Review

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DNA vaccines in veterinary use

Expert Rev. Vaccines 8(9), 1251–1276 (2009)

Laurel Redding and DNA vaccines represent a new frontier in vaccine technology. One important application of this
David B Weiner† technology is in the veterinary arena. DNA vaccines have already gained a foothold in certain
†
Author for correspondence fields of veterinary medicine. However, several important questions must be addressed when
Department of Pathology and developing DNA vaccines for animals, including whether or not the vaccine is efficacious and
Laboratory Medicine, 422 Curie cost effective compared with currently available options. Another important question to
Boulevard – 505 SCL, University consider is how to apply this developing technology in a wide range of different situations,
of Pennsylvania, Philadelphia, from the domestic pet to individual fish in fisheries with several thousand animals, to wildlife
PA 19104, USA programs for disease control. In some cases, DNA vaccines represent an interesting option for
Tel.: +1 215 349 8365 vaccination, while in others, currently available options are sufficient. This review will examine
Fax: +1 215 573 9436
a number of diseases of veterinary importance and the progress being made in DNA vaccine
[email protected]
technology relevant to these diseases, and we compare these with the conventional treatment
options available.

Keywords : cancer • companion animals • cost • DNA vaccine • economic impact • food animal • infectious
disease • veterinary use

Animals play an increasingly important role in DNA vaccines are emerging as a new and
society in both the developed and the develop- important method of vaccination for animals.
ing world. In addition, as the global travel of DNA vaccination involves immunization with
animals, commercial goods and people expands, a plasmid encoding an antigen of the pathogen.
transmission of disease poses a greater threat The gene of interest is inserted into a plasmid,
than ever before. The demand for animal vac- along with appropriate genetic elements such as
cines must be met in a timely, efficient and eco- eukaryotic promoters for transcriptional con-
nomically viable manner. Vaccines for compan- trol, a polyadenylation signal sequence for stable
ion animals, which provide for the health and and effective translation, and a bacterial origin
longevity of pets, are generally well established of replication. The plasmid is transfected into
and affordable; the number of companion ani- host cells via direct injection, or injection with
mals with infectious diseases has decreased sig- electroporation or gene gun. The gene of inter-
nificantly since commercial vaccines were made est then undergoes transcription and translation
available. Vaccines for livestock are essential by host cellular machinery, resulting in the pro-
for herd health, economic survival of farmers, duction of an antigenic protein that can induce
and the maintenance of trade of meat and other cellular and humoral immune responses. DNA
animal products between countries. For live- vaccines have a number of advantages over other
stock, the health of the herd is more important vaccination technologies that are of particular
than that of the individual, and the only way to interest to veterinary medicine. They have the
control outbreaks is to cull and mass slaughter potential to be less expensive than other com-
herds, resulting in significant economic loss. mercial vaccines, as they can be produced in
Vaccination can be an effective preventative large quantities by bacteria and, in the case of
measure against infectious diseases; vaccines are certain pathogens, do not require expensive
therefore required for prophylactic purposes as facilities of a high biosafety level. They are tem-
well as for the control of outbreaks, and need to perature stable and safe to transport, which can
be affordable on a large scale and easily available. be important for farms located in remote areas
For wildlife, the need is for cheap but effective or for wildlife vaccines that need to remain in
vaccines that can ideally be consumed orally in the open for a prolonged period of time. They
the form of bait traps. are able to drive immunity in the presence of

www.expert-reviews.com 10.1586/ERV.09.77 © 2009 Expert Reviews Ltd ISSN 1476-0584 1251

 Review Redding & Weiner

maternal antibodies – an issue of great importance, as neonates leptospirosis and Bordetella) are not required and are used ��������
in situ-
ingesting colostrum are at a high risk of disease. Different genes ations where only some animals truly need to be protected due
can be combined simultaneously, which allows for the develop- to higher exposure or risk. Conventional core vaccines have been
ment of vaccines against multiple strains of a pathogen and for well established and are mostly effective. For diseases such as
combination approaches against multiple pathogens. This aspect rabies where bait vaccines are used for wildlife, a DNA vaccine
is especially attractive for livestock, where vaccine cocktails are could be useful; however, further progress needs to be made in
a useful vaccination option. In addition, because the relevant developing this mode of administration. This is further discussed
protein is produced and presented intracellularly, both a cellular in the review. A number of the core vaccines for dogs and cats are
and humoral immune response are induced, which gives a more summarized in Tables 2 & 3.
efficient immune response when the animal encounters the natu-
ral infection at a later timepoint. DNA vaccines can be adminis- Opportunities for DNA vaccines in infectious diseases
tered via a number of routes and techniques, which can alter the in companion animals
immune response and distribution of the protein. A number of Infectious diseases represent a continuous threat to companion
DNA vaccines have already been approved for commercial use in animals in many places, especially those with limited access to
both companion and food animals. veterinary care. Viral, bacterial and parasitic diseases plague a
A few reviews have been written on the status of DNA vaccines large swathe of animals, from pediatric animals from pet stores
in veterinary medicine, which detail the research and develop- in developed countries to feral animals roaming the streets in
ment of DNA vaccines for a number of veterinary diseases [1–5] . underdeveloped areas. DNA vaccines that have been developed for
This review is unique in that it will examine a number of diseases a number of these diseases are detailed in the following sections.
of veterinary importance, and compare DNA vaccines with cur-
rently available vaccines and treatment options. For some diseases, Feline immunodeficiency virus
DNA vaccines could be very useful and greatly improve upon Feline immunodeficiency virus (FIV) is a lentivirus that causes a
current treatment options, whereas for other diseases, ample com- progressive immune deficiency syndrome in cats. FIV is similar
mercial vaccines are available that have proven efficient and cost to HIV in morphology, genomic organization and pathogenesis,
effective. This review will consider the impact of the disease and and as such, has been of great interest as a model for studying
compare current commercial vaccines and treatment options with HIV [7] . However, FIV also has an important veterinarian applica-
DNA vaccines being developed, both in terms of cost–effective- tion, as FIV affects both feral and domestic cats. The worldwide
ness for those DNA vaccines that are commercially available, prevalence of FIV infection in domestic cats has been reported
ease and frequency of administration, and efficacy of protection. to range from 1 to 28% [8] and is close to 2.5% in the USA [9] .
FIV, like HIV, poses a number of difficulties for vaccine develop-
Companion animals ment: FIV targets its host’s immune cells, impairing the innate
The number of companion animals in the USA is on the rise. In immune response and weakening the adaptive immune system
2007, there were over 43 million households owning dogs, over by depleting CD4 + T cells. Another challenge in vaccine devel-
37 million households owning cats and over 2 million households opment is the genetic diversity of FIV. Six subtypes of FIV have
owning horses. Vaccinations comprised the greatest percentage of been identified, with most diversity found in Env (between 2
veterinary expenditures for dogs and the second largest for cats and and 15% within a particular subtype and 17–26% between sub-
horses after physical exams (Table 1) [6] . The market for companion types) [10] . In addition, an error-prone reverse transcriptase, as well
animal vaccines is therefore substantial and well established. as frequent recombination between feline leukemia virus (FeLV)
Different types of vaccines exist for companion animals. Core and endogenous FeLV-related retroviruses in the cat genome can
vaccines (such as rabies, parvovirus and distemper) are required lead to mutated strains [11] . A number of vaccine approaches have
for all companion animals (dogs and cats), regardless of geo- been tested, including fixed infected cell, inactivated whole virus,
graphic location or risk of exposure. Noncore vaccines (such as subunit, recombinant and dual-subtype vaccines [7] ; however,

Table 1. Pet ownership in the USA and veterinary expenditures.

Parameter Dogs Cats Birds Horses
Number of households owning 43,021,000 37,460,000 4,453,000 2,087,000
Average number owned 1.7 2.2 2.5 3.5
per household
Total number in the USA 72,114,000 81,721,000 11,199,000 7,295,000
Total veterinary expenditure US$16.1 billion US$7.1 billion US$102.8 million US$718.3 million
Expenditure for vaccines 70.2% 63.7% 13% 49.3%
(= US$11.2 billion) (= US$4.52 billion) (= US$13.36 million) (= US$210.46 million)
Data taken from [6].

1252 Expert Rev. Vaccines 8(9), (2009)

 DNA vaccines in veterinary use Review

Table 2. Canine vaccination guidelines of the American Animal Hospital Association.

Pathogen Pathogen Vaccine types Effectiveness Vaccination ages Ref.
family (%) and
duration of
immunity
Canine parvovirus Parvoviridae MLV, killed, >99 Puppies receive three doses between the ages of [85]
(core) recombinant DOI: >7 years 6 and 16 weeks, at 3–4 week intervals. Booster
vaccination 1 year later. Revaccination every
3 years
Canine adenovirus Adenoviridae MLV and killed, >99 Puppies receive three doses between the ages of [85]
(core) MLV parenteral, DOI: >7 years 6 and 16 weeks, at 3–4 week intervals. Booster
killed or MLV vaccination 1 year later. Revaccination every
topical 3 years
Rabies (core) Rhabdoviridae Killed >99 One dose at 3 months. State, provincial and local [85]
DOI: >3 years statutes govern frequency of subsequent
administration
Canine distemper MLV and >99 Puppies receive three doses between the ages of [85]
(core) recombinant DOI: 3–7 years 6 and 16 weeks, with the final dose administered
vectored virus at 14–16 weeks
Parainfluenza Paramyxovirus MLV parenteral Unknown Puppies receive three doses between the ages of [86]
(noncore) DOI: >3 years 6 and 16 weeks, at 3–4 week intervals. Booster
vaccination 1 year later. Revaccination every
3 years
Bordetella Coccobacilli of MLV or 20–24 One dose at 6–8 weeks, one dose at [86,87]
bronchisepta the phylum killed parenteral DOI: 10–12 weeks. Booster 1 year later. Revaccination
(noncore) Proteobacteria <12 months every 3 years
Leptospira Spirochaete Killed <50 One dose at 12 weeks, one dose at 14–16 weeks. [85]
interrogans (noncore) bacteria Annual revaccination
Borrelia burgdorferi/ Spirochaete Recombinant Unknown Initial dose at 9–12 weeks, second dose
Lyme borreliosis bacteria outer surface 2–4 weeks later. Annual revaccination
(noncore) protein A
DOI: Duration of immunity; MLV: Modified live virus.
Data taken from [213].

