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Efficient in vivo delivery of a CRISPR/Cas9 plasmid is of paramount importance for effective therapy. Here, we investigated the usability of Salmonella as a plasmid carrier for in vivo therapy against virus-induced cancer using Marek's disease virus (MDV) as a model for study in chickens. A green fluorescent protein-expressing CRISPR/Cas9 plasmid encoding the virulence gene pp38 was constructed against Marek's disease virus. Therapeutic plasmids were transformed into Salmonella carrying lon and sifA gene deletions. The animals in 5 groups were intraperitoneally inoculated with phosphate-buffered saline, vector control, or Salmonella before or after MDV infection, or left uninfected as a naïve control. Therapeutic effectiveness was evaluated by observing disease outcomes and the viral copy number in peripheral blood mononuclear cells. The efficacy of plasmid delivery by Salmonella was 13 ± 1.7% in the spleen and 8.0 ± 1.8% in the liver on the 6 th day post-infection. The Salmonella-treated groups showed significant resistance to MDV infection. The maximum effect was observed in the group treated with Salmonella before MDV infection. None of the chickens fully recovered; however, the results suggested that timely delivery of Salmonella could be effective for in vivo CRISPR/Cas9-mediated genetic interference against highly pathogenic MDV. The use of Salmonella in CRISPR systems provides a simpler and more efficient platform for in vivo therapy with CRISPR than the use of conventional in vivo gene delivery methods and warrants further development., (© 2021. The Author(s).)

Residual virulence is a major drawback in current Brucella vaccines. Live vaccines induce abortions in pregnant animals. Hence, a novel anti-Brucella vaccine was developed utilizing rough Salmonella delivering four Brucella antigens. Safety implications during pregnancy, humoral immune responses, and protective efficacy against wild type Brucella was investigated in guinea pig model. The vaccine did not induce abortions or severe complications in pregnant guinea pigs when administered 4 × 10 8  CFU via intraperitoneal route. Systemic IgG determination against antigen components reveals induction of immunity via the Salmonella delivery. Protection efficacy against abortions was 33.3% (2/6) when midterm sow challenged with virulent Brucella 544 strain while none was protected in control group. Lower Brucella recovery in spleen and liver and reduced histopathological burden were also noticed. Although abortion induced by Brucella challenge was not completely prevented, the vaccine candidate may perform better with optimization of vaccination such as inoculation dose optimization., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

An anti- Brucella vaccine candidate comprised of purified Brucella lipopolysaccharide (LPS) and a cocktail of four Salmonella Typhimurium (ST)- Brucella vectors was reported previously. Each vector constitutively expressed highly conserved Brucella antigens (rB), viz. , lumazine synthase (BLS), proline racemase subunit A, outer membrane protein-19 (Omp19), and Cu-Zn superoxide dismutase (SOD). The present study determined a relative level of protection conferred by each single strain. Upon virulent challenge, the challenge strain was recovered most abundantly in non-immunized control mice, with the ST-Omp19-, ST-BLS-, LPS-, and ST-SOD-immunized mice showing much less burden. Indirect enzyme-linked immunosorbent assay-based assay also confirmed the induction of antigen-specific immunoglobulin G for each antigen delivered. In a route-wise comparison of the combined vaccine candidate, intraperitoneal (IP), intramuscular (IM), and subcutaneous immunizations revealed an indication of highly efficient routes of protection. Splenocytes of mice immunized via IM and IP routes showed significant relative expression of IL-17 upon antigenic pulsing. Taken together, each of the Brucella antigens delivered by ST successfully induced an antigen-specific immune response, and it was also evident that an individual antigen strain can confer a considerable degree of protection. More effective protection was observed when the candidate was inoculated via IP and IM routes.

The present study aimed to investigate whether the incorporation of flagellin, a TLR5 agonist, in the bacterial ghosts (BGs) of Salmonella Gallinarum can enhance protective immune responses against fowl typhoid, a septicemic disease of poultry, in chickens. BGs are empty cell envelopes derived from Gram-negative bacteria through the bacteriophage phiX174 gene E mediated lysis. In this study, the S. Gallinarum ghosts carrying flagellin were genetically constructed utilizing a lysis plasmid pJHL184-flagellin, designed for the coexpression of the flagellin and the lysis protein E. The adjuvant effect of flagellin was evaluated by immunizing seven day old brown nick layer chicks once orally with either S. Gallinarum-flagellin (SG-fliC) ghosts or S. Gallinarum (SG) ghosts alone. Our results showed that immunization with the SG-fliC ghosts elicited early and higher systemic (IgG) and mucosal (IgA) antibody responses compared to the SG ghosts alone, although not always statistically significant. Flow cytometric analysis of the CD3 + CD4+ and the CD3 + CD8+ T cell populations in peripheral blood mononuclear cells were higher in chickens immunized with the SG-fliC ghosts compared to the chickens vaccinated with the SG ghosts alone. Furthermore, the chickens immunized with SG-fliC ghosts exhibited significantly (p < 0.05) higher IL-6 and IFN-γ responses compared to the chickens vaccinated with the SG ghosts alone. On challenge with the virulent S. Gallinarum wild type strain at 28th day post immunization, 5 of 10 birds died (50%) in case of SG-fliC ghost group while 60% (6 of 10 birds died) mortality was observed in the SG ghost group. Collectively, these results suggest that the expression of flagellin in SG ghosts improves antigen-specific humoral and cell mediated immune responses, and can enhance protective efficacy of the BG-based vaccines against the virulent challenges., (Copyright © 2018 Elsevier B.V. All rights reserved.)

An anti-Brucella vaccine candidate comprising rough Salmonella vector delivering Brucella antigens was developed. This system provides a platform for live Brucella-free vaccine development as it can mimic active-intracellular infection of Brucella organism. Exploiting this phenomenon thus provides significant protection at a single dose and also re-assured the safety. To date, no human anti-Brucella vaccines are available, owing to the lack of safe and effective formulation. This study investigated the safety of the vaccine formulation in mice model and in vitro human cell cultures. The experiment was designed to determine the LD50 of the vaccine formulation. The vaccine formulation did not induce any mortality even when mice were administered at 8 × 10 9 CFU per oral or per subcutaneous (SC), which was 100-times more than the actual vaccine dose intended for mice model. In contrast, wild-type (WT) Salmonella positive control strain induced 100% mortality at 8 × 10 7 CFU per mice via oral or SC routes. Interaction of the vaccine with phagocytic (THP-1 derived macrophage) and non-phagocytic (Caco-2) human cell lines as well as human PBMC was investigated. In in vitro experiments, inflammatory and pyretic cytokines TNF-α, and IL-1β inductions were significantly lower in vaccine group as compared to WT group. Further, apoptosis, nitric oxide synthase and cytotoxicity inductions were comparable and not exacerbated, given that the strain is based on a rough bacterial vector that may have endotoxic lipid-A more readily exposed. These findings corroborated that the vaccine formulation is highly safe in mice model and is relatively mild in the induction of inflammatory cytokines and cellular changes in human cell lines., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

H7N3 and H7N7 are highly pathogenic avian influenza (HPAI) viruses and have posed a great threat not only for the poultry industry but for the human health as well. H7N9, a low pathogenic avian influenza (LPAI) virus, is also highly pathogenic to humans, and there is a great concern that these H7 subtypes would acquire the ability to spread efficiently between humans, thereby becoming a pandemic threat. A vaccine candidate covering all the three subtypes must, therefore, be an integral part of any pandemic preparedness plan. To address this need, we constructed a consensus hemagglutinin (HA) sequence of H7N3, H7N7, and H7N9 based on the data available in the NCBI in early 2012-2015. This artificial sequence was then optimized for protein expression before being transformed into an attenuated auxotrophic mutant of Salmonella Typhimurium, JOL1863 strain. Immunizing chickens with JOL1863, delivered intramuscularly, nasally or orally, elicited efficient humoral and cell mediated immune responses, independently of the route of vaccination. Our results also showed that JOL1863 deliver efficient maturation signals to chicken monocyte derived dendritic cells (MoDCs) which were characterized by upregulation of costimulatory molecules and higher cytokine induction. Moreover, immunization with JOL1863 in chickens conferred a significant protection against the heterologous LPAI H7N1 virus challenge as indicated by reduced viral sheddings in the cloacal swabs. We conclude that this vaccine, based on a consensus HA, could induce broader spectrum of protection against divergent H7 influenza viruses and thus warrants further study.

