serve as a reference summary for the pre-clinical toxicology studies to be performed to support clinical development of vaccines. Furthermore, information on the process of overall product development can be found in the following two documents:

1. The HIV/AIDS vaccine researchers' orientation to the process of preparing a US FDA application for an investigational new drub (IND): what it is all about and how you start by preparing for your pre-IND meeting (PDF), Stuart Shapiro, Vaccine 2001, Oct 18, 20(2002):1261-1280. Reprinted from Vaccine, with permission from Elsevier Science. Single copies of this article can be downloaded and printed for the reader's personal research and study.
2. Toxicity Testing of Vaccines, Polly Y. Chang, Joan Roelands, Janice Schindler-Horvat, and Rebecca L. Sheets. Non-clinical Drug Safety Assessment, Sietsema WK and Schwen R, ed., FDAnews, 2007.

The design of the pre-clinical safety testing protocols and the design of the Phase I clinical trial protocols can occur concurrently and each depends on the other. CBER input may be sought to aid in the filing of the IND by holding a pre-IND meeting between CBER representatives and the vaccine development team. A pre-IND document is submitted to CBER to review and more information can be found on this process in the article and chapter referenced above. Based on CBER’s recommendations at the pre-IND meeting, both pre-clinical toxicology and clinical protocols may need to be modified and then pre-clinical safety testing in animals can be initiated with well-characterized vaccine material. Therefore, it is a good idea not to initiate the pivotal or definitive pre-clinical study(ies) until after the pre-IND meeting.

Considerations for Vaccine Manufacturing

It should be understood that product characterization data and lot release data demonstrating the safety, purity, and potency of the vaccine candidate are critical components to support vaccine safety. Pre-clinical safety assessments may also be gathered in the course of GMP or non-GLP immunogenicity studies (which may be a measure of potency); GMP and/or GLP pyrogenicity studies (as part of the evaluation of vaccine purity); as well as non-GLP challenge/protection studies (if appropriate animal models exist). For guidance in these areas, reference is made to applicable guidance for industry documents. Please see Points to Consider.

Performing pre-clinical safety testing on material that is not sufficiently well characterized may result in invalid studies. It is preferable to perform the pre-clinical studies on the same lot of vaccine that you intend to use in the initial clinical trial(s), although it is acceptable to perform the studies on pilot lots made by the exact same manufacturing process provided product characterization data are available demonstrating comparability of the pilot and clinical lots.

Clinical doses are frequently based on a dose(s) shown to be immunogenic in “proof of concept” or immunogenicity studies conducted in animal models. After the manufacture of the final bulk material under GMP, vaccines are generally filled into vials or syringes for pre-clinical safety studies based on the dose(s) described in the protocols. At a minimum, the highest dose to be used in the proposed clinical trial is usually studied in the animal model. In cases when it is necessary for the pre-clinical safety testing facility to formulate the doses to be given in the pre-clinical study(ies), dose verification (e.g., potency verification), dose stability under the conditions of use, and uniformity testing must be performed to verify the delivered immunization dose(s). Although aliquots of bulk vaccine material can be diluted or formulated into doses within a day or two before animal immunization rather than filled into final containers as single doses of formulated final material, these doses prepared at the pre-clinical safety testing facility must be verified to be what they were supposed to be either at the pre-clinical safety testing site or by the vaccine manufacturer or designee. This testing should include a potency determination (even if a final validated potency test is not developed yet).

Clinical doses proposed in the final clinical protocol should be based on the outcomes of pre-clinical safety (highest safe dose) and immunogenicity (dose giving optimal immunogenicity) studies. Hopefully, the optimal immunogenic dose determined will be well below the highest safe dose [Comparable to the concept of a maximum tolerated dose (MTD) determined for toxic drugs. An MTD may or may not be determined in the pre-clinical toxicology study because often vaccines do not cause significant toxicity at the doses tested in the toxicology study and an MTD cannot be determined. Because of this, generally one does not refer to an MTD for a vaccine. However, it is a concept commonly understood by drug toxicologists].

Pre-clinical Safety Testing of Candidate Vaccines

For a graphical view of this topic, please see the Toxicology Decision Tree.

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 monitored 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/repeated dose 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 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 adjuvant 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 (daily in week post-dose) and histopathology,
4. Body weights (weekly),
5. Food and water consumption, body temperatures (daily in week following inoculations),
6. Ophthalmologic observations (pre-dosing and prior to sacrifice),
7. Clinical pathology at regular intervals for hematology, serum chemistry, serology, urinalysis measurements,
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/or CMI) studies.
11. 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 that do not (yet) meet GLP standards . General recommendations include:
1. Tissue Distribution studies:
2. 1 – 2 doses, paralleling the doses in the systemic toxicity studies, in addition to the vehicle and/or adjuvant control
1. Same route of injection as the proposed clinical route
2. Single administration (not repeated dosing)
3. Time points: from 1 day up to 8-12 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 animals ), 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.
3. Integration studies in tissues where vaccine signals persist if DNA vaccine remains at doses higher than those recommended in the current FDA guidance
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 biodistribution 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.

