1 Department of Medical Microbiology, University Hospital, 20502 Malmö, Sweden
2 Institute of Medical Microbiology and Hygiene, University of Mainz, 55101 Mainz, Germany
3 Department of Microbiology and Immunology, The Milton S. Hershey Center, 500 University Drive, Hershey, PA 17033, USA
Correspondence
Joakim Dillner
joakim.dillner{at}mikrobiol.mas.lu.se
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ABSTRACT |
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INTRODUCTION |
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As HPVs do not grow efficiently in cell culture, systems using HPV VLPs have been used to study viral infectivity (Roden et al., 1996; Rossi et al., 2000
; Stauffer et al., 1998
; Unckell et al., 1997
). Cell surface heparan sulfates are essential for infection by HPV16 and HPV33 pseudovirions (Giroglou et al., 2001
) as well as HPV11 virions (Shafti-Keramat et al., 2003
) and for binding and uptake of VLPs of various HPV-types (Combita et al., 2001
; Drobni et al., 2003
; Joyce et al., 1999
; Selinka & Sapp, 2003
). Heparin, a highly sulfated form of heparan sulfate proteoglycan secreted by mast cells, interferes with pseudoinfection and has been shown to efficiently bind VLPs (Giroglou et al., 2001
; Joyce et al., 1999
). The interaction with heparin requires intact conformation of VLPs (Giroglou et al., 2001
; Rommel et al., 2004
).
VLP-based serology is of widespread importance for studies of HPV epidemiology and vaccinology. VLP ELISAs have been successfully used in epidemiological studies of HPV infections in different populations (Dillner, 1999). However, incorrectly folded VLPs expose cross-reactive epitopes that may compromise the type specificity of VLP-based HPV serological assays (Wang et al., 1997a
), a problem recently highlighted in a study with HPV16/11 hybrid VLPs (Wang et al., 2003
).
An alternative method to avoid background caused by incorrectly folded VLPs is to use type-specific mAbs against conformational epitopes as catching antibodies in the ELISAs (Carter et al., 1994). However, we have previously found that adequate isotype specificity of ELISAs is improved by using mAbs specific for different human Ig isotypes (Heino et al., 1995
; Wang et al., 2000
). Also, two-step detection of bound antibodies (with a mouse mAb and an anti-mouse reagent was important for sensitivity (Heino et al., 1995
). Two-step methods cannot be used if ELISA plates have been coated with mouse mAbs. Finally, type-specific mAbs to HPV are only available for a limited number of HPV-types.
In the present study, we evaluated whether heparin-coated plates could be used to improve performance of VLP serology. HPV16/11 hybrid VLPs that had significant reactivity with children's sera and a batch of HPV18 VLPs that had failed the quality control because of significant reactivity with sera from virginal women were tested in parallel with heparin-based ELISA, direct ELISA and type-specific mAb capture ELISA using positive- and negative-control validation panels of serum samples.
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METHODS |
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Serum samples.
Serum samples that were HPV16+/HPV11 or HPV16/HPV11+ were selected from three previous serological studies. Study 1 investigated attack rates and seroprevalences of human papillomavirus types 11, 16 and 33 among a high-risk population for cervical cancer: in the Shanxi province, China. In total, 1196 high school students/teachers (570 men and 626 women, mean age 16 years, range 1435 years) and 170 couples (mean age 24 years, range 2035 years) attending the mandatory serological screening before marriage donated serum samples at enrolment and 12 months later. Study 2 investigated the human papillomavirus antibody response among 216 women (mean age 53 years, range 2388 years) with untreated primary invasive cervical cancer admitted to the Karolinska Hospital, Stockholm, Sweden. Study 2 also contained 243 age and sex-matched healthy control women (Wang et al., 1997b). Study 3 investigated sexual behaviour and the risk of HPV infection in 188 serum samples from 94 teenage girls (mean age 16·1 years) donated at enrolment and at a follow-up 2 years later. Forty-five samples were from virgins, whereas the other samples were from sexually experienced girls. None of the virgin girls had HPV DNA in the cervix (by PCR) or serum antibodies against HPV16 or HPV33 (Andersson-Ellstrom et al., 1996
). HPV16+/HPV11 and HPV16/HPV11+ samples from these studies were re-tested with HPV16 and HPV11 in parallel with the hybrid VLPs. Besides these, children's serum samples were selected from a study that surveyed the seroprevalence of human papillomavirus types 16, 18 and 33 among 1031 children up to 13 years of age, whose serum samples had been submitted for serological analyses of non-HPV-related diseases and were obtained from the Department of Clinical Virology of the Karolinska Hospital and the Department of Pediatrics of the Huddinge Hospital, both in Stockholm, Sweden (af Geijersstam et al., 1999
). Serum samples from children in the age group 210 years that had been found to be HPV16 seronegative were also tested with HPV11 and re-tested with HPV16 in parallel with the hybrid VLPs to ensure that they were both HPV16 and HPV11 seronegative. In total, 32 HPV16+/HPV11 serum samples, 64 HPV16/HPV11+ serum samples and 23 HPV16/HPV11 serum samples were included in this study. In the case of HPV18, serum samples from 13 women who had tested HPV18 DNA+ by PCR of cervical samples and seven serum samples from HPV18 DNA virginal women were analysed.
