Production Of DNA vaccine, special reference with Zydus Cadila Covid-19 Vaccine

1. MADRAS CHRISTIAN COLLEGE, CHENNAI (AUTONOMOUS)
SEMINAR ON
PRODUCTION OF DNA VACCINES
PRESENTED BY:
ALOK KUMAR
3RD BSc BOTANY

2. WHY VACCINES ?
 In developing countries infectious diseases gene-
rally cause 30% -50% of all deaths.
 SARS CoV-2 ( Covid-19) troubled whole globe.
 There are many such diseases whose effective
chemotherapeutic agents simply don’t exist for.
 Story of some most successful vaccines, like
Small Pox and Diphtheria vaccines tells itself
its importance.

3. PROBLEMS WITH TRADITIONAL VACCINES ?
Traditional vaccines are either live attenuated weak strains or killed microorganisms. Despite
of some very effective traditional vaccines, most of traditional vaccines rise notable concerns-
 Danger of reversion to virulent stage.
 Live attenuated pathogens can produce severe disease in individuals with immune system
deficiencies.
 The principal component bacteria’s outer membrane, lipopolysaccharide (LPS), also called
endotoxin which may elicit a strong toxic response.
 Direct risk to workers who cultivate dangerous pathogens in large amounts for production
of vaccines.
 There is possibility that the organism or toxin may not be completely killed or inactivated.

4. DNA vs RECOMBINANT VACCINE
 A comparison of the steps involved in the production
of a DNA vaccine and a recombinant protein vaccine.
 Initial steps involve the generation of plasmid DNA
encoding the protein gene of interest for both types
of vaccines.
 For DNA vaccines, only expansion and purification
of the plasmid is required prior to immunization.
 The production of recombinant protein vaccines
requires additional steps for the expression and
purification of the protein itself, a much more laborious
process when compared to the preparation of a DNA
Vaccine.

5. DESIGN OF PLASMID DNA CONSTRUCTS FOR VACCINES
 Suitable plasmid for DNA vaccine produc-
tion is which fulfils the required criterion.
 Plasmid shall not be self replicating in
mammalian cell and shall not be integrating to
mammalian genome.
 A DNA vaccine vector shall have very
minimal functions , such that gene expression
in E coli is selectable marker and gene
expression in mammalian cell is antigen.
 Additional plasmid functions such as lac Z
and other genes should be removed.

6. Major steps involved in Vector preparation are-
 Generation of Antigen Insert-
Design primer with preparation of restriction map of antigen sequence and choose to
restriction sites in pVAX1 multiple cloning site. This can be performed by use of restriction
enzymes for isolation of antigen sequence and Pfu polymerase enzyme in PCR to get plenty
copies of antigen sequence. Cut purified DNA fragment by Nhe1 and Xho1.
 Insertion of the Antigen Gene Into the DNA Vaccine Vector-
Cut pVAX1 with NheI and XhoI and purify the vector by agarose gel electrophoresis. Recover
the linearized vector using the QIAquick Gel Extraction Kit. Set up ligation of the vector to the
antigen fragment at a molar ratio of insert : vector of 6–10:1. Ligation can be done by the Roche
Rapid Ligation Kit. Transform competent DH5 cells with the ligation reaction and plate onto L-
kanamycin (50 g/mL) agar plates. Incubate the plates at 37°C for 16–24 h. Select well-isolated
colonies from the transformation plates and grow 5-mL kanamycin cultures. Make plasmid
minipreps from the cultures, using the QIAprep Miniprep Kit (Qiagen) and screen the clones by
restriction digestion with NheI and XhoI.

7.  Verify Clones by Sequence and Expression-
Clones that are identified by restriction mapping should be further verified by sequencing the
insert. This can be done using the T7 promoter-priming site and the BGH reverse priming site
present in the pVAX1 vector. Check for expression of sequence-verified clones in a transiently
transfected mammalian cell line before using the DNA vaccine in animal experiments. A cell
line such as rhabdomyosarcoma (RD) cells is a good choice because it is easily maintained and
transfected.
 Selection of E. coli Strain for Plasmid Production-
Many E. coli strains are commercially available as competent cells and have been developed for
specific needs in molecular biology. Most strains have been characterized for protein
expression, not for plasmid stability and production; therefore, several strains should be tested
prior to choosing a host strain. Once several candidate strains are selected, each should be
tested with the final vaccine clone to determine which will grow at reasonable rates (2 h or less
doubling time), achieve a high cell density, and give the highest plasmid DNA yields in the
fermentation medium in large-scale production.

8. ANTIGEN EXPRESSION SYSTEM FOR PLASMID DNA VACCINES
 Often, foreign proteins, especially small ones, occur in minute quantities when they are
produced in heterologous host cells. This apparently low level of expression is, in many
instances, actually due to degradation of the foreign protein. One way to solve this problem
is to engineer a DNA construct that encodes a target protein that is in frame with a stable
host protein. This combined, single protein, which is called a fusion protein, protects the
cloned gene product from attack by host cell proteases.
 Another way is to genetically engineer the best competent E coli cell so that over expression
of rare t RNA happens. In this way there is high possibility of expressing the antigen gene
because we know cellular incompatibility that can interfere with efficient translation occurs
when a cloned gene has codons that are rarely used by the host cell.

