DNA Vaccine: A Novel Tool For Poultry Disease Prevention
K. Dhama1 and Mahesh Mahendran2
1
Senior Scientist, 2Ph.D. Scholar, Avian Diseases Section, Division of Pathology, Indian Veterinary Research Institute
(IVRI), Izatnagar (U.P.) – 243 122
INTRODUCTION
The Indian poultry sector has become a major contributor to the national economy as a result of
the revolutionary and scientific approaches in avian health care management. Today, India ranks
globally at 4th and 5th in egg and meat production, respectively. As the poultry industry is expanding,
there is much need for improving the efficiency of production, which directly depends on the
prevention of diseases, the flocks often encounter. Presently, the industry is threatened by progressively
more virulent pathogens or by exotic and emerging diseases that can cause catastrophic losses to this
sector. The poultry sector has recognized the importance of health management, especially during the
recent avian influenza outbreaks occurred in the country. The situation thus warrants developing of new
generation preventive measures that could be used alone or in tandem with the conventional
immunizing agents. DNA vaccine, generated using recombination of a pathogen’s immunogenic gene
and an optimized bacterial plasmid, is a novel approach that could ably support the efforts made to the
development of newer immunoprophylactics for controlling infectious diseases of poultry. These third
generation vaccines are having many advantages when compared to the conventional inactivated or live
vaccines, superior cellular immunity generation and non-requirement of cold chain, being few among
those. The concept of DNA vaccine was first evolved during the early 1990’s, when it was found that
intramuscular injection of a recombinant bacterial plasmid DNA yielded the expression of a gene in
mice. This has lead to the research and development of nucleic acid based vaccination technique, which
is currently considered as an effective way for the generation of specific immune response.
For developing DNA vaccines, generally the immunogenic protein of a pathogen which is
protective to the host is incorporated into a suitably designed plasmid which has been derived from
microbial agents. This gene encoded plasmid DNA, when administered to host, is capable of getting
transcribed and translated into a peptide within the host cells to generate protective responses on
encountering with the host immune cells. In human medicine, the application of DNA vaccines have
moved towards second stage clinical trials and has shown promising results in the treatment of diseases
like acquired immunodeficiency syndrome (AIDS), herpes infection, hepatitis B and C, influenza,
rabies, rotaviral infections, Ebola, tuberculosis, malaria, Leishmaniosis, mammary and lung carcinomas
and autoimmune diseases like multiple sclerosis and rheumatoid arthritis. However, a commercial
product has not reached the market yet, mainly due to the safety concerns raised by the international
regulatory organizations due to the suspicion of integration of the inoculated plasmid DNA into host
genome causing activation of oncogenes or inactivation of tumor suppressor genes and also the
generation of anti-DNA antibodies resulting in autoimmune diseases. But this theoretical demerit has
not been proven scientifically. The research for the application of DNA vaccines in veterinary practice,
even though on a lesser pace, have been conducted for developing effective immunizing agents against
various diseases like foot and mouth disease (FMD), infectious bovine rhinotracheitis (IBR), bovine
viral diarrhea (BVD), tuberculosis, brucellosis, Aujeszky's disease, swine fever, rabies and canine
distemper. In poultry, DNA vaccines have been experimentally tried and successfully developed against
avian influenza, infectious bronchitis, infectious bursal disease and coccidiosis. But the doubts
regarding the potential of DNA vaccine at times to develop immune response to sufficient levels has
been a worrying factor. To overcome this lacuna, various technological and immunological approaches
are being employed to improve the efficacy of DNA vaccines to make their practical implementation
and a much faster market entry, which could provide a novel alternative to the conventional vaccines
for the prevention of various infectious as well as emerging diseases of poultry.
2
SALIENT FEATURES OF DNA VACCINES












They comprise of circular bacterial plasmids in which desired antigenic gene of avian pathogens are
encoded.
Modified plasmids used as vaccine vector generally contains a promoter and poly-adenylation (poly
A) signal to facilitate expression of protein in higher eukaryotes.
To facilitate production and selection of plasmid in bacteria during the production process, bacterial
origin of replication and antibiotic resistance gene is incorporated within.
