The authors have declared that no competing interests exist.
Conceived and designed the experiments: MS GB FvdK. Performed the experiments: MS RS. Analyzed the data: MS GB FvdK. Contributed reagents/materials/analysis tools: MM GB FvdK. Wrote the manuscript: MS GB FvdK CM.
In order to assess HPV-specific IgG characteristics, we evaluated multiple aspects of the humoral antibody response that will provide insight in the HPV humoral immune response induced by HPV infection and vaccination.
Cross-reactivity of HPV-specific antibodies induced by infection or vaccination was assessed with VLP16 or 18 inhibition using a VLP-based multiplex immunoassay (MIA) for HPV16, 18, 31, 33, 45, 52 and 58. HPV16/18 specific IgG1-4 subclasses and avidity were determined with the VLP-MIA in sera after HPV infection and after vaccination. Neutralizing antibodies were determined in a small subset of single-seropositive and multi-seropositive naturally derived antibodies.
Naturally derived antibodies from single-positive sera were highly genotype-specific as homologue VLP-inhibition percentages varied between 78-94%. In multi-positive sera, cross-reactive antibodies were observed both within and between α7 and α9 species. After vaccination, cross-reactive antibodies were mainly species-specific. Avidity of vaccine-derived HPV-specific antibodies was 3 times higher than that of antibodies induced by HPV infection (
Naturally derived HPV-specific antibodies from single-positive samples showed different characteristics in terms of cross-reactivity and neutralizing capacity compared with antibodies from multi-positive sera. Post-vaccination, HPV antibody avidity was approximately 3 times higher than antibody avidity induced by HPV infection. Therefore, antibody avidity might be a potential surrogate of protection.
Persistent infection with high-risk (hr) human papillomavirus (HPV) is a necessary event of cervical cancer. Almost all cervical cancers are HPV DNA-positive to at least one of the 15 hr-HPV types that can cause genital infections. Most hr-HPV types cluster together in the α7 and α9 species [
After an incidental HPV infection, only 50-70% of the infected individuals develop detectable HPV-specific antibodies in serum mainly directed against the L1 capsid protein of the virus [
HPV vaccines provide a degree of cross-protection against persistent infection and/or high-grade lesions (CIN2+) attributed to some of these non-vaccine HPV genotypes, particularly HPV31, 33 and 45, but probably not HPV52 and 58 [
At present, no immune parameter or antibody concentration has been defined that correlates with protection. While the HPV-specific antibody levels in response to HPV infection or HPV vaccination have been previously described [
Serum samples were available from a large cross-sectional population-based surveillance study (PIENTER-2), performed in 2006-07 in the Netherlands. This serumbank contained samples from individuals 0-79 years of age and at low risk for HPV infection [
From this serosurveillance study, a panel of naturally HPV infected individuals was selected that included HPV single-positive (
Some infants and children (up to 10 years of age) in the large serosurveillance study were HPV antibody seropositive. To investigate the quality of the antibodies we tested in the PBNA some sera of infants and children, who were part of the previous mentioned selection (n=11).
From a prospective cohort study in Rwanda, Africa [
From an HPV vaccine monitoring study (HAVANA study, HPV amongst vaccinated and non-vaccinated adolescents [
HPV-specific IgG antibodies against L1 VLP16, 18, 31, 33, 45, 52 and 58 were measured using a VLP-MIA [
Homologous inhibition of HPV-specific antibodies induced by infection was assessed for single-positive sera with virus-like particle (VLP) 16, 18, 31, 33, 45, 52, and 58, separately. Inhibition of HPV antibodies in multi-positive sera from the Dutch serosurveillance study and sera from vaccinated girls was determined with VLP16 and 18 separately. VLPs were kindly donated by GlaxoSmithKline Vaccines. Sera were pre-diluted 1/100 and 1/200, respectively, and subsequently added to the same volume of PBS containing 10 µg/ml VLPs. Sera, containing vaccine-derived HPV-specific antibodies, were pre-diluted 1/10,000. After an incubation period at RT for 1 hour, all samples were analyzed using the VLP-based multiplex immunoassay (VLP-MIA). The inhibition index was determined as a percentage of the antibody levels post-inhibition in comparison with the antibody levels pre-inhibition, which were set at 100%.