ELISA methods.
In heparin-based ELISA, intact HPV16, HPV11 capsids and hybrid VLPs H11 : 16[172505] (contains the N-terminal part of HPV11 and the C-terminal part of HPV16), H11 : 16[FG+HI] (two non-contiguous hypervariable regions of HPV16 L1 containing the FG and HI loops transplanted into HPV11 L1 backbone) and H16 : 11[171505] (contains the N-terminal part of HPV16 and the C-terminal part of HPV11) were coated onto heparin-BSA microtitre plates in PBS at pH 7·2. The coating concentrations of VLPs were normalized as described (Wang et al., 2003). The low quality HPV18 batch was coated at 1 µg ml1.
For generation of disrupted VLPs, three different disruption methods were used: (i) alkali treatment: the VLPs were incubated for 4 h at room temperature in 0·1 M carbonate buffer (pH 9·6); (ii) alkali and reducing agent treatment: the VLPs were incubated for 30 min at room temperature in 0·2 M Na2CO3 (pH 10·6) and 0·01 M DTT; (iii) boiling, alkali and reducing agent treatment: the VLPs were heated for 15 min at 96 °C in 0·2 M Na2CO3 (pH 10·6) and 0·01 M DTT. Disrupted bovine papillomavirus (BPV) capsids were used as a negative control in all assays.
After 1 h at 37 °C, plates were washed three times with 150 µl PBS-T and blocked by adding 150 µl 10 % horse serum in PBS (HS-PBS) at 37 °C for 15 min. Serum samples were added in duplicate at a 1 : 30 dilution in HS-PBS for 1 h reaction at 37 °C. Six wells in each plate were incubated with only 10 % HS-PBS. Following three washes with PBS-T, a mAb to human IgG, 1 : 800 diluted in 10 % HS-PBS, was added. The mAb positive controls (V5 at a 1 : 1 000 000 dilution, H11.F1 at a 1 : 100 000 dilution, and H11.H3 at a 1 : 10 000 dilution) were added to the wells that had been incubated only with HS-PBS. The plates were incubated for 45 min at 37 °C. After washing three times, a goat anti-mouse IgG HRP conjugate, 1 : 2000 diluted in HS-PBS, was added for 30 min at 37 °C, followed by the addition of a substrate. The absorbance of each serum with disrupted BPV VLPs was subtracted from the HPV VLP reactivity and the mean value of the duplicates was calculated.
For estimation of antibody levels in units, the serum samples were tested in duplicate with three different dilutions (1 : 10, 1 : 31·6, 1 : 100), the mAb to human IgG 1 : 500 diluted in 10 % HS-PBS was added, and the plates were read after 60 min. The absorbance of each serum with disrupted BPV was subtracted from the VLP reactivity, the mean value of the duplicates was calculated, and then the OD values were transformed into ELISA units using the PLL (parallel line) method (Grabowska et al., 2002).
For capture ELISAs, the HPV16 mAb V5 diluted 1 : 1000 in cold PBS (pH 7·2) or the HPV18 N-mAb R5, diluted 1 : 2000, were coated onto microtitre plates overnight. After the plates were washed once with PBS-T and blocked with 10 % HS-PBS for 1 h at room temperature, VLPs were added for 2 h at room temperature. Following five thorough washings of the plates, serum samples were added either in duplicate at a 1 : 30 dilution or or in duplicate at 1 : 10, 1 : 31·6 and 1 : 100 dilutions for 2 h at room temperature. A rabbit anti-human IgG HRP conjugate (DAKO) was diluted 1 : 1000 in HS-PBS with normal mouse serum (25 µl ml1) and rotated end-over-end for 4 h. In-between washing of the plates, the conjugate was added to the plates and incubated for 1 h at room temperature, followed by addition of substrate. Plates handled identically, except that no VLPs were added, were used as background.
