**2. Safety evaluation**

322 Toxicity and Drug Testing

responses. There is a potential for encoding multiple immunogenic epitopes with the purpose of raising protection against several diseases by a single vaccine. Compared with many conventional vaccines, DNA vaccines are relatively stable. Moreover, DNA vaccines

The above mentioned advantages have resulted attractive for the application of DNA vaccination in the infectious disease field in humans. This immunization strategy has been widely evaluated against a variety of human pathogens; some of them without a current vaccine solution available like hepatitis C virus (HCV) and human immunodeficiency virus (HIV). In fact, DNA immunization has even reached the phase of clinical evaluation in

The mechanism of action for DNA vaccines and their potential use for therapeutic and preventive purposes imposes relevant challenges for the evaluation of their safety. In addition, knowledge about potential undesirable side effects at long term is still limited. So far, all DNA vaccine candidates entering to clinical evaluation in humans have been previously evaluated for immunogenicity and toxicity in animal models with good results. However, immunogenicity in humans of naked DNA vaccine candidates has not generally fulfilled the expectations. Therefore, several strategies are currently being evaluated for enhancing the immune response, but some of them involve incorporation of components which are potentially able to also increase the toxicity, or might raise the risk for noncontrolled or non-desired immune responses. Consequently, evaluation of toxicity related to

are rapid to construct and their manufacture is generic (Liu, 2011).

DNA-based immunization is a continuously challenged field.

HIV Influenza Malaria HBV HCV SARS Marburg Ebola HPV

**Infectious disease** 

West Nile virus

Table 1. Infectious diseases for which DNA vaccines have entered to clinical trials

In this chapter we discuss relevant elements to be considered during the evaluation of toxicity related to DNA vaccines applied to infectious diseases. We will focus on local reactogenicity and systemic toxicity studies, biodistribution, persistence, and integration analysis, as well as immune-related studies for detecting potential adverse events after immunization with DNA-based vaccines candidates against HCV, as a model. We focus on HCV infection since it is a worldwide health problem, causing chronic hepatitis, frequently progressing to cirrhosis and hepatocellular carcinoma. There is no currently available

Dengue HSV Measles

several infectious diseases (Table 1).

In addition to immunogenicity demonstration, regulatory agencies require sufficient preclinical data supporting safety to approve initiation of clinical trials of novel vaccines, including DNA vaccine candidates. The regulatory frame has been abundantly settled (Guidelines for assuring the quality of DNA vaccines, 1998; Guidelines on clinical evaluation of vaccines: regulatory expectations, 2004; Guidelines on nonclinical evaluation of vaccines, 2006). Precisely, the principal aim of non-clinical safety examination is to understand the toxicity of the candidate drug well enough to make judgment that the risk/benefits profile is adequate to initiate clinical trials (Contrera, 1993). Toxicity is complex, and impacted by several factors, such as: the xenobiotic, the dosage, the route, the action mechanism and the products of biotransformation. The distribution of many xenobiotics in the body may only affect certain key organs. Others, however, may damage any cell or tissue it enters in contact with. In addition, the toxicity can result in cellular/biochemical or adverse macromolecular changes. Some examples are: cell substitution, as fibrosis; damage to an enzyme system; interruption of protein synthesis; production of undesired chemical reagents in the cells and damages in the DNA. The distribution of toxic substances and toxic metabolites in the whole body determines the organs and tissues where the toxicity is produced. Many toxic substances are stored in the body, and the most common deposits of storage are fatty structures, the bones and highly vacularized organs involved in blood detoxification, such as the liver and the kidneys.

The safety evaluation involves the experimental studies directed to determine the toxicity, identifying and quantifying effects and establishing parameters (as dose, toxic and lethal concentrations, etc.) of the substances, using *in vivo* or *in vitro* models. With the information provided by these studies and other data, the Evaluation and the Estimate of the Risk are carried out, as determination of the probability and nature of the effects that can be derived from the exposition to the xenobiotics.

As for other vaccination strategies, evaluation of safety in the case of DNA immunization requires several considerations and tests. The lots of vaccine candidates to be used in preclinical studies should have been released according to the specifications required for their use in humans. Manufacturers need to establish a reproducible process for producing the DNA vaccine candidate in a sterile and free of endotoxins condition.

