Adjuvant and immunostimulatory effects of a D-galactose-binding lectin from Synadenium carinatum latex (ScLL) in the mouse model of vaccination against neosporosis.

Vaccination is an important control measure for neosporosis that is caused by a coccidian parasite, Neospora caninum, leading to abortion and reproductive disorders in cattle and serious economic impacts worldwide. A D-galactose-binding lectin from Synadenium carinatum latex (ScLL) was recently described by our group with potential immunostimulatory and adjuvant effects in the leishmaniasis model. In this study, we evaluated the adjuvant effect of ScLL in immunization of mice against neosporosis. First, we investigated in vitro cytokine production by dendritic cells stimulated with Neospora lysate antigen (NLA), ScLL or both. Each treatment induced TNF-α, IL-6, IL-10 and IL-12 production in a dose-dependent manner, with synergistic effect of NLA plus ScLL. Next, four groups of C57BL/6 mice were immunized with NLA + ScLL, NLA, ScLL or PBS. The kinetics of antibody response showed a predominance of IgG and IgG1 for NLA + ScLL group, whereas IgG2a response was similar between NLA + ScLL and NLA groups. Ex vivo cytokine production by mouse spleen cells showed the highest IFN-γ/IL-10 ratio in the presence of NLA stimulation for mice immunized with NLA + ScLL and the lowest for those immunized with ScLL alone. After parasite challenge, mice immunized with NLA + ScLL or ScLL alone presented higher survival rates (70-80%) and lower brain parasite burden as compared to PBS group, but with no significant changes in morbidity and inflammation scores. In conclusion, ScLL combined with NLA was able to change the cytokine profile induced by the antigen or lectin alone for a Th1-biased immune response, resulting in high protection of mice challenged with the parasite, but with low degree of inflammation. Both features may be important to prevent congenital neosporosis, since protection and low inflammatory response are necessary events to guide towards a successful pregnancy.

Vaccination of mice with chitosan nanogel-associated recombinant NcPDI against challenge infection with Neospora caninum tachyzoites.

The effects of nanogel encapsulation of recombinant NcPDI (recNcPDI) following vaccination of mice by intranasal or intraperitoneal routes and challenge infection with Neospora caninum tachyzoites were investigated. Nanogels were chitosan based, with an alginate or alginate-mannose surface. None of the mice receiving recNcPDI intraperitoneal (i.p.) (without nanogels) survived, whereas intranasal (i.n.) application protected 9 of 10 mice from disease. Association of recNcPDI with nanogels improved survival of i.p. vaccinated mice, but nanogels without recNcPDI gave similar protection levels. When nanogels were inoculated via the i.n. route, 80% of the mice were protected. Association of recNcPDI with the alginate-coated nanogels protected all mice against disease. Quantification of the cerebral parasite burden showed a significant reduction of parasite numbers in most experimental groups vaccinated i.n., except those vaccinated with alginate-mannose nanogels with or without recNcPDI. For i.p. vaccinated groups, no significant differences in cerebral infection densities were measured, but there was a reduction in the groups vaccinated with recNcPDI associated with both types of nanogels. Analysis of the immune responses of infected mice indicated that association of recNcPDI with nanogels altered the patterns of cytokine mRNA expression profiles, but had no major impact on the antibody subtype responses. Nevertheless, this did not necessarily relate to the protection.

Neospora caninum: application of apical membrane antigen 1 encapsulated in the oligomannose-coated liposomes for reduction of offspring mortality from infection in BALB/c mice.

Liposomes coated with neoglycolipids constructed with mannopentaose and dipalmitoylphosphatidylethanolamine (M3-DPPE), referred to as M3-DPPE liposomes, have been shown to induce cellular immunity against antigens encapsulated therein. To evaluate whether these M3-DPPE liposomes have an adjuvant capacity against Neospora caninum infection, a novel immunization method utilizing soluble N. caninum apical membrane antigen 1 (NcAMA1) encapsulated in the M3-DPPE liposomes (M3-NcAMA1) was employed. The results revealed that a significant amount of interferon (IFN)-gamma production was detected in culture supernatants of NcAMA1 protein- or N. caninum lysate-stimulated spleen cells obtained from the mice one week after the third immunization with M3-NcAMA1. The parasite burden in the dams' brain tissue was decreased and the survival rate of offspring increased significantly in M3-NcAMA1-immunized mice. Thus, a parasite-specific Th1 immune response was successfully induced in the pregnant mice immunized with M3-NcAMA1, and an effective reduction of offspring mortality from N. caninum infection was triggered.

Neospora GRA6 possesses immune-stimulating activity and confers efficient protection against Neospora caninum infection in mice.

Vaccination has the potential to be the most cost-effective control measure for reducing the economic burden of neosporosis in cattle. In this study, the immune-stimulatory effect of recombinant Neospora caninum dense granule protein 6 (NcGRA6) was confirmed via its triggering of IL-12p40 production in murine macrophages. BALB/c mice were immunized with recombinant NcGRA6 fused with glutathione S-transferase (GST) protein with or without oligomannose-coated-liposomes (OMLs) as the potential adjuvant. Specific IgG1 antibody production was observed from 21 and 35 days after the first immunization in NcGRA6+GST- and NcGRA6+GST-OML-immunized mice, respectively. However, specific IgG2a was detected 1 week after the infection, and IgG2a levels of the NcGRA6+GST- group were higher than those of the NcGRA6+GST-OML-group. Moreover, spleen cell proliferation with concomitant interferon-gamma production was detected in mice immunized with NcGRA6+GST, indicating that a significant cellular immune response was induced. Mouse survival rates against N. caninum challenge infection were 91.7% for NcGRA6+GST and 83.3% for NcGRA6+GST-OML, which were significantly higher than those of control groups (GST-OML: 25%, phosphate-buffered saline: 16.7%). This indicates that naked NcGRA6+GST induced protective immunity. Thus, our findings highlight the immune-stimulating potential of NcGRA6 and the ability to induce protective immunity against N. caninum infection in mice.