Avidity of HPV16 and 18-specific IgG antibodies was assessed by using a modification of the VLP-MIA for determining HPV-specific IgG antibodies. HPV16 and 18-specific antibody concentrations in sera induced by HPV infection (HPV16
Ammonium thiocyanate (NH4SCN; Sigma-Aldrich, St. Louis, Missouri, USA) was used to dissociate low-avidity antigen-antibody binding [
Subsets of single-positive (
Data analyses were conducted using Graphpad Prism version 5. Percentage inhibition was calculated using the antibody concentrations pre- and post-inhibition of each tested sample. Subsequently, an average inhibition percentage was calculated. Significant differences (
Vaccine-derived antibody concentrations (LU/ml) pre- (dark grey dots) and post-inhibition (light grey dots) with VLP16 (A) or VLP18 (B) are shown. Dark grey lines indicate geometric mean concentrations. *
Homologous inhibition of vaccine-derived HPV16 and 18-specific antibodies was nearly complete and amounted to 99.7% and 99.6%, respectively (
After vaccination, cross-reactive antibody levels were mainly species-specific. A significant reduction in antibody levels of phylogenetically related HPV types was observed after VLP16 inhibition with inhibition percentages varying between 76% and 88% for HPV31, 33, 52 and 58 (
Antibody avidity (%) for HPV16 (A) and HPV18 (B) of HPV-specific single-seropositive and multi-seropositive naturally derived antibodies and HPV vaccine-derived antibodies are shown. The dark grey line indicates the mean antibody avidity. ***
2 types | 78 | 42 | 39 | 83 | 76 | 38 |
3 types | 80 | 23 | 44 | 72 | 62 | 55 |
4 types | 75 | 54 | 56 | 85 | 61 | 65 |
5 types | 69 | 47 | 44 | 78 | 48 | 43 |
6 types | 71 | 56 | 57 | 81 | 65 | 56 |
2-6 types | 75 | 45 | 48 | 80 | 62 | 51 |
The percentage of VLP16 an 18 inhibition within and between the α7 and α9 species are shown for sera antibody seropositive for 2 up to 6 HPV types.
Note: inhi = inhibition, α9: HPV16, 31, 33, 52, 58, α7: HPV18, 45.
Homologous inhibition of naturally acquired HPV-specific antibodies against a single HPV type amounted to 94.5% for HPV16 and 83.1% for HPV18 (
In contrast to vaccine-derived and naturally induced antibodies of single-positive sera, antibodies of multi-positive sera were less specific. In sera positive for HPV16 or HPV18 and up to 5 other HPV types, inhibition with VLP16 or 18 amounted to an average percentage of 75% and 80%, respectively (
A subset of sera with high homologous inhibition percentages (98% for HPV16 and 97% for HPV18), indicating that the antibodies were genotype-specific, was selected from the single- and multi-positive samples. In the single-positive sera from this subset, neutralizing antibodies were found in 5 out of 6 for HPV16 and in 4 out of 5 for HPV18 (
1 | 138 | 99 | 591 | 54 | |
2 | 105 | 98 | 297 | 54 | |
3 | 105 | 99 | 110 | 52 | |
4 | 99 | 100 | <40 | 10 | |
5 | 92 | 97 | 263 | 25 | |
6 | 41 | 97 | 100 | 65 | |
7 | 130 | 99 | 94 | 81 | |
8 | 96 | 97 | 108 | 3 | |
9 | 57 | 99 | 187 | 81 | |
10 | 49 | 94 | <40 | 1 | |
11 | 28 | 94 | 62 | 16 |
Antibody concentrations (LU/ml) assessed with the VLP-MIA, homologous inhibition percentages, neutralizing antibody levels (ED50) and antibody avidity indices are presented for HPV16 and HPV18.
Note: Conc, concentration, Inhi, inhibition, PBNA antibody levels <40 were not neutralizing
1 | 224 | 131 | 99 | 74 | <40 | <40 | 63 | |
2 | 142 | 42 | 98 | 98 | <40 | <40 | 4 | |
3 | 27 | 51 | 85 | 77 | <40 | <40 | 1 | |
4 | 27 | 28 | 97 | 68 | <40 | <40 | 37 | |
5 | 19 | 26 | 94 | 74 | 127 | <40 | 12 | |
6 | 9 | 29 | 79 | 69 | <40 | <40 | 24 | |
7 | 121 | 52 | 99 | 96 | 209 | <40 | 1 | |
8 | 40 | 162 | 97 | 94 | <40 | <40 | 53 | |
9 | 37 | 35 | 97 | 76 | <40 | <40 | 30 | |
10 | 25 | 20 | 96 | 61 | <40 | <40 | 15 |
Antibody concentrations (LU/ml) assessed with the VLP-MIA, homologous and heterologous inhibition percentages and neutralizing antibody levels (ED50) are shown for HPV16, 18, 31 and 45. Antibody avidity indices are presented for HPV16 and 18.
