Immunization of animals: from DNA to the dinner plate

Veterinary Immunology and Immunopathology 72 (1999) 189±202

Immunization of animals: from DNA to the dinner plate L.A. Babiuk*, S. van Drunen Littel-van den Hurk, S.L. Babiuk Veterinary Infectious Disease Organization, University of Saskatchewan, 120 Veterinary Road, Saskatoon, Sask., Canada S7N 5E3 Accepted 22 September 1999

Abstract Recently, there has been a great deal of interest in polynucleotide vaccination also referred to as DNA vaccines or genetic immunization for inducing long-term immunity in various animals and humans. The main attraction of this technology is the possibility to induce a broad range of immune responses without the use of conventional adjuvants. To date, most of the studies (>500 reports) have focused on DNA vaccination in mice. The present report summarizes the limited number of trials that have used target animal species to not only test the immune responses but also correlate them to protection. # 1999 Elsevier Science B.V. All rights reserved. Keywords: DNA immunization; Veterinary vaccines; Immunity

1. Introduction The last 200 years have clearly demonstrated that immunization is more effective in preventing economic losses and animal suffering from infectious diseases than all other therapeutic and prophylactic treatments combined. In addition to reducing the severity of disease in vaccinated animals, vaccines also reduce the transmission of infectious agents from vaccinated animals that accidentally become infected with the agent. This results in reduced production losses in all contact animals. These successes have been primarily achieved using live attenuated or killed conventional vaccines. However, even with these successes, death and economic losses due to infectious agents continue in all parts of the world, in all animal species and, therefore, a quest for more effective and safer vaccines continues. *

Corresponding author. Tel.: ‡1-306-966-7475; fax: ‡1-306-966-7478

0165-2427/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 2 7 ( 9 9 ) 0 0 1 3 2 - 4

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Recent advances in our understanding of the pathogenesis of different diseases, combined with the development of immunological reagents to assess immune responses and molecular tools to dissect the pathogen are providing a level of enthusiasm never seen before in the field of vaccinology. As a result, various types of novel vaccines are being developed. These include (1) genetically engineered live gene deleted vaccines, (2) live chimeric vaccines or vectored vaccines, (3) live replication defective vaccines, (4) subunit vaccines produced either as monovalent, multivalent, or chimeric subunits, (5) peptide vaccines, and most recently, (6) polynucleotide vaccines. To further enhance immune responses to some of these vaccines, novel adjuvants, cytokines, and CpG sequences are being incorporated into the vaccines to enhance and focus the immune response in such a way as to ensure that the desired responses are generated. The present review will briefly summarize some of the recent successes in using DNA immunization as a method of inducing immune responses in various animal species. 2. DNA immunization Although transfection of cells in vitro has been employed for some time, it was only recently shown that cells could also be transfected in vivo (Wolff et al., 1990, 1991). This observation rapidly led to the conclusion that if a foreign gene could be expressed in vivo, it should lead to the induction of an immune response (Tang et al., 1992; Ulmer et al., 1993). This proved to be correct and we can now use the animal as the `bioreactor' to produce the proteins required for stimulating immune responses to a variety of pathogens in various animals. This has been shown to be possible with genes from different viruses, bacteria, and parasites (Hoffman et al., 1994; Sedegah et al., 1994; Barry et al., 1995; Cardoso et al., 1996; Davis et al., 1996; Hermann et al., 1996; Boyer et al., 1997; Gerloni et al., 1997; Lewis et al., 1997; Lozes et al., 1997). Table 1 summarizes selective studies where DNA immunization was used in a target species and where, in most cases, animals were challenged to measure the level of protection provided by DNA immunization. Studies with genes from other veterinary pathogens that have been tested in mouse models are not included in this review since in most of these cases, mice were not challenged. Without challenge, it is difficult to draw conclusions as to the efficacy of DNA immunization in inducing protective immune responses. For example, an equine herpesvirus (EHV-1) gene (gD) induced both antibody and lymphocyte proliferative responses as well as induced protection in mice from EHV-1 infection (Ruitenberg et al., 1999). However, whether this has direct relevance to protection in horses remains to be determined. The above table was included to demonstrate the breadth of diseases in species that have been successfully immunized with DNA vaccines. We are aware of other studies where even a greater array of genes are being included in DNA vaccination. These include `concealed' antigens from ectoparasites. These `concealed' antigens normally do not come into contact with the host's immune system and, therefore, do not normally induce an immune response in the animal., However, if animals are exposed to these antigens they do develop immunity to them. For example, if ticks feed on an animal possessing antibodies to antigens of the tick mid-gut, these antibodies interact with the

