Naturally-occurring capsid protein variants of human papillomavirus genotype 31 1
represent a single L1 serotype.
2 3
Sara L. Bissett,1 Anna Godi,1 Maxime J. J. Fleury,2 Antoine Touze,3 Clementina 4
Cocuzza,4 and Simon Beddows1#
5
6
1 Virus Reference Department, Public Health England, London, UK 7
2 GEIHP, UPRES EA 3142, Université d’ Angers, Angers, France 8
3 UMR 1282, Université François Rabelais/INRA, Tours, France 9
4 Department of Surgery and Translational Medicine, University of Milano-Bicocca, 10
Monza, Italy 11
12
# Corresponding author: Simon Beddows, Public Health England, 61 Colindale Avenue, 13
London NW9 5EQ, UK. Tel: +44 (0) 20 8327 6169. simon.beddows@phe.gov.uk 14
15
Running Title: HPV31 capsid protein variants (Max 54 characters including spaces) 16
Key words: Human Papillomavirus, L1, L2, Variant, Antibody, Vaccine, Neutralization, 17
Serotype 18
Abstract: 126 19
Main text: 3,956 20
JVI Accepted Manuscript Posted Online 20 May 2015 J. Virol. doi:10.1128/JVI.00842-15
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
Abstract 21
We investigated naturally-occurring variation within the major (L1) and minor (L2) capsid 22
proteins of oncogenic human papillomavirus (HPV) genotype HPV31 to determine the 23
impact on capsid antigenicity. L1L2 pseudoviruses (PsV) representing the three HPV31 24
variant lineages A, B and C exhibited comparable particle to infectivity ratios and 25
morphology. Lineage-specific L1L2 PsV demonstrated subtle differences in 26
susceptibility to neutralization by antibodies elicited following vaccination, pre-clinical L1 27
VLP immunization or by monoclonal antibodies; however, these differences were 28
generally of a low magnitude. These data indicate diagnostic lineage-specific single 29
nucleotide polymorphisms within the HPV31 capsid genes have a limited effect on L1 30
antibody mediated neutralization and that the three HPV31 variant lineages belong to a 31
single L1 serotype. These data contribute to our understanding of HPV L1 variant 32
antigenicity.
33 34
Importance 35
The virus coat (capsid) of the human papillomavirus contains major (L1) and minor (L2) 36
capsid proteins. These proteins facilitate host cell attachment and viral infectivity and 37
are the targets for antibodies which interfere with these events. In this study we 38
investigated the impact of naturally-occurring variation within these proteins upon 39
susceptibility to viral neutralization by antibodies induced by L1 VLP immunization. We 40
demonstrate that HPV31 L1 and L2 variants exhibit similar susceptibility to antibody- 41
mediated neutralization and that for the purposes of L1 VLP-based vaccines these 42
variant lineages represent a single serotype.
43
Introduction 44
Human papillomaviruses (HPV) comprise a double-stranded DNA genome of 45
approximately 8 kb which is replicated via host cell polymerases with an error rate of ca.
46
2x10-8 base substitutions, per site, per year (1), substantially lower than that found in the 47
majority of single-stranded RNA viruses (ca. 1x10-3 base substitutions/site/year) (2).
48
Despite the low evolutionary rate of the HPV genome, variants have arisen over time 49
leading to the generation of distinct intra-genotype lineages classified by a sequence 50
difference of 1-10% across the whole genome (3). The single nucleotide polymorphisms 51
(SNP) that allow segregation of these variants into distinct lineages can be found in 52
each gene/region, with the highest number accumulating in the noncoding regions 53
(NCR1, NCR2 and URR) and the lowest in structural genes (L1 and L2) (4).
54 55
The HPV structural genes encode the major (L1) and minor (L2) proteins which form the 56
non-enveloped icosahedral viral capsid that comprises 72 pentameric L1 capsomers 57
with upper estimates of one L2 protein per capsomer (5). The L1 protein mediates 58
attachment to host cells (6) whilst the L2 protein is essential for subsequent viral 59
infectivity (7).
60 61
The humoral immune response following natural HPV infection predominately targets 62
conformational epitopes on the surface-exposed loop regions of the L1 protein (8, 9).
