Construction of Neospora caninum stably expressing TgSAG1 and evaluation of its protective effects against Toxoplasma gondii infection in mice

Toxoplasma gondii and Neospora caninum are closely related apicomplexan parasites. The surface antigen 1 of T. gondii (TgSAG1) is a major immunodominant antigen and, therefore, is considered to be a good candidate for the development of an effective recombinant vaccine against toxoplasmosis. In this study, N. caninum stably expressing the TgSAG1 gene (Nc/TgSAG1) was constructed using pyrimethamine-resistant DHFR-TS and GFP genes as double-selection markers. The expression level, molecular weight, and antigenic property of recombinant TgSAG1 expressed by the Nc/TgSAG1 were similar to those of the native TgSAG1. The mice immunized with Nc/TgSAG1 induced TgSAG1-specific Th1-dominant immune responses and protected the mice from a lethal challenge infection with T. gondii. These results indicate that N. caninum may provide a new tool for the production of a live recombinant vector vaccine against toxoplasmosis in animals. To our knowledge, this is the first report to evaluate the usefulness of N. caninum-based live vaccine.     

CpG-ODN combined with Neospora caninum lysate,but not with excreted-secreted antigen,enhances protection against infection in mice

CpG oligodeoxynucleotides (ODN) have shown to be potent immunoadjuvants for several pathogens, but there is limited information concerning their use in immunization protocols against neosporosis. This study aimed to evaluate the potential of CpG-ODN combined with Neospora lysate antigen (NLA) or excreted-secreted antigen (NcESA) to induce protective immune response against Neospora caninum infection in mice. C57BL/6 mice were vaccinated subcutaneously three times at 2-week intervals with NLA, NLA + CpG, NcESA, NcESA + CpG, CpG (adjuvant control) or PBS (infection control). Serological assays showed an increased specific IgG2a response in animals immunized with either antigen plus adjuvant and elevated levels of the IgG1 isotype in those vaccinated with antigens alone. Splenocyte proliferative responses upon antigen stimulation were higher in groups immunized with NLA or NcESA combined with CpG, showing increased IL-12 levels. Also, mice vaccinated with NcESA or NcESA + CpG demonstrated higher IFN-γ levels and IFN-γ/IL-10 ratio. After lethal challenge, mice immunized with NLA + CpG or NLA had lower morbidity score and body weight changes in comparison to other groups, and animals did not succumb during acute infection. In contrast, NcESA + CpG or NcESA groups exhibited the highest morbidity scores, body weight impairment and mortality rates, associated with greatest brain parasite burden and inflammation. In conclusion, CpG-ODN was able to induce a Th1-type humoral immune response with predominant IgG2a levels for either NLA or NcESA, but resulting in an effective Th1-driven cellular immune response and total protection only when combined with NLA. Vaccination with NcESA alone or combined with CpG resulted in a strong cellular immune response associated with high levels of IFN-γ and inflammation, rendering mice more susceptible to parasite challenge.     

Vaccination with profilin encapsulated in oligomannose-coated liposomes induces significant protective immunity against Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular parasite that can infect a variety of mammals and birds, causing toxoplasmosis. Several types of vaccines against T. gondii have been developed, but these have limitations in terms of their safety and inadequate efficacy. T. gondii profilin (TgPF) is a potential immunodominant antigen for a candidate vaccine. In this study, we encapsulated TgPF in oligomannose-coated liposomes (OMLs) to evaluate the immune response induced by this vaccine. C57BL/6 mice were immunized with TgPF-OML three times at 14-day intervals and challenged with T. gondii. TgPF-OML increased the survival of the mice and reduced the parasite burden in their brains after T. gondii infection. Immunization with TgPF-OML also induced TgPF-specific interferon-γ production and IgG antibodies in mice. Our results demonstrate that OML-encapsulated TgPF triggers strong humoral and cellular responses against T. gondii, and that TgPF-OML is a candidate vaccine that warrants further development.     