Recombinant Salmonella strains expressing foreign heterologous antigens have been extensively studied as promising live vaccine delivery vehicles. In this study, we constructed attenuated smooth (S-HA) and rough (R-HA) Salmonella strains expressing hemagglutinin (HA) of H9N2, a low pathogenic avian influenza A virus. We then investigated the HA-specific immune responses following oral immunization with either S-HA or R-HA strain in chicken model. We further examined the effects of the preexisting anti-Salmonella immunity on the subsequent elicitation of the HA and the Salmonella ompA specific immune responses. Our results showed that primary immunization with either the S-HA or the R-HA strain elicited comparable HA-specific immune responses and the responses were significantly (p<0.05) higher compared to the Salmonella vector control. When chickens were pre-immunized with the smooth Salmonella carrier alone and then vaccinated with either S-HA or R-HA strain 3, 6 and 9 weeks later, respectively, significant reductions were seen for HA-specific immune responses at week 6, a point which corresponded to the peak of the primary Salmonella-specific antibody responses. No reductions were seen at week 3 and 9, albeit, the HA-specific immune responses were boosted at week 9, a point which corresponded to the lowest primary Salmonella-specific antibody responses. The ompA recall responses remain refractory at week 3 and 6 following deliberate immunization with the carrier strain, but were significantly (p<0.05) increased at week 9 post-primary immunization. We conclude that preexisting anti-Salmonella immunity inhibits antigen-specific immune responses and this effect could be avoided by carefully selecting the time point when carrier-specific immune responses are relatively low., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Intracellular pathogen Salmonella exhibits natural infection broadly analogous to Brucella, this phenomenon makes Salmonella a pragmatic choice for an anti-Brucella vaccine delivery platform. In this study we developed and formulated a combination of four attenuated Salmonella Typhimurium live vector strains delivering heterologous Brucella antigens (rBs), namely lumazine synthase, proline racemase subunit A, lipoprotein outer membrane protein-19, and Cu-Zn superoxide dismutase. With an aim to develop a cross-protecting vaccine, Brucella pan-species conserved rBs were selected. The present study compared the efficacy of smooth and rough variants of Salmonella delivery vector and also evaluated the inclusion of purified Brucella lipopolysaccharide (LPS) in the formulation. Immunization of SPF-BALB/c mice with the vaccine combinations significantly (P≤0.05) reduced splenic wild-type Brucella abortus 544 colonization as compared to non-immunized mice as well as Salmonella only immunized mice. Increased induction of Brucella specific-IgG, sIgA production, and antigen-specific splenocyte proliferative responses were observed in the mice immunized with the formulations as compared to naïve or vector only immunized mice. Modulatory effects of rB and LPS on production of interleukin (IL)-4, IL-12, and interferon-γ were detected in splenocytes of mice immunized with the formulation. Rough Salmonella variant in combination with LPS could further enhance the efficacy of the delivery when applied intraperitoneally. Taken together, it is compelling that Brucella LPS-augmented Salmonella vector delivering immunogenic Brucella proteins may be more suitable than the current non-ideal live Brucella abortus vaccine. The vaccine system also provides a basis for the development of cross-protecting vaccine capable of preventing multispecies brucellosis., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Salmonella serovar Montevideo (SM) is frequently associated with human Salmonella infections and causes gastrointestinal disease, cases are common particularly among individuals who come in close contact with live poultry or poultry meat products. To characterize SM disease in chickens, the pathogenic traits and tissue predilections of the disease were investigated. Dissemination of fluorescent-tagged SM (JOL1575GFP) was monitored after oral and intramuscular mock infections of specific-pathogen-free chickens. The spleen was predominantly affected by intramuscular infection while the cecum, spleen, and minimally liver were affected by oral infection. No conspicuous illness was observed in infected birds, and histopathological examination showed minimal damage of the intestinal epithelium and splenic parenchyma though SM was readily isolated from these tissues. Levels of SM internalization by primary chicken peritoneal macrophages were similar to that of Salmonella Typhimurium. SM was more sensitive to chicken than rabbit serum complement killing. Internal egg contamination of SM mock infected layers also occurred at trace levels and lasted for a week after inoculation. This study also confirmed that SM infection in chickens is sub-clinical and asymptomatic, which suggests that latent asymptomatic carriers may excrete a large number of bacteria and transmit the pathogen by contaminating water or food sources.

Bacterial ghosts (BGs) are empty cell envelopes derived from Gram-negative bacteria by bacteriophage ɸX174 gene E mediated lysis. They represent a novel inactivated vaccine platform; however, the practical application of BGs for human vaccines seems to be limited due to the safety concerns on the presence of viable cells in BGs. Therefore, to improve the lysis efficiency of the gene E, we exploited the peptidoglycan hydrolyzing ability of the λ phage holin-endolysins to expedite the process of current BG production system. In this report, we constructed a novel ghost plasmid encoding protein E and holin-endolysins in tandem. We observed that sequential expressions of the gene E and the holin-endolysins elicited rapid and highly efficient Salmonella lysis compared to the lysis mediated by gene E only. These lysed BGs displayed improved immunogenicity in mice compared to the gene E mediated BGs. Consequently, seventy percent of the mice immunized with these novel ghosts survived against a lethal challenge while all the mice vaccinated with gene E mediated ghosts died by day 9 post-infection. We conclude that this novel strategy has the potential to generate highly efficient inactivated candidate vaccines that could replace the currently available bacterial vaccines.

