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Senevirathne, Amal,Hewawaduge, Chamith,Hajam, Irshad A.,Lalsiamthara, Jonathan,Lee, John Hwa Elsevier 2019 Veterinary microbiology Vol.228 No.-
<P><B>Abstract</B></P> <P>The present study was aimed to develop a safe and effective anti-<I>Brucella</I> subunit vaccine for mucosal protection against the respiratory exposure of <I>Brucella</I> infection. A chitosan-based <I>Brucella</I> nasal vaccine (BNV) was formulated using well-known <I>Brucella</I> immunogens, sodC, omp19, BLS and PrpA and tested against nasal <I>Brucella</I> challenge in BALB/c mice. The mice were intra-nasally vaccinated with sterile phosphate buffer saline (PBS), BNV or BNV plus <I>Brucella</I> LPS, and humoral (systemic IgG and mucosal IgA) and cell-mediated immune responses were analyzed. Results showed that mice vaccinated with either BNV or BNV plus LPS elicited significantly (p < 0.05) high IgG and IgA responses compared to the PBS control. The IgG responses were significantly (p < 0.05) higher than IgA levels, which showed almost comparable levels observed in either intestines or in lungs. Furthermore, the IgG and IgA responses against each individual component of the BNV formulation indicated that omp19 induced highest levels of both IgG and IgA levels than the other constituents of BNV formulation. Upon re-stimulation of the splenocytes with <I>Brucella</I> whole cell lysate, significantly (p < 0.05) high IFN-γ levels, lymphocyte proliferation, and CD4<SUP>+</SUP> T cell responses were observed in mice vaccinated with BNV or BNV plus LPS. Upon sub-lethal nasal challenge with wild-type <I>Brucella strain</I>, vaccinated mice showed significant reduction of <I>Brucella</I> recovery in lungs and spleen compared to the PBS control. This study indicates that BNV formulation with or without <I>Brucella</I> LPS efficiently induced humoral and cell-mediated immune responses and conferred significant protection against the sub-lethal <I>Brucella</I> challenge.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Development of a safe and effective mucosal vaccine for <I>Brucellosis</I>. </LI> <LI> A chitosan-based vaccine was formulated using <I>Brucella</I> immunogens, sodC, omp19, BLS and PrpA. </LI> <LI> Highly conserved Brucella antigens was able to give significant protection against the nasal challenge. </LI> </UL> </P>
Senevirathne, Amal,Hewawaduge, Chamith,Lee, John Hwa Elsevier 2019 Veterinary immunology and immunopathology Vol.209 No.-
<P><B>Abstract</B></P> <P>We demonstrated the use of attenuated <I>Salmonella</I> strains secreting <I>Brucella</I> antigens SodC, Omp19, BLS, and PrpA as live vaccine candidates against <I>Brucella abortus</I> infection and presented their cross-protection against <I>Salmonella</I> infections using a BALB/c mice model. Here, a single immunization with each individual strain was capable of establishing significantly high (p < 0.05) <I>Brucella</I>-specific systemic immunoglobulin (Ig)G and secretory IgA (sIgA) responses compared to control mice. Upon stimulation of the splenocytes harvested from immunized mice with the respective antigens SodC, Omp19, BLS, and PrpA, significant increases in splenocyte proliferative responses against all four antigens versus PBS and vector controls were observed (p < 0.05). Additionally, interferon-γ and interleukin-4 secretion clearly demonstrated an uplift of these cytokines in all four strains upon immunization compared to the control groups. However, a significantly high response was noted in the mice groups immunized with <I>Salmonella</I> secreting SodC and Omp19 only. Upon virulent <I>Brucella abortus</I> 544 challenge, all four antigens presented a significantly high protection index (PI) in the spleen, as follows: 0.85 for SodC; 0.96 for Omp19; 0.6 for BLS; and 0.66 for PrpA. In contrast, in the liver, the same antigens resulted in PI values of 1.37, 1.14, 1.12, and 1.81, respectively. Immunological profiling of immunized mice against <I>Salmonella</I>-specific immune responses also showed significant elicitation of both humoral and cell-mediated immune responses as measured by IgG, sIgA, splenocyte proliferation, and cytokine induction. In addition, full protection against virulent <I>Salmonella</I> challenge was shown with no mortality in immunized mice, whereas 100% (8/8) mortality was observed in control mice over a two-week post-<I>Salmonella</I> challenge. In conclusion, we show that the live attenuated <I>Salmonella</I> delivering <I>Brucella</I> protective antigens may efficiently confer dual protection against both brucellosis and salmonellosis in immunized mice.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Development of a safe and effective vaccine candidate against Brucellosis. </LI> <LI> <I>Salmonella</I> delivering <I>Brucella</I> antigens sodC, Omp19, BLS and PrpA were evaluated. </LI> <LI> Immunization with attenuated <I>Salmonella</I> conferred protection against virulent <I>Brucella</I> &<I>Salmonella</I> challenge. </LI> </UL> </P>