Administration of the vaccine is performed by intramuscular needle-based injection, nasal and oral
drops or topical application on skin.
Application via intradermal route using ‘gene gun’ and needle free injector system is also practiced.
Contrary to the direct generation of humoral or cellular immunity as seen in case of killed or live
vaccines, the DNA vaccines enter into muscle cells or dendritic cells and produce the immunogenic
pathogen proteins for further action of the immune system.
Activation of immunity more inclined towards cellular immune responses when compared to
humoral responses, especially useful to control intracellular pathogens like viruses and microbes
like Mycobacterium spp.
Professional antigen presenting cells like dendritic cells and macrophages receives antigens from
muscle cells via a phenomenon called ‘cross presentation’.
Various formulations are generated by incorporating gene encoded plasmid in co-polymers,
cochleates and microparticulate compounds
For mucosal or oral immunization, DNA vaccines are formulated in liposomes to get a better
immune response.
Even though adjuvants like calcium phosphate and aluminium hydroxide are commonly used,
cytokines such as interleukin (IL)-2, 12, 15, 18, IFN-γ and GM-CSF are used in combination with
DNA vaccine as new generation adjuvants. Also, the utility of co-stimulatory molecules, which
stimulates the generated specific immune cells, are exploited recently.
Inherent immunity enhancing properties are shown by the plasmid vector itself, primarily due to the
presence of unmethylated CpG sequences, which act as a mitogen for B lymphocytes and a
stimulator of natural killer (NK) cells.
2
3
DNA VACCINES IN POULTRY
Disease prevention by vaccination is an integral part of flock health management programs and
has played an important role in successfully preventing the diseases of poultry to a much desirable
extent in a cost-effective manner. Recently, the advancements in immunology, microbiology and
molecular biology, have created various opportunities for development of highly efficacious vaccines
against various avian pathogens. The application of genetic engineering along with other new
technologies has played crucial roles in introducing novel ideas in vaccinology. New generation
vaccines like subunit vaccines and recombinant DNA vaccines are rapidly gaining acceptance as new
generation vaccines, and considered as alternatives to the conventional vaccines. The last decade have
seen the development of recombinant plasmid based DNA or nucleic acid vaccines, using immunogenic
genes of avian pathogens, to counter diseases like avian influenza, Newcastle disease (ND), infectious
bronchitis (IB), infectious bursal disease (IBD) and coccidiosis. The impetus for avian DNA vaccines
was provided by the landmark studies made in 1993 on genetic immunization of poultry against avian
influenza. Since then, there have been several successful attempts to protect birds against a variety of
viral, bacterial and protozoal diseases, clearly proving the generation of measurable antibody titers and
cell-mediated immune responses resulting in protection during challenge studies. Even though
considerable progress has been made in the avian species, the concept of DNA vaccination in the
poultry sector is still in its infancy, primarily due to variation in immune responses. But many of the
basic immune responses elicited in birds are found to that seen in higher mammals. The avian has
innate, humoral and cellular immune responses mediated by a similar set of cells, as defined in the
mammal. However, there are some discrete and unique lymphatic organs (bursa of Fabricus and
Harderian gland) in the avian that are not found in the mammal and there is a lack of structured
lymphatic system. Therefore, the type and location of some of the early immune responses to DNA
vaccination in avian may be different than that in the mammal. In mammals, after the plasmid is
transfected, the consequent protein production has been tracked to draining lymph nodes after DNA
vaccination. In the avian, the distribution of the plasmid and the sites of the protein production are not
well defined.
Contrary to animal DNA vaccination, in birds, apart from the commonly followed IM and
intradermal routes, in ovo and topical application are also proven to be of much significance. During in
ovo application, 18-day old embryos are used and the plasmid DNA is incorporated in a neutral lipid
formulation along with dimethyl sulphoxide (DMSO) and the mixture is injected through a hole in the
top of egg shell onto shell membrane. For topical application, the plasmid DNA with suitable adjuvants
is applied on to the skin in the wing web region. It has been proven that topical delivery of plasmid
DNA to the skin of the chicken results in a wide distribution of the plasmid with protein production in
several tissues. Alternate delivery systems such as oral delivery through feed and water, spray or
aerosol, and mucosal delivery techniques are also considered nowadays as this will suit well for the
implementation of DNA vaccines in poultry enterprises. Many distinct utilities are also there for the
implementation of nucleic acid vaccines in prevention and control of infectious diseases of poultry. One
among this is it utility in generation of vaccines capable of differentiating infected from the vaccinated
birds (DIVA), by manipulating the genes using recombinant technology, which makes it suitable to
effectively eradicate the pathogen from the environment. Research is underway to exploit this utility
against the much dreaded bird flu. The potential of DNA vaccines to overcome the maternal immunity
is also a remarkable feature, which favors to chalk out an efficacious vaccination program in day old
chicks.
