Listeria: Immunotherapy

Health and its management are often interrupted by the infectious pathogens that exert their prevalence leading to outbreaks associated with mortalities. The ability to withstand such harmful, otherwise detrimental effects of microbial agents relies on the body’s fighting mechanism provided by the Immune system. This machinery is equipped with a variety of cells to facilitate the attacking process in a smooth manner. Exploiting this specific natural mechanism to treat a spectrum of disorders including cancer has become a challenging task to health care researchers under the name of ‘Immunotherapy’. This branch of biomedical science aims at curing diseases by stimulating the immune system to mount a lethal response against the targeted risky foreign bodies. Immunotherapy intended for achieving a pathogenic clearance from the host body would be primarily in need of cytotoxic T lymphocytes, natural killer cells, B cells, and dendritic cells, as they were believed to offer protection against Cancer in addition to their routine immune surveillance role (Jian-Qing Gao, 2008).

This would necessitate a thorough trafficking and biodistribution of immune cells in tissues especially tumor cells. As such, emphasis was laid on the immune cell recruitment and cell-based systems that could potentially control the dynamics of immune cells with regard to the cancer immunotherapy (Jian-Qing Gao, 2008). However, there is a need to highlight various aspects of immunotherapy with regard to other health problems for a better understanding of its anti –cancer strategy.Immune cells have been successfully employed to treat neurodegenerative disorders such as Alzheimer disease (AD) in mouse models (David & David, 2008). It was revealed that certain mechanisms involving antibody-induced phagocytosis of pathological protein deposits, direct antibody-mediated disruption of aggregates, neutralization of toxic soluble proteins, a shift in equilibrium toward efflux of specific proteins from the brain, and cell-mediated immune responses could play important role during the process of immunotherapy (David & David, 2008).

The success of immunotherapy also relies on other factors like co-stimulatory signals which when modulated may help in the management of diseases that have autoimmune etiology like diabetes, rheumatoid arthritis, and systemic lupus erythematosus (Viglietta & Khoury, 2008). Preventing the activity of co-stimulatory signals in animal models was found to diminish autoimmune responses and subsequent progression of autoimmune disease (Viglietta & Khoury, 2008). The co-stimulatory pathway CD28-B7 (cluster of differentiation) has better implications with regard to auto- antigen mediated T-cell activation (Viglietta & Khoury, 2007).Immunotherapeutic approaches utilizing the above strategy could help in understanding the autoimmune responses directed against various tissues that become susceptible to cancer induced damage. Therefore, enhancing the co stimulatory signals may program the body’s defense mechanism to provide significant immunotherapeutic attack.

Treating disorders has also become feasible with cytokines. It was reported that Tumor Necrosis Factor (TNF) and TNF receptor superfamily molecules could play important role in cancer therapy. The potential to generate anti-tumor mechanism has made these molecules as ideal candidates. (Tamada & Chen, 2006). They could operate by exerting their direct actions onto tumor cells and indirect effects through immune or non-immune components of tumor-bearing host. As such, it was described that molecules such as TRAIL (TNF-related apoptosis-inducing ligand), CD40, and 4-1BB (CD137), were reported to offer specific anti-tumor responses (Tamada & Chen, 2006).

This report seems to shed light on the anti-cancer properties of cytokines. So, it is reasonable to assume that this strategy could also offer protection against wide range of disorders which may be in need of TNF.

Therefore, it can be inferred that immunotherapy could be better improved by dissecting the relationship between the pathways linked with the co stimulatory signals or molecules and the target sites, and understanding the efficacy of molecules associated with cytokines.

Special cells such as antigen presenting cells (APC’s) were described to provide better immunotherapy for malignant disorders (Melief, 2008). Activating dendritic cells by Toll-like receptor ligands or CD40 agonists may produce tumoricidal effector cells. This might help to overcome the immunosuppressive effects of tumors and the associated tumor-antigen cross representation enabled T cell anergy or deletion (Melief, 2008).Tumors were also found to produce immunosuppression through cytokines such as TGF-beta and IL-10, (interleukin) (Melief, 2008). As such, this could provide a clue to expand the strategy of cytokine therapy

as achieved with molecules belonging to Tumor Necrosis Factor (TNF) and TNF receptor superfamilies. Hence, oncogene driven cancerous lesions may be inhibited by therapeutic antitumor vaccines that use DCs (Melief, 2008).

