key: cord-0037483-1h84yi2i
authors: Kumar, Sudeep
title: Live-Attenuated Bacterial Vectors for Delivery of Mucosal Vaccines, DNA Vaccines, and Cancer Immunotherapy
date: 2019-01-10
journal: Pharmaceuticals from Microbes
DOI: 10.1007/978-3-030-01881-8_2
sha: 1801513c69b235e2631913f915203bb67a1757e3
doc_id: 37483
cord_uid: 1h84yi2i

Vaccines save millions of lives each year from various life-threatening infectious diseases, and there are more than 20 vaccines currently licensed for human use worldwide. Moreover, in recent decades immunotherapy has become the mainstream therapy, which highlights the tremendous potential of immune response mediators, including vaccines for prevention and treatment of various forms of cancer. However, despite the tremendous advances in microbiology and immunology, there are several vaccine preventable diseases which still lack effective vaccines. Classically, weakened forms (attenuated) of pathogenic microbes were used as vaccines. Although the attenuated microbes induce effective immune response, a significant risk of reversion to pathogenic forms remains. While in the twenty-first century, with the advent of genetic engineering, microbes can be tailored with desired properties. In this review, I have focused on the use of genetically modified bacteria for the delivery of vaccine antigens. More specifically, the live-attenuated bacteria, derived from pathogenic bacteria, possess many features that make them highly suitable vectors for the delivery of vaccine antigens. Bacteria can theoretically express any heterologous gene or can deliver mammalian expression vectors harboring vaccine antigens (DNA vaccines). These properties of live-attenuated microbes are being harnessed to make vaccines against several infectious and noninfectious diseases. In this regard, I have described the desired features of live-attenuated bacterial vectors and the mechanisms of immune responses manifested by live-attenuated bacterial vectors. Interestingly anaerobic bacteria are naturally attracted to tumors, which make them suitable vehicles to deliver tumor-associated antigens thus I have discussed important studies investigating the role of bacterial vectors in immunotherapy. Finally, I have provided important discussion on novel approaches for improvement and tailoring of live-attenuated bacterial vectors for the generation of desired immune responses.

Vaccines provide protection against numerous life-threatening infectious diseases, by activating the adaptive immunity against specific pathogen-derived antigens.

Since the introduction of active immunization, several vaccines have been licensed for human use. These include some subunit vaccines, which are preferred for their superior safety profile. However, their success is limited by their poor immunogenicity, as multiple booster immunizations and adjuvants are required to achieve an adequate level of protective immunity. Moreover, a subunit vaccine is only applicable for pathogens where a well-defined protective antigen has been discovered. Subunit vaccines are also limited in their ability to induce cell-mediated immunity.

In contrast, the live-attenuated/live-inactivated vaccines exhibit superior immunogenicity and induce humoral as well as cell-mediated immunity. Although attenuated viruses and bacteria are both utilized as vaccine vectors, this review will focus only on attenuated bacterial vaccine vectors. Bacteria harbor natural adjuvants in the form of pathogen-associated molecular patterns (PAMPs) (Fig. 2.1) . PAMPs, which are recognized by components of the innate immune system including Tolllike receptors (TLRs), facilitate the release of pro-inflammatory mediators and recruitment of antigen-presenting cells ( Fig. 2.2) . Furthermore, even after attenuation, a limited degree of proliferation and dissemination capacity is retained in the attenuated pathogens. Overall, these factors contribute to the superior immunogenicity of live-attenuated bacteria, which consequently elicit a robust and durable immunity against the cognate antigens.

With the advent of molecular biology and genetics, it is feasible to effectively excise or insert desired genes into bacteria. Bacterial vectors can be engineered to express and deliver heterologous proteins, such as antigens or therapeutic proteins, in mammalian hosts. Moreover, by genetic manipulation, bacterial vectors can be engineered with properties including reduced virulence, high immunogenicity, properties which are desirable in a vaccine vector. Thus far, a variety of liveattenuated bacterial vectors including Mycobacterium bovis strain Bacillus Calmette-Guérin (BCG), Salmonella spp., Listeria monocytogenes (Lm), Vibrio cholerae, Escherichia coli, and Shigella spp. have been utilized for the delivery of heterologous proteins into mammalian hosts as vaccine antigens or therapeutic proteins. Such bacteria are called, live-attenuated bacterial vectors (LABVs).

Vaccines elicit distinct immune responses depending on the route of immunization. Mucosal immunization induces strong systemic as well as mucosal immune (1) is complemented with a functional copy of asd gene, inserted into the plasmid (2); this feature ensures antibiotic-free maintenance of plasmids. The plasmid (2) also carries genes encoding antigenic proteins. Various pathogen-associated molecular patterns including flagellin (3), lipopolysaccharide (4), lipoprotein (5), and peptidoglycan (6) facilitate the interaction with and signal the activation of antigen-presenting cells, while the additional appendages like autotransporters (7) facilitate surface display of antigens response, whereas parenteral immunization induces potent systemic but a poor mucosal immune response. Since birth, mucosal surfaces of the human body are constantly challenged with agents of the external environment that are either completely harmless (food ingredients and nonpathogenic microbes) or pathogenic (pathogenic microbes). Thus, in order to restrict pathogenic insults at mucosal surfaces, mucosa-associated lymphoid tissues (MALT) are organized. In fact, MALT constitutes the largest immune system of the human body. The oral route is the most favored route for mucosal immunization over other mucosal routes including nasal,

Activation of antigen-presenting cells by live-attenuated bacterial vectors leads to adaptive immune response: Various pathogen-associated molecular patterns present in the liveattenuated bacterial vectors interact with Toll-like receptors expressed on the surface or in endosomal membranes. The signaling initiated by this interaction leads to the activation of antigenpresenting cells. Activated antigen-presenting cells express costimulatory molecules CD80, CD86, and CD40 as well as enhance expression of MHC-II. Costimulatory molecules are required to deliver the essential second signal for T-cell activation, while the first signal is received via TCR-MHC-peptide interaction. Importantly, CCR7 expressed by activated APCs help migration to draining lymph node. Moreover, the type of cytokines directs the fate of T-cell polarization to Th1, Th2, or TH17. Cytosolic delivery of antigens gives rise to CTL response vaginal, and rectal. Upon oral administration, antigens travel through the gastrointestinal tract and reach the mucosal inductive sites called Peyer's patches. Peyer's patches are lined with specialized epithelial cells called M cells which serve as a point of entry into the lamina propria. In the lamina propria, dendritic cells take up the antigens and migrate to the draining lymph nodes where they present the antigens to T cells. A specialized feature of dendritic cells from Peyer's patches and mesenteric lymph nodes is that they induce gut-homing receptors α4/β7 and CCR9 on T and B cells. This feature is not found in the dendritic cells from cervical lymph nodes and spleen. Thereby, T and B cells primed at the mucosal sites are destined to migrate to mucosal tissues (Pasetti et al. 2011) (Fig. 2.3 ). Live-attenuated microbes exhibit superior ability to deliver vaccine antigens to the mucosal immune system, as many of them are derived from natural mucosal pathogens, including Salmonella spp., Lm, E. coli, V. cholerae, and Shigella spp. This review explores the current knowledge about the LABV application in the delivery of vaccine antigens (to the mucosal immune system), DNA vaccine, and immunotherapy. Mechanism of immune responses elicited by LABV-based vaccines, the recent advances, and future perspectives have been discussed.

Mucosal immune response elicited by live-attenuated bacterial vectors: Mucosal inductive sites including gut-associated lymphoid tissues and nasal-associated lymphoid tissues facilitate sampling of antigens through M cells. M cells allow the passage of bacteria through the mucosal epithelium, where they are taken up by antigen-presenting cells including DCs and macrophages. APCs undergo activation upon interaction with live-attenuated bacterial vectors, and the associated antigens are presented to T cells in the lymphoid follicles or the draining lymph nodes. Activated T cells help B cells differentiate into IgA-producing plasma cells. The secreted IgA provide effective protection against mucosal pathogens. The T and B cells primed at mucosal sites migrate back to mucosal sites where they perform their effector functions

Nonpathogenic commensals including the lactic acid bacteria and Bacillus subtilis as well as the attenuated versions of the pathogenic bacteria including Salmonella spp., E. coli, Shigella spp., Lm, and V. cholerae have been utilized as LABVs. While the commensal microbes are generally regarded as safe or food grade, the virulence of the pathogenic microbes needs to be significantly attenuated before they can be considered safe to deliver vaccine antigens into humans ( Fig. 2.1) .

Generally, plasmids are employed for the expression of heterologous antigens in LABVs. Plasmids can be easily manipulated in E. coli (a universal tool for genetic engineering) and subsequently introduced into the desired bacterial strains. Shuttle vectors (plasmids) carry genetic elements for replication in E. coli and promoter elements for gene expression in other bacterial or mammalian cells. Thus, mammalian expression vectors can be first manipulated and propagated in E. coli and then delivered via LABVs to mammalian cells, where the desired antigens are expressed ( Fig. 2.1 ).