10. SUCCESSFUL DNA VACCINES TILL DATE
 In August 2021, Indian authorities gave emergency approval to ZyCoV-D. Developed
by Cadila Healthcare, it is the first DNA vaccine approved for humans.
 DNA vaccines against cancer are underdevelopment which can become potential
immunotherapy against various kind of cancers.
 DNA vaccine expression of IL-2 and the HSP65 fusion gene was studied. It elevated the
immunogenicity and protective as well as therapeutic effects of the HSP65-DNA vaccine
against TB in mice.
 HIV-negative people were used to study the effect of preventive vaccine candidates to see if
they can prevent infection. Many scholar see DNA vaccine as future possible immunotherapy
against HIV.
 DNA vaccination is one of the novel approaches for developing new generation vaccines
against malaria. Coated DNA vaccines have been shown to exhibit good immunogenicity and
show protective levels of antigen-specific IgG, an elevated proportion of CD4+, CD8+ T cells,
INF-γ and IL-12 levels in the serum and cultured splenocyte supernatant, as well as INF-γ-
producing cells in the spleen.

12. DNA VACCINE DELIVERY SYSTEM
 Needle-Free Injection of DNA Vaccines.
 Surface-Modified Biodegradable Microspheres for DNA Vaccine Delivery.
 A Dendrimer-Like DNA-Based Vector for DNA Delivery.

13. NEEDLE FREE INJECTION OF DNA VACCINES
 Needle-free injection is accomplished by forcing liquid medications at high speed through a
micro-orifice
 Beclard and Galante of France had developed the first needle-free “aquapuncture” device,
which delivered liquid medications at pressures of 25–30 atmospheres without the need for
a needle
 Needle-free jet injectors are approved for human use. Several different jet injectors have
been used to deliver DNA vaccines
 Mucosal IgA was induced by a HIV-1 DNA vaccine when mice were immunized intra-orally
using a jet injector (SyriJet mark II) designed for the application of dental anesthetics
 Biojector utilizes compressed CO2 to force the liquid through a micro-orifice and it can
deliver the vaccine to traditionally targeted tissues such as skin (ID), muscle (IM), or
adipose tissue (subcutaneous) without the needle

14. SURFACE MODIFIED BIODEGRADABLE MICROSPHERE FOR
DNA VACCINE DELIVERY
 Encapsulating DNA within degradable delivery vehicles such as micro- or nanospheres
provides an effective way to protect the DNA from the surrounding environment prior to
delivery
 One means of highly specific cell targeting is through the addition to the vehicle surface of
ligands that bind specifically to receptors on the surface of the targeted cell type
 Chemical conjugation of ligands to the microsphere surface requires selection of
appropriate conjugation chemistry. When using microspheres that are biodegradable, an
ideal conjugation scheme minimizes premature degradation of the microspheres
 For this method major requirements are Poly(lactic-co-glycolic acid) (PLGA) (store at –20°C,
protected from moisture), dichloromethane,DNA to be encapsulated.

15. A DENDRIMER-LIKE DNA BASED VECTOR FOR DNA DELIVERY
 A synthetic polymer that is anisotropic is needed. The recently developed dendrimer-like
DNA (DL-DNA) is a true dendritic polymer that can be anisotropic, thus providing great
potential as a novel vector to carry multifunctional modules that target specific DNA
delivery obstacles
 DL-DNA is capable of carrying many various peptides, especially those peptides derived
from viruses. These viral peptides can perform desired biological functions including
cellular targeting, DNA condensing, endosome disrupting, and nuclear targeting
 Premade modules will further increase flexibility and make “plug-and-play” possible. Such
flexibility is particularly useful in studying the complex processes of DNA delivery because,
little is known quantitatively about intracellular events and one can easily adjust the
delivery vector based on the experimental outcomes
 In addition, the VNA system is capable of carrying both genes and antigenes (siRNA), as well
as other entities such as enzymes and chemical drugs

17. DNA VACCINE ADJUVANTS AND ACTIVITY ENHANCEMENT
 The activity of bacterial DNA, or the synthetic oligodeoxynucleotides (CpG ODN) that mimic
it, was first characterized in mice.
 Immunomodulatory effects of DNA containing unmethylated CpG motifs on the immune
system. CpG DNA triggers polyclonal activation of B cells and stimulates plasmacytoid
dendritic cells (pDC). The pDC mature and
produce high levels of IFNα/β, which in
turn activate natural killer cells to produce
IFNγ and foster the maturationof mono-
cytes into mDC. These active and mature
mDC present antigen to CD4+, CD8+
T cells increasing the cellular and humoral
immune response.

18. REFERENCES
 DNA Vaccine : Method and Protocols, 2nd Edition by HUMANA EXPRESS.
 DNA Pharmaceuticals by Martin Schleef.
 Current Topics in Microbiology and Immunology
 Molecular Biotechnology by Bernard R. Glick et al.