Vaccination with the recombinant chimeric antigen recNcMIC3-1-R induces a non-protective Th2-type immune response in the pregnant mouse model for N. caninum infection

The major route of transmission of Neospora caninum in cattle is transplacentally from an infected cow to its progeny. Therefore, a vaccine should be able to prevent both the horizontal transmission from contaminated food or water and the vertical transmission. We have previously shown that a chimeric vaccine composed of predicted immunogenic epitopes of NcMIC3, NcMIC1 and NcROP2 (recNcMIC3-1-R) significantly reduced the cerebral infection in BALB/c mice. In this study, mice were first vaccinated, then mated and pregnant mice were challenged with 2 × 10⁶N. caninum tachyzoites at day 7–9 of pregnancy. Partial protection was only observed in the mice vaccinated with a tachyzoite crude protein extract but no protection against vertical transmission or cerebral infection in the dams was observed in the group vaccinated with recNcMIC3-1-R. Serological and cytokine analysis showed an overall lower cytokine level in sera associated with a dominant IL-4 expression and high IgG1 titers. Thus, the Th2-type immune response observed in the pregnant mice was not protective against experimental neosporosis, in contrary to the mixed Th1-/Th2-type immune response observed in the non-pregnant mouse model. These results demonstrate that the immunomodulation that occurs during pregnancy was not favorable for the protection against N. caninum infection conferred by vaccination with recNcMIC3-1-R.     

Recombinant Ehrlichia P29 protein induces a protective immune response in a mouse model of ehrlichiosis

Ehrlichioses are emerging tick-borne bacterial diseases of humans and animals for which no vaccines are available. The diseases are caused by obligately intracellular bacteria belonging to the genus Ehrlichia. Several immunoreactive proteins of ehrlichiae have been identified based on their reactivity with immune sera from human patients and animals. These include the major outer membrane proteins, ankyrin repeat proteins and tandem repeat proteins (TRP). Polyclonal antibodies directed against the tandem repeats (TRs) of Ehrlichia chaffeensis TRP32, TRP47 and TRP120 have been shown to provide protection in mice. In the present study, we evaluated E. muris P29, which is the ortholog of E. chaffeensis TRP47 and E. canis TRP36, as a subunit vaccine in a mouse model of ehrlichiosis. Our study indicated that unlike E. chaffeensis TRP47 and E. canis TRP36, orthologs of E. muris (P29) and E. muris-like agent (EMLA) do not contain tandem repeats. Immunization of mice with recombinant E. muris P29 induced significant protection against a challenge infection. The protection induced by E. muris P29 was associated with induction of strong antibody responses. In contrast to development of P29-specific IgG antibodies following immunization, development of P29-specific IgG antibodies, but not IgM antibodies, was impaired during persistent E. muris infection. Furthermore, our study indicated that CD4+ T cells target P29 during E. muris infection and differentiate into IFN-γ-producing Th1 effector/memory cells. In conclusion, our study indicated that orthologs of E. muris P29 showed considerable variation in the central tandem repeat region among different species, induction of P29-specific IgG antibody response was impaired during persistent E. muris infection, and rP29 induced protective immune responses.     

Immunization of female BALB/c mice with Neospora cyclophilin and/or NcSRS2 elicits specific antibody response and prevents against challenge infection by Neospora caninum

Neospora caninum is the causal agent of bovine neosporosis which results in high levels of abortion. The present study determined the protective efficacy of two Neospora antigens - Neospora cyclophilin (NcCyP) and NcSRS2. The ability of native NcCyP to upregulate mouse IFN-γ was also confirmed in this study. Recombinant NcCyP or NcSRS2 were tested either alone or in combination and formulated with adjuvant ImmuMax-SR and CpG. Female BALB/c mice (n = 15) of 10-12 weeks of age were immunized s.c. twice over a 2-week interval with vaccines containing either NcCyP (20 μg/dose) alone, NcSRS2 (20 μg/dose) alone, NcCyP plus NcSRS2, or non-recombinant bacterial antigen (NR) in 2 separate trials. All mice were challenge-infected 3 weeks following the booster immunization and necropsied 3 weeks after the challenge infection. Brain and serum were collected and Nc-specific DNA sequence in brain tissue and antibodies in serum were analyzed by PCR or ELISA/Western blotting. Results showed that mice vaccinated with rNcCyP, rNcSRS2, or both rNcCyP and rNcSRS2 responded with high levels of NcCyP or NcSRS2 specific antibodies. Overall, mice received vaccines formulated with either rNcCyP or rNcCyP and rNcSRS2 had a higher (p < 0.01) percent protection when compared to the mock- or non-vaccinated mice. The group immunized with rNcSRS2 alone exhibited slightly lower levels of protection, which was higher (p < 0.05) than that of the non-vaccinated group but did not differ (p = 0.06) from that of the mock-vaccinated group. The results of the present study indicate that NcCyP is a highly efficacious vaccine candidate which may be useful in protection against Neospora infection.     

