Elsevier

Experimental Parasitology

Volume 148, January 2015, Pages 56-65
Experimental Parasitology

Full length article
A 78 kDa host cell invasion protein of Neospora caninum as a potential vaccine candidate

https://doi.org/10.1016/j.exppara.2014.10.006Get rights and content

Highlights

  • A T7 phage display cDNA library of Neospora caninum was constructed.

  • NcP78 and NcGRA7 proteins were identified from library screening.

  • NcP78 and NcGRA7 proteins could elicit both Th1 and Th2 immune responses.

  • The immune serum had inhibitory effects on invasion to Vero cells in vitro.

  • NcP78 and NcGRA7 could confer protective efficacies against challenge in mice.

Abstract

Neosporosis is an intracellular protozoan disease caused by Neospora caninum. Until now, there is no effective vaccine to prevent neosporosis. The host cell binding protein has the potential as neosporosis vaccine. In the present study, a T7 phage display library was constructed and screened using Vero cells to obtain host cell binding protein of N. caninum. Two host cell binding proteins, a hypothetical protein of 78 kDa (named as NcP78) homologous to the acylglycerol lipase of Toxoplasma gondii ME49 (XP_002370319.1) and NcGRA7 (known as a dense granules protein that is involved in the invasion of N. caninum to the host cells), were identified. Immune responses induced by recombinant NcP78 and NcGRA7 proteins and their protective efficacies against homologous challenge in BALB/c mice were evaluated respectively. Results showed that recombinant NcP78 and NcGRA7 could elicit both Th1 and Th2 immune responses (with the elevated levels of IgG1 and IgG2a antibody), but predominately a Th2 immune response with a high level of IgG1. The ani-NcP78 and anti-NcGRA7 serum also had inhibitory effects on N. caninum invasion to Vero cells in vitro, which indicated that both NcP78 and NcGRA7 proteins were involved in host cell invasion. Recombinant NcP78 and NcGRA7 could not prolong the survival times and improve the survival rates of dams, but could prolong the survival times and improve the survival rates of offspring significantly. Moreover, the recombinant NcP78 and NcGRA7 could reduce the brain parasite load of dams and offspring. Though these protein vaccines could not effectively alleviate the symptom of abortion, they could increase the number of born offspring significantly, indicating that Nc78 and NcGRA7 recombinant proteins could provide a partial protection against N. caninum infection in mice.

Introduction

Neosporosis is an intracellular protozoan disease caused by Neospora caninum, a parasite closely related to Toxoplasma gondii. Neosporosis has been reported in various species of animals worldwide, including cattle, dog, sheep, goat, deer and horse as reviewed by Dubey and Lindsay (1996). Neosporosis is of significant economic importance because it can cause abortions in cattle. However, ways for prevention and treatment of neosporosis are still limited.

Up to now, various vaccines for neosporosis have been explored, including live vaccines, killed or inactivated parasite vaccines and recombinant vaccines. Though some live vaccines have been developed (Bartley et al, 2008, Marugán-Hernández et al, 2011, Ramamoorthy et al, 2006, Rojo-Montejo et al, 2012, Williams et al, 2007), their general appeal and distribution are limited due to the risks of contamination with other animal pathogens, the higher cost of production, and a limited shelf-life (Reichel and Ellis, 2009). Weber et al. (2013) found that live Nc-Nowra tachyzoites of N. caninum could confer a protection up to 85.2%, indicating an effective method of preventing neosporosis in cattle based on live vaccination. Vaccines based on killed parasite antigens (Moore et al., 2005) or on whole parasite are also developed. Although a vaccine based on whole killed tachyzoites (Bovilis-Neoguard, Intervet) was commercialized, it only prevented 61% abortions in one of five herds and may increase the risk of early embryonic death (Weston et al., 2012). Moreover, this vaccine has been withdrawn from the market for several years.

In recent years, N. caninum recombinant vaccine has been proposed and significant progress has been made. Some N. caninum antigens have been evaluated as vaccine candidates, including the surface protein NcSAG1 and NcSRS2 (Cannas et al., 2003a), dense granules protein NcGRA1 and NcGRA2 (Ellis et al., 2008), NcGRA6 (Ramamoorthy et al., 2006), NcGRA7 (Liddell et al., 2003) and NcMAG1 (Debache et al., 2010), microneme protein NcMIC10 (Ellis et al., 2008), NcMIC1 and NcMIC3 (Alaeddine et al, 2005, Cannas et al, 2003b), rhoptry protein NcROP2 (Monney et al., 2011), apical membrane protein NcAMA1 (Zhang et al., 2007), cyclophilin (Tuo et al., 2011) and so on. Ellis et al. (2008) evaluated recombinant proteins (GRA1, GRA2, MIC10, and p24B) of N. caninum as vaccine candidates in a mouse model, and found that the mixture of MIC10 and a novel protein p24B could produce a partial protection against transplacental transmission of N. caninum. However, the protective immunity was not as potent as live vaccine based on a naturally attenuated isolate (NC-Nowra), and the author doubted the protective efficacy of recombinant protein vaccine against abortion.

