Human Papillomavirus Antibody Reference Reagents for Use in
Postvaccination Surveillance Serology
Sara L. Bissett,aDianna Wilkinson,bKate I. Tettmar,cNicky Jones,bElaine Stanford,dGitika Panicker,eHelena Faust,fRay Borrow,d
Kate Soldan,aElizabeth R. Unger,eJoakim Dillner,fPhilip Minor,band Simon Beddowsa
Centre for Infections, Health Protection Agency, London, United Kingdoma; National Institute for Biological Standards and Control, Potters Bar, United Kingdomb; NHS
Blood and Transplant, London, United Kingdomc; Vaccine Evaluation and Seroepidemiology Unit, Health Protection Agency, Manchester, United Kingdomd; Global WHO
HPV Reference Laboratory, Centers for Disease Control and Prevention, Atlanta, Georgia, USAe; and Global WHO HPV Reference Laboratory, Malmö University Hospital,
established. The development of highly efficacious vaccines
against the two most prevalent genotypes, HPV16 and HPV18
(13), represents one of the most significant advances in human
vaccination for many years (12, 15, 19). Emerging data from effi-
cacy trials also suggest some degree of cross-protection against
nonvaccine types, including HPV31 and HPV45 (1, 15).
Serologic assays for the evaluation of HPV vaccine responses
are currently limited to an enzyme-linked immunosorbent assay
(ELISA) (9), three multiplex assay systems (4, 6, 14), and a pseu-
dovirus neutralization assay (2), and emerging data suggest that
each system has some utility for characterizing HPV vaccine anti-
to be mediated by neutralizing antibodies (17), and while the
mechanism of vaccine-induced cross-protection is uncertain, the
measurement of antibodies against nonvaccine types (5, 11) may
be useful as a potential correlate or surrogate of cross-protection
(16). The only internationally available serologic standard is a
WHO International Standard (IS) for HPV16 antibodies, derived
from subjects with natural HPV16 infection (7), although a can-
didate IS for HPV18 antibodies, derived from subjects with natu-
ral HPV18 infection, is currently being characterized.
for use as quality controls in postimmunization serosurveillance
surveys able to control for responses against vaccine (HPV16 and
HPV18) and nonvaccine (HPV31 and HPV45) types. While ISs
are essential for assigning an international unitage of antibody
levels, the daily quality control of serological tests needs to have
access to secondary standards that are available in larger amounts
than the IS itself. Such secondary standards should preferably be
characterized by analysis of antibody level in parallel with the IS,
to assign a traceable international unitage to them (21). The ref-
erence reagents described in this paper have the high antibody
levels that are typical of vaccinated subjects, and this makes them
easier to use as reference standards for laboratories that perform
serology mostly on vaccinated subjects, who have antibody levels
substantially higher than found in the IS.
he link between persistent infection with high-risk human
papillomavirus (HPV) and the development of cervical can-
Twenty-seven citrated plasma packs not required for transfu-
sion were obtained from NHS Blood and Transplant and tested
negative for anti-HIV antibodies, anti-hepatitis C virus (HCV)
antibodies, and HBsAg. The plasma packs were selected from fe-
United Kingdom National HPV Immunization Programme
for HPV neutralization assays, due to the potential for heparin to
collected as citrated plasma packs, this is not expected to be an
A plasma panel containing one aliquot of each coded sample
Health Protection Agency, United Kingdom) for testing in a neu-
resenting HPV16, HPV18, HPV31, HPV45, and the control bo-
vine papillomavirus (BPV) made by transfection of 293TT cells
with the appropriate bicistronic psheLL L1-L2 plasmid and the
secreted alkaline phosphatase (SEAP) reporter vector (http:
reporter gene assay (Roche) and Glomax multidetection system
(Promega), (ii) laboratory B (Global WHO HPV Reference Lab-
for testing in the pseudovirus neutralization assay containing
HPV16, HPV18, and the control BPV and detected using the
SEAP reporter gene assay (BD Biosciences) and a Victor 2 lumi-
nometer (Perkin Elmer), and (iii) laboratory C (Global WHO
Received 22 November 2011 Returned for modification 29 December 2011
Accepted 14 January 2012
Published ahead of print 25 January 2012
Address correspondence to Simon Beddows, firstname.lastname@example.org.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
1556-6811/12/$12.00 Clinical and Vaccine Immunologyp. 449–451cvi.asm.org
for testing in a multiplex serology assay with the following non-
reporter-containing HPV L1-L2 pseudoviruses: ?1 (HPV32), ?2
(HPV3), ?7 (HPV18, HPV45, and HPV68), ?9 (HPV16, HPV31,
HPV33, HPV52, and HPV58), ?10 (HPV6 and HPV11), ?1
(HPV5), ?2 (HPV15 and HPV38), and ?3 (HPV76) according to
published methodology (6).
against any of the four HPV types tested and 18 (66.7%) neutral-
ized both HPV16 and HPV18 (12 of these also neutralized both
HPV31 and HPV45), while 1 sample (3.7%) was positive for
HPV16 alone, suggesting a natural HPV16 infection (Fig. 1). No
neutralization of the control BPV pseudovirus was seen (all ti-
B. In addition, there was also very good agreement between the
magnitudes of neutralizing antibody titers obtained by both lab-
oratories for HPV16 (96% concordance, ? ? 0.945) and HPV18
(85% concordance, ? ? 0.797) when stratified by discrete titer
A comparison of neutralization (laboratory A) and multiplex
serological (laboratory C) assay data sets showed 100% concor-
dance (? ? 1.000) for HPV16 and HPV18, while for HPV31 and
HPV45, concordance was lower, at 96.3% (? ? 0.922; McNemar
test for discrepancies, P ? 1.000 [Stata 10.1]) and 85.2% (? ?