In early days, attenuation of the pathogenic microbes was achieved by in vitro cultivation for several generations, followed by the evaluation of virulence in successive generations. The classic example is BCG, where Albert Calmette and Camille Guerin, by culturing a virulent strain of M. bovis for more than 230 serial passages in vitro (between 1908 and 1921) , generated the attenuated strain BCG. BCG is still the only vaccine available for prevention against tuberculosis. Subsequent genetic analysis revealed that BCG lack multiple virulence factors associated with M. bovis (Zheng et al. 2015) . Similarly, the search for vaccines against typhoid fever led to the generation of attenuated live vaccine strains of Salmonella. Salmonella enterica serotype Typhi (ST) strain Ty21a (ST-Ty21a) was generated by chemical mutagenesis of wild-type ST strain Ty2. The ST-Ty21a is considerably attenuated which is now licensed for humans use as an oral vaccine (Wang et al. 2001 ). However, the strain exhibits low immunogenicity, as 3-4 doses are required to achieve adequate levels of protection. With the advances in microbial genetics and genetic engineering techniques, it has now become routine to identify and inactivate virulence genes. Interestingly, various auxotrophic mutant Salmonella strains, which lack the ability to synthesize aromatic amino acids, were found to be avirulent (Hoiseth and Stocker 1981) . It is known that in Salmonella, the gene products of aroA, aroC, and aroD are required for the biosynthesis of aromatic amino acids, as well as several essential vitamins. Hoiseth and Stocker note that these factors are not found in mammalian hosts in sufficient amount; thus Salmonella aroA mutants cannot proliferate in mammalian hosts (Hoiseth and Stocker 1981) . Harnessing this knowledge, various Salmonella enterica serovar Typhi (ST) or Typhimurium (STm) have been created, with mutations in aroA (Dalla Pozza et al. 1998; Roberts et al. 2000; Arnold et al. 2004) , aroC (Khan et al. 2003; Capozzo et al. 2004) , aroD (Capozzo et al. 2004; Sevil Domènech et al. 2008) , or aroAD (Strugnell et al. 1992; Roberts et al. 2000) . Notably, the ST strain CVD908 which carries aroC and aroD mutations exhibits residual virulence in humans (Wang et al. 2001) , while other investigators have also targeted genes in nucleotide biosynthesis pathways for creating attenuated Salmonella. Wang et al. engineered a Salmonella strain with a mutation in guaBA operon, which interferes in the guanine nucleotide biosynthesis. The resultant strain, called CVD 915, exhibits safety profile comparable to that of the typhoid vaccine strain ST-Ty21a. Importantly, ST-Ty21a, CVD-908-htrA (harboring mutations at aroC, aroD, and htrA), and CVD 915 all exhibit a high level of immunogenicity (Wang et al. 2001) . Not surprisingly, ST strains with guaBA mutations have been widely utilized as LABV (Pasetti et al. 1999 (Pasetti et al. , 2000 Wang et al. 2001) . Another approach of attenuation of Salmonella is to introduce mutations in cya (adenylate cyclase) and crp (cyclic AMP) receptor genes. These proteins are transcriptional regulators of many important genes. Although cAMP is found in mammalian cells, their concentrations in gastrointestinal tissues are below the requirement of Salmonella. Thus cya and crp mutants show reduced virulence (Tacket et al. 1992; Chen and Schifferli 2003; Wyszyńska et al. 2004; Ferreira Oliveira et al. 2012) . Mutations in the two-component regulatory system PhoP/PhoQ, which controls more than 40 virulence genes involved in resistance to antimicrobial peptides, nutrient scavenging, and lipid A modifications, significantly decrease Salmonella virulence (Raupach and Kaufmann 2001) . Salmonella strains with PhoP/PhoQ mutations have been used in a number of studies as LABV (Angelakopoulos and Hohmann 2000; Kotton et al. 2006; Galen et al. 2009; Wang et al. 2013) . RpoS is an alternate sigma factor that regulates resistance under stress induced during gastrointestinal infection such as pH, nutrient starvation, change in osmolarity, and temperature. ST-Ty21a contains multiple mutations including rpoS (Wang et al. 2001) . SsaV is a component of Salmonella type III secretion system, which is required for secretion of SPI-2 genes (essential for growth in macrophages). The ST strain ZH9 which contains aroC and ssaV mutations is highly attenuated and immunogenic in humans (Hindle et al. 2002) . Tacket et al. generated mutations in htrA gene, which encodes a heat shock protein. The resultant strain was avirulent because of reduced ability to survive and replicate in host tissues (Tacket et al. 2000) . HtrA mutant Salmonella strains have been used in multiple studies as LABVs (Galen et al. 1999; Roberts et al. 2000; Pasetti et al. 2002; Capozzo et al. 2004; Fraillery et al. 2007) .

Similarly, for attenuation of Lm, multiple virulence factors have been targeted. ActA which encodes for a surface protein required for actin polymerization in host cells and helps in intracellular migration has been a prominent target for Lm attenuation. Together with the mutation in internalin B (inlB), the actA mutation renders Lm unable to infect hepatocytes; thus these strains are highly attenuated . Phospholipase-C B (PlcB) is required for efficient escape from phagosomal vacuoles. PlcB mutants are thus attenuated due to defect in escape from secondary vacuoles (Peters et al. 2003; Starks et al. 2004; Stevens et al. 2004; Johnson et al. 2011; Jia et al. 2012; Liang et al. 2014) . Cell wall biosynthesis genes specifically dal (alanine racemase) and dat (D-amino acid aminotransferase) have been mutated in several attenuated Lm vaccine vectors (Friedman et al. 2000; Verch et al. 2004; Jiang et al. 2007; Im et al. 2013) . The double mutant of Lm requires D-alanine for cell wall biosynthesis and is highly attenuated. Recently McLaughlin et al. demonstrated that deletion of Lm fur-regulated virulence factor A (frvA) results in attenuation in murine models of infection, due to the inability of iron homeostasis (McLaughlin et al. 2013) .

Attenuated strains of Shigella are also being used as LABV. Noriega et al. (Noriega et al. 1996) generated guaBA mutant of Shigella CVD1204, which is highly attenuated in animals and is widely used as LABV. Other approaches of mutations include SC602, with deletions on icsA (mediate intra-and intercellular spread) and iucA (aerobactin); this strain is highly attenuated and immunogenic (Ranallo et al. 2005 ).

Introduction of heterologous genes into bacterial vectors is facilitated by plasmids. Plasmids are extrachromosomal circular DNA, which are introduced into bacteria by a process called transformation. Generally, bacteria maintain the plasmids utilizing antibiotic resistance mechanism. During in vitro growth, antibiotic selection pressure ensures stable plasmid maintenance; however, in the in vivo conditions, the lack of antibiotic selection pressure plasmid-less bacteria outgrows plasmid-bearing bacteria. Moreover, the use of antibiotic markers are also discouraged, due to the risk of horizontal gene transfer to other microbes with pathogenic potential (Lin et al. 2015; Mignon et al. 2015) . Novel antibiotic-free approaches of plasmid maintenance have been devised to mitigate these concerns. One such approach, known as the balanced lethal system, utilizes mutation in an essential gene in the bacterial chromosome, while the plasmid carries the functional copy of the same gene, thereby ensuring its maintenance by the bacteria (Fig. 2 .1). Galen et al. generated a balanced lethal system for STm based on mutation in asd gene. Asd encodes aspartate semialdehyde dehydrogenase, an enzyme required in the biosynthesis pathway of DAP (diaminopimelic acid), which is an essential component of bacterial cell wall. DAP is needed for growth and maintenance of asd mutants. A copy of asd gene is inserted into the plasmid; thus asd-deficient bacteria are forced to maintain the plasmid in order to survive in DAP-deprived conditions, such as in the mammalian tissues. The resultant Salmonella typhimurium (STm)-based balanced lethal system exhibits high degree of plasmid stability. This system also exhibits stable expression of the associated heterologous genes (Galán et al. 1990 ). Balanced lethal system has been most widely used in various LABVs including ST (Tacket et al. 1997) , STm (Kang et al. 2002) , and S. flexneri (Zheng et al. 2005) . Similarly, thymidine auxotrophy has also been utilized in ST (Bumann et al. 2010) , STm (Mignon et al. 2015) , and lactic acid bacteria (Bermúdez-Humarán et al. 2011) for balanced lethal system approach of plasmid maintenance. Glutamine auxotroph V. cholerae complemented with glnA gene is another example of the balanced lethal system utilized for antibiotic-free plasmid maintenance (Ryan et al. 2000 ).