Monoclonal antibodies (mAb) have provided another option as reliable immunotherapeutic agents. This became evident when a murine asthma model injected with anti-CD137 (4-1BB) mAb was found with inhibitory effects on the development of airway hyperreactivity, eosinophilic airway inflammation, excessive mucus production, and elevated IgE(Tobias Polte et al., 2006).

As asthma was believed to result due to Th2 cell–driven immune response, this strategy was reported to induce reduced Th2 cytokine production and increased secretion of the Th1 cytokine IFN-γ, and CD4+ T cell anergy (Tobias Polte et al., 2006).This mode of alleviating a disorder that has wide spread prevalence may possibly open the doors for many other health complaints that have allergic etiology. This approach has also strengthened the role of co-stimulatory molecules, cytokines in the scenario of immunotherapy thus making them ideal candidates. This may indicate that a cascade of molecular events may be involved in the process of immune cell mediated protection against pathogenic microorganisms. Here, it can also be inferred that developing techniques aimed at immunotherapy could ensure smooth research such that reliable information may be obtained.

Next, exploitation of T-cells has offered a promising approach for treating patients. By employing method of adoptive immunotherapy, researchers were able to produce new T cells from the hematopoietic stem cells when T cell – depleted human bone marrow cells were introduced into swine (Ogle et al., 2008). These cells detected in thymus and blood stream of treated swine have various characteristic features enough to recognize and respond to antigen presented by human antigen-presenting cells (Ogle et al., 2008).This method could better serve to treat individuals who may be devoid of tumor- or virus-specific T cell clones (Ogle et al., 2008).The application of hematopoietic stem cells for inducing immunity by adoptive transfer could make them as potential targets for therapeutic research. Here, it is reasonable to assume that since T- cells constitute the essential infection fighting cells , this approach may not only become a helpful tool to treat patients but also may especially serve to assist immune suppressed individuals who often become often to infectious agents.

Recent developments in the molecular biology have increased the reliability of immunotherapeutic discipline. Harrison et al. (2008) described that viruses would serve as vectors or vehicles to enable the gene to be delivered to the targeted site with objective of conferring protection against cancers. As many viruses were believed to mediate long term gene expression, they may have better therapeutic potential (Collins et al., 2008). This strategy when exploited in animal and human experiments has made obvious the anti-tumour responses against cancerous cells. The other implications of this approach may be development of vaccines. Hence, facilitating the direct transfer of immunostimulatory molecules to tumour cells could be achieved by the use of viral vectors (Collins et al., 2008).

So, keeping in view of the above information the following sections of this description would be focused on Listeria Immunotherapy.

Listeria scientifically known as Listeria monocytogenes belongs to the category of Gram -positive facultative intracellular pathogens (Ramaswamy et al., 2007). As it was considered as food borne agent, it could cause a disease Listerosis among pregnant women and could also reduce the immune potential which might lead to brain infection and death in severe cases (Ramaswamy et al., 2007). Considerable research interest was centered on this microbe to explore the intracellular parasitism and gastrointestinal phase of L. monocytogenes infection (Ramaswamy et al., 2007). This made it a model organism and finally it was revealed from the epidemiological studies that outbreaks of human disease have connection with Listeria induced gastroenteritis. Hence, understanding the pathogenesis of L. monocytogenes was given paramount importance with regard to its whole genome sequences and virulence determinants (Ramaswamy et al., 2007).

This organism has a complicated evolutionary framework. This was revealed by determining the phylogenetic structure of this microbe by sequencing the internal portions of seven housekeeping genes (3,288 nucleotides) in 360 representative isolates (Marie Ragon et al., 2008).They have described that each clone had a unique or dominant serotype with no association of clones with clinical forms of human listeriosis. This microbe has long-term genetic stability of multilocus genotypes because of its limited homologous recombination.

This has further strengthened its utility as a model organism for exploring its cellular microbiology and host–pathogen interactions (Marie Ragon et al., 2008).As it is omnipresent in the environment, this pathogen could offer another platform for addressing its various clinical aspects.

There has been contribution from the genetic engineers and health care professionals who could better evaluate the pros and cons of employing Listeria as a therapeutic tool.

