Reactivity pattern of 92 monoclonal antibodies with 15 human papillomavirus types

13 Pages·2007·0.18 MB·English

JournalofGeneralVirology(2008),89,117–129 DOI10.1099/vir.0.83145-0
Reactivity pattern of 92 monoclonal antibodies with
15 human papillomavirus types
Raeda Z. Rizk,1 Neil D. Christensen,2 Kristina M. Michael,1 Martin Mu¨ller,1
Peter Sehr,13 Tim Waterboer1 and Michael Pawlita1
Correspondence 1DepartmentofGenomeModificationsandCancer,InfectionandCancerProgram,GermanCancer
MichaelPawlita Research Center (DKFZ), Im Neuenheimer Feld242, 69120 Heidelberg, Germany
[email protected] 2DepartmentsofPathologyandMicrobiologyandImmunology,TheJakeGittlenCancerResearch
Foundation, Collegeof Medicine, Pennsylvania State University, Hershey, PA17033,USA
Most anti-humanpapillomavirus (HPV) capsid antibody assaysarebased on virus-likeparticles
(VLP).We evaluated glutathione S-transferase (GST)–L1fusion proteins as ELISAantigens for
determiningtypespecificityandcross-reactivityof92VLP-specificmonoclonalantibodies(mAb)
generated against ninemucosal alphapapillomavirus types ofspecies 7, 9and10.The
antibodypanelincluded25newmAb,and24previouslypublishedmAbarefurthercharacterized.
Wedeterminedthecross-reactivitypatternswith15differentHPVtypesrepresenting6species
(alpha1, 2,4,7, 9and 10)andneutralization andcross-neutralization propertieswith HPV
types 6,11, 16,18and45. Eighty-nine (97%) oftheantibodies including 34,71 and14
recognizing neutralizing, conformational and linearepitopes, respectively, reacted withthe
GST–L1proteinoftheHPVtypeusedasimmunogen,withlogtitresrangingfrom2.0to7.3.Of
these89antibodies, 52%weremonotypic, 20%showed intra-species and28%inter-species
cross-reactivity. Log neutralization titresto theimmunogen HPVranged from1.7 to5.6. A
single cross-neutralizing mAb(H6.L12) wasfound. ELISAtitres werealwayshigher than
neutralization titres. All neutralizing epitopes wereconformational andmostlytype-specific. Our
data show thatbacterially expressed, affinity-purified GST–L1fusion proteins displaya broad
variety ofepitopes andthusare wellsuited for detection ofHPVantibodies. Cross-reactivity is
associatedwithlinearaswellasconformationalepitopes.Distantlyrelatedmucosalandskinalpha
Received 7May2007 papillomaviruses share someconformational epitopes andthephylogenetic L1-basedspecies
Accepted 23August2007 definition may not definea serological unitsince no species-specific epitope wasfound.
INTRODUCTION mucosa, some can additionally (species 2 and 8) or
exclusively (species 4) infect the skin. Many alpha HPV
Humanpapillomaviruses(HPV)aresmall(about55 nmin
can induce benign genital or common skin warts, while so
diameter) non-enveloped DNA tumour viruses with a
called high-risk types (most frequently HPV types 16 and
tropism for squamous epithelia. The icosahedral protein
18)(Munozetal.,2003)belongingtospecies5,6,7,9and
capsid is composed of 72 capsomeres (pentamers of the
11, can induce intraepithelial neoplasia, the precursor of
majorcapsidproteinL1)andcontainsasinglemoleculeof
cervical cancer (Bosch & de Sanjose, 2003; Clifford et al.,
about 8 kbp closed circular double-stranded DNA.
2003; de Villiers et al., 2004).
So far, more than 100 HPV types have been fully
Upon infection with HPV, serum antibodies to L1 protein
characterized by cloning and complete sequencing of their
can develop (Dillner, 1999; van Doornum et al., 1998;
genomes. Classification of HPV is based on the major
Wang et al., 1996; Wideroff et al., 1999). This immune
capsidproteinL1openreadingframenucleotidesequence:
response is highly type-specific (Carter et al., 1996, 2000;
HPV of the same genus show at least 60% sequence
Giroglou et al., 2001; Wang et al., 1997) and persists for
identity,thoseofthesamespeciesatleast70%,thoseofthe
years (Carter et al., 2000; Dillner, 1999). L1 antibodies are
same type at least 90% and those of the same subtype
considered markers for current and past infection (Carter
(variants)atleast98%(deVilliersetal.,2004).HPVofthe
etal.,1996;Carter&Galloway,1997;Kirnbauer,1996)and
genus alpha (15 species) mostly infect anogenital and oral
areweaklyassociatedwithcervicalcancer(vanDoornumet
3Present address: Joint DKFZ-EMBL Chemical Biology Core Facility, al.,2003).Incontrast,antibodiestotheearlyoncoproteins
E6 and E7 that are consistently expressed in HPV-
EuropeanMolecularBiologyLaboratory(EMBL),Meyerhofstr.1,69117
Heidelberg,Germany. transformed cells are strongly associated with cervical
0008-3145G2008SGM PrintedinGreatBritain 117
R.Z.Rizkandothers
carcinoma (Lehtinen et al., 2003; Meschede et al., 1998; (Roden et al., 1996; Touze & Coursaget, 1998; Unckell
Silins et al., 2002; Zumbach et al., 2000). et al., 1997). Pastrana and colleagues (Buck et al., 2005;
Pastrana et al., 2004) have developed an in vitro
Analysis of HPV humoral immune responses faces some
neutralization assay utilizing HPV pseudovirions carrying
limitations due to the large number of HPV types
a secreted alkaline phosphatase (SEAP) reporter plasmid.
associated with the different diseases and to difficulties in
producingsufficientquantitiesofinfectiousvirusparticles. Thisstudyaimed,first,toevaluateGST–L1fusionproteins
Asanalternativetovirionproduction,effortsweremadeto as ELISA antigens for detecting capsid-specific antibodies
reproduce the antigenic properties of virions by virus-like againstalphapapillomaviruses,andsecond,touseGST–L1
particles (VLP). VLP are formed spontaneously after proteinsof15differentalphaHPVtypestodeterminetype
overexpressing L1 by vaccinia virus (Hagensee et al., specificityandcross-reactivityof92mAbrecognizingHPV
1993), plasmid vectors (Pastrana et al., 2004) or baculo- capsid epitopes. We show that GST–L1 fusion proteins
virus (Kirnbauer et al., 1992) in yeast (Sasagawa et al., display almost all epitopes previously defined on VLP,
1995), mammalian, or insect cells (Le Cann et al., 1994), includingconformational,linearandneutralizingepitopes.
respectively. VLP resemble papillomavirus virions in Additionally, we describe a series of intra- and/or inter-
morphology and display conformational and neutralizing species cross-reactive epitopes and show that cross-
epitopes(Christensen etal.,1996a,b,2001;LeCannetal., reactivity only loosely follows phylogenetic relationships.