Note: Conc, concentration, Inhi, inhibition, PBNA antibody levels <40 were not neutralizing
Naturally derived HPV16-specific antibodies in serum samples of children 1-10 years of age (n=8) showed no neutralizing capacity while from the samples of 3 infants tested (0-6 months of age) in 1 case HPV-specific neutralizing antibodies were found.
HPV16 and 18-specific antibodies induced by HPV infections are mainly of low-avidity (
The most abundant antibody subclass of IgG induced after HPV16 and 18 infection was IgG1 (78% and 92%, respectively), followed by IgG3 (20% and 6%, respectively) (
% | 95%CI | % | 95%CI | % | 95%CI | % | 95%CI | ||
---|---|---|---|---|---|---|---|---|---|
IgG1 | 78 | 71-84 | 92 | 86-98 | 80 | 73-87 | 70 | 61-78 | |
IgG2 | 2 | 1-4 | 1 | 0-1 | 0 | 0-1 | 1 | 0-3 | |
IgG3 | 20 | 13-26 | 6 | 0-11 | 19 | 13-26 | 26 | 19-34 | |
IgG4 | 1 | 0-1 | 2 | 0-6 | 0 | 0-1 | 3 | 1-4 |
Proportions of IgG isotypes are expressed as percentages towards total HPV-specific IgG.
Note: CI, Confidence interval
Here we describe HPV-specific antibody characteristics after HPV infection and vaccination. Cross-reactivity of HPV antibodies derived after HPV vaccination was mainly species-specific. Naturally induced HPV-specific antibodies from single-positive sera were genotype-specific as expected and tended to be neutralizing. In contrast, antibodies of multi-positive sera were less genotype-specific, cross-reactive, and tended to be non-neutralizing. IgG1 was found the predominant subclass induced after HPV infection and vaccination, followed by IgG3. Moreover, post-vaccination antibody avidity was approximately 3 times higher than after HPV infection.
Vaccine efficacy against HPV16 and 18 infection sustains over 8 years post-vaccination [
It is unclear whether naturally derived HPV-specific antibodies can protect against subsequent homologous HPV infection or infection with phylogenetically related HPV types. In women, the protective role of HPV16 serum antibodies induced by HPV infection has been inconsistent in the literature, with moderate protection observed in a limited number of studies [
Cross-sectional serosurveillance studies [
With the HPV vaccine clinical trials performed so far, a level of HPV-specific antibodies that can be used as a correlate of protection is still not established as too few disease cases occurred within these trials and breakthrough infections could not be clearly distinguished from the reactivation of prevalent HPV infections [
Other immune parameters than antibody levels, that might correlate with protection, have not been defined and data on antibody characteristics such as avidity are scarce [
We found that antibody avidity after vaccination was 3 times higher than after HPV infection. After HPV infection, we observed a wide range of antibody avidity levels in sera of naturally infected individuals reflecting a great biological diversity in individual responses to HPV infection. Naturally induced HPV16 antibodies of low avidity have been associated with possible susceptibility to infection with other HPV types [
Our findings on IgG subclasses are in line with Harro et al. reporting that IgG1 was the predominant subclass induced after VLP vaccination [
In conclusion, we showed that vaccine-derived antibodies were mainly genotype-specific and cross-reacted only for a smaller part with other HPV types within the species. After HPV infection, single-seropositive antibodies were highly type-specific and neutralizing whereas multi-positive sera were less specific and tended to be non-neutralizing. This might mean that naturally induced HPV-specific antibodies in multi-HPV positive individuals could not or only partially protect against subsequent HPV infection and these individuals are still at risk.
Although sample sizes were small, neutralizing antibodies in single-positive sera inclined to be associated with higher antibody avidity indices than non-neutralizing antibodies. Further, we found the avidity of vaccine-derived antibodies to be approximately 3 times higher than after HPV infection. These results imply that the avidity of HPV antibodies might be used as a potential surrogate of protection. However, more studies are needed to establish the role of HPV antibody avidity as a potential surrogate of protection and the use of this immunological tool in sero-epidemiological and vaccine monitoring studies.
The Rwandan samples, used for the determination of IgG subclasses, were kindly supplied by the Project Ubuzima group, in Kigali, Rwanda. We would like to thank Sylviane Poncelet and Francis Dessy (GlaxoSmithKline vaccines, Belgium) who coordinated the PBNA analysis.