63
Seroconversion generally occurs 6-18 months after infection with low levels of L1 64
antibodies detected in 50-70% of individuals (10, 11). It is not clear whether antibodies 65
induced by natural infection protect against subsequent reinfection by the same HPV 66
genotype but increasing evidence indicates that high antibody titers can be associated 67
with a reduced risk of reinfection (12-15).
68 69
The L1 protein can self-assemble into virus-like particles (VLP) which are the basis of 70
the current prophylactic HPV vaccines, Cervarix® and Gardasil® (16). Clinical trials have 71
demonstrated the high efficacy of both vaccines against infection and cervical disease 72
associated with vaccine genotypes HPV16 and HPV18. A degree of vaccine-induced 73
cross-protection has also been demonstrated against closely-related genotypes, 74
particular HPV31, HPV33 and HPV45 (16-18). HPV vaccine type-specific protection is 75
assumed to be mediated by L1 neutralizing antibodies which can be detected in the 76
serum and cervicovaginal secretions of vaccinees (16, 19-22). The role of L1 77
neutralizing antibodies in mediating cross-protection is less clear although a recent 78
study reported an association between the presence of HPV31 cross-neutralizing 79
antibodies and a reduced risk of HPV31 infection (23). Next generation L1 VLP-based 80
vaccines aim to extend the breadth of coverage by incorporating an increased number 81
of L1 VLP (24, 25).
82 83
Intra-genotype variation within the L1 protein is generally localized to the surface- 84
exposed loop domains (26), akin to the majority of inter-genotype variation (27, 28).
85
Data informing the potential impact of such variation on L1 antigenicity are limited to 86
HPV16 where L1L2 pseudoviruses (PsV) representing lineage-specific L1 variants were 87
neutralized to a similar extent by antibodies elicited against a single L1 VLP (29).
88
However, the impact of a common Thr to Ala switch at amino acid residue position 266 89
within the FG loop of HPV16 was not evaluated, nor was variation within the 90
corresponding L2. Another study found that an FG loop specific neutralizing monoclonal 91
antibody (MAb), H16.E70, had reduced recognition for HPV16 L1 VLP bearing a Thr at 92
amino acid residue 266 indicating that variation within this region can impact upon 93
antigenicity (30).
94 95
HPV31 is closely related to HPV16 within the Alpha-9 species group and is associated 96
with ca. 3.8% of cervical cancer cases worldwide (31). The full genome sequence 97
analysis of HPV31 has led to the delineation of three distinct variant lineages: A, B and 98
C (4). Infections due to HPV31 lineage variants A or B have been associated with an 99
increased risk of developing cervical intraepithelial neoplasia grades 2–3 (CIN2/3) yet 100
somewhat paradoxically infections with lineage variant C appear to persist for longer 101
(32, 33). Differences in the natural history between variants of other HPV genotypes 102
have also been observed (34, 35). The relative infectivity of variants or the ability of 103
variants to differentially disrupt cellular differentiation are possible virological factors 104
which can contribute to the disparities in variant pathology (3). One study suggested 105
that the genetic background of the host may also play a role since African-American 106
women were found to be less likely to clear a HPV31 lineage variant C infection than a 107
lineage variant A infection, yet there was no difference in the likelihood of clearing 108
lineage variant A over C in Caucasian women (33).
109 110
Two non-synonymous, lineage-specific SNP within the L1 of HPV31 are located within 111
the FG loop at positions 267 and 274 (26). The FG loop of HPV31 has been shown to 112
be an important antigenic domain targeted by both type-specific and cross-reactive L1 113
MAbs (36, 37). In the present study we generated additional HPV31 L1 and L2 114
sequences and synthesized representative antigens in order to investigate the potential 115
impact of this variation. Such data should improve our understanding of the potential 116
biological impact of naturally-occurring HPV31 variation.