these have mostly been unable to offer effective protection. A Feline leukemia virus
commercial vaccine (Fel-O-Vax® by Fort Dodge, KS, USA) is Feline leukemia virus, like FIV, is a retrovirus that can cause
available against subtype B of FIV, and effectively protects against fatal disease in cats, including leukemia, lymphoma, anemia and
subtype B and slightly against subtype A, the two most prevalent immunodeficiency. There are three subtypes of FeLV – A, B and
subtypes in the USA. Several DNA vaccines against FIV, consist- C, with A being the most prevalent and contagious but the least
ing of mutated whole genomes or individually mutated genes, pathogenic. Oronasal exposure to the virus can lead to a tempo-
have also been attempted, and these offer limited protection. A rary infection with transient viremia (65% of cases) or persistent
representative sample of these vaccines is provided in Table 4. viremia (30–40%), which causes more severe illnesses, especially
Cats diagnosed with FIV require extensive supportive care, in kittens [12] . The prevalence of FeLV in a healthy cat population
therefore pet cats that are at risk of infection would benefit from is less than 0.5% [13] but can range from 0.35% in a free-roaming
being vaccinated. The available commercial vaccines are likely population to 32.6% in a high-risk multicat household [14] . The
sufficient to offer protection, as the costs are not prohibitive. virus is shed in saliva, nasal secretions, urine, feces and milk. Cats
Feral cats represent more of a problem. Shelters that practice trap- with the greatest risk of infection are those living with infected
neuter-release (TNR) programs for feral cats usually test all cats cats, cats that venture or live outdoors and may be bitten by an
for FIV, vaccinate seronegative adults and euthanize seropositive infected cat, and kittens born to infected mothers.
adults and kittens. TNR programs are expensive and contro­versial Both FeLV-specific cytotoxic T-lymphocyte activity and virus-
in their effectiveness. If DNA vaccines could be produced more neutralizing antibodies contribute to limiting the infection within
cheaply than current commercial vaccines, they would be a bet- 4–8 weeks of infection [15] . Vaccines against type A are consid-
ter choice for feral cats. DNA vaccines should also be studied ered effective against all subtypes [16] . A number of commercial
for this disease in this species, as they represent an interesting vaccines are available with varying efficacies. Treatment options
model for HIV. are mostly palliative and usually require weekly to daily doses,

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and are therefore expensive. In most veterinary clinics, the FeLV in producing a rabies vaccine lies in inducing enough immunity
vaccine is not considered a core vaccine, but is highly recom- to fully protect the animal while being in a form that is able to
mended. In 2003, sales of vaccines for one of the commercial be widely distributed at a relatively inexpensive cost. Vaccines for
vaccines (Leucogen, Virbac, France) amounted to €13 million wild animals present a particular problem in that it is impossible
(US$20 million) [17] . A DNA vaccine against FeLV was devel- to know if animals obtain the full, necessary dosage of a vaccine
oped and tested in kittens and adult cats by Hanlon et al., and and that it is very difficult to revaccinate animals. Vaccination
was found to provide robust protection against persistent viremia programs for wild animals frequently use vaccines that are not
(Table 5) [18] . approved for rabies protection in domestic animals, distributed via
Considering the number of commercial vaccines available for bait units. The development of a DNA vaccine that would be safe
FeLV, the relatively low prevalence of the disease and the rela- and effective for both wild and domestic animals could be useful,
tively low cost of the vaccine, the development of a DNA vac- particularly if the duration of immunity can be increased com-
cine against FeLV is not an urgent necessity for strictly clinical pared with commercial vaccines. Currently available ­commercial
purposes. A cheaper vaccine could be useful for feral cat popula- vaccines and DNA vaccines for rabies are listed in Table 6.
tions, but for cat owners, prophylactic and treatment options are There are numerous effective and inexpensive vaccines available
available. FeLV as a model for DNA vaccines could be of interest for rabies protection for domestic animals. Since rabies vaccines
from a basic scientific point of view, as the virus belongs to the are mandatory, the market has responded with a wide choice of
same family as human T-lymphotropic viruses [12] . vaccines that offer vaccine protection for similarly aged animals
with similar vaccination protocols. DNA vaccines, however, offer
Rabies the potential advantage of a more rapid induction of neutralizing
Rabies virus is a lyssavirus of the Rhabdoviridae family, which antibodies [21] . If DNA vaccines can be produced inexpensively,
constitutes a serious health threat for domestic animals, humans they may also be useful in rabies control programs in develop-
and wildlife. The virus kills 55,000 people each year and costs the ing countries, since vaccination using inactivated live viruses can
global community over US$583 million [19] . Rabies is transmitted be expensive.
when the virus is introduced into bite wounds, open cuts in skin, For the inoculation of wildlife against rabies, recombinant vac-
or onto mucous membranes from saliva or other potentially infec- cines encoding rabies virus glycoproteins have been shown to
tious material such as neural tissue. The rabies vaccine is required provide protection in wildlife such as raccoons, foxes and ferrets
for all domestic animals in the USA and a number of large-scale in a cost-effective manner [22–24] . DNA vaccines could also be
rabies control vaccination programs for wild animals are in place potentially used in a similar manner, as a number of studies have
in a number of states and countries. The most important viral produced orally administered DNA vaccines [25,26] . The hope
reservoir for the rabies virus is in developing countries, where only is for a DNA vaccine against rabies that could be inexpensively
30–50% of the canine population is vaccinated [20] . The difficulty produced and effective in both wild and domestic animals.

Table 3. Feline vaccination guidelines of the American Association of Feline Practitioners.

Pathogen Vaccine types Effectiveness Vaccination ages Ref.
(%)
Feline panleukopenia virus MLV and killed, >99 Kittens beginning at 6 weeks of age, then every [85,86]
(core) adjuvanted and DOI: 7 years 3–4 weeks until 16 weeks. Booster 1 year later,
nonadjuvanted then every 3 years
FHV and FCV (core) MLV and killed, FHV: 52 Kittens beginning at 6 weeks of age, then every [85,86,219]
adjuvanted and DOI: 5 years 3–4 weeks until 16 weeks. Booster 1 year later,
nonadjuvanted FCV: <75 then every 3 years
DOI: 5 years
Rabies (core) Killed, canarypox >99 Single dose as early as 8 weeks, 1 year booster. [85,86]
virus-vectored DOI: 3 years State, provincial and local statutes govern frequency
recombinant of subsequent administration
Feline leukemia virus Killed, canarypox Unknown Single dose at 8 weeks, second dose 3–4 weeks
(noncore) virus-vectored later. Annual revaccination for cats at risk
recombinant
Bordetella bronchisepta Avirulent live Unknown Single dose at 8 weeks. Annual booster for cats [86]
(noncore) DOI: 1 year at risk
Chlamydophila felis Avirulent live, killed Unknown Single dose at 9 weeks, second dose 3–4 weeks [86]
(noncore) DOI: 1 year later. Annual booster for cats at risk
DOI: Duration of Immunity; FCV: Feline calcivirus; FHV: Feline herpes virus; MLV: Modified live virus.
Data taken from [214].

1254 Expert Rev. Vaccines 8(9), (2009)

 DNA vaccines in veterinary use Review

Table 4. Feline immunodeficiency virus vaccines.

Treatment Vector Dosage/price Immune response Protection Ref.
Fel-O-Vax Inactivated whole viruses of Three initial doses at Antibodies against whole Protected 82% preventable
(Fort Dodge) subtype A and subtype D 2–3-week intervals with FIV antigen and r-Gag fraction
with adjuvant annual revaccination;
US$599/50 doses
(US$11.98/dose)
DNA vaccine Whole FIV genome with 100 µg im. CTL responses to FIV Gag Lower viral load and [122]
(Hosie et al. in-frame deletion in pol and Env in the absence of protection for 4/10 vaccinated
1998) (FIVDRT) administered a serological response
with IFN-g
DNA vaccine FIV-pPPRDvif vif deletion 600 µg im. Increased FIV-specific T-cell Similar plasma virus loads in
(Gupta et al. mutant carrying a 375-bp proliferation, no antiviral vaccinated and control [123]
2007) deletion within the vif gene antibodies infected cats
+ IFN-g
DNA vaccine FIV with in-frame deletion 100 µg im. Slightly increased CTL 5/18 DIN and 2/12 DRT [124]
(Dunham in either DRT or DIN genes responses, no antiviral protected against challenge
et al. 2002) ± IL-12/18 antibodies with low-virulence FIV;
lower viral loads in vaccinated
cats infected with more
virulent FIV
Lymphocyte Glycoprotein that increases Weekly injections during Regulator of CD4 Unknown
T-cell leukocyte number and first month, every other lymphocytes and increases
immune IL-2 production week during second IL-2 production
modulator month, every 4–6 weeks
(Imulan) afterwards
CTL: Cytotoxic T lymphocyte; FIV: Feline immunodeficiency virus; im.: Intramuscular; DIN: Integrase; DRT: Reverse transcriptase.

West Nile virus all three resulting in 100% survival against a severe challenge
West Nile virus (WNV) disease has emerged as an important model of encephalomyelitis, whereby all controls exhibited clini-
disease in horses. The disease is caused by WNV, a flavivirus cal disease (fever, viremia, onset of grave neurological disease and
transmitted to animals by mosquitoes that have acquired the histopathologic lesions in the CNS) and 100% mortality [29] .
infection by feeding on viremic birds [27] . While the number of These vaccines are detailed in Table 7, alongside the DNA vaccine.
cases in the USA has decreased over the past few years, it still Commercial vaccines for WNV are available for varying prices.
represents a significant health threat. During an outbreak in 2002, The largest demand for WNV vaccines is most likely from pri-
over 15,000 cases of WNV disease in horses were reported, with vate horse owners, where efficacy of the vaccine is of the greatest
only a third of affected horses having recovered. In the states importance. Studies show that the DNA vaccine offers compa-
of Colorado and Nebraska alone, for example, 1478 cases of rable protection and should therefore be an acceptable choice
WNV were reported, with a mortality rate of 29%, which cost for horse owners. Another key attraction of a DNA vaccine for
the states US$600,660 and horse owners US$163,659 [201] . In a performance horses is the ability to differentiate vaccinated ani-
study following the outbreak, veterinarians estimated the costs to mals from infected animals [3] . High titers of certain diseases
treat affected horses: US$200 to treat animals with mild disease, such as WNV can prevent horses from being entered into shows;
US$400 for those with moderate disease and US$250 for equids an animal vaccinated with a DNA vaccine would not face this
with severe disease [201] . No effective prophylactic treatments are particular problem. Ease of transport and stability of the vaccine is
available for WNV; mosquito control has been the most common of further importance, as farms and ranches can require veterinar-
method of preventing contraction of the disease, although pro- ians to travel long distances with such vaccines. The cost of the
tective antibodies can control viremia upon infection. Successful DNA vaccine is comparable to the cost of conventional vaccines
vaccination requires the induction of both neutralizing antibodies against WNV. It will be interesting to follow the performance of
and cell-mediated immunity [28] . this first commercial DNA vaccine.
Three vaccines against WNV are currently commercially avail-
able, with a DNA vaccine recently developed by Fort Dodge that Opportunities for DNA vaccines for cancer in
received US Department of Agriculture (USDA) approval and was companion animals
released on the market in December 2008. A study by Seino et al. In addition to targeting pathogenic agents, DNA vaccines are also
in 2007 found that all three commercial vaccines were efficacious being used as cancer vaccines for companion animals. These vac-
in the prevention of WNV-induced encephalitis in horses, with cines incorporate plasmids encoding tumor antigens that induce

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 Review Redding & Weiner

the formation of antibodies that are expected to target tumor cells Canine oral melanoma
in the vaccinated animals, leading to the regression of tumors. One of the DNA vaccines that have been conditionally licensed
DNA vaccines are, overall, an exciting prospect in the field of by the USDA is canine melanoma vaccine (Merial Animal Health
companion animal oncology. Many examples of DNA vaccination Ltd, UK). Canine melanoma is the most common oral tumor in
against tumor antigens in animal models have been developed [30] . the dog but can also occur in the toes and footpads. The biologi-
Some of these antigens include: a human tyrosinase antigen for cal behavior of the tumor is closely related to site, size, stage and
canine melanoma (detailed in the following section); the a-folate histologic parameters. Site, for example, influences invasiveness
receptor for ovarian carcinoma [31] ; HER-2/neu associated with and metastatic properties: tumors in haired skin are not as malig-
breast cancer [32] ; the paraneoplastic encephalomyelitis antigen nant as tumors in the mucosa or oral cavity. Size of tumors is also
Hu D associated with small-cell lung cancer [33] ; tyrosine hydroxy- prognostic, as is the stage (I, II or III, and IV) [36] .
lase of neuroblastoma [34] ; and prostate-specific antigens for pros- Traditional treatment of canine melanoma involves either
tate cancer [35] . Many of these have resulted in significant tumor surgery (tumor excision), radiation therapy, chemotherapy or
protection or the reduction of tumor development and tumor size immuno­t herapy, all with fairly poor results. Tumor excision
in mice. Aside from the canine melanoma vaccine, no experi- either runs the risk of not removing the entire tumor or is not
ments have yet been conducted in companion animals. However, possible if the tumor has spread to lymph nodes and other areas.
since many of these cancers do affect dogs and cats, these vaccines Radiation therapy or radiation therapy with chemotherapy can
­represent an interesting possible application of DNA technology. also be used to achieve slightly better results, but treatment is

Table 5. Feline leukemia virus vaccines.