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Table 1 Vaccination of veterinary species with DNA vaccinesa Target species

Pathogen/antigen

Method/dose boost

Immune response

Challenge outcome

References

Cattle

BHV-1 gD

i.m./500 mg or 125 mg 4 boosts

decreased viral shedding

(Cox et al., 1993)

BHV-1 gD and tgDb

i.m./l mg 2 boosts

moderate serum ELISA and s.n. Ab at 20 weeks in high dose animals low Ab in low dose animals low ELISA and s.n. Ab antigen specific proliferation of PBMC's moderate ELISA and s.n. Ab antigen specific proliferation of PBMC's moderate to high serum ELISA Ab

n.d. with i.m. vaccines

(van Drunen Littel-van den Hurk et al., 1998)

i.d. (ear)/500 mg 1 boost

Sheep

BRSV G protein

i.d. or i.m. 400 mg 2 boosts

Theileria annulata Tams 1-1,1-2

i.m./500 mg of each antigen 2 boosts

no serum ELISA Ab

Taenia ovis 45W

i.m./200 mg 2 boosts

low serum ELISA Ab with DNA only moderate serum ELISA Ab with DNA followed by rec45W/QuilA boost long-term CMI moderate ELISA Ab antigen specific proliferation of PBMC's Ab detected no response with 10 mg

i.d./200 mg 1 boost

BHV-1 tgD

i.d. 250 mg or gene gun 2.5 mg 1 boost

Cryptosporidium parvum (surface antigen)

i.m. jet injection 10, 100, 1000 mg 2 boosts

decreased clinical signs; decreased viral shedding; tgD offered greater protection decreased viral shedding at day 7 but not at Day 5 50±60% decrease in mortality following lethal challenge

(Schrijver et al., 1997) (d'Oliviera et al., 1998)

n.d.

(Rothel et al., 1997)

animals were not challenged

(Braun et al., 1997)

n.d.

(Jenkins et al., 1995)

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Table 1 (Continued ) Target species

Pigs

Pathogen/antigen

Method/dose boost

Immune response

Challenge outcome

References

BHV-l tgD

i.d. 500 mg to neonates 1 boost at 5 months of age

Moderate ELISA and s.n. Ab Antigen specific proliferation of PBMC's

Animals were not challenged

(van Drunen Littel-van den Hurk et al., 1998)

PRV gD

i.m./400 mg 1 boost

no significant clinical protection

(Monteil et al., 1996)

PRV gD

i.m./370 mg 1 boost with commercial vaccine

no serum ELISA or s.n. Ab preboost low serum ELISA and s.n. Ab post boost no s.n. Ab preboost substantial increase in s.n. Ab post-boost

PRV gD

i.m. i.d. 400 mg total

tgB/tgC/gD

id. 1.2 mg total, 2 boosts

PRV gD/gI gC

i.m./i.d., 1.50 mg 3 boosts

PRRS ORF 5 (gp5)

i.m. 100 mg 2 boosts

Swine influenza virus HA (HINI)

i.d. gene gun, 1.25 mg tongue, 1 boost

NP FMDV infectious clone

Horses

Influenza HA

Poultry Influenza H1, H7

i.d. or i.m. 200 mg 3 boosts

high s.n. Ab moderate antigen specific proliferation moderate s.n. Ab high antigen specific proliferation moderate s.n. Ab antigen specific proliferative responses in PBMC's Ab response 2±3 weeks after last boost/lymphocyte proliferation Ab response

