Pathogenic Mycobacteria

Mycobacterium is a bacterial genus containing more than 30 different species including M. tuberculosis and M. bovis causing human and bovine tuberculosis, respectively, and M. leprae the causative agent of leprosy in humans. Other mycobacterial human pathogens comprise M. avium, a pathogen causing opportunistic infections in immuno-compromised individuals like AIDS patients, and M. ulcerans, which causes buruli ulcer, an emerging health problem in developing countries.

The Challenges Presented by Tuberculosis

Human tuberculosis, caused by M. tuberculosis, is a major global human health problem that claims more than 2 millions lives every year.¹ The advent of the HIV pandemic has tragically increased the incidence of tuberculosis in developing countries, e.g., sub-Saharan Africa, where AIDS-related tuberculosis is becoming the main cause of mortality in young adults. Indeed, it has been estimated that M. tuberculosis is responsible for the deaths of more youths and adults than any other infectious agent. The WHO has therefore declared tuberculosis a global health emergency.²

BCG (M. bovis Bacille Calmette Guerin), the TB vaccine, is a major component of many national vaccination programs. About 90 million doses are supplied by the World Health Organization (WHO) every year. However, trials performed in a number of countries have revealed that BCG imparts a very heterogeneous degree of protection, ranging from 0 to about 80%.^3–5 Although there is usually quite good protection imparted against the severe childhood forms of tuberculosis, such as tuberculous meningitis, there can be poor protection against the infectious adult form.⁶ Hence a better vaccine is needed, or one that can be given in addition to BCG and results in better protection against both childhood and adult disease. Critically, vaccination must prevent the establishment of the persistent latent state of infection that can follow primary infection and provides a major source of adult disease many years later.

A vaccine against tuberculosis in cattle is also needed. Bovine tuberculosis, caused by the closely related organism M. bovis, can be a significant source of disease in man. For example, around years 1930–1940 about 40% of cows in the UK were infected and 6–7% of total human deaths due to tuberculosis were caused by M. bovis.⁷ BCG can be effective in cattle, but as in man, the efficacy varies widely and the vaccine response interferes with the hypersensitivity tests deployed in control programs. The strategy of detecting (by skin test hypersensitivity) and slaughtering infected animals is being used to reduce and contain the problem (in conjunction with pasteurization of raw milk) but is not universally applicable especially in the developing world and cannot eliminate the disease in the face of reservoirs of infection in wildlife.⁸ A recent review of the situation of bovine TB in cattle and wildlife in England and Wales for example has concluded that the development of an effective cattle vaccine against bovine TB has the best prospect of TB control in the National herd.⁹

The Nature of Protective Immunity Against Tuberculosis

Most constituents of the cellular and humoral immune system have been documented to be involved following infection with M. tuberculosis and several comprehensive review papers have been published recently.^10–13 The view that antibody has no role to play in protection may be changing slightly,^14,15 but as yet the paradigm of protection in tuberculosis remains cellular immunity. Unfortunately, the disease is also entirely a consequence of the cellular immune response. Accordingly, vaccination is required to enhance the protective and not the harmful aspects of cellular immunity. Numerous studies have shown that DNA vaccination is able to achieve this, although we do not fully understand how, and most remarkably DNA vaccines are even able to exert a therapeutic effect in infected animals.

The general principles characterizing protective immune responses are represented graphically in Figure 1. Tubercle bacilli reside and multiply within infected macrophages. Antigen-specific T lymphocytes produce lymphokines including macrophage activating factors such as IFN-γ that enable the macrophages to at least restrict growth of the bacteria and maybe even to kill them. In 90% of people who become infected the organisms are eliminated without ever causing disease, but it has been hard to establish that activated macrophages actually kill the bacteria. However, it may be significant that killing of tubercle bacteria can occur when infected macrophages are themselves killed by the cytotoxic lymphocytes either through induction of apoptosis or by granule-mediated lysis.

Figure 1

Immune responses against tuberculosis. The upper panel represents the induction phase of the immune response involving infection of dendritic cells by tubercle bacilli and presentation of mycobacterial antigens to naive T cells. The lower panel depicts (more...)

TB Vaccines for Prophylactic Vaccination and Identification of Protective Antigens

The era of DNA vaccination against mycobacterial diseases began in 1994 when Silva and Lowrie demonstrated protection against M. tuberculosis after vaccination of mice with the macrophage cell line J774 that had been stably transfected with the heat-shock protein HSP65 from M. leprae.¹⁶ This provided proof of principle that a mycobacterial antigen expressed by a eukaryotic expression vector could induce protective immunity and was quickly followed by the successful use of a naked DNA vaccine expressing the same antigen.¹⁷ Similar protection could be obtained with DNA vaccines expressing either the secreted fibronectin binding protein antigen 85A (Ag85A)¹⁸ of M. tuberculosis or the M. leprae HSP65.¹⁹ This was followed soon after by a report indicating that another secreted TB antigen, the phosphate transporter PstS-1 (38 kDa antigen) also induced protective immunity.²⁰ Since then a large number of antigens have been expressed as DNA vaccines and tested in mouse, guinea pig, or cattle models of tuberculosis. Antigens, which in at least one of these animal models have been proven protective, are listed in Table 1. The overwhelming majority of antigens are secreted antigens. This is consistent with (and in part a consequence of) the long-held belief that such antigens will be the earliest targets seen by the immune system upon infection and therefore likely to be protective.²¹ Nevertheless, a number of other antigens that are probably not secreted also impart protection when given as DNA vaccines. Examples are the heat-shock protein HSP65, or the cytoplasmic enzyme KatG (Table 1, see refs. ^19,22, ²³). Although immune responses to the antigens listed in Table 1 conferred protection against M. tuberculosis to some degree, only very rarely did the protection surpass that observed after BCG vaccination (Table 1). We will now briefly discuss the results obtained so far for several of the more intimately studied antigens, namely HSP65, Ag85A, ESAT-6, and phosphate transporters.

Table 1

Mycobacterial antigens protecting against tuberculosis when applied as prophylactic vaccine.

