Bacterial toxin's DNA vaccine serves as a strategy for the treatment of cancer, infectious and autoimmune diseases

Accepted Manuscript Bacterial toxin's DNA vaccine serves as a strategy for the treatment of cancer, infectious and autoimmune diseases Elham Behzadi, Raheleh Halabian, Hamideh Mahmoodzadeh Hosseini, Abbas Ali Imani Fooladi PII:

S0882-4010(16)30387-4

DOI:

10.1016/j.micpath.2016.09.017

Reference:

YMPAT 1957

To appear in:

Microbial Pathogenesis

Received Date: 16 July 2016 Revised Date:

18 September 2016

Accepted Date: 21 September 2016

Please cite this article as: Behzadi E, Halabian R, Hosseini HM, Imani Fooladi AA, Bacterial toxin's DNA vaccine serves as a strategy for the treatment of cancer, infectious and autoimmune diseases, Microbial Pathogenesis (2016), doi: 10.1016/j.micpath.2016.09.017. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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1

Bacterial Toxin’s DNA Vaccine Serves as a strategy for the Treatment of Cancer,

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Infectious and Autoimmune diseases

3 Elham Behzadi1, Raheleh Halabian1*, Hamideh Mahmoodzadeh Hosseini1, Abbas Ali Imani

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Fooladi1*1

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Applied Microbiology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran

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* Corresponding Authors:

Abbas Ali Imani Fooladi, Applied Microbiology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran. Email: [email protected] and [email protected]

Raheleh Halabian, Applied Microbiology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran. Email: [email protected] and [email protected]

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17 Absract

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DNA vaccination –a third generation vaccine- is a modern approach to stimulate humoral and

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cellular responses against different diseases such as infectious diseases, cancer and

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autoimmunity. These vaccines are composed of a gene that encodes sequences of a desired

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protein under control of a proper (eukaryotic or viral) promoter.

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Immune response following DNA vaccination is influenced by the route and the dose of

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injection. In addition, antigen presentation following DNA administration has three

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different mechanisms including antigen presentation by transfected myocytes, transfection

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of professional antigen presenting cells (APCs) and cross priming.

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Recently, it has been shown that bacterial toxins and their components can stimulate and

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enhance immune responses in experimental models. A study demonstrated that DNA

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fusion vaccine encoding the first domain (DOM) of the Fragment C (FrC) of tetanus

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neurotoxin (CTN) coupled with tumor antigen sequences is highly immunogenic against

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colon carcinoma. DNA toxin vaccines against infectious and autoimmune diseases are less

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studied until now.

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All in all, this novel approach has shown encouraging results in animal models, but it has to go

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through adequate clinical trials to ensure its effectiveness in human. However, it has been proven

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that these vaccines are safe, multifaceted and simple and can be used widely in organisms which

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may be of advantage to public health in the near future.

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This paper outlines the mechanism of the action of DNA vaccines and their possible application

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for targeting infectious diseases, cancer and autoimmunity.

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Key words: DNA vaccines; infectious diseases; cancer; autoimmunity

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40 41 High Light:

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• DNA Toxin Vaccine and Bacterial Toxin

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• DNA Toxin Vaccine as a Treatment Option

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Contents 1. Introduction 2. Bacterial toxins and immune system responses 3. Mechanism of action of DNA toxin vaccine 4. DNA toxin vaccine for treatment of infectious diseases 5. DNA toxin vaccine for cancer therapy 6. DNA toxin vaccine for autoimmune diseases 7. Concluding remarks and future perspectives 8. Acknowledgement

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1. Introduction

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The recent concern of researchers is treating the infectious diseases, cancer and autoimmunity by

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developing new vaccines that help to elicit stronger immune system responses [1-3]. The novel

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strategy is to use genetic vaccines composed of DNA or RNA, which have immune stimulating

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properties with no infection risk. On the other hand, the production of new vaccines must be

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economical and safe. Great improvements in biotechnology and vaccine production have opened

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the way to construct "smart vaccines" in the coming future [4]. Innovation of naked plasmid

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vectors expressing desired gene in the host cells encouraged the scientists to use this approach

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for curing patients, but naked DNA is poorly immunogenic and it is important to be injected in

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an efficient way that delivers into the target cells [5]. Investigations have shown that there are

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some parasitic sequences in vertebrates' genome revealing the occurrence of genomic invasions

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in their ancestors. This finding shows that human immune system may not recognize naked

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delivered DNA in vaccine unless it is translated into specific proteins; which must be processed

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and presented by the professional antigen-presenting cells (APCs) to elicit an antigen-specific

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immune response [6]. One of the advantages of DNA vaccination is providing sustained

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exposure of APCs to low levels of antigen to develop appropriate responses [7].

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A number of research studies in 1990s [8, 9] showed the growing interest for DNA vaccine

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technology, but the most prominent research made by Wolff et al. proving that direct

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intramuscular administration of DNA and genetic information led to expression of transgene and

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stimulated immunological response within the muscle cells [10]. Since then the naked plasmid

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DNA has been used as a tool to treat many diseases. This approach can induce antibodies as

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well as cellular responses and can alter cytokine profile. This technology has not the

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shortcomings of viral vectors in stimulating unfavorable immune response and the risk of

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mutagenesis [11].

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On the other hand, the delivery mode of DNA vaccines determines the level of induced

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immunization in an individual. Therefore, new generation of DNA vaccines are co-delivered

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with Micro- or Nano-particles [11]. Recently DNA vaccine encoding certain domain of a

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bacterial exotoxin has been demonstrated to enable translocation from the extracellular part into

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the cytoplasm and therefore greatly enhance vaccine potency for curing diseases such as cancer

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[12], infectious diseases [13] and autoimmune diseases [14]. In some cases the injection of DNA

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and sequential expression of the gene may induce cytotoxic T lymphocytes (CTLs) to demolish

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transfected cells [15] or in some cases may cause anti-DNA antibody response and lead to Lupus

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like syndrome [16].

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This review focuses on the opportunities offered by DNA toxin vaccines for the treatment of

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diverse diseases. The applications and the mechanisms of action of some most promising

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therapeutic DNA toxin vaccines are also described in this review to open the way for designing

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new toxin-derived molecules for medical treatments.