Heat shock protein 70 from Trichinella spiralis induces protective immunity in BALB/c mice by activating dendritic cells

Trichinella spiralis heat shock protein 70 (Ts-Hsp70) is a protective antigen that induces partial protective immunity against T. spiralis infection in mice. To determine whether dendritic cells are involved in the mechanism responsible for the protection induced by Ts-Hsp70, mouse bone marrow-derived dendritic cells (DCs) were incubated with recombinant Ts-Hsp70 (rTs-Hsp70), and the DC-secreted cytokines and expressed surface markers were measured. The results demonstrated that rTs-Hsp70 activated DC maturation that was characterized by the secretion of IL-1β, IL-12p70, TNF-α, and IL-6 and the increased surface expression of CD11c, MHC II, CD40, CD80, and CD86. The rTs-Hsp70-activated DCs enabled the stimulation, proliferation and secretion of Th1/2 cytokines (i.e., INF-γ, IL-2, IL-4 and IL-6) in CD4⁺ T cells from T. spiralis-infected mice. The mice that received rTs-Hsp70-activated DCs exhibited a 38.4% reduction in muscle larvae upon larval challenge with T. spiralis compared to the group that received PBS-incubated DCs. This partial protection was correlated with Th1 and Th2 mixed anti-Ts-Hsp70-specific immune responses that included high titers of total IgG, IgG1 and IgG2a and increased levels of Th1/2 cytokines (i.e., IFN-γ, IL-2, IL-4, IL-6). These results indicate that the rTs-Hsp70-induced protective immunity was mediated by the activation of the DCs and that rTs-Hsp70-loaded DCs could be an alternative vaccine approach against trichinellosis.     

Attenuated Francisella asiatica iglC mutant induces protective immunity to francisellosis in tilapia

Francisella asiatica is a Gram-negative, facultative intracellular bacteria that causes fish francisellosis. Fish francisellosis is a severe sub-acute to chronic granulomatous disease with high mortalities and high infectivity rates in cultured and wild fish. To date, there is no approved vaccine for this widespread emergent disease. The goal of this study was to characterize the efficacy of a defined F. asiatica mutant (ΔiglC) as a live attenuated vaccine against subsequent immersion challenge with the wild-type (WT) organism. In previous work, the ΔiglC was found to be attenuated upon intraperitoneal injection and immersion challenges. In vitro, the ΔiglC exhibited reduced growth in tilapia head-kidney derived macrophages, and was significantly attenuated (p < 0.001) as demonstrated by cytopathogenic and apoptosis assays. In this study, the ΔiglC was tested to determine its ability to protect tilapia against challenge with high doses (lethal dose 80) of WT bacteria. Naïve tilapia vaccinated by immersion with a suspension of the ΔiglC and subsequently challenged with WT F. asiatica were protected (90% mean percent survival) from the lethal challenges. F. asiatica-specific antibodies produced in response to immunization with the ΔiglC were subsequently found to protect naïve tilapia against high-dose F. asiatica challenge in passive immunization experiments. Significant protection (p < 0.001) was obtained when fish were passively immunized and challenged with 10⁴ and 10⁵ CFU/fish of WT F. asiatica; but not when challenged with 10⁶ CFU/fish. This is the first report of a defined live attenuated strain providing protection against F. asiatica in fish.     