117
Materials and Methods 118
119
Study samples 120
Residual vulva-vaginal samples collected from 16-24 year old females undergoing 121
chlamydia testing in England which had previously been confirmed as HPV31 DNA 122
positive using the Hybrid Capture 2 HPV DNA test (Qiagen) and the Linear Array HPV 123
Genotyping test (Roche) (38) were selected for L1 and L2 sequencing. Cervical cell 124
samples from HPV31 DNA positive women (aged 19 – 76 years) attending 125
gynaecological care at the San Gerardo hospital (Milan, Italy: Ethical approval study 126
code: 08/UNIMIB-HPA/HPV1) following a cytological diagnosis of ASCUS (atypical 127
squamous cells of undetermined significance) or LSIL (Low grade squamous 128
intraepithelial lesion) were available for L1 and L2 sequencing. Serum samples were 129
available from HPV31 DNA positive women within this cohort. Serum samples were 130
available from 12-15 year old girls one month after receiving three doses of Cervarix® or 131
Gardasil® HPV vaccine (20). A panel of HPV31 MAbs were available as either ascites 132
fluid or tissue culture supernatant (39).
133 134
Sequencing of HPV31 capsid genes 135
The L1 (base pair 5443-7119 (40) or FG loop region 6141-6476; numbered according to 136
the HPV31 reference, J04353) and L2 (3921-5725) genes were amplified with Platinum 137
Taq® High Fidelity DNA polymerase (Life Technologies) and sequenced using the ABI 138
Genetic Analyzer 3730. Sequence data were collated using DNASTAR Lasergene® 9.0 139
software (DNASTAR, Inc). The HPV31 L1 and L2 sequences generated in this study 140
were assigned the following GenBank accession numbers: KJ754561 – KJ754580.
141
Additional HPV31 L1 and L2 sequences were downloaded from the National Center for 142
Biotechnology Information [NCBI: http://www.ncbi.nlm.nih.gov/, accession numbers:
143
HQ537666 - HQ537687 (4), U37410 (41) and J04353 (42)] and analyzed using the 144
Neighbour-Joining tree algorithm with bootstrap values (n=500 iterations) generated 145
using MEGA v6 (43). HPV31 L1 variant residues were mapped to the surface of the 146
HPV16 capsomer crystal structure (PDB code: 2R5H) and analyzed using Swiss-PDP 147
viewer v4.0 (Deep View) (44).
148 149
L1 VLP 150
HPV31 L1 VLP were expressed using the Bac-to-Bac® Baculovirus System (Life 151
Technologies) and purified on a iodixanol (Sigma-Aldrich) gradient as previously 152
described (45). The L1 protein was visualized by SDS-PAGE stained with SimplyBlue™
153
SafeStain (Life Technologies) alongside a standard curve derived from known input 154
concentrations of bovine serum albumin. Gel analysis was carried out using ImageJ 155
software (U. S. National Institutes of Health, http://imagej.nih.gov/ij) to determine the L1 156
concentration of the gradient fractions. VLP formation was confirmed by electron 157
microscopic analysis of negatively stained particles. The HPV31 L1 VLP shared a 100%
158
amino acid sequence identity with the L1 gene of the HPV31 reference sequence 159
(J04353) of lineage variant A. QuikChange® Site-Direct Mutagenesis (Stratagene) was 160
employed to generate L1 sequences representing lineage variants HPV31 B and 161
HPV31 C. The L1 VLP were used as target antigens in an ELISA, as previously 162
described (20, 45). The panel of HPV31 MAbs were tested at a standardised input 163
concentration of 250µg/mL of mouse IgG, except 31.D24 (starting input 20µg/mL). The 164
MAbs were subjected to serial dilutions and the IgG concentration which resulted in 165
50% maximal binding optimal density (OD) was estimated by interpolation and the 166
results presented as a 50% binding concentration.
167 168
Mouse immunizations 169
VLP were adsorbed onto aluminium hydroxide (Alhydrogel, Brenntag Biosector) before 170
addition of the Monophosphoryl Lipid A (MPL) based Sigma Adjuvant System (Sigma- 171
Aldrich). BALB/c mice were injected intramuscularly with 2μg of VLP on Day 0 and Day 172
14, before a terminal bleed was taken at Day 21. Pre-bleeds were taken from all mice 173
prior to initial immunization. A total of ten mice were immunized with either HPV31 A 174
VLP, HPV31 B VLP or HPV31 C VLP over three separate immunization schedules. All 175
animal husbandry and procedures were carried out in strict accordance with UK Home 176
Office guidelines and governed by the Animals (Scientific Procedures) Act 1986 and 177
performed under licences PPL 70/7412 & 70/7414.