1994).VLP-basedELISAisthestandardtechniqueinHPV We also show that neutralizing epitopes are always
serology. It was developed to screen for antibodies to conformational and mostly, but not always, type-specific.
HPV1(Carteretal.,1993),HPV16(Kirnbaueretal.,1994),
canine oral PV (CoPV) (Suzich et al., 1995), cottontail
METHODS
rabbit PV (CRPV) and bovine PV1 (BPV1) (Christensen
et al., 1996a), BPV4 (Kirnbauer et al., 1996), HPV45
Cloningand expressionof recombinant HPV L1 proteins.The
(Touze et al., 1996), HPV6 and 11 (Touze et al., 1998), generation of GST–L1 fusion proteins from HPV6, 16 and 18 (all
HPV5 (Favre et al., 1998), HPV8 (Bouwes Bavinck et al., threelacking10aaattheNterminus)hasbeenpreviouslydescribed
2000), HPV31, 33, 35, 18, 39 (Giroglou et al., 2001), (Sehr et al., 2002). Similarly, the complete L1 open reading frames
(ORF) of HPV2, 3, 10, 11, 31, 32, 33, 35, 45, 52, 57 and 58 were
HPV59 (Combita et al., 2002) and HPV15, 20 and 24
expressed as double fusion proteins with N-terminal GST and a C-
(Feltkamp et al., 2003).
terminal undecapeptide (tag) of the SV40 large T-antigen. These
L1ofHPV11andHPV16expressedasfusionproteinswith constructs and characterization of the fusion proteins will be
describedindetailelsewhere(manuscriptinpreparation).
glutathione S-transferase (GST) in E. coli spontaneously
formhomogeneouscapsomeres(Chenetal.,2000,2001;Li Transformation of the modified pGEX plasmids into E. coli strain
et al., 1997). Capsomeres generated from bacterially BL21RosettaDE3(Invitrogen)andfurtherexpressionandtreatment
expressed L1 of HPV11 and CoPV display neutralizing, of the fusion proteins was performed as previously described (Sehr
etal.,2002).
linear and conformational epitopes, and induce neutral-
izingantibodiesuponexperimentalimmunization(Roseet
Anti-HPV VLP monoclonal antibodies. Altogether, 89 tissue
al., 1998; Yuan et al., 2001). culture supernatants and two ascites preparations of 91 mAb were
analysed (Tables 1, 2). Yet unpublished mAb were generated and
Recently, GST–HPV L1 fusion protein-based antibody
characterizedaspreviouslydescribed(Christensenetal.,1990,1994a,
detection systems have been developed for many HPV b,1996a,b;Mulleretal.,1997;Sehretal.,2002).Seventy-threemAb
types (Karagas et al., 2006; Sehr et al., 2002; Waterboer wereraisedagainstVLPofninedifferentHPVtypes(HPV6,16,11,
et al., 2005). 18,45,31,33,52and35).SixmAb(theH263series)weregenerated
against a hybrid containing residues 1–168 of HPV11 and residues
Monoclonalantibodies(mAb)havebeengeneratedagainst 172–505ofHPV16.Another12mAbweregeneratedagainstHPV11
VLPofdifferentHPVincludingtypes6,11,16,18,31,33, VLP containing a G131S substitution (Tables 1, 2). All antibody
35 and 45 (Christensen et al., 1996a, b; Combita et al., preparationsusedhadanIgGconcentrationofatleast800ngml21
2002;Ludmereretal.,2000).TheyareusedtodefineHPV (Easy-TiterIgGkit;Pierce).
capsid epitopes.
Detectionoflinearepitopes.Detection of epitope type was done
Epitopes on HPV capsids can be experimentally classified by an immunodot blot method. Bacterial lysates containing over-
expressed GST–L1 were diluted to 1mg ml21 total lysate protein
asconformational(presentonintactVLPandcapsomeres)
either in PBS (native conformation) or in 0.2M Na CO , pH10,
or linear (displayed by synthetic peptides and denatured 2 3
1mM DTT (denaturing conditions) and incubated at room
L1) (Christensen et al., 1996a, b). Neutralizing epitopes temperature for 30min. For each mAb a nitrocellulose membrane
have also been identified in the L1 major capsid protein waspreparedwith2mlaliquotseachofnativeanddenaturedlysates.
(Christensenetal.,1994b;Giroglouetal.,2001;Roseetal., After drying, membranes were blocked (room temperature, 1h) in
1998; White et al., 1998, 1999). To analyse HPV 10% milk-PBS buffer, incubated (room temperature, 1h) with the
mAb preparations diluted 1:50 in blocking buffer, washed
neutralization properties, many systems have been estab-
(6610min) in PBS and incubated (room temperature, 1h) with
lished using athymic mouse xenograft (Kreider et al.,
peroxidase-conjugated goat anti-mouse antibody (Dianova). After
1987), raft culture systems (Meyers & Laimins, 1994) or washing,antibodyreactionsweredetectedusingAmershamECLPlus
production of infectious HPV pseudovirions in vitro WesternBlottingDetectionSystem(GEHealthcare).Theepitopetype
118 JournalofGeneralVirology89
Reactivitypatternof92mAbwith15HPVtypes
of four antibody preparations that reacted neither with native nor comparison to the reactivity of the pseudovirions added without
withdenaturedGST–L1couldnotbedetermined. antibody.Forcross-neutralization,allmAbwereanalysedinduplicate
cultures.