Enhancement of protective immune responses induced by Toxoplasma gondii dense granule antigen 7 (GRA7) against toxoplasmosis in mice using a prime-boost vaccination strategy

Effective vaccines against Toxoplasma gondii may contribute to preventing and controlling the spread of toxoplasmosis, which is important for improving outcomes of infections in humans and livestock animals. The dense granule antigen 7 (GRA7) of T. gondii might be an immunodominant antigen for a vaccine candidate. In the present study, a further exploration of its vaccine effect, a heterologous prime-boost vaccination strategy with a recombinant eukaryotic plasmid pEGFP-GRA7 and a recombinant protein GRA7 expressed from a prokaryotic plasmid pET30-GRA7, was performed in BALB/c mice. The data reveal that a DNA prime-protein boost vaccination induces both humoral and cellular immune responses against T. gondii associated with high levels of total IgG, IgG2a isotype and gamma interferon (IFN-γ). Challenge experiments further show that the DNA prime-protein boost vaccination significantly increases survival rate (60%), compared with controls in which all died within 8 days of challenge. Therefore, the DNA prime-protein boost vaccination based on GRA7 might be a promising regimen for further development of an effective vaccine against T. gondii.     

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.     

Recombinant gp90 protein expressed in Pichia pastoris induces a protective immune response against reticuloendotheliosis virus in chickens

Reticuloendotheliosis virus (REV) causes an oncogenic, immunosuppressive and runting syndrome in multiple avian hosts worldwide. In this study, the gp90 protein of REV was secretory expressed in Pichia pastoris with high production level and good antigenicity. To fully utilize the expression potential of the P. pastoris expression system, a panel of Pichia clones carrying increasing copies of the gp90 expression cassette was created using an in vitro multimerization approach and the effects of gene dosage on gp90 expression were investigated. Results demonstrated that an increase in gp90 copy number can significantly improve the yields of gp90 protein. Following expression and scale-up, the gp90 protein production level could reach up to 400mg/L, and the protein could be detected by gp90-specific monoclonal antibody. Investigations of its vaccine efficacy demonstrated that the recombinant gp90 protein was able to induce sustained high levels of antibodies against REV as being detected by ELISA and virus neutralizing test. Furthermore, immunization of chickens with the recombinant gp90 vaccine fully protected the animals from viremia after REV infection. Overall, the yeast-expressed gp90 protein retains good immunogenicity and could be used as a potential subunit vaccine candidate for REV prevention.     

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.     

RecNcMIC3-1-R is a microneme- and rhoptry-based chimeric antigen that protects against acute neosporosis and limits cerebral parasite load in the mouse model for Neospora caninum infection

In order to achieve host cell entry, the apicomplexan parasite Neospora caninum relies on the contents of distinct organelles, named micronemes, rhoptries and dense granules, which are secreted at defined timepoints during and after host cell entry. It was shown previously that a vaccine composed of a mixture of three recombinant antigens, corresponding to the two microneme antigens NcMIC1 and NcMIC3 and the rhoptry protein NcROP2, prevented disease and limited cerebral infection and transplacental transmission in mice. In this study, we selected predicted immunogenic domains of each of these proteins and created four different chimeric antigens, with the respective domains incorporated into these chimers in different orders. Following vaccination, mice were challenged intraperitoneally with 2 × 10⁶N. caninum tachzyoites and were then carefully monitored for clinical symptoms during 4 weeks post-infection. Of the four chimeric antigens, only recNcMIC3-1-R provided complete protection against disease with 100% survivors, compared to 40-80% of survivors in the other groups. Serology did not show any clear differences in total IgG, IgG1 and IgG2a levels between the different treatment groups. Vaccination with all four chimeric variants generated an IL-4 biased cytokine expression, which then shifted to an IFN-γ-dominated response following experimental infection. Sera of recNcMIC3-1-R vaccinated mice reacted with each individual recombinant antigen, as well as with three distinct bands in Neospora extracts with similar Mr as NcMIC1, NcMIC3 and NcROP2, and exhibited distinct apical labeling in tachyzoites. These results suggest that recNcMIC3-1-R is an interesting chimeric vaccine candidate and should be followed up in subsequent studies in a fetal infection model.     