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2. Bacterial Toxins and Immune System responses

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Bacterial toxin is a virulence factor, mostly deleterious in human and other bacterial host

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produced by both gram positive and gram negative bacteria. These toxins target surface receptors

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on cells and injure cells or sometimes kill the host [17]. Bacterial toxins can modulate signaling

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pathways implicated in different physiological events [18]. The host immune system adopts

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different strategies to combat the effect of toxins and most efficient mode is producing the

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antitoxin antibodies. These antibodies are of essential importance against the bacteria. Normally,

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detoxified bacterial toxins are treated with chemical agents or modified by genetic handling to develop various vaccines [19].

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Bacterial toxins can also elevate the immune responses to other irrelevant antigens specifically

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when delivered by mucosal routes [20]. Nowadays bacterial toxins and their nontoxic

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components are used as DNA fusion vaccines and mucosal adjuvant in subunit vaccines against

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various diseases such as infections [21], autoimmune diseases [22] and cancer[23].

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Bacterial toxin affects immediately innate or non-specific immune system but can also activate

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adaptive or specific (antigen-specific T and B cells) immune system [19].

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Immunogenicity of killed and attenuated bacterial vaccines is partly assigned to adjuvant activity

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of toxins in association with other cell wall components of bacteria [24].

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DNA fusion gene vaccines are poorly immunogenic but by the fusion to the bacterial toxin

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genes, their immunogenicity can be enhanced and stimulate the innate immune system,

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effectively. In this approach, the main effort is to modify toxin gene in a way that their toxicity

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diminish but still are able to elicit immune responses [25]. In a study, Pizza et al. produced

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several Escherichia coli heat-labile enterotoxin (LT) mutants with site directed mutagenesis

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which were completely non-toxic or with highly reduced toxicity but their adjuvanticity

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remained very strong at mucosal level [26]. Then Arrington et al. designed two plasmid vectors

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encoding the A and B subunits of LT for assessing their potential as a genetic adjuvant for fusion

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DNA vaccine which strongly enhanced T helper 1 (Th1) and T helper 2 (Th2) cytokine

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responses in animal models [27].

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The innate immune system recognizes and activates by the interaction of specific molecules on

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microbial pathogens (pathogen-associated molecular patterns or (PAMPs)) with the

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recognition receptors on macrophages and dendritic cells (DCs) called pattern recognition

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receptors (PRRs). These cells function as antigen-presenting cells (APCs) for T cells [28]. Some

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microbial pathogen receptors are CD14 receptors, Toll-like receptors (TLRs) [29] (Fig 1.), Fc

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receptors, complement receptors, mannose binding protein, serum amyloid P and C-reactive

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proteins [30]. Each microbial product is recognized by a specific receptor; for instance NOD-like

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receptor (NLR) recognizes bacterial toxins [31] (Fig 1.) and TLR9 recognizes CpG motifs in

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bacterial plasmid DNA [32].

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As it is shown in figure 1, PAMPs are recognized by TLRs, and then the synthesis of Pro-

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Interleukin (IL)-1β is stimulated through nuclear factor kappa-light-chain-enhancer of

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activated B cells (NF-κB). One of the means that bacteria and bacterial products can go through

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cell membrane is via pore-forming toxin. Cytosolic PAMPs activates NOD-like receptors

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(NLRs) which cause formation of caspase-1-activating inflammasomes. The inflammasome

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adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC) is

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recruited to activate caspase-1. The role of caspase-1 is to induce the cell necrosis or pyroptosis

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and usually interfered with various developmental stages [31].

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When bacterial toxin binds to NLR, signals convert through an adaptor protein and some

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immune response genes and co-stimulatory molecules are transcribed by transcription factors.

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When PRRs like toxins ligate on APCs, the intensity of antigen presentation in these cells

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enhance and elicit the T cell-mediated responses. As the protective immunity depends on

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induction of T helper (Th1 or Th2) responses, the Th2 cells mediate immunity against

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extracellular bacteria and function as helper for the production of neutralizing antibodies such as

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IgG, IgE and IgA against bacterial toxins. Macrophages and DCs secrete IL-12, IL-18, IL-23 and

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IL-27 in response to some microbial products [33].

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When DCs are activated by bacterial products especially gram negative bacterial

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lipopolysaccharides (LPS) the differentiation of T helper cells from immature precursors occur

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[34]. In response to some bacterial toxins, DCs may release IL-10 and IL-12 production is

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hindered which subsequently induce IL-10-secreting regulatory T cells (Tregs). Tregs are a kind

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of T cells with regulatory and suppressive function that control the Th cells and inflammatory

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responses through attacking the foreign antigen. In this case, the signal for maturation of the

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DCs may be supplied by interaction between bacterial toxins and PRR and activation of

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signaling pathways launch the transcription of genes involved in the immune responses. The

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maturation of DCs and activation of signaling pathways are essential for the onset of T cell

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responses [35]. Immature DCs in contact with foreign antigens in tissues produce chemokine

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receptors CCR5, CCR6, CD80, CD86 and class II major histocompatibility complex (MHC) and

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in this level these cells catch the foreign antigens and engulf them which cause to

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downregulation of CCR5 and CCR6 expression and CCR7 upregulation. In this stage DCs are

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placed in the lymph nodes and become mature [36]. CpG motifs which are prevalent in bacterial

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DNA or LPS may serve as maturation signal for these cells [37]. As DCs mature, the uptake and

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process the antigen by the DCs diminish but the expression of CD80, CD86 and class II MHC

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molecules elevate the antigen presentation potency. For performing as APCs for Th1 and Th2,

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DCs must be influenced by the activation signals from pathogens [6]. Some toxins like cholera

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toxin (CT) produced by Vibrio cholera composed of an enzymatic part (A subunit) and a

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receptor binding part (five copies of the B subunit) can activate DCs which in turn induce

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Th2 cells while LPS initiate maturation of DCs that activate Th1. Some bacterial products which

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work as immunomodulatory molecules can elicit acquired immune responses. These molecules

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can control cytokine production and maturation of DCs by directing T cell differentiation [38].