Vaccine Vector Dosage Price Vaccination Immune Protection Ref.
ages response
Leucogen p45 envelope antigen Two initial doses at US$99.99/ten 8 weeks Neutralizing 80% preventable [88]
(Virbac, with adjuvant 3–4-week intervals doses antibodies fraction against
France) (aluminium hydroxide injected im., annual (US$9.99/dose) within 9 weeks persistent viremia
gel, extract of Quillaja revaccination
saponaria) with single dose
Purevax® / Canarypox virus with Two initial doses of US$121.99/25 8 weeks Cytotoxic T-cell 78% preventable [89]
Eurifel env and gag genes and 0.25 ml 3–4 weeks doses response, very fraction for
(Merial part of the pol gene of apart sc., annual (US$4.88/ limited persistent viremia
Animal subgroup A (no revaccination with dose) neutralizing
Health Ltd, adjuvant) single dose antibody
UK) production
Leukocell 2 Inactivated, Two initial doses of US$50.70/ten 9 weeks Neutralizing Protects more [90]
(Pfizer, NY, adjuvanted, mixed 1 ml 3–4 weeks doses antibodies than 70% of
USA) subunits from apart sc., annual (US$5.07/dose) within 7 weeks artificially
FeLV strains A, B and revaccination with immunosuppressed
C, with sterile adjuvant single dose cats against
persistent viremia
Fel-O-Vax Inactivated whole virus Two initial doses of US$74.00/ten 9 weeks Neutralizing 44–100%
LvK (Fort of subgroups A and B 1 ml 3–4 weeks doses antibodies preventable
Dodge, KS, with adjuvant apart sc. or (US$7.40/dose) within fraction
USA) im., annual 11 weeks
revaccination with
single dose
Fevaxyn Inactivated whole virus Two initial doses of US$119.99/25 9 weeks Unknown 90.4–100%
(Schering- of subgroups A and B 1 ml 3–4 weeks doses preventable
Plough, NJ, with adjuvant apart sc., annual (US$4.80/ fraction
USA) revaccination with dose)
single dose
DNA vaccine Two plasmids 100 µg of each Unknown 13–15 weeks Unknown 100% protection [18]
(Hanlon expressing gag/pol and DNA construct against transient
et al. 2001) env gene with adjuvant injected im. and persistent
plasmids encoding viremia, 5/6 kittens
feline IL-12, IL-18 protected against
and/or IFN-g latent infection
FeLV: Feline leukemia virus; im.: Intramuscular; sc.: Subcutaneous.

1256 Expert Rev. Vaccines 8(9), (2009)

 DNA vaccines in veterinary use Review

Table 6. Commercial and DNA rabies vaccines.

Vaccine Vector Dosage Price Vaccination Protection Ref.
ages
Rabvac™ Killed virus One 1-ml dose sc. or im. US$74.99/50 3 months Dogs, cats and horses [20]
(Fort Dodge, with adjuvant Revaccinate 1 year later doses ($1.50/ Neutralizing antibodies in dogs
KS, USA) and every 3 years dose) within 2 months, 100% against
thereafter lethal challenge
Defensor 3® Chemically One 1-ml dose sc. US$21.95/ten 3 months Dogs, cats, sheep and cattle,
(Pfizer, NY, inactivated rabies Revaccinate 1 year later and doses (US$2.20/ but extends to raccoons
USA) virus with adjuvant every 3 years thereafter dose) and bats
Prorab® Killed virus One 1-ml dose sc. or im. US$13.85/ten 3 months Dogs, cats and sheep
(Intervet, The with adjuvant with annual revaccination doses (US$1.39/
Netherlands) dose)
Imrab® Inactivated One 1-ml dose sc. or im. US$102.97/50 3 months Cats, dogs, sheep, cattle,
(Merial rabies virus Revaccinate 2 year later doses horses and ferrets
Animal and every 3 years (US$2.06/dose) Virus-neutralizing antibodies
Health Ltd, thereafter
UK)
Raboral Nonpathogenic 2 ml of vaccine in fish US$1.30/ For adult wild Protects coyotes and raccoons
V-RG® virus with small meal or dog food bait vaccine-bait unit animals within 14 days
(Merial portion of viral
Animal RNA encoding
Health Ltd) G protein of the
rabies virus
DNA vaccine Plasmid encoding 100 µg of DNA im., Unknown 1–2 years Neutralizing antibodies in cats [91]
(Tesoro-Cruz glycoprotein of intranasally and within 15 days and in mice via
et al. 2008) rabies virus intradermally with passive transfer of cat sera/
booster 30 days later antibodies. 100% protection in
mice. Protection from lethal
challenge for cats (100% for
intradermal vaccination and
67% for im. route)
DNA vaccine Plasmid encoding 100-µg DNA Unknown 12–14 months Neutralizing antibodies within [20]
(Lodmell glycoprotein of intradermally in ear 2 months in dogs, persisting for
et al. 2006) rabies virus pinnae 6 months. 100% protection
against lethal challenge
im.: Intramuscular; sc.: Subcutaneous.

expensive and causes significant temporary discomfort to the Considering the difficult nature of malignant melanoma and
animal. Nonspecific immunotherapy is also being investigated, the limited effectiveness of available treatment options, the advent
with techniques such as allogeneic tumor cell vaccines, dendritic of a DNA vaccine is a welcome development. Unfortunately, the
cell vaccines and the use of immune adjuvants (interleukins and vaccine can only be used in more advanced stages of the cancer,
granulocyte–macrophage colony-stimulating factor). once surgical control of the tumor has been achieved. However,
DNA vaccination has proven, under certain circumstances, to the vaccine is easy to administer and less harsh for the patient
be safe and effective in combating melanoma. Dogs in stage II or than chemotherapy or radiation treatment, with fewer side effects.
III, where local tumor control has been achieved, respond well to While administration of the DNA vaccine itself may be inexpen-
this treatment. The DNA vaccine consists of a plasmid express- sive, additional costs associated with administering it, such as
ing xenogeneic human tyrosinase. Melanoma tumor cells overex- blood tests, radiographs, initial surgical treatment and vaccine
press tyrosine proteins. The human tyrosinase protein is different administration, add up to a cost similar to that of chemotherapy or
enough from the canine tyrosinase protein that it will stimulate radiation therapy. Likewise, administration of the vaccine requires
an immune response, yet similar enough to the canine tyrosinase four injections at 2-week intervals, which is a similar time require-
that the immune response is effective against canine melanoma ment to that of traditional treatment options. Therefore, in terms
cells that express tyrosinase. Table 8 details the cost and efficacies of cost to the client, the vaccine does not significantly reduce this
of current treatment options against those of the DNA vaccine at cost, but can reduce discomfort to the patient. The vaccine has
a veterinary teaching hospital, as well as the mean survival time been granted a conditional license. It will be interesting to follow
for various modalities. its performance on the market once it achieves full license.

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Opportunities for DNA vaccines in livestock Bovine respiratory disease complex

While DNA vaccines represent an interesting possibility for tar- The cattle industry is a large and growing industry both in terms
geting infectious disease and cancer in companion animals, their of dairy and beef cattle. Growing demand for beef, especially
potential use in livestock is even greater. Livestock production in developing countries, has led to increased and more intensi-
in the USA alone is significant in scale: the USA is the world’s fied production. Infectious diseases represent a serious threat,
largest beef producer, second largest beef export and second larg- especially in large-scale productions. One of the most important
est pork producer. Details of the livestock industry are listed diseases is the undifferentiated fever/bovine respiratory disease
in Table 9. complex (BRDC), representing a US$1 billion economic loss to
Most livestock operations incorporate large numbers of ani- the industry and a US$3 billion cost in preventative and cura-
mals, where herd immunity is of greater importance than the tive treatments [37] . BRDC can cause a large range of clinical
health of individual animals. Herd immunity involves confer- symptoms, including subclinical benign infection, fatal mucosal
ring protection to a large population of animals by vaccinating a disease, hemorrhagic disease, reproductive failure, congenital
significant percentage of the whole group. Herd immunity pre- abnormalities [38] , acute infections with immunosuppression,
vents the rapid spread and decreases the persistence of a disease. pyrexia, anorexia, depression, bronchitis, pneumonia and
This is especially important for diseases with longer incubation death [39] . BRDC can be caused by a number of pathogens,
periods, where infected animals cannot readily be identified such as bovine herpes virus, Mannheimia haemolytica, Pasteurella
and can transmit disease to other members of a herd. While a multocida, parainfluenza type-3 (PI3), and bovine respiratory
large number of commercial vaccines are available for the wide synctitial virus (BRSV). The economic cost of the disease is
span of livestock diseases, DNA vaccines possess an inherent significant: a study by Gunn et al. estimated costs of a disease,
advantage in that vaccinated animals can be distinguished with losses stemming from the abortion of calves to lost produc-
from infected animals, and since they have the potential to be tion to heifer death (Table 10) . In addition, vaccinated calves sell
less expensive to produce, they could be of value in large-scale for a higher price than unvaccinated calves. Therefore, livestock
animal operations. Furthermore, many commercial vaccines producers should have a vested interest in vaccinating calves
come in the form of cocktails, offering protection against several efficiently and effectively.
diseases in one vaccine. DNA vaccines are particularly able to A large number of commercial vaccines are available for each
provide such an option, as several plasmids encoding different infectious agent. For example, there are over 180 USDA-licensed
genes can be incorporated into a DNA vaccine. A number of bovine diarrhea virus (BVDV) vaccines [40] , all with varying
diseases of veterinary importance in livestock are detailed in the efficacies and for varying prices. For cattle, herd health is more
following sections. important than the health of individual cattle. Vaccines will be

Table 7. West Nile virus vaccines.

Vaccine Vector Dose Immunity Vaccination ages Price
West-Nile Formalin-inactivated Two doses at 3–6 week Within 2 weeks Nonvaccinated dam: US$138.30/ten
Innovator® whole virus with intervals, annual after second dose 3–4 months doses
(Fort Dodge, MetaStim adjuvant revaccination with a Vaccinated dam: (US$13.83/dose)
KS, USA) single dose 5–7 months
Recombitek Lyophilized Two im. doses at 4–6-week Within 28 days, Unknown US$169.99/ten
(Merial recombinant canarypox intervals, annual for 12 months doses
Animal vectored West Nile revaccination with a (US$16.99/dose)
Health Ltd, virus vaccine expressing single dose
UK) membrane and
envelope proteins,
plus a sterile liquid
diluent
PreveNile™ Lyophilized yellow fever Single im. dose Within 28 days, 4 months or older US$199.99/ten
(Intervet, The West Nile chimera virus for 12 months doses
Netherlands) vaccine expressing (US$19.99/dose)
membrane and
envelope proteins
without adjuvant
West-Nile Plasmid encoding PrM Two doses 3 weeks apart Within 28 days Foals 8–9 months old US$199.99/ten
Innovator® and E gene with were found to be doses
DNA (Fort adjuvant (SP oil, protected against viremia (US$19.99/dose)
Dodge) MetaStim)
im.: Intramuscular.