Various mucosal pathogens and nonpathogenic food grade microbes have been extensively utilized for LABV development. Salmonella infect via orogastric route and enter the intestinal lamina propria by transcytosis via M cells, which are present in the mucosal inductive sites (Peyer's patches). In the lamina propria, Salmonella is taken up by various phagocytes including neutrophils, macrophages, and dendritic cells. The infected phagocytes then carry Salmonella to various organs including the liver and spleen via blood or to the mesenteric lymph nodes via lymph. The virulence factors, clustered in Salmonella pathogenicity islands (SPI-1 and SPI-2), facilitate invasion, survival, and proliferation in the intracellular spaces of macrophages (Pham and McSorley 2015) . Salmonella possess a variety of pathogen-associated molecular patterns (PAMPs) including lipoprotein, lipopolysaccharide (LPS), flagellin (FliC), and CpG. These PAMPs are recognized by host pattern recognition receptors (PRRs) including TLR 2(1/6) (lipoproteins), TLR4 (LPS), TLR 5 (FliC), or TLR9 (CpG). Activation of these PRRs leads to the expression and secretion of cytokines such as TNFα, IL1β, IL6, IL8, IL12, IL-18, and IL-23 (Broz et al. 2012 ). These pro-inflammatory factors recruit neutrophils, macrophages, and dendritic cells. On the other hand, upon interaction with Salmonella LPS and flagellin, dendritic cells increase the expression of CCR7, CD80, CD86, and CD40. These mature dendritic cells with enhanced capability to process and present antigens can migrate to T-cell areas and initiate adaptive immune responses to cognate antigens. Studies suggest that Salmonella induces humoral as well as CD4 + -, CD8 + -, and Th17-dependent cellmediated immune responses (Pham and McSorley 2015 (Angelakopoulos and Hohmann 2000) , L. monocytogenes (Igwe et al. 2002) , Pseudomonas aeruginosa (Bumann et al. 2010) , Streptococcus pneumoniae , and Yersinia pestis . While antigen-specific Th1 responses are generated against some parasitic pathogens including Leishmania mexicana (González et al. 1998) , Schistosoma japonicum (Chen et al. 2011) , and Taenia solium (Ding et al. 2013) , mucosal IgG and IgA are generated against Giardia lamblia (Abdul-Wahid and Faubert 2007) and Cryptosporidium parvum (Benitez et al. 2009 ) by ST-and STm-based LABVs carrying related antigens. Besides Salmonella, Listeria is the most extensively studied bacteria as LABV. Similar to Salmonella, Listeria infection begins with orogastric infection. Lm moves across gastrointestinal epithelial barrier by first attaching to and invading epithelial cells. Adhesion and internalization require Lm protein Ami and internalin A (inlA), respectively. Once in the lamina propria Lm rapidly spreads systemically. Lm primarily targets liver with the help of fibronectin binding protein (FbpA). FbpA recognizes fibronectin on the surface of hepatocytes, and at this point another molecule, called internalin (inlB), facilitates Lm internalization. On the other hand, phagocytes specifically macrophages and monocytes recognize lipoteichoic acid via scavenger receptors. After the phagocytosis Lm escapes phagocytic vesicles by synergistic activities of listeriolysin O (LLO) and two phospholipase C (PlcA and PlcB). Another virulence factor is ActA, which has actin polymerization activity and helps Lm migrate from cell to cell (Liang et al. 2014) . Lm expresses various TLR agonists including peptidoglycan, flagellin, and bacterial DNA, which induces pro-inflammatory cytokines including TNFα, IFNγ, IL1β, and IL12. Lm-induced cell death results in secretion of IL6, which helps in recruitment of neutrophils. IL12 helps in induction of IFNγ by NK cells and CD8 + T cells. Lm can also induce type-I IFNs (IFNαand IFNβ), which is desirable as antiviral immunity. Evidences suggest that Lm induces both CD4 + and CD8 + T-cell-mediated immune response (Zenewicz and Shen 2007; Liang et al. 2014) . Lm-carrying viral antigens such as HIV/SIV-gag (Frankel et al. 1995; Friedman et al. 2000; Im et al. 2013) , HPV-E7 (Jia et al. 2012) , and LCMV-NP118-126 (Tvinnereim et al. 2002) induces CTL immune response. Lm-based LABV also induces neutralizing antibody against HIV-gp160 . However, there are limited reports on Lm as LABV against bacterial and parasitic pathogens. In one study, Lm-carrying Coxiella burnetii antigen T4SS (epitopes) induced CD8 + T-cell immune response (Xiong et al. 2017) . In another study Lm-carrying Francisella tularensis antigen IglC induced IFNγ producing CD4 + and CD8 + T-cell-mediated immune response.

BCG, an attenuated M. bovis, has also exhibited potential as LABV. Following immunization, BCG interacts with phagocytes such as macrophage, dendritic cells, and neutrophils. Various PRRs of macrophages involved in interaction with BCG include CR3, TLR2 (1/6) and TLR-4. However, dendritic cells utilize a different set of phagocytic receptors including CR3, CR4, DC-SIGN (CD209), and DEC 205. Infected dendritic cells upregulate expression of MHC-II and costimulatory markers CD80, CD86, CD40, and CD54 which are involved in activation of adaptive immune response (Moliva et al. 2017) . BCG is known to induce humoral as well as T-cell-mediated immune response (Abomoelak et al. 1999) . The T-cell responses induced by BCG include polyfunctional CD4 + T cells that secret TNF, IL-2, and IFNγ (Moliva et al. 2017) . BCG expressing IL12 and two M. tuberculosis (Mtb) antigens (secreting antigen Ag85B and culture filtrate antigen CFP10) induce antigen-specific Th1-type immune response including IFNγ-producing cells and IgG2a (Chen et al. 2017) . Antigen-specific humoral immune response is induced by BCG expressing a hepatitis-B surface antigen (Rezende et al. 2005) . BCG-induced CD8 + T cells also secrete IFNγ (Moliva et al. 2017) . In a mouse model, BCG carrying Mtb antigen (Ag85B) exhibit antigen-specific Th17 immune response (Hatano et al. 2016) . Pertussis toxin subunit S1 expressed by BCG induces IFNγ producing CD4 + T cells which completely protects against lethal Bordetella pertussis challenge (Nascimento et al. 2008) . Furthermore, in a mouse model of an intracellular pathogen Lm, BCG expressing Lm antigen p60 induced CD4 + and CD8 + T-celldependent protection (Grode et al. 2002) . Studies have demonstrated that BCG induces long-lived mycobacteria-specific memory B cells. Moreover, following BCG immunization, hosts secrete robust Mtb-specific serum antibodies including IgG isotypes IgG1, IgG2, and IgG3. However, it is not known if specific mucosal IgA is induced by BCG (Moliva et al. 2017) .

S. flexneri infection occurs through orogastric route. Once in the colon, S. flexneri crosses epithelial layer through highly endocytic M cells. S. flexneri then adhere to and infect colonic epithelium through the basolateral surface. Colonic epithelial cells engulf S. flexneri by macropinocytosis, and through the activity of IpaB and IpaC, they are released from macropinocytic vacuoles to the cytosol (Mellouk and Enninga 2016) . S. flexneri invasion causes activation of innate immune system and release of a variety of cytokines including IL-1, TNF-α, IL6, TGF-β, and IL-8 (Fernandez and Sansonetti 2003; Jennison and Verma 2004) . Though Shigella has the capacity of cell-to-cell translocation, its infection is limited to lamina propria of the intestine, and it doesn't migrate to other organs (Maurelli and Sansonetti 1988) . In the lamina propria S. flexneri is phagocytosed by macrophages and dendritic cells. Infected macrophages undergo apoptosis, which leads to the release of proinflammatory cytokine IL1, IL18, and IFNγ (Fernandez and Sansonetti 2003) . S flexneri induces both systemic and mucosal antibody response including IgM, IgG, and secretory IgA (Jennison and Verma 2004) . S. flexneri 2a with guaBA mutation (CVD 1204) has limited invasiveness, and proliferative capacity. Attenuated S. flexneri expressing ETEC antigens CFA-I, LTB, CS2, CS3, and CS4 induce antigenspecific serum IgG and mucosal IgA (Koprowski et al. 2000; Barry et al. 2003; Strain et al. 2003; Ranallo et al. 2005; Zheng et al. 2005) .