DNA Vaccines as Post-Exposure Vaccines or Immunotherapeutic Agents

As we have pointed out earlier, there is also a need to develop novel tuberculosis vaccines that are effective in latently infected individuals (post-exposure vaccine), or are effective as immunotherapeutic reagents to assist conventional chemotherapy. Effective treatment with chemotherapy requires large doses of costly antibacterial drugs that have to be taken for at least 6 months, which is difficult to achieve in many developing countries. In addition, immunotherapy could improve the treatment of multidrug-resistant tuberculosis. Addressing these objectives, Lowrie and colleagues demonstrated that DNA vaccines that were effective as prophylactic vaccines against tuberculosis also had therapeutic effects against an established, chronic M. tuberculosis infection by reducing the bacterial burdens in both the lungs and spleens of treated mice.⁴⁵ They tested DNA vaccines expressing M. leprae HSP65, M. tuberculosis HSP70, ESAT-6, and another secreted antigen, MPT70 (see Table 1 for details of antigens) and could show strong therapeutic activity, mainly with HSP65 and MPT70. Interestingly, injection of DNA encoding IL-12 alone was as beneficial as injection of HSP65 or MPT70 although coinjection of IL-12 and HSP65 DNA resulted in reduced therapeutic efficacy.⁴⁵ The administration of BCG had no effect in these experiments. The authors' experiments suggested that the herapeutic effect of HSP65 was due to a reprogramming of the immune responses characterized by a switch from IL-4 responses towards predominant IFN-γ responses.⁴⁵

A frequent result of incomplete drug treatment of tuberculosis is the regrowth of bacteria that have persisted in a nonreplicating and physiologically drug-resistant form. Lowrie and colleagues used a mouse relapse model to show that HSP65 DNA vaccination given at the end of incomplete chemotherapy could prevent the corticosteroid-induced regrowth of M. tuberculosis in the lungs of 8/8 mice and in the spleens of 6/8 mice.⁴⁵ Significantly, BCG was ineffective in this experimental model of relapsing tuberculosis. These results raised the possibility that therapeutic DNA vaccination could be used as a postexposure vaccine in man to eliminate latent or persistent infection prior to disease.

Turner et al⁴⁶ conducted similar immunotherapeutic vaccination experiments in mice with an Ag85A DNA vaccine. They were unable to demonstrate any effect of vaccination on the course of infection in the lung of aerosol-infected mice but could demonstrate a reduction in bacterial loads in their spleens.

DNA Vaccines Protecting Against Mycobacterial Diseases Other Than Tuberculosis

Mycobacteria are the causative agents of several other human diseases apart from tuberculosis. For example, M. leprae is the causative agent of leprosy in humans, M. avium is a common opportunistic pathogen of immuno-compromised individuals like AIDS patients, and buruli ulcer, an emerging health problem of developing countries is caused by M. ulcerans. DNA vaccines are being developed to protect against all three of these diseases. This is being facilitated by the availability of suitable murine models to test vaccine efficacy. For example, DNA vaccination with M. tuberculosis Ag85B has been shown to impart protection against M. leprae growth on challenge of mouse footpads,⁴⁷ as did a DNA vaccine expressing the M. leprae 35 kDa antigen (ORF designation ML0841^47,48). The degree of M. leprae growth reduction was similar to that achieved with BCG, which appears to confer protection against leprosy in man. DNA vaccination with the M. avium homologues of either the M. leprae 35 kDa antigen,^47,49 Ag85A,⁵⁰ or of HSP65⁵⁰ protected mice against M. avium infection to a degree similar to, but not exceeding, BCG vaccination. In addition, DNA vaccination with M. tuberculosis Ag85A protected mouse footpads against M. ulcerans infection almost as well as BCG vaccination.⁵¹ Interestingly, “vaccination” of mice, either before or after M. avium infection using a construct expressing IL-18 reduced the bacterial loads and increased the survival period of treated mice.⁵²

Strategies to Improve the Efficacy of Tuberculosis DNA Vaccines

As shown in Table 1 and emphasized throughout this chapter, protection levels with DNA vaccination against challenge with tuberculosis has been generally less effective than BCG vaccination alone. Our own DNA vaccination studies in cattle, which, unlike mice and guinea pigs, are natural hosts for M. bovis, have confirmed this observation. We have tested 5 different DNA vaccine constructs (M. leprae HSP65, M. tuberculosis HSP70, Apa, MPB83, MPB70), either individually or as cocktails, and, to date, we have not been able to protect cattle against M. bovis infection (Buddle, Vordermeier, Hewinson, unpublished data) despite the proven effectiveness of these vaccines in rodents.^32,52–54 Therefore, it is important to improve the efficacy of DNA vaccines against tuberculosis and several strategies have been applied to address this objective.

Use of Vaccine Cocktails

Several groups have applied DNA vaccines in pools of up to 4 different vaccines (e.g., ESAT-6, MPT43, Ag85B, or ESAT-6, MPT63, MPT64, KatG,^22,55). Some combinations gave improvements in protection compared to the individual vaccines. However, BCG was still the most protective vaccine in these studies (Table 2A). Interestingly, formulating Ag85B and MPT64 together with the cytokine GM-CSF did not further improve protection, despite increases in cellular immunity.⁵⁶

Table 2

Strategies to increase efficacies of tuberculosis DNA vaccines.

Characterization of Immune Responses after DNA Vaccination

It is not known with certainty what immune responses equate with protection in tuberculosis. As highlighted in Table 1, DNA vaccination generally induces strong antigen-specific T cell proliferative and type 1 cytokine responses and IFN-γ and class I-restricted CD8 CTL responses tend to be prominent. The level of IFN-γ produced early after infection is certainly one parameter observed in protective immunity resulting from DNA vaccination of mice.^65,66 The breadth of the profile of epitopes recognized by both CD4 and CD8 T cells may be another. For example, CD4 T cells from PstS-1 DNA vaccinated BALB/c mice recognized epitopes on 7 different peptides compared to 5 recognized following M. tuberculosis infection;^20,67 CD4 T cells from M. tuberculosis-infected mice recognized a single Ag85A-derived epitope whereas 8 were recognized in DNA vaccinated mice.⁶⁸ Similarly, the CD8 T cell epitope repertoire of DNA vaccinated mice was also strikingly different and broader than that of M. tuberculosis-infected mice (Ag85A: 3 vs. 0 epitopes, PstS-1: 3 vs. 1 epitope, in DNA vaccinated and M. tuberculosis-infected mice, respectively^20,67,68). The induction, through DNA vaccination, of T cells that are specific for epitopes that are subdominant or cryptic during natural infection, can have a significant impact on protective immunity. For example, it has been recently shown that effective protection could be obtained after vaccination with a peptide comprising a subdominant epitope of ESAT-6 that gave only negligible responses during M. tuberculosis infection, whereas vaccination with a peptide that was a dominant epitope of ESAT-6 during infection was not protective.⁶⁹