178 179
L1L2 pseudoviruses 180
A bicistronic psheLL vector (46) containing codon-optimized HPV31 L1 and L2 genes 181
representing the HPV31 reference (J04353) of lineage variant A was expressed and 182
purified on a iodixanol (Sigma-Aldrich) gradient as previously described (47). The L1 183
and L2 gene representing lineage variants HPV31 B and HPV31 C were either 184
synthesisedby GeneArt® (Life Technologies) or generated by QuikChange® Site-Direct 185
Mutagenesis (Stratagene). Particle formation and size were determined by electron 186
microscopic analysis of negatively stained particles. The L1 concentrations of PsV 187
stocks were estimated by semi-quantitative L1 western blot using CamVir-1 (abcam, 188
U.K.) and the Tissue Culture Infectious Dose 50% (TCID50) was estimated using the 189
Spearman-Karber equation as previously described (47). Particle to infectivity (PI) ratios 190
were determined based on an estimated 3x107 particles per ng L1 protein 191
(http://home.ccr.cancer.gov/lco/production.asp) with the ratio normalized for input 192
volume and TCID50. The presence of L2 protein and reporter gene (Luciferase) in 193
purified PsV stocks was confirmed by qualitative L2 western blot using HPV16 L2 anti- 194
peptide sera (amino acids 17-36) and qualitative PCR (base pair 1222-1641; pGL4.51, 195
Promega) following DNA extraction (QIAamp DNA Blood Mini Kit, Qiagen), respectively.
196
The PsV neutralization assay was performed as previously described (48) with minor 197
modifications (47). A standardized input of 100 TCID50 was used for all PsV and 198
samples were subjected to serial dilutions with the antibody titer or concentration 199
resulting in a 80% reduction of the luciferase signal (RLU) produced by the control wells 200
containing PsV only estimated by interpolation. HPV antibody control reagents were 201
included in each assay run (49) alongside Heparin (H-4784, Sigma-Aldrich) which was 202
used as a positive inhibitor control. The median neutralization titer and inter-quartile 203
range for the positive antibody control reagent (High HPV16/18) were as follows: HPV31 204
A PsV 231 (173 – 337; n=14), HPV31 B PsV 462 (387 – 671; n=10) and HPV31 C PsV 205
500 (354 – 589; n=12). The negative antibody control reagent (HPV negative) had a titer 206
of <40 in all assays (n=42).
207
208
Statistical analysis 209
The Wilcoxon paired signed rank test was used to compare neutralization titers using 210
Stata 12.1(Statacorp, College Station, TX).
211
Results 212
213
HPV31 L1 and L2 amino acid variation 214
Full length HPV31 L1 and L2 sequences were represented by contemporary English 215
(n=17) and Italian (n=3) sequences, in addition to available NCBI database sequences 216
(n=24). Analysis of the aligned, concatenated L1 and L2 sequences demonstrated three 217
distinct clusters consistent with the lineages HPV31 A, HPV31 B and HPV31 C (Figure 218
1A). Lineage A contained 14 sequences, including the HPV31 reference sequence 219
(J04353), 7 of which demonstrated variation from the reference in the L1 (T432S) and a 220
single sequence demonstrated variation within the L2 (I270M) (Figure 1B). The 13 221
sequences in lineage B all demonstrated variation from the reference sequence at L1 222
amino acid position T274N within the FG loop whilst 4 sequences also varied at position 223
S194T and 1 varied at position T432A. Five sequences exhibit variation from the 224
reference at L2 position T362A. Lineage C contained 17 sequences all of which 225
demonstrate variation from the reference at L1 amino acid position T267A and T274N 226
within the FG loop and L2 positions V115I, I270M, V377L, 5 sequences also varied from 227
the reference at L2 amino acid position N269D.