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Interaction of the antigen-specific T cell receptor (TCR) with peptide-MHC and co-

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stimulation of CD28 molecules on the T cells and CD80 or CD86 on APCs activate T cells

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of the acquired immune system [39]. As TCRs have not enough intracellular domains, they

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associate with CD3 molecules and form TCR/CD3 complex. This complex activates

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transcription of cytokine related genes such as IL-2. For the production of IL-10, inducible co-

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stimulator (ICOS) that is expressed on activated T cells binds to B7-H2 on APCs. Other

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interactions between activated T cells and other co-stimulatory molecules on APCs are possible

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to form immune synapse. Some bacterial derived molecules can elevate or demolish the co-

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stimulation of T cells by APCs. Therefore for increasing immune system responses, detoxified

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bacterial components such as bacterial toxin are employed as adjuvants in biotechnology and

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vaccine development. Bacterial toxins bind to the T cells and activate them in a polyclonal

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fashion. Studies show that AB toxins like Pertussis Toxin (PT) and CT can interact with T cells

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and modify cytokine secretion [40].

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Moreover, CTLs that are important functional immune effectors which recognize bacterial

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components on the surface of contaminated cells play significant role in elimination of bacteria.

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In contrast to CD4+ T cells that identify exogenous antigen in cooperation with class II MHC

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molecules, CTL identify endogenous antigen in cooperation with class I MHC molecules. To

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elicit CTL response, exogenous antigens have to go through an endogenous processing pathway

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to be presented to class I-restricted T cells. Studies have shown that bacterial toxins fused with

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foreign antigens can activate class I processing pathway [40, 41].

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As mucosal tissues are the main routes of pathogen entry into the host, nasal or oral delivery of

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vaccines and induction of local immunity at these sites is the best way to prevent some infectious

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diseases. Mucosal vaccination improves local immune responses and the effect of parenteral

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immunization. Mucosal defense is including the epithelium, mucus, innate and adaptive immune

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responses. Secretory IgA is important for defending against invasive pathogens and serum IgG is

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necessary for the prevention of systemic infection [42]. When an antigen carries across the

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mucosal surface into the mucosal lymphoid tissue, the IgA is secreted. When the antigen is

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processed, the IgA producing cells will locate in mucosal effector sites and will hinder entry,

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attachment, and replication of pathogens or neutralizing microbial toxins in the next encounters.

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As mucosal surface is in the constant exposure to the foreign antigens, it becomes unresponsive

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to prevent unnecessary reactions to benign antigens [43].

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For producing mucosal vaccines the antigen delivery system must keep antigen from enzymes

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and improve the acquisition by Mauthner cell (M-cell) and epithelial cells. Addition of an

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adjuvant such as bacterial toxin elicits innate and adaptive immune responses against antigens.

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As the most of bacterial toxins are toxic for the human body, they must be detoxified but keep

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their immunogenic properties [43].

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3. Mechanism of action of the DNA toxin vaccine

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One strategy to deliver antigens into cells of individuals is by using viral and bacterial plasmid

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vectors. They function as a shuttle system to deliver and express desired antigen for stimulating

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cellular and humoral responses. Genetic vaccines consist of DNA vaccine, RNA vaccine and

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viral vaccine [44]. Without considering the mode of action of a DNA vaccine, it can be

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employed against various tumor cells, or bacterial infectious diseases [45] or autoimmune

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diseases [46]. In this paper we just review the properties of DNA vaccine.

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Nowadays, new approaches such as DNA vaccines are available for more specific treatment of

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diseases [47]. To clinically use these vaccines, the mechanism of action of the naked DNA

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vaccines and the adjuvant coupled with DNA must be understood. If the desired protein is not

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immunogenic, DNA vaccine encoding this antigen may not produce ideal immune responses.

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But DNA vaccination is a proper approach to manipulate the antigen sequences to improve the

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immunogenic property of the desired antigen [48].

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Generally, DNA vaccines transfect DCs, muscle cells and keratinocytes and activate the innate

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immune system with the help of PAMPs such as unmethylated CpG-containing sequences.

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Recently, researchers consider improving four characteristics of DNA vaccines to enhance the

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immunogenicity including improving stability and potency of transfection, eliciting proper

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immune response and augmenting the antigen presentation by DCs [49].

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Mammalian expression plasmids consist of a viral promoter (cytomegalovirus (CMV)

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immediate-early promoter) to manage transcription and translation in vivo, an adjacent intron A

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sequence to improve transcription efficacy and stability of mRNA then enhance protein

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expression, a transcription terminator like bovine growth hormone or rabbit beta-globin

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sequence, which contain strong polyadenylation signal and usually an antibiotic resistance gene

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for bacterial selection. For expressing more than one immunogen, multi cistronic vector is

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constructed. The best immune responses with DNA vaccine is achieved if a very active

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expression vector is utilized [48].

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The bacterial DNA performs as PAMPs which react with TLRs and other immunity related

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molecules to activate the immune cells. As plasmid has the ability to express a desired

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immunogenic protein, amelioration of the vector design for high protein expression is necessary

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[49]. One approach to increase protein expression is to make the codon usage of bacterial mRNA

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as optimal as possible for eukaryotic cells. For optimizing the expression the best method is to

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change the gene sequence of immunogenic protein and design a more common codon which is

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used in target cell. The most important concern to induce protein expression is the type of

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promoter. Previously the Simian virus 40 (SV40) promoter was usually used but recently it has

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been shown that the Rous Sarcoma Virus (RSV) has a much better expression rate and CMV

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function even better than those two mentioned before. For improving expression rate some

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modifications such as insertion of enhancer sequences, modifications of interons, transcriptional

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termination sequences and polyadenylation signal are required [50].

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This kind of treatment has some advantages such as multiple genes incorporation into a vector to

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ensure the modulation of intracellular ways and modification of antigens. By adding a leader

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sequence, the antigen can target endoplasmic reticulum (ER) and induce humoral immunity. The

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reverse transfer of antigen from ER to cytosol and delivery of DNA to APC may increase the

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possibility to produce CD8+ T cell responses. Some studies show that constructing a recombinant

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vaccine by fusing single-chain Fv (scFv) of Idiotypic immunoglobulin (Id) to tetanus

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neurotoxin (CTN) fragment C can stimulate high level production of anti-idiotopic antibodies

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and give immune protection against lymphoma. CTN is composed of a heavy (B chain) and a

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light chain (A chain) linked through a disulfide bridge. The digestion of toxin by papain

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yields two fragments; one of them is fragment C. The non-toxic carboxy-terminal fragment

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of CTN heavy chain (fragment C) has been used as a valuable tool is construction of DNA

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vaccine [51, 52].