Several studies have attempted to define the T cell subsets responsible for protecting mice after DNA vaccination. D'Souza and co-workers⁷⁰ vaccinated a range of CD4, β2-microglobulin and IFN-γ gene-disrupted mouse strains with an Ag85A DNA vaccine. They demonstrated that mice lacking CD4 T cells or unable to produce IFN-γ could not be protected against tuberculosis, whereas β2-microglobulin knock-out mice effectively controlled the disease when vaccinated with the DNA vaccine. These experiments therefore confirmed the importance of IFN-γ produced by CD4 T cells in providing protective immunity against tuberculous infection in mice. The authors concluded that such cells mediate the protective effect of this vaccine independently of CD8 T cells. However, their experiments did not rule out a role for CD8 T cells in Ag85A-mediated protection. Mice with an MHC H-2^b were used and no Ag85A-specific CD8 T cell responses were demonstrable, even in wild-type mice.⁷⁰ This experiment should therefore be repeated in mice of H-2^d background where strong CD8 T cell responses were reported after Ag85A vaccination.

The importance of CD8 T cells that both secrete IFN-γ and lyse M. tuberculosis-infected cells in vitro, has been demonstrated in a series of experiments analyzing T cell clones prepared from M. leprae HSP65 DNA or J774-HSP65 vaccinated mice.^71–73 These CD8CD44^hi activated/memory T cells were able to adoptively transfer protection into naïve recipients. IFN-γ producing, cytolytic CD4CD44^hi clones were also able to transfer protection, although they were less potent than the CD8CD44^hi cells. In contrast, CD8CD44^hi T cells that produced IFN-γ but were not cytolytic were unable to transfer protection. Furthermore, adoptive transfer of protection correlated with the ability of the clones to use the cytotoxic granule-mediated pathway to lyse infected targets; in other systems, T cells that lyse targets by apoptosis may also contribute to mycobacterial killing.⁷⁴

A key question to be addressed is how long protective immunity will persist after DNA vaccination. The rest periods between vaccination and mycobacterial infection, in most cases, do not exceed 10 weeks, and are more frequently around 4–6 weeks. However, a study by Silva and colleagues indicated that mice were still protected after long periods post-DNA vaccination prior to challenge.⁷³ They vaccinated mice with a DNA vaccine expressing mycobacterial HSP65 and challenged them after rest periods of 1, 4, 8, and 15 months. Encouragingly, they demonstrated that mice were significantly protected even if they were challenged after a 15 months rest period. The same was observed for BCG vaccinated mice but the degree of protection after either DNA or BCG vaccination was lower than expected at all intervals. However, CD44^hi T cells that produced high levels of IFN-γ and were cytolytic were found in these mice throughout the 15 months rest period in the DNA vaccinated mice. It is encouraging to demonstrate such long-lived memory in rodent models, but it will obviously be important to determine how DNA-vaccine induced memory responses can be sustained in target species. We have addressed this recently in cattle by studying the kinetics of the immune response following immunization with DNA encoding the M. leprae HSP65. In these studies we determined T cell proliferation and IFN-γ production after vaccination and demonstrated potent proliferative T cell and IFN-γ responses in vitro 2 weeks after the final DNA vaccination (Fig. 2). However, after a 6 weeks rest period, we detected only low frequencies of IFN-γ producing cells and low levels of in vitro produced IFN-γ, whereas we were still able to demonstrate strong HSP65-specific T cell proliferation. This seemingly paradoxical finding is completely in line with current concepts of memory T cell development in mice and humans (e.g., see refs. 75–77): The frequency of IFN-γ producing effector cells declines with the decrease in antigen stimulus over time, to be replaced with low frequencies of effector memory cells producing cytokines like IFN-γ, and central memory cells producing mainly IL-2. It is likely that the latter subset of T cells were those that proliferated but did not produce IFN-γ after the prolonged rest periods. Consistent with this hypothesis, IFN-γ producing effector cells were induced within 3 days of the subcutaneous injection of HSP65 protein (as judged by the amount of and frequency of IFN-γ cells found in the draining lymph nodes of these cows, Fig. 2). Therefore DNA vaccination against tuberculosis in rodent models as well as in a target species is able to induce long-lasting memory responses of the expected type. In conclusion, DNA vaccination against mycobacterial infections has been a potent tool to define protective antigens and to study aspects of protective immunity. It has now reached a stage of development where studies in target species are opportune. In particular we look forward to the exploitation in target host species of strategies like heterologous prime-boost protocols that have improved vaccine efficacies compared to BCG and to the immunotherapeutic applications of DNA vaccines in which role BCG vaccination is not effective.

Figure 2

T cell memory responses in draining lymph nodes in M. leprae HSP65 DNA vaccinated cattle. Six cattle were vaccinated at weeks 0 and 3 with 1 mg of a M. leprae HSP65 expressing DNA vaccine. Blood samples were taken 2 weeks after the second vaccination (more...)

Bacillus anthracis

Bacillus anthracisis the causative agent of anthrax, a potentially lethal gastrointestinal and respiratory disease of domesticated and wild animals, as well as humans. Virulent strains of B. anthracis are characterized by their expression of a polyglutamic acid capsule and the production of a protein toxin. The anthrax toxin has three components (protective antigen, lethal factor and edema factor), although toxicity can be mediated experimentally using a combination of protective antigen (PA) and lethal factor (LF) alone. The PA of anthrax toxin binds to the target cell permitting access of the lethal and edema factors to the cytosol. The current licensed vaccine against anthrax contains PA adsorbed onto aluminum hydroxide. Antibodies to PA prevent binding to the target cell and confer protection from anthrax.

