Protection of cattle against a natural infection of Fasciola hepatica by vaccination with recombinant cathepsin L1 (rFhCL1)

The liver fluke, Fasciola hepatica causes liver fluke disease, or fasciolosis, in ruminants such as cattle and sheep. An effective vaccine against the helminth parasite is essential to reduce our reliance on anthelmintics, particularly in light of frequent reports of resistance to some frontline drugs. In our study, Friesian cattle (13 per group) were vaccinated with recombinant F. hepatica cathepsin L1 protease (rFhCL1) formulated in mineral-oil based adjuvants, Montanide™ ISA 70VG and ISA 206VG. Following vaccination the animals were exposed to fluke-contaminated pastures for 13 weeks. At slaughter, there was a significant reduction in fluke burden of 48.2% in the cattle in both vaccinated groups, relative to the control non-vaccinated group, at p ≤ 0.05. All vaccinated animals showed a sharp rise in total IgG levels to rFhCL1 post-vaccination which was maintained over the course of the 13-week challenge infection and was significantly higher than levels reached in the control group. Arginase levels in the macrophages of vaccinated cattle were significantly lower than those of the control cattle, indicating that the parasite-induced alternative-activation of the macrophages was altered by vaccination. The data demonstrate the potential for recombinant FhCL1 vaccine in controlling fasciolosis in cattle under field conditions.     

The recombinant gut-associated M17 leucine aminopeptidase in combination with different adjuvants confers a high level of protection against Fasciola hepatica infection in sheep

Fasciola hepatica M17 leucine aminopeptidase (FhLAP) is thought to play a role in catabolizing peptides generated by the concerted activity of gut-associated endopeptidases on host polypeptides, thus releasing amino acids to be used in protein anabolism. In this study, a recombinant functional form of this homo hexameric metallopeptidase produced in Escherichia coli was used in combination with adjuvants of different types in a vaccination trial in Corriedale sheep against experimental challenge with F. hepatica metacercariae. The experimental assay consisted of 6 groups of 10 animals; 5 of the groups (1-5) were subcutaneously inoculated at weeks 0 and 4 with100 μg of rFhLAP mixed with Freund's complete plus incomplete adjuvant (group 1), Alum (group 2), Adyuvac 50 (group 3), DEAE-D (group 4) and Ribi (group 5); the adjuvant control group (group 6) received Freund's adjuvant. Two weeks after the booster, the sheep were orally challenged with 200 metacercariae. Immunization with rFhLAP induced significant reduction in fluke burdens in all vaccinated groups: 83.8% in the Freund's group, 86.7% in the Alum group, 74.4% in the Adyuvac 50 group, 49.8% in the Ribi group and 49.5% in the DEAE-D group compared to the adjuvant control group. Morphometric analysis of recovered liver flukes showed no significant size modifications in the different vaccination groups. All vaccine preparations elicited specific IgG, IgG1 and IgG2 responses. This study shows that a liver fluke vaccine based on rFhLAP combined with different adjuvants significantly reduced worm burden in a ruminant species that was high in animals that received the enzyme along with the commercially approved adjuvants Alum and Adyuvac 50.     

Immunization of mice with a novel recombinant molecular chaperon confers protection against Brucella melitensis infection

