CVI Accepted Manuscript Posted Online 13 May 2015 Clin. Vaccine Immunol. doi:10.1128/CVI.00799-14 Copyright © 2015, American Society for Microbiology. All Rights Reserved.
Wang et al
Page 1
1
Sero-epidemiology of HPV16 L2 and generation of papillomavirus L2-
2
specific human chimeric monoclonal antibodies
3
Joshua W. Wanga, Subhashini Jagua, Wai-Hong Wua, Raphael P. Viscidib, Anne
4
Macgregor-Dasa, Jessica M. Fogela, Kihyuck Kwaka, Sai Daayanae, Henry Kitchenere,
5
Peter L. Sternf, Patti E. Gravittg, Cornelia L. Trimblea,c,d and Richard B.S. Rodena,c,d*
6
Departments of Pathologya, Pediatricsb, Oncologyc, Gynecology and Obstetricsd, The
7
Johns Hopkins University, Baltimore, MD, USA; Woman's Cancer Centre , St Mary’s
8
Hospital e and Paterson Buildingf Institute of Cancer Sciences, University of Manchester,
9
Manchester, UK; Department of Pathologyg, University of New Mexico School of
10
Medicine, Albuquerque, NM, USA.
11
Running Title: Human/chimeric antibodies to papillomavirus L2
12
Key Words: Antibody; serology; HPV; L2; Biological Reference Standard; vaccine;
13
papillomavirus
14
*Corresponding Author. Department of Pathology, Johns Hopkins University, Room
15
308, Cancer Research Building 2, 1550 Orleans St., Baltimore, MD 21231, USA. Tel.: +1
16
410 502 5161; fax: +1 443 287 4295; E-mail address:
[email protected] 17
Funding: The study was funded by Public Health Service grants P50 CA098252 and
18
RO1 CA118790 from the National Cancer Institute, and the V foundation
19
(http://www.jimmyv.org/). Sai Daayana was funded by Wigan Cancer Research Fund;
20
Henry Kitchener and Peter Stern were funded by Cancer Research UK
21
(http://www.cancerresearchuk.org/).
22
Competing Interests: Richard Roden and Subhashini Jagu are inventors of L2-related
23
patents licensed to Shantha Biotechnics Ltd., GlaxoSmithKline, PaxVax, Inc. and
24
Acambis, Inc. Richard Roden has received research funding from Sanofi Pasteur,
25
Shantha Biotechnic and GlaxoSmithKline and is a founder of Papivax LLC and a
Wang et al
Page 2
26
scientific advisor to Papivax Biotech Inc.. The terms of these arrangements are
27
managed by Johns Hopkins University in accordance with its conflict of interest policies.
Wang et al
Page 3
28
ABSTRACT (225 WORDS)
29
Presently, the sero-prevalence of human papillomavirus (HPV) minor capsid antigen L2-
30
reactive antibody is not well understood, and no serologic standard exists for L2-specific
31
neutralizing antibodies. Therefore, we screened for HPV16 L2-reactivity a total of 1078
32
sera obtained from four prior clinical studies: a population-based (n=880) surveillance
33
study with 10.8% high risk HPV DNA prevalence, a cohort study of women (n=160) with
34
high-grade cervical intraepithelial neoplasia (CIN), and two phase II trials in women with
35
high-grade vulvar intraepithelial neoplasia (VIN) testing imiquimod therapy combined
36
with either photodynamic therapy (PDT, n=19) or vaccination with a fusion protein
37
comprising HPV16 L2, E7, and E6 (TA-CIN, n=19). Sera were screened sequentially by
38
HPV16 L2 ELISA, then Western blot. Seven of 1078 sera tested had L2-specific
39
antibody, but none were detectably neutralizing for HPV16. To develop a standard, we
40
substituted human IgG1 sequences into conserved regions of two rodent monoclonal
41
antibodies (mAb) specific for neutralizing epitopes at HPV16 L2 residues 17-36 and 58-
42
64, creating JWW-1 and JWW-2 respectively. These chimeric mAbs retained
43
neutralizing activity and together reacted with 33/34 clinically-relevant HPV types tested.
44
In conclusion, our inability to identify an HPV16 L2-specific neutralizing antibody
45
response even in sera of patients with active genital HPV disease suggests sub-
46
dominance of L2 protective epitopes and the value of the chimeric mAbs JWW-1 and
47
JWW-2 as standards for immunoassays to measure L2-specific human antibodies.
Wang et al
Page 4
48
INTRODUCTION
49
Persistent infection with a high risk human papillomavirus (hrHPV) is a necessary,
50
although insufficient cause of cervical cancer and subsets of other anogenital and oral-
51
pharyngeal cancers (1, 2). Despite the licensure of two HPV vaccines based on L1 virus-
52
like particles (VLP) that have been shown to be highly effective, cervical cancer remains
53
as the third most common cancer worldwide, with 80% of cases occurring in the
54
developing world (3). This disparity reflects both limited vaccine implementation and
55
certain technical and logistic issues in cervical cancer screening in the developing world.