Mucosal vaccination with a codon-optimized hemagglutinin gene expressed by attenuated Salmonella elicits a protective immune response in chickens against highly pathogenic avian influenza

The purpose of this study was to evaluate clinical protection from challenge conferred by two attenuated Salmonella enteria serovar typhimurium vaccine strains expressing the hemagglutinin (HA1) gene from a highly pathogenic avian influenza (HPAI) H5N1 (A/whooper swan/Mongolia/3/2005), under control of the anaerobically inducible nir15 promoter. Two-week-old White Leghorn chickens were immunized by oral gavage with one milliliter doses of >109 Salmonella colony-forming units once weekly for 4 weeks prior to challenge. Expression of recombinant protein was confirmed via Western blot. Serum and mucosal gavage samples were collected prior to, and following immunization and antibodies against avian influenza HA were confirmed by Western blot and hemagglutination-inhibition (HI) assay. Chickens were challenged with homologous (A/whooper swan/Mongolia/3/2005), or heterologous (A/Chicken/Queretaro/14588-19/95) HPAI virus strains. Chickens immunized with attenuated Salmonella strains containing plasmid expression vector (pTETnir15HA) demonstrated a statistically significant increase in survival compared to control groups. Results provide evidence of effectiveness of attenuated Salmonella strains for delivery of recombinant avian influenza HA antigens and induction of mucosal and systemic immune responses protective against lethal challenge with HPAI.     

Oral immunization with Lactococcus lactis-expressing EspB induces protective immune responses against Escherichia coli O157:H7 in a murine model of colonization

Enterohemorrhagic Escherichia coli (EHEC) have been responsible for several outbreaks of hemolytic–uremic syndrome (HUS) worldwide. HUS is the most common cause of acute renal failure in children and results in fatalities as high as 50% in the elderly. Currently, neither a specific treatment nor a vaccine is available for EHEC. Lactococcus lactis is a generally regarded as safe “GRAS” bacterium that offers a valuable platform for oral vaccine delivery. Toward the development of an oral vaccine against EHEC, we have previously constructed a recombinant L. lactis strain expressing the EHEC antigen, EspB in the cytoplasmic compartment. However, oral immunization of mice with this strain induced weak priming of the immune system. This outcome was attributed to the rather low levels of EspB expressed by this recombinant strain. Therefore, in the present study we optimized the expression of EspB in L. lactis by secreting the antigen either under constitutive or nisin-inducible control. Indeed, oral immunization of mice with the EspB-secreting strains successfully induced specific mucosal and systemic antibody responses. These responses were associated with mixed Th1/Th2 cell responses in Peyer's Patches and mesenteric lymph nodes. Moreover, immunized mice exhibited significant protection against E. coli O157:H7 colonization, as indicated by the reduced amount and/or duration of the bacterial fecal shedding. Our results demonstrate the protective potential of EspB as an oral vaccine against EHEC infection. Additionally, the study demonstrates the efficient delivery of recombinant EspB by the “GRAS” bacterium, L. lactis. The safety profile of L. lactis as a vaccine vehicle can particularly be beneficial to children and elderly as high-risk groups for HUS incidence.     

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 Leptospirainterrogans 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 × 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-γ) 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.     

Novel influenza virus vectors expressing Brucella L7/L12 or Omp16 proteins in cattle induced a strong T-cell immune response,as well as high protectiveness against B. abortus infection

This paper presents the results of a study of the immunogenicity and protectiveness of new candidate vector vaccine against Brucella abortus – a bivalent vaccine formulation consisting of a mixture of recombinant influenza A subtype H5N1 or H1N1 (viral constructs vaccine formulation) viruses expressing Brucella ribosomal protein L7/L12 and Omp16, in cattle. To increase the effectiveness of the candidate vaccine, adjuvants such as Montanide Gel01 or chitosan were included in its composition. Immunization of cattle (heifers aged 1–1.5 years, 5 animals per group) with the viral constructs vaccine formulation only, or its combination with adjuvants Montanide Gel01 or chitosan, was conducted via the conjunctival method using cross prime (influenza virus subtype H5N1) and booster (influenza virus subtype H1N1) vaccination schedules at an interval of 28 days. Vaccine candidates were evaluated in comparison with the positive (B. abortus S19) and negative (PBS) controls. The viral constructs vaccine formulations, particularly in combination with Montanide Gel01 adjuvant promoted formation of IgG antibodies (with a predominance of antibodies of isotype IgG2a) against Brucella L7/L12 and Omp16 proteins in ELISA. Moreover, these vaccines in cattle induced a strong antigen-specific T-cell immune response, as indicated by a high number of CD4⁺ and CD8⁺ cells, as well as the concentration of IFN-γ, and most importantly provided a high level of protectiveness comparable to the commercial B. abortus S19 vaccine and superior to the B. abortus S19 vaccine in combination with Montanide Gel01 adjuvant. Based on these findings, we recommended the bivalent vaccine formulation containing the adjuvant Montanide Gel01 for practical use in cattle.     