To our knowledge there has only been one report of a DNA immunization approach to anthrax vaccination.⁷⁸ Mice immunized with a plasmid encoding the immunogenic portion of PA-generated splenocytes that secreted IFN-γ and IL-4, consistent with the generation of a mixed Th1/Th2-like response. Immunized mice produced a serum IgG response to PA able to neutralize the biological activity of the protein in vitro and were protected from lethal challenge with a combination of PA and LF. The level of protection varied from 75–100%, depending on the dose of lethal factor administered. Complete protection to five lethal doses of toxin was obtained. It is hoped that a DNA vaccine against anthrax may be safer and more efficacious than the current licensed vaccine.

Borrelia burgdorferi

Borrelia burgdorferi is one of the members of the Borrelia genus of spirochetes responsible for Lyme disease, a chronic disease with arthritic and neurological sequelae if not diagnosed and treated adequately early in infection. The Outer Surface Protein A (OspA) of B. burgdorferi is the most frequent antigen component of vaccines under development for Lyme disease. Mice immunized with DNA expressing OspA generated a serum immunoglobulin response^79,80 that was dependent on both the dose of DNA administered and the promoter used to drive expression of OspA.⁸⁰ Interestingly, antibodies to OspA were generated following immunization with plasmid containing OspA in the reverse orientation,⁸⁰ suggesting that the upstream control region of OspA was active in eukaryotic cells, a fact confirmed in vitro.⁸⁰ All isotypes of murine IgG were generated to OspA DNA immunization, consistent with the proliferative response of spleen and lymph node T-cells to OspA and the production of IFN-γ.⁸⁰ Vaccinated mice were protected from spirochete challenge.^79,80 Immune serum was demonstrated to inhibit growth of B. burgdorferi in culture,⁷⁹ as well as to confer protection to SCID mice against the onset of clinical arthritis following spirochete challenge.⁸⁰ Compared with OspA, DNA vaccination with OspB stimulated a lower titer of serum immunoglobulin and failed to protect mice from challenge, correlating with the inability of the immune serum to inhibit spirochete growth in culture.⁷⁹

Brucella abortus

Brucella sp. are Gram-negative facultative intracellular coccobacilli that infect both animals and humans. Brucella may enter the body via the skin, respiratory tract, or digestive tract. Once there, they can enter the blood and the lymphatics where they multiply inside phagocytes and eventually cause bacteraemia. Symptoms of Brucellosis vary from patient to patient but can include high fever, chills, and sweating. The four species of this genus that can infect humans are principally named after the animal in which they are most commonly found: B. abortus (cattle), B. suis (swine), B. melitensis (goats), B. canis (dogs). B. abortus causes spontaneous abortion and infertility in cattle and is the most common species associated with Brucellosis.

There is currently only one report of a DNA vaccination approach to Brucellosis.⁸¹ In that study, mice were vaccinated once with DNA expressing the ribosomal protein L7/L12, the immunodominant protein of B. abortus identified in primed cattle.⁸² Mice responded to vaccination by splenocyte proliferation and a serum IgG response specific for L7/L12. When challenged 28 days later, vaccinated mice had 0.47 to 1.26 log less bacteria in their spleens compared with controls, depending on the time postinfection. Thirty days postinfection, the protection induced by DNA vaccination was equivalent to that achieved with the live Brucella vaccine-B. abortus strain 19.

Chlamydia spp

There are four species of Chlamydia recognized to date: Chlamydia pecorum, Chlamydia pneumoniae, Chlamydia psittaci and Chlamydia trachomatis. All are obligate intracellular pathogens, causing disease primarily through induction of the host immune response. This feature has hampered efforts to develop a protective vaccine against chlamydial infection. Humans are the predominant host for C. pneumoniae and C. trachomatis, whereas C. pecorum and C. psittaci can infect a variety of hosts, including humans. At least C. trachomatis and C. psittaci can be subdivided into biovariants, depending on both the disease they cause and the host they infect. The four biovariants of C. trachomatis cause trachoma or sexually transmitted infections in humans, mouse pneumonitis, or infections of swine, respectively. Similarly, four biovariants of C. psittaci have been defined, responsible for infections in guinea pigs, cats, ruminants, or birds, respectively. Most chlamydial species can infect the respiratory tract, although C. pneumoniae is the most common agent of chlamydial pneumonia, a potentially life-threatening condition with systemic sequelae. Infection of birds and farm mammals with Chlamydia can result in significant economic losses, as well as presenting an occupational zoonotic hazard. The prevalence of Chlamydia, together with the emergence of antibiotic-resistant isolates, provides the impetus to develop vaccines against chlamydial disease. Candidate antigens for incorporation into a vaccine, include the Major Outer Membrane Protein (MOMP), a cysteine rich outer membrane protein (Omp2), Heat Shock Protein 60 (HSP60), and ADP/ATP translocase (Npt1Cp). Determination of the genome sequences of C. trachomatis and C. pneumoniae ^55,83 has resulted in opportunities for the empirical testing of open reading frames (ORFs) in the context of DNA immunization.⁸⁴

Clostridium tetani

Two studies report the evaluation of DNA immunization for protection against tetanus toxin, the potent neurotoxin synthesized by the anaerobic bacterium Clostridium tetani.^96,97 In both studies, BALB/c mice were immunized intramuscularly with plasmids encoding the non-toxic C-terminal domain of tetanus toxin (fragment C). Anti-fragment C serum immunoglobulin responses were obtained, as well as proliferative responses by cultured splenocytes. Evaluation of the IgG subclasses and cytokines produced by the splenocyte cultures suggested that DNA immunization induced a Th1-like response. In contrast, immunization with tetanus toxoid or a polypeptide of fragment C induced a Th2-like response. In most cases, the serum immunoglobulin response following DNA immunization was sufficient to protect 70–100% of mice from lethal challenge with tetanus toxin. The level of protection was dependent on the amount of DNA used for immunization and the challenge dose. Whilst encouraging, the level of protection conferred by DNA immunization was inferior to that achieved with conventional toxoid or a polypeptide of fragment C.

