Jonathan Lalsiamthara

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

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.

Immunization with Salmonella Enteritidis secreting mucosal adjuvant labile toxin confers protection against wild type challenge via augmentation of CD3+CD4+ T-cell proliferation and enhancement of IFN-γ, IL-6 and IL-10 expressions in chicken

The protective efficacy and immunological profiles of chickens immunized with an attenuated Salmonella Enteritidis (SE) constitutively secreting double mutant heat labile enterotoxin (dmLT) were investigated. The dmLT is a detoxified variant of Escherichia coli heat labile toxin and is a potent mucosal adjuvant capable of inducing both humoral and cell-mediated immunity. In this study, four-week-old chickens were inoculated with SE-dmLT strain JOL1641, parental SE strain JOL1087 or phosphate buffered saline control. Peripheral blood mononuclear cells of SE-dmLT inoculated birds showed significant proliferation upon stimulation with SE antigens as compared to the control and JOL1087 groups (P⩽0.05). One week post-challenge, the ratio of CD3+CD4+ to CD3+CD8+ T-cells showed a significant increase in the immunized groups. Significant increases in IFN-γ levels were observed in JOL1641 birds immunized via oral and intramuscular routes. While immunizations with the JOL1087 strain via the intramuscular route also induced significant increases in IFN-γ, immunization via the oral route did not trigger significant changes. Pro-inflammatory cytokine IL-6 was also elevated significantly in immunized birds; a significant elevation of IL-10 was observed only in oral immunization with JOL1641 (P⩽0.05). JOL1641 immunized birds showed significant reduction of challenge bacterial-organ recovery as compared to JOL1087 and non-immunized birds. Collectively, our results revealed that immunization with the adjuvant-secreting S. Enteritidis confers protection against wild type SE challenge via induction of strong cell proliferative response, augmentation of CD3+CD4+: CD3+CD8+ T-cells ratio and enhancement of IFN-γ, IL-6 and IL-10 cytokine secretion.

Pathogenic traits of Salmonella Montevideo in experimental infections in vivo and in vitro

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.

Virulence Associated Genes-Deleted Salmonella Montevideo Is Attenuated, Highly Immunogenic and Confers Protection against Virulent Challenge in Chickens.

To construct a novel live vaccine against Salmonella enterica serovar Montevideo (SM) infection in chickens, two important bacterial regulatory genes, lon and cpxR, which are associated with invasion and virulence, were deleted from the wild type SM genome. Attenuated strains, JOL1625 (Δlon), JOL1597 (ΔcpxR), and JOL1599 (ΔlonΔcpxR) were thereby generated. Observations with scanning electron microscopy suggested that JOL1625 and JOL1599 cells showed increased ruffled surface which may be related to abundant extracellular polysaccharide (EPS) production. JOL1597 depicted milder ruffled surface but showed increased surface corrugation. ConA affinity-based fluorometric quantification and fluorescence microscopy revealed significant increases in EPS production in JOL1625 and JOL1599. Four weeks old chickens were used for safety and immunological studies. The mutants were not observed in feces beyond day 3 nor in spleen and cecum beyond day 7, whereas wild type SM was detected for at least 2 weeks in spleen and cecum. JOL1599 was further evaluated as a vaccine candidate. Chickens immunized with JOL1599 showed strong humoral responses, as indicated by systemic IgG and secretory IgA levels, as well as strong cell-mediated immune response, as indicated by increased lymphocyte proliferation. JOL1599-immunized groups also showed significant degree of protection against wild type challenge. Our results indicate that Δlon- and/or ΔcpxR-deleted SM exhibited EPS-enhanced immunogenicity and attenuation via reduced bacterial cell intracellular replication, conferred increased protection, and possess safety qualities favorable for effective vaccine development against virulent SM infections.

Development and trial of vaccines against Brucella

The search for ideal brucellosis vaccines remains active today. Currently, no licensed human or canine anti-brucellosis vaccines are available. In bovines, the most successful vaccine (S19) is only used in calves, as adult vaccination results in orchitis in male, prolonged infection, and possible abortion complications in pregnant female cattle. Another widely deployed vaccine (RB51) has a low protective efficacy. An ideal vaccine should exhibit a safe profile as well as enhance protective efficacy. However, currently available vaccines exhibit one or more major drawbacks. Smooth live attenuated vaccines suffer shortcomings such as residual virulence and serodiagnostic interference. Inactivated vaccines, in general, confer relatively low levels of protection. Recent developments to improve brucellosis vaccines include generation of knockout mutants by targeting genes involved in metabolism, virulence, and the lipopolysaccharide synthesis pathway, as well as generation of DNA vaccines, mucosal vaccines, and live vectored vaccines, have all produced varying degrees of success. Herein, we briefly review the bacteriology, pathogenesis, immunological implications, candidate vaccines, vaccinations, and models related to Brucella.