Host cell binding protein is involved in adhesion and invasion to host cells. Vaccine based on host cell binding protein could inhibit the invasion of N. caninum to host cells and offer protection against Neosporosis (Hemphill et al., 2013). Up to now, the identified adhesion or invasion proteins of N. caninum include: NcMIC3 (Naguleswaran et al., 2001), NcSRS2 (Haldorson et al, 2005, Haldorson et al, 2006), disulfide isomerase (Naguleswaran et al., 2005), apical membrane antigen 1 (Zhang et al., 2007), NcROP2 (Debache et al., 2008), a microneme adhesive repeat (MAR) containing protein (Friedrich et al., 2010), NcMIC2-like1 (Pereira et al., 2011), immune mapped protein 1 (Cui et al., 2012), Hsp20 (Coceres et al., 2012), NcROP2Fam-1 (Alaeddine et al., 2013), Nc-p43 (Hemphill et al., 1997), Nc-p33 (Hemphill et al., 1998), a cell surface-associated glycoprotein (p36) (Sonda et al., 1998), Nc-70 kDa protein (Nishikawa et al., 2000), and NcMIC1 (Keller et al., 2002). Among them, SRS2 and NcROP2 have been evaluated as protein vaccine candidates. SRS2 could decrease congenital transmission among immunized mice by eliciting a predominately Th2 immune response (Haldorson et al., 2005), while the vaccine based NcROP2 could reduce mortality and cerebral infection in mice by inducing a protective Th-1- or Th-2-biased immune response.

Phage display library has been an important tool for the study of proteins involved in the interactions between parasite and the host cells (Guo et al, 2009, Keizer et al, 2003, Ren et al, 2013). This technique has facilitated in the discovery of the binding targets (Tonelli et al., 2012) or vaccine antigens in parasites, such as nematode (Ellis et al., 2012), Trichinella spiralis (Cui et al., 2013), Schistosoma mansoni (Rao et al., 2003), T. gondii (Hoe et al., 2005) and malaria (Fu et al, 1997, Keizer et al, 2003). As for N. caninum, Dong et al. (2013) selected two murine antibodies against NcSAG1 using phage-display technology that can be applied on the detection of N. caninum.

In the present study, a T7 phage display library of N. caninum tachyzoite was constructed and then screened. Recombinant protein vaccines based on two host cell binding proteins (NcP78 and NcGRA7) from the screening were then prepared using pGEX-4T-1 and pET-28a expression vectors. After immunization in mice, the inhibitory effects of ani-NcP78 and anti-NcGRA7 serum on invasion to the Vero cells in vitro were examined. Moreover, the immune responses and protective efficacies of the two recombinant protein vaccines against homologous challenge were also evaluated.

Section snippets

Cultivation and purification of N. caninum tachyzoites

N. caninum (NC-1 isolate) tachyzoites were maintained in Vero cells and purified as previously described (Tuo et al., 2005). Briefly, N. caninum tachyzoites infected cells were collected and passed through a 20-gauge and a 27-gauge needles. Tachyzoites were then purified by centrifugation (2000 × g, 30 min, 4 °C) over a 40% Percoll gradient (Sigma-Aldrich, USA). The tachyzoite pellet was washed using PBS (pH 7.4) and then kept at −80 °C.

The construction of T7 phage display library of N. caninum tachyzoite

The T7 phage display library of N. caninum tachyzoite was

The construction and biopanning of T7 phage display library of N. caninum tachyzoite

A T7 phage display library of N. caninum with a titer of 5.24 × 1012 pfu/ml was constructed. PCR screening showed that all of the randomly picked clones contained a cDNA insert ranging from 500 to 2000 bp in size.

After screening of the library, two positive plaques were obtained. DNA Blast analyses of the two positive plaques indicated that one sequence had 99% homology with the N. caninum liverpool hypothetical protein mRNA (XM 003879607.1, named NcP78 in this paper) and the other had 100%

Discussion

Phage display library has been an important tool for the study of the interactions between parasite and the host cells and for the screening of vaccine antigens of parasites. Though some vaccine antigens of N. caninum were obtained by cDNA expression library, such as AMA1 (Zhang et al., 2007), GRA1 and NcP20 (Atkinson et al., 2001), MIC3 (Sonda et al., 2000), GRA2 (Ellis et al., 2000), protein disulfide isomerase (PDI) and ribosomal protein 1 (RP1) (Liao et al., 2005), heat-shock protein 70

Acknowledgments

Project support was provided by the “National Key Basic Research Program (973 program) of China” (Grant No. 2015CB150300) and Department of Jilin Provincial Science and Technology of China (no. 20100222 and no. 20130521005JH).

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