Brucella spp. are zoonotic Gram-negative intracellular pathogens with the ability to survive and replicate in phagocytes. It has been shown that bacterial proteins expressed abundantly in this niche are stress-related proteins capable of triggering effective immune responses. BMEI1549 is a molecular chaperone designated DnaK that is expressed under stress conditions and helps to prevent formation of protein aggregates. In order to study the potential of DnaK as a prospective Brucella subunit vaccine, immunogenicity and protective efficacy of recombinant DnaK from Brucella melitensis was evaluated in BALB/c mice. The dnak gene was cloned, expressed in Escherichia coli, and the resulting recombinant protein used as subunit vaccine. DnaK-immunized mice showed a strong lymphocyte proliferative response to in vitro antigen stimulation. Although comparable levels of antigen-specific IgG2a and IgG1 were observed in immunized mice, high amounts of IFN-γ, IL-12 and IL-6, no detectable level of IL-4 and very low levels of IL-10 and IL-5 were produced by splenocytes of vaccinated mice suggesting induction of a Th1 dominant immune response by DnaK. Compared to control animals, mice vaccinated with DnaK exhibited a significant degree of protection against subsequent Brucella infection (p < 0.001), albeit this protection was less than the protection conferred by Rev.1 (p < 0.05). A further increase in protection was observed, when DnaK was combined with recombinant Omp31. Notably, this combination, as opposed to each component alone, induced statistically similar level of protection as induced by Rev.1 suggesting that DnaK could be viewed as a promising candidate for the development of a subunit vaccine against brucellosis.     

Toxoplasma gondii microneme protein 6 (MIC6) is a potential vaccine candidate against toxoplasmosis in mice

Infection with the intracellular protozoan parasite Toxoplasma gondii causes serious public health problems and is of great economic importance worldwide. Microneme proteins which are responsible for adhesion and invasion have been implicated as vaccine candidates. In this study, we constructed a DNA vaccine expressing microneme protein 6 (MIC6) of T. gondii, and evaluated the immune response it induced in Kunming mice. The gene sequence encoding MIC6 was inserted into the eukaryotic expression vector pVAXI. We immunized Kunming mice intramuscularly. After immunization, we evaluated the immune response using lymphoproliferative assay, cytokine and antibody measurements, and the survival times of mice challenged lethally. The results showed that the group immunized with pVAX-MIC6 developed a high level of specific antibody responses against T. gondii lysate antigen (TLA), a strong lymphoproliferative response, and significant levels of IFN-γ, IL-2, IL-4 and IL-10 production, compared with the other groups immunized with empty plasmid or phosphate-buffered saline, respectively. These results demonstrate that pVAX-MIC6 induces significant humoral and cellular Th1 immune responses. After lethal challenge, the mice immunized with the pVAX-MIC6 showed an increased survival time (13.3 ± 1.2 days) compared with control mice died within 7 days of challenge. Our data demonstrate, for the first time, that MIC6 triggered a strong humoral and cellular response against T. gondii, and that the antigen is a potential vaccine candidate against toxoplasmosis, worth further development.     

Evaluation of a multisubunit recombinant polymorphic membrane protein and major outer membrane protein T cell vaccine against Chlamydia muridarum genital infection in three strains of mice

An efficacious vaccine is needed to control Chlamydia trachomatis infection. In the murine model of Chlamydia muridarum genital infection, multifunctional mucosal CD4 T cells are the foundation for protective immunity, with antibody playing a secondary role. We previously identified four Chlamydia outer membrane proteins (PmpE, PmpF, PmpG and PmpH) as CD4 T cell vaccine candidates using a dendritic cell-based immunoproteomic approach. We also demonstrated that these four polymorphic membrane proteins (Pmps) individually conferred protection as measured by accelerated clearance of Chlamydia infection in the C57BL/6 murine genital tract model. The major outer membrane protein, MOMP is also a well-studied protective vaccine antigen in this system. In the current study, we tested immunogenicity and protection of a multisubunit recombinant protein vaccine consisting of the four Pmps (PmpEFGH) with or without the major outer membrane protein (MOMP) formulated with a Th1 polarizing adjuvant in C57BL/6, Balb/c and C3H mice. We found that C57BL/6 mice vaccinated with PmpEFGH + MOMP elicited more robust cellular immune responses than mice immunized with individual protein antigens. Pmps elicited more variable cellular immune responses than MOMP among the three strains of mice. The combination vaccine accelerated clearance in the three strains of mice although at different rates. We conclude that the recombinant outer membrane protein combination constitutes a promising first generation Chlamydia vaccine construct that should provide broad immunogenicity in an outbred population.     