1258 Expert Rev. Vaccines 8(9), (2009)

 DNA vaccines in veterinary use Review

Table 8. Treatment options for canine oral melanoma.

Treatment Dosing/administration Cost/availability Results Ref.
Surgery Excision surgery and biopsy ~US$3000 MST of 17–18 months for stage I (<2 cm [95]
diameter tumor), 5–6 months for stage II
(2–4 cm diameter tumor) and 3 months for
stage III (>4 cm diameter tumor)
Radiation therapy Total dose of 24 Gy over three doses ~US$2000 53% complete remission, 30% [92,93]
36 Gy in varying doses partial remission, MST of 7 months
Chemotherapy Carboplatin administered at doses of US$3000–4000 28% overall response rate, 24% partial [94]
300 or 350 mg/m2 of body surface area (carboplatin is response rate, MST of 165 days
US$1.90–1.95/mg)
Merial DNA Administered after surgical excision Available to MST of 569 days (19 months) for [96]
vaccine (Merial of tumor board-certified stage II–III dogs
Animal Health Four 0.4-ml doses administered veterinary
Ltd, UK) transdermally biweekly. Booster dose oncologists
every 6 months thereafter US$500/dose
MST: Mean survival time.

important for disease prevention and disease control in the event DNA vaccine was able to reduce lung lesions and viral shedding
of an outbreak. The cost and longevity of vaccines will be as [42] , while the Taylor et al. DNA vaccine stimulated humoral
important as their efficiency. and cell-mediated immunity within 9 weeks of vaccination [41] .
Details of current vaccination options are compared with two
Bovine respiratory synctitial virus DNA vaccines in Table 11.
Bovine respiratory synctitial virus causes 60–70% of respira-
tory disease in cattle and mortality of BRSV can reach 20% Bovine viral diarrhea
in herd outbreaks [39] . Vaccination against BRSV is therefore Bovine viral diarrhea virus is a small, enveloped, single-stranded
essential for the industry and further represents a model for the RNA pestivirus of the Flaviviridae family. Relative antigenic
study of human respiratory synctitial virus (RSV). BRSV is an heterogeneity exists among the different strains, and isolates are
enveloped, single-negative strand RNA virus of the Pneumovirus divided into two biotypes – cytopathic, which cause changes in
genus and Paramyxoviridae family. A number of commercial host cells, and noncytopathic, which do not cause host cellular
vaccines are available for BRSV and are routinely administered changes. Both types can cause disease in cattle, but only the
to calves, including combinations of vaccines for BRSV and noncytopathic type causes persistently infected animals that con-
other disease agents. However, there are several problems with tinually shed the virus throughout their lives. The virus is also
these vaccines. First, peak incidence of BRSV occurs between subdivided into two different genotypes – type 1 and type 2.
2 and 7 months of age; the immaturity of the young calves’ Both cause disease, but type 2 is associated with high mortal-
immune system as well as the immunosuppressive effects of ity and acute, fulminating infections [40] . Despite the genetic
maternal antibodies found in the colostrum make vaccination heterogeneity, it has been suggested that subgenotypes tend to
difficult [41] . In addition, these vaccines generally do not induce be herd-specific and geographically clustered, which could poten-
long-term immunity and have been shown to exacerbate sub- tially reduce the need for expanded crossreactivity in vaccines.
sequent respiratory disease, probably due to the elicitation of Bovine virus diarrhea vaccine (BVDV) not only is a causative
a Th2 response upon vaccination [39] . Since most BRSV vac- agent of BRDC, but is also associated with enteritis in calves
cines are modified live viruses, they can also trigger abortion in and can cause acute hemorrhagic disease [43] . In general, vaccines
pregnant cows. DNA vaccines represent a promising approach developed for bovine diarrhea are modified live or inactivated
to the vaccination of cattle against BRSV. The Hamers et al. viruses. The former induces longer lasting and broader immune

Table 9. US livestock industry in 2007 according to the US Department of Agriculture.

Industry Amount produced Number of animals Economic value Ref.
Beef 30 million tons of meat 104.8 million US$89 billion [215,216]

Dairy 185.6 million tons of milk 9.25 million US$35.5 billion [215]

Poultry 40 million tons of meat 806 million broilers US$35.1 billion [203,215]
42.5 million layers
Pork 22 million tons of meat 62 million US$5.5 billion [203,215]

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Vaccination of cattle is controversial, as

Table 10. Costs of bovine respiratory disease complex.
it is generally not very effective: it does not
Event Cost eliminate or prevent infection of the cow,
Immunosuppression of calves US$6.36/calf/year and only reduces the development of clini-
Congenital defects, growth retardation, and so on US$61.5/calf/year
cal disease and shedding of the virus. It also
can induce the formation of granulomatous
Aborted calf US$711/cow/year lesions in the site of vaccination [48] . Two
Delayed breeding of cow due to infection US$148.5/cow/year commercial vaccines are available, only one
Infected heifer US$148.5/heifer/year of which is licensed in the USA (Mycopar
[Fort Dodge], available in the USA, and
Death of a cow or heifer US$1179
Silirum® [Pfizer, NY, USA], available in
responses (including T-cell-mediated responses since replication Australia). An ana­lysis by van Schaik et al. in 1996 determined
of the virus amplifies antigens, while the latter induces shorter and that there was a cost–benefit advantage for vaccinating, since
sometimes inadequate immunity but is safe to use). Two DNA vaccinating a cow cost US$15 and total return of vaccination
vaccines, described in Table 12, have been developed against BVDV, amounted to US$142 per cow due to reduction of clinical disease
including one that induces strong immunity when boosted with (although, interestingly enough, vaccination did not prevent loss
the relevant viral proteins [44] . of milk production) [49] . However, vaccination is not effective in
preventing an outbreak or spread of the disease. Furthermore, vac-
Johne’s disease cinated animals cannot be distinguished from infected animals,
Bacterial as well as viral diseases represent a serious threat to the which further complicates the detection of a disease that is already
livestock industry. One of particular importance is that of Johne’s difficult to detect. The development of a DNA vaccine that could
disease. Johne’s disease is an intestinal infection of ruminants, prevent disease, especially in very young animals, would be of
affecting cattle, goats and sheep, and is caused by Mycobacterium great interest. A number of experimental DNA vaccines have been
avium subsp. paratuberculosis, a bacillus closely related to the developed for cattle and sheep and are compared with commercial
causative agent of TB in both cattle and humans (Mycobacterium vaccines in Table 13.
tuberculosis). The USDA estimates that 22% of dairy operations DNA vaccines offer a very possible improvement upon cur-
in the USA are infected with M. paratuberculosis and have an rent vaccination and prevention of infection with Johne’s disease.
infection rate of at least 10% [202] . A study by Ott et al. in 1996 Commercial vaccines are at best effective in reducing but not pre-
estimated that the cost to the dairy industry amounted to approxi- venting clinical disease, and at worst can cause lesions in the site
mately US$200–250 million annually (US$22–27 per cow) of vaccination. Current methods of preventing infection include
owing to decreased milk production, higher cow-replacement removing calves from their dams at birth, which is stressful to
costs and lower cow-cull revenues [45] . Worldwide, the disease is the animals and can lead to the possibility of calves not getting
estimated to cost the industry over US$1.5 billion [46] . enough immunoglobulin-rich colostrum. Both DNA vaccines
Like M. tuberculosis, M. paratuberculosis is an extremely cited in Table 13, elicited strong immune responses, and seem to be
hardy bacterium, able to survive a wide range of temperatures able to prevent disease in light of the absence of lesions in tissues of
in water, soil and the air. It is excreted in the feces and milk certain animals after challenge. Unlike the commercial vaccines,
of infected cattle and generally infects calves at an early age. DNA vaccines would allow differentiation between vaccinated
However, the development of clinical disease, which includes and infected animals, therefore limiting the likelihood of an out-
chronic diarrhea, weight loss, intestinal lesions and reduced milk break if the detection of infected animals did not work. DNA
production, usually does not occur until 2 years of age, which vaccines would also prevent the problem of lesions at the site of
makes detection difficult. Further complicating detection efforts infection, as well as transmission of bacteria to calves via lactation.
is the fact that current detection methods detect less than 50% of
infected animals, requiring multiple testing, which can become Foot-and-mouth disease
expensive [47] . The foot-and-mouth disease (FMD) virus is an apthovirus of the
Treatment for Johne’s disease is expensive, both in terms of cost Picornaviridae family, which causes FMD, a highly contagious
of drugs and forfeited income, due to drug traces in milk and and devastating disease of cloven-hoofed animals, such as cattle,
meat. Treatment also only treats clinical signs, and is therefore sheep, goats, buffalo and pigs. The disease is characterized by fever,
not a valid option for herd treatment. Currently, avoiding infec- lameness, vesicular lesions of the mouth, tongue and feet, and acute
tion of calves is the most practiced way of controlling disease myocarditis in young animals. The virus can be excreted in lesions,
– calves are birthed in as clean, manure-free environments as saliva, exhaled air, milk, urine, feces, and semen, leading to rapid
possible and removed immediately after birth from their dams. and easy transmission between animals [203] . FMD represents a seri-
Some cattle practices use milk replacers instead of allowing calves ous economic and public-health problem: outbreak of the disease
to milk from their dams, and obtain essential colostrums from can decimate a nation’s livestock industry, such as happened in
Johne’s-free cows. Culling and isolation of infected animals is Taiwan in 1997, where 4 million animals were culled, costing the
also widely practiced. country US$5 billion. In addition, the presence of FMD, especially

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 DNA vaccines in veterinary use Review

Table 11. Vaccines against bovine respiratory synctitial virus.

