Anja Holm Danish Medicines Agency. Genetic vaccines Oslo, 2008

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1 Anja Holm Danish Medicines Agency Genetic vaccines Oslo, 2008

2 Ministry of Food, Agriculture and Fisheries Danish Veterinary Institute Technical review & discussion of safety issues Author: Anja Holm. Anja Holm - DNA-vaccines - Oslo

3 A: Technical description B: Exposure assessment Distribution and persistence after injection and ingestion C: Hazard identification Potential consequences Probability assessment Risk reduction measures D: Perspectives / conclusions Anja Holm - DNA-vaccines - Oslo

4 Written in , based on published literature and research performed at DVI. Focused on naked, non-replicable DNA-vaccines We tried to identify and discuss all potential concerns that had been proposed by our own or other research groups or authorities. The report is freely available on the website of the Danish Veterinary Institute (google for the title). _Final1.doc Anja Holm - DNA-vaccines - Oslo

5 Circular, double-stranded bacterial DNA (plasmid). Comprises the (viral) gene to be expressed, plus genes for expression, production, construction etc. After injection, body cells of the host produce the gene-product (= immunogenic protein of virus). Immunity against the intracellularly produced protein is more precise due to correct structural folding. Induces both antibody and T-cell response Anja Holm - DNA-vaccines - Oslo

6 Approximate figures: A standard plasmid consists of ~ 5 x 10 3 base-pairs (bp) A bacterial genome consists of ~ 5 x 10 6 bp, which is 1000 x the size of a plasmid A genome of a mammalian cell consists of ~ 5 x 10 9 bp, which is 1,000,000 x the size of a plasmid. The PCR methods can detect down to 1 plasmid per µg DNA, or 1 plasmid/150,000 cells! Anja Holm - DNA-vaccines - Oslo

7 IM-injections to mice, doses µg ~ plasmids Distal tissue: After 1 day: ~ % of injected dose dispersed in total mouse After 1 week: 0-20 plasmids/150,000 cells Injection site: After 2 days: < 0.01% of injected dose left After 6 weeks: ~ 5000 plasmids/150,000 cells After 23 weeks ~ 500 plasmids/150,000 cells plasmids persist > a year, probably in nucleus of cells. Gene-gun: doses 1-10 µg ~ plasmids ~ no distal distribution, exfoliation in ~ 1 month Anja Holm - DNA-vaccines - Oslo

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9 Consumption of 200 g meat 1 week after vaccination or the entire injection site 6 weeks after injection: plasmids, which corresponds to approximately % of total daily DNA intake for a consumer Effect of cooking:?, - i.e. degradation from 0 to 100% Gastric passage: Degradation > 95 % Total uptake: max. 1 % of ingested: plasmids/meal Can be found in various organs for 24 hours, predominantly in leukocytes Fragment size: basepairs; which means that one gene may be intact but not the entire expression-cassette. No expression has been shown after ingestion of naked DNA. Anja Holm - DNA-vaccines - Oslo

10 DNA-vaccines: up to plasmids/dose ~ µg DNA/dose Inactivated bacterial vaccines: up to 10 9 bacterial cells/dose ~ 10 µg DNA/dose Inactivated viral vaccines: up to 10 8 viral particles/dose ~ 0.2 µg DNA/dose Live vaccines: reproduction inside host: exposure unknown the exposure to foreign DNA is probably much higher than for inactivated vaccines Active infection: exposure unknown Human parvovirus infection: virus particles / ml blood. Anja Holm - DNA-vaccines - Oslo

11 Post-vaccination reactions Adverse immune effects Undesired immune response, auto-antibodies, tolerance, allergy Tumor development Integration, methylation patterns, probability Transmission to offspring Egg-cell, sperm, transplacental to fetus Environmental concerns Spread of antibiotic resistance genes Anja Holm - DNA-vaccines - Oslo

12 Concern: Injection site reactions? IM: almost nothing Gene-gun: brown-colouration from gold, slight redness 5-10 days. (many other administration techniques exist) Concern: Systemic reactions? None Endotoxin levels should be controlled as for other vaccines Anja Holm - DNA-vaccines - Oslo