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Classical adjuvants such as alum commonly used in vaccines to induce high Th2 response with

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a small amount or no Th1 response. For improving natural ligands and synthetic agonists for

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PRRs such as TLRs agonists are novel adjuvants which elicit pro-inflammatory cytokines,

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chemokines, and type I Interferons (IFNs) which enhance the ability of the host immune

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system to defeat pathogens [53]. Thus for improving the host immunological responses, DNA

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vaccines can be fused with immunostimulatory molecules such as TLR agonists [54]. In a study

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TLR7/8 agonists were used as adjuvants in combination with cancer vaccines that greatly

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postponed the occurrence of spontaneous mammary tumors and reduced their incidence

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compared to the DNA vaccine alone [53].

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It has been proposed that DNA vaccines provide immunity by three different mechanisms: (1)

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Somatic cells such as myocytes present DNA-encoded antigens through MHC class I pathway to

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CD8+ T cells, (2) Professional antigen presenting cells (APCs) such as DCs are transfected by

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DNA and (3) Transfected somatic cells phagocytized by professional APCs present antigens to T

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cells to cross-prime immune responses. As myocytes cannot effectively present antigens through

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MHC class I, therefore the two other mechanisms seem to be of more importance in DNA

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vaccination. Here, antigen presentation is mediated by MHC class I and MHC class II pathways

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which induce both cellular and humoral immune responses against antigen. Besides the antigen,

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unmethylated CpG motifs which present in the plasmid backbone of DNA vaccine are

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recognized by TLR9 and can provoke DCs to promote efficient cell mediated immune response

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[3].

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The route of administration is also of importance in the efficacy of DNA vaccines. Either

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intramuscular (i.m.) needle immunization or intradermal (i.d.) injection using “gene gun” may be

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used. Gene gun is a needle free apparatus which is used to inject gold beads coated with DNA

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vaccine plasmids into the epidermal layer of skin. Studies show that the development of immune

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response and the extent of transfected cell types are influenced by the mean of injection [55].

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For summarizing this issue, most common advantages and disadvantages of DNA vaccine and its

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immunological properties in comparison with traditional vaccines are mentioned in the table 1

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and Fig 2.

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Although DNA vaccination has numerous advantages, some issues have been raised with regard

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to this approach. Occasionally the integration of vaccine DNA into the genome of the host cell is

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seen, which may cause mutagenesis and activation of oncogenes. Also the repeated DNA

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vaccination can prompt autoimmunity. On the other hand, further researches have shown that the

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possibility for integration is very lower than naturally occurring mutations and increase in anti-

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DNA antibodies and changes in clinical markers for autoimmunity were not detected [13].

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As shown in table 1., the DNA vaccine provides an attractive approach over other vaccines with

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several advantages. Although the major problem to develop favorable DNA vaccines is its low

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efficacy to induce the immune responses, but its production is very cost-effective, has longer life

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time, has no risk of infection associated with live-attenuated vaccines or pathogens, well

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tolerated and has an excellent safety record. Therefore, DNA vaccines are a promising modern

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approach to protect humans and animals from various diseases.

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To overcome the low efficacy of DNA vaccines, vector and antigen design, delivery methods

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have been improved and incorporation of adjuvants and prime boosting strategy have

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significantly enhanced the immunogenicity of these vaccines.

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4. DNA toxin vaccine for treatment of infectious diseases

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Vaccination is one of the best means to overcome infectious diseases, which is named after

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Vaccinia virus. In 1798, Edward Jenner attempted to prevent smallpox after the injection of

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obtained cowpox pus to healthy people [56].

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The first bacterial vaccine was discovered by Louis Pasteur in 1880 that inoculated an old culture

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of Pasteurella multicida (formerly known as Pasteurella septica) to the chickens and protected

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them against the aforementioned bacteria [57].

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Conventional vaccines in use are usually consist of killed, inactivated or live, attenuated

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microorganisms, some microbial metabolisms such as detoxified toxins, purified antigens or

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conjugated polysaccharides. Although these vaccines have eradicated many infectious diseases

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such as smallpox in many countries [58] but they still have some limitations in handling the

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virulent organisms. In this context, new approaches are under way to develop subunit and DNA

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vaccines for smallpox typically composed of vaccinia virus (VACV or VV) membrane proteins

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that stimulate neutralizing antibodies against the intracellular mature virus and extracellular

310

enveloped virus forms [58].

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The purified microbial components vaccine which introduced in 1920s was used against

312

diphtheria and tetanus and the toxin of both microorganisms was modified by chemical agents to

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obtain non-toxic toxoid. These early diphtheria and tetanus toxoids should be considered as the

314

first subunit vaccines. Filtration of broth cultures of Corynebacterium diphtheria (C. diphtheria)

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and Clostridium tetani (C. tetani) resulted in sterile filtrates substance that were toxic for animals

316

and caused diseases in both animals and human. Then the specific antitoxins produced in horses

317

for passive protection and treatment and toxin/antitoxin mixture was used to produce active

318

protection. Sometimes accidental errors in the preparation of mixture caused terrible outcomes

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such as contamination of mixture with Staphylococcus aureus caused several deaths. Finally, it

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was found that formalin can detoxify diphtheria toxin (DT) and CTN yet preserving its

321

antigenicity stimulate neutralizing antitoxin [59]. Early was shown that proteins presented in a

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solution were less immunogenic than protein particles. That’s why the soluble toxoids of

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diphtheria and tetanus were absorbed to an alum gel to perform as an adjuvant. Many bacterial

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components such as LPS, toxin and killed bacteria have been used in animal models but because

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of safety issues, they have not been approved for use in human beings [60]. Recently, for

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improving the quality of this toxin considering safety, efficacy and stability researchers have

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made some effort for genetic inactivation of diphtheria toxoid [61]. C. diphtheria strain C7-

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(β197) has a non-toxic mutant form of DT called cross-reacting material (CRM197) which is a

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potent vaccine candidate. DT is a single polypeptide chain A-B toxin consists of two subunits

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coupled with disulfide bonds; the B subunit is binding fragment and A subunit is the active

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site of the toxin. When in the active site a glycine is substituted with a glutamic acid, its

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toxicity abolishes. Thereby, CRM197 is promising option to diphtheria toxoid [62].