Vaccine Vector Dosage Price Vaccination ages Immunity Ref.
Bovi-Shield MLV for BRSV, IBR, PI3 Two 2-ml im. US$25.99/50 doses No age restriction, Serum antibodies within [97,98]
BRSV (Pfizer, and BVD doses (US$0.52/dose) but calves vaccinated 2 weeks and neutralizing
NY, USA) 3–4 weeks apart before 6 months antibodies within
with annual should be 5 weeks
revaccination revaccinated after
with single dose 6 months of age
Cattlemaster® MLV BRSV, chemically Two 5-ml sc. US$59.99/25 doses No age restriction, Serum antibodies in 89% [99]
Gold™ 5LP5 altered strains of IBR doses 3 weeks (US$2.40/dose) but calves vaccinated of calves within 2 weeks
(Pfizer) and PI3 and inactivated apart with before 6 months
BVD with aluminum annual should be
hydroxide adjuvant revaccination revaccinated after
with single dose 6 months of age
Jencine 4 MLV for BRSV, One 2-ml im. or US$46.96/50 2 weeks old; calves Unknown
(Schering- noncytopathic BVD sc. dose doses (US$0.94/ vaccinated before
Plough, NJ, virus with annual dose) 6 months of age
USA) revaccination should be
revaccinated after
6 months of age
Pyramid 4 MLV for BRSV, IBR, PI3 One 2-ml dose US$77.99/50 doses No age restriction, Unknown
(Fort Dodge, and BVD with sc. with annual (US$1.56/dose) but calves vaccinated
KS, USA) MetaStim adjuvant revaccination before 6 months of
age should be
revaccinated after
6 months of age
Vira Shield Inactivated BRSV, IBR, Two 5-ml doses US$62.90/50 No age restriction, Cell-mediated and
(Novartis, UK) BVD, PI3 viruses with sc. 4–5 weeks doses (US$1.26/ but calves vaccinated humoral immunity
Xtend® SP adjuvant apart with dose) before 6 months of
annual age should be
revaccination revaccinated after
6 months of age
DNA vaccine Two plasmids 2 ml of DNA Unknown 3–6 weeks (plus No antibodies, reduced [41]
BRSV expressing the vaccine im. maternal antibodies) lung lesions, reduced
(Hamers et al. BRSV F and N antigens 4 weeks apart or virus shedding correlated
2007) single dose with IFN-g producing
followed by T-cell response
vaccination with
inactivated virus
DNA vaccine Plasmid expressing Two doses of Unknown 2 weeks (no Antibodies within [41]
BRSV (Taylor F gene of the Snook 0.25-mg DNA maternal antibodies) 9 weeks and BRSV-
et al. 2005) strain of BRSV im., specific lymphocyte
intradermally proliferative responses
and within 2–4 weeks
intratracheally No virus found in lungs
5 weeks apart of 9/11 challenged calves
BRSV: Bovine respiratory synctitial virus; BVD: Bovine virus diarrhea; IBR: Infectious bovine rhinotracheitis; Im.: Intramuscular; MVL: Modified live virus;
PI3: Parainfluenza type-3; sc.: Subcutaneous.

in developing countries, can greatly affect the export market of cat- banks exist that provide different strains of the virus that can be
tle to FMD-free countries too. Vaccination campaigns in Western used for outbreak scenarios (Table 14) . Prophylactic vaccination is
Europe, parts of South America, North America, New Zealand performed in countries where FMD is enzoonotic (especially in
and Australia have rendered these areas FMD-free. However, these Southern Africa and South America); however, there are significant
campaigns are expensive and certain outbreaks have been linked to problems associated with vaccine production and development.
the presence of residual live virus in chemically inactivated vaccines No universal prophylactic vaccine currently exists for FMD.
[50] . Prophylactic vaccination is now prohibited in the EU and the Seven very different types of FMDV are believed to exist and
USA, and a number of other FMD-free countries; however, antigen there is no cross-protection between serotypes. Another difficulty

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Table 12. Vaccines against bovine viral diarrhea disease.

Vaccine Vector Dosage Price Vaccination Immunity Contraindication Ref.
ages
Bovilis Inactivated BVD virus Two 2-ml doses im. US$21/25 8 months Reduced viral Fetal protection [102]
(Intervet, The 4 weeks apart with doses excretion and can be achieved if
Netherlands) annual revaccination (US$0.84/ neutralizing administered
with single dose dose) antibodies 4 weeks before
start of gestation
Should not be
administered
within 21 days
to slaughter
Breed-back™ BVD Types 1 and 2, Two 2-ml doses sc. US$9.75/five No age 91–100% Should not be [103]
FP 10 MLV, IBR, PI3 and 14–28 days apart doses restriction, protection from vaccinated within
(Boehringer- BRSV, MLV with with annual (US$1.95/ but calves fetal infection, 21 days before
Ingelheim, adjuvant revaccination with dose) vaccinated 14/22 calves free slaughter
UK) single dose before of clinical signs Should not use in
6 months of pregnant cows or
age should be in calves nursing
revaccinated pregnant cows
after
6 months
of age
DNA vaccine Plasmid encoding a 1 mg of DNA Unknown 8–9 months Virus-neutralizing Unknown [100]
(Liang et al. truncated secreted delivered antibodies and
2008) version of E2 with a transdermally via INF-g-producing
tissue plasminogen needle-free CD4 + T cells,
activator signal injection, followed reducing
sequence, followed by 50 µg of protein leucopenia, no
by boosting with weight loss or
E2 protein formulated temperature
with 10% Emulsigen, response
a mineral oil in
water emulsion,
and CpG
oligodeoxynucleotide
DNA vaccine Plasmid expressing E2 500 µg DNA Unknown 3–7 months Low levels of Unknown [101]
(Nobiron glycoprotein of BVDV intradermally neutralizing
et al. 2003) antibodies,
elevated T-cell
proliferation,
reduced febrile
responses
BRSV: Bovine respiratory synctitial virus; BVD: Bovine virus diarrhea; BVDV: Bovine virus diarrhea vaccine; IBR: Infectious bovine rhinotracheitis; im.: Intramuscular;
MLV: Modified live virus; PI3: Parainfluenza type-3; sc.: Subcutaneous.

associated with the development of a vaccine for FMD is that the DNA vaccination, while still not perfected, represents a poten-
virus is able to persist in cattle and small ruminants, irrespective tial solution to FMD. Countries that are FMD-free generally do
of vaccination status [51] . Given this and the fact that the virus not want to prophylactically vaccinate against FMD, as not only
can persist outside of a host for more than a month and is easily are vaccines costly and induce short-lived protection, but they
transmitted, vaccination becomes even more difficult. could also cause an outbreak. A DNA vaccine could address both
Vaccine banks generally either hold reserves of fully formulated problems – it would be cheaper than conventional vaccines and
and tested vaccines that can be used immediately but have a short would eliminate the need to introduce the virus itself into a dis-
shelf life, or reserves of antigens, which have longer shelf-lives but ease-free location. In addition, DNA vaccination allows discrimi-
need to be formulated into a vaccine before it can be shipped out nation between vaccinated and infected animals, which would
for use. Characteristics of the three principal vaccine banks are circumvent the problem of the export of meat from countries with
listed in Table 15, as are comparisons of commercial vaccines with FMD. In addition, DNA vaccines elicit both humoral and cellular
DNA vaccines. immunity, which are both essential for control of the disease.

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 DNA vaccines in veterinary use Review

Table 13. Vaccines against Johne’s disease.

Vaccine Vector Species Dosage Vaccination Immunity Ref.
ages
Mycopar® (Fort Whole-cell bacterin Cattle, Cattle: Calves: less than Cattle: ~90% effective in [106,107]
Dodge, KS, containing inactivated sheep 0.5 ml sc. 35 days eliminating clinical disease
USA) Mycobacterium Sheep: Sheep: 3 months Sheep: specific humoral and
paratuberculosis bacteria 1 ml sc. cellular responses elicited
suspended in oil
Silirum® (Pfizer, Killed bacteria in mineral Cattle, One dose sc. Calves: 14 days Cattle: no antibodies. Reduced [108,109]
NY, USA) oil adjuvant deer for cattle and Deer: 5 months colonization of tissues by
deer pathogen
Deer: reduced severity of disease
DNA vaccine Four rAgs (85A, 85B, 85C Cattle 100 µg of 5–10 days Antibodies within 3 weeks; [104]
(Kathaperumal and superoxide dismutase) each antigen significant IFN-g production
et al. 2008) with two adjuvants and 100 µg within 11 weeks
(monophosphoryl lipid A of IL-12 im. Significant increases in CD4 + and
and bovine IL-12) CD8 + T cells against all four rAgs;
rAg-specific expression of IL-2,
IL-12 and TNF-α. 4/8 animals did
not show bacteria in tissue
DNA vaccine Three rAgs (Mycobacterium Sheep Three doses 5 months Increased IFN-g and IL-10 [105]
(Sechi et al. avium 85A, BCG 85A of 1 mg of expression, increased CD4 + T cells
2005) and 65K) each antigen Absence of lesions and bacteria
im. 20 days in tissues
apart
Im.: Intramuscular; rAg: Recombinant antigen; sc.: Subcutaneous.

DNA vaccines effectively adjuvanted – such as with interleukins or Porcine reproductive & respiratory syndrome virus,
complement components [52] – could constitute powerful vaccines. & swine influenza
More work needs to be completed on these vaccines, especially in The worldwide pork industry is a fast-growing industry, with
a more diverse range of species; most research has been carried intensive animal husbandry practices. According to the USDA,
out in swine [53–56] . Attention should also be paid to cattle, goats the world production of pork in 2008 was forecast to reach
and sheep, which are also affected by this disease. 97 million tons of pork [204] . Porcine reproductive and respiratory

Table 14. Vaccine banks for foot-and-mouth disease.

Vaccine bank Total vaccine stock Provenance of Antigen subtypes Manufacturing time
vaccines
International Antigens equivalent to Manufactured A: 1,500,000 Capability of formulating, filling and
Vaccine Bank 3.5 million doses of on site O: 1,000,000 dispatching up to 500,000 doses of
(Pirbright, UK) finished vaccine of each C: 500,000 vaccine within 3 days
subtype Asia1: 500,000
European Union Antigen equivalent to Purchased from O1 Tur178: 5,000,590 5 days to release inactivated antigen.
Vaccine Bank 5 million doses of vaccine European C1 Europe: 2,500,000 Manufacture of vaccines from raw
(Pirbright, UK; of each subtype manufacturers Asia1: 2,500,000 materials takes aproximately 10 weeks
Lyon, France; O1 BFS: 5,000,692
di Brescia, Italy) A24 Cruzeiro: 5,000,874
A22 Iraq: 3,887,124
North American 38,417,720 antigens Purchased from O: 10,778,718 Can obtain hundreds of thousands of
Vaccine Bank available manufacturers A : 13,599,002 doses of FMD vaccine within days
(Plum Island, C : 6,800,000
USA) Asia1: 5,240,000
SAT1: 1,000,000
SAT2: 1,000,000
FMD: Foot-and-mouth disease; SAT: Southern African Territory.
Data taken from [113].

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Table 15. Vaccines against foot-and-mouth disease.