Ab response Ab response

highest level of (Le Potier clinical protection et al., 1997) as compared to a variety of vaccination regimes decreased (van Rooij clinical signs et al., 1998) decreased viral shedding partial protection in gC plasmid immunized pigs

(Gerdts et al., 1997)

Protection after intratracheal challenge

(Pirzadeh and Dea, 1998)

Protection/ accelerated clearance of homologous challenge No protection Partial protection. No direct correlation to level of Ab

(Macklin et al., 1998)

(Lunn et al., 1997; Soboll et al., 1998)

(Ward et al., 1997)

i.d. gene gun, mucosal surfaces of tongue and eyelids

Ab response/ lymphocyte proliferation Il-6 co-administration elevated immune response

Protection and reduced vinis shedding

i.m., i.v., i.n., i.t./100±200 mg 1 boost

n.d.

increased protection (Fynan et al., in 25±63% chickens 1993)

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Table 1 (Continued ) Target species

Pathogen/antigen

Method/dose boost

Immune response

Challenge outcome

References

NDV HA

i.m. 100 mg

Ab detected

Protection best with linearized DNA in lipofectin. Protection correlated with Ab.

(Sakaguchi et al., 1996)

Ducks

DHBV pre S/S and S protein

i.m./250 or 750 mg 2 boosts

high serum ELISA Ab after 3rd immunization

increased rate of systemic viral clearance in animals immunized with S protein reduced viral replication in hepatocytes increased in vivo neutralization with anti-S sera but not with anti pre-S/S

(Triyatni et al., 1998)

Fish

IHNV NP and Gi.m./10 mg protein

increased serum ELISA Ab at 8 weeks. with G protein and at 4 weeks. with G protein ‡ NP increased s.n. Ab at 6 weeks with G protein and G protein ‡ NP

50±60% decrease in mortality following lethal challenge

(Anderson et al., 1996) also see (Winton, 1997)

Cats

FIV env

low to undetectable serum ELISA Ab Moderate ELISA Ab

enhancement of infection Partial protection

(Richardson et al., 1997) (Cuisinier et al., 1997)

n.d.

(Keller et al., 1996)

Protection present Protection correlated with rapid anamnestic response

(Jiang et al., 1998) (Schultz, personal communication)

FIV gp120 p10

Dogs

IL-2, IL-6, GM-CSF

CPV VP1 CPV VP1

i.m./200 mg 2 boosts i.m., 600 mg 2  gp120 or 1 boost gp120 ! p10 ballistic/0.5 mg buccal mucosa or epidermis

increased infiltration of neutrophils at epidermal injection site with GM-CSF no change at buccal mucosa i.m. 200±800 mg Ab detected in boost at 16 weeks animals i.m. 50±200 mg Low to no Ab detected

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Table 1 (Continued ) Target species

Pathogen/antigen

Rabbits CRPV major capsid protein (L1)

Method/dose boost

Immune response

Challenge outcome

References

ballistic/1.0 mg over 30 sites 3 boosts

high serum ELISA and s.n. Ab antigen-specific proliferation of PBMCs

90±100% protection following challenge

(Sundaram et al., 1997)

a i.m.: intramuscular; i.d.: intradermal; i.n.: intranasal; i.t.: intratracheal; n.d.: not done; s.n.: serum neutralization. b truncated, secreted version of bovine herpesvirus-l glycoprotein D.

