Immunology Letters, 33 (1992) 289-294 0165 - 2478 / 92 / $ 5.00 © 1992 Elsevier Science Publishers B.V. All rights reserved IMLET 01834

A monoclonal antibody directed against the sporozoite stage of Plasmodium vivax binds to liver parenchymal cells Pascal Millet a, Carlo Chizzolini a, Norman J. Pieniazek, a Yupin Charoenvit b, Kei-ichiro Nakamura c, Masamichi Aikawa c, Trevor R. Jones b, Stephen L. H o f f m a n b a n d William E. Collins a aDivision of Parasitic Diseases, Centerfor Infectious Diseases, Centersfor Disease Control, Atlanta, GA, USA; bMalaria Program, Infectious Diseases Department, Naval Medical Research Institute, Bethesda, MD, USA; and eDepartment of Pathology, Case Western University, Cleveland, OH, USA (Received 12 June 1992; accepted 17 June 1992)

1.

Summary

The circumsporozoite (CS) protein of malaria parasites is a major surface protein of the sporozoite stage. In the process of investigating the immunogenicity of this protein in the Plasmodium vivax complex, we found that a monoclonal antibody (mAb) directed against the CS protein of isolates of P. vivax recognizes New World monkey hepatocytes and human hepatoma cells HepG2A16 in Western blot and by immunoelectron microscopy. The mAb NVS3 binds to the amino acid sequence AGDR, which is also shared with the ~t3 domain of the human and primate major histocompatibility complex class I. In addition, in vitro experiments suggest that the binding of the mAb NVS3 to hepatocytes from Saimiri monkey enhances the invasion or development of malaria sporozoites. These results form the basis for investigating the relationships between parasite surface proteins and host-cell receptors.

Key words: Plasmodium vivax; Major histocompatibility complex Class I; Hepatocyte; Monoclonal antibody

Correspondence to: Pascal Millet, Division of Parasite Diseases, Center for Infectious Diseases, Centers for Disease Control, Atlanta, GA 30333, USA.

2.

Introduction

The circumsporozoite (CS) protein of the sporozoite stage of malaria parasites is a target for malaria vaccine development [1]. The central third of all CS protein consists of repetitive amino acid sequences that contain immunodominant B cell epitopes [2]. The repeat region of the CS protein of most of the isolates of P. vivax, including Salvador I (Sal I), Chesson, and Belem strains, is characterized by the repeated sequences D R A D G Q P A G or DRAAGQPAG [3]. Passive transfer to Saimiri monkeys of NVS3, an IgG3 monoclonal antibody (mAb) directed against the A G D R sequence contained within the nine-amino acid repeated sequences, protects against sporozoite-induced malaria [4]. Moreover, sera obtained from these animals just prior to challenge inhibit the in vitro development of P. vivax liver-stage parasites in primary culture of Saimiri monkey hepatocytes [4]. These findings have led to efforts to construct synthetic peptide vaccines designed to produce antibodies to AGDR. However, a recent study has shown that the repeat region of the CS protein of the VK247 strain of P. vivax from Thailand does not include the sequence A G D R [5]. If antibodies to A G D R do not inhibit invasion of hepatocytes by VK247 sporozoites, a multi-epitope vaccine may be necessary. Here we report an enhancing effect of NVS3 on the development 289

of P. vivax, VK247' strain, sporozoites in primary culture of Saimiri monkey hepatocytes, and demonstrate that the MAb NVS3 interferes between the parasite and the hepatocyte by binding to hepatocyte molecules. 3.

3.1.