Vaccine Vector Dosage Price
Decivac FMD DOE
®
Antigens of chemically inactivated FMDV Cattle, buffalo, pigs: 2 ml US$0.05/cow
(Intervet, The types O, A, C, Asia1 and SAT1, SAT2, SAT3, Sheep, goats: 1 ml US$0.01/sheep
Netherlands) with DOE. Combinations of antigens depend sc. or im. or goat
on local situation
Aftopor® (Merial Purified, inactiaved FMDV types O, A, C, 3–15 µg of antigen/strain for pigs and US$2/dose for
Animal Health Ltd, Asia1, SAT1, SAT2, SAT3 in a DOE adjuvant. ruminants trivalent vaccine
UK) Combinations of antigens depend on local Large ruminants and pigs: 2 ml
situation Small ruminants: 1 ml
im.
Bayovac (Bayer, UK) Trivalent inactivated virus of O, A and C Cattle and buffalo: 5 ml Unknown
strains in oil emulsion Sheep and goats: 3 ml im. or sc.. Revaccinate
90 days after and thereafter every 6 months
DNA vaccine for 1. Plasmid encoding all viral structural 5 µg of DNA with 25 mg gold, by gene gun Unknown
swine (Benvenisti proteins with viral protease of strain O1(G) and sc.
et al. 2001) (pKVI335). Booster inoculation after 3 weeks
2. Construct with viral P1, 2A and 3CD
sequences (pKVI326)
DNA vaccines for 1. Plasmid encoding entire FMDV genome Two injections of 3 µg of DNA bound to Unknown
swine (Beard et al. with mutation at cell-binding site (pWRMHX) 1.5 mg of gold 4 weeks apart, via im.,
1999) 2. Plasmid encoding viral capsid gene P1 and intradermal and gene gun inoculations
processing proteinase 3C (iP12X3C)
DNA vaccine for Plasmid expressing two FMDV VP1 epitopes Two injections of 200 µg of DNA im. Unknown
swine (Wong et al. (pCEIS) coadministered with IL-2 plasmid 4 weeks apart
2002)
DNA vaccine 1. Plasmid encoding VP1 and a 3D Three injections of 300 µg of DNA Unknown
for swine polymerase (pcDNA3.1/3D15) from type O 3–4 weeks apart im. and intradermally
(Cedillo-Barron FMDV
et al. 2001) 2. Plasmid encoding VP1 and NS2B
(pcDNA3.1/2B15) from type O FMDV
DNA vaccine Plasmid encoding FMDVP1 of O1 strain with Three injections of 400 µg of DNA coated on Unknown
against sheep ovine GM-CSF with adjuvant PLG PLG 3 weeks apart intradermally and im.
(Niborski et al. microparticles
2006)
DOE: Double-oil emulsion; FMD: Foot-and-mouth disease; FMDV: Foot-and-mouth disease virus; Im.: Intramuscular; PLG: Poly ( d,l-lactide-coglycolide);
SAT: Southern African Territory; sc.: Subcutaneous.

syndrome is a disease that has recently emerged as one of the most many infections are subclinical, thus rendering detection of the
important pathogens in countries with intensive swine industries. disease unreliable [61] . A number of commercial vaccinations
The disease is characterized by reproductive disorders in pregnant are available for PRRSV with varying efficacy, which generally
sows, perinatal losses and respiratory distress in piglets, causing reduce clinical disease rather than preventing infection [62] . A
the industry an estimated US$66.75 million annually in breed- number of DNA vaccines have also been tested that achieve vary-
ing herds and US$493.57 million in growing pig populations ing results (Table 16) . DNA vaccines would be particularly valuable
[57] . The causative agent of porcine reproductive and respiratory for this disease if a cocktail vaccine against different strains of
syndrome (PRRS) is a virus of the Arteriviridae family of the the virus could be achieved without the possibility of causing
Nidovirales order (porcine reproductive and respiratory syndrome an outbreak.
virus [PRRSV]), which shows high sequence variation between Given the relative inefficacy of commercial vaccines and the
European and North American isolates [58] . contraindications of some of these, there is much room for
The disease is particularly difficult to treat and detect. improvement in finding a solution to PRRSV. DNA vaccine
Following infection, the induction of neutralizing antibodies is development for PRRSV is still only in the preliminary stages,
slow, which enables the virus to persist in the host and propagate but offers an interesting alternative to commercial vaccines. In
to other animals through contaminated mucus, saliva, excrement, particular, the fact that certain commercial vaccines cannot be
semen and possibly via the airborne route [59,60] . Heterogeneity used for prophylactic purposes provides a potential DNA vaccine
of isolates is a complicating factor for achieving protection via with a distinct advantage, as it would be effective for prophylactic
vaccination, as is the fact that in the endemic phase of the disease, use. Indeed, in Denmark, for example, a national eradication

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 DNA vaccines in veterinary use Review

Table 15. Vaccines against foot-and-mouth disease (cont.).

Vaccination ages Protection Ref.
Young animals without maternal antibodies: 2 weeks old, Adult animals: every 6 months
revaccination 4–6 months later Immunity within 10 days lasting for 6 months in cattle,
Young animals with maternal antibodies: 4 months of age, buffalo, sheep and goats; 4 months in pigs
revaccination 4–6 months later
Ruminants: starting at 2 weeks of age followed by second Antibodies that reduce clinical signs and mortality following [54]
vaccination 4 weeks later, with booster every 6 months exposure to FMDV in 92% of animals within 16 weeks
Pigs: starting at 8 weeks of age followed by second vaccination
4 weeks later, with booster every 4 weeks

Until 4 months of age Confers immunity within 21 days, lasting 6 months

3 weeks old (8.9–13.3 kg) Vaccination with pKVI326 induced partial protection but no [111]
detectable neutralizing antibodies
Vaccination with pKVI335 provided no protection

Unknown Low levels of neutralizing antibody within 4 weeks [50]

Pigs vaccinated with pWRMHX were protected from
challenge, those vaccinated with piP12X3C were not

30–40 kg 10–20-fold T-cell proliferation, low levels of neutralizing [111]

antibodies, 100% protection from challenge

20–25 kg B-cell, T-cell and antibody response to nonstructural [112]

proteins 2B and 3D, but no immunity induced to FMDV15
peptide, no protection against challenge

6–12 months Transient T-cell response, some humoral immunity [113]

Protection against FMD symptoms, inhibition of viral
replication

DOE: Double-oil emulsion; FMD: Foot-and-mouth disease; FMDV: Foot-and-mouth disease virus; Im.: Intramuscular; PLG: Poly ( d,l-lactide-coglycolide);
SAT: Southern African Territory; sc.: Subcutaneous.

vaccination program was unsuccessful, as the vaccine virus was Laddy et al. [64,65] and Chen et al. [66] , using consensus-based
found to be transmitted between boars for a period of 8 weeks and immunogens, appear particularly promising in the H5 system
was introduced to herds via semen [63] . Similarly, a DNA vaccine (see the section on avian influenza later). Furthermore, the use of
would be more advantageous in achieving herd immunity, as it electroporation to improve DNA delivery in pig populations has
would be able to be administered to all animals, including boars already been licensed for clinical application. Such combination
and pregnant sows. Further research must be undertaken to study DNA vaccine approaches may be attractive for the prevention of
the immune mechanisms of these DNA vaccines and of the virus swine influenza in commercial farming. As this DNA approach
itself in order to achieve a viable vaccination option. is a non-live, nonspreading, nonreplicating delivery platform,
The recent human outbreak of H1N1 swine flu, which was first concerns over attenuation and inadvertent spread are avoided.
reported in 2009 in human populations in Mexico, has caused Furthermore, vaccination can be easily distinguished from true
great alarm. This hemagglutinin has not been circulating rou- infection, thus allowing for protection of herds in case of exposure
tinely for close to half a century. As such, human immune resist- to this pathogen.
ance to this strain is currently low, so the infections of pigs with
influenza as a component of translation of infection to human Opportunities for DNA vaccines in birds:
populations has become of increased importance, and suggests avian influenza
that a renewed focus on such vaccination for porcine populations Avian zoonoses have only fairly recently emerged as a public
is in order. The variability of H1N1 is particularly amenable health issue, yet avian diseases have been a prevalent and con-
for approach by DNA vaccine technology. Recently, studies by stant threat to birds of all species, from poultry to zoo birds to

www.expert-reviews.com 1265
 Review Redding & Weiner

Table 16. Vaccines against porcine reproductive and respiratory syndrome.

Vaccine Vector Dose Vaccination ages Price
Ingelvac RespPRRS/Repro™ Modified live virus 2 ml im. 3 weeks and US$57.99/50
(Boehringer-Ingelheim, UK) nonpregnant females doses
(US$1.16/dose)
Suvaxyn® PRSS (Fort Dodge, European-type modified 2 ml im. 4 weeks Unknown
KS, USA) live virus
Progressis (Merial Animal Inactivated virus with oil Two 2-ml doses im. at 3–4-week 2–3 weeks Unknown
Health Ltd, UK) excipient intervals with revaccination
within 60–70 days
Porcilis® PRRS Attenuated live European Two 2-ml doses im. 2 weeks US$16.64/25
(Schering-Plough, NJ, USA) strain with Diluvac Forte doses
adjuvant (US$0.66/dose)

DNA vaccine (Hou et al. Plasmid coexpressing GP5 Three 500-µg doses im. at 30 days Unknown
2008) gene of PRRSV and swine 3-week intervals
ubiquitin

DNA vaccine Plasmid coexpressing Four 500-µg doses im. at 30 days Unknown
(Xue et al. 2004) PRRSV ORF5, ORF7 genes 2-week intervals
and porcine IL-2 or IFN-g

DNA vaccine (Jiang et al. Plasmid coexpressing GP5 Two doses of 100 µg im. 3 weeks Unknown
2006) and M gene of PRRSV 4 weeks apart
PRRSV: Porcine reproductive and respiratory syndrome virus; im.: Intramuscular; ORF: Open-reading frame.

wild birds. Birds represent a significant part of agriculture and six human deaths [69] . Several subtypes of AIV exist, classified by
the economy: approximately 850 million poultry birds contribute the antigenicity of the surface proteins hemagglutinin (H1–16)
US$35.1 billion to the US economy annually, while 11 million and neuraminidase (N1–9). Certain subtypes, when introduced
pet birds constitute the third most popular companion pet after into a host, can become highly pathogenic, thus switching from
dogs and cats [6] . Poultry in large-scale operations are routinely low-pathogenic avian influenza (LPAI) to highly pathogenic avian
vaccinated against a number of diseases: day-old chicks are vac- influenza (HPAI). Wild waterfowl are considered carriers of LPAI
cinated against Marek’s disease, and can be vaccinated against viruses. HPAI viruses not only cause up to 100% mortality in a
Newcastle disease and infectious bronchitis. Older birds can be wide range of avian species, but can also infect mammalian spe-
vaccinated against fowl pox, fowl cholera and avian encephalo- cies [70] . Symptoms of HPAI in poultry range from cessation of
myelitis. Poultry in smaller scale operations and in developing egg laying, to loss of appetite and depression, to sudden death.
countries, however, are not vaccinated as often, as vaccines can be The virus is shed in saliva, nasal secretions and feces.
expensive and are usually produced in large-dose vials intended The vaccination of poultry and zoo birds has been undertaken
for commercial use. In such operations, culling is the most fre- in a number of countries, subject to strict risk assessment and
quent method of disease control. Vaccination of wild birds is, for surveillance requirements by national authorities. The EU, for
obvious reasons, impractical and frequently impossible. Currently example, which initially had a nonvaccination policy, subse-
available avian vaccines are not always effective and some only quently passed Avian Influenza Directive 2005/94/EC, which
treat symptoms rather than preventing disease [67,68] . DNA vac- allowed for both the emergency and preventative vaccination of
cines therefore have a potential application in avian disease. One zoo birds and poultry. In Hong Kong, the USDA developed an
such disease, which represents a grave threat to avian and human AI Hong Kong H5N1 Response Plan that allowed vaccination to
health, is avian influenza. be used in eradication program for HPAI [71] . China has recently
Avian influenza virus (AIV) is an orthomoxyvirus that has announced plans to vaccinate all 4 billion of its chickens [72] .
garnered a great deal of attention in the past few years. Its viru- Concomitantly, a number of differentiating infected from vac-
lence and its potential to spread to humans have generated fears cinated animal (DIVA) techniques have been introduced, with
of pandemics among humans and panzootics among birds. varying advantages and disadvantages [73] .
Outbreaks of avian influenza, in particular of the H5N1 strain, Vaccination has several advantages, including reducing the
have occurred in a number of countries, such as in Hong Kong risk of birds becoming infected, reducing mortality and reduc-
in 1997, 2001 and 2002, resulting in the slaughter of over 3 mil- ing shedding of the virus in the event of an outbreak, which can
lion chickens, a loss of HK$200 million (~US$25 million) and help prevent spread of the disease. Several commercial vaccines