`concealed' antigens of the gut and in combination with complement, lead to lysis of intestinal cells or disrupt gut cell function. This results in death of the ticks or at least a dramatic reduction in the reproductive capacity of the ticks (Willadsen et al., 1993; Opdebeeck, 1994). It is based on this observation that a subunit vaccine directed against an 86k glycoprotein is being used in cattle to reduce tick populations and infestation of pastures with ticks (Willadsen et al., 1988, 1989). Similar studies are being performed with salivary glands of other ectoparasites (Allen and Humphreys, 1979). These have not been included in the summary table since many of these studies are preliminary and have not been published to date. However, now that the principle of immunization against ectoparasites has been established, it is anticipated that in the near future reports will be forthcoming regarding the use of DNA immunization to induce immune responses to these `concealed' antigens. Finally, DNA immunization is also being used to develop reagents, monoclonal antibodies for diagnostic tests and most recently for producing antibodies to cytokines (Weynants et al., 1998). One major conclusion that can be drawn from the above-mentioned studies is that it is important to test a variety of immune parameters in addition to just looking for antibody to determine whether vaccination protocols are successful. It is clear that, in addition to testing for antibody, it is critical to look at cell-mediated immunity, cytokine production, antibody isotypes, and, most importantly, to challenge the animals. We feel that protection from challenge is the most appropriate test to determine efficacy of DNA vaccination. This is especially important since many DNA vaccines do not produce very high levels of antibody, but they are extremely efficient primers of the immune response. Thus, upon exposure to the pathogen there is a rapid anamnestic response which rapidly clears the infectious agent. This is best illustrated by canine parvovirus where puppies have low or no detectable levels of antibody after immunization, but are protected from challenge as a result of a very rapid anamnestic antibody response (Schultz, personal communication). In some cases, DNA vaccines may be the only vaccine required to protect the animals, whereas in other cases they may be used to prime the animals' immune systems and possibly even polarize the response followed by a boost with other types of vaccines (Rothel et al., 1997). In addition to the broad applicability of this technology to various pathogens in animal species, DNA immunization has some real or perceived advantages over currently used

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Table 2 Advantages of DNA vaccines Safety

Disease monitoring Induction of broad range of immune responses

Delivery Neonatal immunization Cost

Others

does not require cultivation of dangerous pathogens. no risk of causing disease since only a single gene is introduced. no injection site reactions. can be used as marker vaccines. CMI, antibody, mucosal immunity duration of immunity is long ability to `tailor' the immune response to be primarily cellular, humoral, or both induction of memory cells multiple modes of delivery Ð IM, ID, mucosal, transdermal, needleless injection. This area still presents a challenge. minimal interference with passive maternal antibody. no threat to young as immunocompromised animals. able to overcome immature neonatal immune system. potential for low cost production and administration. potential for multi-component/multivalent single shot vaccines. no need for cold chain can produce vaccines against organisms which are difficult to culture in vitro or to attenuate. can be used to identify potentially protective antigens of various pathogens.

conventional vaccines. Because of these perceived advantages, DNA immunization has been referred to as the `third generation of vaccinology' (Dixon, 1995). Some of these advantages are summarized in Table 2. The first advantage is the safety of the vaccines. Since the vaccine consists of a single gene, there is no opportunity to induce an infection or subclinical disease by vaccination. Associated with the fact that the vaccine contains only a single protein is the possibility of developing companion diagnostic tests to differentiate vaccinated animals from animals that are carriers of the disease (van Oirschot et al., 1996). This requirement for differentiation of vaccinated animals from those that have been exposed to the pathogen and could be potential carriers of the agent is becoming increasingly important as countries try to eradicate specific diseases from their borders. As this technology becomes perfected and accepted by the regulatory agencies and the public, it will play an important role in non-tariff trade barriers to control exportation and importation of animals from infected regions. The basis for marker vaccines is that animals develop immune responses to the antigen present in the vaccine but not to the other antigens of the pathogen. Fig. 1 illustrates the marker vaccine concept for a herpesvirus vaccine. Animals vaccinated with a plasmid construct encoding glycoprotein gD will only produce antibodies to gD and not to any other protein in the virus. Although herpesvirus was used as an example, since such differential diagnostic tests are being used today to help reduce the spread of BHV-1 in herds as well as help eventually eradicate the disease in some countries, the concept could

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Fig. 1. The conventional vaccine produces antibodies to all the viral proteins, whereas the plasmid-based vaccone only produces a single protein which then induces antibodies only to that protein. Using an antibody ELISA it is possible to differentiate the plasmid immunized animals from those that are infected with field strains of virus.