GST–L1 capture ELISA. Ninety-six-well PolySorp plates (Nunc)
were coated overnight at 4uC with glutathione-casein and blocked
the next day as previously described (Sehr et al., 2001). The plates RESULTS
were incubated (room temperature, 1h) with the cleared GST–L1
fusionproteinlysatesdilutedincaseinblockingbufferto0.25mgml21
total protein (saturating concentration) and washed. As a negative ReactivityofVLP-specificmAbwithGST–L1ofthe
control,GST-taglysatewasusedinparallel.Fortitrations(eachmAb respective HPV type
against the immunogen HPV L1), plates were incubated (room
Eighty-nine(97%)ofthe92mAbinvestigatedreactedwith
temperature, 1h) with threefold dilutions (starting concentration
1:10) of the mAb in casein blocking buffer (Sehr et al., 2001). For the GST–L1 fusion protein of the respective immunogen
cross-reactivity analyses, mAb were incubated (room temperature, HPV type (Tables 1, 2). The six mAb (H263.A2, H263.C3,
1h) with the 15 GST–L1. Plates were washed, incubated (room H263.F4, H263.G2, H263.H9 and H263.D1) generated
temperature, 1h) with biotin-conjugated goat anti-mouse Ig, against a hybrid VLP containing residues 1–168 of HPV11
incubated (room temperature, 30min) with streptavidin–poly-HRP
and residues 172–505 of HPV16 were analysed with both
(horseradish peroxidase), washed again and the colour reaction was
HPV16 and HPV11 GST–L1. H263.A2 reacted only with
developedaspreviouslydescribed(Sehretal.,2001).For28HPV16
HPV16whileH263.G2reactedonlywithHPV11(Table1).
mAbanalysedontwoseparatedays,thecoefficientofdetermination
ofthelinearregressionoftheabsorptionvalueswasR250.78. The other four mAb reacted more strongly with HPV11
than with HPV16 (Table 2). All 12 mAb generated against
HPV16 VLP-capture ELISA. HPV16 VLP-capture ELISA was VLP of HPV11 L1 mutant G131S reacted with HPV11
performed as previously described (Muller et al., 1997). Briefly, GST–L1.
plates(Nunc)werecoatedovernightat4uCwithrabbitanti-HPV16
VLPpolyclonalantibodydiluted1:200inPBSandblockedthenext Of the 89 reactive mAb, 82 showed A values above
max
day with 5% milk, PBS, 0.05% Tween 20 (blocking buffer). As 1500 mAU with log end-point dilution titres (the last
antigen,HPV16VLP(3.5mgml21inblockingbuffer)generatedfrom dilution giving a signal ¢110 mAU) varying from 3.3 to
thesameparentalplasmidasGST16L1andpurifiedbytwogradient
6.3fortissueculturesupernatantsandupto7.3forascites
centrifugations as previously described (Muller et al., 1997) were
as antibody source (Tables 1, 2). The titration curves of
used. Further assay procedures were asdescribed above for GST–L1
these mAb showed steep slopes, i.e. A values decreasing
captureELISA. 450
from 80 to 20% of A within 10-fold dilution. For the
max
Neutralizationassay.In vitro neutralization assays based on HPV remainingsevenmAb(H45.N5,H35.Q8,H35.H9,H35.N6,
pseudovirionscarryingaSEAPreportergeneweredoneaspreviously H33.E12,H33.B6andH18.A7),theA valueswerebelow
max
described (Buck et al., 2005; Pastrana et al., 2004). Briefly, 300000 1100 mAU, log titres were between 2.0 and 4.3 and the
293TT cells ml21 (293 cells transfected with an expression plasmid
titration curves were flat, i.e. A values decreasing from
encoding a cDNA for SV40 large T-antigen) were seeded, infected 450
80 to 20% of A only within more than 100-fold
after 5h with a mixture of serial mAb dilutions (starting max
dilution. Fig. 1 shows six representative examples of mAb
concentration 1:50) with the pseudovirions of the immunogen
HPV type and incubated (5days, 37uC). Cell supernatants were titration curves.
assayed for SEAP using a chemiluminescent SEAP Reporter Gene
ThreemAbpreparations,despiteestimatedIgGconcentra-
Assay(RocheDiagnostics).PseudovirionsofHPV16,18,45,6and11
tions of .800 ng ml21, were either very weakly reactive
wereavailable.Forcross-neutralizationanalyses,allneutralizingmAb
wereinvestigatedat1:50dilutionwiththeHPVpseudovirionsofthe (H6.C6 and H35.O3) or non-reactive (H16.L4) with the
othertypes. respective GST–L1 protein (A , 110 mAU at 1:90
450
dilution) (Table 2) and were not analysed further. For
Data analysis and statistics. In all ELISA binding experiments, H6.C6 and H16.L4, the L1 N terminus is known to be an
lysatefrombacteriaexpressingGST-tagalonewasanalysedinparallel
essentialpartoftheepitope(Christensenetal.,1996b),and
todefinethereactionbackground.Themeasuredabsorbances(A)at
the HPV6 and 16 GST–L1 proteins used here lack the 10
450nmwereexpressedinmilliunits(mAU).ThenetA valueofa
450 N-terminal amino acid residues (Sehr et al., 2002). The
mAbwasobtainedbysubtractingthebackgroundreactivityfromthe
absorbance with the GST–L1-tag fusion protein. Monoclonal reasonfortheveryweakreactivityofmAbH35.O3remains
antibodies were arbitrarily classified as reactive when the A value unclear. However, the reactivity of the other three mAb
450
at1:90dilutionwasequaltoorgreaterthan110mAU.Theantibody generated against HPV35 VLP with HPV35 GST–L1 was
titre was defined as the last dilution yielding readings equal to or ratherweakwithlowA andflattitrationcurves(Table1,
max
greater than 110mAU. For cross-reactivity experiments, a mAb
Fig. 1).
concentration close to saturation (mean 79% of A with the
max
immunogenHPVtype;SD15%)wasused.AllmAbweremeasuredin
duplicatewellsandthemeanofthespecificreactivityoftheduplicate HPV type specificity of mAb
valueswastakenasthefinalreadout.