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For enhancing the immunogenicity of some vaccines bacterial polysaccharides were conjugated

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to a protein antigen to stimulate T cells. The first attempt for producing conjugated vaccines was

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made on H. influenza type b and then on pneumococci which showed great success [63]. A

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recent study on DNA vaccine expressing Pneumococcal surface protein A (PspA) has shown

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proper protection of mice against an intranasal pneumococcal infection [64]. PspA4 is a protein

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composed of leader sequence, α-helical region, clade-defining region (CDR), proline-rich

339

region (P – block of proline repeats and N – block without proline) and choline-binding

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region. This protein is a promising candidate against pneumococcal infection. pspA4A and

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pspA4Pro were two fragments of PspA4 which were used in this study. pAE vector and

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DNA vaccine vector pSec-Tag2A were used for expression in E. coli. Mice were immunized

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with PspA as protein adjuvant with alum or DNA vaccine and IgG2a bound to bacteria for DNA,

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but not protein immunization. Both protein and DNA vaccines were protected the mice against

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intranasal lethal bacteria. Antibodies induced by protein and DNA immunizations were distinct

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both in Fc and Fab.

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As some microbes cannot be grown easily in vitro, they cause a problem in vaccine development

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with conventional methods. Then the best procedure to prepare vaccines against these microbes

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was recombinant technology. In this approach desired genes are transferred to the genome of an

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easily grown microorganism such as Escherichia coli, Bacillus subtilis or yeast to express a

351

recombinant product. The most used recombinant product is a surface protein of HBV produced

352

in yeast cells that purified and formed as viral particles which stimulate antibodies to protect

353

against infection. The advantages of these vaccines are ease of growing of the hosts, the

354

possibility to upregulate the production and ease of purification [65].

355

Today modern vaccine approaches have substituted traditional methods and we still expect much

356

more advances in this area. The first attempt to demonstrate the effectiveness of a recombinant

357

DNA plasmid in animals was made during the 90's. The first attempt regarding infectious

358

diseases was made by two separate research teams by Fynan et al. and Ulmer et al. in 1993. They

359

found that immunization of mice with a plasmid DNA could protect them against influenza virus

360

[66].

361

Indeed, the appearance of antibiotic resistant bacteria emerged the need of new generations of

362

vaccines. Although some bacterial vaccines have been developed and are widely used for

363

prevention and treatment in humans; the mortality and morbidity rate among ill human being

364

populations still is high. Therefore, an urgent need is seen for new vaccine approaches [67].

365

Since then, diverse studies have shown that DNA vaccines have many advantages over

366

conventional types of vaccines (Table 1.) but they are barely used in infectious diseases. The

367

results of experiments show that bacterial DNA vaccines are going to become reality in the

368

coming future [13].

369

For the elimination of the endogenous bacteria, the immune responses are commonly cellular and

370

against exogenous bacteria antibodies are produced which are necessary for controlling almost

371

any bacterial infection. The antibody usually produced against bacterial toxins and capsule and

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structural proteins. For developing an effective DNA vaccine, the bacterial antigenic epitopes

373

must be determined. For designing a proper structure for the vaccine against bacterial infections

374

the relevant antigens must be selected [13]. The schematic immunological mechanism of DNA

375

vaccine in respiratory infections is demonstrated in Fig 3.

376

The occurrence of multidrug resistant mycobacteria, low efficiency of bacillus calmette-guerin

377

(BCG) vaccine and co-infection with HIV-1, a novel DNA vaccine incorporating HIV-1 p24

378

protein is designed, which provides protection to Mycobacterium tuberculosis (M. tuberculosis)

379

and concurrently stimulate humoral and cellular response against HIV-1 in BALB/c mice. In this

380

experiment four undefined epitopes belonging to T cell including Ag85A, Ag85B, MPT64, and

381

TB10 were investigated and 4 antigens of M. tuberculosis were designated [68].

382

Since constructed bacterial DNA must use the eukaryotic cells' machinery and have to be

383

expressed in a host cell, some problems may occur. These obstacles may arise during the

384

folding, translocation and post translational modifications and cause some undesired

385

results. The main and the most important problem concerning these kinds of vaccines are

386

autoimmune responses following vaccination with capsular polysaccharides of some

387

bacteria which have similar structure to the host cellular carbohydrates. To solve this

388

problem, scientists have developed peptides that can mimic polysaccharide antigens and can

389

elicit T cell-dependent polysaccharide immune responses. But these peptides have some

390

limitations; for instance they are not a good immunogenic molecule and have a low stability.

391

Furthermore, small peptides can break down and become ineffective [45].

392

Also it has been demonstrated that during infection or after vaccination, efficient immune

393

responses are produced against bacterial toxins and these immune responses are usually able to

394

protect individuals against toxin-generating bacteria. Scott et al. have shown that plasmids

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encoding the receptor binding domains (RBDs) from Clostridium difficile (C. difficile) toxin A

396

(TcdA) and toxin B (TcdB) were able to generate both anti-RBD antibodies and RBD-specific

397

antibody-secreting cells (Table 2) TcdA and TcdB both have enzymatic, translocation, and

398

receptor-binding domains and each domain has a specific role in cell intoxication [69].

399

In summary, naked DNA has the ability to elicit the immune system against an expressed

400

antigen in infectious diseases, and induced immunity has ability to give protection against

401

infection and the foreign antigens. But there are only a limited number of vaccines available for

402

some bacteria and mostly cannot give complete protection [13]. Moreover, some bacteria toxins

403

such as CT B subunit is able to be used as an adjuvant in combination of another bacterial

404

vaccine toxin [70].

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405

5. DNA toxin vaccine for cancer therapy

407

Although conventional vaccines are usually administrated to healthy people to prevent different

408

types of diseases, cancer vaccines are used as therapeutic agents to treat cancer patients and

409

fortify their immune system to combat the disease [71].