Evaluation of hepatic changes and local and systemic immune responses in goats immunized with recombinant Peroxiredoxin (Prx) and challenged with Fasciola hepatica

Protection against Fasciola hepatica in goats immunized with Peroxiredoxin (Prx) was assessed. The experimental trial consisted of three groups of seven animals; group 1 were unimmunized and uninfected, group 2 were immunized with adjuvant only and group 3 were immunized with recombinant Prx in adjuvant (immunized and infected). Immunization with Prx in Quil A adjuvant, group 3, induced a reduction in fluke burden of 33.04% when compared to adjuvant control, group 2, although this difference was not significant. The hepatic gross and microscopical morphometric study revealed lower damage in the Prx-immunized compared to group 2 (p < 0.05). Furthermore, immunohistochemical studies revealed that the Prx-immunized group exhibited reduced infiltration of CD4⁺, CD8⁺, IFN-γ⁺ and TCR⁺ (p < 0.05); and CD2⁺ and IL-4⁺ (p < 0.001) in hepatic lesions. Levels of anti-Prx serum IgG in group 3 showed a significant increase at the 4th week after challenge infection compared with group 2 (p < 0.0001). This is the first report of ruminant immunization with recombinant Prx of F. hepatica. The study shows that this vaccine significantly reduces hepatic damage and encourages further studies to improve the vaccine efficacy.     

Immune responses of mice against recombinant bovine herpesvirus 5 glycoprotein D

Glycoprotein D (gD) is essential for attachment and penetration of Bovine herpesvirus 5 (BoHV-5) into permissive cells, and is a major target of the host immune system, inducing strong humoral and cellular immune responses. The aim of this study was to evaluate in mice the immunogenicity of recombinant BoHV-5 gD (rgD5) expressed in Pichia pastoris. Vaccines formulated with rgD5 alone or adjuvanted with Montanide 50 ISA V2; Emulsigen or Emulsigen-DDA was administered intramuscularly or subcutaneously. Almost all formulations stimulated a humoral immune response after the first inoculation. The only exception was observed when the rgD5 was administered subcutaneously without adjuvant, in this case, the antibodies were observed after three doses. Higher titers of neutralizing antibodies were obtained with the three oil-based adjuvant formulations when compared to non-adjuvanted vaccine formulations. The rgD5 vaccine stimulated high mRNA expression levels of Th1 (INF-γ) and pro-inflammatory cytokines (IL-17, GM-CSF). The results demonstrated that the recombinant gD from BoHV-5 conserved important epitopes for viral neutralization from native BoHV-5 gD and was able to elicit mixed Th1/Th2 immune response in mice.     

PCR-RFLP Analysis of 28 SrDNA for Specification of Fasciola gigantica (Cobbold, 1855) in the Infected Lymnaea auricularia (Linnaeus, 1785) Snails from Northwestern Iran

Background

Fasciolosis in livestock is a crucial concern in the globe, mainly due to its impact on human health. The aim of this study was to determine the rate of infection with Fasciola gigantica (Cobbold, 1855) larvae in the field-collected snails of Lymnaea auricularia (Linnaeus, 1785) from northwestern Iran using a molecular approach.

Methods

A total of 6,759 pond snails were collected from 28 freshwater bodies in West Azarbaijan. PCR was performed to amplify a 618-bp fragment of the nuclear 28 SrRNA gene of Fasciola. The PCR products were digested by AvaII restriction enzyme to create restriction fragment length polymorphism (RFLP) patterns specific for the detection of F. gigantica.

Results

Of the total collected snails 496 (7.34 %) were L. auricularia, among which 4.64% (23 out of 496) were infected with a Fasciola species according to the PCR analysis. Only 2.22% (11 out of 496) of the infected snails were from the mountainous areas. The highest Fasciola infection rate recorded in the snails of a single site was 1.81% (9 out of 496 snails). Based on the RFLP pattern, F. gigantica accounted for 2.42% of the infection rates in the study sites.

Conclusion

Application of PCR-RFLP was proven to be a useful approach for valid and robust detection of the infection with F. gigantica in its intermediate host snails. These findings may therefore be applicable for establishment of the control programs against dissemination of the infection in different regions.     

