Toxiology Studies

Numerous studies have addressed public concern and scepticism regarding health risks of DNA vaccines; the results so far have demonstrated that DNA vaccines are both safe and well-tolerated. Below are some case studies.

A PICTURE

Study 1 Extract

A study conducted by Pilling et al. in 2002 revealed the biodistribution and persistence of the plasmid was within acceptable limits for a DNA Hepatitus B vaccine as well as its delivery method of particle-mediation.

4 groups of minipigs were sacrificed at 2, 28, 56, or 141 days after treatment. The delivery method carried out was particle-mediated delivery, in this case gold nanoparticles using the gene gun. The procedure was well tolerated with mild local skin reactions at 2 days postdosing and no evidence of systemic toxicity. By 28 days the skin lesions had regressed and all disappeared in 141 days.

Polymerase chain reaction (PCR) was used to assay treatment sites and selected internal organs to evaluate biodistribution and persistence of the DNA plasmid. At 2 days the plasmid was detected in the treatment sites and also in the inguinal lymph nodes. At day 57 it was present in the treatment sites only and by day 141 appeared to have cleared.

MINIPIG PICTURE

As the minipig is regarded as a good model for humans these data support the concept that particle-mediated DNA delivery (gene gun delivery method) will be safe in human clinical applications.

Study 2 Extract The Vaccine Research Center has developed a number of vaccine candidates for different disease/infectious agents (HIV-1, Severe Acute Respiratory Syndrome virus, West Nile virus, and Ebola virus) based on a DNA plasmid vaccine platform. To support the clinical development of each of these vaccine candidates, preclinical studies have been performed in mice or rabbits to determine where in the body these plasmid vaccines would biodistribute and how rapidly they would clear.

In the course of these studies, it has been observed that regardless of the gene insert (expressing the vaccine immunogen) and regardless of the promoter used to drive the expression of the gene insert in the plasmid backbone, the plasmid vaccines do not biodistribute widely and remain essentially in the site of injection, in the muscle and overlying subcutis. This removes any concerns that they might biodistribute in the gonads. Even though ~1014 molecules are inoculated in the studies in the rabbits, by day 8 or 9 (~1 week postinoculation), already all but on the order of 104-106 molecules per microgram of DNA extracted from the tissue have been cleared at the injection site.

Over the course of 2 months, the plasmid clears from the site of injection with only a small percentage of animals (generally 10-20%) retaining a small number of copies (generally around 100 copies) in the muscle at the injection site. This pattern of biodistribution (confined to the injection site) and clearance (within 2 months) is consistent regardless of differences in the promoter in the plasmid backbone or differences in the gene insert being expressed by the plasmid vaccine.

In addition, integration has not been observed with plasmid vaccine candidates inoculated intramuscularly by Biojector 2000 (gene gun delivery) or by needle and syringe. These data build on Study 3 to demonstrate the safety and suitability for investigational human use of DNA plasmid vaccine candidates for a variety of infectious disease prevention indications.

Study 3 Extract (companion study with Study 2) To support the clinical development of each of these vaccine candidates, preclinical studies were performed to screen for potential toxicities (intrinsic and immunotoxicities). All treatment-related toxicities identified in these repeated-dose toxicology studies have seen confined primarily to the sites of injection and seem to be the result of both the delivery method (as they are seen in both control and treatment animals) and the intended immune response to the vaccine (as they occur with greater frequency and severity in treated animals).

The results of these safety studies have been submitted to the Food and Drug Administration to support the safety of initiating clinical studies with these and related DNA plasmid vaccines. Thus far, standard repeated-dose toxicology studies have not identified any target organs for toxicity (other than the injection site) for our DNA plasmid vaccines at doses up to 8 mg per immunization, regardless of disease indication (i.e., expressed gene-insert) and despite differences (strengths) in the promotors used to drive this expression.

Such toxicology studies have permitted an evaluation to be made that the candidates were safe to enter human clinical trials. Given the urgency of public health crises, such as the HIV/AIDS epidemic with 14,000 new infections daily, and the threat of global spread of newly emerging viral infections like SARS, this shortened development time is crucial.

Key summary

Research efforts to develop DNA vaccine products have already progressed to the clinical evaluation (under regulatory policies that have guided their development) of several vaccine candidates, which assess safety and immunogenicity and whether different routes and schedules of administration can improve immune response. These include those for hepatitis B, HSV-2, HIV-1, influenza and malaria.

The unique safety concerns include the potential for genomic integration, biodistribution, tolerance and auto immunity. Agencies such as the Center for Biologics Evaluation and Research (CBER) has issued a Points to Consider (PTC) document outlining regulatory policy, recommendations for the development of consistent manufacturing processes, and the design of preclinical studies to establish the safety of DNA vaccine products.