HPV type specificity of the 89 reactive mAb was
In SEAP neutralization assays, net relative light units (RLU) were
investigatedwithGST–L1fusionproteinsof15HPVtypes
calculated by subtracting RLU of cells without mAb and pseudo-
representing six alpha papillomavirus species (a1, a2, a4,
virions (background) from RLU of cells treated with the mAb/
pseudovirionmixture.Theend-pointneutralizationtitrewasdefined a7,a9anda10).Toallowcross-reactivityanalysisinsimilar
asthelastdilutionyielding¢70%reductionintheSEAPactivityin conditionsforallantibodies,weusedamAbconcentration
http://vir.sgmjournals.org 119
R.Z.Rizkandothers
Table1. Reactivity ofmono-reactive HPV antibodies
Species ImmunogenHPV mAb A *(mAU) mAbtitre(log ) Publisheddata
max 10
type
ED Nd E/N§ Igisotype Epitope
Type|| Essentialaa Ref#
a9 16 H16.5A 2417 6.2 3.8 2.4 G2a C+ 266–297339–365 AP
H16.14J 2299 5.3 2.9 2.4 C 172–505 A
H16.3A 2283 3.8 3.5 0.3 C+ 172–505 A
H16.1A 2292 5.3 2.9 2.4 G2 C 266–297339–365 AP
H16.6F 2276 6.2 3.5 2.7 G2a C+ 266–297339–365 AP
H16.15G 2284 5.3 2 L 172–505 A
H16.U4 1976 6.8 G2a C+ 172–505 AB
H16.V5 1978 7.3 G2b C+ 266–297339–365 AB
H16.E70 1645 3.8 2.3 1.5 G2b C+ 282 ABK
H16.4A 1942 5.8 3.5 2.3 C+ 172–505 A
H16.L4DD 164 1.0 G2a L Nterminus ANC
H11:16** H263.A2 2332 5.8 2.9 2.9 C+ 266–297339–365 A
31 H31.A6 2171 4.3 G1 C+ AB
33 H33.J3 2372 4.8 NC
H33.B6 1576 4.3 C NC
H33.E12 1355 4.3 C+ B
35 H35.H9 658 2.3 NC
H35.Q8 1166 3.8 C NC
H35.N6 340 2.9 NC
H35.O3DD 125 1.0 NC
52 H52.C1 2095 4.8 C NC
H52.B4 1854 4.3 C NC
H52.A7 2047 4.8 C NC
H52.D11 1980 4.8 C NC
a7 18 H18.K2 2531 6.2 2 M C B
H18.J4 2326 5.8 5.6 0.2 G2a C B
H18.R5 2371 4.8 2 G2b C B
H18.U15 2413 4.8 2 G1 C B
H18.G10 2384 5.3 4.1 1.2 G2b C B
45 H45.N5 429 3.8 2.9 0.9 C G
H45.L10 2102 4.8 3.8 1.0 C G
H45.C1 2188 5.3 3.2 2.1 M C B
a10 6 H6.M48 2105 3.8 2.9 0.9 G1 C+ 4653169–178 FI
H6.B10.5 1856 2.9 G2b C+ 4653169–178 BI
H6.N8 2347 4.3 2.3 2.0 G1 C+ 4653 FI
H6.C6DD 288 1.5 G2a L2 Nterminus ANC
11 H11.H3 2145 5.8 4.7 1.1 G2b C+ 132246345–348 ADJ
H11.F1 2178 5.3 4.4 0.9 G2a C+ 131132246278 HJP
H11.B2 2249 6.2 4.7 1.5 G2b C+ 131132246278 BJ
H11.A3.2 2130 4.8 2.9 1.9 G2a C2 49–54170–179246278 DIJ
IV17B6(16) 1643 5.8 3.5 2.3 C MM
IV8D3(7) 1644 5.8 3.5 2.3 C MM
IV12D9(11) 1637 4.8 3.8 1.0 C MM
IV4A1(3) 1613 4.8 2.6 2.2 C MM
IV12D3(12) 1632 5.3 2.9 2.4 C MM
11G131Sdd G131S11.I1 2289 5.3 4.1 1.2 G1 C+ 262–289 D
G131S11.G3 2396 5.8 4.7 1.1 M C+ 262–289345–348 D
G131S11.C2 2121 5.3 2.9 2.4 G1 C+ D
H11:16 H263.G2 1685 3.8 2.9 0.9 C 1–168 A
*A at1:10dilutioninELISAwithGST–L1ofimmunogenHPVtype;mAU,absorbancemilliunits.
450
DELISAtitre.
dNeutralizationtitre.
120 JournalofGeneralVirology89
Reactivitypatternof92mAbwith15HPVtypes
Table1.cont.
§Log ofratioofELISAtoneutralizationtitre.
10
||Reportedepitopeandneutralizationtype;C,conformational;L,linear;+,neutralizing;2,nonneutralizing;
,epitopetypeisdefinedinthisstudy.
#References:A,Christensenetal.,2001;B,Christensenetal.,1996a;D,Ludmereretal.,2000;F,Christensenetal.,1996b;G,Combitaetal.,2002;
H,Ludmereretal.,1997;I,McClementsetal.,2001;J,Ludmereretal.,1996;K,Rodenetal.,1997;MM,MartinMu¨ller,unpublished;NC,NeilD.
Christensen,unpublished;P,Culpetal.,2007.
**H11:16,hybridcontainingaa1–168ofHPV11and172–505ofHPV16.
DDmAbnotanalysedfurther.
ddMutantHPV11withG131Ssubstitution.
belowbutclosetosaturationfortheimmunogenHPVtype HPV31, all from its own a9 species. In addition, a strong
(Fig. 1). but isolated cross-reaction was seen with the phylogeneti-
cally distantly related HPV32.
Undertheseconditions,46(52%)ofthemAbreactedonly
with the GST–L1 fusion protein of the HPV type used for Ofthe11HPV18mAb,6(55%) werecross-reactive.They
immunization (Table 1), while the other 43 mAb (48%) all showed mixed intra- and inter-species cross-reactivity
showeddifferentlevelsofintra-and/orinter-speciescross- and reacted with the closely related HPV type 45. Five of
reactivity (Table 2). them reacted also with skin HPV types, i.e. HPV2 and
HPV57, and four also with HPV3 and HPV10. Fourteen
Of the 43 cross-reactive mAb, 23 (54%) showed strong
out of the 18 cross-reactions with skin types were strong
(A relative to the type used for immunization .80%),
450 (n511) or intermediate. Antibody H18.A7, which reacted
11(26%)showedintermediate(relativeA 50–80%)and
450 only weakly with HPV18, showed very broad although
9 (20%) showed weak (relative A , 50% but absolute
450 mostly weak (n57) or intermediate (n56) cross-reactions
A . 110 mAU) cross-reactivity with at least one other
450 with all other HPV tested. The other five mAb cross-
HPV type. Of the 153 cross-reactions, 66 (43%) were
reacted with neither the mucosal high-risk HPV of the a9
strong, 34 (22%) were intermediate and 53 (37%) were
northelow-riskHPVofthea10anda1species,whichisin
weak. This suggests that the cross-reactive epitopes
contrast to the frequent cross-reactions of HPV16 mAb
recognized by about half of the cross-reactive antibodies
with HPV18.
and in about half of all cross-reactions are very similar or
even identical to the specific epitope. Of the five HPV45 mAb, two showed strong and
intermediate intra-species cross-reactivity while inter-
All mAb reactive with HPV31 (n51), HPV35 (n53) and
species cross-reactivity was absent.
HPV52 (n54) were monotypic.
