Vaccination with recombinant NcROP2 combined with recombinant NcMIC1 and NcMIC3 reduces cerebral infection and vertical transmission in mice experimentally infected with Neospora caninum tachyzoites

Debache, Karim; Alaeddine, Ferial; Guionaud, Christophe; Monney, Thierry; Müller, Joachim; Strohbusch, Maria; Leib, Stephen L.; Grandgirard, Denis; Hemphill, Andrew

doi:10.1016/j.ijpara.2009.04.006

Cited by 68 publications

(74 citation statements)

References 67 publications

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“…This observation provides further support that the protein may be accessible on the surface of parasites during the invasion process. RecNcROP2Fam-1 is an efficient antigen against cerebral infection by N. caninum in adult mice and against a vertical transmission of the infection from the mother to the offspring (Debache et al 2008(Debache et al , 2009(Debache et al , 2010. Classically, Th1 responses are elicited against intracellular antigens while Th2 responses are elicited against extracellular antigens.…”

Section: Discussionmentioning

confidence: 99%

“…Details on the construction of the recombinant protein, on the production and affinitypurification of rabbit α-NcROP2Fam-1 polyclonal Abs and use thereof to neutralize invasion in vitro can be found elsewhere (Debache et al 2008). The recombinant protein, previously dubbed recNcROP2, was used in a number of vaccination trials in mice (Debache et al 2008(Debache et al , 2009(Debache et al , 2010.…”

Section: Quantitative Reverse Transcription-pcrmentioning

confidence: 99%

“…However, several studies from our group demonstrated that a recombinant, bacterially expressed C-terminal moiety of NcROP2Fam-1 (formerly designated recNcROP2) induced highly effective immunity against challenge infection with N. caninum tachyzoites in both cerebral and fetal infection mouse models (Debache et al 2008(Debache et al , 2009(Debache et al , 2010.…”

Section: Introductionmentioning

confidence: 99%

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Molecular cloning and characterization ofNcROP2Fam-1, a member of the ROP2 family of rhoptry proteins inNeospora caninumthat is targeted by antibodies neutralizing host cell invasionin vitro

et al. 2013

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S U M M A R YRecent publications demonstrated that a fragment of a Neospora caninum ROP2 family member antigen represents a promising vaccine candidate. We here report on the cloning of the cDNA encoding this protein, N. caninum ROP2 family member 1 (NcROP2Fam-1), its molecular characterization and localization. The protein possesses the hallmarks of ROP2 family members and is apparently devoid of catalytic activity. NcROP2Fam-1 is synthesized as a pre-pro-protein that is matured to 2 proteins of 49 and 55 kDa that localize to rhoptry bulbs. Upon invasion the protein is associated with the nascent parasitophorous vacuole membrane (PVM), evacuoles surrounding the host cell nucleus and, in some instances, the surface of intracellular parasites. Staining was also observed within the cyst wall of 'cysts' produced in vitro. Interestingly, NcROP2Fam-1 was also detected on the surface of extracellular parasites entering the host cells and antibodies directed against NcROP2Fam-1-specific peptides partially neutralized invasion in vitro. We conclude that, in spite of the general belief that ROP2 family proteins are intracellular antigens, NcROP2Fam-1 can also be considered as an extracellular antigen, a property that should be taken into account in further experiments employing ROP2 family proteins as vaccines.

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Section: Discussionmentioning

confidence: 99%

Section: Quantitative Reverse Transcription-pcrmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Molecular cloning and characterization ofNcROP2Fam-1, a member of the ROP2 family of rhoptry proteins inNeospora caninumthat is targeted by antibodies neutralizing host cell invasionin vitro

et al. 2013

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“…Quantification of parasite loads in the brains of nonpregnant mice and dams was done as described previously (10,27). Seropositivity for N. caninum was assessed as described previously (28,29).…”

Section: Methodsmentioning

confidence: 99%

In Vitro and In Vivo Effects of the Bumped Kinase Inhibitor 1294 in the Related Cyst-Forming Apicomplexans Toxoplasma gondii and Neospora caninum