410

The first attempt for improving immune system in cancer patients is made by Dr. William Coley

411

in 1891. He injected Streptococcus pyogenes and Serratia marcescens into the cancer tumors.

412

Until now, some bacteria such as BCG which is similar to Coley’s toxin are used to treat cancer

413

patients [72].

414

At the present time, efforts for developing an efficient, safe and long-lasting therapeutic cancer

415

vaccine are ongoing. Cancer vaccines are classified into different groups on the basis of their

416

compositions including tumor cell vaccines, protein/peptide-based cancer vaccines, DC vaccines,

417

and genetic vaccines [73]. Peptide plus adjuvant vaccine has the noncomplex cancer vaccine

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formulation which is composed of peptide injected intradermal with an immunogenic adjuvant.

419

To produce this kind of vaccine the relevant tumor antigens and their related MHC Class I and

420

MHC Class II epitopes must be determined.

421

Plasmid DNA immunization is another simple strategy for developing cancer vaccine. Similar to

422

peptide vaccines, related target antigens must be detected to constitute the plasmid vector to

423

carry the proper gene sequences. Contrary to peptide vaccines the related epitopes are not

424

required to be defined. Another strategy is recombinant viral vectors which are used to deliver

425

determined tumor antigens through gene transfer. The most important recombinant viral vectors

426

are replication-defective adenoviruses (rAd) and adeno-associated viruses (AAV). The

427

immunogenic ability of these vectors is high. Using recombinant bacteria to deliver determined

428

tumor antigens for gene transfer is another strategy in cancer vaccine production. Salmonella

429

typhimurium and Listeria monocytogenes are commonly used as antigen delivery platforms in

430

this strategy. DCs are professional APCs, and strongly stimulate the naïve T cells. MHC class I

431

and MHC class II molecules are highly expressed by activated DCs to excite CD8+ and CD4+ T

432

cells and also provide co-stimulatory signals for activating T cells. So, DCs represent a very

433

attractive vaccine option for cancer vaccines [74].

434

Conventional vaccines are prepared by killed or attenuated microorganisms, antigens, peptides

435

and tumor cell extracts, but most recently used methods for cancer treatment are based on genetic

436

approaches [75] which are discussed in this paper.

437

In a cancer therapy strategy, autologous tumor antigens are used but they are not capable to

438

induce efficient T helper responses, and CD8+ effector T cells are useless in this context. To

439

conquer this deficiency, Rice et al. co-operated sequences derived from CTN in the same vector

440

as tumor antigen to augment tumor related immune responses [76].

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Rice et al. in 2002 have described a DNA fusion vaccine encoding the first domain (DOM) of

442

the Fragment C (FrC) of CTN fused to specific MHC class I-binding epitope sequences in the

443

C-terminus. This structure stimulates high levels of CD41 T cell and the insertion of the

444

tumor-derived epitope help to prime epitope-specific CTLs. In addition, epitope-specific

445

CD81 T cells protect individual from tumor challenge (Fig 4.) [77].

446

Most recently, Stevenson et al. have made the similar trial on DNA vaccines using tumor-derived

447

peptide sequences fused to an engineered domain of CTN was able to suppress tumor growth in

448

prostate cancer patients (Table 3) [78].

449

As numerous of cancer tumors are resistant to conventional anticancer therapies, novel

450

approaches have been developed to overcome the problem. In addition, these new strategies must

451

selectively act on cancer cell and have minimal toxicity for normal cells. The application of

452

bacterial toxins is proved to be an effective strategy for cancer treatment [76, 78].

453

Bacterial toxins are able to destroy cells or modify some activities such as cellular divisions,

454

apoptosis and cellular differentiations. These modifications are related to carcinogenesis and may

455

cause cellular abnormality or corrupt the function of normal cells. Cytolethal distending toxins

456

(CDTs) which consider as cell-cycle inhibitors are found in gram negative bacteria such as

457

Campylobacter jejuni and Salmonella typhi and cycle inhibitor factors (Cif) which cause the

458

inhibition of mitosis are found in enteropathogenic (EPEC) and enterohaemorrhagic (EHEC) E.

459

coli. It has been suggested that aforementioned toxins can interfere with clonal expansion of

460

lymphocytes to balance the immune system. On the other hand, cytotoxic necrotizing factor

461

(CNF) induces DNA replication and proliferation of cells but prevent cell differentiation. Some

462

bacteria such as E. coli can release CNF. When CNF influence, the number of tumor cells

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does not increase, instead poly-nuclear cells occur. This event appears because of the ability

464

of toxin to prevent cell differentiation and apoptosis [79].

465

One advantage of bacterial toxins is its less side-effect in comparison with other anti-cancer

466

therapies therefore toxin encoding genes can be applied in DNA vaccine technology to augment

467

the efficacy of this approach [80].

468

Some bacterial exotoxins such as Pseudomonas exotoxin A [81], DT [82] and Clostridium

469

perfringens (C. perfringens) enterotoxin [83] have the ability to bind to the surface antigens of

470

tumor cells and be used as anti-cancer vaccines.

471

DT binds to the heparin-binding epidermal growth factor-like growth factor (HB-EGF) precursor

472

on the surface of cells. Internalization of DT-HB-EGF complex through endocytosis via clathrin-

473

coated vesicles occurs. Following DT post-translational process, catalytically active toxin

474

produced. This toxin ribosylates elongation factor-2 (EF-2) and inhibits protein synthesis. This

475

event leads to cell lysis or apoptosis DT post-translational process catalytically active toxin is

476

produced. The action of the toxin is to ribosylate elongation factor-2 (EF-2). Pseudomonas

477

exotoxin also can ribosylate EF-2 and inhibits the protein synthesis [84]. Denis-Mize et al. have

478

shown that vaccination with DNA encoding Pseudomonas aeruginosa (P. aeruginosa) exotoxin

479

A elicit specific immunity and protected mice from the lethal doses of exotoxin A producing

480

from Pseudomonas [85].