Materials and Methods

In vitro culture o f Siamiri monkey hepatocytes and mAbs

The liver-stage parasite assay was performed using previously developed techniques [6]. Sporozoites were obtained from dissected salivary glands of infected Anopheles dirus mosquitoes previously fed on blood from chimpanzees (Pan troglodytes) infected with gametocytes of P. vivax, Sal I or VK247 strains. Sporozoites were counted and suspended in culture medium (Gibco, Grand Island, New York; formula #850227AK) supplemented with 10% fetal bovine serum, 2 g/1 bovine serum albumin, 10 mg/1 bovine insulin, 0.1 mM nonessential amino acids, 292 mg/1 L-glutamine, 150 U/ml penicillin, and 150 #g/ml streptomycin. 30 #1 of sporozoite suspension containing approximately 60000 sporozoites was incubated with 30 #1 of the following mAbs diluted in phosphate-buffered saline (PBS) for 15 min at room temperature: NVS3 (IgG3), directed against the repeat region of the CS protein of P. vivax, Salvador I strain [4]; 3G6 (IgM), directed against the repeat region of the CS protein of P. vivax, VK247 strain [5]; 2A10 (IgG2a), recognizing the sequence NANP on P. falciparum sporozoites [6]; and W6/32 (IgG2a), reactive with a monomorphic determinant on the Major Histocompatibility Complex (MHC), HLA-A, -B, and -C molecules [7]. The sporozoites were then overlaid on hepatocyte monolayers (30000 sporozoites per monolayer containing 50000 hepatocytes). Hepatocytes were obtained from a liver biopsy from an uninfected Saimiri monkey. After 2 h, sporozoites and plasma were removed, and medium was added to culture dishes. This medium was replaced 1 h later and then changed daily. After incubation for 7 days at 37°C in 5% CO2, the cultures were fixed with methanol and Giemsa-stained. Schizonts were counted with the identity of each plate concealed. 290

3.2.

Western blot

Western blotting [8] was performed as follows. Freshly dissociated hepatocytes were solubilized in reducing sample buffer (0.06 M Tris-HC1, 5% sodium dodecyl sulfate (SDS), 0.7 M 2-mercaptoethanol) and then incubated for 15 rain at 75°C. After vertical electrophoresis in SDS gel (3.3% - 20%), the polypeptides were electrophoretically transferred to nitrocellulose paper. Strips of nitrocellulose paper were reacted overnight at 4°C with mAb NVS3 (A,D) or with two unrelated mAbs from other malarial parasites: 2A10 (P. falciparum) directed against the sequence (NANP)n (B,E), and 12D6 (P. cynomolgi, B strain) directed against the sequence (NAGG)n (C,F), diluted at 1:100 in PBS containing 0.3% Tween-20. After being incubated for 1 h at room temperature with peroxidase-conjugated goat antibodies to mouse IgG diluted in PBS/Tween, the strips were washed, and the antibodies bound to the strips were detected by staining with diaminobenzidine and hydrogen peroxide. Molecular weights were estimated from control-labeled proteins (Bethesda Research Laboratory, Gaithersburg, MD).

3.3.

Immunoelectron microscopy

Immunoelectron microscopy [9] was carried out as follows. HepG2/A16 cells and Aotus hepatocytes were fixed with 1% paraformaldehyde and 0.2% glutaraldehyde in PBS for 40 min at 4°C. After dehydration, samples were embedded in LR Gold resin (Polyscience, N J) at -20°C. Sections mounted on nickel grids were incubated for 30 min with PBS containing 5% non-fat dry milk (Camation) and 0.01% Tween before being immunostained, first with NVS3 (500 #g/ml) overnight at 4°C and then with a goat anti-mouse IgG conjugated with 15 nm gold particles as a secondary antibody. Grids were examined with a Zeiss CEM902 electron microscope after being double stained with uranyl acetate and lead citrate. Sequence similarity searches with the amino acid sequence AGDR were done using the Fasta program [10] to scan the SwissProt data bank [11], release 17.0 as of March 1991.

Mabs PB8 control NVSS (soo ug/ml) age (0.0S ug/m0

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A monoclonal antibody directed against the sporozoite stage of Plasmodium vivax binds to liver parenchymal cells.

The circumsporozoite (CS) protein of malaria parasites is a major surface protein of the sporozoite stage. In the process of investigating the immunog...
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