The development of naked DNA immunization as third generation vaccines has been well studied
and recently a variety of such vaccines are in clinical trials for their use against infectious diseases of
poultry. Plasmid DNA vaccine encoding the enterotoxigenic Escherichia coli K88 fimbrial protein has
3
4
been found to stimulate the production of antibodies against the protein and further protected the birds
from challenge infection. Chlamydophila psittaci is another pathogen, causing psittacosis in birds,
against which DNA vaccine expressing its major outer membrane protein (MOMP) has been developed
and found effective. Recently, cytokine adjuvant IFNγ, and Vitamin D have been used as
immunomodulators along with DNA vaccine against C. psittaci, the use of which elicited an augmented
immune response with increased serum and mucosal antibody titer in birds. Plasmid DNA based
vaccines that are capable of eliciting desired immune response has been developed against major viral
infections of poultry like avian influenza utilizing the hemagglutinin (HA) gene of influenza A virus.
Against ND, nucleic acid vaccine based on haemagglutinin (HN) and fusion (F) gene induced higher
level of antibodies for protection against velogenic Newcastle disease (NDV) in chickens. Against
Marek’s disease MD, whole genome clone of virulent serotype-1 is used as a DNA vaccine to protect
against the disease in birds. Utility of plasmid DNA vaccines using N protein and S1 glycoprotein gene
of infectious bronchitis virus (IBV) after in ovo and intramuscular immunization has been exploited to
develop vaccines that could effectively protect the birds from infection. Against infectious bursal
disease (IBD), VP2 gene of the IBD virus or the VP2/4/3 polyprotein coding gene was used in plasmid
backbone to develop efficacious DNA vaccines. Against chicken infectious anemia (CIA), the
simultaneous in vivo expression of viral proteins VP1 and VP2 has been studied for developing a DNA
vaccine that has been found capable of generating neutralizing antibodies in vaccine administered
experimental birds. Similarly DNA vaccines has been developed against avian reovirus and egg drop
syndrome (EDS-76) virus, using the σC protein gene and penton fiber gene fragment, respectively and
both these vaccines were found effective in protecting the birds during respective challenges. DNA
vaccination against duck hepatitis B virus has also shown efficacy as it facilitated the rapid removal of
the virus from host after the challenge. In another study, DNA vaccination with a mutated and nononcogenic v-src gene derived from avian leucosis virus (ALV) was found to be efficient for induction
of cytotoxic T-lymphocyte (CTL) response and protection of birds from tumors. Recently, the plasmid
DNA coding for the VP3 gene (apoptin) of chicken anemia virus was found to regress the RSV induced
tumor in birds. Against protozoan infections, especially coccidiosis, development of successful DNA
vaccines, utilizing the 3-1E and EtMIC2 genes in combination with an array of cytokines has resulted in
significant reduction of fecal oocysts, reduced body weight loss and protected the birds from coccidial
infection.
4
5
ADVANTAGES OF DNA VACCINES IN POULTRY















Non-requirement of cold chain when compared to commonly used live vaccines in poultry.
Useful in producing vaccines against deadly viruses and microbes that are unsafe to handle
under laboratory conditions.
Able to produce vaccines that differentiate infected from vaccinated birds, especially useful
in containing diseases like avian influenza.
A single vaccine for multiple pathogens (multigenic vaccine) can be produced.
Provides long-lived cellular and humoral immune response.
Majority of the viral diseases of poultry can be controlled due to the prominent cellular
immunity generation potential of DNA vaccines.