Identification and characterization of OmpL as a potential vaccine candidate for immune-protection against salmonellosis in mice

Salmonella is gram-negative flagellated bacteria that can cause food and waterborne gastroenteritis and typhoid fever in humans. Despite the importance of Salmonella infections in human and animal health, the target antigens of Salmonella-specific immunity remain poorly defined, the effectiveness of the currently available vaccines is also limited. Outer membrane proteins (OMPs) of Salmonella have been considered possible candidates for conferring protection against salmonellosis. OMPs interface the cell with the environment, thus representing important potential vaccine candidate for pathogen infection. We showed that the outer membrane porin L (OmpL) is a transmembrane β barrel (TMBB) protein, which forms 12 transmembrane β-strands. OmpL of S. Typhimurium is highly immunogenic, OmpL could evoke humoral and cell-mediated immune responses, and confer 100% protection to immunized mice against challenge with very high doses of S. Typhimurium. Besides, very efficient clearance of bacteria from the reticuloendothelial systems of immunized mice was seen. The homology search further revealed that OmpL is widely distributed and conserved, homologous proteins were identified in S. Typhi and Paratyphi by RT-PCR and western blot. We also found that anti-rOmpL serum harber a high bactericidal activity for Salmonella serovars tested in this study. Therefore, OmpL provide a promising target for the development of a candidate vaccine against Salmonella infection.     

Immunization with recombinant Lactobacillus casei strains producing K99, K88 fimbrial protein protects mice against enterotoxigenic Escherichia coli

To exploit a safe and effective vaccine for the prevention against K99 or K88 infections of enterotoxigenic Escherichia coli (ETEC), we have developed a mucosal delivery vehicle based on Lactobacillus casei CICC 6105 using poly-γ-glutamate synthetase A (PgsA) as an anchoring matrix. To evaluate the immunization effect of the recombinant strains (harboring plasmids pLA-K99-K88-LTB, pLA-K99, and pLA-K88), anti-ETEC K99 or K88 antibody responses, T-cell proliferation, and cytokines by intracellular staining (ICS) were investigated after specific pathogen-free (SPF) C57BL/6 mice orally inoculated with these recombinant strains. After oral vaccination into C57BL/6 mice, all recombinant strains were proved to be immunogenic and able to elicit high levels of mucosal immunoglobulin A (IgA) titers in bronchoalveolar lavage fluids, intestinal fluids and prominent systemic immunoglobulin G and IgG subclasses (IgG1, IgG2b, and IgG2a) responses in sera. Using the T-cell proliferation assay, the stimulation index (SI) of groups immunized with pLA-K99/L. casei and pLA-K88/L. casei reached to 2.73 and 2.64, respectively, versus 2.56 in a group immunized with pLA-K99-K88-LTB/L. casei. A detailed analysis of the cell-mediated immune responses by ICS showed the number of specific CD8(+) T cells expressing cytokines (IFN-γ, TNF-α, and IL-2) and granule-associated proteins (CD107a) was higher than that of specific CD4(+) T cells secreted by immune spleen cells upon restimulation in vitro with peptides. Next, the results showed that DCs activated in vitro with recombinant L. casei enhance specific T-cell proliferation and promote T cells to produce both Th1 and Th2 cytokines. More than 80% of the vaccinated mice were protected after challenge with a lethal dose of standard strains C83912 and C83902. These results demonstrate that recombinant L. casei can induce specific humoral and mucosal antibodies and cellular immune response against protective antigens upon oral administration.     