be used with any pathogen. In addition to helping eradicate a disease which is endemic in a country, marker vaccines could also be used in instances where an exotic disease, such as classical swine fever (hog cholera) was accidentally introduced into a country. In this instance, vaccination with marker vaccines could be introduced in the periphery of the outbreak to restrict its spread while quarantine and slaughter activities in the infected area are being carried out. This approach of disease control and eradication should prove to be a much more cost effective one than is presently being used. Another advantage of having the host respond to a single protein rather than the myriad of proteins present in a pathogen is that many pathogens have proteins that can downregulate the immune response to the desired protein (Harland et al., 1992; Hengel and Koszinowski, 1997; Ploegh, 1998). Thus, the immune response to the protective components of the pathogen should be elevated using a single DNA construct. Since each vaccine is directed against a single protein, it should also be possible to combine plasmids encoding genes of various pathogens into a single vaccine. This has already been shown to be possible with at least a number of different genes encoding protective proteins from bovine herpesviruses and parainfluenza-3 (Braun et al., 1998). Possibly the most complex DNA vaccine being contemplated is one to control malaria where 15 different genes are being designed to target various malaria antigens (Hoffman, personal communication). Possibly one of the major advantages of using DNA vaccines is that this form of immunization appears to stimulate both humoral and cellular immunity. Although there are still limited reports of the types of immune responses induced by DNA immunization in various veterinary species, as compared to the type of immune response induced following natural infection, in cattle immunized with BHV-1 gD encoding plasmids, the

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immunoglobulin isotype of DNA immunized animals is very similar to that induced by live vaccines or in animals recovering from virulent virus (van Drunen Littel-van den Hurk et al., 1998). The possible reason for this similarity in immune responses between plasmid immunization and exposure to virulent virus is that the glycoproteins are produced in mammalian cells and are processed in a manner similar to those in a true viral infection. This leads to correct processing and presentation of the antigen and, therefore, a similar immune response. Although DNA immunization, in most cases, induces a strong Th1-like response, if a Th2-like response is required such a response can be induced by altering the route or method of DNA administration, altering the form of the antigen, or by co-administration of genes encoding various cytokines or co-stimulatory molecules (Lewis et al., 1997). Thus, it should be possible to polarize the immune response to ensure the desired immunity is achieved by manipulating the types of gene constructs or mode of administration. In addition, it is possible to deliver the plasmids to mucosal surfaces so as to induce local mucosal immunity as well as systemic immunity (Ban et al., 1997; Darji et al., 1997; Jones et al., 1997; Kuklin et al., 1997). This is critical in most disease situations since most pathogens enter at mucosal surfaces. By inducing mucosal immunity, it will not only reduce the chance of initial infection, but it will also reduce horizontal transmission if a vaccinated animal is infected. In livestock and pets a major concern is being voiced with regards to injection site reactions following immunization. These are very common with killed vaccines where adjuvants are used. Such injection site reactions can range from simple granulomas to osteosarcomas in cats (Kessler et al., 1997). Since DNA vaccines do not use adjuvants, such reactions should be eliminated or at least dramatically reduced. This should ensure better meat quality in food producing animals and the trauma associated with osteosarcoma in pets. Meat quality is becoming an extremely important issue for consumers and the export market. Another major problem in vaccinology is the inability to elicit active immunity in neonatal animals born to immune mothers. Since these neonates possess maternallyderived antibodies, they generally do not respond to conventional vaccines. Unfortunately, the level of maternal antibody which inhibits the response to conventional vaccines is often lower than that required for protection. Thus, there is a `window of susceptibility' where animals do not respond to vaccines but are susceptible to infection. Although it is possible to test each individual animal for the presence of passively acquired maternal antibody, this is an extremely expensive exercise. Even in a single litter, all animals do not obtain the same amount of maternal antibody. Thus, some animals will respond to vaccination at 4 weeks after birth, whereas others will still have sufficient levels of antibody to be refractory to the vaccination. To overcome this problem, veterinarians generally recommend multiple vaccination of young pets to avoid unnecessary long periods of disease susceptibility in neonates. Fortunately, using DNA immunization it is possible to circumvent this interference by maternal antibodies (Monteil et al., 1996; Hassett et al., 1997; van Drunen Littel-van den Hurk et al., 1999). An additional advantage of being able to immunize animals at birth is that most large animals are handled at this time. Thus, the added expense of frequent handling of livestock is reduced.