The main challenge in establishing a predictive non-clinical safety assessment comes from the fact that vaccines act through complex multi-stage mechanisms. Thus, the detection of the toxicity of vaccines is likely to be more complex than for conventional chemicallyderived drug products, because safety concerns regarding the immune response to the vaccine add to the general concerns related to exogenous substances administration. Thus, toxicity testing programs recommended for conventional drug products may not always be applicable to vaccine products.

The non-clinical safety assessment of vaccines represents a new and evolving field. And clearly, consensus is needed among industry, academia, and regulatory authorities regarding the most appropriate approaches to this area. Depending on the target population

Evaluation of Drug Toxicity for DNA

humans.

administration.

Vaccine Candidates Against Infectious Diseases: Hepatitis C as Experimental Model 325

The toxicological studies in animals constitute one of the main sources of information to study the toxicity of chemical compounds and biotechnology products, including vaccines. DNA vaccines evaluated in toxicological studies should comply with good manufacturing practices (Good manufacturing practices for biological products, 1992; Good manufacturing practices for pharmaceutical products, 2003). In these studies, even the less evident effects of the acute and chronic exposition can be generally evaluated easily. In these assays, the capacity to manipulate the experimental conditions allows the evaluation of many variables in response to toxic substances. These studies are very important to predict the toxicity effects in human susceptible populations. However, important limitations should be remarked regarding to the uncertainty of extrapolation from animals to humans. Particularly, it is difficult to extrapolate data obtained with high dose in animals, to the prospective toxicity of the relatively much smaller administration of therapeutic dose in

The selection of the doses, duration and frequency of the dosage should be based on the proposed clinical regime; the levels and duration of the genetic expression in animal experimental models and in humans should also be considered. Typically, pre-clinical studies are carried out in rodents (mice or rats) and rabbits in a general toxicology "screening" base. Such studies are designed to identify both, intrinsic toxicity of the vaccine candidate, as well as immunotoxicity arising from the host immune response after its

Accumulated data uniformly suggest that DNA vaccines are safe (reviewed by Liu, 2011). Mice and rats have been usually used as "first" species to study the toxicity of DNA vaccines. However, the reliability of a particular animal model in predicting an adverse clinical outcome cannot be established. In addition, the effect of vaccines does not exactly scale up directly on body weight or body surface area, since DNA vaccine candidates are expected to act mostly at the local site of administration to induce an immune response that traffics systemically. For this reason, rabbits are frequently used as "confirmatory" species to evaluate acute and chronic toxicity of DNA vaccine candidates since they are animals large

According to current Guidance, local reactogenicity and systemic toxicity studies should test the highest dose of the vaccine candidate planned for human use. In addition, N+1 administrations of the vaccine candidate should be delivered in these studies, with respect to the planned number of immunizations to be applied in humans. Recommended analysis include serum chemistry, haematology and coagulation test, in addition to gross and microscopic histology of different organs, particularly those potentially targeted by DNA immunization. Short-term and persistent toxicity are suggested to be evaluated in separate

The U.S. Pharmaceutical Research and Manufacturers Association has recommended that non-clinical toxicologic evaluations should be decided case by case (Stoll, 1987) and regulatory and industry representatives attending the first International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human

From the regulatory point of view, the information provided by the study of acute toxicity is essential for the classification, manipulation and transportation of a product. From the

enough to receive a full human dose of the vaccine candidate.

cohorts of animals 2-3 days and 2-3 weeks after final vaccination.

Use also supported this position (ICH, 1997).

**2.1 Studies of acute toxicity** 

and vaccine indication, it may be necessary to conduct special non-clinical safety assessments. In particular, if a target population for the product includes pregnant women or females of reproductive age, reproductive toxicity studies should be considered. A global picture of the pre-clinical studies suggested for DNA vaccines is shown in Table 2.



and vaccine indication, it may be necessary to conduct special non-clinical safety assessments. In particular, if a target population for the product includes pregnant women or females of reproductive age, reproductive toxicity studies should be considered. A global

investigate potential efficacy and toxicological consequences where

intended. The route, mode, frequency and duration of administration in the animal studies should mimic the clinical dosing regimen. Where the duration of treatment of patients is long-term, toxicity studies should generally be of 6 months duration. The duration of the recovery phase investigations should be based on the persistence of

the nucleic acid, the vector-derived material (e.g. viral protein) or the

The potential production of anti-DNA antibodies upon nucleic acid administration should be addressed because they could mediate resistance to treatment and/or signal the development of autoimmunity. Formation of neutralizing antibodies to the gene construct, its vector or the expressed gene product should be studied

Standard genotoxicity or life-time rodent carcinogenicity studies are not generally required. Depending on the extent of integration of DNA into the host genome and the clinical indication, studies may be required to investigate the potential for tumor formation or disruption

Observation time should cover persistence of signal (i.e. duration of transgene expression and activity) and include time-points for which there is no signal detection, if applicable. The dosing should mimic the

disease or pediatric use). The likelihood and the possible consequences of vector integration should be evaluated and measures to control potential associated risks should be described and justified.