Immunological responses induced by a DNA vaccine expressing RON4 and by immunogenic recombinant protein RON4 failed to protect mice against chronic toxoplasmosis

The development of an effective vaccine against Toxoplasma gondii infection is an important issue due to the seriousness of the related public health problems, and the economic importance of this parasitic disease worldwide. Rhoptry neck proteins (RONs) are components of the moving junction macromolecular complex formed during invasion. The aim of this study was to evaluate the vaccine potential of RON4 using two vaccination strategies: DNA vaccination by the intramuscular route, and recombinant protein vaccination by the nasal route. We produced recombinant RON4 protein (RON4S2) using the Schneider insect cells expression system, and validated its antigenicity and immunogenicity. We also constructed optimized plasmids encoding full length RON4 (pRON4), or only the N-terminal (pNRON4), or the C-terminal part (pCRON4) of RON4. CBA/J mice immunized with pRON4, pNRON4 or pCRON4 plus a plasmid encoding the granulocyte-macrophage-colony-stimulating factor showed high IgG titers against rRON4S2. Mice immunized by the nasal route with rRON4S2 plus cholera toxin exhibited low levels of anti-RON4S2 IgG antibodies, and no intestinal IgA antibodies specific to RON4 were detected. Both DNA and protein vaccination generated a mixed Th1/Th2 response polarized towards the IgG1 antibody isotype. Both DNA and protein vaccination primed CD4+ T cells in vivo. In addition to the production of IFN-γ, and IL-2, Il-10 and IL-5 were also produced by the spleen cells of the immunized mice stimulated with RON4S2, suggesting that a mixed Th1/Th2 type immune response occurred in all the immunized groups. No cytokine was detectable in stimulated mesenteric lymph nodes from mice immunized by the nasal route. Immune responses were induced by both DNA and protein vaccination, but failed to protect the mice against a subsequent oral challenge with T. gondii cysts. In conclusion, strategies designed to enhance the immunogenicity and to redirect the cellular response towards a Th1 type response against RON4 could lead to more encouraging results.     

Protection against Fasciola gigantica infection in mice by vaccination with recombinant juvenile-specific cathepsin L

Fasciola gigantica cathepsin L1H (FgCatL1H) is one of the major cathepsin L released by juveniles of F. gigantica to aid in the invasion of host's tissues. Due to its high sequence similarity with other cathepsin L (CatL) isoforms of late stage F. gigantica, it was considered to be a good vaccine candidate that can block all CatL-mediated protease activities and affect juveniles as well as adult parasites. In this study, recombinant proFgCatL1H protein expressed in yeast, Pichia pastoris, system was mixed with Freund's adjuvants and used to subcutaneously immunize mice that were later challenged with metacercariae of F. gigantica. The percentage of worm protection in the rproFgCatL1H-vaccinated mice compared to the non-immunized and adjuvant control mice were approximately 62.7% and 66.1%, respectively. Anti-rproFgCatL1H antisera collected from vaccinated mice reacted specifically with rproFgCatL1H and other cathepsin L isoforms of F. gigantica, but the antibodies did not cross react with antigens from other trematode and nematode parasites, including Eurytrema pancreaticum, Opisthorchis viverrini, Fischoederius cobboldi, Cotylophoron cotylophorum, Gigantocotyle explanatum, Paramphistomum cervi, and Setaria labiato-papillosa. The levels of IgG1 and IgG2a in mouse sera increased significantly at two weeks after immunization and were highest during the sixth to eighth weeks after immunization. The IgG1 level was higher than IgG2a at all periods of immunization, implicating the dominance of the Th2 response. The levels of IgG1 and IgG2a in the immune sera were shown to be strongly correlated with the numbers of worm recovery, and the correlation coefficient was higher for IgG1. The levels of serum aspartate aminotransferase and alanine transaminase were significantly lower in the sera of rproFgCatL1H-vaccinated mice than in the infected control mice indicating a lower degree of liver damage. This study demonstrated a high potential of FgCatL1H vaccine, and its efficacy is currently being studied in the larger economic animals.     