Characterization of possible correlates of protective response against Brucella ovis infection in rams immunized with the B. melitensis Rev 1 vaccine

Vaccination with the live attenuated Brucella melitensis Rev 1 vaccine is used to control ovine brucellosis caused by Brucella ovis in sheep. The objective of this study was to identify possible correlates of protective response to B. ovis infection through the characterization by microarray hybridization and real-time RT-PCR of inflammatory and immune response genes differentially expressed in rams previously immunized with B. melitensis Rev 1 and experimentally challenged with B. ovis. Gene expression profiles were compared before and after challenge with B. ovis between rams protected and those vaccinated but found infected after challenge. The TLR10, Bak and ANXI genes were expressed at higher levels in vaccinated and protected rams. These genes provide possible correlates of protective response to B. ovis infection in rams immunized with the B. melitensis Rev 1 vaccine.     

Characterization of protective immune responses promoted by human antigen targets in a urogenital Chlamydia trachomatis mouse model

A vaccine against genital tract infections caused by Chlamydia trachomatis is urgently needed. We have previously identified a number of immunodominant human T- and/or B-cell antigen targets in patients with a C. trachomatis infection. Herein we use a urogenital C. trachomatis mouse model to investigate the protective efficacy of these antigens. C3H/HeN mice were immunized with recombinant antigens formulated in the adjuvant CAF01. Immunity post vaccination was analyzed and the protective efficacy against vaginal challenge with C. trachomatis was monitored by vaginal swabbing. All antigens elicited a significant immune response when administered in CAF01 but the balance between CMI and humoral responses differed markedly for the different antigens. Six (CT443, CT043, CT858, CT610, CT004 and CT681) antigens were found to be protective. We demonstrated by T-cell depletion studies that the protection promoted by the two antigens CT043 and CT004 was mediated by CD4⁺ T-cells. Both antigens are frequently recognized by T-cells during a natural Chlamydia infection in humans and if included in a future multi-component Chlamydia vaccine probably would operate mainly through the induction of a CMI response.     

Development of an Indirect ELISA Using Different Fragments of Recombinant Ncgra7 for Detection of Neospora caninum Infection in Cattle and Water Buffalo

Background:

Dense granules are immunodominant proteins for the standardization of immunodiagnostic procedures to detect neosporosis. In the presented study different fragment of a dense-granule protein was evaluated for serodiagnosis of Neospora caninum in cattle and water buffalo.

Methods:

NcGRA7, from N. caninum tachyzoites was amplified. PCR product and pMAL-c2X plasmid were digested with EcoR1 restriction enzyme and expressed in Escherichia coli to evaluate its competence for detection of anti- N. caninum antibodies with ELISA in comparison with commercial IDEXX ELISA. Furthermore, 230 sera of presumably healthy cattle and water buffaloes (108 cattle and 122 water buffaloes) were analyzed by both tests to determine the agreement of these two procedures.

Results:

Sensitivities and specificities of NcGRA7-based ELISA were 94.64% and 90.38% respectively using sera of cattle, but were 98.57% and 86.54% in the case of buffaloes respectively. A good correlation between the results of IDEXX ELISA and ELISA based on recombinant NcGRA7 for detecting N. caninum antibodies was appeared. Analyzing by Mc Nemar′s showed that NcGRA7-based ELISA has acceptable capability to differentiate the positive results in comparison with IDEXX ELISA.

Conclusion:

NcGRA7-based ELISA considering utilized new fragment of genomic DNA is a good tool for serodiagnosis of anti- N. caninum antibodies for screening and epidemiological purposes on cattle herd and water buffaloes as well.