Clinically Listeria was proven to induce antigen-presenting cell maturation accompanied with strong innate immunity (Paterson & Maciag, 2005). This could elicit immune response to poorly immunogenic antigens, such as tumor-associated antigens As the presentation of any antigen depends on its association with MHC (Major Histocompatibility Complex) molecules, Listeria better enables the antigen to be processed through MHC class I and II pathways (Paterson & Maciag, 2005). This strategy helps it generate virulence factors such that host cell colonization would be achieved. It was revealed that Listeria obtained from recombinant DNA technology could provide therapeutic immunity directed towards established tumors. It could harbor number of tumor-associated antigens and may ultimately become a vaccine vector for tumor-associated antigens (Paterson & Maciag, 2005).This has provided the opportunity for Listeria to serve as a cancer immunotherapeutic tool intended for human use. It may indicate that Listeria’s vector driven virulence and unique antigen processing mechanism make it a suitable candidate for immunotherapy (Paterson & Maciag, 2005).

L. monocytogenes was reported to induce cellular immunity which is essential for HIV positive individuals (Jiang et al., 2007).It was revealed that live attenuated Listeria monocytogenes induces cellular and humoral immune responses to HIV Gag through the production of T-helper type 1 (Th1) or Th2 cells and systemic as well as mucosal anti-Gag antibodies, respectively (Jiang et al., 2007). This became evident when Rhesus macaques co-administered with D-alanine were immunized with Lmdd-gag that expresses HIV gag. This has indicated the safety and immunogenic potential of live attenuated, recombinant Lmdd-gag in primates (Jiang et al., 2007).

Hence, it is reasonable to assume that L. monocytogenes offers protection against HIV when it is injected as AIDS vaccine. This approach may also help to withstand the detrimental effects of opportunistic infections in HIV positives. This information may help to gain further insights on the efficacy of Listeria vaccines.

However, the vaccine strategy of L. monocytogenes was been under investigation as far as cancer is concerned. Hussain et al. (2004) previously described that CD4+CD25+ T cells generated in a vaccine could restrict the growth of tumors. After constructing the vaccine with Human Papilloma Virus (HPV-16 E7) protein, a potent cellular immunity to HPV-E7-expressing tumors was observed (Hussain & Paterson et al., 2004). These cells were reported to induce tumor regression after their number was significantly increased in spleen and tumor-infiltrating lymphocytes in contrast to Lm-LLO-E7-vaccinated mice. Hence, this demonstrated their suppressor function by the production of suppressor cytokines interleukin-10 (IL) and transforming growth factor beta (TGF-) (Hussain & Paterson et al., 2004). This could reflect the efficacy of tumor-infiltrating CD4+CD25+ regulatory T cells in lessening the tumor progression. Therefore, Listeria vaccine with this cancer fighting potential may work well for wide range of disorders that rely on T -cell stimulation.

In addition, the virulence factor identified was listeriolysin O (LLO) that induces the immunogenicity and antitumor efficacy of the tumor antigen when delivered by Listeria or by vaccinia (Sewell et al., 2004). As Listeria was reported to contain PEST(proline (P), glutamic acid (E), serine (S), and threonine (T) )sequence at the NH (2) terminus, it was significant in retarding the progress of established Human papilloma virus (HPV-16) immortalized tumors in a mice model (Sewell et al., 2004).This also enhanced its ability to generate CD8 (+) T-cells and its tumor homing and the antitumor efficacy (Sewell et al., 2004). Berraondo et al. (2007) described that HPV -E7 antigen in combination with adenylate cyclase (CyaA) of Bordetella pertussis could become a potent vaccine vector when it was co-administered with CpG complexed with a cationic lipid and low-dose cyclophosphamide. This approach known as tritherapy was found to induce tumor-associated immunosuppression and completely inhibited the growth of established tumors in majority of treated animals (Berraondo et al., 2007).

This has also resulted in the expansion of regulatory T cells in tumor, spleen, and tumor-draining lymph nodes and of splenic neutrophils. This has indicated that targeting the innate, adaptive, and regulatory components of the immune system could lead to destruction of large tumors (Berraondo et al., 2007). Listeria vaccine was earlier reported to induce potent cellular immunity to HPV-E7-expressing tumors (Hussain & Paterson et al., 2004). So, this may indicate the necessity of utilizing HPV –E7 protein for Listeria based immunotherapies.