Corynebacterium pseudotuberculosis

Corynebacterium pseudotuberculosis is the causative agent of caseous lymphadenitis (CLA); a chronic infectious disease of sheep and goats that results in abscesses of the lymph nodes, and occasionally, the internal organs. C. pseudotuberculosis may also cause ulcerative lymphangitis, a mildly contagious disease of horses characterized by inflammation of the lymphatic vessels of the lower limbs. CLA is highly contagious and causes significant economic loss to affected premises. Infection of humans with C. pseudotuberculosis is rare and is most often associated with skinning infected animals or the consumption of unpasteurized milk from such animals.

A DNA vaccination trial against CLA was recently conducted in sheep⁹⁸ and used the novel approach of directing the antigen to sites of immune induction by expressing it as a fusion protein with bovine CTLA-4. This approach had previously been shown to enhance both the speed and magnitude of the immune response to human immunoglobulin.⁹⁹ The phospholipase D gene of C. pseudotuberculosis was genetically detoxified (PLD) and cloned into two DNA plasmids, designed (and confirmed in vitro) to secrete PLD with or without targeting to CD80/86-positive, antigen-presenting cells. A significant antibody response was generated only in those sheep vaccinated with DNA expressing the targeted CTLA-4-PLD fusion. Animals were challenged above the coronet with 10⁶ C. pseudotuberculosis six weeks after the primary immunization and killed six weeks later. The lymph nodes draining the site of challenge were inspected post mortem for the presence of abscesses. A proportion of animals that received either PLD construct as DNA vaccine were completely protected from abscess formation: 45% in the case of the non-targeted vaccine; 70% with the targeted vaccine. This compared favorably to the level of protection (90%) obtained with a commercial vaccine comprised of formalinin-activated toxins and PLD.

Enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) is one of the most common causes of acute diarrhea in children in developing countries and in travelers who visit those areas. ETEC is also one of the most important pathogens of pigs, causing diarrhea, edema disease or colisepticaemia. Essential virulence factors for the pathogenicity of ETEC include the enterotoxins and the Colonisation Factor Antigens (CFAs), which comprise fimbrial and nonfimbrial adhesins responsible for the adhesion of ETEC to enterocyte receptors. Studies of DNA vaccination for ETEC are restricted to the CFA/I fimbriae of human ETEC serogroups (encoded by cfaB) and FaeG, the major protein subunit of K88 fimbriae expressed by a subset of pig ETEC serogroups. Mice given a single inoculation of DNA expressing the CFA/I fimbrial adhesin mounted a serum IgG response, although the antibodies were unable to agglutinate bacterial cells or inhibit the haemagglutination (HA) promoted by CFA/I-bearing ETEC.¹⁰⁰ The serological response to CFA/I DNA vaccination was subsequently shown to be predominantly of the IgG2a isotype, compared with the predominantly IgG1 response induced by immunization with purified CFA/I protein subunits.¹⁰¹ This group went on to show that the magnitude, isotype, and specificity of the serological response to CFA/I was dependent on the number of DNA inocula administered¹⁰² as well as the plasmid used. Fusion of the cfaB gene to the tissue plasminogen activator protein (tPA) signal sequence resulted in the secretion of CFA/I protein from transfected cells¹⁰³ and vaccination with this construct induced serum IgG capable of inhibiting the HA of CFA/Ipositive bacteria.^103,104 Cellular responses to CFA/I protein were demonstrable with all plasmids, but not IgA in gut mucosa or feces.¹⁰⁴ In order to generate mucosal IgA mice were primed with CFA/I plasmid DNA, followed by intragastric inoculation with recombinant Salmonella expressing CFA/I protein. The combined regime of DNA prime-Salmonella boost, generated serum IgG and fecal IgA responses greater than achieved with either DNA or recombinant Salmonella alone.¹⁰⁵

In the pig ETEC study,¹⁰⁶ mice immunized with DNA encoding the faeG gene of ETEC K88ab produced a serum IgG2a response. Three pregnant sows were subsequently immunized with 200μg, 400μg or 1128μg total DNA. Neither of the sows receiving the lower doses of DNA seroconverted, although an antibody response was reported in the colostrum of the first sow. A serum IgG response was detected in the sow that received the highest dose of DNA, although it aborted 48 hours after the second DNA inoculation. Antibody titers in the surviving offspring were reportedly higher than those of their respective mothers, although these data were not presented in the paper.

Francisella tularensis

Francisella tularensis is the causative agent of tularaemia. There are five forms of clinical tularaemia. The most common (80% of cases) is ulceroglandular tularaemia with symptoms of fever, headache, lymphadenopathy and characteristic ulceration of the site of inoculation. Tularaemia carries a mortality rate of 5–15%, even higher with the typhoidal form, but antibiotics lower this rate to about 1 %. Many wild animals and some domestic animals can harbor F. tularensis, although the rabbit is the species most often involved in transmission. Infection can be acquired through numerous routes. The most common routes include: inoculation of the skin or mucous membranes with blood or tissue while handling, dressing or skinning infected animals, contact with fluids from infected flies or ticks, the bite of infected ticks or handling or eating insufficiently cooked contaminated meat.

An attenuated F. tularensis live vaccine strain (LVS) is available for those most at risk of infection, so there has been little effort to develop a DNA vaccine for tularaemia. However, lethal infection of mice with F. tularensis LVS has been used as a model with which to study the protective host response to noneukaryotic DNA.¹⁰⁷ BALB/c mice were injected with 0.01–20 μg chromosomal DNA isolated from F. tularensis and then challenged with different doses of F. tularensis at various time points after immunization. Protection was dependent on the dose of DNA administered, the challenge dose and time-to-challenge, but not the route of immunization. Protection was shown to be dependent on the unmethylated state of the bacterial DNA. Synthetic unmethylated CpG-rich oligonucleotides (CpG.ODN) are common in prokaryotic genomes but rare in eukaryotes. Their ability to induce the innate immune response is well documented, and as such, CpG.ODN have attracted much interest as mediators of vaccine enhancement (reviewed in 108; see section on Listeria monocytogenes). The importance of unmethylated CpG in F. tularensis DNA for the induction of short-term protection was demonstrated using CpG-containing ODN. Immunization with unmethylated CpG-ODN, but not methylated, conferred 100% protection against lethal challenge with either F. tularensis or L. monocytogenes. Using IFN-γ gene-knockout (GKO) mice, the protection obtained with either unmethylated F. tularensis DNA or CpG-ODN was shown to be dependent on IFN-γ, T-cells and critically, B-cells. Importantly, long-term pathogen-specific immunity was demonstrated in mice that had survived a previous lethal challenge through immunization with bacterial DNA.

