**2.8 Final general recommendations for safety evaluation of DNA vaccine candidates**

A generic protocol is not provided, but general recommendations are described below:

Evaluation of Drug Toxicity for DNA

Infectious Disease Indications, 2007).

2007).

**3. Conclusions** 

Vaccine Candidates Against Infectious Diseases: Hepatitis C as Experimental Model 339

Biodistribution studies may be waived for DNA vaccines produced by inserting a novel gene into a plasmid vector previously documented to have an acceptable biodistribution/integration profile. Biodistribution studies will still be necessary for DNA vaccines utilizing novel vectors, formulations, methods of delivery, routes of administration, or any other modifications expected to significantly impact cellular uptake and/or biodistribution. In every case, the decision should be consulted with the regulatory authorities. It is recommendable that manufacturers provide the complete sequence of the plasmid before initiating phase 1 clinical studies. 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

Consideration must be given to the possibility that the in vivo synthesized antigen may exhibit unwanted biological activity. If necessary, appropriate steps must be taken, e.g. by deletion mutagenesis, to eliminate this activity while retaining the desired immune response. If other gene constructs are included in the plasmid, such as antibiotic resistance genes for manufacturing reasons, then the possibility of expression of such gene sequences in mammalian cells or in micro-organisms which are potentially pathogenic, and the

When more than one type of vaccine is used in a sequential immunization protocol, if information supporting the safety and tolerability of the dose, schedule, and route of administration of each component proposed for use in the heterologous prime-boost regimen exist and data are deemed adequate to characterize the potential risks of the primeboost regimen to study participants, additional toxicology studies may not be necessary. However, this information should be submitted for consideration to the regulatory authorities that will evaluate the need for additional toxicology information to support the clinical plans (Guidance for Industry Considerations for Plasmid DNA Vaccines for

If modifications to the manufacturing process or the DNA product are made during the development programme, the potential impact on the product should be considered. Modifications of the genetic sequence, the use of alternative promoter/enhancer sequences, or other changes to the product, may require additional non-clinical safety evaluation. Equally, if aspects related to the immunization protocol like the route of administration are changed, then additional non-clinical test should be done to assess the impact of these modifications. The scientific rationale for the approach taken should be provided (Guidance for Industry Considerations for Plasmid DNA Vaccines for Infectious Disease Indications,

The risk/benefit evaluation for a product is related to the actual product and its intended use. For example, a prophylactic DNA vaccine for use in healthy children will have a different risk/benefit ratio compared to a therapeutic DNA vaccine against cancer or a persistent pathogen like HCV, for which there is no other available treatment or the efficacy of therapy is limited. Thus, for these and other reasons, it is likely that a flexible approach

DNA vaccination is a continuously evolving and exciting field with many challenges to face. Methodological and regulatory frames are also developing every day. One important issue

will be necessary for the non-clinical safety evaluation of DNA vaccines.

may be required if the vaccine is demonstrated to be integrated in tissues.

possible clinical consequences of such expression, should be considered.

	- Daily clinical observations
	- Weekly physical examinations
	- Evaluation of injection site(s) for irritation (daily in the post-dose week) and histopathology
	- Weekly body weights assessment
	- Food and water consumption, body temperatures (daily in the week following inoculations)
	- Ophthalmologic observations (pre-dosing and prior to sacrifice)
	- Clinical pathology at regular intervals for hematology, serum chemistry, serology, urinalysis measurements
	- Gross observations and organ weights at necropsy
	- 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 is examined histopathologically)
	- Relevant immunogenicity (humoral and/or cell mediated immune responses) studies
	- Additional endpoints may be included to address therapeutic-specific concerns. Here, specialized studies to examine genetic toxicology (e.g., biodistribution) are strongly recommended. These are studies that may incorporate the use of assays that do not (yet) meet good laboratory practices standards. General recommendations include: tissue distribution studies, integration studies in tissues where the DNA vaccine remains at doses higher than those recommended guidances, immunotoxicity studies if repeated doses are planned in the clinical evaluation.

Biodistribution studies may be waived for DNA vaccines produced by inserting a novel gene into a plasmid vector previously documented to have an acceptable biodistribution/integration profile. Biodistribution studies will still be necessary for DNA vaccines utilizing novel vectors, formulations, methods of delivery, routes of administration, or any other modifications expected to significantly impact cellular uptake and/or biodistribution. In every case, the decision should be consulted with the regulatory authorities. It is recommendable that manufacturers provide the complete sequence of the plasmid before initiating phase 1 clinical studies. 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.

Consideration must be given to the possibility that the in vivo synthesized antigen may exhibit unwanted biological activity. If necessary, appropriate steps must be taken, e.g. by deletion mutagenesis, to eliminate this activity while retaining the desired immune response. If other gene constructs are included in the plasmid, such as antibiotic resistance genes for manufacturing reasons, then the possibility of expression of such gene sequences in mammalian cells or in micro-organisms which are potentially pathogenic, and the possible clinical consequences of such expression, should be considered.

When more than one type of vaccine is used in a sequential immunization protocol, if information supporting the safety and tolerability of the dose, schedule, and route of administration of each component proposed for use in the heterologous prime-boost regimen exist and data are deemed adequate to characterize the potential risks of the primeboost regimen to study participants, additional toxicology studies may not be necessary. However, this information should be submitted for consideration to the regulatory authorities that will evaluate the need for additional toxicology information to support the clinical plans (Guidance for Industry Considerations for Plasmid DNA Vaccines for Infectious Disease Indications, 2007).

If modifications to the manufacturing process or the DNA product are made during the development programme, the potential impact on the product should be considered. Modifications of the genetic sequence, the use of alternative promoter/enhancer sequences, or other changes to the product, may require additional non-clinical safety evaluation. Equally, if aspects related to the immunization protocol like the route of administration are changed, then additional non-clinical test should be done to assess the impact of these modifications. The scientific rationale for the approach taken should be provided (Guidance for Industry Considerations for Plasmid DNA Vaccines for Infectious Disease Indications, 2007).

The risk/benefit evaluation for a product is related to the actual product and its intended use. For example, a prophylactic DNA vaccine for use in healthy children will have a different risk/benefit ratio compared to a therapeutic DNA vaccine against cancer or a persistent pathogen like HCV, for which there is no other available treatment or the efficacy of therapy is limited. Thus, for these and other reasons, it is likely that a flexible approach will be necessary for the non-clinical safety evaluation of DNA vaccines.