481

C. tetani cleaves VAMP-2 and thereby prevents neurotransmitter to be released from pre-

482

synaptic nerve endings in central nervous system (CNS). Above mentioned bacterial toxins with

483

anti-cancer effects can be employed as therapeutic agents in DNA vaccine strategies and be

484

produced within target cells [86]. Chudley et al. have demonstrated that a fusion DNA

485

vaccine which encoded a DOM of fragment C of CTN which was linked to an HLA-A2-

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binding epitopes from prostate-specific membrane antigen (PSMA) was safe and well

487

tolerated. The findings and clinical effects were promising for coming research [52].

488

The main goal of cancer vaccine research is obtaining an effective and long-lasting CTL

489

response. CD4+ T cell is produced through a MHC-dependent pathway which is essential for

490

reproduction of CD8+ T cell responses and sustaining memory throughout DNA vaccination. In

491

the previous study the vaccine produced DOM-specific-CD4+and PSMA27-specific CD8+ T cells

492

which were quite significant.

493

Tang et al. found that administration of plasmids containing human growth hormone (hGH)

494

gene was able to provoke humoral immunity against hGH proposing that pDNA can elicit

495

specific immune responses [66].

496

The results of these findings are encouraging which strongly imply that DNA immunization can

497

be an efficient approach to overcome many types of cancers. But, before becoming widely used,

498

it must still undergo further experiments, such as different second generation DNA vaccine

499

optimization strategies.

500

6. DNA toxin vaccine for autoimmune diseases

501

Autoimmune diseases are conditions that the antigen-specific and organ-specific immune invade

502

on their own and autoreactive immune cells are achievable from these patients. It is supposed

503

that the action of specific T cells and/or antibodies is the cause of this disease, therefore

504

inhibiting the production of these cells and antibodies are of therapeutic value. If the

505

autoimmune disease is caused by the action of specific T cells associated with class II antigens,

506

on the antigen-presenting cell, immunomodulators can compete with the self-antigen causing

507

autoimmune disease and if it is at the level of the T cell receptor, an immune response to the

508

variable α and β chains of these receptors can eliminate the autoimmune reaction[87].

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An infection can also induce or launch an autoimmune disease. Studies show that most

510

pathogens produce Tregs that sustain tolerance and prevent autoimmune disease. One of the

511

most studied autoimmune diseases is multiple sclerosis (MS). Multiple sclerosis is a progressive

512

autoimmune disorder that damages the myelin sheath of motor neurons in central nervous

513

system. The injury arises as a result of the inflammation and may cause loss of nerve signal

514

conduction and develop secondary disordered repair or gliosis. Four types of MS are

515

recognized until now: Relapsing-Remitting MS (RRMS), Secondary-Progressive MS (SPMS),

516

Primary-Progressive

517

manifestations in all four types are different which indicate localization, the nature and mode of

518

activation of invading cells and recovery process of diseases [88].

519

Smith et al. compared M. tuberculosis that induces a prototypical Th1-mediated immune

520

response with Citrobacter rodentium (C. rodentium), a bacterium that requires IL-23-dependent

521

Th17 cell response during infection to produce experimental autoimmune encephalomyelitis.

522

Mice immunized with C. rodentium adjuvant showed classical signs of experimental

523

autoimmune encephalomyelitis (EAE), similar to the mice immunized with M. tuberculosis, but

524

the disease was not as severe as the latter with moderate progression and the number of Th1 and

525

Th17 cells was reduced in the central nervous system (CNS) in C .rodentium immunized mice

526

[89].

527

In a recent study, Philips et al. have induced experimental autoimmune encephalomyelitis (EAE)

528

in mice with myelin antigen and demonstrated that DAB389IL-2 (denileukin diftitox;

529

ONTAK™)- a recombinant fusion toxin- targeting IL-2R bearing cells, suppressed the γσT cell

530

subpopulation in spinal cord lesions and subsequently overcome the disease [88] (Table 4).

(PPMS)

and

Progressive-Relapsing

MS

(PRMS).

Clinical

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Recently, some drugs have been approved for the treatment of autoimmune diseases, but their

532

therapeutic efficacy is controversial. DNA vaccines are a novel method for treating autoimmune

533

diseases in a highly specific manner [88].

534

In some cases, administration of DNA vaccines against tumor cells may cause autoimmunity

535

which is a disadvantage to the application of DNA as a vaccine.

536

Actually there are several claims and counter claims in association with DNA vaccinations.

537

In 1997, Mor et al. analyzed the potential of DNA vaccines to induce or enhance autoimmunity

538

by administrating DNA vaccine to the mice. Normal mice immunized several times did not

539

develop clinical nor serological signs of autoimmune disease [90].

540

However, complementary investigations are needed for identifying the certain effect of DNA

541

vaccination on autoimmune disease.

542

The results obtained from experimental models of infectious diseases, cancer and

543

autoimmunity suggest that it is only the matter of time that clarifies the complete safety

544

and efficacy of DNA toxin vaccines. Until now, there are limited studies concerning DNA

545

vaccine associated with bacterial toxins and still a number of questions must be answered

546

to assess its complete safety and effectiveness for prevention and treatment of

547

aforementioned diseases.

548

7. Concluding remarks and future perspectives

549

The discovery of the conventional vaccines -first generation vaccines- was the most

550

important development in the last century which rescued millions of people from different

551

diseases. Subunit vaccines –second generation vaccines- developed to decrease the risks of

552

live attenuated vaccines and third generation vaccines namely DNA vaccines produced to

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overcome the shortage of other vaccines including problems in transportation and its high

554

production costs [91].

555

The most important feature of DNA vaccines is the ability to develop either humoral or

556

cellular immune response or both that makes them convenient for the treatment of

557

different diseases. In many cases of infectious diseases, DNA vaccine gives proper

558

protection against foreign antigens but only a limited number of vaccines exist for some

559

bacteria and the use of bacterial toxin sequences is somehow proved to be a useful

560

approach [91].

561

In cancer therapy, bacterial DNA toxin vaccines proved to have less side-effects and more

562

effective compared to conventional cancer therapy [92].

563

Findings about the treatment of autoimmune diseases using DNA vaccines are

564

controversial and bacterial toxins are less studied in this case and complementary studies

565

are needed.

566

All in all, DNA vaccine is a revolutionary technology in the field of preventive immunology,

567

still new approaches such as implementation of bacterial toxin sequences as an adjuvant

568

for optimization and augmentation of its immunogenicity is needed to be used.