228 229
Sensitivity of variant HPV31 L1L2 PsV to antibody-mediated neutralization 230
L1L2 PsV representing lineage variants HPV31 A (Ref: J04353), HPV31 B and HPV31 231
C were generated from the consensus L1 and L2 sequences representing each lineage 232
and bear the major L1 (267 and 274) and L2 (115, 270 and 377) variant residues 233
(Figure 2A). All three lineage variant PsV, herein referred to as HPV31 A PsV, HPV31 234
B PsV and HPV31 C PsV, generated similar sized PsV particles of around 50nm and 235
produced comparable PI ratios of ca. 102 (Figure 2B). The PsV preparations also 236
contained L2 protein and luciferase reporter plasmid (data not shown).
237
238
The HPV31 variant PsV were tested against sera from girls who received either the 239
Cervarix® or Gardasil® (n=46) HPV vaccine (Table 1). Both HPV31 B and C PsV were 240
more sensitive to neutralization when compared to HPV31 A PsV. HPV31 B PsV 241
displayed a median 1.7-fold (inter-quartile range, IQR, 1.1 – 2.4) (Wilcoxon paired 242
signed rank test p <0.001) increased sensitivity to vaccine-induced cross-neutralizing 243
antibodies whilst HPV31 C PsV displayed a 1.4-fold (1.1 – 1.6) (p <0.001) increase in 244
sensitivity compared to HPV31 A PsV. The increased sensitivity of HPV31 B and C PsV 245
to cross-neutralizing antibodies was independent of the HPV vaccine received (Table 246
247 1).
248
All three HPV31 variant PsV were susceptible to neutralization by a small panel of 249
longitudinal (month 0, 6, 12 and 18) serum samples from women naturally infected with 250
HPV31 (Table 2). Median antibody neutralization titers were 576 (391 – 1,144), 839 251
(587 – 1,899) and 882 (337 – 1,895) against HPV31 A, B and C PsV, respectively.
252
253
Immunogenicity of variant HPV31 L1 VLP 254
HPV31 L1 VLP bearing the L1 variant residues T267A and T274N (Figure 2A), herein 255
referred to as HPV31 A, HPV31 B and HPV31 C VLP were expressed and used to 256
immunize BALB/c mice. All three preparations contained VLPs of various sizes, ranging 257
from ca. 20 nm up to 60 nm in diameter with VLPs of >40 nm in diameter constituting 258
30% of the HPV31 B VLP, 37% of the HPV31 A VLP and 57% of HPV31 C VLP 259
preparations (Figure 2C).
260 261
The three variant PsV demonstrated differential susceptibility to neutralization by HPV31 262
VLP mouse polyclonal serum (Figure 3). Both HPV31 B PsV (median log10
263
neutralization titer, 4.27; IQR, 3.75 – 4.55) (Wilcoxon paired signed rank test p = 0.008) 264
and HPV31 C PsV (4.18; 3.86 – 4.49) (p = 0.007) were more sensitive to neutralization 265
by HPV31 A VLP sera compared to the homologous HPV31 A PsV (4.09; 3.54 – 4.26).
266
HPV31 A PsV was less sensitive to neutralization by HPV31 B VLP sera (3.74; 3.53 – 267
4.07) (p = 0.031) whilst HPV31 C PsV demonstrated increased sensitivity (4.12; 3.96 – 268
4.53) (p = 0.028) compared to HPV31 B PsV (3.98; 3.69 – 4.22). Both HPV31 B and C 269
PsV had similar sensitivity to neutralization by HPV31 C VLP sera (B PsV: 4.42 [4.04 – 270
4.35]; C PsV: 4.34 [4.08 – 4.55]) whilst HPV31 A PsV demonstrated a reduced 271
sensitivity (4.01; 3.84 – 4.14) (p = 0.021). There were also differences in the magnitude 272
of the antibody response based upon reactivity against homologous PsV with HPV31 C 273
VLP (4.34; 4.08 – 4.55) being slightly more immunogenic than HPV31 A VLP (4.09; 3.54 274
– 4.26) (p = 0.018) and HPV31 B VLP (3.98; 3.69 – 4.22) (p = 0.006).