1266 Expert Rev. Vaccines 8(9), (2009)

 DNA vaccines in veterinary use Review

Table 16. Vaccines against porcine reproductive and respiratory syndrome (cont.).
Immunity Contraindication Ref.
Vaccination of infected animals: reduced duration of viral shedding, but no Boars should not be vaccinated. Not [116]
reduction of viral load in tissues recommended for use in naive herds.
no change in proportion of persistently infected pigs Duration of protection is ~4 months
Reduced preweaning mortality of piglets but no prevention of clinical signs or None [117]
viremia
Induction of PRRSV-specific antibodies None
Reduction of the reproductive disorders and of the number of early farrowings
still-births
Reduced morbidity in fattening pigs, improvement of reproductive performance Pregnant sows should only be
and reduced transplacental virus transmission in breeding pigs vaccinatedafter previous exposure to
European
PRRSV
Enhanced T-cell proliferation Unknown [59]
No neutralizing antibodies
Lower viral replication and distribution in tissues
Lack of lesions in 4/6 vaccinated pigs.
Reduced viral replication Unknown [115]
Absence of lesions in 2/3 pigs vaccinated with ORF5/IFN-g, 1/3 vaccinated with
ORF5/IL-2 and 1/3 vaccinated with ORF7/IL-2

Neutralizing antibodies within 10 weeks. Unknown [116]

Enhanced splenocyte proliferation activity
PRRSV: Porcine reproductive and respiratory syndrome virus; im.: Intramuscular; ORF: Open-reading frame.

are available, including inactivated vaccines, recombinant fowl injections. DNA vaccines would also have a ‘built-in’ DIVA, which
pox H5 vector, combination vaccines of different AI strains and would require fewer steps in the vaccine program. The safety of
AI with Newcastle disease [74] . A number of DNA vaccines are DNA vaccines would prevent the possibility of an outbreak result-
also being developed for both poultry and humans. Table 17 exam- ing from vaccination, and perhaps most promising of all is the fact
ines both commercial and experimental DNA vaccines for use that DNA vaccines can elicit both humoral and cellular immune
in poultry. responses. Protective antibodies must be matched to the strain of
Vaccination of fowl for AIV, while permitted in many coun- AI, whereas cellular immunity can achieve greater cross-protection
tries, is still not always encouraged. The Avian Influenza Directive – a particularly important prospect given the number of strains of
2005/94/EC, for example, limits preventative vaccination pro- virus and the ability of the virus to mutate [76] . The DNA vaccines
grams to programs authorized only for specific birds in specified by Laddy et al. [64] or Chen et al. [66] , for example, are consensus-
regions, and are subjected to rigorous surveillance and control based vaccines that provide protection against multiple strains of
requirements. For example, in 2006 France vaccinated 900,000 AI. Furthermore, the use of microelectrodes and electorporation
ducks and geese, while Germany vaccinated three commercial as shown by Laddy et al. to induce strong HAI and broad micro­
poultry holdings of ducks, geese and layer chickens. However, neutralization titers in several species of animals, including non­
the standard practices of keeping poultry separated from wild human primates [76] , using consensus DNA immunogens appear
birds and, in the event of an outbreak, the culling of birds, are to be a highly attractive option for avian influenza, as well as
much preferred. Concerns about outbreaks due to vaccines are perhaps for swine flu as previously discussed.
not unfounded, and DIVA is not always highly effective, which
can have an impact on the import/export of poultry markets. Opportunities for DNA vaccines in fish: infectious
Vaccination can also mask the occurrence of disease in a farm, hematopoietic necrosis
delaying its detection. Furthermore, the virus has a tendency Another target for DNA vaccines today is fish. As the oceans
to mutate, which may render commercial vaccines ineffective. become depleted of fish due to overfishing and pollution, intensive
Vaccination is also difficult and expensive, as commercial vaccines farming of fish is becoming more widespread and economically
must generally be administered one-to-three times. Vaccination important. The scale of these fisheries is expanding: for exam-
trials of 400–600 commercial farms in Italy cost €2–4 million a ple, in the 1970s, there were 70,000 acres of catfish farm ponds
year, with the vaccine costing €0.10–0.15 per bird [205] . DNA vac- in the USA, with approximately 20,000 fish per acre. In 1996,
cines could most likely provide a less expensive option, and, in the US catfisheries occupied over 90,000 acres, producing a total of
case of vaccines such as that of Jiang et al. [75] , would require fewer 270,000 tons of fish annually [206] . The Alaskan salmon market

www.expert-reviews.com 1267
 Table 17. Commercial and DNA vaccines for avian influenza.

1268
Vaccine Species Vaccine vector Dosage Vaccination age Protection Ref.
Poulvac FluFend (Fort Chickens, ducks Inactivated AIV, H5N9 Chickens: two doses of 0.5 ml im. Chickens: from 2 weeks Protection against clinical signs and
Review

Dodge, KS, USA; and turkeys strain A/CK/ltaly/22A/ within 14 days Ducks: from 1 day mortality and reduced viral excretion
product label) H5N9/1998 with oil Ducks: two doses of 0.2 ml sc. Turkeys: from 8 days within 3 weeks
emulsion adjuvant within 3 weeks Significant antibody titers persist in
Turkeys: three doses of 0.5 ml sc. at chickens for at least 22 weeks
21-day intervals

Nobilis Influenza H5 Poultry and ducks Inactivated AIV for Chicken: two doses of 0.5 ml within Chickens: from 1 day Complete protection in chickens and [67]
(Intervet; product various subtypes with 4–6 weeks im. or sc. ducks against virus transmission
brochure, summary of oil emulsion adjuvant Ducks: 1-ml dose sc. from infected birds and
Redding & Weiner

product trials) clinical disease

Trovac AI H5 (Merial; Chickens, Live recombinant Chickens: 0.2 ml sc. Chickens: from 1 day Protection within 1 week, lasting [68]
product label and potentially fowlpox recombinant 20 weeks, significantly decreased
Bublot et al. 2006) mammals expressing the HA virus shedding
(immunogenic in gene of AI H5 subtype
cats) isolate

DNA vaccine (Laddy Mice, ferrets and Set of synthetic Mice: three doses of 25 µg 2 weeks Mice: 6–8 weeks Macaques: unknown [64]
et al. 2008) macaques consensus DNA apart im. with electroporation Ferrets: 4–6 months cellular and humoral immune
constructs encoding Ferrets: three doses of 200 µg responses, protection from
AIV A antigens H5HA, 1 month apart im. with morbidity and mortality, and
H1NA, NP electroporation reduction of viral shedding in mice
Macaque: two doses of 1 mg and ferrets
1 month apart im.
with electroporation
DNA vaccine (Kodihalli Chickens Plasmids encoding HA Two 10-µg doses 3 weeks apart im. 3 weeks HA plasmids: protection of 86% of [119]
et al. 2000) and NP genes chickens
transfixed to gold NP plasmids: protection of 42% of
particles, delivered by chickens
gene gun

DNA vaccine (Jiang Chickens Plasmids expressing One 100-µg dose im. 3 weeks Antibodies within 1 week, complete [75]
et al. 2007) optiHA and wild-type protection against challenge
HA genes
DNA vaccine Chickens Prime–boost: plasmid Two 100-µg doses 3 weeks apart 4 weeks Significant decrease in cloacal and [120]
(Le Gall-Reculé et al. encoding H7 and M1 tracheal shedding of virus
2007) viral proteins from an
Italian H7N1 LPAI virus
AIV: Avian influenza virus; HA: Hemagglutinin; im.: Intramuscular; LPAI: Low pathogenic avian influenza; NP: Nuclear protein; sc.: Subcutaneous.

Expert Rev. Vaccines 8(9), (2009)

 DNA vaccines in veterinary use Review

produces close to US$900 million worth of fish annually [207] . in the order of US$200 million Canadian dollars (~US$198 mil-
Fish are raised for food and sport, and many fisheries are also lion) [208] . A DNA vaccine against IHN virus for Atlantic farmed
involved in conservation efforts. salmon was developed by an affiliate of Novartis (Aqua Health
Owing to intensive fishing practices, fish are particularly vulner- Ltd, PE, USA), with permission from appropriate Canadian regu-
able to a number of bacterial and viral diseases. Previous methods latory authorities, and granted a commercial license. The vaccine
of disease control included the introduction of bacterins and vac- has been shown to provide strong protection against the virus and
cines in feed over prolonged periods of time, with varying degrees will hopefully be able to be used in other countries soon (Table 18) .
of success. Attenuated or modified viral and bacterial organisms A number of other DNA vaccines for fish have been tested
have also been introduced directly into the water, but this method (Table 18) including for viral hemorrhagic septicemia virus, infec-
runs the risk of viruses reverting to virulent forms and contaminat- tious pancreatic necrosis virus (Birnaviridae family), infectious
ing rivers, lakes, other hatcheries and wildlife. Recently, fisheries salmon anemia virus and Mycobacterium marinum. While these
have had to resort to individually injecting fish with vaccines. A vaccines have achieved varying degrees of success, DNA vaccines
number of vaccines for fish are available today, such as for Koi for fish remain an attractive option for several reasons. Given
herpes virus, pasteurellosis, vibriosis, enteric septicemia of catfish, the size of hatcheries and the number of fish in them, a cheap
piscirickettsiae, infectious salmon anemia, Moratella viscosis, and vaccination option is necessary; inactivated viruses would be pro-
a number of streptococci and mycobacteria [208] . hibitively expensive and attenuated viruses can cause disease. In
One disease that has been a scourge of the industry has been addition, the introduction of viral pathogens in water could lead
infectious hematopoietic necrosis (IHN). This disease, caused by to contamination of other bodies of water and the fish in them.
the IHN virus of the Rhabdoviridae family, results in symptoms DNA vaccines are safer for fish as they are not formulated with
characterized by extensive necrosis of hematopoietic tissues (spleen, an oil adjuvant that can cause peritonitis [77] . They are also safe
kidney and liver). Transmission occurs horizontally between for the consumer, as the fish are consumed months or even years
fish, as the virus is shed in feces, urine, sexual fluids and external after vaccination, and the quantity of DNA used is very small. At
mucus. The virus can also be transmitted from farmed popula- this point, immune mechanisms of protection achieved by DNA
tions to wild populations. The severity of outbreaks depends on a vaccines are poorly known; however, the effectiveness of these
number of factors, including species and age of fish, rearing con- vaccines is fairly well established.
ditions and water temperature [209] . Previously, the only method
of preventing disease was to avoid exposure to the virus through Future directions for DNA vaccines
hygienic rearing practices, such as disinfection of fertilized eggs, DNA vaccines are currently mainly being developed to vaccinate
incubating eggs, and raising fry and young fish in isolated sites. against diseases. However, alternate uses of DNA inoculation are
The Canadian salmon industry, which produced 98,441 tons also being considered. One that has enjoyed success in terms of
of fish valued at US$543,634,000 in 2006 [210] , was particularly efficacy is the inoculation of pigs with plasmids encoding the
vulnerable to IHN. From 2001 to 2003, the virus caused losses gene for porcine somatotropin. Vaccination of gilts or pregnant

Table 18. DNA vaccines for fish.