Porcine edema disease (ED) caused by F18 + Shiga toxin 2e-producing Escherichia coli (STEC) has imposed significant economic losses in the swine industry worldwide, resulting in sudden deaths in post-weaned piglets. The flagellin protein of F18 + STEC, a structural component of the flagellar filament, is a known virulence factor that mediates adhesion and invasion to porcine epithelial cells. In this study, Salmonella inactivated by the E lysis gene and expressing the flagellin (fliC) antigen was genetically engineered utilizing a plasmid (pMMP184) carrying an efficient heterologous antigen delivery system. The resulting strain JOL1485 producing FliC was successfully inactivated by the E lysis gene cassette. Following the lysis procedure, FliC secretion and production of JOL1485 was validated by immunoblot analysis. To evaluate protective immunogenicity elicited by the constructed strain, BALB/c mice were injected with 1×10 8 lysed cells via the intramuscular route. The markedly elevated titers of FliC-specific IgG, IgG1 and sIgA antibodies were observed, indicating a robust Th2-associated humoral immune response was raised in the immunized mice. The proportion of CD3 + CD4 + splenic T cells and proliferative activity were also elevated in in vivo and in vitro stimulated mice splenocytes. Further, JOL1485 successfully elicited upregulated gene expression of cytokines IL-6, IL-8, IL17, IL-21, IFN-γ and TNF-α in naïve porcine peripheral blood mononuclear cells (PBMCs). The overall immune response elicited by JOL1485 conferred a significant rise of protection against a lethal virulent F18 + STEC challenge whereas all non-immunized mice died following the challenge. Our results demonstrate that fliC efficiently expressed in the genetically inactivated Salmonella strain has immunostimulatory and protective effects against a F18 + STEC lethal challenge, and may be promising as a potential vaccine candidate against ED infection., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

In this study, the immune responses to and protective efficacy of a live attenuated Salmonella-delivered vaccine candidate secreting the papA, papG, iutA, and clpG antigens of Escherichia coli were evaluated against infection with avian pathogenic E. coli (APEC) in layer chickens. Primary vaccination was done at age 7 d and booster vaccination at age 5 wk. The levels of intestinal secretory immunoglobulin A specific to the 4 antigens were significantly higher in the vaccinated group than in the control group. A potent lymphocyte-proliferation response and increased levels of interferon-γ, interleukin-2, and interleukin-6 in the plasma and in culture supernatants of antigen-stimulated lymphocytes from the vaccinated group suggested significant induction of the cell-mediated immune response in this group compared with the control group. Upon challenge with a virulent APEC strain at 8 wk of age, the vaccinated group had no deaths, whereas the control group had a 15% mortality rate. In addition, the morbidity rate was significantly higher in the control group (55%) than in the vaccinated group (15%). Thus, giving primary and booster vaccination with the Salmonella-delivered APEC vaccine candidate significantly elevated both mucosal and cellular immune responses, which protected the chickens against colibacillosis.

A major limiting issue of bacterial ghost technology involves the stable maintenance of Phix174 lysis gene E expression. Unwanted leaky expression of gene E in the absence of induction temperature results in reduced biomass production of host bacterium, consequently leading to the lower yield of bacterial ghost. To mitigate the leaky expression status of lysis gene E, we utilized a novel E-lysis system in which gene E is located between sense λpR promoter with a CI857 regulator and antisense ParaBAD promoter with the AraC regulator. In the presence of L-arabinose at 28 C, unwanted transcription of lysis gene E from λpR promoter is repressed by a simultaneous transcription event from ParaBAD promoter by means of anti-sense RNA-mediated inhibition. Tight repression of lysis gene E in the absence of induction temperature resulted in higher bacterial cell number in culture suspension and, consequently, higher production of Salmonella Gallinarum (SG) ghost biomass. The safety and protective efficacy of the SG ghost vaccine were further examined in chickens. All of the immunized chickens showed significantly higher mucosal and systemic antibody responses accompanied by a potent antigen-specific lymphocyte proliferative response. Vaccination of chickens with SG ghost preparation offered efficient protection against wild-type SG challenge.

In this study, a comparative evaluation between a Salmonella ghost vaccine expressing enterotoxigenic Escherichia coli (ETEC) fimbrial antigens and a commercial ETEC vaccine was conducted in neonatal piglets. Genes encoding the ETEC K88ab, K88ac, K99, FasA, and F41 fimbrial proteins were individually cloned into an expression/ghost plasmid (pJHLP184) carrying the pBR origin, asd, the ompA signal sequence to direct antigens to the cell membrane, cI857/λPR promoter, araC ParaBAD, and the phiX174 lysis gene E. Individual clones were subsequently used to electroporate a Δasd S. Typhimurium strain, and positive clones were used to generate Salmonella ghosts. Pregnant sows (n=12) were equally divided into four groups. Pregnant sows were primed and boosted at 11 and 14 weeks of pregnancy, respectively. Group A, B, and C sows were intramuscularly inoculated with PBS as a control, a commercial vaccine, and with 2×10(10) ghost cells, respectively, while group D sows were orally immunized with 2×10(10) ghost cells. All the vaccinated sows and their offspring exhibited increased ETEC fimbrial antigen antibody levels relative to those in the unimmunized group A. Especially, serum IgG and colostrum IgA levels in the group D sows and serum IgG and IgA levels in their piglets were significantly higher than those in control group sows and offspring, respectively. The levels of interleukin-4 against all the fimbrial proteins in peripheral blood lymphocytes of group D sows also increased significantly, whereas no difference between the control group and any of the immunized groups was detected for interferon-γ. In addition, the piglets of group D did not experience diarrhea following challenge with virulent ETEC strains. Diarrhea was observed in 88.9%, 30.4%, and 23.5% of the piglets in groups A, B and C, respectively. Mortality was observed in 16.7% and 5.9% of the piglets in groups A and C, respectively. These findings indicate that immunization of sows with the ghost vaccine candidate, especially by the oral route, can effectively protect their offspring against E. coli colibacillosis., (Copyright © 2015 Elsevier B.V. All rights reserved.)

We previously reported JOL916, a live attenuated Salmonella gallinarum (SG), as a vaccine candidate for protection from fowl typhoid (FT). In the present study, we evaluated JOL1355, an SG that secretes heat-labile enterotoxin B subunit protein, for safety, immunogenicity, and protective efficacy against FT. In a single intramuscular inoculation, live attenuated SG (JOL916) and commercial live attenuated SG9R immunized groups showed gross lesions and bacterial persistence up to 21 days post-immunization. However, the JOL1355 immunized group showed gross lesions and bacterial persistence up to only 3 and 7 days post-immunization, respectively. In addition, several birds in the JOL916 and SG9R immunized group shown clinical signs after immunization, while JOL1355 immunized birds did not show any adverse effects. In a subsequent study, birds were primed and boosted at 4 and 8 weeks of age, respectively, and compared with control birds inoculated with sterile phosphate-buffered saline. The immunized groups B (JOL916), C (SG9R), and D (JOL1355) exhibited significantly higher humoral and cellular immune responses compared to those in the unimmunized control group A. In addition, the birds of each group were challenged with virulent SG at 11 weeks of age, and significantly increased survival rates were observed in all immunized groups compared with the control group. These results indicated that JOL1355 was able to efficiently induce an acquired immune response to protect birds after challenge, and may be safer than JOL916 and the commercial vaccine SG9R in chickens., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Mice were intranasally inoculated at various times to optimize the vaccination strategy with a new live candidate vaccine expressing the antigens CP39, FimA, PtfA, and ToxA of Pasteurella multocida and F1P2 of Bordetella bronchiseptica in an attenuated live Salmonella system to protect against progressive atrophic rhinitis (PAR). Sixty BALB/c mice were divided equally into 4 groups. The group A mice were vaccinated only at 12 wk of age, the group B mice received a primary vaccination at 9 wk of age and a booster at 12 wk of age, the group C mice received a primary vaccination at 6 wk of age and boosters at 9 and 12 wk of age, and the group D mice were inoculated intranasally with sterile phosphate-buffered saline as a control. The humoral and mucosal immune responses of groups A, B, and C increased significantly compared with those of the control group. Expression of the cytokines interleukin-4 and interferon-γ in splenocytes also increased significantly. In addition, the group B mice exhibited significantly fewer gross lesions in lung tissue compared with the other vaccinated groups after challenge with a virulent P. multocida strain. These results indicate that a strategy of double intranasal vaccination can optimize protection against PAR.

