Weiwei Yan

Immunogenicity and protective efficacy of recombinant Leptospira immunoglobulin-like protein B (rLigB) in a hamster challenge model

Leptospiral immunoglobulin-like protein (LigB) was truncated into conserved (LigBcon) and variable (varB1, varB2) fragments and expressed as GST/His-tag fusion proteins. Four-week-old hamsters were immunized with equal amounts of each fragment individually or combined in alum adjuvant at days 0 and 21 and subsequently challenged three weeks after the booster with 2.5 LD(50) live virulent Leptospira interrogans serovar Pomona. Our results demonstrate that immunization with LigB produced strong humoral immune responses as revealed by high titers against each fragment and significant enhancement in Th2 cytokines (IL-4, IL-10). A significant activation of CMI is revealed by enhanced proliferation of lymphocytes and up regulation of Th1 cytokines (IL-12p40, IFN-gamma) was also noted. Of the peptides studied, rLigBcon was able to impart maximum protection (71%), followed by rVarB1 (54%), whereas rVarB2 was not able to impart a significant level of protection (33%) against lethal infection as revealed by enhanced survival and reduced severity of histopathological lesions in vital organs (viz. kidney, liver, spleen) of the immunized animals. Moreover, concurrent administration of all three fragments significantly enhanced the protective efficacy of the vaccine (83%). Overall, our results clearly demonstrate that LigB has emerged as novel protective antigen that can be used in future subunit vaccines against leptospirosis.

Leptospira immunoglobulin-like protein A variable region (LigAvar) incorporated in liposomes and PLGA microspheres produces a robust immune response correlating to protective immunity.

Subunit vaccines are attractive as an intervention strategy against leptospirosis, an important zoonotic disease afflicting both humans and livestock. However, the success of subunit vaccines has been hampered by weak or short-term immunity and unavailability of nontoxic, potent adjuvants. In the present study, the variable region of recombinant Leptospira immunoglobulin like protein A (LigAvar) incorporated into conventional liposomes and PLGA microspheres produced robust immune responses that induced significant protection against virulent Leptospira interrogans serovar Pomona challenge in hamsters. Four-week-old hamsters were immunized subcutaneously with LigAvar incorporated into conventional liposomes or adsorbed on aluminum hydroxide (alum) and subsequently boosted after 3 weeks. Additionally, LigAvar incorporated into PLGA microspheres was evaluated as a single dose vaccine. All animals were challenged intraperitoneally 3 weeks after booster with a lethal dose (10 x MLD50) of virulent L. interrogans serovar Pomona. Animals were bled at various time points to evaluate antibody response, then sacrificed. Splenocytes were isolated and assayed for lymphocyte proliferation and cytokine profiles in response to recall antigen. Our results indicate that both liposomes and microspheres prove to be better adjuvants compared to conventional alum as revealed by enhanced antibody titers, lymphocyte proliferation and significant enhancement in both Th1(IL-12, IFN-gamma) and Th2 (IL-4, IL-10) cytokines. Moreover, LigAvar associated with liposomes and microspheres is able to provide better protection than LigAvar with alum as revealed by enhanced survival and reduced histopathological lesions in vital organs. Taken together, the data of the present study suggests that both liposomes and PLGA microspheres are promising adjuvants for use with future subunit vaccines for prevention of leptospirosis.

Identification and characterization of OmpA-like proteins as novel vaccine candidates for Leptospirosis