Cytokine and new generation immunoadjuvants can be used along with DNA vaccines to
potentiate its effect.
Bacterial plasmids used as vector have inherent immunogenic properties.
Have ability to over come effects of maternal antibodies, in day-old chicks.
Can be administered as in ovo vaccination, spray vaccine or by topical application in wing
web region.
Normal saline or phosphate buffered saline (PBS) can be used as vaccine vehicle.
Much easier bulk production process in bacteria.
Cost effective.
Shows enormous stability as a vaccine.
Can be easily transported in lyophilized form.
Ways to improve the efficacy of DNA vaccines











By co-administering new generation adjuvants like cytokines.
Use of co-stimulatory molecules and immune cell targeting ligands.
Delivery of DNA vaccines using liposomes has to be standardized.
Linking the genes to ubiquitin molecules to assist in endogenous peptide release.
Linking the genes to nuclear localization signals.
Targeting the dendritic cells with heat shock proteins.
Use of intacellular bacteria as bacterial vectors for plasmid DNA delivery.
Utility of toll like receptor (TLR) adaptor molecules to be exploited.
Electroporation (electric impulses) to increase inflammatory responses in muscle cells.
Modification of plasmid DNA to prevent its in vivo degradation.
Suitable optimization of the vector DNA (eg. Addition of poly A signal enhancer).
Future directions
The ability of killed or live vaccines in preventing and controlling various diseases of birds has
been well established. Yet, a complete prevention or eradication of many infectious agents is a distant
dream. Moreover, the drawbacks of the conventional vaccines have forced researchers to evolve newer
alternatives, among which nucleic acid vaccines holds much promise. Vaccination with DNA is one of
the promising novel immunization techniques against pathogens, for which conventional vaccines have
5
6
been less effective. The utility of DNA vaccines and their advantages have been proven beyond doubt.
But its efficacy largely depends on proper optimization of the plasmid vector, the route of delivery and
formulation. Further research on the various routes like in ovo administration and topical application
has to be undertaken, and suitable delivery vehicles has to be used to enable effective antigen
presentation to enhance the immunity towards DNA vaccines and make them practical for
administration in poultry. Exploiting the utility of a variety of immune modulators like cytokines and
co-stimulatory molecules is another option to develop safer, cheaper and efficacious DNA vaccines. In
future, research should also be targeted towards ways to protect the plasmid DNA from in vivo
degradation and to improve the transfection efficiency in host cells. Using such multivariate options, if
potency can be improved, DNA vaccines are capable to induce longer immune response that may be
useful for the active immunization against various infectious diseases of birds.
SUGGESTED READING
Babiuk LA, Lewis PJ, van Drunen Little-van den Hurk S, Tikoo S and Liang X. 1998. Nucleic acid vaccines:
veterinary applications. Curr. Topics Microbiol. Immunol., 226: 90-106.
Brandsma JL. 2006. DNA vaccine design. Methods Mol. Med., 127: 3-10.
Clark KR and Johnson PR. 2001. Gene delivery of vaccines for infectious disease. Curr. Opin. Mol. Ther., 3:
375-384.
Daudel D, Weidinger G and Spreng S. 2007. Use of attenuated bacteria as delivery vectors for DNA
vaccines. Expert Rev. Vaccines, 6: 97-110.
Dhama K, Singh SD, Mahendran M and Tomar S, 2006. Immunity & disease resistance strategies in poultry:
current and future prospects. Lead Paper presented in National Seminar on Poultry Research Priorities
to 2020, Nov., 2006 at CARI, Izatnagar (U.P.), India. Souvenir, pp, 200-211
Dhama K, Chauhan RS, Mahendran M, Tomar S and Singhal L. 2007. DNA vaccines and prevention of
infectious diseases in bovines: A Review. Int. J. Cow Sci., 3(1): 1-15. (In Press).
Dhama K, Chauhan RS, Kataria JM, Mahendran M and Tomar S. 2005. Avian Influenza: The current
perspectives. J. Immunol. Immunopathol., 7(2): 1-33.