A safety enhanced Salmonella Gallinarum (SG) ghost was constructed using an antibiotic resistance gene free plasmid and evaluated its potential as fowl typhoid (FT) vaccine candidate. The antibiotic resistance free pYA3342 plasmid possesses aspartate semialdehyde dehydrogenase gene which is complimentary to the deletion of the chromosomal asd gene in the bacterial host. This plasmid was incorporated with a ghost cassette containing the bacteriophage PhiX174 lysis gene E, designated as pJHL101. The plasmid pJHL101 was transformed into a two virulence genes-deleted SG. The SG ghosts with tunnel formation and loss of cytoplasmic contents were observed by scanning electron microscopy and transmission electron microscopy. The cell viability of the culture solution was decreased to 0% at 24h after the induction of gene E expression by an increase in temperature from 37°C to 42°C. The safety and protective efficacy of the SG ghost vaccine was further examined in chickens which were divided into three groups: group A (non-immunized control), group B (orally immunized), and group C (intramuscularly immunized). The birds were immunized at 7d of age. No clinical symptoms associated with FT such as anorexia, depression and greenish diarrhea were observed in the immunized chickens. Upon challenge with a virulent SG strain at 3 week post-immunization, the chickens immunized with the SG ghost via various routes were efficiently protected, as shown by significantly lower mortality and post-mortem lesions in comparison with control group. In addition, all the immunized chickens showed significantly higher antibody responses accompanied by a potent antigen-specific lymphocyte proliferative response along with significantly increased numbers of CD4⁺ and CD8⁺ T lymphocytes. Overall, our results provide a promising approach of generating SG ghosts using the antibiotic resistance free plasmid in order to prepare a non-living bacterial vaccine candidate which could be environmentally safe yet efficient to prevent FT in chickens., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

In our previous study, we constructed a vaccine candidate (JOL916) for fowl typhoid (FT). A live adjuvant Salmonella Gallinarum (SG) strain was generated in the present study to facilitate efficacious oral vaccination with this vaccine. The Escherichia coli eltB gene secreting heat-labile enterotoxin B subunit (LTB) was cloned into an Asd(+) plasmid pJHL65. This was transformed into a Δlon ΔcpxR Δasd SG strain and the resulting strain was designated JOL1229. Secretion of LTB from JOL1229 was confirmed with an immunoblot assay. To determine the optimal dose of the strain, 50 six-week-old female chickens were divided into five groups (Groups A-E, n=10 per group) and orally inoculated with various doses of JOL1229 and JOL916. In Group B (consisting of four parts JOL916 and one part JOL1229), significant cell-mediated immune responses, plasma IgG levels and intestinal secretary IgA levels were induced after inoculation with both strains. On challenge with the wild-type strain, significant reductions in mortality were observed in the group. In addition, after inoculation the LTB strain was not recovered in feces samples, and resulted in no, or very mild, gross lesions in the liver and spleen. Both CD4(+) and CD8(+) T-cells were significantly increased in peripheral blood samples from the chickens immunized with the LTB strain. Expression of the interleukin-6 (IL-6) gene in splenocytes was induced in the chickens immunized with the LTB strain. These results suggest that oral immunization with the LTB-adjuvant strain, in particular with the four parts JOL916 and one part JOL1229 mixture, increased the immune response and provided efficient protection against FT in chickens., (Copyright © 2012 Elsevier B.V. All rights reserved.)

In order to construct a conditional lethal Salmonella mutant, an arabinose-regulated recombinant genetic system was used. The Salmonella aspartate semialdehyde dehydrogenase (asd) gene was localized under the control of araC P(araBAD) in a plasmid to create the araC P(araBAD)::asd cassette. The cassette was cloned into a plasmid carrying a p15A replication origin to create the recombinant plasmid pMMP55. The growth of Salmonella MMP10 harboring pMMP55 was dependent on the presence of arabinose. In the presence of arabinose, the Asd deficiency due to chromosomal deletion of asd in the Salmonella host was complemented by the asd gene transcribed and translated under the P(araBAD) promoter and araBAD Shine-Dalgarno (SD) sequence in pMMP55. Growth inhibition of the strain was demonstrated by arabinose depletion in M9 minimal medium, indicating that the strain were unable to grow in an arabinose-limited environment. In addition, the analysis of a 50% lethal dose (LD50) using mice revealed that the strain MMP10 exhibited attenuation by approximately 100-fold relative to that of the unmodified strain. In conclusion, these data suggest that the araC P(araBAD)::asd system developed in this study can be used to construct conditional lethal Salmonella mutants for application as safe, live-attenuated Salmonella vaccines., (Copyright © 2011 Elsevier B.V. All rights reserved.)

We evaluated a recently developed live vaccine candidate for fowl typhoid (FT)-JOL916, a lon/cpxR mutant of Salmonella Gallinarum (SG)-by comparing its safety and efficacy with that of the well-known rough mutant strain SG9R vaccine in 6-wk-old Hy-Line hens. Forty-five chickens were divided into three groups of 15 chickens each. The chickens were then intramuscularly inoculated with 2 x 10(7) colony-forming units (CFUs) of JOL916 (JOL916 group), 2 x 10(7) CFUs of SG9R (SG9R group), or phosphate-buffered saline (control group). After vaccination, no clinical symptoms were observed in any of the groups. No differences in body weight increase were detected among the three groups postvaccination. A cellular immune response was observed at 2 wk postvaccination (wpv) in the JOL916 group with the peripheral lymphocyte proliferation assay, whereas no response was detected in the SG9R group. Elevation of SG antigen-specific plasma immunoglobulin was observed 2 and 3 wpv in the JOL916 and SG9R vaccine groups, respectively. After virulent challenge on day 25 postvaccination, 0, 1, and 15 chickens in the JOL916 group, SG9R group, and control group, respectively, died by 12 days postchallenge; the death rate of the SG9R vaccine group was statistically similar to that of the JOL916 group. Postmortem examination revealed that the JOL916 vaccine offered more efficient protection than the SG9R vaccine, with significantly decreased hepatic necrotic foci scores, splenic enlargement scores, necrotic foci scores, and recovery of the challenge strain from the spleen. Vaccination with JOL916 appears to be safe and offers better protection than SG9R against FT in chickens.

In seeking to develop a safe fowl typhoid (FT) vaccine, a novel candidate lacking cpxR, lon, and asd Salmonella Gallinarum (SG) genes was constructed with the plasmid-containing araC::P(araBAD)::asd system. A balanced-lethal host-vector system based on the essential bacterial gene for aspartate beta-semialdehyde dehydrogenase (asd) was used to construct the SG mutant strain. A plasmid (p15A ori) with an araC::P(araBAD)::asd cassette was introduced into an auxotrophic mutant to prevent ex vivo survival. The safety, immunity, and protective properties of the SG mutant were evaluated. Inoculation of the mutant at 10(6) colony-forming units (CFU) did not result in recovery in feces and internal organs, whereas inoculation at 10(8) and 10(10) CFU resulted in moderate bacterial recovery from feces and organs. Birds immunized with the mutant were challenged with a virulent SG strain at day 14 postimmunization; significantly reduced mortality and induced plasma immunoglobulin (Ig)G and mucosal IgA responses were noted. Cellular immune responses as evaluated by a peripheral lymphocyte proliferation assay were also significantly induced. The balanced-lethal host-vector system for construction of SG mutants is an effective and improved approach for safe vaccine construction against FT.