Lalsiamthara, Jonathan,Senevirathne, Amal,Lee, John Hwa Elsevier 2019 Vaccine Vol.37 No.7
<P><B>Abstract</B></P> <P>Residual virulence is a major drawback in current <I>Brucella</I> vaccines. Live vaccines induce abortions in pregnant animals. Hence, a novel anti-<I>Brucella</I> vaccine was developed utilizing rough <I>Salmonella</I> delivering four <I>Brucella</I> antigens. Safety implications during pregnancy, humoral immune responses, and protective efficacy against wild type <I>Brucella</I> was investigated in guinea pig model. The vaccine did not induce abortions or severe complications in pregnant guinea pigs when administered 4 × 10<SUP>8</SUP> CFU via intraperitoneal route. Systemic IgG determination against antigen components reveals induction of immunity via the <I>Salmonella</I> delivery. Protection efficacy against abortions was 33.3% (2/6) when midterm sow challenged with virulent <I>Brucella</I> 544 strain while none was protected in control group. Lower <I>Brucella</I> recovery in spleen and liver and reduced histopathological burden were also noticed. Although abortion induced by <I>Brucella</I> challenge was not completely prevented, the vaccine candidate may perform better with optimization of vaccination such as inoculation dose optimization.</P>
Lalsiamthara, Jonathan,Senevirathne, Amal,So, Mi Young,Lee, John Hwa Elsevier 2018 Vaccine Vol.36 No.14
<P><B>Abstract</B></P> <P>An anti-<I>Brucella</I> vaccine candidate comprising rough <I>Salmonella</I> vector delivering <I>Brucella</I> antigens was developed. This system provides a platform for live <I>Brucella</I>-free vaccine development as it can mimic active-intracellular infection of <I>Brucella</I> organism. Exploiting this phenomenon thus provides significant protection at a single dose and also re-assured the safety. To date, no human anti-<I>Brucella</I> 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<SUP>9</SUP> 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) <I>Salmonella</I> positive control strain induced 100% mortality at 8 × 10<SUP>7</SUP> 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.</P>
Sungwoo Park,Eunseok Cho,Amal Senevirathne,Hak-Jae Chung,Seungmin Ha,Chae-Hyun Kim,Seogjin Kang,John Hwa Lee 대한수의학회 2024 Journal of Veterinary Science Vol.25 No.1
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 × 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.
Chandrapala, Dilini,Kim, Kyumson,Choi, Younho,Senevirathne, Amal,Kang, Dong-Hyun,Ryu, Sangryeol,Kim, Kwang-Pyo American Society for Microbiology 2014 Infection and immunity Vol.82 No.5
<P><I>Cronobacter sakazakii</I> is an opportunistic pathogen that causes neonatal meningitis and necrotizing enterocolitis. Its interaction with intestinal epithelium is important in the pathogenesis of enteric infections. In this study, we investigated the involvement of the <I>inv</I> gene in the virulence of <I>C. sakazakii</I> ATCC 29544 <I>in vitro</I> and <I>in vivo</I>. Sequence analysis of <I>C. sakazakii</I> ATCC 29544 <I>inv</I> revealed that it is different from other <I>C. sakazakii</I> isolates. In various cell culture models, an Δ<I>inv</I> deletion mutant showed significantly lowered invasion efficiency, which was restored upon genetic complementation. Studying invasion potentials using tight-junction-disrupted Caco-2 cells suggested that the <I>inv</I> gene product mediates basolateral invasion of <I>C. sakazakii</I> ATCC 29544. In addition, comparison of invasion potentials of double mutant (Δ<I>ompA Δinv</I>) and single mutants (Δ<I>ompA</I> and Δ<I>inv</I>) provided evidence for an additive effect of the two putative outer membrane proteins. Finally, the importance of <I>inv</I> and the additive effect of putative Inv and OmpA were also proven in an <I>in vivo</I> rat pup model. This report is the first to demonstrate two proteins working synergistically <I>in vitro</I>, as well as <I>in vivo</I> in <I>C. sakazakii</I> pathogenesis.</P>