Food grade bacteria including B. subtilis and Lactobacillus lactis are considered important candidates for LABV, due to their superior safety profile. Upon oral administration, B. subtilis spores can safely transit through the stomach, germinate, and proliferate in the upper intestine and finally undergo re-sporulation in the colon (Cutting et al. 2009 ). Nevertheless, the mechanism of immune response in response to B. subtilis delivered antigens is not fully understood. Antigens delivered by B. subtilis have been shown to induce humoral as well as Th1-mediated immune response (Cutting et al. 2009 ). B. subtilis has been used as LABV for various bacterial and parasitic pathogens including pathogenic E. coli, H. pylori, Mtb, Clonorchis sinensis, and S. japonicum. B. subtilis induces systemic IgG (Amuguni and Tzipori 2012; Zhou et al. 2015) , mucosal IgA (Amuguni and Tzipori 2012; Zhou et al. 2015) , and Th1/Th17 (Sibley et al. 2014; Stasilojc et al. 2015) immune response against cognate antigens. The lactic acid bacteria are among the microbes, which occur physiologically in animal digestive tracts and like other natural microflora through their metabolites and interaction with macrophages can stimulate cytokine production. Peptidoglycan of the lactic acid bacteria induces secretion of IL1, IL6, and TNF, by monocytes (Bermúdez-Humarán et al. 2011; Szatraj et al. 2017) . Unlike attenuated strains of otherwise pathogenic microbes used as LABV, B. subtilis and L. lactis do not invade through the gut mucosa and serve mainly as protein (antigen) factories, which supply vaccine antigens to gut-associated lymphoid tissue (GALT). Protective antigens of pathogenic viruses H1N1 (HA) and H5N1 (HA) expressed by L. lactis induce mucosal antibodies. L. lactis expressing bacterial antigens Campylobacter jejuni (cjAD) (Kobierecka et al. 2016) , Clostridium difficile (TcdA) (Yang et al. 2013) , Clostridium perfringens (epsilon toxoid) (Alimolaei et al. 2016) , H. pylori (omp22, HpaA, cag12, and UreaseB) (Kim et al. 2006; Gu et al. 2009; Zhang et al. 2016b) , and V. cholerae (WZM) (Zamri et al. 2012 ) also induce mucosal antibodies.

In preclinical models, DNA vaccines have proven to confer protective immunity against a variety of infectious agents including HIV, herpes simplex virus (HSV), Plasmodium spp., and Mtb (Schoen et al. 2004 ). An attractive feature of DNA vaccine is that it can induce humoral as well as cell-mediated immune response. While antibodies alone can protect against many pathogens and toxins, cell-mediated immunity is required for protection against intracellular pathogens and cancer. The DNA vaccines in the form of eukaryotic expression plasmids are delivered either by intramuscular injection of naked DNA, intradermal bombardment using DNA coated on gold particles with help of a gene gun, or electroporation following needle injection. However, most of these methods induce only moderate levels of protection in animal models and fail to show efficacy in clinical trials (Schoen et al. 2004 ). In recent years many bacterial vectors have been utilized to deliver plasmids into the host cells (Schoen et al. 2004 ). As many attenuated strains are being developed for delivery of vaccine antigens, similar strains can also be utilized to deliver plasmids as DNA vaccines. Attenuated strains of gut pathogens including ST, STm, or L. monocytogenes are of particular importance, as they colonize and infect mucosal epithelial cells. As discussed above, Listeria infection begins at gastrointestinal tract, and after invasion through intestinal mucosa, Listeria migrate through blood vessels and lymph to other organs. Listeria can infect a wide array of cell types including intestinal epithelial cells, hepatocytes, dendritic cells, and macrophages. Listeria escape phagocytic vesicles and multiply in cytosol where they release the plasmids. Listeriolysin O helps Listeria lyse and escapes the phagosomal vacuoles (Liang et al. 2014 ). Miki et al. engineered a self-destructing Lm-based vaccine delivering a eukaryotic expression plasmid encoding Mtb antigens Ag85a/Ag85b and MPB/ MPT51. The vaccine induced protective immune response against Mtb in a mouse model (Miki et al. 2004) .

Salmonella also infects via gastrointestinal tract, and after crossing epithelial barrier through M cells, Salmonella is taken up by macrophages (Pham and McSorley 2015) . Salmonella has the capability of surviving and replicating in phagocytic vacuoles (Pham and McSorley 2015) . However, through unknown mechanisms, they can release plasmid DNA into the cytosol (Schoen et al. 2004) . Salmonella strains expressing listeriolysin O have been shown to escape the phagosome vesicles to the cytosol, thus making gene transfer by Salmonella more efficient (Schoen et al. 2004 ). HIV-1 T-cell epitopes in the form of eukaryotic expression plasmid delivered by attenuated STm induced CTL as well as antibody immune response (Karpenko et al. 2004) . Another study targeting an S. pneumoniae protective antigen PsaA and PspA delivered by STm induced mucosal IgA against both antigens. Thus immunized mice were protected against nasopharyngeal colonization by S. pneumoniae (Zhang et al. 2011) . Pathogenic parasites Trichinella spiralis and Trypanosoma cruzi have also been targeted for STm-mediated DNA vaccination. Yang et al. constructed a DNA vaccine against T. spiralis using antigen Ts87 and STm as the delivery vehicle. Mice immunized orally with this vaccine induced antigen-specific mucosal IgA which correlated with protection against T. spiralis larval challenge. Salmonella-delivered T. spiralis DNA vaccine induced a Th1-/ Th2-type immunity and IL5, IL6, and IL10 cytokines (Yang et al. 2010) . In another study, Matos et al. using STm delivered T. cruzi antigens (Tc-52) into mice via the oral route. Immunized mice elicited specific antibodies with higher IgG2a/IgG1 ratio, suggesting a Th1 bias. The vaccinated group also induced strong cell-mediated immunity and mucosal IgA (Matos et al. 2014 ).

Most bacteria used as DNA delivery vehicles were designed to disintegrate after infecting host cells. If the bacterial DNA vaccine vectors are destroyed in the phagolysosomes, before reaching the cell cytoplasm, it will lead to inefficient delivery of the plasmid. To circumvent this problem various approaches have been devised. One such approach takes advantage of phage lysin to disintegrate ∆aroA-Lm after reaching host cell cytosol. The inclusion of phage lysin significantly improved bactofection (bacteria-mediated delivery of plasmid DNA into mammalian cells) efficiency in phagocytic as well as non-phagocytic cells (Pilgrim et al. 2003) . Recently, Kong et al. developed a universal DNA vaccine delivery platform, which includes several modalities for enhanced delivery and immune response to cognate antigens. The attenuated STm includes the capability to escape the phagosomal compartment to the cytosol of the host cells, before phagolysosomal degradation (Kong et al. 2012) . SifA proteins direct Salmonella-induced filament formation when Salmonella is contained in the endosomal vacuoles, and the deletion of sifA gene results in the release of Salmonella into the cytosol. Hence, mutation of sifA gene in Salmonella plasmid carriers allowed successful transfer of plasmid DNA into the cytosol of the host cells (Kong et al. 2012) . Kong et al. also incorporated elements that guide the plasmid into the nucleus. Transcription factors such as NF-κB and AP2 bind to plasmids carrying NF-κB and AP2 binding sequences and transport them to the nucleus where the desired antigens are transcribed (Kong et al. 2012) . Salmonella induces apoptosis/pyroptosis in infected cells that diminishes the overall transfection efficiency. Deletion of tlpA and sseL genes significantly reduces apoptosis in host cells (Kong et al. 2012) . Moreover, Salmonella degradation is delayed due to the regulated expression of the Salmonella lysis program. This allows a limited number of replication and invasiveness, thereby ensuring optimal delivery of plasmids. An influenza antigen (HA) delivered by this platform induced enhanced HA-specific IgG, which correlated with protection against influenza virus challenge (Kong et al. 2012 ).

A nineteenth-century physician, William B. Coley, for the first time observed regression of malignant tumor in one of his patients after a bacterial infection. Coley went on to develop the first bacterial therapy against cancer using killed gram-positive bacteria streptococci and a gram-negative bacteria Serratia marcescens. This mixture called "Coley's toxins" when injected into patients suffering from various forms of cancer resulted in partial to complete regression. In cases of soft tissue sarcoma, long-term disease-free survival was achieved in approximately 50% of the patients. Nevertheless, despite the remarkable success of "Coley's toxins," with the advent of chemotherapy and radiotherapy, this line of investigation was prematurely abandoned (Bickels et al. 2002) . However, in recent years this approach is regaining attention. In fact, BCG is currently being used as immunotherapy for bladder cancer and exhibits superiority over epirubicin and IFNα2b, mitomycin, and epirubicin alone (Fuge et al. 2015) . Since the first report of BCG's use in cancer treatment in 1936, preclinical and clinical investigations of BCG have also been reported for other forms of cancer. Mice preimmunized with BCG exhibited slower tumor growth compared to control (Zheng et al. 2015 (Zheng et al. 2015) . Bacteria, specifically anaerobes, exhibit natural tropism toward solid tumors. This phenomenon, although poorly understood, is theorized that certain characteristics of tumor microenvironment facilitate this phenomenon. The deeper pockets of tumors, which are devoid of new blood vessels, are poorly oxygenated and show limited accessibility to chemotherapeutic drugs (Lee 2012; Lin et al. 2015) . Forbes et al. demonstrated that STm accumulate at a rate of 2000-fold more compared to other organs including the liver, spleen, lung, heart, and skin (Forbes et al. 2003) .