Helicobacter pylori

Helicobacter pylori colonizes the gastric mucosa of more than half the world's population, causing asymptomatic gastritis in the majority. However, up to 10% of infected individuals develop clinical disease, ranging from gastric and duodenal ulcers to adenocarcinoma of the stomach and lymphoma of mucosaassociated lymphoid tissue. The current standard treatment for H. pylori infection consists of antibiotics administered with an inhibitor of gastric acid secretion. The increasing resistance of H. pylori to antibiotics makes vaccination an attractive alternative, although complete elimination of the organism may be undesirable given the possible association of H. pylori colonization with reduced incidence of esophageal diseases. The latter observation has likely tempered enthusiasm for H. pylori vaccination and, in fact, only one study has been published on a DNA approach to vaccination.¹⁰⁹ Immunization of mice with plasmid encoding either the Heat Shock Protein (Hsp) A or B of H. pylori stimulated an antigen-specific serum IgG and IgA response. Differences in the relative levels of IgG to IgA and of IgG2a to IgG1 isotypes were observed between the response to Hsp A and Hsp B. DNA vaccination conferred protection against H. pylori replication in the stomachs of mice infected three months after vaccination and examined six months after challenge. Vaccinated mice also had less antral gastritis, as determined histologically. The protection afforded by Hsp B was greater than with Hsp A by both criteria.

Leptospira interrogans

Leptospirosis is the most widespread bacterial infection in the world, affecting both humans and animals. Zoonotic infection of humans with Leptospira interrogans responds well to treatment, but rarely infection may cause the acute life-threatening (Weil's) disease, resulting in jaundice and severe damage to the body organs. The protection conferred to guinea pigs by DNA vaccination with the seroreactive P68 antigen¹¹⁰ is, to our knowledge, the only published report of a DNA vaccine approach to leptospirosis. In that study, 77% of vaccinated guinea pigs were protected from death and 85% from pulmonary diffuse hemorrhaging (PDH), following challenge with L. interrogans. In contrast, 75% of guinea pigs vaccinated with vector plasmid alone died and nine out of ten had PDH post mortem.

Listeria monocytogenes

Listeria monocytogenes is the only one of seven species of Listeria that is pathogenic to humans. Diarrhea can follow ingestion of organisms via contaminated food, although infection is rare in normal populations. However, in individuals made susceptible by age, pregnancy, or immunosuppression, infection can lead to encephalitis, meningitis, stillbirth, and high mortality.

Infection of mice with L. monocytogenes is an established model for studying host antibacterial defense mechanisms (reviewed in ref. 111). The most extensively studied protective antigen in murine listeriosis is Listeriolysin O (LLO). Protection of BALB/c mice against challenge with L. monocytogenes can be mediated by CD8 T cytotoxic lymphocytes that recognize an H-2K^d restricted epitope from amino acids 91 – 99 of LLO.¹¹² It is not surprising, therefore, that the majority of studies on DNA vaccination against L. monocytogenes have included this antigen. However, with the possible exception of Mycobacteria, the strategies adopted for DNA vaccination against L. monocytogenes are amongst the most diverse and novel.

The first publication of a DNA vaccination approach to L. monocytogenes described the novel use of an attenuated strain of Salmonella to deliver DNA vaccine plasmids via the oral route.¹¹³ In this way, plasmids encoding truncated L. monocytogenes genes actA and hly (encoding LLO) were most likely transferred from the Salmonella organisms to host antigen-presenting cells, resulting in CD8CTL activity, CD4 T-cell proliferation and the release of IFN-γ, as well as serum IgG and IgA responses.¹¹³ Mice vaccinated with recombinant Salmonella containing the hly plasmid, but not the actA plasmid, were protected from a 10 × LD₅₀ challenge with L. monocytogenes.

Despite the protective nature of the 91–99 epitope of LLO, vaccination of BALB/c mice with DNA encoding the epitope failed to induce an immune response or confer protection against challenge.¹¹⁴ However, when the plasmid was reconstructed so that the codon usage of the epitope was optimized for expression in mice, a CTL response was induced, mediated by IFN-γ-secreting CD8 T lymphocytes.¹¹⁴ Furthermore, immunization with the optimized 91–99 epitope plasmid gave partial protection against sublethal challenge with L. monocytogenes. This study emphasized that the optimization of codon usage to improve the efficiency of antigen gene translation in the target species is an important consideration in constructing DNA vaccines against nonviral pathogens. Using DNA constructs containing other epitopes of LLO, these workers demonstrated further that the magnitude of protection was related to the level of CTL induction.¹¹⁵

In contrast to the protective efficacy observed in the studies using Salmonella as a surrogate vector for DNA vaccines described above, a study using the whole hly gene for intramuscular DNA vaccination found the immune response to be poor and unable to confer significant protection against challenge.¹¹⁶ Since LLO encoded by hly is a cytotoxin, these workers reasoned that expression of whole LLO by antigen-presenting cells might result in autotoxicity and failure to induce protective immunity. A variety of plasmids were therefore constructed, expressing either mutant versions of LLO with reduced toxicity, or engineered to secrete the wild-type or mutant LLO (by virtue of fusion to the murine tPA signal sequence). In adoptive transfer protection experiments, the mutant form of LLO gave a ˜0.6 log improvement in protection, the secreted wild-type form a ˜0.3 log improvement, and the secreted mutant form a ˜1.9 log improvement compared with the nonsecreted wild-type form of LLO.¹¹⁶ However, in the vaccinated animal itself, only the secreted mutant form of LLO conferred protection to challenge.