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3. Future challenges and opportunities Although there are many advantages to DNA immunization, before it can be widely used in veterinary medicine, the issue of delivery needs to be addressed. Using intramuscular injection, the majority of the DNA never enters cells to produce protein. Although the efficiency is slightly higher using gene gun administration, the limitations of the quantity of DNA that can be administered in a single `shot' with a gene gun dictates that multiple shots must be administered to ensure that sufficient quantities of DNA are introduced in the animal to stimulate an immune response. Thus, methods of effective DNA delivery are crucial and will be the Achilles heel of this exciting technology if this impediment is not overcome. We are confident that investigators will find ways around this problem since numerous groups are aggressively pursuing different forms of needleless injection for DNA. As we develop these novel delivery systems, we must always be cognizant of the fact that many of these delivery methods cannot be used in areas of the body containing hair. Unfortunately, few areas are devoid of hair in livestock and normal management systems will resist shaving areas of the animal prior to vaccine administration since this is labor intensive and expensive. One of the potential sites of administration of DNA vaccines are mucosal surfaces of the oral and nasal cavities. In addition to being free of hair, immunization at these sites should also lead to induction of mucosal immunity. Delivery of DNA vaccines using a gene gun, needleless injection systems, or liposomes have already been reported to be successful in inducing immune responses in at least some instances. A second challenge with DNA immunization is that of integration of the DNA into the transfected cells and especially cells of the germ line. This is especially important since it is clear that the DNA does not reside at the site of injection. Although most evidence to date has not shown the presence of integrated plasmid DNA (Nichols et al., 1995), the observation that injected DNA can be present in the semen of DNA vaccinated pigs requires further analysis to investigate the state of the DNA in the semen (Lutz-Wallace, personal communication). This is obviously of concern to the regulators and all the proponents of DNA immunization. Time and research will, hopefully, determine whether these concerns are legitimate and whether ways can be developed to reduce the probability of this occurring. Further regulatory concerns were expressed regarding the possibility of developing anti-DNA antibodies which could lead to autoimmunity. This might be especially critical if animals were exposed to multiple DNA vaccines over their lifetime. To-date, there is no evidence of the presence of anti-DNA antibodies following DNA immunization, even when animals were injected with milligram quantities of DNA multiple times (van Drunen Littel-van den Hurk et al., 1998). However, one must always interpret these results with caution since animals were only observed for relatively short periods of time. Since food-producing animals generally have a short life span, this would be of less concern than in pets or humans where life spans may approach 80±100 years. A second reason for optimism is that attempts to develop anti-DNA antibodies have shown that this requires strong adjuvants. Since it is not anticipated that strong adjuvants will be used with DNA vaccines, this may be a mute point. However, we must continue to monitor these developments as we transfer this technology from food-producing animals with a

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short life span to other animals and humans where anti-DNA antibodies could have devastating effects. The recent discovery that specific oligonucleotide sequences (CpG) can have immunomodulatory activity (Krieg, 1996; Krieg et al., 1995, 1998; Klinman et al., 1996, 1997) is providing insights into how immune responses can be increased with properly constructed plasmids. Even more exciting is the observation that these CpG motifs can enhance both non-specific responses and provide early protection after vaccination before the specific immune responses are present and to also enhance immune responses to conventional vaccines (Klinman, personal communication). It are these types of serendipitous findings that provide us with the confidence that DNA vaccination holds great promise in helping us develop more effective vaccines and that we will be able to address all of the regulatory issues surrounding this new technology. Acknowledgements Work in the authors' laboratories is supported by the Medical Research Council and Natural Sciences and Engineering Research Council of Canada. The authors thank Michelle Balaski for excellent secretarial assistance.

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