Leptospira is an important infectious Gram-negative bacterium causing Leptospirosis in mammals. Outer membrane proteins (OMPs) are key molecules in the interface between the cell and its environment. A group of putative leptospiral outer membrane proteins with an OmpA-like domain, comprising Lp0056, Lp0222, Lp3615, Lp3685, Lp4337 and Lbp328, were identified by bioinformatic methods and expressed as GST-tag fusion proteins. All these recombinant proteins were screened for immune-protective potential in hamsters challenged with Leptospira interrogans serovar Pomona. Out of these six proteins, three fusion proteins including Lp4337, Lp3685 and Lp0222 were found to be protective on the basis of survival. The immune response generated against these three recombinant proteins was evaluated on the basis of antibody production, lymphocyte proliferation and cytokine profiles in response to recall antigens whereas protective efficacy was assessed on the basis of survival and histopathological lesions in the vital organs (kidney, liver, and lung). Our results show that all three recombinant proteins generated strong immune responses, enhanced survival and reduced the severity of histopathological lesions. Of the proteins studied, Lp4337 generated the strongest immune response and was able to impart maximum protection (75%), followed by Lp3685 (58%), whereas Lp0222 generated lowest immune response correlating to protection (42%) against lethal infection of leptospira in the immunized animals. In contrast, control animals injected with PBS demonstrated low survival and had significant lesions. All these results clearly indicate that these three OmpA-like proteins may serve as novel vaccine candidates for leptospirosis.

Analysis of Ultra Low Genome Conservation in Clostridium difficile

Microarray-based comparative genome hybridisations (CGH) and genome sequencing of Clostridium difficile isolates have shown that the genomes of this species are highly variable. To further characterize their genome variation, we employed integration of data from CGH, genome sequencing and putative cellular pathways. Transcontinental strain comparison using CGH data confirmed the emergence of a human-specific hypervirulent cluster. However, there was no correlation between total toxin production and hypervirulent phenotype, indicating the possibility of involvement of additional factors towards hypervirulence. Calculation of C. difficile core and pan genome size using CGH and sequence data estimated that the core genome is composed of 947 to 1,033 genes and a pan genome comprised of 9,640 genes. The reconstruction, annotation and analysis of cellular pathways revealed highly conserved pathways despite large genome variation. However, few pathways such as tetrahydrofolate biosynthesis were found to be variable and could be contributing to adaptation towards virulence such as antibiotic resistance.

Evaluation of novel fusion proteins derived from extracellular matrix binding domains of LigB as vaccine candidates against leptospirosis in a hamster model.

Leptospira binds to host extracellular matrix (ECM) through surface exposed outer membrane proteins called adhesin in order to initiate infection. Of various adhesins present on the surface of the spirochete, Leptospira-immunoglobulin like proteins (Lig proteins) and LipL32 are most abundant, widely distributed among pathogenic serovars and well characterized. Various fragments of Lig proteins (Ligcon4, Ligcon4-7.5, LigBcen2) and C-terminus fragment of LipL32 all of that bind to host ECM were fused, expressed and purified in soluble form as fusion proteins. Four week hamsters were immunized subcutaneously with various fusion proteins emulsified in EMULSIGEN-D adjuvant and subsequently boosted at 3 weeks. The protective efficacy of these novel fusion proteins was evaluated against subsequent challenge with highly virulent L. interrogans serovar Pomona (MLD50-100). Our results indicate that fusion protein based vaccine induced significant protection against acute infection with respect to PBS-adjuvant vaccinated controls as revealed by enhanced survival and reduced pulmonary hemorrhage. Moreover, the protection mediated by these novel proteins was higher than that of conserved region of Lig protein (Ligcon, established protective antigen) and correlated to the level of antibodies. LipL32 failed to impart significant protection, however fusing its immunogenic C-terminus domain to Lig fragments slightly delayed the morbidity of the infected animals. Our results demonstrate that this novel strategy could be promising in developing effective subunit vaccine to combat this zoonotic infection.

A novel fibronectin type III module binding motif identified on C-terminus of Leptospira immunoglobulin-like protein, LigB.