However, if the proposed clinical formulation and route of administration have been examined in other animal studies then

separate local tolerance studies are not necessary.

the gene therapy product and expression of gene product

Embryo-fetal and perinatal toxicity studies may be required depending on the disease and clinical population to be treated, if women of child-bearing potential are to be exposed to gene therapy

picture of the pre-clinical studies suggested for DNA vaccines is shown in Table 2.

Single dose toxicity Should incorporate some safety pharmacology endpoints, and

Immunotoxicity The potential for stimulating cell mediated or humoral immunity to

expressed protein should be investigated.

systemic exposure is maximized. Repeated dose toxicity It will be required where multiple dosing of human subjects is

Type of study By guideline (EMEA. CPMP/SWP/112/98, 1998)

as it may reduce efficacy.

of normal gene expression.

Distribution studies Studies should provide data on all organs, whether target or not.

clinical use with appropriate safety margins. Integration studies Depending on the proposed clinical use (e.g., non-life threatening

Local tolerance A local tolerance study may be required in an appropriate species.

products.

Table 2. Pre-clinical studies indicated for DNA vaccines

Reproduction and developmental toxicity

Genotoxicity and Carcinogenicity/ oncogenicity/tumorigeni

city studies

studies

The toxicological studies in animals constitute one of the main sources of information to study the toxicity of chemical compounds and biotechnology products, including vaccines. DNA vaccines evaluated in toxicological studies should comply with good manufacturing practices (Good manufacturing practices for biological products, 1992; Good manufacturing practices for pharmaceutical products, 2003). In these studies, even the less evident effects of the acute and chronic exposition can be generally evaluated easily. In these assays, the capacity to manipulate the experimental conditions allows the evaluation of many variables in response to toxic substances. These studies are very important to predict the toxicity effects in human susceptible populations. However, important limitations should be remarked regarding to the uncertainty of extrapolation from animals to humans. Particularly, it is difficult to extrapolate data obtained with high dose in animals, to the prospective toxicity of the relatively much smaller administration of therapeutic dose in humans.

The selection of the doses, duration and frequency of the dosage should be based on the proposed clinical regime; the levels and duration of the genetic expression in animal experimental models and in humans should also be considered. Typically, pre-clinical studies are carried out in rodents (mice or rats) and rabbits in a general toxicology "screening" base. Such studies are designed to identify both, intrinsic toxicity of the vaccine candidate, as well as immunotoxicity arising from the host immune response after its administration.

Accumulated data uniformly suggest that DNA vaccines are safe (reviewed by Liu, 2011). Mice and rats have been usually used as "first" species to study the toxicity of DNA vaccines. However, the reliability of a particular animal model in predicting an adverse clinical outcome cannot be established. In addition, the effect of vaccines does not exactly scale up directly on body weight or body surface area, since DNA vaccine candidates are expected to act mostly at the local site of administration to induce an immune response that traffics systemically. For this reason, rabbits are frequently used as "confirmatory" species to evaluate acute and chronic toxicity of DNA vaccine candidates since they are animals large enough to receive a full human dose of the vaccine candidate.

According to current Guidance, local reactogenicity and systemic toxicity studies should test the highest dose of the vaccine candidate planned for human use. In addition, N+1 administrations of the vaccine candidate should be delivered in these studies, with respect to the planned number of immunizations to be applied in humans. Recommended analysis include serum chemistry, haematology and coagulation test, in addition to gross and microscopic histology of different organs, particularly those potentially targeted by DNA immunization. Short-term and persistent toxicity are suggested to be evaluated in separate cohorts of animals 2-3 days and 2-3 weeks after final vaccination.

The U.S. Pharmaceutical Research and Manufacturers Association has recommended that non-clinical toxicologic evaluations should be decided case by case (Stoll, 1987) and regulatory and industry representatives attending the first International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use also supported this position (ICH, 1997).