Using an in vitro model, Kasinkas and Forbes demonstrated that STm exhibits chemotaxis. Depending on the availability of specific receptors (tsr, tar, and trg), STm were differentially attracted to corresponding chemoattractants expressed in the tumor microenvironment (serine, aspartate, and ribose/glucose), while the wildtype strains accumulate around necrotic zones inside tumors (Kasinskas and Forbes 2007) . Moreover, various immunosuppressive mechanisms manifested by the tumor microenvironment also support the proliferation of microbes (Lin et al. 2015) .

Distinct tumor-homing property of microbes, including Lm and Salmonella, has been harnessed to deliver various tumor therapeutic modalities, including therapeutic vaccine antigens, DNA vaccines, and anticancer drugs. Various tumor-associated antigens (TAAs) have been targeted for therapeutic vaccines using LABV as delivery vehicles. PSA (prostate-specific antigen) is secreted by prostate epithelial cells and is overexpressed in malignant prostate cells. Attenuated Lm expressing PSA (Lm-LLO-PSA) antigen was tested as therapeutic vaccine in mouse tumor models expressing human PSA. Immunization with Lm-LLO-PSA completely regressed tumors in five out of eight mice and induced PSA-specific cellular immune response. Immunization of Lm-LLO-PSA significantly increased infiltration of PSA-specific CD8 + T cells in tumors and decrease in CD4/CD25/FoxP3 + T reg cells (Wallecha et al. 2009 ). HER2/neu is overexpressed in about 25-30% of breast cancers and is a potential target for immunotherapy. Shahabi et al. engineered an Lm-based vaccine incorporating HER2/neu as antigen (ADXS31-1642). ADXS31-164 elicited HER2specific CD8 + T cells. The vaccine caused a significant delay in the formation of mammary tumors, and 50% of mice were tumor-free till 45 weeks of the experiment, whereas all sham-treated mice developed tumors and succumbed to the disease. This vaccine also resulted in significant increase in tumor-infiltrating CD8 + T cells and a decrease in the intratumoral FoxP3 + T reg cells (Shahabi et al. 2011) . P. aeruginosa can also deliver heterologous antigens using its type III secretion system. In an experimental model of B-cell melanoma expressing ovalbumin (OVA), Chauchet et al. demonstrated antitumor efficacy of P. aeruginosa-based vaccine expressing OVA. P. aeruginosa induced a long-lasting and polyfunctional CD8 + T-cell immune response against the cognate antigen, wherein antigen-specific CD8 + T cells expressed IFNγ, TNFα, and IL2 simultaneously. These CD8 + T cells also showed enhanced tumor infiltration property and a greater ratio between effector versus regulatory T cells (Chauchet et al. 2016) . Recently Mei et al. utilized a composite approach of DNA vaccine and bacterial surface expression to achieve CD8 + and CD4 + T-cell-mediated immunity targeted to a tumor-associated antigen. The Salmonella-based vaccine included AIDA-I autotransporter-Melan A (a murine melanoma antigen) fusion protein and a DNA vaccine element encoding two murine melanoma epitopes (Mei et al. 2017 ).

High levels of antigen synthesis by multicopy plasmids exert metabolic burden to LABV, which results in hyperattenuation, low colonization, loss of viability, and most importantly poor immunogenicity. Various strategies have been adapted to circumvent this problem including the use of low-copy plasmid, use of in vivo inducible promoters (IVIP), and use of arabinose-inducible promoters (Loessner et al. 2007 ). Among the first promoters introduced in LABV is P nir B, which is activated under anaerobic conditions. P pag C and P ssa G are macrophage-inducible promoters from Salmonella. Dunstan directly compared the immunogenicity of antigens upon expression of antigens regulated by P nir B and P pag C and found significantly higher antibody response with P pag C compared to P nir B (Dunstan et al. 1999 ). Arnold et al. achieved differential antigen expression in vivo using in vivo inducible promoters P pag C, comprising variable ribosomal binding site (RBS). By this approach, strains with a high level of expression of heterologous protein exhibited low level of colonization, while a moderate amount of expression resulted in a significantly improved infection rate in mesenteric lymph nodes. A very low level of in vivo inducible antigen expression resulted in unhampered infectivity compared to the parent strain. Immunogenicity was dependent on the rate of infection, as well as the level of antigen expression. Notably, the best immune response was achieved with moderate level of antigen expression and infectivity, while high antigenexpressing strain resulted in little to no immune response. On the other hand, a moderate level of immune response was generated with high infectivity and low antigen expression (Arnold et al. 2004 ). Wang et al. developed a regulated delayed antigen synthesis system, consisting of LacI repressor to repress transcription from P trc during in vitro cultivation. The arabinose-regulated promoter P BAD drives LacI expression in vitro in medium supplemented with arabinose. Upon immunization and lack of external arabinose supplementation, P trc is derepressed, leading to the synthesis of antigens. The regulated delayed antigen synthesis system induced equivalent levels of antibody and protection to that of P pag C-controlled antigen synthesis and better than that of P ssa G-controlled antigen synthesis ).

Upon oral immunization, LABV must withstand acidic environment of the stomach for successful colonization. Enteric pathogens including E. coli, L. monocytogenes, Shigella spp., and L. lactis can tolerate extreme acidic pH (below pH 2.5) because they possess the most potent acid resistance (AR) system known as GDAR (glutamatedependent acid resistance) pathway. Attenuated strains of ST and STm have limited acid tolerance and exhibit moderate immunogenicity (Dharmasena et al. 2016a 

LPS plays important role in survival and infectivity of bacteria. However, it is also involved in toxicity to the host. Various attempts at the use of LPS O-antigen mutants of STm resulted in poor attachment and intestinal invasion and survival following oral immunization. By regulated expression of LPS O-antigen components such that they are expressed in vitro and at the time of immunization, but soon after colonization their synthesis is stopped, it is expected to achieve maximal infectivity and minimal toxicity . Kong et al. engineered a Salmonella strain where LPS O-antigen synthesis genes rfc and rfaH are kept under the control of the promoter araC-P BAD , which is tightly regulated by arabinose. This strain is highly attenuated nevertheless exhibits superior immunogenicity (Kong et al. 2009 . Another approach of detoxification of Salmonella LPS included removal of 1-phosphate group from lipid A of LPS. Kong et al. introduced 

Many methods employed for attenuation, although make the LABV strains less pathogenic and safe to administer at high doses, it often renders them poorly immunogenic due to their inability to circumvent physicochemical defense of the host. Moreover, inability of penetration through mucosal barrier also makes them poorly immunogenic. To circumvent this problem, Curtiss et al. generated a regulated delayed attenuation system (RDAS), which retains full virulence till the passage through gastrointestinal tract and infection of epithelial cells. In the modified RDAS strains, Salmonella virulence genes fur, PhoP/Q, rpoS, and crp are expressed under the control of araC-P BAD promoter. Arabinose concentration in human tissues is very less. Thus, in vitro these strains express all the virulence genes in medium supplemented with arabinose, whereas in vivo under the arabinose deprivation, many virulence genes are suppressed, resulting in attenuation of Salmonella. This approach results in high immunogenicity combined with tolerance at high doses (Curtiss 3rd et al. 2009 ).

In order to evoke CD8 + T-cell (CTL) response, antigens need to be delivered into the cytoplasm of host cell. Various approaches are in use to accomplish the cytosolic delivery of antigens including the use of a type III secretion system that can directly deliver vaccine antigens into the host cell cytoplasm and use of a-hemolysin (HlyA) secretion system of E. coli which is fully active in Salmonella (Gentschev et al. 1996) . On the other hand, escape from endocytic vacuoles is also a feasible approach. Unlike Lm, ST and STm do not reach cytoplasm of infected cells and elicit CD4 + T-cell response more effectively compared to CD8 + T-cell response to cognate antigens. Chen et al. used secretion signal of a type III secretion system Salmonella outer protein E (SopE) and HlyA (secretion signal) to deliver S. japonicum antigen Sj23-LHD-GST. The Salmonella vaccine constructs carrying Sj23 LHD-GST fused to HlyA (secretion signal) or SopE effectively expressed and delivered antigens into cytoplasm of murine macrophages in vitro. This vaccine construct induced Sj23-LHD-GST-specific Th1 type response and protected against S. japonicum infection (Chen et al. 2011). Gentschev et al. reported that two Listerial antigens delivered by STm using HlyA (secretion signal) generated protection against Listeria infection (Gentschev et al. 1996) . Simultaneous delivery of two Listerial antigens (LLO and p60) by STm using Yersinia outer protein E (YopE) as a carrier molecule for Salmonella type III secretion system developed LLO-and p60-specific T cells and protection against murine listeriosis (Igwe et al. 2002) . SopE-mediated delivery of Listerial antigen p60 generated CD8 + T-cell-mediated protection against Listeria infection (Berchtold et al. 2009 ).