HPV6 and 11 are very closely related low-risk types of the
Of the 27 mAb generated against HPV16 VLP, 16 (59%)
a10 species. Of the 34 mAb raised against these two types,
werecross-reactive.TwomAbshowedstrictlyintra-species
18 (53%) were cross-reactive; all recognized both HPV
and four showed strictly inter-species cross-reactivity. The
types.Inter-speciescross-reactivitywasrare,withonlyfour
other 10 mAb showed mixed intra/inter-species cross-
mAb additionally reacting weakly or intermediately with
reactivity. Most frequentintra-speciescross-reactionswere
one or two types of the high-risk HPV.
with HPV35 (n510), HPV31 and HPV58 (both n59),
followed by HPV33 (n57). HPV52 L1 (n54) showed the
leastfrequentcross-reactivity.Ontheotherhand,themost Direct comparison of mAb reactivity with HPV16
frequent inter-species cross-reactions were with HPV18 GST–L1 and VLP
(n512), followed by HPV45 (n58), HPV11 (n57) and
Inadirectexperimentalcomparison,32mAb,encompass-
HPV6(n56).Cross-reactivitywaslessfrequentwithHPV2
ing28raisedagainstHPV16andthefourgeneratedagainst
and HPV32 (both n54), HPV10 (n53), and HPV57 and
other HPV types but cross-reactive with HPV16 GST–L1,
HPV3 (both n52). Five HPV16 mAb cross-reacted also
were also analysed by HPV16 VLP ELISA. All HPV16-
with skin alpha papillomavirus types; in this group, cross-
specific mAb reacted in VLP-ELISA, including H16.L4,
reactivity was weak or absent with low-risk mucosal HPV
which was non-reactive in GST–L1 ELISA; this mAb is
types 6, 11 and 32, but strong or intermediate with the
known to detect an epitope located within the N terminus
high-risk HPV types 18 and 45. Antibody H16.11B, which
ofL1(Christensenetal.,1996a;Christensen,unpublished)
recognized a conformational epitope, showed the broadest
that is deleted in HPV16 GST–L1. GST–L1 and VLP titres
cross-reactivity. It reacted with all 15 alpha papilloma-
weresimilar(GST–L1toVLPtitreratio:median2.9,range
viruses analysed, strongly with 12 HPV types and weakly
0.04–27) for the 14 neutralizing mAb, but GST–L1 titres
with types 6, 11 and 32.
were much higher for the 13 non-neutralizing mAb (222,
From the four HPV33 mAb, only one cross-reacted 3–6000). Also the 11 monospecific mAb had similar GST–
strongly with HPV52 and HPV58 and weakly with L1 and VLP titres (1, 0.05–10), whereas the GST–L1 titres
http://vir.sgmjournals.org 121
1 R
2 Table2. Reactivity ofcross-reactive HPVantibodies .
2 Z
.
R
Dil.,Dilution. iz
k
a
n
Species/ mAb A * Abtitre ELISAReactivity(mAU)withGST–L1ofspecificHPVtypes Publisheddata d
max o
HPVtype (mAU) (log10) the
rs
ED Nd E/N§ Dil. a9 a7 a10 a1 a4 a2 Ig HPVVLPbinding Epitope
isotype
16 31 33 35 52 58 18 45 6 11 32 2 57 3 10 + ” Type||EssentialRef#
aa
a9
16 H16.7E 1835 5.8 2.6 3.2 2500 1782 2 2 177 2 2 2 2 2 2 2 2 2 2 2 G2b 16 11 C+ 38–65 AP
H16.13D 2148 4.8 2 2000 1433 1842 115 2 209 111 2 2 2 2 2 2 2 2 2 C* NC
H16.H5 2198 6.2 2 2500 2153 2 2 2 2 2 582 2 2 2 2 2 2 2 2 G2b 16 6113133 L2 174–185B
351845
H16.9A 2290 6.2 2 2500 2052 2 2 2 2 2 168 2 2 2 2 2 2 2 2 16 11 C 1–173A
H16.2F 1849 6.2 3.8 2.4 2500 1939 2 2 2 2 2 624 162 2 2 2 2 2 2 2 G2 16 11 C+ 266–297AP
339–365
H16.C2 1983 6.2 2 3000 1949 2 2 2 2 2 1124 2 2 121 2 2 2 2 2 G1 16183133 L 174–185B
3545
Ritti01 2054 5.3 2.3 3.0 1000 903 748 382 471 2 490 2 460 2 2 2 2 2 2 2 C O
V16H9 1969 5.3 2 1000 972 747 2 2 2 626 119 2 364 700 2 2 2 2 2 C MM
H16.D9 2316 6.2 2 1000 2123 2049 2 1486 2 2 276 2 2285 2314 2 2 2 2 2 G1 611 16313335 L2 B
1845
H16.B20 1910 4.8 2 1000 1598 2 2 575 2 145 144 2 131 170 2 2 2 2 2 G2a 16 6111831 L2 396–415B
333545
MM07 2040 5.3 2.0 3.3 1000 844 837 549 615 2 619 2 591 2 2 2 117 2 2 2 L O
H16.S1 1871 5.3 2 2500 1838 2 2 1511 2 2 1785 1645 2 2 2 2 2 1701 176G1 16183545 6113133 L 111–130B
H16.J4 2265 6.2 2 2500 2118 2139 2011 2046 2 2087 376 2063 2 2 261 2 2 2 2 G2a 16313335 61118 L 261–280B
45
MM08 1646 3.3 2 200 301 454 232 194 412 335 274 229 188 236 153 241 2 2 179 L MM
H16.8B 2342 6.2 2 2500 2295 2114 2042 2190 2154 2155 2106 1986 123 135 466 556 221 2 2 16 11 C A
H16.11B 1925 6.2 2 5000 1877 1771 1764 1811 1847 1824 1724 1878 129 394 405 1763 1876 1722 1834 16 11 C A
33 H33.L11 2308 3.8 200 2 126 1654 2 1826 1842 2 2 2 2 1388 2 2 2 2 L NC
a7 H18.B1 2444 6.2 2 1000 2 2 2 2 2 2 2319 1253 2 2 2 1714 1296 2 2 G1 61116 C B
J
ou 1845
rna H18.Q2 1740 6.2 2 2500 2 2 2 2 2 2 1784 1845 2 2 2 1827 1855 2 2 G1 61116 L 397–416B
l
o 1845
f
G H18.F8 2464 6.2 2 1000 2 2 2 2 2 2 2268 2171 2 2 2 2 2 1475 1995G1 1845 61116 C B