Gene-gun immunization using LLO and p60 of L. monocytogenes has been used to examine the ability of CpG-ODN to act as an adjunct to DNA immunization, in addition to boosting or co-administration of plasmid encoding GM-CSF.¹¹⁷ An optimal vaccination schedule utilized LLO with or without p60 plus co-injection of CpG-ODN. With this schedule over 3 log protection was obtained, representing sterile elimination of L. monocytogenes from the spleen of challenged mice¹¹⁷ and demonstrating that CpG-ODN can improve suboptimal vaccination protocols. In another study, administration of CpG-ODN alone to mice was sufficient to confer 100 % protection against lethal challenge with L. monocytogenes three days later.¹⁰⁷

Mycoplasma pulmonis

Mycoplasma are minute pleomorphic Gram-negative microorganisms without cell walls that are intermediate in some respects between viruses and bacteria.Mycoplasma pneumoniae is an important human pathogen and mycoplasmoses among farm animals are responsible for considerable economic losses. Murine respiratory mycoplasmosis caused by M. pulmonis is one of the most important pathologies for laboratory rodents and is comparable to the pathological manifestations of M. pneumoniae in humans and of other animal respiratory mycoplasmoses. The experimental infection of mice with M. pulmonis has been used as a model for two novel approaches to DNA vaccination.^118,119 The first was termed Expression Library Immunization (ELI), in which an expression library of M. pulmonis DNA was used to vaccinate mice in order to identify protective antigens in an empirical fashion. The small genome size of M. pulmonis was ideal to prove the principle behind this approach. The second, and only other report of DNA vaccination against Mycoplasma, was to our knowledge the first to demonstrate the therapeutic potential of DNA immunization.

Using the ELI approach, the entire M. pulmonis genome was represented in each reading frame by nine libraries of about 3000 members. Injection of two of the nine libraries intradermally into groups of mice resulted in serum antibody and DTH responses to M. pulmonis antigens. Complete protection to M. pulmonis challenge was obtained with either library.¹¹⁸ A Listeria monocytogenes ELI library was also constructed and used a negative control. Protection against L. monocytogenes challenge was not examined. The advantage of the ELI approach is that a DNA vaccine can be constructed without any prior knowledge of the pathogen's biology or of the suitable target genes to use for immunization. However, the approach may only be suitable for pathogens with genomes of manageable size and ultimately the genes responsible for protection must be dissected from the library and tested individually.

The therapeutic potential of DNA immunization in bacterial infections was reported twice, first for M. pulmonis¹¹⁹ and subsequently, for Mycobacterium tuberculosis.^45,46 The latter papers are reviewed earlier in this chapter. In the M. pulmonis study, cellular and humoral immune responses were generated in mice immunized with DNA encoding M. pulmonis antigens A7-1 or A8-1, administered either singly or in combination. For the protection study, mice received a combination of A7-1 and A8-1 either one week prior to M. pulmonis challenge (vaccination) or one week after (therapy). Further immunizations were administered two more times at two-week intervals after the first immunization. Mice were killed monthly after infection and the extent of disease determined histopathologically and by enumerating bacteria in tracheo-lung lavage. Following either vaccination regime, the number of M. pulmonis recovered by lavage decreased monthly until no bacteria were cultured at four months. The continued reduction in CFU was accompanied by reduction in the severity of lung inflammation.

Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen, commonly associated with cystic fibrosis patients, immunocompromised individuals, including AIDS patients, and humans and animals that have a breach in their skin barrier caused by burn or wounding. Infection with P. aeruginosa is a significant cause of death in domestic animals. A number of virulence factors are secreted by Pseudomonas. The most toxic protein produced by Pseudomonas is Exotoxin A, which shares its mode of action (ADP-ribosylation) with diphtheria toxin. Neutralization of Exotoxin A toxicity by DNA immunization was the aim of two recent studies in which the coding sequence for non-toxic mutant forms of P. aeruginosa Exotoxin A were cloned into DNA expression vectors.^120,121 Both studies reported a serum immunoglobulin response to Exotoxin A following DNA immunization. Analysis of the IgG isotype and cytokine secretion by splenocytes suggested that vaccination favored a Th1-type response.¹²¹ DNA vaccinated mice in both studies were completely protected from the lethal effect of intraperitoneal injection with wild-type Exotoxin A, consistent with the demonstration of Exotoxin A neutralization by immune sera in vitro.¹²¹

While neutralization of Exotoxin A activity following DNA immunization would not be expected to prevent infection with P. aeruginosa, it might prevent certain pathology associated with infection. The coding sequence for a non-toxic mutant form of Exotoxin A could, therefore, be an important constituent of a multicomponent P. aeruginosa DNA vaccine. An additional candidate for such a vaccine cocktail could be the oprF gene of P. aeruginosa, encoding Outer Membrane Protein F. Immunization with this protein has been shown to confer some protection in a variety of rodent models of P. aeruginosa infection.^122–124 Mice immunized with a DNA plasmid encoding oprF generated an IgG1 serum response to both OprF and OprH of the homologous strain of P. aeruginosa, and to heterologous immunotypes.¹²⁵ The immune serum also mediated opsonophagocytic uptake of P. aeruginosa by peripheral blood mononuclear cells in culture. In a chronic pulmonary infection model, DNA vaccinated mice showed a significant reduction in the number of severe lung lesions and the number of mice with lungs yielding >5000 CFU of P. aeruginosa compared with controls.¹²⁵

Salmonella typhi

Salmonella typhi causes typhoid fever, a severe systemic disease characterized by fever and abdominal symptoms, including hemorrhage, which is the principle cause of death in such infections. If untreated, typhoid fever has a 10–20% mortality rate. S. typhi infects only humans and chimpanzees, making study of the infection difficult. However, under certain experimental conditions it is possible to infect animals such as mice and mimic some aspects of the natural infection. The murine model has been used to evaluate a DNA immunization approach to prevent S. typhi infection.¹²⁶ In these studies, BALB/c mice injected with DNA expressing the Outer Membrane Protein C (OmpC) Porin of S. typhi produced a serum IgG response specific to the protein. Responses were greater to OmpC expressed with its leader sequence than without. Since vaccination of mice with recombinant OmpC induces protection from S. typhi in mice.¹²⁷ The preliminary DNA immunization results were taken as encouraging, although to our knowledge, no further studies have been reported.