569

On the other hand, we know that bacteria are not just single-celled organisms that cause diseases

570

in human and other animals and bacterial toxins are not toxic for all cells and the host organism.

571

In some cases toxin may adjust the function of the target cell and still little is known about their

572

mechanisms. Some toxins target the molecular machinery of the cell cycle. Therefore these toxin

573

producing genes can be implemented in DNA toxin vaccine development. But still, further

574

research for more complete understanding of the molecular action of bacterial toxins in vivo is

575

needed. It seems that some bacterial toxins play no role in pathogenesis and some have different

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effects on various cell types because although toxin molecules act in the same mode in different

577

cell types, the activated signaling pathways differ from one cell to another, which result in

578

various cellular consequences.

579

The characteristics of some toxins such as Pasteurella multocida toxin (PMT) and Cytotoxic

580

necrotizing factor 1 (CNF-1) produced by E. coli make them function as tumor inducers which

581

their properties must be studied carefully in vaccine development [93]. As some toxins are

582

effective for treating cancerous cells but exposing cells to the high quantity of toxins may have a

583

reverse effect on them. Low quantities might still induce signaling pathway, for this reason the

584

mode and amount of injection are of importance [94].

585

The DNA toxin vaccines needed more attention and the research on this area may bring

586

considerable promotions in curing cancer, autoimmune and infectious patients in the future. In

587

addition, some experimental and clinical evidences show the efficacy of engineered toxin

588

vaccines against certain tumors and infectious diseases.

589

In conclusion, DNA vaccines possess significant advantages including rapid manipulation, easy

590

translocation via plasmids, applicable as multifunctional vaccines, stability with the least

591

adverse effects in humans, cost-effectiveness and reliability which show a bright promising

592

future relating to DNA vaccines [95].

593

8. Funding

594

This study was not funded by any grant from institute.

595

9. Conflict of Interest

596

No conflict of interest declared.

597

10. Informed consent

598

All of the authors had the same contribution in the article and are agreed to submit manuscript.

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11. Figures:

600

Fig 1. Schematic representation of PAMPs recognition by TLRs and NLRs, respectively.

601

Figure 2. Schematic representation of immunological mechanism of DNA vaccine in

602

comparison with traditional vaccines.

603

Figure 3. Schematic representation of immunological mechanism of DNA vaccine in

604

respiratory infections.

605

Figure 4. Schematic representation of immunological mechanism of DNA vaccine in cancer

606

treatment.

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AC C

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803 804 805

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Table 1. Advantages and disadvantages of DNA vaccine vs. traditional vaccine

Safety


No risk of infection

Immunogenicity


Long lasting immunity
Well tolerated
No adverse effect

Cost

AC C

Spectrum

EP

TE D

Development Production


Thermal stability
Longer life time
Easy to store and transport
Rapid to produce
Can encodes for several antigens and
Simple design with possibility of further modification
Easy and large-scale production
Relatively inexpensive to produce Several plasmids could be used to make a broad spectrum vaccine

M AN U

Stability

Disadvantages
Induce immunologic tolerance
Can affect the genes which control the growth of cells
Induce antibody production
Antibiotic resistance
Relatively poor immunogenicity
Not useful against non-protein based antigens
Immunotoxicity

RI PT

Advantages

SC

Characteristics

-

-

Difficulty/cost of delivery Not effective for all common pathogens/diseases

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Adjuvant

Host

Refs.

Clostridium difficile

Alum compounds

Mice, Hamster

(70)

Clostridium tetani toxoid

Alum compounds

Human

(59)

Corynebacteriumdiphtheriae toxoid

Alum compounds

Human

(59)

Bordetellapertusis toxin

Alum compounds with others such as liposomes and monophosphoryl lipid A, Gamma Inulin Alhydrogel

Human

(59)

Mice

(71)

Mice

(13, 69)

Pseudomonas aeruginosa Exotoxin A

Vibrio cholerae toxin

T domain belonging to diphtheria toxin Cholera toxin b subunit (CTB) CT, IL-1α, LPS, CpG, Pam3CSK4, 3M-019, resiquimod/R848 or c48/80 HemolysinCyclodextrin complex CTB

TE D

Bacillus anthracis toxin

M AN U

Clostridium botulinum toxoid

Staphylococcus aureus α-toxin

AC C

EP

Escherrichia coli (ETEC) toxoid

RI PT

Infectious disease

SC

Table 2.DNA toxin vaccines for infectious diseases

Different animals (72) and Human Mice (73)

Mice

(69)

Human

(69)

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Table 3.DNA tetanus toxin vaccines for cancer therapy Cancer Genetic encoding

Host

Refs.

DNA Plasmid/HER2 ICD

Human

(54)

Cervical cancer

DNA Plasmid/mutated E7 HPV antigen

Human

(54)

Liver cancer

DNA Plasmid/Murine α-fetoprotein,

RI PT

Breast cancer

Human-

Adenoviral vector

Melanoma

DNA Plasmid/Synchrotope MA2M DNA Plasmid/pSEM Ex-DNA/rhPSA DNA Plasmid/PSMA DNA Plasmid/Bacterial HSP90

Prostate cancer Tumors

Human

SC

DNA idiotopicscFv and TTFrC

Mice

M AN U

Lymphatic cancer

Table 4.DNA toxin vaccines for autoimmune diseases therapy Host Allergy Vaccine elements

Adjuvant arthritis

(54)

Human

(54)

Mammals (91)

Refs.

Lewis Rat

(91)

Mycobacterial &Chlamydia HSP60 diphtheria toxin

Human

(91)

Mice

(93)

AC C

Multiple sclerosis (MS)

Human

Lewis Human

EP

Atherosclerosis

(54)

Mycobacteria and Escherichia coliHSP60& HSP70/plasmid MycobacteriaHSP60/plasmid

TE D

Rheumatoid arthritis

(54)

Rats, (91, 96)

AC C

EP

TE D

M AN U

SC

RI PT

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AC C

EP

TE D

M AN U

SC

RI PT

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AC C

EP

TE D

M AN U

SC

RI PT

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AC C

EP

TE D

M AN U

SC

RI PT

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