To construct a novel live vaccine candidate for fowl typhoid (FT) caused by Salmonella Gallinarum (SG), the lon and cpxR genes that are related to host-pathogen interaction were deleted from a wild type SG using the allelic exchange method. The mutants were grown normally, as was the wild type. The biochemical properties of the mutants remained very similar to those of the wild-type, while JOL914 (Deltalon) and JOL916 (DeltalonDeltacpxR) were mucoid. Extracellular polysaccharide increased 30.6-, 1.3-, and 46.2-fold in JOL914, JOL915 (DeltacpxR), and JOL916, respectively. Dot-blot analysis demonstrated significant increases of FimA expression at 6.77-, 2.33-, and 3.90-fold for JOL914, JOL915, and JOL916, respectively. Internalizations of JOL914, JOL915, and JOL916, in chicken abdominal macrophages, were increased at 4.65-, 0.50-, and 2.72-fold, respectively. Virulences of JOL914, JOL915 and JOL916, analyzed by LD50 using 1-week-old chickens, were attenuated approximately at 10(1)-, 10(1)-, and >10(3)-fold, respectively. The oral inoculations of 2x10(7) cfu of the wild type, JOL914, JOL915 and JOL916 caused 55.6, 16.7, 22.2, and 0.0% mortality, respectively. Significantly moderate gross lesions of the liver and spleen were observed in the JOL916 group compared to the other groups. An induced immune response and significant peripheral mononuclear proliferation reaction were observed in the JOL916 group. At the protection against the wild type challenge, JOL916 offered 100% protection. Thus, the results of this study suggest that JOL916 among the mutants studied represented the safest and most effective live vaccine candidate against FT., (© The authors, published by INRA/EDP Sciences, 2010.)

This study was carried out to examine a novel Salmonella Typhimurium (S. Typhimurium) vaccine for protection of suckling piglets against salmonellosis by immunization of pregnant sows using various administration routes. The vaccine strain was constructed by deletion of cpxR and lon from a wild type S. Typhimurium and the S. Typhimurium Delta cpxR Delta lon Delta asd secreting the B subunit of the Escherichia coli heat-labile enterotoxin were used as a live form of mucosal adjuvant for this study. Pregnant sows were divided into 4 groups of 3 sows a piece. Sows were primed at 8 weeks of pregnancy and were boosted 11 weeks of pregnancy. Group A sows were primed intramuscularly with the formalin-inactivated vaccine and boosted orally with the live vaccine and mucosal adjuvant, group B sows were orally primed with the live vaccine and mucosal adjuvant and boosted orally with live vaccine, group C sows were orally primed with live vaccine and mucosal adjuvant and intramuscularly boosted, and group D sows were primed and boosted with phosphate-buffered saline as controls. Piglets were orally challenged with a virulent S. Typhimurium strain at day 6 after birth. Sows from group A and B had significantly increased IgG levels compared to control group sows (P<0.05), and group C sows had lower IgG levels compared to group A and B sows. Mucosal sIgA and IgG levels in group A and B sow colostrums were significantly increased as compared to those of group D sows (P<0.05). Serum IgG and IgA levels in group A and B piglets were also significantly increased as compared to those of group D piglets (P<0.001). These data suggested that systemic and mucosal immune responses were highly induced by the vaccine candidate, especially when this was administered by both routes of intramuscular-prime and oral booster, and oral prime and booster. Furthermore, clinical signs such as diarrhea and weight loss were not observed after virulent Salmonella strain challenge in group A and B suckling piglets, however, group C (41.7%) and group D (83.3%) piglets suffered from diarrhea. This data suggested that sow immunized by intramuscular-prime and oral booster and oral prime and oral booster with this vaccine candidate effectively protected piglets from salmonellosis., ((c) 2009 Elsevier B.V. All rights reserved.)

The antibacterial potential of a preparation of medicinal herbs for the treatment of salmonellosis was evaluated. The preparation contained a combination of methanolic extracts from Mume Fructus, Coptidis Rhizoma and Schizandrae Fructus, and is designated NP532. NP532 was tested against 26 different Salmonella stains. All these strains were affected by NP532 even though many of the salmonella strains were resistant to multiple antibiotics. The MIC values of this combination against the Salmonella strains varied from 0.49 to 7.8 mg/ml. The in vivo antibacterial activity of NP532 was examined using a S. Gallinarum infection chicken model. The chickens were initially infected with S. Gallinarum, and NP532 was then administered. This preparation was found to have major effects on mortality. The histopathological observations of the NP532 treated animals did not show any of the clinical signs and rarely showed histological damage associated with the disease. On the other hand, the untreated controls showed the clinical signs, e.g. congestion and necrotic changes in the liver, kidney, and spleen. This suggests that NP532 might be an effective treatment for salmonellosis.

The therapeutic potentials of twenty-two medicinal herb species traditionally used in Korea to treat gastrointestinal infections were evaluated for the treatment of salmonellosis. Candidates were primarily screened using the disk-agar method for antibacterial activity against three different Salmonella serotypes. Of the herbs tested, the aqueous and methanolic extracts of Schizandrae Fructus exhibited antibacterial activity against all three Salmonella. The extracts of this herb were further tested against 13 additional Salmonella strains of 6 different serotypes. All of these strains were also affected by these extracts, though the methanolic extract had slightly higher activity. The MIC values of this extract against the 16 Salmonella strains varied from 15.6 to 125 microg/ml. Nine of the 16 strains tested had MIC values of <31.3 microg/ml for the methanolic extract of Schizandrae Fructus. The in vivo antibacterial activity of Schizandrae Fructus extract was examined in a S. Typhimurium infection mouse model. Mice were initially infected with S. Typhimurium, and then administered with Schizandrae Fructus extract. The extract was found to have major effects on mortality and on the numbers of viable S. Typhimurium recovered from feces. Clinical signs and histological damages were rarely observed in the treated mice, whereas the untreated controls showed clinical signs, e.g., lethargy, and histological damage in the kidney, liver, intestine, and spleen. We conclude that Schizandrae Fructus has the potential to provide an effective treatment for salmonellosis.

Salmonella serovar Pullorum is a causative agent of pullorum disease (PD) in poultry and is responsible for severe economic losses to the poultry industry in many parts of the world. A definitive detection of Pullorum requires culture followed by serotyping and biochemical identification, a process that is tedious and takes several weeks to accomplish. We have developed a rapid allele-specific polymerase chain reaction (PCR) method based on the nucleotide polymorphism in rfbS gene sequence for the serotype-specific detection of Pullorum and its differentiation from the closely related Gallinarum. The specificity of this PCR assay was tested using DNA samples from Pullorum (n = 13), Salmonella serotypes other than Pullorum (n = 19), and closely related non-Salmonella organisms (n = 5). The PCR assay was highly serotype-specific as the PCR amplicon of 147 base pairs was observed only in the case of Pullorum, while all the other DNA samples tested PCR negative. A definitive identification of Pullorum cultures was possible in less than 3 hr. As little as 100 pg of SP DNA was detected. This allele-specific PCR method is highly specific as well as sensitive and may be an effective molecular tool in the rapid and serotype-specific detection of Pullorum and differentiation from other Salmonella species.