Infection by pathogenic strains of Leptospira hinges on the pathogens ability to adhere to host cells via extracellular matrix such as fibronectin (Fn). Previously, the immunoglobulin-like domains of Leptospira Lig proteins were recognized as adhesins binding to N-terminal domain (NTD) and gelatin binding domain (GBD) of Fn. In this study, we identified another Fn-binding motif on the C-terminus of the Leptospira adhesin LigB (LigBCtv), residues 1708-1712 containing sequence LIPAD with a beta-strand and nascent helical structure. This motif binds to 15th type III modules (15F(3)) (K(D)=10.70 microM), and association (k(on)=600 M(-1)s(-1)) and dissociation (k(off)=0.0129 s(-1)) rate constants represents a slow binding kinetics in this interaction. Moreover, pretreatment of MDCK cells with LigB(1706-1716) blocked the binding of Leptospira by 39%, demonstrating a significant role of LigB(1706-1716) in cellular adhesion. These data indicate that the LIPAD residues (LigB(1708-1712)) of the Leptospira interrogans LigB protein bind 15F(3) of Fn at a novel binding site, and this interaction contributes to adhesion to host cells.

Leptosome-entrapped leptospiral antigens conferred significant higher levels of protection than those entrapped with PC-liposomes in a hamster model

We prepared novel liposomes from total polar lipids of non-pathogenic Leptospira biflexa serovar Potac (designated leptosomes) and evaluated their vaccine delivery/adjuvant potential with novel protective antigens (Lp0607, Lp1118 and Lp1454) of L. interrogans serovar Pomona in a hamster model. The immune response induced by three individual antigens and protective efficacy were evaluated and compared to those induced by same antigens entrapped with PC-liposomes and E. coli lipid liposomes (escheriosomes). Four-week-old hamsters were immunized subcutaneously twice at a 3-week interval, bled at various time points to evaluate antibody response and sacrificed to isolate splenocytes for lymphocyte proliferation and cytokine profiles in response to recall antigen. For the challenge test, 10x MLD(50) (modified lethal dose 50%) of virulent L. interrogans serovar Pomona were administered intraperitoneally. Our results demonstrate that leptosome are better adjuvant than PC-liposomes as revealed by enhanced long term antibody response, lymphocyte proliferation and significant enhancement of both Th1 (IFN-gamma) and Th2 (IL-4 and IL-10) cytokines. Additionally, leptosomes and escheriosomes induced significantly higher level of memory responses than PC-liposome did. Moreover, the novel leptosomal vaccine induced significantly higher levels of protection than those prepared with PC-liposomes as revealed by enhanced survival, reduced histopathological lesions in vital organs and reduced leptospiral load in kidneys. Taken together, the results of the present study clearly reveal that both leptosomes and escheriosomes have emerged as promising delivery vehicles/adjuvants that can be widely exploited with newly discovered antigens in future leptospira vaccines.

Immune response and prophylactic efficacy of smegmosomes in a hamster model of leptospirosis.

Leptospirosis is an important zoonotic disease worldwide. Subunit vaccines are an attractive intervention strategy against this disease, but potent, non-toxic adjuvants are necessary components to any effective vaccine. Among various adjuvant candidates, liposomes have garnered recent attention for their capacity as carriers of vaccines. In the present study we prepared novel liposomes using total polar lipids from the nonpathogenic bacterium, Mycobacterium smegmatis (designated smegmosomes). The potential for smegmosomes as a vaccine delivery/adjuvant system was evaluated with novel leptospira protective antigens (Lp0607, Lp1118, Lp1454) and compared with conventional aluminum hydroxide adjuvant (alum) in a hamster model of leptospirosis. Four-week-old hamsters were immunized subcutaneously twice at three weeks intervals and either bled at various time points to evaluate antibody responses, sacrificed to isolate splenocytes for lymphocyte proliferation and cytokine profiles in response to recall antigen, or challenged intraperitoneally with a modified lethal dose (10X MLD(50)) of virulent Leptospira interrogans serovar Pomona. Our results demonstrate that smegmosomes carrying antigens are better adjuvants than alum as revealed by enhanced and long term antibody response, lymphocyte proliferation and significant enhancement in both Th1 (IFN-gamma) and Th2 (IL-4, IL-10) cytokine production. Additionally, smegmosomes were found to induce memory responses that are significantly higher than those of alum. Above all, smegmosomes were observed to impart a significantly higher level of protection than alum as revealed by enhanced survival, reduced histopathological lesions and bacterial load in vital organs. Taken together, the data of the present study suggests that smegmosomes will serve well as a promising delivery vehicle/adjuvant system that can induce both Th1 and Th2 type immune responses and provide a novel tool in development of improved vaccines for leptospirosis and other infectious diseases.