Winzer

Müller

Aguado‐Martínez

et al. 2015

Antimicrob Agents Chemother

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eWe report on the in vitro effects of the bumped kinase inhibitor 1294 (BKI-1294) in cultures of virulent Neospora caninum isolates Nc-Liverpool (Nc-Liv) and Nc-Spain7 and in two strains of Toxoplasma gondii (RH and ME49), all grown in human foreskin fibroblasts. In these parasites, BKI-1294 acted with 50% inhibitory concentrations (IC 50 s) ranging from 20 nM (T. gondii RH) to 360 nM (N. caninum Nc-Liv), and exposure of intracellular stages to 1294 led to the nondisjunction of newly formed tachyzoites, resulting in the formation of multinucleated complexes similar to complexes previously observed in BKI-1294-treated N. caninum beta-galactosidase-expressing parasites. However, such complexes were not seen in a transgenic T. gondii strain that expressed CDPK1 harboring a mutation (G to M) in the gatekeeper residue. In T. gondii ME49 and N. caninum NcLiv, exposure of cultures to BKI-1294 resulted in the elevated expression of mRNA coding for the bradyzoite marker BAG1. Unlike in bradyzoites, SAG1 expression was not repressed. Immunofluorescence also showed that these multinucleated complexes expressed SAG1 and BAG1 and the monoclonal antibody CC2, which binds to a yet unidentified bradyzoite antigen, also exhibited increased labeling. In a pregnant mouse model, BKI-1294 efficiently inhibited vertical transmission in BALB/c mice experimentally infected with one of the two virulent isolates Nc-Liv or Nc-Spain7, demonstrating proof of concept that this compound protected offspring from vertical transmission and disease. The observed deregulated antigen expression effect may enhance the immune response during BKI-1294 therapy and will be the subject of future studies. N eospora caninum is a cyst-forming apicomplexan parasite that is closely related to Toxoplasma gondii but exhibits distinct differences in transmission patterns, virulence, host specificity, immunogenetic aspects, and the pathology it induces. T. gondii causes toxoplasmosis in humans and many domestic and wildlife animals, with great economic impact especially in sheep but also in many other animal species (1). Human toxoplasmosis causes serious pathology in immune-suppressed individuals. In addition, if a seronegative mother acquires primary infection during pregnancy, human toxoplasmosis can lead to abortion, microcephalus and hydrocephalus, and other fetal abnormalities causing intellectual disability (2). N. caninum is a veterinary health problem and represents one of the most important infectious causes of bovine abortion, stillbirth, and the birth of weak calves, with an economic impact of over $1.3 billion (3-5). In addition, N. caninum causes neuromuscular disease in dogs, and neosporosis has also been detected in a wide range of other species of livestock and wild animals worldwide.Despite their differences, an important common feature of these parasites is their ability to invade and replicate within a wide range of cell types and tissues, where they reside in an intracellular parasitophorous vacuole, surrounded by a parasitophorous vacuole me...

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“…RecNcROP2 was cloned into pQE-30 (Qiagen, Hilden, Germany) as described by Debache et al 2008Debache et al , 2009, and expressed in E. coli BL21 (Novagen-EMD Biosciences, Madison, WI, USA) harbouring the pREP4 repressor plasmid (Qiagen).…”

Section: Neospora Caninum Tachyzoite Culture and Purificationmentioning

confidence: 99%

Intraperitoneal and intra-nasal vaccination of mice with three distinct recombinantNeospora caninumantigens results in differential effects with regard to protection against experimental challenge withNeospora caninumtachyzoites

et al. 2009

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S U M M A R YRecombinant NcPDI(recNcPDI), NcROP2(recNcROP2), and NcMAG1(recNcMAG1) were expressed in Escherichia coli and purified, and evaluated as potential vaccine candidates by employing the C57Bl/6 mouse cerebral infection model. Intraperitoneal application of these proteins suspended in saponin adjuvants lead to protection against disease in 50 % and 70 % of mice vaccinated with recNcMAG1 and recNcROP2, respectively, while only 20 % of mice vaccinated with recNcPDI remained without clinical signs. In contrast, a 90 % protection rate was achieved following intra-nasal vaccination with recNcPDI emulsified in cholera toxin. Only 1 mouse vaccinated intra-nasally with recNcMAG1 survived the challenge infection, and protection achieved with intra-nasally applied recNcROP2 was at 60 %. Determination of cerebral parasite burdens by real-time PCR showed that these were significantly reduced only in recNcROP2-vaccinated animals (following intraperitoneal and intra-nasal application) and in recNcPDI-vaccinated mice (intra-nasal application only). Quantification of viable tachyzoites in brain tissue of intra-nasally vaccinated mice showed that immunization with recNcPDI resulted in significantly decreased numbers of live parasites. These data show that, besides the nature of the antigen, the protective effect of vaccination also depends largely on the route of antigen delivery. In the case of recNcPDI, the intra-nasal route provides a platform to generate a highly protective immune response.

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Vaccination with recombinant NcROP2 combined with recombinant NcMIC1 and NcMIC3 reduces cerebral infection and vertical transmission in mice experimentally infected with Neospora caninum tachyzoites

Cited by 68 publications

References 67 publications

Molecular cloning and characterization ofNcROP2Fam-1, a member of the ROP2 family of rhoptry proteins inNeospora caninumthat is targeted by antibodies neutralizing host cell invasionin vitro

Molecular cloning and characterization ofNcROP2Fam-1, a member of the ROP2 family of rhoptry proteins inNeospora caninumthat is targeted by antibodies neutralizing host cell invasionin vitro

In Vitro and In Vivo Effects of the Bumped Kinase Inhibitor 1294 in the Related Cyst-Forming Apicomplexans Toxoplasma gondii and Neospora caninum

Intraperitoneal and intra-nasal vaccination of mice with three distinct recombinantNeospora caninumantigens results in differential effects with regard to protection against experimental challenge withNeospora caninumtachyzoites

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