Corrigendum: Pathogenic traits of Salmonella Montevideo in experimental infections in vivo and in vitro

Scientific Reports 7: Article number: 46232; published online: 07 April 2017; updated: 04 May 2017 The Acknowledgements section in this Article has been omitted. It should read: “Acknowledgements This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MISP) (No.

A live attenuated mutant of Salmonella Montevideo triggers IL-6, IFN-γ and IL-12 cytokines that co-related with humoral and cellular immune responses required for reduction of challenge bacterial load in experimental chickens

A live attenuated Salmonella enterica serovar Montevideo (SM) mutant JOL1599 was constructed by deletion of virulence-associated genes. The protective efficacy and immune response profiles of chickens immunized with JOL1599 were investigated. Immunized chickens demonstrated significant increases in plasma IgG and lymphocyte proliferative responses (P≤0.05). Increased levels of IL-6, INF-γ, and IL-12 were also observed. Immunized birds strongly responded to infection by rapid stimulation of a CD4+ subset of T cells. Organ bacterial recovery assay revealed a significant reduction in the challenge strain among immunized birds. Multiple doses of JOL1599 enhanced the immune responses of the birds as revealed by ascending trends of the immunological profiles. These findings indicate that immunization of chickens with JOL1599 may provide protection against Salmonella Montevideo infection via induction of IL-6, INF-γ, and IL-12 protective cytokines, which in turn triggers conducive humoral and cell-mediated immune responses.

Immunization of guinea pigs with Salmonella delivered anti-Brucella formulation reduces organs bacterial load and mitigates histopathological consequences of Brucella abortus 544 challenge

With an objective to generate safe and effective anti-Brucella vaccine, an attenuated live Salmonella Typhimurium vector delivering heterologous Brucella immunogenic proteins SOD, Omp19, BLS, and PrpA formulated with purified Brucella abortus lipopolysaccharide was evaluated on a guinea pig model. This model represents high susceptibility to Brucella infections and showed similarities in reproducing human pathologies. On safety perspectives, the vaccine formulation induced no observable alterations on general health and histology of the vaccinated guinea pigs. Upon virulent strain 544 challenge, a protective index of 1.52 was observed based on differential splenic counts. Post-challenge histopathology revealed that Brucella induced microgranulomas and fatty degenerations were prominent in the organs of non-immunized animals as compared to immunized animals. With these findings, it is suggestive that this live Brucella-free vaccine formulation is safe and protective on a sensitive guinea pig model and may be suitable for further human-related vaccine trials.

Safety implication of Salmonella based Brucella vaccine candidate in mice and in vitro human cell culture

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 × 109 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 × 107 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.

Engineering of a rough auxotrophic mutant Salmonella Typhimurium for effective delivery

Live Salmonella vaccine vectors offer a remarkable platform for delivering immunogens and therapeutic molecules by mimicking natural intracellular infections; however, pre-existing anti-vector immunity can impede effective deployment. Measures to alleviate pre-existing immunity include the use of heterologous vectors, development of highly attenuated strain enabling greater payload, removal of major immunoreactive components from the vector, and/or augmentation of delivered antigens via increased presentation in antigen presenting cells. Here we report a Salmonella Typhimurium (ST) vector-JOL1800 that embodies these requisite properties. JOL1800 is a highly attenuated, auxotrophic, and O-antigen deficient rough-mutant strain. Heterologous bacterial and viral antigens were expressed and delivered using JOL1800 in mice, irrespective of the inoculation route successful inductions of the mucosal and systemic humoral responses were observed. Compared to smooth LPS vector delivery, we observed an increased fraction of delivered-antigen presenting dendritic cells and a higher frequency of delivered-antigen displayed per macrophage. Upon post-priming with JOL1800 delivery, efficacy of the delivery was minimally affected as indicated by insignificant decrease in colonization, humoral and cellular responses. Our results show that the generated vector is capable of remote antigen delivery, manifests higher antigen presentation, is Differentiating Infected from Vaccinated Animals (DIVA) capable, evades normal pre-existing immunity, and can be deployed for effective delivery.