These reports may strongly indicate the reliable nature of Listeria-E7 vaccines intended for treating cancer patients.

Immunotherapies that rely on tumor -antigen enabled strategies could better address the complications associated with the decline of humoral and cellular immunity which constitute the essential defense branches of the immune system. Hence, there is a need to further explore the medical applications of Listeria.

It was described that a new form of killed but metabolically active (KBMA) recombinant Listeria monocytogenes could ensure antigen delivery and maturation of human dendritic cells DCs (Mojca Skoberne et al., 2008). This was revealed when highly attenuated KBMA

L. monocytogenes was genetically modified to express an epitope of the melanoma-associated antigen MelanA/Mart-1.This strain of Listeria could induce human dendritic cell enabled upregulation of costimulatory molecules and secretion of pro-Th1 cytokines and type I interferons. (Mojca Skoberne et al., 2008). This resulted in priming of Mart-1–specific human CD8+ T cells and lysis of patient-derived melanoma cells. Similarly, this recombinant strain harbouring a melanoma-associated antigen, full-length NY-ESO-1 protein, was reported to deliver the antigen for presentation by MHC class I and II molecules (Mojca Skoberne et al., 2008). This has indicted the efficient antigen delivery of KBMA L. monocytogenes to dendritic cells for presentation thus making it an ideal immunotherapeutic tool for cancer.

It can be inferred that L. monocytogenes may have a diverse antigen presenting potential with regard to the expression of various genes tailored into its genome. This has also strengthened the role of dendritic cells as the reliable antigen presenting cells in the process of immunotherapy.

Schoen et al. (2008) described that the novel strains of L. monocytogenes would play vital role in the delivery of antigens or antigen-encoding DNA and RNA to eukaryotic host cells.

To this end, a novel balanced-lethal plasmid system in L. monocytogenes was built that could enable the cytosolic expression of phage lysin (Schoen et al., 2008). Listeria thus modified became a self-destructing carrier strain and was used for the delivery of antigens and antigen-encoding plasmid DNA and particularly mRNA. This strategy has better implications for bacteria-mediated DNA delivery for the development of live vaccines (Schoen et al., 2008).

Listeria monocytogenes has better addressed the problems concerned with prostate cancer.

The target antigen having immunotherapeutic potential in this context is a Prostate specific antigen (PSA) (Shahabi et al., 2008).Recent workers demonstrated that a recombinant live attenuated L. monocytogenes/PSA (Lm-LLO-PSA) developed by cloning human PSA gene in an attenuated Lm strain has increased its inhibitory effect on tumor infiltrating T regulatory cells leading to the regression of human PSA induced tumors in mouse cell lines(Shahabi et al., 2008). Immunogenicity of Lm-LLO-PSA was revealed in immunized C57BL/6 mice when its splenoytes showed higher number of IFN-gamma secreting cells. The T- cells produced in response to PSA immunization developed the potential to lyse PSA-peptide pulsed target cells. This approach was found to be much more reliable than that observed with PSA H2Db-specific peptide. These findings may indicate that immunization with the new strain Lm-LLO-PSA would be essential to resist the deadly attack of prostrate cancer (Shahabi et al., 2008).

Listeria monocytogenes may also contribute to the management of head and neck cancer.

This could be due to a significant progress achieved with DNA, bacterial vector, viral vector, peptide, protein, dendritic cell, and tumor-cell based vaccines intended for antigen-specific immunotherapy (Wu, Niparko, & Pai, 2008). This strategy may also work as an adjuvant to cancer therapy to destroy regionally localized metastases without inducing damage to the healthy cells in the vicinity (Wu, Niparko, & Pai, 2008).

From the previous information, it is reasonable to mention that as Listeria vector was being tailored to meet the various clinical tasks, it may offer an efficacious immunotherapy for the head and neck cancer. However, understanding the origin and dynamics of cancerous lesions may furnish better insights on the specific antigen to be delivered to the target site using Listeria vector. Frick et al. (2005) previously described that heat killed Listeria monocytogenes (HKL) may provide therapy against allergens. They described that dogs with high food allergy (peanut) have shown improved health with HKL plus allergen treatment indicating the implications of this approach in humans sensitive to food allergy and anaphylaxis. It is believed that heat-killed Listeria monocytogenes when used as an adjuvant was found to reduce immunoglobulin (Ig) E production and reverse allergen-induced airway hyperreactivity (AHR) in a murine model of asthma(Frick et al., 2005).

