Serum antibody response to Human papillomavirus (HPV) infections detected by a novel ELISA technique based on denatured recombinant HPV16 L1, L2, E4, E6 and E7 proteins

Abstract

Background: Human papillomaviruses (HPVs) are the primary etiological agents of cervical cancer and are also involved in the development of other tumours (skin, head and neck). Serological survey of the HPV infections is important to better elucidate their natural history and to disclose antigen determinants useful for vaccine development. At present, the analysis of the HPV-specific antibodies has not diagnostic value for the viral infections, and new approaches are needed to correlate the antibody response to the disease outcome. The aim of this study is to develop a novel ELISA, based on five denatured recombinant HPV16 proteins, to be used for detection HPV-specific antibodies.

Methods: The HPV16 L1, L2, E4, E6 and E7 genes were cloned in a prokaryotic expression vector and expressed as histidine-tagged proteins. These proteins, in a denatured form, were used in ELISA as coating antigens. Human sera were collected from women with abnormal PAP smear enrolled during an ongoing multicenter HPV-PathogenISS study in Italy, assessing the HPV-related pathogenetic mechanisms of progression of cervical cancer precursor lesions. Negative human sera were collected from patients affected by other infectious agents. All the HPV-positive sera were also subjected to an avidity test to assess the binding strength in the antigen-antibody complexes.

Results: Most of the sera showed a positive reactivity to the denatured HPV16 proteins: 82% of the sera from HPV16 infected women and 89% of the sera from women infected by other HPV genotypes recognised at least one of the HPV16 proteins. The percentages of samples showing reactivity to L1, L2 and E7 were similar, but only a few serum samples reacted to E6 and E4. Most sera bound the antigens with medium and high avidity index, suggesting specific antigen-antibody reactions.

Conclusion: This novel ELISA, based on multiple denatured HPV16 antigens, is able to detect antibodies in women infected by HPV16 and it is not genotype-specific, as it detects antibodies also in women infected by other genital HPVs. The assay is easy to perform and has low cost, making it suitable for monitoring the natural history of HPV infections as well as for detecting pre-existing HPV antibodies in women who receive VLP-based HPV vaccination.

Figure 1

Analysis of purified proteins by SDS-PAGE. The purified HPV16 proteins E4, E6, E7, L1 and L2 were run on a polyacrilamide gel electrophoresis and stained by Coomassie blue. Each protein is indicated on the top of the corresponding lane. The weight of the molecular mass markers (lane M) is indicated on the left of the figure.

Figure 2

ELISA based on HPV16 L1, L2, E4, E6 and E7 coating antigens. Detection of specific immunoglobulins to the HPV16 proteins in sera of women infected by HPV 16 (empty dots) or by other HPVs (black dots). The protein used as coating antigen is indicated on each panel. In abscissa is reported the number of each serum as listed in Tab.2; in ordinate is reported the optical density of each reaction.

Figure 3

Comparison of the percentages of sero-reactivity. The percentages of sera reacting to the HPV16 L1, L2, E4, E6 and E7 proteins are shown; the percentages of the HPV16 serum group are in white bars and those the other HPVs serum group are in black bars. The percentages of negative sera in the two groups (Neg) are also given.