13 Concern:Undesired immune response in consumer? expression was never found after oral intake. Concern: Will injection of DNA-vaccines induce antibodies against DNA itself? Studies show a slight transient increase in antimammalian-dna antibody secreting cells, but less than after injection of bacterial (E.coli) DNA. No induction or acceleration of autoimmune disease. If the expressed gene is a self-antigen: more studies needed. Anja Holm - DNA-vaccines - Oslo

14 Concern: Induction of tolerance in neonates? Tolerance has been shown occasionally in newborn mice. However, most studies show immune response also in neonates of several species. Tolerance is probably the exception rather than the rule. Concern: Induction of allergy? No evidence found. The main problem would be residues of antimicrobial substances from production, which should be controlled like for other vaccines. Anja Holm - DNA-vaccines - Oslo

15 Concern: Integration into genomic DNA? At the injection site: less than 8 plasmids/ cells remain (co-migrate) after intensive purification of the genomic DNA Studies performed in mice with very large doses DNA-vaccine Integration was not confirmed Tumor induction has not been shown However, for new plasmids: a test for integration enhancing sequences should be performed Anja Holm - DNA-vaccines - Oslo

16 Concern: Changes in methylation patterns of cellular DNA? Requires integration Methylation of integrated foreign sequences may silence the new gene, but also change distant cellular genes -> relevance in oncogenesis? Concern: Increased population cancer frequency in the vaccinated animals? Probability estimation based on integration of retrovirus. Theoretical calculations regarding activation of protooncogenes or inactivation of tumor suppresser genes in a multistep cancer development process: 1 cancer per 10 million vaccinations? Anja Holm - DNA-vaccines - Oslo

17 Concern: Transmission in the germline? ( i.e., if foreign DNA integrates in the egg or sperm cells = the foreign DNA is inherited in all cells of the offspring) egg-cells seem protected against foreign DNA uptake sperm-cells may transfer foreign DNA to egg-cell after chemical and heat-chock treatment. Relevance in natural breeding? 8 generations of mice continuously fed plasmid-dna: no germline transmission could be shown. Several studies confirmed that no integration could be shown in ovaries and testes after IM injection. Anja Holm - DNA-vaccines - Oslo

18 Concern: Transplacental transmission? Pregnant mice injected IV: transfer to fetus after placental development. Intact, non-integrated plasmids were recovered in offspring before and after birth. Expression of gene-product in offspring was shown. No apparent abnormalities in dam or offspring. Pregnant mice fed plasmids: plasmid fragments found in several organs of fetuses. No expression was seen. Integration of foreign DNA in offspring after feeding? May occur but rare event. (The study model used M13 bacteriophage DNA, not specific for DNA-vaccines) Significance? More studies needed. Anja Holm - DNA-vaccines - Oslo

19 Spread of antibiotic resistance genes Concern: Will plasmids spread their genes to the environment? The only identified hazard is the antibiotic resistance gene, which may be taken up by bacteria. Frequency of gene transfer is low. May occur in intestines food environment May be relevant if the plasmid carries an antibiotic resistance gene, which codes for a clinically important (therapeutic) antibiotic. Must be evaluated in relation to the existing pool of antibiotic resistance genes in environmental bacteria. Anja Holm - DNA-vaccines - Oslo

20 Acceptable risk depends on who decides and who takes the risk. For example: Small risk from food is unacceptable to consumer, if the benefit is only economical for the producer, whereas large risk from smoking is acceptable because decision and benefit is personal. Nothing is completely free of risks, but science must communicate risks and benefits for the society and for individuals. Anja Holm - DNA-vaccines - Oslo

21 Both CVMP and FDA have published guidelines on DNAvaccines Are MRLs needed for DNA-vaccines?: active principle of biological origin intended for induction of immunity: MRL not required Excipients etc: If pharmacologically active in the intended concentration: MRL required or Out of scope -list. Ask EMEA. Operator safety: risk if self-injected? Other? GMO-legislation: Not applicable. Are DNA-vaccinated animals genetically modified organisms (GMOs)?: In Denmark: No. If in doubt: ask the competent authority Anja Holm - DNA-vaccines - Oslo

22 DNA-vaccines are generally safe The consumer s safety is not at risk Some issues need more research Very important: Every vaccine must be evaluated by the Competent Authority based on its specific contents, risks and benefits! Communication is important for a balanced risk perception. Anja Holm - DNA-vaccines - Oslo

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