Evaluation of a Mycobacterium avium subsp. paratuberculosis leuD Mutant as a Vaccine Candidate against Challenge in a Caprine Model

ABSTRACT Johnes disease (JD) is prevalent worldwide and has a significant impact on the global agricultural economy. In the present study, we evaluated the protective efficacy of a leuD (Δleud) mutant and gained insight into differential immune responses after challenge with virulent M. avium subsp. paratuberculosis in a caprine colonization model. The immune response and protective efficacy were compared with those of the killed vaccine Mycopar. In vitro stimulation of peripheral blood mononuclear cells with johnin purified protein derivative showed that Mycopar and ΔleuD generated similar levels of gamma interferon (IFN-γ) but significantly higher levels than unvaccinated and challenged phosphate-buffered saline controls. However, only with ΔleuD was the IFN-γ response maintained. Flow cytometric analysis showed that the increase in IFN-γ correlated with proliferation and activation (increased expression of CD25) of CD4, CD8, and γδT cells, but this response was significantly higher in ΔleuD-vaccinated animals at some time points after challenge. Both Mycopar and ΔleuD vaccines upregulated Th1/proinflammatory and Th17 cytokines and downregulated Th2/anti-inflammatory and regulatory cytokines at similar levels at almost all time points. However, significantly higher levels of IFN-γ (at weeks 26 and 30), interleukin-2 (IL-2; week 18), IL-1b (weeks 14 and 22), IL-17 (weeks 18 and 22), and IL-23 (week 18) and a significantly lower level of IL-10 (weeks 14 and 18) and transforming growth factor β (week 18) were detected in the ΔleuD-vaccinated group. Most importantly, ΔleuD elicited an immune response that significantly limited colonization of tissues compared to Mycopar upon challenge with wild-type M. avium subsp. paratuberculosis. In conclusion, the ΔleuD mutant is a promising vaccine candidate for development of a live attenuated vaccine for JD in ruminants.

The recombinant Lactococcus lactis oral vaccine induces protection against C. difficile spore challenge in a mouse model

Clostridium difficile infection (CDI) causes nosocomial antibiotic-associated diarrhea and colitis in the developed world. Two potent cytotoxins, toxin A (TcdA) and toxin B (TcdB) are the virulence factors of this disease and can be a good vaccine candidate against CDI. In the present study, we genetically engineered Lactococcus lactis to express the nontoxic, recombinant fragments derived from TcdA and TcdB C-terminal receptor binding domains (Tcd-AC and Tcd-BC) as an oral vaccine candidate. The immunogenicity of the genetically engineered L. lactis oral vaccine delivery system (animal groups LAC and LBC or the combination of both, LACBC) was compared with the recombinant TcdA and TcdB C-terminal receptor binding domain proteins (animal groups PAC and PBC or the combination of both, PACBC), which were expressed and purified from E. coli. After the C. difficile challenge, the control groups received PBS or engineered L. lactis with empty vector, showed severe diarrhea symptoms and died within 2-3 days. However, both the oral vaccine and recombinant protein vaccine groups had significantly lower mortalities, body weight decreases and histopathologic lesions than the control sham-vaccine groups (p<0.05) except group LBC which only had a 31% survival rate after the challenge. The data of post infection survival showed that an average of 86% of animals survived in groups PAC and PACBC, 75% of animals survived in group LACBC, and 65% of animals survived in group LAC. All of the vaccinated animals produced higher titers of both IgG and IgA than the control groups (p<0.05), and the antibodies were able to neutralize the cytopathic effect of toxins in vitro. The results of this study indicate that there is a potential to use L. lactis as a delivery system to develop a cost effective oral vaccine against CDI.