e
ne H18.E20 1875 5.3 2 2500 2 2 2 2 2 2 1741 1624 2 2 2 1750 247 1593 1787G2a 45 6111618 L B
ral H18.L9 1824 6.2 2 2500 2 2 2 2 2 2 1696 1532 2 2 2 1768 174 1495 1668G2a 45 6111618 L B
V
iro H18.A7 1079 2.0 2 20 356 613 199 239 409 337 625 334 163 362 218 666 187 197 394 NC
lo 45 H45.E30 2226 4.8 2 270 2 2 2 2 2 2 1354 1891 2 2 2 2 2 2 2 C+ B
g
y H45.H11 2169 4.3 2 270 2 2 2 2 2 2 1908 1942 2 2 2 2 2 2 2 C GNC
8
9
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ttp
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v
ir.s
g Species/ mAb A * Abtitre ELISAReactivity(mAU)withGST–L1ofspecificHPVtypes Publisheddata
m max
jou HPVtype (mAU) (log10)
rn
als ED Nd E/N§ Dil. a9 a7 a10 a1 a4 a2 Ig HPVVLPbinding Epitope
.org 16 31 33 35 52 58 18 45 6 11 32 2 57 3 10 isotype + ” Type||EssentialRef#
aa
a10
11G131S**G131S11.E38 2167 4.3 2 1000 2 2 2 2 2 2 2 2 804 1053 2 2 2 2 2 A 611 C 2 D
G131S11.F6 2222 4.3 2 1000 2 2 2 2 2 2 2 2 428 694 2 2 2 2 2 G2b 611 C 2 D
G131S11.H5 2296 5.3 2 1000 2 2 2 2 2 2 2 2 1938 2136 2 2 2 2 2 G1 611 C+ D
G131S11.B8 2206 4.3 2 1000 2 2 2 2 2 2 2 2 591 1004 2 2 2 2 2 G2a 611 C*- D
G131S11.D2 2270 4.3 2 1000 2 2 2 2 2 2 2 2 343 769 2 2 2 2 2 G2a 611 C+ D
G131S11.A10 2075 3.3 2 300 2 2 2 2 2 2 2 2 204 885 2 2 2 2 2 G1 11 6 C+ D
G131S11.L14 2127 4.8 2 1000 2 2 2 2 2 2 2 2 900 1424 2 2 2 2 2 C NC
G131S11.N10 2152 4.8 2 1000 2 2 2 2 2 2 2 2 1133 1386 2 2 2 2 2 C NC
G131S11.J1 1973 4.8 2 1000 2 2 2 2 2 2 2 2 451 996 2 2 2 2 2 C NC
H11:16DD H263.H9 1831 3.3 2 100 2 2 2 2 2 2 2 2 1457 1633 2 2 2 2 2 611 16 C A
H263.D1 1852 3.8 2 2500 304 2 2 2 2 2 1441 2 2006 1889 2 2 2 2 2 611 C A
H263.F4 1720 3.3 2 500 682 2 2 814 2 2 2 2 1463 1501 2 2 2 2 2 611 16 C A
H263.C3 1862 4.3 2 500 1442 2 2 1459 2 2 579 142 1669 1836 2 2 2 2 2 61116 C A
6 H6.F62 2228 5.3 1.7 3.6 1000 2 2 2 2 2 2 2 2 2159 321 2 2 2 2 2 M 6 111618 C+ F
H6.K57 2251 4.8 2 1000 2 2 2 2 2 2 2 2 1791 2160 2 2 2 2 2 G2b 611 1618 C2 F
H6.L12dd 2143 5.8 2.3 3.5 1000 2 2 2 2 2 2 2 2 2247 2257 2 2 2 2 2 M 611 1618 C+ F
H6.H9 1825 3.3 2 100 2 2 2 2 2 2 2 2 626 853 2 2 2 2 2 G2a 611 1618 C2 F
H6.D10 2181 4.8 2 1000 2 772 2 2 2 2 2 2 1839 1850 2 2 2 2 2 G3 6111618 L2 F
R
e
*A at1:10dilutioninELISAwithGST–L1ofimmunogenHPVtype;mAU,absorbancemilliunits. a
450 c
DELISAtitre. tiv
dNeutralizationtitre. ity
p
§||LRoegp1o0rtoefdreaptiiotoopfeEaLnIdSAnetuittrrealtiozantieountrtaylpizea;tiCo,nctointrfeo.rmational;L,linear;+,neutralizing;2,nonneutralizing; ,epitopetypeisdefinedinthisstudy. attern
#References:A,Christensenetal.,2001;B,Christensenetal.,1996a;D,Ludmereretal.,2000;F,Christensenetal.,1996b;G,Combitaetal.,2002;MM,MartinMu¨eller,unpublished;NC,NeilD. o
f
Christensen,unpublished;O,Mulleretal.,1997;P,Culpetal.,2007. 9
2
**MutantHPV11withG131Ssubstitution. m
DDH11:16,hybridcontainingaa1–168ofHPV11and172–505ofHPV16. Ab
ddAlsocross-neutralizesHPV11withlog of2.6. w
10 ith
1
5
H
P
V
12 type
3 s
R.Z.Rizkandothers
previously published mAb. It reproduced published data
for 36 out of 42 mAb and the only differences observed
were the extremely weak binding reactivity of H35.O3 and
H6.C6, and the absence of binding (H16.L4) or neutral-
ization activity of mAb H16.J4 with HPV16, and
G131S11.H5, -.D2 and -.A10 with HPV11.
The majority of published HPV serology data has been
established using VLP-based ELISA assays. Here, we used
HPV L1 antibody assays based on bacterially expressed,
affinity-purified glutathione S-transferase L1 (GST–L1)
fusion proteins as antigens (Sehr et al., 2002). This assay
hasbeenpreviouslyvalidatedforHPV16and18withhuman
and experimental mouse sera by comparison with VLP-
Fig.1. Representativedose–responsecurvesforbindingofHPV ELISA data (Davidson et al., 2003; Sehr et al., 2002). The
VLP-specific mAb to GST–L1 fusion proteins. ELISA analysis of bacterialexpressionsystemallowedanalysingninemucosal
serialdilutionsofmAbpreparationsincubatedwithGST–L1fusion HPV types that had been available before as VLP and six
proteins immobilized via glutathione-casein to the ELISA plates. HPV types, i.e. skin HPV2, 3, 10 and 57 as well as the
Each mAb was analysed with the L1 protein of the immunogen mucosal high-risk HPV type 58 and low-risk HPV type 32
HPV type. Circles indicate the dilution used for cross-reactivity that had not been available before as VLP. These numbers
analyses.
reflect the ease with which GST–L1 fusion proteins can be
generated,purifiedandappliedinantibodyassays.