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RESULTS |
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Reactivity of HPV16/11 hybrid VLPs with human serum samples in heparin-based and capture ELISAs
Thirty-two HPV16+/HPV11 serum samples, 64 HPV16/HPV11+ serum samples and 23 HPV16/HPV11 children's serum samples were tested in heparin-based ELISA in comparison with direct ELISA for reactivity with HPV16 and HPV11 VLPs and with the three hybrid VLPs. Similar experiments with direct ELISAs have been published previously (Wang et al., 2003).
The antibody responses among HPV16/HPV11+ human sera to hybrid VLPs were rather similar in heparin-based and direct ELISAs (Table 2). The HPV11+/ HPV16 sera had very similar reactivity with HPV11 and hybrids that contained the HPV11 C terminus (H11 : 16[FG+HI] and H16 : 11[171505]) (Fig. 1
). There were minor differences in correlation depending on whether heparin-based or direct ELISAs had been used. For example, H11 : 16[FG+HI] versus HPV11 had a correlation coefficient in direct ELISA of 0·58 (Wang et al., 2003
), in heparin-based ELISA of 0·68. The correlation coefficient between hybrid H16 : 11[171-505] to HPV11 was 0·71 for direct ELISA (Wang et al., 2003
) and 0·63 for heparin-based ELISA (Fig. 1
).
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The level of the antibody responses among HPV16+/HPV11 sera to all three hybrid VLPs were remarkably decreased and the correlation of the reactivity of HPV16+/HPV11 human sera against HPV16 as compared with the reactivity against all three hybrids to HPV16 was lower in heparin-based ELISA than in direct ELISA (Table 2). For example, H11 : 16[172505] versus HPV16 had a correlation coefficient in direct ELISA of 0·78 (Wang et al., 2003
), in heparin-based ELISA of 0·60 (Fig. 2
).
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DISCUSSION |
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The HPV11+/HPV16 human sera had very similar reactivity with HPV11 and HPV16/11 VLP hybrids that contained the HPV11 C terminus (H11 : 16[FG+HI] and H16 : 11[171505]) in both heparin-based and direct ELISAs, suggesting that the major human serum-reactive HPV11 epitope(s) is present in these VLPs and that VLPs containing this epitope are heparin-binding.
In contrast, the data for HPV16 were much less clear. The reactivities were much lower in the heparin-based ELISAs and showed lower correlations with the reactivities against the parent virus when heparin-based ELISAs were used. The hybrid virus H11 : 16[FG+HI] was unique in that adequate disruption appeared to require exceptionally harsh disruption methods. Also, this VLP was the only one from which the results of heparin-based and capture ELISAs where distinctly different. Capture ELISAs did not improve background reactivity with children's sera, whereas heparin ELISA did. Conceivably, the artificial addition of the V5-binding site to an HPV11 backbone may have also resulted in the V5 epitope being exposed on incorrectly folded H11 : 16[FG+HI] VLPs.
Quality control of ELISAs depends on maintaining intact VLPs of good quality and a quality assurance system. We found that the mAbs that reacted with disrupted virus in direct ELISAs did not react with the same disrupted viruses if heparin-coated plates were used, suggesting that the use of heparin-coated microtitre plates could add a quality control component to the VLP-based ELISAs by preventing binding of denatured L1 antigen and contaminating protein. Numerous approaches towards decreasing non-specific binding to ELISA plates have been described, notably the use of blocking with vinyl polymers (Studentsov et al., 2002). The approach with heparin-coated plates has the advantage that a specific biological property of the virus is exploited to increase the specificity of binding. VLPs of all HPV-types tested so far bind to heparin with similar efficiency (Rommel et al., 2004
), suggesting that heparin can be used as a universal capture molecule for HPV serology.
In summary, heparin-coated plates will select a subset of VLPs with intact heparin-binding activity. At least for some HPVs, intact heparin-binding ability was found to be correlated with serological specificity, allowing for a straightforward and simple improvement of the performance of HPV serology.
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ACKNOWLEDGEMENTS |
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Received 26 July 2004;
accepted 1 October 2004.