Salmonella gallinarum (SG) is a non-motile host-adapted salmonella that causes fowl typhoid, a severe systemic disease responsible for significant economic losses to the poultry industry worldwide. This study describes the application of a PCR-based signature-tagged mutagenesis system to identify in vivo-essential genes of SG. Ninety-six pools representing 1152 SG mutants were screened in a natural-host chicken infection model. Twenty presumptive attenuated mutants were identified and examined further. The identity of the disrupted gene in each mutant was determined by cloning of the DNA sequences adjacent to the transposon, followed by sequencing and comparison with the bacterial genome database. In vitro and in vivo competition indices were determined for each identified mutant and a total of 18 unique, attenuating gene disruptions were identified. These mutations represented six broad genomic classes: Salmonella pathogenicity island-1 (SPI-1), SPI-2, SPI-10, SPI-13, SPI-14 and non-SPI-encoded virulence genes. SPI-13 and SPI-14 are newly identified and designated in this study. Most of the genes identified in this study were not previously believed or known to play a role in the pathogenesis of SG infection in chickens. Each STM identified mutant showed competitiveness and/or virulence defects, confirmed by in vitro and in vivo assays, and challenge tests. This study should contribute to a better understanding of the pathogenic mechanisms involved in progression of disease caused by SG, and identification of novel live vaccine candidates and new potential antibiotic targets.

Introduction: The limitations of conventional cancer therapies necessitate target-oriented, highly invasive, and safe treatment approaches. Hence, the intrinsic anti-tumor activity of Salmonella can offer better options to combat cancers. Objectives: This study aims to utilize attenuated Salmonella and deliver cytolytic protein cytolysin A (ClyA) under quorum sensing (QS) signaling for precise localized expression in tumors but not in healthy organs. Methods: The therapeutic delivery strain was imposed with tryptophan auxotroph for selective colonization in tumors by trpA and trpE deletion, and lipid-A and O-antigen were altered by pagL and rfaL deletions using lambda red recombination method. The strain was transformed with the designed QS-controlled ClyA expression vector which was validated by western blot. The in vivo passaged therapeutic strain was used for treatment four times at a weekly interval, with a dose of 5 × 106 CFU/mouse for cancer therapy. Results: The attenuated strain induced minimal endotoxicity-related cytokines TNF-α, IL-1β, and IFN-γ and exhibited adequate colonization despite earlier exposure in mice. The QS-controlled ClyA expression was confirmed by western blot from bacterial cultures grown at different cell densities. The results demonstrated that the in vivo passaged strain preferentially colonized the tumor after vacating the spleen, liver, and lung, leaving no outward histological scars. The anti-cancer effect of the designed construct was evaluated in the murine CT26 colon cancer model. The expression of ClyA increased tumoricidal activity by 67 % compared to vector control. Conclusion: Hence, the anti-tumor effect of the engineered Salmonella strain was improved by ClyA expression via QS activation after achieving the threshold bacterial cell density. Further, immunohistochemical staining of the tumor and other organs corroborated the QS-controlled tumor-specific expression of ClyA. Overall, the results imply that the developed anti-cancer Salmonella has low endotoxicity and QS-controlled expression of ClyA as beneficial safety elements and supports recurrent Salmonella inoculation by O-antigen deficiency.

A universal vaccine is in high demand to address the uncertainties of antigenic drift and the reduced effectiveness of current influenza vaccines. In this study, a strategy called computationally optimized broadly reactive antigen (COBRA) was used to generate a consensus sequence of the hemagglutinin globular head portion (HA1) of influenza virus samples collected from 1918 to 2021 to trace evolutionary changes and incorporate them into the designed constructs. Constructs carrying different HA1 regions were delivered into eukaryotic cells by Salmonella-mediated bactofection using a Semliki Forest virus RNA-dependent RNA polymerase (RdRp)-based eukaryotic expression system, pJHL204. Recombinant protein expression was confirmed by Western blot and immunofluorescence assays. Mice immunized with the designed constructs produced a humoral response, with a significant increase in immunoglobulin G (IgG) levels, and a cell-mediated immune response, including a 1.5-fold increase in CD4+ and CD8+ T cells. Specifically, constructs #1 and #5 increased the production of interferon-γ (IFN-γ) producing CD4+ and CD8+ T cells, skewing the response toward the T helper type 1 cell (Th1) pathway. Additionally, interleukin-4 (IL-4)-producing T cells were upregulated 4-fold. Protective efficacy was demonstrated, with up to 4-fold higher production of neutralizing antibodies and a hemagglutination inhibition titer > 40 against the selected viral strains. The designed constructs conferred a broadly protective immune response, resulting in a notable reduction in viral titer and minimal inflammation in the lungs of mice challenged with the influenza A/PR8/34, A/Brisbane/59/2007, A/California/07/2009, KBPV VR-92, and NCCP 43021 strains. This discovery revolutionizes influenza vaccine design and delivery; Salmonella-mediated COBRA-HA1 is a highly effective in vivo antigen presentation strategy. This approach can effectively combat seasonal H1N1 influenza strains and potential pandemic outbreaks.

Abstract In the present study, two prospective Salmonella delivery strains, JOL2782 and JOL2837, were developed by gene deletions of lon and cpxR, which are related to cellular adhesion and intracellular survival. Additionally, sifA deletion was introduced for JOL2782, which confers immune susceptibility and improves antigen delivery. Similarly, the rfaL deletion and lpxE substitution for pagL were accomplished in JOL2837 to reduce virulence and endotoxicity. Thus, enhanced adhesion and invasion and reduced intracellular survival were attained. Furthermore, aspartic acid auxotrophic (asd) was deleted to impose Darwinian selection on retention of the foreign antigen-expressing plasmid. Both delivery strains induced sufficient cytokine expression, but the level was significantly lower than that of the wild-type strain; the lowest cytokine expression was induced by the JOL2837 strain, indicating reduced endotoxicity. In parallel, IgG production was significantly enhanced by both delivery strains. Thus, the innate and adaptive immunogenicity of the strains was ensured. The environmental safety of these strains was ascertained through faecal dissemination assays. The nonpathogenicity of these strains to the host was confirmed by body weight monitoring, survival assays, and morphological and histological assessments of the vital organs. The in vitro assay in murine and human cell lines and in vivo safety assessments in mice suggest that these novel strains possess safety, invasiveness, and immunogenicity, making them ideal delivery strains. Overall, the results clearly showed that strain JOL2782 with sifA deletion had higher invasiveness, demonstrating superior vaccine deliverability, while JOL2837 with lpxE substitution for pagL and rfaL deletion had outstanding safety potential with drastically abridged endotoxicity.

The global cancer burden is growing and accounted for 10 million deaths in 2020. The resurgence of chemo- and radiation resistance have contributed to the treatment failures in many cancer types. Therefore, alternative strategies are desired for the effective cancer therapy. Bacteria-mediated cancer therapy presents an attarctive alternative option for the treatment and diagnosis of cancers. Herein, we describe an engineered Salmonella Typhimurium (ST) auxotrophic for tryptophan as a cancer therapeutic. The tryptophan auxotrophy was sufficient to render ST avirulent and highly safe to mice. The auxotroph recovered from the infected tumors had improved ability to target and colonize the tumors. We show that tryptophan auxotrophy reduced the fitness of ST in healthy tissues, but not in the tumors. We evaluated the auxotroph in highly aggressive metastatic 4T1 breast cancer model to inhibit primary tumor growth and lung metastases. The therapy greatly suppressed the primary growth with tumor-free survival of 40% mice. Importantly, therapy abolished the metastatic dissemination of tumor to lungs. Further, therapy markedly diminished the macrophage population in the tumors that may have contributed to the therapeutic benefit recorded. Collectively, results highlight the therapeutic efficacy of the tryptophan auxotrophic ST in an aggressive metastatic cancer model.