This may indicate that L. monocytogenes could offer better remedy to allergic disorders when administered with other allergens thus indicating the efficacy of adjuvant approach.

This could also support the report of Wu and his associates who emphasized on the role of adjuvant approaches in managing the cancerous lesions. Hence, this may suggest further exploration of the adjuvant properties of L. monocytogenes that might improve its immunotherapeutic potential. Previously, it was described that a live-attenuated vaccine strain of L. monocytogenes developed by selectively deleting the two virulence factors, Act A (_actA) and Internalin B (_inlB) was able to maintain the immunopotency (Dirk et al., 2004).

This strain was further made to loose its toxicity by blocking the direct internalin B-mediated infection of nonphagocytic cells, such as hepatocytes, and the indirect ActA-mediated infection by cell to- cell spread from adjacent phagocytic cells. Mice model (BALB_c) bearing murine CT26 colon tumor lung metastases vaccinated with recombinant Listeria _actA__inlB expressing an endogenous tumor antigen was found with reduced self-tolerance and long-term survival (Dirk et al., 2004). This strategy has made it an efficient therapeutic tool for cancer vaccine which when administered may readily induce potent innate and adaptive immunity. Listeria _actA__inlB may have better implications if further investigations were carried out with special emphasis on human clinical trials (Dirk et al., 2004).Plasmids intended for Recombinant vaccine technology need to be retained by antibiotic resistance markers which could improve their suitability for various clinical applications (Thorsten Verch, Zhen-Kun Pan &, Yvonne Paterson, 2004). For this purpose, they have developed a shuttle plasmid constructed with Listeria monocytogenes and Escherichia coli that was made to retain the complementation of D-alanine racemase-deficient mutant strains both in vitro and in vivo(Thorsten Verch, Zhen-Kun Pan & Yvonne Paterson, 2004). This approach resulted in the regression of established tumors as observed with a vector harboring a conventional plasmid carrying a tumor-specific antigen. This may indicate that the DNA vaccines and bacterial vaccine vectors may be produced without incorporating antibiotic resistance genes by choosing D-alanine racemase complementation system as the essential Listeria cancer vaccine platform (Thorsten Verch, Zhen-Kun Pan & Yvonne Paterson, 2004). This strategy may alleviate problems concerned with the utility of antibiotic resistance markers. Hence, further investigations may be required. As there were concerns regarding the efficacy of therapeutic vaccines to induce cancer-specific immunity, L. monocytogenes was engineered to express a plasmid encoding a tumor antigen under the control of a mammalian promoter (Souders, 2006).

This approach was based on plasmid release mechanism that involves the suicide of the carrier bacteria with the objective of providing anti-tumor immunity to the cervical cancer oncoprotein, E7. The vaccine thus developed was termed bactofection vaccine’. (Souders, 2006) This was also reported to lessen the tumor growth and facilitate a cytotoxic CD8 + T cell response against the RAHYNIVTF epitope for E7. It was also revealed that Listeria -based vaccines against E7 may dominate the central tolerance by increasing the low avidity CD8 + T cells specific for E7 such that they develop the potential to kill solid tumors. This strategy was exploited to target the tumor associated antigen, telomerase as it is prevalent in 85-90% of all human tumors that express it (Souders, 2006).

Hence, Listeria -based bactofection vector may have better implications for cancer research. Likhite and Vilas (1989) patented Listeria monocytogenes akka, considered as a novel mutant strain. It was reported to possess dual functions by inducing significant titers of Ig M antibodies and as an immunopotentiating agent when conjugated to a sensitizing antigen, for example, living tumor cells and herpes simplex virus (Likhite & Vilas, 1989). The antibodies obtained in this approach were found to have mimicked the specificity as of IgM immunoglobulins (Likhite & Vilas, 1989). The novel strain could serve as a potent immunotherapeutic agent in cancer immunotherapy because of its conjugate nature (Likhite & Vilas, 1989). This may indicate that patenting of Listeria strains has ensured the understanding of potent cancerous agents through humoral and cellular branches of the immune system. Therefore, it is a reliable immunotherapeutic tool.