Ding X, Lillehoj HS, Dalloul RA, Min W, Sato T, Yasuda A and Lillehoj EP. 2005. In ovo vaccination with
the Eimeria tenella EtMIC2 gene induces protective immunity against coccidiosis. Vaccine, 23: 37333740.
Donnelly JJ, Wahren B and Liu MA. 2005. DNA Vaccines: Progress and Challenges. J. Immunol., 175: 633639.
Dufour V. 2001. DNA vaccines: new applications for veterinary medicine. Vet. Sci. Tomorrow, 1: 1-19.
Dunham SP. 2002. The application of nucleic acid vaccines in veterinary medicine. Res. Vet. Sci., 73: 9-16.
Fynan EF, Robinson HL and Webster RG. 1993. Use of DNA encoding influenza hemagglutinin as an avian
influenza vaccine. DNA Cell Biol., 12: 785-789.
Gurunathan S, Klinman DM and Seder RA. 2000. DNA vaccines: immunology, application, and
optimization. Annu. Rev. Immunol., 18: 927-974.
Jang JH and Shea LD. 2006. Intramuscular delivery of DNA releasing microspheres. Microparticulate
properties and transgene expression. J. Control. Release, 112: 120-128.
Kataria, J.M., Dey, S., Dhama, K. and Mohan, C.M., 2005. Newer perspectives in the biotechnological
approaches towards control of avian diseases. Presented in XII Annual Convention of ISVIB and
National Symposium held on Nov., 2005 at PAU, Ludhiana (Punjab), India, Souvenir, pp: 59-66
Kodihalli S, Kobasa DL and Webster R.G. 2000. Strategies for inducing protection against avian influenza A
virus subtypes with DNA vaccines. Vaccine, 18: 2592-2599.
Krieg, A.M., 2002. CpG motifs in bacterial DNA and their immune effects. Annu. Rev. Immunol., 20: 709760.
Lewis PJ and Babiuk LA. 1999. DNA vaccines. a review. Adv. Virus Res., 54: 129-188.
6
7
Lillehoj HS, Ding X, Dalloul RA, Sato T, Yasuda A and Lillehoj EP. 2005. Embryo vaccination against
Eimeria tenella and E. acervulina infections using recombinant proteins and cytokine adjuvants. J.
Parasitol., 91: 666-673.
Liu MA, Wahren B and Karlsson-Hedestam GB. 2006. DNA vaccines: recent developments and future
possibilities. Hum. Gene Ther., 17: 1051-1061.
Manoj S, Babiuk LA and van Drunen Littel-van den Hurk S. 2004. Approaches to enhance the efficacy of
DNA vaccines. Crit. Rev. Clin. Lab. Sci., 41: 1-39.
Oshop GL, Elankumaran S and Heckert RA. (2002). DNA vaccination in avian. Vet. Immunol.
Immunopathol., 89: 1-12.
Senthilkumar N, Kataria, JM, Dhama K, Bhardwaj N, Sylvester SA and Rahul S (2004). Development of
DNA vaccine against chicken anaemia virus simultaneously using it’s VP1 and VP2 proteins. Paper
Presented in XII Conference of Indian Poultry Science Association and National Symposium, April 79, 2004 at HPKVV, Palampur (HP), Souvenir, pp: 152.
Shams H. 2005. Recent developments in veterinary vaccinology. Vet. J., 170: 289-299.
Stevenson FK. 2004. DNA vaccines and adjuvants. Immunol. Reviews, 199: 5-8.
Tischer BK, Schumacher D, Beer M, Beyer J, Teifke JP, Osterrieder K, Wink K, Zelnik V, Fehlero F and
Osterrieder N. 2002. A DNA vaccine containing an infectious Marek’s disease virus genome can
confer protection against tumorigenic Marek’s disease in chickens. J. Gen. Virol. 83: 2367-2376.
Ulmer JB, Wahren B and Liu MA. 2006. DNA vaccines: recent technological and clinical advances. Discov.
Med., 6: 109-112.
Vannier P and Martignat L. 2005. New vaccines and new veterinary therapies derived from biotechnologies:
examples of applications. Rev. Sci. Tech., 24: 215-229.
Wolff JA and Budker V. 2005. The mechanism of naked DNA uptake and expression. Adv. Genet., 54: 3-20.
7