275
276
Antigenicity of variant HPV31 L1 VLP and L1L2 PsV 277
HPV31 L1 MAbs were previously (39) generated against immunogens representing the 278
HPV31 reference sequence (J04353). The majority were raised against L1L2 VLP apart 279
from 31.D24 and 31.A19 where L1 VLP immunogens were used. Generally all the type- 280
specific MAbs bound L1 VLP and L1L2 PsV representing the lineages variants A, B and 281
C at similar 50% binding concentrations (µg/mL) by ELISA; the exceptions to this were 282
31.F16 which demonstrated a higher binding concentration against the HPV31 B PsV in 283
comparison the HPV31 A PsV whilst both 31.H12 and 31.H17 bound L1 VLP 284
representing HPV31 lineage variants B and C at lower concentrations compared to A.
285
Although the cross-reactive MAbs (31.D24, 31.B5, 31.C19 and 31.E22) bound the L1 286
VLP they did not bind or neutralize the L1L2 PsV (Table 3). All type-specific MAbs were 287
able to neutralize the three variant PsV to similar orders of magnitude (Table 3). HPV31 288
C PsV demonstrated an increased susceptibility, ca. 3.5-fold, to neutralization by FG 289
loop MAb 31.F16 and a ca. 4.0-fold increase in sensitivity to MAb 31.H17 (epitope 290
unknown) compared to HPV31 A and B PsV (Table 3).
291 292
The three neutralizing FG loop MAbs (31.B1, 31.F16 and 31.H12) recognize 293
conformational epitopes which encompass variant amino acid position 267 and are 294
adjacent to amino acid 274 (Figure 4A). The FG loop of monomer 1 (FG1) is adjacent to 295
the BC5, DE1, DE5, EF1, HI4 andHI5 loops within the capsomer (Figure 4B) and residues 296
267 and 274 are within close proximity (within 10Å) to residue positions predominantly 297
within the adjacent BC5, FG1,and HI5 loops (Figure 4C and 4D). These include Lysine 298
residues at position 279 within the FG loop and 362 within the HI loop.
299
Discussion 300
This study attempted to evaluate the potential impact of non-synonymous SNP within 301
the HPV31 L1 and L2 genes on capsid protein antigenicity and immunogenicity. We 302
generated additional HPV31 L1 and L2 sequences to supplement those already 303
available and created L1L2 PsV and L1 VLP representing lineage variants A, B and C to 304
evaluate lineage-specific immunogenicity and antigenicity, including susceptibility to 305
antibody-mediated neutralization.
306
307
All three variant L1L2 PsV were susceptible to neutralization by vaccine-induced cross- 308
neutralizing antibodies. HPV31 B and C PsV demonstrated an increased sensitivity to 309
neutralization compared to HPV31 A PsV but the difference was of a low magnitude.
310
The cross-protection afforded by the current prophylactic vaccines is an unexpected 311
additional benefit, although no correlate of protection has been defined (10). If cross- 312
neutralizing antibodies are determined to be the immune effectors of vaccine-induced 313
cross-protection it is important to demonstrate that the contemporary circulating HPV31 314
variants, represented by L1L2 PsV, do not exhibit resistance to cross-neutralization by 315
such antibody specificities.
316 317
The increased sensitivity of HPV31 B and C PsV to cross-neutralization suggests that 318
the Asn residue at position 274, which is common to both variants, enhances the 319
recognition of HPV31 L1 epitopes by cross-neutralizing antibodies produced against 320
vaccine HPV16 L1 VLPs. It is unlikely that the Asn residue in itself has a critical role 321
within a cross-neutralizing epitope since the switch from Thr and Asn is a relative subtle 322
change as both amino acids have polar uncharged side chains. However, the change of 323
residue at position 274, near the tip of the FG loop, may result in local structural 324
changes which increase recognition of more distal epitope residues.
325 326
Subtle differences in variant antigenicity were identified when a panel of HPV31 MAbs 327
were tested in a neutralization assay against the HPV31 lineage variants. HPV31 C PsV 328
demonstrated increased sensitivity to neutralization by the FG loop MAb 31.F16 in 329
comparison to both HPV31 A and B PsV indicating that the double residue switch at 330
positions T267A and T274N impacts upon 31.F16 epitope recognition. It has previously 331
been demonstrated by comparison of L1 pentamer crystal structures from different 332
genotypes (HPV11, HPV16, HPV18 and HPV35) that L1 antigenic determinants can be 333
altered by a shift of a few angstroms within the loop as a result of a single residue 334
substitution (28).