What makes bacteria an excellent vaccine delivery vehicle is their natural ability to induce potent and long-lasting immune response. LABVs possess the capacity to induce humoral as well as cell-mediated immune response. While the humoral immune response includes serum IgG and mucosal IgG and IgA, the cell-mediated immunity is characterized by Th1-, Th2-, and Th17-type CD4+ T cells and CD8+ CTLs. IgA and IL17 have been specifically implicated in mucosal protection against various mucosal pathogens. The cell-mediated immunity is required for intracellular pathogens. It should be noted that subunit vaccines have a poor capacity to evoke mucosal as well as cell-mediated immunity. LABVs have also shown the capacity to overcome immunosuppressive nature of various forms of tumors. These characteristic of LABVs, together with their tumor-tropic capacity, makes them a highly suitable vector for cancer immunotherapeutic vaccines. In the past two decades, tremendous progress has been made regarding LABV-mediated delivery of vaccine antigens for prevention of a variety of viral, bacterial, and parasitic diseases. Recent advances have further improved the safety and immunogenicity profile of several LABV platforms. The new-generation LABVs can withstand harsh physicochemical conditions of gastrointestinal tract, exhibit regulated attenuation, regulated antigen expression, and targeted antigen delivery. LABVs have exhibited effectiveness in various preclinical and preliminary clinical trials (Table 2 .1). However, a limited number of clinical trials have been conducted to date using LABVs, due to potential safety concerns. Further optimization would result in a versatile, safe, and highly immunogenic vaccine delivery platforms. Ding et al. (2013) 

Mucosal delivery of a transmission-blocking DNA vaccine encoding Giardia lamblia CWP2 by Salmonella typhimurium bactofection vehicle

A Listeria monocytogenes-based vaccine that secretes sand fly salivary protein LJM11 confers longterm protection against vector-transmitted Leishmania major

Humoral and cellular immune responses in mice immunized with recombinant Mycobacterium bovis Bacillus Calmette-Guérin producing a pertussis toxin-tetanus toxin hybrid protein

Oral immunization of mice against Clostridium perfringens epsilon toxin with a Lactobacillus casei vector vaccine expressing epsilon toxoid

Kinetics of the mucosal antibody secreting cell response and evidence of specific lymphocyte migration to the lung after oral immunisation with attenuated S. enterica var. typhimurium

Expression of fibronectin binding protein A (FnBPA) from Staphylococcus aureus at the cell surface of Lactococcus lactis improves its immunomodulatory properties when used as protein delivery vector

Bacillus subtilis: a temperature resistant and needle free delivery system of immunogens

Sublingually administered Bacillus subtilis cells expressing tetanus toxin C fragment induce protective systemic and mucosal antibodies against tetanus toxin in mice

Pilot study of phoP/phoQ-deleted Salmonella enterica serovar typhimurium expressing Helicobacter pylori urease in adult volunteers

Enhanced immunogenicity in the murine airway mucosa with an attenuated Salmonella live vaccine expressing OprF-OprI from Pseudomonas aeruginosa

Protective cellular responses elicited by vaccination with influenza nucleoprotein delivered by a live recombinant attenuated Salmonella vaccine

Lactococcus lactis-expressing listeriolysin O (LLO) provides protection and specific CD8(+) T cells against Listeria monocytogenes in the murine infection model

Construction of attenuated Salmonella typhimurium strain expressing Helicobacter pylori conservative region of adhesin antigen and its immunogenicity

Immune responses elicited against multiple enterotoxigenic Escherichia coli fimbriae and mutant LT expressed in attenuated Shigella vaccine strains

Oral immunization with attenuated Salmonella enterica serovar typhimurium encoding Cryptosporidium parvum Cp23 and Cp40 antigens induces a specific immune response in mice

Superior protective immunity against murine listeriosis by combined vaccination with CpG DNA and recombinant Salmonella enterica serovar typhimurium

Lactococci and lactobacilli as mucosal delivery vectors for therapeutic proteins and DNA vaccines

Coley's toxin: historical perspective

A clinically relevant, syngeneic model of spontaneous, highly metastatic B16 mouse melanoma

Evaluation of Psn, HmuR and a modified LcrV protein delivered to mice by live attenuated Salmonella as a vaccine against bubonic and pneumonic Yersinia pestis challenge

Listeriabased cancer vaccines that segregate immunogenicity from toxicity

Innate immune response to Salmonella typhimurium, a model enteric pathogen

Systemic, nasal and oral live vaccines against Pseudomonas aeruginosa: a clinical trial of immunogenicity in lower airways of human volunteers

Attenuated Escherichia coli strains expressing the colonization factor antigen I (CFA/I) and a detoxified heat-labile enterotoxin (LThK63) enhance clearance of ETEC from the lungs of mice and protect mice from intestinal ETEC colonization and LT-induced f

Mucosally delivered Salmonella live vector vaccines elicit potent immune responses against a foreign antigen in neonatal mice born to naive and immune mothers.pdf

Poly-functional and long-lasting anticancer immune response elicited by a safe attenuated Pseudomonas aeruginosa vector for antigens delivery

Construction, characterization, and immunogenicity of an attenuated Salmonella enterica serovar typhimurium pgtE vaccine expressing fimbriae with integrated viral epitopes from the spiC promoter

Comparison of a fimbrial versus an autotransporter display system for viral epitopes on an attenuated Salmonella vaccine vector

A recombinant live attenuated strain of Vibrio cholerae induces immunity against tetanus toxin and Bordetella pertussis tracheal colonization factor

Oral delivery of the Sj23LHD-GST antigen by Salmonella typhimurium type III secretion system protects against Schistosoma japonicum infection in mice

Recombinant bacille Calmette???Guerin coexpressing Ag85b, CFP10, and interleukin-12 elicits effective protection against Mycobacterium tuberculosis

Recombinant Salmonella bacteria vectoring HIV/AIDS vaccines

Salmonella enterica serovar typhimurium strains with regulated delayed attenuation in vivo

Oral vaccine delivery by recombinant spore probiotics

Construction and characterisation of Salmonella typhimurium aroA simultaneously expressing the five pertussis toxin subunits

Stable expression of Shigella sonnei form I O-polysaccharide genes recombineered into the chromosome of live Salmonella oral vaccine vector Ty21a

Development of an acid-resistant Salmonella Typhi Ty21a attenuated vector for improved oral vaccine delivery

Stable expression of Shigella dysenteriae serotype 1 O-antigen genes integrated into the chromosome of live Salmonella oral vaccine vector Ty21a

Oral vaccination of BALB/c mice with Salmonella enterica serovar Typhimurium expressing Pseudomonas aeruginosa O antigen promotes increased survival in an acute fatal pneumonia model

Immune responses to a recombinant attenuated Salmonella typhimurium strain expressing a Taenia solium oncosphere antigen TSOL18

Use of in vivo-regulated promoters to deliver antigens from attenuated Salmonella enterica var. typhimurium

Shigella interaction with intestinal epithelial cells determines the innate immune response in shigellosis

Oral immunization with attenuated Salmonella vaccine expressing Escherichia coli O157: H7 intimin gamma triggers both systemic and mucosal humoral immunity in mice

Sparse initial entrapment of systemically injected Salmonella typhimurium leads to heterogeneous accumulation within tumors 1

Salmonella enterica serovar Tphi Ty21a expressing human papillomavirus type 16 L1 as a potential live vaccine against cervical cancer and typhoid fever

Induction of cell-mediated immune responses to human immunodeficiency virus type 1 Gag protein by using Listeria monocytogenes as a live vaccine vector

A Phase I, dose-escalation trial in adults of three recombinant attenuated Salmonella Typhi vaccine vectors producing Streptococcus pneumoniae surface protein antigen PspA

Induction of human immunodeficiency virus (HIV)-specific CD8 T-cell responses by Listeria monocytogenes and a hyperattenuated Listeria strain engineered to express HIV antigens

Cloning and characterization of the asd gene of Salmonella typhimurium: use in stable maintenance of recombinant plasmids in Salmonella vaccine strains

Optimization of plasmid maintenance in the attenuated live vector vaccine strain Salmonella typhi CVD 908-htrA

Adaptation of the endogenous Salmonella enterica serovar Typhi clyA-encoded hemolysin for antigen export enhances the immunogenicity of anthrax protective antigen domain 4 expressed by the attenuated live-vector vaccine strain CVD 908-htrA