Protection against an intranasal challenge by vaccines formulated with native and recombinant preparations of the Chlamydia trachomatis major outer membrane protein

To compare the ability of a native and a recombinant preparation of the major outer membrane protein of Chlamydia trachomatis mouse pneumonitis (MoPn; Ct-nMOMP and Ct-rMOMP) to protect against an intranasal (i.n.) challenge, BALB/c mice were vaccinated by the intramuscular (i.m.) and subcutaneous (s.c.) routes using CpG-1826 and Montanide ISA 720 as adjuvants. Animals inoculated i.n. with live elementary bodies (EB) of Chlamydia served as a positive control. Negative control groups were immunized with either Neisseria gonorrhoeae recombinant porin B (Ng-rPorB) or with minimal essential medium (MEM-0). Mice immunized with Ct-rMOMP, Ct-nMOMP and EB developed a strong immune response as shown by high levels of Chlamydia specific antibodies in serum and a strong T-cell lymphoproliferative response. Following the i.n. challenge with 10⁴ inclusion forming units (IFU) of C. trachomatis, mice immunized with Ct-nMOMP or Ct-rMOMP lost significantly less weight than the negative control animals immunized with Ng-rPorB or MEM-0 (P < 0.05). However, mice vaccinated with the Ct-nMOMP lost less weight than those immunized with the Ct-rMOMP (P < 0.05). Mice were euthanized at 10 days following the challenge, their lungs weighed and the number of IFU of Chlamydia determined. Based on the lung weight and number of IFU recovered, significant protection was observed in the groups of mice immunized with both Ct-nMOMP and the Ct-rMOMP (P < 0.05). Nevertheless, significantly better protection was achieved with the Ct-nMOMP in comparison with the Ct-rMOMP (P < 0.05). In conclusion, vaccination with a preparation of the nMOMP elicited a more robust protection than immunization with rMOMP, suggesting that the conformational structure of MOMP is critical for inducing strong protection.     

Protective immunity against Toxoplasma challenge in mice by coadministration of T. gondii antigens and Eimeria profilin-like protein as an adjuvant

Toll-like receptor (TLR) ligands are attractive adjuvant candidates in vaccine development. Eimeria tenella profilin-like protein has recently been shown to be a potent agonist of the innate immune system through its recognition by Toll-like receptor-11. In this report, we studied the systemic and mucosal adjuvant activity of Eimeria profilin-like protein within a vaccinal strategy against Toxoplasma gondii in mice. Using intraperitoneal (i.p.) immunization, we observed that coadministration of the recombinant Eimeria antigen (rEA) with T. gondii antigen (TAg) effectively elevates plasma levels of IL-12p70 and consequently induced both enhanced specific humoral and Th1 cellular immune responses. The co-administration of TAg plus rEA by i.p route significantly enhanced the protection against T. gondii infection (62% brain cyst reduction) in comparison with control mice and with mice immunized with TAg alone (only 36% brain cyst reduction). After intranasal immunization, humoral and cellular responses were weak. However mice immunized nasally with TAg plus rEA were significantly protected with 50% of brain cyst reduction, conversely TAg immunized mice did not present any brain cyst reduction.These results indicate that Eimeria profilin-like protein would serve as an efficacious systemic and mucosal adjuvant inducing protective immune response against chronical stage of T. gondii infection through TLR11 activation.     

Intraperitoneal immunization of recombinant HSP70 (DnaK) of Salmonella Typhi induces a predominant Th2 response and protective immunity in mice against lethal Salmonella infection

Heat shock proteins serve as important antigens in defense against infectious diseases. Members of HSP70 family, particularly microbial HSP70s have acquired special significance in immunity. In the present study, we evaluated the immunogenicity and protective efficacy of HSP70 of Salmonella enterica serovar Typhi against lethal infection by Salmonella in mice with or without adjuvants. The HSP70 gene was cloned and expressed in Escherichia coli BL21 and purified by affinity chromatography. Immunization of mice with HSP70 either alone or in combination with complete Freund's adjuvant (CFA) resulted in a significant increase in antibody titers as compared to control. Antibody isotyping revealed that HSP70 immunization induces both IgG1 and IgG2a antibodies to a significant extent but a higher IgG1/IgG2a ratio indicates a predominant Th2 response. There was a significant increase in lymphocyte proliferation, and levels of both Th2 and Th1 cytokines in cells isolated from immunized mice as compared to control. Immunization of mice with recombinant HSP70 either alone or in combination with CFA conferred 70-90% protection against lethal infections by Salmonella Typhi Ty2 or Salmonella Typhimurium. However, passive immunization with anti-HSP70 sera induced only partial protection in the immunized mice.     

