Vaccine discovery DNA vaccine, mechanism methods of delivery Main concerns: do DNA vaccines cause insertional mutation and elicit anti-DNA antibodies in the body? completed and ongoing trials. new strategies: Prime Boost vaccines Future prospects

1. DNA VACCINES: PAST, PRESENT
& FUTURE POSSIBILITIES
W Roseybala Devi & Gareth Lawrence,
MSc Bioinformatics, First Semester
JSS Academy of Higher Education and Research.

2. Vaccine?
■ Discovered in 1793 by Edward Jenner
■ First vaccine: Small pox vaccine.
■ Vaccine: biological preparation (live, killed/attenuated pathogens or proteins)
that mimics the pathogen; no virulence; has antigenic properties & evokes
immune response.
■ Immunization: controlled exposure to pathogen; Adaptive immune system gains
immunological memories for quick response in future encounters.
■ Jenner’s Expt: prior exposure of James Phipps to cow pox virus – develops
immunity to small pox virus. (Variolation)
inoculation of cowpox blister fluid into cut skin -> mild infection -
>recovery&immunity

3. Types of Vaccine
■ Live vaccines; weakened/attenuated pathogens; Strong & long lasting immunity.
disadvantages: revert mutation; expensive purification techniques &
cold strorage. (Rubella vaccine)
■ Killed vaccines: killed pathogens; safer & stable.
disadvantages: weak immune response, endotoxins,
hypersensitivity reactions; multiple doses required. (thyphoid vaccine.)
■ Sub-unit vaccines: a part of the pathogen (antigenic proteins) .
safe and applicable for immuno-compromised people.
disadvantages: conformational changes of antigens. Weak immunity.
(Hepatitis B vaccine).
■ Toxoids: toxins – low concentration – sufficient to elicit an immune response.
safe but produces weak immunity – multiple doses required.
(Tetanus Toxoid)

4. An Alternative to traditional vaccines
■ Live vaccine: risk – contamination & Failure to maintain proper storage
conditions -> reverse mutation. Less preferable in developing countries.
■ Killed vaccines: endotoxins may cause hypersensitivity reaction.
■ Consequences: outbreak of infectious diseases, already eradicated in the
developed countries. Fear of vaccines.
■ Urgent need: a vaccine as safe as sub-unit vaccines and as effective and strong
as life vaccines + cheap production cost & easy to handle and transport.
■ The Solution: DNA vaccine;

5. Why DNA vaccines?
Traditional vaccines
■ Uses weakened/killed pathogen
■ Risk of being infected
■ Provides cell mediated immunity
■ Refrigeration required
DNA vaccines
■ Uses only the pathogen’s DNA
■ No risk of infection
■ Provides both humoral and cell
mediated immunity
■ Refrigeration not required
■ Low production cost, easy handling, high effectivity: assumptions:- potential
solution to most clinical problems & great significance -> draws attention of many
researchers

6. DNA vaccine?
■ Primarily recombinant plasmids (circular, double stranded)
■ Expression plasmids with regulatory elements are used to ensure that the foreign gene
inserts are expressed after injected to eukaryotic animals.
■ Genes isolated from a pathogen encodes antigenic protein of that particular pathogen.
■ The gene is inserted downstream to the promoter so that the RNA polymerase binds to
the promoter and transcribe mRNA which in turn translates protein.
■ The foreign gene inserted depends on the target pathogen.

7. Construction
■ Constructed using recombinant DNA technology.

8. Mechanism of Action of vaccines
■ Induction of immune response.
■ Types: cell mediated or cellular immune response
and humoral immune response.
Humoral IR: found in body fluids. Binds to pathogen >
prevent entry into the cells.
Cellular IR: does not involve antibodies. Involves
antigen presenting cells (macrophages), B cells, T
cells.
Mechanism:
(1)Antigen exposure (2)macrophage engulfs &
degrades the antigen (3)fragments of antigen
displayed on the macrophage cell membrane by MHC
(4)T cells recognize the antigen (5)activates B cells
(6) B cell differentiation (7)antibody production and
immunogenic memory.

9. DNA vaccine mechanism
■ Complex & one of the challenges in DNA vaccine
research.
■ 3 mechanisms of action have been proposed:
■ (i) DNA encoded antigens are presented by somatic
cells (myocytes or keratinocytes) through MHC to
the T cells.
■ (ii) direct transfection of Antigen Presenting cells
(e.g. Dendritic cells).
■ (iii) cross-priming results from transfected somatic
cells being phagocytosed by APCs (monocytes)
presenting the cells to the T cells.

10.  Direct injection: using syringe (using a standard
hypodermic needle .Injection in saline is normally
conducted intramuscularly (IM) in skeletal muscle, or
intradermally (ID).
 Gene gun: shooting DNA into the cells.
 Epidermal powder: DNA plasmids linked to gold
particles injeted into the skin in dry powder formulation.
 Jet injector: liquid under high pressure through a tiny
orifice, producing a focused stream that penetrates the
skin to deliver.
Methods of delivery

11. Safety concern
■ Insertional mutation: will the plasmid from the DNA vaccine integrate into the
genome
■ Lupus erythematosus :will the foreign DNA activate ani-DNA antibodies,
attacking even the organism’s own DNA?