Salmonella enterica serovar Typhi live vector vaccines finally come of age

A new generation of stable, nonantibiotic, low-copy-number plasmids improves immune responses to foreign antigens Salmonella enterica serovar typhi live vectors

A bivalent typhoid live vector vaccine expressing both chromosome-and plasmid-encoded Yersinia pestis antigens fully protects against murine lethal pulmonary plague infection

Development of antigen-delivery systems, based on the Escherichia coli hemolysin secretion pathway

Simultaneous expression of CFA/I and CS3 colonization factor antigens of enterotoxigenic Escherichia coli by ΔaroC, ΔaroD Salmonella typhi vaccine strain CVD 908

Immunogenicity of a Salmonella typhi CVD 908 candidate vaccine strain expressing the major surface protein gp63 of Leishmania mexicana mexicana

A combination of recombinant BCG expressing pneumococcal proteins induces cellular and humoral immune responses and protects against pneumococcal colonization and sepsis. Clin Vaccine Immunol CVI

Cell-mediated immunity induced by recombinant Mycobacterium bovis Bacille Calmette-Guérin strains against an intracellular bacterial pathogen: importance of antigen secretion or membrane-targeted antigen display as lipoprotein for vaccine efficacy

Oral vaccination of mice against Helicobacter pylori with recombinant Lactococcus lactis expressing urease subunit B

An influenza HA and M2e based vaccine delivered by a novel attenuated Salmonella mutant protects mice against homologous H1N1 infection

Stable expression of lentiviral antigens by quality-controlled recombinant mycobacterium bovis BCG vectors

Recombinant Mycobacterium bovis bacillus Calmette-Guérin expressing Ag85B-IL-7 fusion protein enhances IL-17A-producing innate γδ T cells

Characterization of Salmonella enterica derivatives harboring defined aroC and Salmonella pathogenicity island 2 type III secretion system (ssaV) mutations by immunization of healthy volunteers

Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines

Concomitant cytosolic delivery of two immunodominant Listerial antigens by Salmonella enterica serovar typhimurium confers superior protection against murine listeriosis

An attenuated Listeria monocytogenes vector primes more potent simian immunodeficiency virusspecific mucosal immunity than DNA vaccines in mice

Expression of a Porphyromonas gingivalis hemagglutinin on the surface of a Salmonella vaccine vector

1-giardin based live heterologous vaccine protects against Giardia lamblia infection in a murine model

Shigella flexneri infection: pathogenesis and vaccine development

Prophylactic and therapeutic efficacy of an attenuated Listeria monocytogenes-based vaccine delivering HPV16 E7 in a mouse model

Live attenuated Listeria monocytogenes expressing HIV Gag: immunogenicity in rhesus monkeys

Oral vaccine of Lactococcus lactis harbouring pandemic H1N1 2009 haemagglutinin1 and nisP anchor fusion protein elevates anti-HA1 sIgA levels in mice

Attenuated Listeria monocytogenes vaccine vectors expressing influenza A nucleoprotein: preclinical evaluation and oral inoculation of volunteers

Immune responses to recombinant pneumococcal PspA antigen delivered by live attenuated Salmonella enterica serovar typhimurium vaccine

Comparative analysis using a mouse model of the immunogenicity of artificial VLP and attenuated Salmonella strain carrying a DNA-vaccine encoding HIV-1 polyepitope CTL-immunogen

Salmonella typhimurium lacking ribose chemoreceptors localize in tumor quiescence and induce apoptosis

Salmonella typhi and S. typhimurium derivatives harbouring deletions in aromatic biosynthesis and Salmonella Pathogenicity Island-2 (SPI-2) genes as vaccines and vectors

Expression of Helicobacter pylori cag12 gene in Lactococcus lactis MG1363 and its oral administration to induce systemic anti-Cag12 immune response in mice

Oral administration of Lactococcus lactis expressing Helicobacter pylori Cag7-ct383 protein induces systemic anti-Cag7 immune response in mice

Comparison between immunization routes of live attenuated Salmonella typhimurium strains expressing BCSP31, Omp3b, and SOD of Brucella abortus in murine model

Cell wall anchoring of the Campylobacter antigens to Lactococcus lactis

Regulated delayed expression of rfaH in an attenuated Salmonella enterica serovar typhimurium a vaccine enhances immunogenicity of outer membrane proteins and a heterologous antigen

Regulated delayed expression of rfc enhances the immunogenicity and protective efficacy of a heterologous antigen delivered by live attenuated Salmonella enterica vaccines

Salmonella synthesizing 1-monophosphorylated Lipopolysaccharide exhibits low endotoxic activity while retaining its immunogenicity

Turning self-destructing Salmonella into a universal DNA vaccine delivery platform

Attenuated shigella flexneri 2a vaccine strain CVD 1204 expressing colonization factor antigen I and mutant heat-labile enterotoxin of enterotoxigenic escherichia coli

Safety and immunogenicity of attenuated Salmonella enterica serovar Typhimurium delivering an HIV-1 Gag antigen via the Salmonella Type III secretion system

Autodisplay: development of an efficacious system for surface display of antigenic determinants in Salmonella vaccine strains

Prime-boost vaccination with heterologous live vectors encoding SIV gag and multimeric HIV-1 gp160 protein: efficacy against repeated mucosal R5 clade C SHIV challenges

Brucella lipopolysaccharide reinforced Salmonella delivering Brucella immunogens protects mice against virulent challenge

Engineering bacteria toward tumor targeting for cancer treatment: current state and perspectives

Immunogenicity of self-adjuvanticity oral vaccine candidate based on use of Bacillus subtilis spore displaying Schistosoma japonicum 26 KDa GST protein

Oral immunization with recombinant Lactococcus lactis delivering a multi-epitope antigen CTB-UE attenuates Helicobacter pylori infection in mice

Live attenuated Salmonella displaying HIV-1 10E8 epitope on fimbriae: systemic and mucosal immune responses in BALB/c mice by mucosal administration

Listeria monocytogenes: a promising vehicle for neonatal vaccination

Live-attenuated bacterial vectors: tools for vaccine and therapeutic agent delivery

Construction and characterization of recombinant attenuated Salmonella typhimurium expressing the babA2/ureI fusion gene of Helicobacter pylori

Remote control of tumour-targeted Salmonella enterica serovar Typhimurium by the use of l-arabinose as inducer of bacterial gene expression in vivo

Type IVB pilus operon promoter controlling expression of the severe acute respiratory syndrome-associated coronavirus nucleocapsid gene in Salmonella enterica serovar Typhi elicits full immune response by intranasal vaccination

A fusogenic peptide expressed on the surface of Salmonella enterica elicits CTL responses to a dengue virus epitope

Preclinical development of BCG.HIVA2auxo.int, harboring an integrative expression vector, for a HIV-TB Pediatric vaccine. Enhancement of stability and specific HIV-1 T-cell immunity

Protection in a gerbil model of amebiasis by oral immunization with Salmonella expressing the galactose/N-acetyl D-galactosamine inhibitable lectin of Entamoeba histolytica

Mucosal vaccine made from live, recombinant Lactococcus lactis protects mice against pharyngeal infection with Streptococcus pyogenes

Tc52 aminoterminal-domain DNA carried by attenuated Salmonella enterica serovar typhimurium induces protection against a trypanosoma cruzi lethal challenge

Genetic determinants of Shigella pathogenicity

A mutant in the Listeria monocytogenes furregulated virulence locus (frvA) induces cellular immunity and confers protection against listeriosis in mice

Combining DNA vaccine and AIDA-1 in attenuated Salmonella activates tumor-specific CD4 + and CD8 + T-cell responses

Cytosolic access of intracellular bacterial pathogens: the Shigella paradigm

Antibiotic-free selection in biotherapeutics: now and forever

Induction of protective cellular immunity against Mycobacterium tuberculosis by recombinant attenuated selfdestructing Listeria monocytogenes strains harboring eukaryotic expression plasmids for antigen 85 complex and MPB/MPT51

Immune responses to Bacillus Calmette-Guerin vaccination: why do they fail to protect against mycobacterium tuberculosis? Front Immunol

Neonatal immunization with a single dose of recombinant BCG expressing subunit S1 from pertussis toxin induces complete protection against Bordetella pertussis intracerebral challenge

Construction of an unmarked recombinant BCG expressing a pertussis antigen by auxotrophic complementation: protection against Bordetella pertussis challenge in neonates

Engineered deltaguaB-A deltavirG Shigella flexneri 2a strain CVD 1205: construction, safety, immunogenicity, and potential efficacy as a mucosal vaccine

Novel use of anaerobically induced promoter, dmsA, for controlled expression of fragment C of tetanus toxin in live attenuated Salmonella enterica serovar Typhi strain CVD 908-htrA

Attenuated deltaguaBA Salmonella typhi vaccine strain CVD 915 as a live vector utilizing prokaryotic or eukaryotic expression systems to deliver foreign antigens and elicit immune responses