Structural optimisation of a conformational epitope improves antigenicity when expressed as a recombinant fusion protein

A conformationally restricted B cell epitope has been identified as a potential safe vaccine candidate from the major group A streptococcal virulence factor, the M protein. To maintain α-helical secondary structure, the minimal epitope is flanked with heterologous sequences to produce the chimeric vaccine candidate called J14. As a strategy toward developing an affordable multivalent GAS vaccine, we have expressed J14 recombinantly with a second GAS protective antigen H12 (rJ14H12). When administered to mice sub-cutaneously, the fusion protein stimulated a strong serum IgG response to the H12 component, but J14 was poorly immunogenic. To increase the immunogenicity of J14 when expressed with the model fusion partner, amino acid modifications were made to the initial recombinant J14 construct to produce rJJo. These changes stabilised the α-helical conformation of the recombinant antigen as assessed by circular dichroism. Mice immunised with rJJoH12, the fusion protein incorporating JJo, effectively stimulated a humoral response to both of the included antigens. These data support the feasibility of developing a multivalent vaccine incorporating the conformationally restricted protective antigen J14.     

Dual mechanism of protection by live attenuated Bordetella pertussis BPZE1 against Bordetella bronchiseptica in mice

Bordetella bronchiseptica, a gram-negative bacterium, causes chronic respiratory tract infections in a wide variety of mammalian hosts, including man, and no human vaccine is currently available. Acellular pertussis vaccines protect poorly against B. bronchiseptica, although they contain cross-reactive antigens. We have recently developed Bordetella pertussis BPZE1, a novel, live attenuated pertussis vaccine, currently completing phase I clinical trials in humans, and found that it protects against both B. pertussis and Bordetella parapertussis in mice. Here, we show that a single nasal administration of BPZE1 protects mice against lethal infection with B. bronchiseptica. After challenge, the vaccinated animals displayed markedly reduced lung inflammation and tissue damage, decreased neutrophil infiltration and increased levels of CD4(+)CD25(+)FoxP3(+) regulatory T cells in the lungs compared to non-immunized mice. Depletion of these cells abolished BPZE1-induced protection, indicating that BPZE1 protects against lethal inflammation through the recruitment of regulatory T cells. In addition, the B. bronchiseptica load was significantly decreased in the vaccinated animals. Using passive transfer experiments, protection was found to be essentially cell mediated, and BPZE1-induced Th1 and Th17 T cells recognize whole B. bronchiseptica extracts, although the participation of antibodies in protection cannot be discounted. Thus, a single administration of BPZE1 can confer protection against B. bronchiseptica in mice by a dual mechanism.     

Molecular Differentiation of Fasciola Species and Characterization of Genetic Diversity of F. gigantica Using NADH Dehydrogenase I (ND1) Gene in the Endemic Areas of Iran

Background:

Fasciola hepatica and F. gigantica are the causative agents of fasciolosis in domestic animals and humans. Based on the morphometric criteria, differential diagnosis between them is problematic. In addition, intermediate forms of Fasciola have been found in Iran, which makes the differentiation more difficult. The aim of the present study was to provide molecular evidence for the existence of F. gigantica in Iran using sequencing analysis of ND1 and PCR-RFLP analysis of ITS2 regions and to study the intraspecies variations of F. gigantica based on mitochondrial ND1 gene polymorphism.

Methods:

Forty Fasciola spp. samples collected from four distinct provinces (Fars, Khuzestan, Gilan, Khorasan Razavi) in Iran were collected for morphological and molecular characterization. In molecular method, PCR-RFLP analysis of ITS2 using pagI restriction enzyme was used as a screening approach for F. gigantica differentiation. Then mitochondrial DNA sequence variations in the ND1 gene were used for phylogenetic analysis.