Introduction: The emergence of SARS-CoV-2 variants has raised concerns on future vaccine efficacy as most vaccines target only the spike protein. Hence, vaccines targeting multiple SARS-CoV-2 proteins will offer broader protection and improve our preparedness to combat the pandemic. Objectives: The study aimed to develop a novel vaccine strategy by combining a eukaryotic vector expressing multiple SARS-CoV-2 genes and Salmonella-mediated in vivo DNA delivery. Methods: The eukaryotic vector was designed to function as a DNA-launched RNA replicon in a self-replicating and self-amplifying mRNA mechanism. By exploiting the self-cleaving peptide, P2A, we fused four SARS-CoV-2 targets, including receptor-binding domain (RBD), heptad repeat domain (HR), membrane protein (M) and epitopes of nsp13, in a single open reading frame. Western blot and immunofluorescence assays were used to determine protein expression. In mice, the vaccine's safety and immunogenicity were investigated. Results: Western blot analysis revealed co-expression all four proteins from the vaccine construct, confirming the efficiency of Salmonella-mediated gene delivery and protein expression. The vaccine candidate was safe and elicited robust antigen-specific antibody titers in mice, and a recall response from splenocytes revealed induction of strong cell-mediated immunity. Flow cytometry demonstrated an increase in sub-populations of CD4+ and CD8+ T cells with the highest CD4+ and CD8+ T cells recorded for HR and RBD, respectively. Overall, humoral and cellular immune response data suggested the induction of both Th1 and Th2 immunity with polarization towards an antiviral Th1 response. We recorded a potent SARS-CoV-2 neutralizing antibody titers in the immunized mice sera. Conclusions: The Salmonella bactofection ensured optimum in vivo gene delivery, and through a P2A-enabled efficient multicistronic expression, the vaccine candidate elicited potent anti-SARS-CoV-2 immune responses. These findings provide important insight into development of an effective multivalent vaccine to combat SARS-CoV-2 and its variants.

This study investigates the anticancer properties of the α7-nAChR antagonist SLURP1 with a specific focus on its effect as an inflammation modulator on human colorectal cancer cell lines Caco2, Colo320DM, and H508 cells. The investigation includes the evaluation of cell cycle arrest, cell migration arrest, endogenous expression of SLURP1 and related proteins, calcium influx, and inflammatory responses. The results demonstrate that SLURP1 not only inhibits cell proliferation but also has the potential to arrest the cell cycle at the G1/S interface. The impact of SLURP1 on cell cycle regulation varied among cell lines, with H508 cells displaying the strongest response to exogenous SLURP1. Additionally, SLURP1 affects the nuclear factor kappa B expression and effectively reverses inflammatory responses elicited by purified lipopolysaccharides in H508 and Caco2 cells. This study further confirmed the expression of human SLURP1 by Salmonella, under Ptrc promoter, through Western blot analysis. Moreover, Salmonella secreting SLURP1 revealed a significant tumor regression in a mouse CT26 tumor model, suggesting the cross-species anticancer potential of human SLURP1. However, further investigations are required to fully understand the mechanisms underlying SLURP1’s ability to prevent cancer proliferation and its protective function in humans.

Abstract Efficient in vivo delivery of a CRISPR/Cas9 plasmid is of paramount importance for effective therapy. Here, we investigated the usability of Salmonella as a plasmid carrier for in vivo therapy against virus-induced cancer using Marek’s disease virus (MDV) as a model for study in chickens. A green fluorescent protein-expressing CRISPR/Cas9 plasmid encoding the virulence gene pp38 was constructed against Marek’s disease virus. Therapeutic plasmids were transformed into Salmonella carrying lon and sifA gene deletions. The animals in 5 groups were intraperitoneally inoculated with phosphate-buffered saline, vector control, or Salmonella before or after MDV infection, or left uninfected as a naïve control. Therapeutic effectiveness was evaluated by observing disease outcomes and the viral copy number in peripheral blood mononuclear cells. The efficacy of plasmid delivery by Salmonella was 13 ± 1.7% in the spleen and 8.0 ± 1.8% in the liver on the 6th day post-infection. The Salmonella-treated groups showed significant resistance to MDV infection. The maximum effect was observed in the group treated with Salmonella before MDV infection. None of the chickens fully recovered; however, the results suggested that timely delivery of Salmonella could be effective for in vivo CRISPR/Cas9-mediated genetic interference against highly pathogenic MDV. The use of Salmonella in CRISPR systems provides a simpler and more efficient platform for in vivo therapy with CRISPR than the use of conventional in vivo gene delivery methods and warrants further development.

In the present study, we characterized the involvement of Lon protease in bacterial virulence and intracellular survival in Salmonella under abiotic stress conditions resembling the conditions of a natural infection. Wild type (JOL401) and the lon mutant (JOL909) Salmonella Typhimurium were exposed to low temperature, pH, osmotic, and oxidative stress conditions and changes in gene expression profiles related to virulence and metal ion uptake were investigated. Expression of candidate genes invF and hilC of Salmonella Pathogenicity Island (SPI)-1 and sifA and sseJ of SPI-2 revealed that Lon protease controls SPI-1 genes and not SPI-2 genes under all stress conditions tested. The lon mutant exhibited increased accumulation of hydroxyl (OH·) ions that lead to cell damage due to oxidative stress. This oxidative damage can also be linked to an unregulated influx of iron due to the upregulation of ion channel genes such as fepA in the lon mutant. The deletion of lon from the Salmonella genome causes oxidative damage and increased expression of virulence genes. It also prompts the secretion of host pro-inflammatory cytokines leading to early clearance of the bacteria from host cells. We conclude that poor bacterial recovery from mice infected with the lon mutant is a result of disrupted bacterial intracellular equilibrium and rapid activation of cytokine expression leading to bacterial lysis.

Bacteria-mediated cancer therapy has become a topic of interest under the broad umbrella of oncotherapy. Among many bacterial species, Salmonella remains at the forefront due to its ability to localize and proliferate inside tumor microenvironments and often suppress tumor growth. Salmonella Typhimurium is one of the most promising mediators, with engineering plasticity and cancer specificity. It can be used to deliver toxins that induce cell death in cancer cells specifically, and also as a cancer-specific instrument for immunotherapy by delivering tumor antigens and exposing the tumor environment to the host immune system. Salmonella can be used to deliver prodrug converting enzymes unambiguously against cancer. Though positive responses in Salmonella-mediated cancer treatments are still at a preliminary level, they have paved the way for developing combinatorial therapy with conventional chemotherapy, radiotherapy, and surgery, and can be used synergistically to combat multi-drug resistant and higher-stage cancers. With this background, Salmonella-mediated cancer therapy was approved for clinical trials by U.S. Food and Drug Administration, but the results were not satisfactory and more pre-clinical investigation is needed. This review summarizes the recent advancements in Salmonella-mediated oncotherapy in the fight against cancer. The present article emphasizes the demand for Salmonella mutants with high stringency toward cancer and with amenable elements of safety by virulence deletions.