Staphylococcus aureus

Contamination of food by toxins produced by Staphylococcus aureus is the major cause of food-borne intoxication. Symptoms are associated with acute gastroenteritis and mortality is less than 1%. Since S. aureus is part of the normal respiratory and skin flora of approximately 30–40% of people collectively, contamination of food with S. aureus most commonly originates from the foodhandler. More significantly, such transmission may occur nosocomially from hospital personnel to susceptible patients by direct contact. This is of particular concern with regard to the transmission of methicillin-resistant S. aureus (MRSA) to immunocompromised individuals. More than 90% of MRSA isolates produce the penicillin-binding protein PBP2', encoded by the mecA gene and carried on a foreign region of DNA integrated into the chromosome of MRSA. The source of mecA is not known but it may originate from other genera of bacteria or from within the Staphylococci.¹²⁸ Possession of mecA and expression of PBP2' distinguishes MRSA from methicillin-susceptible isolates.

A DNA vaccination approach to prevent MRSA infection was reported recently.¹²⁹ In this study, DNA encoding the mecA gene was used to immunize BALB/c mice three times at two-week intervals. Immunization resulted in a serum immunoglobulin response to PBP2', demonstrated by immunoblotting and ELISA. The immune serum enhanced the phagocytosis of PBP2'-positive MRSA by peritoneal macrophages. Fourteen days after the final immunization, the mice were challenged intravenously with 10⁸ CFU MRSA 1191. Eight days after challenge, the kidneys were removed and the bacterial load determined. Vaccination with mecA plasmid gave a 0.4 log reduction in kidney CFU compared with controls, although this difference was significant statistically. This preliminary study suggests that PBP2' might be a target molecule for vaccination against MRSA.

Streptococcus pneumoniae

Despite the introduction of antibiotics, infection with Streptococcus pneumoniae still causes significant human mortality and morbidity through the induction of pneumococcal pneumonia, septicaemia, meningitis and otitis media. Most clinical isolates of S. pneumoniae express capsular polysaccharide that protects the organism from phagocytosis and represents the major virulence determinant of the organism. Ninety chemically and antigenically distinct capsular polysaccharides have been identified, forming the basis of the serotypic classification of S. pneumoniae. Recent efforts at vaccination have focused predominantly on the production of conjugate vaccines in which polysaccharide antigens are linked to a protein carrier in order to generate protective antibodies in recipients. DNA immunization against S. pneumoniae has not been rigorously pursued, although one study has reported the protection of mice from lethal pneumococcal septicaemia following immunization with a plasmid expressing pneumococcal surface protein A (PspA).¹³⁰ PspA is expressed on the surface of all serotypes of S. pneumoniae and is required for full virulence. Only 30% of BALB/c mice immunized with the plasmid expressing PspA produced a serum immunoglobulin response to PspA protein, although the isotype of immunoglobulin was not reported. Interestingly, the generation of this response did not correlate with protection from challenge with 20 lethal doses of virulent S. pneumoniae. None of the mice generated a mucosal response to PspA, as determined serologically using saliva and feces. Immunization with the plasmid protected 40% of mice from death for up to 150 hours after challenge. All of the control mice were dead before 100 hours following challenge. Protection was also expressed in a significant reduction in the number of S. pneumoniae cultured from the blood. The efficacy of DNA immunization was less than that achieved with PspA protein, which had been shown to protect >90% of mice from challenge with more than one serotype of S. pneumoniae.^131,132

Yersinia spp

The genus Yersinia contains three pathogenic species: Yersinia enterocolitica, Yersinia pseudotuberculosis and Yersinia pestis. The first two species cause food-borne illness. Y. enterocolitica causes gastroenteritis characterized by fever and abdominal pain often accompanied by diarrhea and/or fever. Y. pseudotuberculosis typically causes an acute mesenteric lymphadenitis. Y. pestis is the causative agent of ‘the plague’, one of the most devastating infectious diseases known to man. Even though the three species use different routes to infect their host and cause diseases of different severity, they share a common tropism for the lymphoid tissue and are able to resist the primary immune defense mechanisms of the host. Only one immunogenicity study has been reported using DNA encoding the V antigen of Y. pestis.¹³³ Two challenge studies have been reported for Y. enterocolitica,^134,135 the latter study also investigated the outcome of challenge with Y. pseudotuberculosis.¹³⁵

Y. enterocolitica and Y. pseudotuberculosis

The Heat-shock Protein 60 (HSP60) of Y. enterocolitica is a protective antigen in mouse models of yersiniosis and invokes both the humoral and cellular arms of the immune response.¹³⁶ Vaccination of mice with a DNA plasmid encoding HSP60 induced a Th1-type response characterized by an IgG2a serum antibody response, antigen-specific splenocyte proliferation and the liberation of IFN-γ, but not IL-4 or IL-10.^134,135 Mice immunized with the HSP60 DNA vaccine were protected from challenge with a lethal dose of Y. enterocolitica administered either intravenously^134,135 or orally¹³⁵ For those mice infected orally, protection was expressed at the level of a reduction in bacterial burden in the spleen but not locally at the site of mucosal entry. By using knockout mice, protection was found to be dependent on both CD4 and CD8 T-cells.¹³⁵

Table 3

Protection of mice against yersiniosis requires a Th1-type response, characterized by the interplay of T-cells and macrophages, and the production of IFN-γ. Therefore, in an effort to augment the response of mice to DNA vaccination, the HSP60 DNA plasmid was engineered to coexpress the gene for IL-2, IL-4 or IFN-γ.¹³⁴ In each case, the cytokine gene was separated from hsp60 by an internal ribosomal entry sequences (IRES) to facilitate equimolar expression of both the cytokine and HSP60. Coexpression of IFN-γ with HSP60 enhanced both splenocyte proliferation and the production of IgG. Coexpression of IFN-γ, but not IL-2 or IL-4, also enhanced protection against challenge by a further 1.58 log, compared with the HSP60 plasmid alone.

Since the HSP60 is highly conserved across bacterial genera, mice vaccinated with the HSP60 of Y. enterocolitica were also challenged with a variety of different bacteria, including Y. pseudotuberculosis, to determine whether any cross-protection had been generated following vaccination. No cross-protection was observed, even to Y. pseudotuberculosis.¹³⁵