Results:

Based on the morphometric criteria and RFLP analysis, 14 parasitic samples were initially identified to be F. gigantica. Phylogenetic results showed that there are at least 10 different genotypes of F. gigantica in Iran, which are different from those existing in the GenBank. Twenty-six points out of 410 base pairs of sequenced ND1 gene in 10 varieties of F. gigantica were diagnosed to be polymorphic. From 26 points of polymorphism, only eight resulted in the post-translational amino acid changes in ND1 gene product structure.

Conclusion:

Data revealed noticeable genetic diversity (up to 4.63%) between different varieties of F. gigantica in Iran.     

Protective effect against toxoplasmosis in mice induced by DNA immunization with gene encoding Toxoplasma gondii ROP18

Toxoplasma gondii is an obligate intracellular protozoan parasite infecting mammals and birds including humans. Rhoptry protein 18 has been implicated as an important virulence factor. In this study, we constructed a DNA vaccine expressing rhoptry protein 18 (ROP18) of T. gondii, and evaluated the immune response and protective immunity in Kunming mice. The gene sequence encoding ROP18 was inserted into the eukaryotic expression vector pVAX I. Intramuscular immunization of mice with pVAX-ROP18 elicited specific humoral responses and stimulated lymphoproliferation (P < 0.05). The cellular immune response was associated with the production of IFN-γ, indicating that a Th1 type response was elicited, which was confirmed by the production of large amounts of IgG2a (P < 0.05). By the expression of the CD69, an activation marker of CD4+ and CD8+ T cells, we found that pVAX-ROP18 enhanced the activation of CD4+ and CD8+ T cells in lymphoid in mice. After lethal challenge, the mice immunized with the pVAX-ROP18 showed a significantly increased survival time (27.9 ± 15.1 days) compared with control mice which died within 7 days of challenge (P < 0.05). Our results show for the first time, that a ROP18 vaccine construct can enhance the T. gondii-specific CTL. Th1 responses and increased survival suggested that ROP18 is a promising vaccine candidate against infection with T. gondii.     

Immunization with recombinant Chaperonin60 (Chp60) outer membrane protein induces a bactericidal antibody response against Neisseria meningitidis

Sera from individuals colonized with Neisseria meningitidis and from patients with meningococcal disease contain antibodies specific for the neisserial heat-shock/chaperonin (Chp)60 protein. In this study, immunization of mice with recombinant (r)Chp60 in saline; adsorbed to aluminium hydroxide; in liposomes and detergent micelles, with and without the adjuvant MonoPhosphoryl Lipid A (MPLA), induced high and similar (p > 0.05) levels of antibodies that recognized Chp60 in outer membranes (OM). FACS analysis and immuno-fluorescence experiments demonstrated that Chp60 was surface-expressed on meningococci. By western blotting, murine anti-rChp60 sera recognized a protein of Mr 60 kDa in meningococcal cell lysates. However, cross-reactivity with human HSP60 protein was also observed. By comparing translated protein sequences of strains, 40 different alleles were found in meningococci in the Bacterial Isolate Genome Sequence database with an additional 5 new alleles found in our selection of 13 other strains from colonized individuals and patients. Comparison of the non-redundant translated amino acid sequences from all the strains revealed ≥97% identity between meningococcal Chp60 proteins, and in our 13 strains the protein was expressed to high and similar levels. Bactericidal antibodies (median reciprocal titres of 32–64) against the homologous strain MC58 were induced by immunization with rChp60 in liposomes, detergent micelles and on Al(OH)₃. Bactericidal activity was influenced by the addition of MPLA and the delivery formulation used. Moreover, the biological activity of anti-Chp60 antisera did not extend significantly to heterologous meningococcal strains. Thus, in order to provide broad coverage, vaccines based on Chp60 would require multiple proteins and specific bactericidal epitope identification.