A comparison of immunogenicity and in vivo distribution of Salmonella enterica serovar Typhi and Typhimurium live vector vaccines delivered by mucosal routes in the murine model

Salmonella enterica serovar Typhi live vector vaccines delivered intranasally elicit regional and systemic specific CD8+ major histocompatibility class I-restricted cytotoxic T lymphocytes

Immunology of gut mucosal vaccines

Effect of expression level on immune responses to recombinant oral Salmonella enterica serovar Typhimurium vaccines

Oral delivery of a novel attenuated salmonella vaccine expressing influenza a virus proteins protects mice against H5N1 and H1N1 viral infection

Tailoring host immune responses to Listeria by manipulation of virulence genes -the interface between innate and acquired immunity

Protective host immune responses to Salmonella infection

Bactofection of mammalian cells by Listeria monocytogenes: improvement and mechanism of DNA delivery

Mucosally delivered Salmonella typhi expressing the Yersinia pestis F1 antigen elicits mucosal and systemic immunity early in life and primes the neonatal immune system for a vigorous anamnestic response to parenteral F1 boost

Construction and characterization of bivalent Shigella flexneri 2a vaccine strains SC608(pCFAI) and SC608(pCFAI/ LTB) that express antigens from enterotoxigenic Escherichia coli

Bacterial virulence, proinflammatory cytokines and host immunity: how to choose the appropriate Salmonella vaccine strain?

Systemic immunity and mucosal immunity are induced against human immunodeficiency virus Gag protein in mice by a new hyperattenuated strain of Listeria monocytogenes

Safety and immunogenicity in neonatal mice of a hyperattenuated Listeria vaccine directed against human immunodeficiency virus

Humoral response and genetic stability of recombinant BCG expressing hepatitis B surface antigens

Comparison of abilities of Salmonella enterica serovar Typhimurium aroA aroD and aroA htrA mutants to act as live vectors

Intranasal vaccination in mice with an attenuated Salmonella enterica Serovar 908htr A expressing Cp15 of Cryptosporidium: impact of malnutrition with preservation of cytokine secretion

Salmonella enterica serovar typhimurium vaccine strains expressing a nontoxic shigalike toxin 2 derivative induce partial protective immunity to the toxin expressed by enterohemorrhagic escherichia coli

Protective immunity against Clostridium difficile toxin A induced by oral immunization with a live, attenuated Vibrio cholerae vector strain

In vivo expression and immunoadjuvancy of a mutant of heat-labile enterotoxin of Escherichia coli in vaccine and vector strains of Vibrio cholerae

Development of a ??glnA balanced lethal plasmid system for expression of heterologous antigens by attenuated vaccine vector strains of Vibrio cholerae

Enteral immunization with attenuated recombinant Listeria monocytogenes as a live vaccine vector: organ-dependent dynamics of CD4 T lymphocytes reactive to a Leishmania major tracer epitope

Bacteria as DNA vaccine carriers for genetic immunization

A live attenuated Listeria monocytogenes vaccine vector expressing SIV Gag is safe and immunogenic in macaques and can be administered repeatedly

Heterologous prime-boost immunizations with different Salmonella serovars for enhanced antigen-specific CD8 T-cell induction

Development of a live and highly attenuated Listeria monocytogenes-based vaccine for the treatment of Her2/neuoverexpressing cancers in human

Live recombinant Salmonella typhi vaccines constructed to investigate the role of rpoS in eliciting immunity to a heterologous antigen

Recombinant Bacillus subtilis spores expressing MPT64 evaluated as a vaccine against tuberculosis in the murine model

Construction and screening of attenuated ??phoP/Q Salmonella typhimurium vectored plague vaccine candidates

Effect of intragastric and intraperitoneal immunisation with attenuated and wild-type LACKexpressing Listeria monocytogenes on control of murine Leishmania major infection

Salmonella vaccines secreting measles virus epitopes induce protective immune responses against measles virus encephalitis

Listeria monocytogenes as a vaccine vector: virulence attenuation or existing antivector immunity does not diminish therapeutic efficacy

Recombinant Bacillus subtilis spores elicit Th1/Th17-polarized immune response in a Murine model of Helicobacter pylori vaccination

Oral immunization with recombinant Listeria monocytogenes controls virus load after vaginal challenge with feline immunodeficiency virus

Genetic characterization and immunogenicity of coli surface antigen 4 from enterotoxigenic Escherichia coli when it is expressed in a Shigella live-vector strain

Characterization of a Salmonella typhimurium aro vaccine strain expressing the P.69 antigen of Bordetella pertussis

Evaluation of YadC protein delivered by live attenuated Salmonella as a vaccine against plague

Lactic acid bacteria -promising vaccine vectors: possibilities, limitations, doubts

Comparison of the safety and immunogenicity of DaroC DaroD and & cya Acrp Salmonella typhi strains in adult volunteers

Safety and immunogenicity in humans of an attenuated Salmonella typhi vaccine vector strain expressing plasmid-encoded hepatitis B antigens stabilized by the Asd-balanced lethal vector system

Phase 2 clinical trial of attenuated Salmonella enterica serovar Typhi oral live vector vaccine CVD 908-htrAin U.S. volunteers

Surface expression of Helicobacter pylori HpaA adhesion antigen on Vibrio cholerae, enhanced by co-expressed enterotoxigenic Escherichia coli fimbrial antigens

Fine-tuning synthesis of yersinia pestis lcrv from runaway-like replication balanced-lethal plasmid in a salmonella enterica serovar typhimurium vaccine induces protection against a lethal y. pestis challenge in mice

CD8 + -T-cell response to secreted and nonsecreted antigens delivered by recombinant Listeria monocytogenes during secondary infection

Improving Mycobacterium bovis bacillus calmette-Guèrin as a vaccine delivery vector for viral antigens by incorporation of glycolipid activators of NKT cells

Listeria monocytogenes-based antibiotic resistance gene-free antigen delivery system applicable to other bacterial vectors and DNA vaccines

Construction and characterization of an attenuated Listeria monocytogenes strain for clinical use in cancer immunotherapy

Construction, genotypic and phenotypic characterization, and immunogenicity of attenuated Δ guaBA Salmonella enterica Serovar Typhi S

An attenuated Salmonella-vectored vaccine elicits protective immunity against Mycobacterium tuberculosis

Salmonella vaccine vectors displaying delayed antigen synthesis in vivo to enhance immunogenicity

Comparison of a regulated delayed antigen synthesis system with in vivo-inducible promoters for antigen delivery by live attenuated Salmonella vaccines

New technologies in developing recombinant attenuated Salmonella vaccine vectors

Oral immunization of chickens with avirulent Salmonella vaccine strain carrying C. jejuni 72Dz/92 cjaA gene elicits specific humoral immune response associated with protection against challenge with wild-type Campylobacter

PspA family fusion proteins delivered by attenuated Salmonella enterica serovar typhimurium extend and enhance protection against Streptococcus pneumoniae

Identification of Coxiella burnetii CD8 + T-cell epitopes and delivery by attenuated Listeria monocytogenes as a vaccine vector in a C57BL/6 mouse model

Construction of a recombinant attenuated Salmonella typhimurium DNA vaccine carrying Helicobacter pylori hpaA

Oral vaccination with Ts87 DNA vaccine delivered by attenuated Salmonella typhimurium elicits a protective immune response against Trichinella spiralis larval challenge

The protective effect of recombinant Lactococcus lactis oral vaccine on a Clostridium difficile-infected animal model

Oral delivery of Bacillus subtilis spore expressing enolase of Clonorchis sinensis in rat model: induce systemic and local mucosal immune responses and has no side effect on liver function

Oral vaccination with Lactococcus lactis expressing the Vibrio cholerae Wzm protein to enhance mucosal and systemic immunity

Innate and adaptive immune responses to Listeria monocytogenes: a short overview

Enhanced protection against nasopharyngeal carriage of Streptococcus pneumoniae elicited by oral multiantigen DNA vaccines delivered in attenuated Salmonella typhimurium

Construction of an oral vaccine for transmissible gastroenteritis virus based on the TGEV N gene expressed in an attenuated Salmonella typhimurium vector

Construction of a recombinant Lactococcus lactis strain expressing a fusion protein of Omp22 and HpaA from Helicobacter pylori for oral vaccine development

Construction of a novel Shigella live-vector strain co-expressing CS3 and LTB/STm of enterotoxigenic E.coli

Applications of bacillus Calmette-Guerin and recombinant bacillus Calmette-Guerin in vaccine development and tumor immunotherapy

Expression of Helicobacter pylori urease B on the surface of Bacillus subtilis spores

Delivery of heterologous protein antigens via hemolysin or autotransporter systems by an attenuated ler mutant of rabbit enteropathogenic Escherichia coli