335 336
In contrast the other two FG loop MAbs (31.B1 and 31.H12) neutralized all variants to a 337
similar extent. These data corroborate previous data from a bacterial cell surface display 338
model which demonstrated that these three MAbs recognise overlapping, yet distinct FG 339
loop epitopes (36). Residues 267 and 274 are in close proximity to (Lys279 & Lys362), 340
or near (Lys54, Asn57, Lys60 & Lys367) residues, the corresponding residues of 341
HPV16 are involved in HPV binding to heparin sulfate (50) which is an essential step for 342
a successful HPV infection and the FG loop MAbs may neutralize by abrogating this 343
345
The panel of HPV31 MAbs bound all three variant L1 VLP, when used as target 346
antigens in an ELISA format, indicating that the residues at positions 267 and 274 were 347
not critical in the epitope footprints recognized by this panel of MAbs. However, when 348
variant L1L2 PsV were used as target antigens, the 50% binding concentration of the 349
four cross-reactive MAbs (31.D24, 31.B5, 31.C19 and 31.E22) could not be determined 350
due to the reduction in epitope recognition. This observation implies that inclusion of the 351
L2 protein within the PsV capsid alters L1 epitope exposure and therefore impacts upon 352
L1 protein antigenicity. It has been reported that a subset of HPV16 MAbs demonstrated 353
reduced binding to L1L2 PsV compared to L1 VLP with the differential binding thought 354
to be as a result of L2 altering the conformation or availability of L1 epitopes (51). These 355
findings imply that the capsids of native HPV virions, represented by L1L2 PsV, and the 356
L1-only VLP within the prophylactic vaccine preparations differ in their L1 antigenicity.
357 358
All three variant L1L2 PsV were susceptible to neutralization by HPV31 VLP antibodies 359
generated in mice; however, differences in neutralization sensitivity were evident. Both 360
HPV31 B and C PsV, in comparison to HPV31 A PsV, demonstrated increased 361
sensitivity to antibody-mediated neutralization irrespective of the variant L1 VLP used as 362
the immunogen. These findings are in line with a previous study of HPV16 variants 363
which demonstrated that HPV16 VLP sera raised against a European variant was able 364
to neutralize pseudoviruses representing a range of geographical variants of HPV16 365
with ≤4-fold difference in neutralization titer between the homologous and heterologous 366
types, leading to the conclusion that HPV16 variants belong to a single serotype (29).
367
368
The criterion used to designate serotypes is generally based upon an fold difference in 369
antibody-mediated neutralization titers between viral types, which differ in magnitude 370
and range between virus families: Adenovirus 8- to 16-fold (52), Rotavirus ≥20-fold (53), 371
Polyomavirus 4- to 100-fold (54). For HPV there are no currently defined criteria with 372
which to designate L1 serotypes. It is reasonably clear that HPV genotypes induce high 373
titer, type-specific neutralizing antibody responses which represent different serotypes 374
(55-57). However, for lineage variants the relationship between L1 sequence and 375
antigenicity is less clear (29, 58).
376 377
Although HPV31 lineage variants demonstrated differences in susceptibility to antibody- 378
mediated neutralization by vaccine, pre-clinical and MAb panels the difference was <4- 379
fold and under this criterion, as defined for HPV16 (29), HPV31 variants should be 380
considered as belonging to a single serotype. This implies that the choice of a 381
representative HPV31 L1 sequence for VLP based vaccines is not critical.
382
383
Given the relatively low prevalence of HPV31 (59), only a small panel of serum samples 384
from HPV31 DNA positive women were available. These data suggest that all three 385
HPV31 lineage variants were susceptible to neutralization by antibodies derived from 386
natural infection. However, further work will be required to address this issue 387
appropriately by utilizing a larger panel of samples with an equal representation of 388
variant lineages.
