Partial protection induced by <i>Salmonella</i> based <i>Brucella</i> vaccine candidate in pregnant guinea pigs

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>

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

Lalsiamthara, Jonathan,Lee, John Hwa Elsevier 2018 Veterinary immunology and immunopathology Vol.195 No.-

<P><B>Abstract</B></P> <P>With an objective to generate safe and effective anti-<I>Brucella</I> vaccine, an attenuated live <I>Salmonella</I> Typhimurium vector delivering heterologous <I>Brucella</I> immunogenic proteins SOD, Omp19, BLS, and PrpA formulated with purified <I>Brucella abortus</I> lipopolysaccharide was evaluated on a guinea pig model. This model represents high susceptibility to <I>Brucella</I> 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 <I>Brucella</I> 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 <I>Brucella</I>-free vaccine formulation is safe and protective on a sensitive guinea pig model and may be suitable for further human-related vaccine trials.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Assessment of <I>Salmonella</I> vectored anti-<I>Brucella</I> vaccine formulated with lipopolysaccharide in guinea pigs. </LI> <LI> The formulation exhibited safety on general health, postmortem, and histological standpoints. </LI> <LI> The formulation reduces organ challenge bacterial loads and mitigations of histopathological consequences. </LI> </UL> </P>

A live attenuated <i>Salmonella</i> Enteritidis secreting detoxified heat labile toxin enhances mucosal immunity and confers protection against wild-type challenge in chickens

Lalsiamthara, Jonathan,Kamble, Nitin Machindra,Lee, John Hwa BioMed Central 2016 Veterinary research Vol.47 No.-

<P>A live attenuated <I>Salmonella</I> Enteritidis (SE) capable of constitutively secreting detoxified double mutant <I>Escherichia coli</I> heat labile toxin (dmLT) was developed. The biologically adjuvanted strain was generated via transformation of a highly immunogenic SE JOL1087 with a plasmid encoding dmLT gene cassette; the resultant strain was designated JOL1641. A balanced-lethal host-vector system stably maintained the plasmid via auxotrophic host complementation with a plasmid encoded aspartate semialdehyde dehydrogenase (<I>asd</I>) gene. Characterization by western blot assay revealed the dmLT subunit proteins in culture supernatants of JOL1641. For the investigation of adjuvanticity and protective efficacy, chickens were immunized via oral or intramuscular routes with PBS, JOL1087 and JOL1641. Birds immunized with JOL1641 showed significant (<I>P</I> ≤ 0.05) increases in intestinal SIgA production at the 1<SUP>st</SUP> and 2<SUP>nd</SUP> weeks post-immunization via oral and intramuscular routes, respectively. Interestingly, while both strains showed significant splenic protection via intramuscular immunization, JOL1641 outperformed JOL1087 upon oral immunization. Oral immunization of birds with JOL1641 significantly reduced splenic bacterial counts. The reduction in bacterial counts may be correlated with an adjuvant effect of dmLT that increases SIgA secretion in the intestines of immunized birds. The inclusion of detoxified dmLT in the strain did not cause adverse reactions to birds, nor did it extend the period of bacterial fecal shedding. In conclusion, we report here that dmLT could be biologically incorporated in the secretion system of a live attenuated <I>Salmonella</I>-based vaccine, and that this construction is safe and could enhance mucosal immunity, and protect immunized birds against wild-type challenge.</P>

Safety implication of <i>Salmonella</i> based <i>Brucella</i> vaccine candidate in mice and in vitro human cell culture

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>

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

Lalsiamthara, J.,Lee, J.H. Elsevier Scientific Pub. Co 2017 Veterinary microbiology Vol.205 No.-

<P>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 naive or vector only immunized mice. Modulatory effects of rB and LPS on production of interleukin (IL)-4, IL-12, and interferon-gamma 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.</P>

Immuno-profiles of BALB/c mice inoculated with <i>Salmonella</i> vector delivering B-cell mitogen hydroxyproline epimerase

Lalsiamthara, Jonathan,Lee, John Hwa Elsevier 2018 Molecular immunology Vol.95 No.-

<P>The enzyme 4-hydroxyproline 2-epimerase (PrpA) involves in modulation of host immunity and is also reported as a potent B-cell mitogen. Live attenuated Salmonella Typhimurium (ST) vector constitutively expressing heterologous Brucella abortus PrpA protein (ST-PrpA) was inoculated in BALB/c mice in order to investigate the influence of the enzyme, on safety aspects, humoral and cellular immunity as well as protective efficacies against wild type challenges. No aggravation of morbidity was observed upon mice inoculation of ST-PrpA. Immunized mice showed significantly quicker anti-Salmonella IgG responses as compared to ST only immunization. This finding is in congruency with the increase production evident in in vitro pulsed mice splenocytes. Increase protection against Salmonella challenge was also observed. These findings suggest that PrpA can be used as a protein adjuvant in a live Salmonella delivery system, in order to increase humoral responses effectively without major interference on the cell mediated immunity.</P>

Effect of immunization routes and protective efficacy of Brucella antigens delivered via Salmonella vector vaccine

Jonathan Lalsiamthara,Gayeon Won,John Hwa Lee 대한수의학회 2018 Journal of Veterinary Science Vol.19 No.3

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.

Development and trial of vaccines against Brucella

Jonathan Lalsiamthara,이존화 대한수의학회 2017 Journal of Veterinary Science Vol.18 No.-

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.

Intranasally administered anti-<i>Brucella</i> subunit vaccine formulation induces protective immune responses against nasal <i>Brucella</i> challenge

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>