12. Insertional mutation
■ Experimental studies have been conducted on animal models to detect
integration of plasmids after DNA vaccine administration.
■ Injection of a single dose of DNA vaccine to Mus musculus (mouse) results in
association of at least 30 copies of plasmid (covalently or simple association)
with the genome.
■ Assuming that 30 copies of plasmid are integrated into the genome, the
calculated rate of insertional mutation was found to be 3,000 times less than the
natural mutation rate of mammalian genes.
■ Insertional mutation: not a serious safety concern.

13. Lupus erythematosus
■ Experimental studies led to observations that in mammals, anti-DNA antibodies
are produced during infections and the antibodies are specific to the pathogens.
■ Anti-DNA antibodies targets pathogenic DNA and do not react with mammalian
DNA.
■ Lupus prone mice animal models are used to test the action of DNA vaccine
■ Concluded that purified plasmid or DNA does not elicit anti-DNA
antibodies production.

14. Completed trials & Current status of
DNA vaccine
■ Phase I trials of the First generation DNA vaccine: 2 decades ago.
■ Evaluated the efficacy of DNA vaccine targetting HIV type I for therapeutic and
prophylactic purposes.
■ It was followed by influenza, Human Papilloma Virus(HPV), Hepatitis and
malaria.
■ Failure of first generation DNA vaccines due to:
■ poorly immunogenic
■ low antibody titer
■ T cell response was low (low cellular immune response).
■ No DNA vaccines have been approved for human use but it is approved for
veterinary practice. (canine melanoma, west nile virus).

15. Ongoing trials
■ Second generation DNA vaccines
■ Improvements in humoral and cellular immune response in both small and large animal
models.
■ Activates the cytotoxic T cells in larger amount than the first generation DNA vaccines.
■ Low immunogenicity of First genertion vaccines was due to insufficient uptake of
plasmids by the cells (inefficient delivery)
■ Second gen vaccines: new strategies to enhance delivery of plasmids into the cells
■ (i) particle bombardment: highly pressurized steam delivers plasmid linked to
microscopic metal particles.
■ (ii) formulation and molecular adjuvants: inclusion of additional plasmids that encodes
molecular adjuvants.

16. Antigen design
■ Most recent approach
■ Sequences are selected from a collection of target antigen protein sequences.
■ From a phylogenetic tree of pathogens, the most commonly occuring sequence
is selected from the root of the tree.
■ This approach is to develop vaccines that provide immunity to diverse
pathogens.

17. Clinical targets
■ 43 clinical trials have been done for viral and non viral diseases.
■ 33% : vaccine for HIV
■ 29%: vaccine for cancer(mostly melanoma)
■ 38%: influenza, Hepatitis C, HPV and malaria.
■ DNA Vaccine Strategies for the Treatment of Cancer
DNA vaccines directed at eliciting an immune response toward tumors appear to
offer promise for both the prophylactic and therapeutic treatment of cancer.

18. Ongoing trials
■ DNA vaccine for influenza
■ - stage I clinical trial
■ - a completed phase I trial by PowerMed demonstrated reduction in symptoms & viral
shedding in subjects who received the vaccine delivered by gene gun.
■ HIV type I vaccine
■ - for prophylaxis; most elusive & challenging goal
■ - complexity of HIV I
■ - vaccine target: to induce broadly neutralizing antibodies against HIV I to reduce viral
loads.
■ - completed trial: recombinant Adenovirus serotype 5 vaccines and recombinant protein
gp120 vaccines: not effective at prevention.

19. Prime-boost
■ Latest vaccine strategy targeting HIV I
■ Administration of different vectors that presents the same
antigen.
induce broad and high-level T-cell immunity
■ Combination of a DNA-based (viral based) and a protein-
based
DNA-based/Prime: a fragment of DNA which carries a gene coding
for the antigenic protein > translated to the antigen and activate T-
cells & produce the corresponding antibodies.
Protein based/booster: It is the antigen, administered directly in the
form of the protein.

20. Prime-boost vaccines
 A new multi-clade DNA prime/recombinant MVA modified vaccinia virus Ankara
boost vaccine induces broad and high levels of HIV-1-specific CD8(+) T-cell and
humoral responses in mice.
 Proved to induce extraordinarily strong cellular responses, with more than 80%
of the CD8(+) T cells specific for HIV-1 antigens.
 After booster vaccine administration, a significant proportion of T cells were
stained positive for both interferon-gamma and interleukin-2 (IL-2), a feature that
has been associated with control of HIV-1 infection.
 The results from this study demonstrate the potency of this combination of DNA
plasmids and MVA construct to induce broad and high levels of immune
responses against several HIV-1 proteins of different subtypes.

21. Future prospects
 Limiting feature of DNA vaccine: low level of antigen expression. Solution:
altering plasmid construction & improved engineering techniques.
 Low immune response; solution: targeting APCs (e.g. dendritic cells) for better
MHC loading.
 The fist DNA vaccine will be most likely a part of the DNA prime-protein boost
vaccine.
 Although quality & safety guidelines for veterinary use and human use are
different, experiences with vet. DNA vaccines can provide valuable informations
for control and use of human DNA vaccines.
 Gaining full understanding of DNA uptake by the cells will help in improving
applications for gene therapy.
 Success in DNA vaccines --> development of genomic vaccines against
microbial infections. (expression library immunization).