HPV antibody detection by ELISA with capsid protein L1 fused to glutathione S-transferase

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Abstract

An alternative enzyme linked immunosorbent assay (ELISA) system was developed to analyze antibodies to human papillomavirus capsid antigens. The assay uses glutathione crosslinked to casein to capture the major capsid protein L1 from human papillomavirus (HPV) types 6b, 16 and 18 fused to glutathione S-transferase (GST) as antigen. The method allows efficient one-step purification of L1 fusion protein from crude bacterial lysates on ELISA plates coated with glutathione casein. The GST-L1 capture ELISA detected HPV 16 antibodies with high type specificity. Comparison with the current “gold-standard” for L1-serology that uses virus-like particles (VLP) as antigen demonstrated similar assay sensitivity. Pairwise comparison of the absorbance values of 105 human sera obtained in the two ELISA formats for HPV 16 showed a R2 value of linear regression of 0.68. Conformity of the two ELISAs in classification of sera as HPV 16 L1 antibody-positive or -negative was verified with Cohen's kappa test, yielding a value of 0.62. These data indicate that the GST-L1 capture ELISA is similar in performance to the VLP ELISA. The ease of antigen production and purification in the GST-based ELISA will be advantageous to screen large sample numbers in vaccine trials or epidemiological studies examining immune responses to many HPV types in parallel.

Introduction

Human papillomaviruses (HPV) comprise a family of at least 82 different types (van Regenmortel et al., 2000) that infect epithelial cells. This restriction of viral replication to external differentiated epithelial cells may shield these infections from efficient interference by the host's immune system. Nevertheless, the majority of HPV-infections are cleared spontaneously. With most HPV types, persistent infection only causes a benign proliferative lesion but some HPV types, termed “high risk” types, can lead to the development of invasive cancer (zur Hausen, 1996).

Acute or persistent HPV infection is identified mainly by detection of HPV DNA in clinical specimens. Acute as well as past, cleared infection can be assessed by testing for antibodies to HPV capsid antigens, which probably measures cumulative exposure. Furthermore, in the case of transient infections, prior exposure to virus is not necessarily reflected in the current presence of HPV DNA. Thus, a valid approach to assess previous HPV infection is the detection of an immune response to HPV capsid antigens (van Doornum et al., 1998). Prophylactic vaccination based on HPV capsid antigens is currently developed for several HPV types and induction of capsid antibodies is used as a surrogate marker to evaluate vaccine efficacy (Schiller and Hildesheim, 2000).

HPV capsids consist of 72 capsomers which are pentamers of the major capsid protein L1 and can contain one minor capsid protein L2 in their center (Baker et al., 1991). The infection with HPV is followed by a serologic immune response to viral capsid proteins (for review see Dillner, 1999). Virus-like particles (VLP) self-assemble from L1 protein expressed in insect cells (Kirnbauer et al., 1992) or yeast (Sasagawa et al., 1995) and are morphologically similar to virions. Enzyme linked immunosorbent assays (ELISA) based on VLP were developed initially for HPV 16 (Kirnbauer et al., 1994) and since then this technique has been extended to other HPV types (Dillner, 1999, Combita et al., 2002). VLP-based ELISA require complex preparation and purification procedures for high quality VLP.

Recently, it was shown that L1 from HPV 11 and HPV 16 expressed in Escherichia coli forms capsomers that can be assembled in vitro into VLP (Chen et al., 2001, Chen et al., 2000, Li et al., 1997). Capsomers of HPV 11 were shown to react with conformation-specific antibodies and to induce neutralizing antibodies in rabbits (Rose et al., 1998) which indicates that capsomers present conformational immunogenic epitopes of virions. L1 proteins with glutathione S-transferase (GST) fused to their N-terminus (GST-L1) can form proper pentamers (Chen et al., 2000) and immunization with GST-L1, in which L1 was derived from the canine oral papillomavirus, protects dogs from high-dose infection of their oral mucosa with this virus (Yuan et al., 2001).

We developed recently a generic ELISA that uses glutathione crosslinked to casein as capture protein to purify and immobilize bacterially expressed GST fusion proteins as antigens in a single step to the solid phase. Using the early proteins E6 and E7 of HPV types 16 and 18 as model antigens the sensitivity and specificity of this technique to detect specific antibodies was demonstrated (Sehr et al., 2001). This simple and reliable GST capture ELISA was adapted to L1 proteins of HPV types 6b, 16 and 18 and show sensitive and HPV type-specific detection of vaccination-induced mouse antibodies to HPV 16 L1. Good agreement for the detection of infection-induced human antibodies was found for the established VLP-based and the new GST-based ELISA.

Section snippets

Recombinant proteins

A modified pGEX vector (Pharmacia) was constructed for expression of GST fusion proteins with an additional C-terminal fusion tag in E. coli. Therefore an oligonucleotide encoding the C-terminal undecapeptide (“tag”, amino acid sequence: KPPTPPPEPET) of the SV40 large T-antigen with a 5′-SalI-site and a 3′-NotI-site was cloned in an appropriately digested pGEX4T3 plasmid. HPV type 6b, 16 and 18 L1 coding sequences, all lacking the 10 N-terminal residues were amplified by PCR with SmaI/SalI ends

Results and discussion

L1 forms pentamers when its N-terminus is fused to GST (Chen et al., 2000). The GST-L1 proteins from HPV types 6b, 16 and 18 were expressed and treated further as described by Chen et al., 2000, Chen et al., 2001 to obtain pentameric L1 in a native form which presents conformational and intermolecular epitopes. To demonstrate efficient and specific binding of GST-L1 from the E. coli lysates to glutathione casein-coated ELISA plates, the plates were first incubated with the lysates of bacteria

Acknowledgements

We thank Elisabeth Schwarz for providing the genomic plasmid of HPV 6b and Robert Garcea for helpful suggestions on expression of GST-L1.

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