of the 16 cross-reactive mAb were always higher (50, 2.5– The binding of 89 (97%) of the 92 mAb indicates that
6500). Finally, the GST–L1 to VLP titre ratios were higher GST–L1 fusion proteins (with the exception of HPV35)
for the 9 mAb recognizing linear (27, 8–6000) than for presentmost(ifnotall)oftheantigenicepitopespresented
those9mAbrecognizingconformationalepitopes(3,0.04– by VLP. Only two mAb reacted extremely weakly and one
800). In summary, GST–L1 display neutralizing, mono- did not react with the GST–L1 protein of the respective
specificandconformationalepitopeslikeVLP,butGST–L1 HPVtype.H6.C6andH16.L4detectanepitopelocatedon
appears to display non-neutralizing, cross-reactive and the N terminus of L1 (Christensen et al., 1996a, b), and
linear epitopes in larger quantity than highly purified, GST–L1preparationsofHPV6and16lackaa1–10.Fusion
antibody-captured VLP. of GST to the L1 N terminus apparently did not inhibit
binding of any other mAb, indicating that all of them are
eithertargetingepitopeselsewhereorthatGSTfusiondoes
Neutralization activity of VLP-specific mAb
not affect N-terminal epitope conformation (Sehr et al.,
Seventy four mAb were analysed for neutralization of 2002).
HPV6, 11, 16, 18 and 45 (Tables 1, 2). Thirty four (46%)
Four HPV35 specific mAb reacted very weakly, suggesting
mAb neutralized pseudovirions of the immunogen HPV
that the HPV35 GST–L1 preparation lacked most of the
type.End-pointneutralizationlogtitresrangedfrom1.7to
VLP-displayedepitopesforyetunknownreasons.However,
5.6. Thirty three of the neutralizing mAb recognized a
thepresenceofthreeconformationalcross-reactiveepitopes
conformational epitope and one (MM07) recognized a
(H16.8B,H16.11BandH263.C3)indicatesatleastpartially
linear epitope. Among mAb recognizing monospecific
correctconformationofHPV35GST–L1.H18.R5,reported
epitopes,28outof42(67%)wereneutralizing,incontrast
to recognize a conformational epitope on HPV18 and
to only five out of 29 (17%) mAb recognizing cross-
HPV45VLP,reactedonlywithHPV18GST–L1,suggesting
reactive epitopes.
asubtleepitopedifferenceofHPV45GSTL1.
All neutralizing mAb were further investigated at a 1:50
Our results show that GST–L1 fusion proteins have the
dilution for cross-neutralization with pseudovirion prep-
epitoperepertoireofintactVLP(conformational),butalso
arations of HPV6 and 11 (a10), 16 (a9), as well as 18 and
display epitopes presented by denatured VLP (linear).
45(a7).Cross-neutralizationwasobservedonlyonce;mAb
LinearepitopesarealsopresentonintactVLPpreparations
H6.L12neutralizedbothHPV6andthemostcloselyrelated
either because they are surface-exposed on capsids or
HPV11 pseudovirions.
becausetheVLPpreparationalsocontainssomedenatured
L1, but reactions with intact VLP are usually weaker than
with denatured proteins (Christensen et al., 1996a). Our
DISCUSSION
direct comparison using twofold gradient-purified and
This study represents the largest and most comprehensive antibody-captured HPV16 L1 VLP as ELISA targets also
analysis conducted so far on the presence and type demonstrated the presence of all linear and all cross-
specificity of epitopes displayed by HPV capsid proteins. reactiveepitopesinVLPELISA,butinclearlylowerdensity
It introduced 25 new mAb and further characterized 24 than in GST–L1 ELISA.
124 JournalofGeneralVirology89
Reactivitypatternof92mAbwith15HPVtypes
Of the 89 mAb reacting with GST–L1, 71 are known to mucosal and skin alpha papillomaviruses. In contrast, the
recognize conformational epitopes, including 34 neutral- cross-reactivity to HPV11 GST–L1, which was not seen
izing epitopes, and 14 are known to recognize linear with HPV11 VLP, was very weak. For mAb H16.7E we
epitopes, of which eight were reported also to react with confirm the absence of cross-reactivity to HPV11
someintactVLPpreparations(see Tables1and2 formAb (Christensen et al., 2001), but observed a weak cross-
and references). These findings suggest that the GST–L1 reactivitytoHPV35.However,wedidnotseeanyreactivity
fusion protein preparations display neutralizing and con- to the four other HPV types in the same species (a9). For
formationalaswellaslinearepitopes.TwomAbrecognizing mAb H16.2F we confirm the absence of cross-reactivity to
linearepitopes(H16.B20,H16.S1)didnotreactwithGST– HPV11(Christensenetal.,2001),butweadditionallyfound
L1ofHPV33or31,respectively,incontrasttotheirreported weakcross-reactivitywithHPV18andHPV45(botha7).
cross-reactivity with denatured VLP of these types, indic-
Wedescribenewcross-reactivityfor12additionalantibod-
atingthattheGST–L1preparationsofthesetypespresented
ies,includingfiveraisedagainstHPV16,oneagainstHPV18,
theselinearepitopesininsufficientamounts.
two against HPV45, one against HPV33 and three against
The titres of the different mAb preparations used in this the HPV11 G131S L1 mutant. Moreover, we extend the
study varied by several orders of magnitude. Choosing of cross-reactivitypatternfor12mAb.Altogetherwehavedata
the first dilution below saturation for cross-reactivity for 43 cross-reactive monoclonal antibodies, encompassing
experiments allowed comparison of the strength of the a total of 153 individual cross-reactions. This provides a
cross-reactivity signals and their classification into three comprehensive description of some features of cross-
categories, strong, intermediate or weak. In a previous, reactiveepitopesamonghumanpapillomaviruses.
smalleranalysisofVLPcross-reactivityofsomeofthemAb
Cross-reactivity follows the phylogenetic grouping only
usedhere,onlyauniformdilutionof1:50oftissueculture
loosely. Among very closely related HPV types, i.e. HPV6
supernatants irrespective of the effective antibody concen-
and 11 (.92% aa identity in L1) as well as 18 and 45
tration had been applied (Christensen et al., 1996a).