Potential areas for streamlining the study methods include proposing to OVRR to only perform the histopathology on recovery sacrifice tissues in the event that treatment-related findings are noted at the immediate sacrifice timepoint or only in those tissues in which findings are noted to demonstrate recovery or reversibility of the findings. In addition, additional sites might be injected in the biodistribution study and collected for integration analyses, and only analyzed in the event it is required to do so. Doing so may prevent the additional time and resources needed to repeat the in-life portion of the study, although it may require utilizing more of the vaccine product than conducting a biodistribution only study.

A detailed summary of the designed protocol is included in the pre-IND document and frequently will 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 (and within 120 days of submission of the draft reports).

Please see our Glossary for Pre-clinical Toxicology Testing of Vaccines.

Summary

Pre-clinical toxicology for vaccines is a necessary step in product development to assess what may be a safe starting dose for clinical trials and to identify potential toxicities to monitor in the clinical safety studies. Safety in animals is an important tool to help clinical investigators, an Investigational New Drug (IND) sponsor, and the FDA evaluate the potential risk of a novel product that has never before been in humans. These webpages provide a generic protocol design for repeated (episodic, not daily) dose toxicity studies applicable to most vaccine modalities and for genetic toxicity studies applicable to DNA vaccines and some vector-based vaccine approaches. Specific guidance on your vaccine candidate should be sought from the FDA and both these webpages and the other cited references provide recommendations on how to go about getting that specific guidance from FDA.

Acknowledgements

The authors would like to acknowledge co-authors of the prior version of this web-based information, specifically, Drs. Stuart Shapiro (NIH/NIAID/DAIDS), Sally Hargus (formerly at FDA/CBER/OVRR/DVRPA), and Marion Gruber (FDA/CBER/OVRR).

About the authors

Polly Chang, Ph.D., Director of the Molecular & Genetic Toxicology at the Biosciences Division, SRI International (http://www.sri.com/biopharm/), currently has served as Principal Investigator (PI) on a number of NIAID vaccine contracts and has supervised the successful preclinical safety testing of more than 6 candidate HIV vaccines. Working closely with the project management and regulatory compliance teams in the preparation of the INDs, several of these vaccines have been approved by the FDA for Phase I clinical trials. Dr. Chang received her B.A. in mammalian physiology and her M.S. in Bioradiology/Biophysics, both from the University of California, Berkeley. She worked as a researcher at Lawrence Berkeley National Laboratory (LBNL), eventually attaining the position of Principal Research Associate before returning to the University of California at Berkeley to obtain her Ph.D. in Biophysics. The focus of her research was on the interactions of two clinical treatment modalities (hyperthermia and radiation) and the impact of de novo protein synthesis in in vitro cellular responses. Her global scientific experience during the 1990s includes working as a Visiting Scientist at the National Cancer Center, in Tokyo, Japan, and as a NIH Fogarty Fellow at the Cancer Biology Department, University of Tokyo Medical Center in Japan, examining the differential expression of a conserved family of homeotic genes in multi-step chemical carcinogenesis. In 1997 Dr. Chang joined SRI where, in addition to her vaccine work, she is currently the PI on a number of grants from NASA to study the effect of charged particle radiation that mimics exposure that space travelers will receive in animal models. She is examining the expression of key genes that are involved in cell functions, linking perturbations in cellular systems after treatments with genotoxic agents to the misregulation of normal differentiation and pathophysiology. She also supervises a variety of molecular biology-related projects and serves as Study Director on several NCI studies to evaluate chemopreventive therapeutics. She is author or coauthor of over 30 publications.

CAPT Rebecca Sheets, Ph.D., (USPHS)

Rebecca Sheets serves as the Vaccine Scientific and Regulatory Specialist in the National Institute of Allergy and Infectious Diseases at the National Institutes of Health. In this role, she formulates regulatory strategy for the Division of AIDS and the Vaccine Research Center for the pre-clinical development of research concepts into HIV vaccine candidates suitable for human clinical trials. Her background in virology, and specifically, retrovirology, serves to address HIV vaccine development and adventitious agent testing in vaccines.

Rebecca Sheets obtained her B.S. degree in Biology from the California Institute of Technology; M.S. degree in Cellular, Viral, and Molecular Biology from the University of Utah School of Medicine, and Ph.D. in Pathology from the University of Southern California School of Medicine.

Dr. Sheets served for 9 years as a Scientific Reviewer in the Viral Vaccines Branch of the Division of Vaccines and Related Products Applications, Office of Vaccines Research and Review, CBER/FDA. She came to FDA in 1993. In 1994, to foster her commitment to public health, she became a Commissioned Officer in the U.S. Public Health Service (Scientist Category), in which she has been promoted to the rank of Captain (CAPT). Her review and evaluation responsibilities at CBER included the review and evaluation of INDs and Product/Biological License Applications for candidate vaccines to prevent or treat Human Immunodeficiency Virus, rotaviruses, adenoviruses, polioviruses, parainfluenza viruses, Herpes Simplex Virus Type 2, Cytomegalovirus, and influenza viruses. She also evaluated two PMAs for HIV-1 viral load test kits. In addition, she was involved in several policy working groups on HIV vaccines, HIV/AIDS immunotherapeutics, vaccine cell substrates, retrovirus testing, and the joint working group between OVRR and the Division of AIDS at NIAID for HIV vaccine development.

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