[Display omitted] • Attenuated auxotrophic Salmonella Typhimurium was utilized for the anti-tumor effect. • Reduction of endotoxicity was rendered by lipid A modification. • Regulated expression of ClyA at higher cell density under QS signaling. • Engineered Salmonella Typhimurium enhanced safety and tumor invasion. • ClyA expressing Salmonella reduced 67 % of tumors in mouse CT 26 cancer model. The limitations of conventional cancer therapies necessitate target-oriented, highly invasive, and safe treatment approaches. Hence, the intrinsic anti-tumor activity of Salmonella can offer better options to combat cancers. This study aims to utilize attenuated Salmonella and deliver cytolytic protein cytolysin A (ClyA) under quorum sensing (QS) signaling for precise localized expression in tumors but not in healthy organs. The therapeutic delivery strain was imposed with tryptophan auxotroph for selective colonization in tumors by trpA and trpE deletion, and lipid-A and O-antigen were altered by pagL and rfaL deletions using lambda red recombination method. The strain was transformed with the designed QS-controlled ClyA expression vector which was validated by western blot. The in vivo passaged therapeutic strain was used for treatment four times at a weekly interval, with a dose of 5 × 106 CFU/mouse for cancer therapy. The attenuated strain induced minimal endotoxicity-related cytokines TNF-α, IL-1β, and IFN-γ and exhibited adequate colonization despite earlier exposure in mice. The QS-controlled ClyA expression was confirmed by western blot from bacterial cultures grown at different cell densities. The results demonstrated that the in vivo passaged strain preferentially colonized the tumor after vacating the spleen, liver, and lung, leaving no outward histological scars. The anti-cancer effect of the designed construct was evaluated in the murine CT26 colon cancer model. The expression of ClyA increased tumoricidal activity by 67 % compared to vector control. Hence, the anti-tumor effect of the engineered Salmonella strain was improved by ClyA expression via QS activation after achieving the threshold bacterial cell density. Further, immunohistochemical staining of the tumor and other organs corroborated the QS-controlled tumor-specific expression of ClyA. Overall, the results imply that the developed anti-cancer Salmonella has low endotoxicity and QS-controlled expression of ClyA as beneficial safety elements and supports recurrent Salmonella inoculation by O-antigen deficiency. [ABSTRACT FROM AUTHOR]

The lipopolysaccharide (LPS) that resides on the outermost surface and protects Gram-negative bacteria from host defenses is one of the key components leading to Salmonella infection, particularly the endotoxic lipid A domain of LPS. Lipid A modifications have been associated with several genes such as the arnT that encodes 4-amino-4-deoxy-L-arabinose transferase, which can be critical for bacteria to resist cationic antimicrobial peptides and interfere with host immune recognition. However, the association of arnT with virulence is not completely understood. Thus, this study aimed to elucidate the interrelationship of the major lipid A modification gene arnT with Salmonella Typhimurium virulence. We observed that the arnT-deficient S. Typhimurium (JOL2943), compared to the wild type (JOL401), displayed a significant decrease in several virulence phenotypes such as polymyxin B resistance, intracellular survival, swarming, and biofilm and extracellular polymeric substance (EPS) production. Interestingly, the cell-surface hydrophobicity, adhesion, and invasion characteristics remained unaffected. Additionally, LPS isolated from the mutant induced notably lower levels of endotoxicity-related cytokines in RAW and Hela cells and mice, particularly IL-1β with a nine-fold decrease, than WT. In terms of in vivo colonization, JOL2943 showed diminished presence in internal organs such as the spleen and liver by more than 60%, while ileal infectivity remained similar to JOL401. Overall, the arnT deletion rendered the strain less virulent, with low endotoxicity, maintained gut infectivity, and reduced colonization in internal organs. With these ideal characteristics, it can be further explored as a potential attenuated Salmonella strain for therapeutics or vaccine delivery systems. [ABSTRACT FROM AUTHOR]

Background: Lawsonia intracellularis is the causative agent of proliferative enteropathy and is associated with several outbreaks, causing substantial economic loss to the porcine industry. Objectives: In this study, we focused on demonstrating the protective effect in the mouse model through the immunological bases of two vaccine strains against porcine proliferative enteritis. Methods: We used live-attenuated Salmonella Typhimurium (ST) secreting two selected immunogenic LI antigens ( Lawsonia autotransporter A epitopes and flagellin [FliC]- peptidoglycan-associated lipoprotein-FliC) as the vaccine carrier. The constructs were cloned into a Salmonella expression vector (pJHL65) and transformed into the ST strain (JOL912). The expression of immunogenic proteins within Salmonella was evaluated via immunoblotting. Results: Immunizing BALB/c mice orally and subcutaneously induced high levels of LI-specific systemic immunoglobulin G and mucosal secretory immunoglobulin A. In immunized mice, there was significant upregulation of interferon-γ and interleukin-4 cytokine mRNA and an increase in the subpopulations of cluster of differentiation (CD) 4+ and CD 8+ T lymphocytes upon splenocytes re-stimulation with LI antigens. We observed significant protection in C57BL/6 mice against challenge with 106.9 times the median tissue culture infectious dose of LI or 2 x 109 colony-forming units of the virulent ST strain. Immunizing mice with either individual vaccine strains or co-mixture inhibited bacterial proliferation, with a marked reduction in the percentage of mice shedding Lawsonia in their feces. Conclusions: Salmonella-mediated LI gene delivery induces robust humoral and cellular immune reactions, leading to significant protection against LI and salmonellosis. [ABSTRACT FROM AUTHOR]

Oral vaccines are highly envisaged for veterinary applications due to their convenience and ability to induce protective mucosal immunity as the first line of defense. The present investigation harnessed live-attenuated Salmonella Typhimurium to orally deliver novel expression vector systems containing the Cap and Rep genes from porcine circovirus type 2 (PCV2), a significant swine pathogen. The antigen expression by the vaccine candidates JOL2885 and JOL2886, comprising eukaryotic pJHL204 and pro-eukaryotic expression pJHL270 plasmids, respectively, was confirmed by Western blot and IFA. We evaluated their immunogenicity and protective efficacy through oral vaccination in a mouse model. This approach elicited both mucosal and systemic immunity against PCV2d. Oral administration of the candidates induced PCV2-specific sIgA, serum IgG antibodies, and neutralizing antibodies, resulting in reduced viral loads in the livers and lungs of PCV2d-challenged mice. T-lymphocyte proliferation and flow-cytometry assays confirmed enhanced cellular immune responses after oral inoculation. The synchronized elicitation of both Th1 and Th2 responses was also confirmed by enhanced expression of TNF-α, IFN-γ, IL-4, MHC-I, and MHC-II. Our findings highlight the effectiveness and safety of the constructs with an engineered-attenuated S. Typhimurium, suggesting its potential application as an oral PCV2 vaccine candidate. [ABSTRACT FROM AUTHOR]

This study investigates the anticancer properties of the α7-nAChR antagonist SLURP1 with a specific focus on its effect as an inflammation modulator on human colorectal cancer cell lines Caco2, Colo320DM, and H508 cells. The investigation includes the evaluation of cell cycle arrest, cell migration arrest, endogenous expression of SLURP1 and related proteins, calcium influx, and inflammatory responses. The results demonstrate that SLURP1 not only inhibits cell proliferation but also has the potential to arrest the cell cycle at the G1/S interface. The impact of SLURP1 on cell cycle regulation varied among cell lines, with H508 cells displaying the strongest response to exogenous SLURP1. Additionally, SLURP1 affects the nuclear factor kappa B expression and effectively reverses inflammatory responses elicited by purified lipopolysaccharides in H508 and Caco2 cells. This study further confirmed the expression of human SLURP1 by Salmonella, under Ptrc promoter, through Western blot analysis. Moreover, Salmonella secreting SLURP1 revealed a significant tumor regression in a mouse CT26 tumor model, suggesting the cross-species anticancer potential of human SLURP1. However, further investigations are required to fully understand the mechanisms underlying SLURP1's ability to prevent cancer proliferation and its protective function in humans. [ABSTRACT FROM AUTHOR]