Vaccination against Eimeria tenella infection using a fraction of E. tenella sporozoites selected by the capacity to activate T cells.

At 8 days after a primary Eimeria tenella infection, a subset of T cells, of which the protective role is as yet unclear, circulates in the peripheral blood. In order to investigate this, the in vitro cellular responsiveness of these peripheral blood lymphocytes has been used as selection criterion to identify potentially protective E. tenella sporozoite antigens. The hydrophilic protein phase of purified E. tenella sporozoite homogenates obtained by Triton X-114 extraction was fractionated using preparative gel electrophoresis. Nine fractions, separated according to different molecular weight, were tested for their ability to stimulate T-cell responses. Both the proliferation of peripheral blood lymphocytes and the macrophage activating activity released in the culture supernatants were measured. On the basis of this responsiveness, four fractions were selected and used to vaccinate chickens. All vaccine preparations induced strong T-cell responses. One fraction immunised chickens against subsequent challenge infection, in that the caecal lesion scores were significantly lower as compared with that of the unvaccinated controls. This fraction contained hydrophilic polypeptides with a molecular mass that ranged from 26 to 30 kDa.

Oligomannose-coated liposome-entrapped dense granule protein 7 induces protective immune response to Neospora caninum in cattle.

Neospora caninum is an intracellular protozoan parasite that causes abortion in cows. Vaccination is an important strategy for control of neosporosis, and a safe and effective vaccine suitable for cattle is required. Dense granule protein 7 of N. caninum (NcGRA7) is a secretory protein with high antigenicity in hosts. We demonstrated previously that NcGRA7 entrapped in liposomes coated with mannotriose (M3-NcGRA7) could induce a parasite-specific T-helper type 1 immune response and produce humoral antibodies that resulted in increased offspring survival and decreased infection in the brains of mice dams. In the present study, the efficacy of M3-NcGRA7 as a vaccine candidate against N. caninum has been evaluated in cattle (n=12). Cattle were immunized with M3-NcGRA7 containing 50 µg (n=4) or 200 µg NcGRA7 (n=4) subcutaneously twice with a 4-week interval and all cattle including the non-immunized controls (n=4) were inoculated with 10(7) tachyzoites of Nc-1 strain 27 days after the second immunization and euthanized at 85-87 days post infection (dpi). In immunized cattle, NcGRA7-specific antibody production and IFN-γ production in PBMC was induced before challenge. At 3 dpi, body temperature and concentration of serum IFN-γ tended to be higher in control cattle than in the immunized cattle. Furthermore, the parasite load in the brain significantly decreased in cattle immunized with 50 µg M3-NcGRA7 compared with controls. These results suggest that M3-NcGRA7 can induce protective immune responses to N. caninum tachyzoites in cattle, which could lead to practical application of safe and effective subunit vaccines.

Low efficacy of NcGRA7, NcSAG4, NcBSR4 and NcSRS9 formulated in poly-ε-caprolactone against Neospora caninum infection in mice.

The protective efficacy of vaccination with Neospora caninum recombinant antigens was evaluated in Balb/c pregnant and non-pregnant mouse models of neosporosis. A major immunodominant dense granule protein (NcGRA7) and three bradyzoite-specific surface antigens (NcSAG4, NcBSR4 and NcSRS9) were expressed in Escherichia coli and encapsulated within poly-ε-caprolactone (PCL) nanoparticles for the first time. Good efficiencies of entrapment (greater than 50%) were obtained for all encapsulated proteins. Moreover, antigenicity was unaffected after formulation. Afterwards, separate groups of mice were immunised with the nanoparticles and were then challenged with N. caninum tachyzoites. High IgG1 and IgG2a antibody levels of anti-N. caninum and specific antibodies directed against recombinant proteins were developed by all of the immunised groups. Mice previously inoculated with encapsulated rNcGRA7 produced significant levels of IFN-γ. However, in general, a low production of IFN-γ was detected. This may indicate a failure in the complete liberation of antigens after immunisation or an incorrect balance of the Th1/Th2 response to combat acute neosporosis during pregnancy. In fact, high morbidity and mortality rates were observed in dams. Moreover, vertical transmission was not prevented, and high neonatal mortality rates occurred similarly among the groups. Despite the global absence of efficacy, the study reveals some results of positive efficacy regarding dams and pups' survival and parasite presence for NcSRS9 recombinant protein. Furthermore, vaccination with rNcGRA7 encapsulated alone or combined with rNcSAG4 resulted in a slight decrease of parasite presence in non-pregnant mice. These promising results are further discussed to suggest new approaches that may be more suitable to test vaccine formulations based on bradyzoite stage-specific proteins.

Immunization with oligomannose-coated liposome-entrapped dense granule protein 7 protects dams and offspring from Neospora caninum infection in mice.

The present study demonstrates that the subcutaneous administration of Neospora caninum dense granule protein 7 (NcGRA7) entrapped in liposomes coated with mannotriose strongly induces the parasite-specific T-helper type 1 immune response and humoral antibody in mice. Although anti-NcGRA7 immunoglobulin G1 antibody production was induced in mice injected with NcGRA7 alone, the dams and offspring were never protected from N. caninum infection. The immunization of mice with liposome-entrapped NcGRA7 before pregnancy resulted in increased offspring survival and decreased the infection rates in the brains of dams after parasite infection at 6 to 9 days of gestation. In conclusion, oligomannose-coated liposome-entrapped NcGRA7 can be used as a new type of effective vaccine to control neosporosis.