0.705; P ? 0.617), respectively, although these discrepancies were
ization activity was detected, four of these had serological reactiv-
ity against one or more ? or ? HPV types not included in the
neutralization panel (P07 [HPV6 and HPV32], P16 [HPV6], P20
[HPV68 and HPV76], and P27 [HPV38]).
These data were used to assemble HPV-negative, intermediate
HPV16/18 antibody, and high HPV16/18 antibody plasma pools
whose specificity was confirmed by laboratory A in the pseudovi-
rus neutralization assay (Table 1). The WHO International Stan-
dard for HPV16 antibodies (IS16; code 05/134, 10 international
units [IU]/ml) demonstrated type-specific neutralization of
HPV16 at levels consistent with natural infection (Table 1) (7).
The HPV-negative plasma pool (Table 1) comprised four
plasma samples (P03, P04, P12, and P19) that were negative for
neutralizing antibodies against HPV16, HPV18, HPV31, and
HPV45 (Fig. 1) and negative for binding antibodies against all of
the ? and ? HPV types by multiplex serology.
The intermediate HPV16/18 antibody plasma pool (Table 1)
comprised two plasma samples (P18 and P21) that displayed in-
FIG 1 Neutralization of HPV16 (filled circles), HPV18 (filled triangles), HPV31 (open circles), HPV45 (open triangles), and control BPV (asterisks) pseudo-
viruses by using individual plasmas. HPV16, HPV18, and BPV neutralization titers were assembled using data generated by both laboratories A and B, while
intermediate HPV16/18 (P18 and P21), and HPV-negative (P03, P04, P12, and P19) antibody pools.
TABLE 1 Neutralization of vaccine and nonvaccine HPV pseudoviruses by plasma pools and IS16
HPV antibody reagent
Avg neutralization titer (%CV) against indicated HPV pseudovirusa
aAverage neutralization titers derived from three experiments (with percent coefficient of variation [%CV] of the log10titers in parentheses).
bIS16, International Standard for HPV16 antibodies.
Bissett et al.
cvi.asm.orgClinical and Vaccine Immunology
termediate levels of neutralizing antibody against HPV16 and
HPV18; plasma sample P18 also displayed some neutralizing an-
tibody activity against HPV31 (Fig. 1). Both plasma samples were
positive for binding antibodies against HPV16 and HPV18, and
apart from P18 having some reactivity against HPV32 (?1), these
samples were negative for binding antibodies against other ? and
? HPV types tested by multiplex serology. The median HPV16
IU per milliliter was 187 IU/ml (interquartile range [IQR], 126 to
211 IU/ml; n ? 3), consistent with a low-level to intermediate
vaccine response (7).
The high HPV16/18 antibody plasma pool (Table 1) com-
prised seven plasma samples (P02, P06, P09, P14, P22, P24, and
P25) that displayed high levels of neutralizing antibody against
HPV16 and HPV18 and intermediate levels against HPV31 and
? and ? HPV types tested by multiplex serology, including ?1
(HPV32), ?2 (HPV3), ?7 (HPV18, HPV45, and HPV68), ?9
(HPV16, HPV31, HPV33, HPV52, and HPV58), ?10 (HPV6),
?1 (HPV5), ?2 (HPV15 and HPV38), and ?3 (HPV76). The me-
dian HPV16 neutralizing antibody level was 6,668 IU/ml (IQR,
5,356 to 8,023 IU/ml; n ? 3), consistent with a high-level vaccine
antibody response (7).
Overall, the results obtained from testing the plasma pools
were similar to those expected from the averages of the responses
for the individual plasma samples.
lower than from vaccinees (8), and HPV16, HPV18, HPV31, and
Howell-Jones, N. de Silva, M. Akpan, P. Oakeshott, C. Carder, L.
Coupland, M. Sillis, H. Mallinson, V. Ellis, D. Frodsham, T. I.
Robinson, O. N. Gill, S. Beddonis, and K. Soldan, submitted for
with high-titer antibodies against both HPV16 and HPV18 (in-
cluding those with reactivity against HPV31 and HPV45) are al-
most certainly a result of vaccination. Our panel is not perfectly
representative of most vaccinees, due to the age of vaccination in
the catch-up cohort being rather older than the target age for
routine immunization. However, the neutralization responses
responses seen with sera taken from 13- to 14-year-old vaccinees
multiplex serology, cannot be ruled out.
Surveillance studies are important for estimating the preva-
lence of an infectious agent in, or for monitoring the impact of a
vaccine on, a population or demographic group. With the recent
postvaccine surveillance, including seroepidemiology surveys.
These plasma pools will be useful as reference reagents. They are
currently available as 250-?l aliquots of liquid plasma archived at
?80°C and can be obtained from the National Institute for Bio-
logical Standards and Control.
This work was supported by the United Kingdom Medical Research
Council (grant number G0701217). The work performed in Sweden and
the United States was supported by the WHO.
The findings and conclusions in this report are ours and do not nec-
essarily represent the views of the funding agency.
in this study.
We declare no conflict of interest.
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Plasma Pools for HPV Serologic Quality Control
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