389
There are potential shortcomings to this work. L1 VLP immunizations were carried out 391
using a relative small number of animals and while all three constructs induced 392
neutralizing antibodies against all three variant PsV, the variability inherent in using 393
small groups of animals may have concealed subtle differences in immunogenicity.
394
Although HPV L1L2 PsV have been used widely to monitor antibody responses to 395
vaccine and natural infection (22, 47, 48, 60), as well as elucidate steps in the entry 396
process (61-64), there are likely to be some differences between how these behave in 397
vitro and how authentic HPV31 lineage variants behave in vivo, although this is a 398
limitation of most PsV-based systems.
399 400
Despite these caveats, these data suggest that HPV31 lineage variant PsV display 401
similar sensitivity to recognition by antibodies elicited following vaccination with the 402
current HPV vaccines and after pre-clinical HPV31 L1 VLP immunization indicating that 403
HPV31 variants belong to a single L1 serotype. Such data may be useful to guide 404
impact modelling of the current L1 VLP vaccines and informing post-vaccine 405
surveillance programmes. These data also inform our understanding of the antigenicity 406
of the HPV structural proteins.
407
Acknowledgments 408
The sera from young girls who received Gardasil® or Cervarix® came from a study 409
funded by the Department of Health Policy Research Programme (National Vaccine 410
Evaluation Consortium, 039/0031). The views expressed in this publication are those of 411
the author(s) and not necessarily those of the Department of Health.
412 413
We are indebted to Prof. John T. Schiller and Dr. Chris Buck (National Cancer Institute, 414
Bethesda, U.S.A.) for access to the p31sheLL clone. We thank Ilaria Epifano from the 415
University of Milano-Bicocca, Monza, Italy for the natural infection HPV31 DNA and 416
antibody status data.
417 418
The authors of this paper declare no conflicts of interest.
419
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644 645
Figure 1. HPV31 L1 and L2 variation. (A) Phylogenetic tree constructed from 646
concatenated L1 and L2 nucleotide sequences, labelled with designated lineages (A, B 647
& C) (51) and bootstrap values >95%. (B) Site-specific amino acid co-variation within 648
the L1 and L2 proteins. N indicates the number of sequences in the phylogentic tree 649
represented by the each L1 and L2 combination.
650 651
Figure 2. HPV31 L1 and L2 variants. (A) Graphical representation of L1 and L2 variant 652
protein combinations. (B) L1L2 pseudovirus preparation characterized for particle 653
dimension in nm (median, inter-quartile range [IQR]), infectivity and L1 concentration.
654
(C) L1 VLP dimensions in nm (median, IQR for particles >40 nm in diameter) and L1 655
concentration.
656
657
Figure 3. Heatmaps representing the neutralization potential of variant HPV31 L1 VLP 658
immunization serum against variant HPV31 L1L2 PsV. Log10 neutralization titers of sera 659
from BALB/c mice (n = 10) following variant HPV31 VLP immunization carried out over 660
three separate schedules and presented as the average of two data sets per sample.
661
Key indicates log10 heatmap gradient. p values represent difference in median 662
neutralization titers from homologous variant VLP and PsV pair: *p < 0.05; **p < 0.01;
663
***p < 0.001; NSp > 0.05 using the Wilcoxon paired signed rank test.
664
665
Figure 4. Crystal model surface highlighting HPV31 FG loop variant residue locations.
666
(A) Linear amino acid epitope footprint of HPV31 FG loop MAbs. (B) Side view 667
highlighting loops in close proximity to FG loop variant residues 267 and 274 (C and D).
668
Top view of loop ribbons, circled areas indicate regions within a 10Å radius of residues 669
267 (C) and 274 (D) as determined by Swiss-PDP viewer algorithm. Lysine residues at 670
positons 279 and 362 are highlighted in blue. The FG loop of monomer 1 (FG1) is 671
coloured orange and residues 267 and 274 are highlighted in black. Neighbouring loops 672
on the same (DE1 - dark pink; EF1 - red) or adjacent monomers (HI4 -dark greeen; BC5 - 673
yellow; DE5 - light pink; HI5 - light green) are indicated. The remaining surface exposed 674
regions of the capsomer are coloured in light grey and core regions are coloured in dark 675
grey.
676