L. monocytogenes was genetically engineered to express high molecular weight melanoma-associated antigen (HMW-MAA), known as melanoma chondroitin sulfate proteoglycan (Paulo Cesar Maciag et al., 2008).This approach was intended for the immunotherapy of melanoma.

Here, the efficacy of using this antigen is that it expresses on cell surface in a variety of transformed cells with limited distribution in healthy tissues, and its presence in pericytes whch makes it a suitable candidate to meet the requirements of tumor angiogenesis (Paulo Cesar Maciag et al., 2008). Mice immunized with recombinant Lm-LLO-HMW-MAA-C suppressed the tumor growth in its earlier stages indicating the requirement of CD4+ and CD8+ T cells for therapeutic efficacy. They have also observed immune responses to a known HLA-A2 epitope present in the HMW-MAA2160-2258 fragment in the HLA-A2/Kb transgenic mice. This has indicated the potency of Lm-based vaccine in eliciting cell-mediated immune responses to HMW-MAA antigen that can target not only tumor cells but also pericytes in the tumor vasculature (Paulo Cesar Maciag et al., 2008).

This recombinant vaccine was also found to inhibit the progress of tumorigenic cell lines, such as melanoma, renal carcinoma, and breast tumors, which were not engineered to express HMW-MAA. Further, this microbe was also exploited for targeting breast cancer metastases. The expression of overlapping fragments and protein encoding regions of Mage-b (Melanoma antigen) as a fusion protein with a truncated non-cytolytic form of listeriolysin O (LLO) in recombinant L.monocytogenes has improved its therapeutic efficacy in a syngeneic mouse tumour model 4T1(Kim et al., 2008).This became evident when LM-LLO-Mage-b/2nd enabled vaccination produced a relatively low number of metastases that was in agreement with the Mage-b-specific CD8 T-cell responses in the spleen, after restimulation with Mage-b. This has strengthened the role of Listeria vaccine as an efficient immunotherapeutic tool to prevent metastases and residual tumor cells. In contrast, 4T1 primary tumors were insensitive to the effect of LM-LLO-Mage-b/2, not indicating any presence of Mage-b-specific immune responses (Kim et al., 2008). Hence, it can be inferred that there may be a need to monitor the efficacy of this vaccine after the removal of primary tumors in breast cancer individuals.

In view of the above information, therapeutic aspects of Listeria monoctyogenes have addressed the problems concerned with the immunology of cancer and the associated infectious disorders. This bacterium considered as the chief agent in inducing food borne disease, was reported to contain virulence factors that facilitate the antigen processing and presentation (Paterson & Maciag, 2005).Interest was centered on its molecular biology as it has strong evolutionary background that made it achieve genetic stability with regard to its cellular and host–pathogen interactions (Marie Ragon et al., 2008). Since years investigators have been developing various strains of Listeria due to the fact that it could harbor genes or antigenic products of various cancers. Tumor protein of Human Papilloma virus such as HPV-E7, was proven to generate significant cellular immune responses through the development of vaccine vectors such as Lm-LLO-E7. The virulence factor listeriolysin O (LLO) has contributed to the better understanding of antitumor potential of Listeria (Sewell et al., 2004).This might have improved its utility and consistency till the recent development of L. monocytogenes recombinants such as Lm-LLO-PSA, Lm-LLO-HMW-MAA-C and LM-LLO-Mage-b/2^nd.

Antigen delivery was made much efficient with killed but metabolically active (KBMA) recombinant Listeria monocytogenes (Mojca Skoberne et al., 2008).Adjuvant approaches of Listeria immunotherapy may also have better implications for treating head and neck cancer (Wu, Niparko, & Pai, 2008).Various animal models have been exploited to study the immunotherapeutic potential of Listeria based vaccine vectors. As findings revealed that Listeria monocytogenes based therapy could stimulate both humoral and cellular branches of the immune system, it might retard the early progress of cancerous lesions that may strive to overcome the vital immunological barriers. On the whole Listeria monocytogenes could become an essential immunotherapeutic tool in light of growing concerns over the cancer induced depletion of normal cellular and molecular machinery.

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