Pathogen Species Vaccine Dosage Vaccination Protection Conventional Ref.
vector age treatment
IHNV (Apex-IHN by Atlantic G-protein Two 20-µg 30 g or larger 73% protection Avoiding exposure to [208]
Novartis, UK) salmon gene doses im. the virus
IPNV (Mikalsen Atlantic VP2 gene 25 µg of Postmolts of RPS* of 84% Commercial vaccines: [124]
et al. 2004) salmon DNA im. 20 g or larger inactivated virus or
structural virus proteins
expressed in
Escherichia coli
VHSV (Lorenzen Rainbow G-protein 1 µg of DNA Fry 0.5 g RPS of 98% [125]
et al. 2001) trout gene im. (3 months after
hatching)
Mycobacterium Striped bass AG85A 25 or 50 µg 40–50 g Low levels of AG85A- No chemotherapeutic [126]
marinum (Pasnik gene doses with (~5 months) specific antibodies, agents; avoiding
and Smith 2004) (secreted booster lymphoproliferative exposure to bacteria
fibronectin- 14 days later responses within
binding im. 42 days, 80% RPS for
protein) 25-µg dose, 90% for
50-µg dose
*
Relative percent survival (= one cumulative mortality vaccinated group/cumulative mortality control group).
im.: Intramuscular; RPS: Relative percent survival.

www.expert-reviews.com 1269
 Review Redding & Weiner

sows results in increased piglet viability, size at birth, growth In terms of large food-animal production, vaccines are essential
and performance, via the upregulation and increased activity of not only for individual animal health, but also for herd health and,
IGF [78,79] . by extension, human health. The large number of commercial
A commercial growth hormone-releasing hormone vaccine for vaccines available for a number of large animal diseases allows
pigs has been developed and approved for use in Australia by VGX breeders and producers ample choice for vaccination strategies.
Pharmaceuticals (LifeTide SW5; PA, USA). A single vaccination, In addition, an advantage of many commercial vaccines is that
delivered via electroporation to pigs, resulted in increased weights they provide a cocktail of immunizations for a relatively low price.
and increased growth rate of piglets, reduced mortality in piglets If DNA vaccines are to compete with commercially available
and dams, and an increased number of offspring in sows [80] . vaccines, the price of production will need to be minimal. The
Neither the USDA nor the US FDA have yet approved the use advantage of a DNA vaccine, especially in large herds or produc-
of such vaccines, nor of any form of growth hormone treatment tion establishments, is its ease of transport and storage compared
for the swine industry. However, such DNA vaccines have been with commercial vaccines. In addition, a DNA vaccine specific
approved in other countries and may be of great value in countries to a certain disease could be more useful in an outbreak scenario
such as China, which is the world’s largest consumer and producer where control of the spread of the disease is desired. An additional
of pork. Indeed, according to a report by the USDA, the Chinese significant advantage is the potential ability of DNA vaccines to
consumed 51 million metric tons of pork in 2006, roughly half of act in the face of maternal antibodies [3] . For neonates ingesting
the world’s total pig consumption (versus 8.6 million metric tons colostrums and maternal antibodies, commercial vaccines are
in the USA) [211] . This technology could also be useful in develop- generally inefficacious, yet young calves are very susceptible to
ing countries where animal owners are predominantly resource- a number of infectious diseases. The current method of disease
poor, small-scale operators with little land and few animals, who prevention for calves is immediate postnatal separation of the calf
must operate on few resources and capital. Similar to the use of from its dam; therefore, the ability to induce immunity in young
bovine somatotropin in dairy cows to increase milk production, animals in the face of maternal antibodies could be very valuable.
the use of porcine somatotropin has many advantages. Pigs are
not only healthier at birth and beyond, but also leaner, which Expert commentary & five-year view
translates into healthier meat for consumption. Somatotropin is DNA vaccines should be developed for diseases where traditional
species specific and studies have shown that porcine somatotropin vaccination is not very effective (such as PRRSV or in diseases
is active only in swine, not cattle or humans, thereby guarantee- where various regional serotypes exist) or only treats clinical signs
ing its safety for human consumption [212] . Furthermore, if more and does not prevent disease (such as FMD). DNA vaccines should
meat can be derived from a smaller number of animals, then fewer also be looked into as an option if there is concern about tradi-
resources will be required for pork production, which will make tional vaccination causing an outbreak in a herd or causing adverse
pork production less environmentally detrimental. effects on the animal (such as a vaccine-induced sarcoma in cats or
lesions at the site of vaccination in pigs). However, improvements
Conclusion must be made in the degree of protection induced by DNA vac-
Ultimately, the factors that will make a DNA vaccine attrac- cines, as well as in the delivery method. Several methods of delivery
tive for a certain disease will include its reduced cost, its ease have been developed, including naked DNA delivery by injection,
of transport and administration, its ability to act in the face of gene gun delivery, lipid-based and polylactide-­coglycolide (PLG)
maternal antibodies, the ability to differentiate diseased animals microparticles (which allow for oral delivery but can damage
from vaccinated animals, and the reduced likelihood of the vac- delivered DNA), mucosal delivery (suppository or oral delivery),
cine to cause an outbreak. In addition, DNA vaccines have the intramuscular or intradermal injection followed by electropora-
ability to induce both cellular and humoral protection since anti- tion, or via a transdermal device, a spring-powered device used to
gens can be delivered and processed intracellularly, which can inject the vaccine intramuscularly without a needle. The naked
improve upon certain traditional vaccinations. DNA vaccines are DNA delivery has a low rate of uptake; gene-gun delivery limits
also powerful in that they have extended boosting capability for the amount of DNA that can be incorporated in the vaccine and
continued immunotherapy, since the vaccine can be administered is expensive due to the use of gold beads, the encapsulation proc-
repeatedly without inducing neutralizing antibodies to the plas- ess of PLG microparticles is harsh and can potentially damage
mid. However, all of these factors must be weighed against the DNA, mucosal delivery is able to effectively prime but not induce
ability of the vaccine to offer protection to the host. If the number a satisfactory immune response [81] . Electroporation is a preferred
of currently commercially available DNA vaccines is low, it is method for administering DNA vaccines and has shown to be
because prior DNA vaccines did not induce a high enough degree effective for both intradermal and intramuscular injections [83,84] ,
of protection in larger animals and humans. The improvement of and newer and simpler electroporation systems could become very
DNA vaccine immune potency must be achieved, through prom- practical in terms of vaccinating large numbers of animals, and in
ising technologies as improved formulations or simple electropo- lowering the level of discomfort to the vaccinated animal.
ration, or alternate vaccination strategies should be considered, DNA vaccines are starting to gain a foothold in the veterinary
such as prime–boost approaches, cytokine gene adjuvants or other commercial market. A number of properties discussed in the
adjuvant formulations.
­
review make DNA vaccines a safe, efficient and attractive option

1270 Expert Rev. Vaccines 8(9), (2009)

 DNA vaccines in veterinary use Review

for veterinary use, making the future use of these vaccines highly trials for both animal and human DNA vaccines are ongoing for
likely. Further development of DNA vaccines will also improve a wide range of infectious diseases and cancers. For now, DNA
production methods, which will decrease the costs of these vac- vaccines have made more headway in their use in animals than
cines. As a result, we expect that the use of DNA vaccines will in humans; however, it is likely that they will prove possible and
increase within the next 5 years. However, improvements need to efficacious for both continued animal and human use.
be made in terms of boosting immunogenicity, improving delivery
methods and enhancing formulations. Electroporation, for exam- Financial & competing interests disclosure
ple, has significantly improved efficacy of DNA vaccines, while This work was supported in part by NIH grants awarded to David Weiner.
other methods of delivery, such as the use of lentiviral vectors, The authors note possible commercial conflicts associated with this work,
show promise too. Diverse methods of purifying DNA and pro- which may include Wyeth, VGX, BMS, Virxsys, Ichor, Merck, Althea and
ducing vaccines have also been developed, including fermentation Aldeveron. The authors have no other relevant affiliations or financial
methods, gene recombination processes and PCR scale-ups, which involvement with any organization or entity with a financial interest in or
will allow for improved production capability. New methods of financial conflict with the subject matter or materials discussed in the
vaccination, such as priming with a DNA vaccine and boosting ­manuscript apart from those disclosed.
with a protein vaccine, have been shown to be very effective and No writing assistance was utilized in the production of this
might be an avenue of further exploration. A large number of manuscript.

Key issues
• Although tremendous strides have been made in terms of fighting infectious disease using vaccines in both companion and
production animals, infectious disease still poses a considerable threat to the health, wellbeing, and economic value of animals and
production systems.
• There are a number of important animal diseases for which conventional vaccination is not possible, not permitted or ineffective.
• DNA vaccines have a number of advantages for veterinary use, including safety and ease of transport, the ability to drive immunity in
the presence of maternal antibodies, the ability to differentiate vaccinated from infected animals, and the potential to be cost effective
to produce.
• DNA vaccines have been shown to induce a potent immune response for a number of diseases of veterinary importance, and four DNA
vaccine-based products have been made commercially available for equids, swine, canines and fish.
• However, improvements in immunogenicity, plasmid delivery and production methods are important to allow DNA vaccines to become
more widely applicable.
• DNA vaccines have the potential to expand beyond the realm of infectious disease to the fields of cancer or the enhanced production
capacity of food animals.

References •• Provides a good overview of the 8 Uhl EW, Heaton-Jones TG, Pu R,

Papers of special note have been highlighted as: mechanism of DNA vaccination and good Yamamoto JK. FIV vaccine development
• of interest examples of its use in a number of and its importance to veterinary and
•• of considerable interest different species. human medicine: a review FIV vaccine
2002 update and review. Vet. Immunol.
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•• Provides a good overview of some DNA 5 Meeusen EN, Walker J, Peters A, Pastoret
Crawford PC. Seroprevalence of
vaccine trials from 2000. PP, Jungersen G. Current status of
feline leukemia virus and feline
2 Babiuk LA, van Drunen Littel-van den veterinary vaccines. Clin. Microbiol. Rev.
immunodeficiency virus infection
Hurk, Babiuk SL. Immunization of 20(3), 489–510 (2007).
among cats in North America and
animals: from DNA to the dinner plate. •• Comparative examination of the risk factors for seropositivity. J. Am.
Vet. Immunol. Immunopathol. 72(1–2), variety of different vaccines used in the Vet. Med. Assoc. 228(3), 371–376
189–202 (1999). veterinary field. (2006).
•• Provides a good overview of the principles 6 AVMA. U.S. Pet Ownership & 10 Lecollinet S, Richardson J. Vaccination
of DNA vaccination in veterinary use and Demographics Sourcebook (2007 Edition). against the feline immunodeficiency virus:
a limited number of trials in place at the American Veterinary Medical Association, the road not taken. Comp. Immunol.
time. IL, USA (2007). Microbiol. Infect. Dis. 31(2–3), 167–190
3 Dhama K, Mahendran M, Gupta PK, Rai 7 Kusuhara H, Hohdatsu T, Okumura M (2008).
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215 Livestock, dairy, and poultry outlook Affiliations • David B Weiner

http://usda.mannlib.cornell.edu/usda/ers/ Department of Pathology and Laboratory
• Laurel Redding
LDP-M//2000s/2008/ Medicine, 422 Curie Boulevard – 505
University of Pennsylvania School of
LDP-M-07-18-2008.pdf SCL, University of Pennsylvania,
Veterinary Medicine, 3800 Spruce Street,
216 The Royal Society. Infectious diseases Philadelphia, PA 19104, USA
University of Pennsylvania, Philadelphia,
in livestock: summary and main Tel.: +1 215 349 8365
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