(88%), cross-reactivity is frequent (Table 3). Since no
Amongthe89reactivemAb,46reactedonly withGST–L1 otherHPVtypesfromspeciesa10anda7wereanalysed,it
proteins of the specific HPV type used for immunization, isunclearwhethertheremightbemoreintra-speciescross-
indicatingthatGST–L1fusionproteinsaredisplayingtype- reactivity among these antibodies. Only two of the 17
specific epitopes. Although we analysed cross-reactivity cross-reactive mAb raised against a HPV type of the a9
with 15 alpha papillomavirus types, as compared to a species were strictly intra-species cross-reactive. These two
maximumofninetypesinpreviousVLPstudies,wecannot mAb both recognized only a subset of the six HPV types
excludethatsomeofthemAbclassifiedhereasmonotypic analysed from this species, suggesting that species-specific
might turn out to be cross-reactive when analysed with epitopes are rare or even do not exist. Furthermore, most
additional types. Previously published data suggested that mAb with inter-species cross-reactivity did not react with
conformation-dependentmAbH16.11BandH16.8Bmight all members of the species of the HPV type against which
be specific for HPV16 alone (Christensen et al., 2001), but they were raised, indicating again that HPV species may
here we describe strong cross-reactivity to many other not share common epitopes.
Table3. Summary ofmAb reactivities
Immunogen* Reactivity Cross-reactivitypattern
HPVtype
All Mono- Cross- All Intra-species Inter-species Mixed
All 89 46 43 43 18 4 21
a9
16 27 11 16 16 2 4 10
31 1 1
33 4 3 1 1 1
35 3 3
52 4 4
a7
18 11 5 6 6 6
45 5 3 2 2 2
a10
6 8 3 5 5 4 1
11 26 13 13 13 10 3
*ForgroupingofmAbagainsthybridH11:16andmutant11G131SseeTables1and2.
http://vir.sgmjournals.org 125
R.Z.Rizkandothers
There were no mAb specific for all mucosal alpha phylogenetic L1-based species definition may not define a
papillomaviruses analysed that also lacked reactivity to serologicalunitsincenospecies-specificepitopewasfound.
anyofthefouralphaskinHPV.Manyothercross-reactive
ForH16.J4,neutralizingactivitywasdescribedbyCombita
mAb did not recognize some of the most closely related
et al. (2002), whereas in our experiments this antibody
HPV, but rather showed cross-reactions with individual,
showed no HPV16 neutralization.
distantlyrelatedHPVofotherspecies,includingalphaskin
HPV described here for the first time. Two of the HPV16 For five of the six cross-reactive neutralizing mAb, cross-
mAb (H16.H5 and H16.9A) did not react with any of the neutralization could be assessed. H6.L12 was strongly
fiveotherHPVinspeciesa9butinsteadreactedselectively cross-reactive with the very closely related HPV11 and
with HPV18 L1. The weak cross-reaction suggests that the neutralizedHPV11withasimilartitreasHPV6,whichisin
epitope recognized on HPV18 is not identical to that on agreement with previously published data (Christensen
HPV16. Antibody H16.H5 has been described to bind et al., 1996b). Antibodies H16.2F, H6.F62, Ritti01 and
HPV16 L1 peptide aa 174–185 (Christensen et al., 1996a). MM07wereweaklycross-reactivewithHPV18/45,HPV11,
HPV18hassevenmismatchesinthehomologoussequence, andHPV45(Ritti01andMM07),respectively, but showed
suggesting that weak cross-reactivity is not mediated by no cross-neutralization with these types. The weak cross-
this sequence. On the other hand, two other HPV16 mAb reaction indicates lower affinity of the mAb to the cross-
(Ritti01 and MM07) reacted with five of the six a9 HPV reactive epitope, which might be insufficient to induce
types and very selectively also with HPV45. One of them neutralization on the cross-reactive epitope; alternatively,
reacted marginally even with skin HPV2, but not with the absence of cross-neutralization might indicate that the
closely related skin HPV57. Monoclonal antibody H16.S1 cross-reactive epitope is functionally different, i.e. that
cross-reacted with mucosal HPV types, including HPV35 antibody binding does not lead to neutralization.
(a9), HPV18 and 45 (a7) and additionally with skin HPV
GST–L1 ELISA titres were always higher than neutraliza-
types 3 and 10 (a2). It has been described to bind HPV16
tiontitres,ashasbeendescribedpreviously(Combitaetal.,
L1 peptide aa 111–130 (Christensen et al., 1996a). Leucine
2002; White et al., 1999) (Tables 1, 2). ELISA log titres on
residues 122 and 126 appear to be critical since all non-
average were similar among monospecific (median 5.3,
reactiveHPVtypeshavereplacedatleastoneofthembyan
range 3.8–6.2) and cross-reactive mAb (5.0, 2.0–6.2).
aromatic residue and the weakly cross-reactive HPV10
However, cross-reactive mAb rather consistently showed
differs at the neighbouring position (D127E).
lower neutralization log titres (2.5, 1.7–3.8) than the
While HPV18 was the type most frequently recognized by monospecific mAb (3.5,2.3–5.6). Thus, theratio ofELISA
cross-reactive HPV16 mAb (12 out of 16), only one (pan- to neutralization titres was on average about 100-fold
reactive H18.A7) of the six cross-reactive HPV18 mAb higher among the cross-reactive (log median 3.3, range
recognized HPV16. Most frequently observed inter-species 2.4–3.6)thanamongthemonospecificmAb(1.5,0.2–2.9).
cross-reactivity was directed against skin HPV3, 10 and/or This may indicate that neutralization epitopes are highly
2,57.AntibodyH18.Q2cross-reactedwithHPVtypes45,2 type-specific. The cross-reactive antibodiesthat aremono-
and 57. specific in neutralization might recognize the L1 surface
involved in neutralization (functional structure) only
Of14mAbdetectingalinearepitope,13wereinter-species
partially because they also recognize neighbouring, non-
cross-reactiveandone(H16.15G)monotypic(Table4).Of
functional structures shared by other types; thus their
71mAbdetectingconformationalepitopes,29(41%)were
ability to induce a structural change or to block a surface
cross-reactive. While 16 of these recognized only closely
essential for the infection process is reduced, but could be
related HPV types (6/11 and 18/45), three showed weak
overcome in higher concentrations, resulting in lower
and four strong inter-species cross-reactions, even includ-
neutralization titres.
ing skin HPV types. In conclusion, the cross-reactivity
observed here suggests that distantly related mucosal and In conclusion, HPV capsid epitopes defining neutralizing
skinalphaHPVshareconformationalepitopesandthatthe sites are always conformational and most of the mAb
Table4. Association ofcross-reactivity ofHPVmAb withlinear andconformational epitopes
Epitope Reactivity Neutralizing
activity
All Monotypic Cross-reactive
Intra-speciesonly Inter-speciesormixed
Total 85 43 16 26 34
Conformational 71 42 16 13 33
Linear 14 1 0 13 1
126 JournalofGeneralVirology89