56 57
The first L1 VLP vaccines licensed (Gardasil®, Merck & Co. and Cervarix®, GSK)
58
targeted the two most problematic hrHPV genotypes, HPV16 and HPV18, which
59
together cause ~70% of all cervical cancer cases. Gardasil® also contains L1 VLP types
60
derived from HPV6 and HPV11, and provides protection from benign genital warts
61
caused by these viruses (4). Recognizing the importance of greater breadth of coverage,
62
a nonavalent vaccine targeting 7 of the 15 high risk HPV types (5) and has recently been
63
approved by the FDA. Emerging data suggest that the genotype distribution of HPV that
64
causes cervical pathology differs in different countries, ethnicities and in HIV+ individuals
65
(6-8). However, increasing vaccine valency to further expand coverage will raise
66
manufacturing costs and complexity. Importantly, >85% of the cervical cancer disease
67
burden lies in low resource settings, reflecting in part insufficient resources for screening
68
programs and HPV vaccination (9). Although some cross-protection against very closely
69
related HPV types has been identified in the current vaccines, it does not cover all
70
hrHPV and its longevity is uncertain (10, 11). Thus, there remains a clear need to
71
develop an affordable vaccine that broadly protects against all hrHPV types. Finally none
72
of these vaccines target cutaneous HPV genotypes associated with common warts or
Wang et al
73
beta papillomaviruses associated with non-melanoma skin cancer in persons with
74
epidermodysplasia verruciformis (EV) or who are immunocompromised.
Page 5
75 76
Vaccination with the minor capsid protein L2 has potential as an approach to
77
comprehensive and inexpensive HPV vaccination as the N-terminal protective epitope
78
sequences are well conserved. Preclinical vaccine studies demonstrate that this region
79
of L2 can elicit protection against diverse papillomavirus types (12-14). Further, the
80
passive transfer of L2-specific neutralizing antibodies into naïve animals is sufficient for
81
protection from experimental challenge, providing evidence to support their central role
82
in protective immunity. Additionally, because the neutralizing epitopes of L2 are linear
83
and conserved, in contrast to the type-restricted conformational epitopes of L1-VLP
84
vaccines, cross-reactive L2 vaccines can be produced as a single antigen in bacteria,
85
which may reduce manufacturing costs compared to the licensed L1 VLP vaccines.
86
However, while L2-specific antibodies are broadly reactive, their ability to neutralize less
87
evolutionarily-related types is weaker than for the cognate type. Therefore, we and
88
others have sought to enhance cross-protection by either the concatenation of L2
89
epitopes derived from several HPV types or displaying L2 epitopes on VLPs. Both
90
approaches can broadly protect vaccinated animals against experimental challenge with
91
diverse HPV types (15-19).
92 93
To lay the groundwork for L2 vaccine trials, the prevalence of L2-specific neutralizing
94
antibodies should be understood in both healthy patients and those with HPV disease.
95
Pre-clinical studies suggest that L2 is immunologically subdominant to L1 in the context
96
of the capsid suggesting that responses to infection with genital HPV are likely to be rare
97
and/or weak when they are detectable. To date, studies addressing L2-specific antibody
98
responses to HPV infection are both limited in number and inconsistent with respect to
Wang et al
Page 6
99
sero-prevalence (20-26). Some of these inconsistencies may reflect reliance upon a
100
single assay format and/or utilizing only bacterially-expressed L2 antigens (20-26). To
101
address this, here we examined the serologic responses to HPV16 L2 in both an
102
unscreened healthy population at risk for HPV infection and in patients with high grade
103
cervical or vulvar intraepithelial neoplasia (CIN2/3 or VIN2/3 respectively), using HPV16
104
L2 ELISA followed by a confirmatory Western blot analysis (i.e. a two-step approach).
105
We also examine whether topical application of a Toll-like receptor 7 (TLR7) agonist,
106
imiquimod, at the lesion site and either ablation of the lesion with photodynamic therapy
107
or intramuscular vaccination with HPV16 L2E7E6 fusion protein (TA-CIN) elicit serum
108
antibody responses to L2.
109 110
In addition to sero-prevalence, validated serologic assessment methods that are both
111
functionally sensitive as well as high-throughput will be required to assess neutralizing
112
antibody responses of patients. Recently, several new in vitro assays with enhanced
113
sensitivity to L2-specific neutralizing antibodies have been developed (27-29). A key
114
requirement for their routine validation is a reproducible positive control (30). Indeed, the
115
need for standardization of assays for assessing responses to HPV L1-VLP vaccination
116
led the World Health Organization (WHO) to develop international serologic standards
117
for HPV16 and HPV18 L1-VLP reactive antibodies respectively, using serum pooled
118
from HPV-infected women (30, 31). Here, we sought to follow a similar approach in
119
generating a standard for future L2-based vaccination validation studies, by identifying
120
patient sera with L2-reactive neutralizing antibody.
121 122
MATERIALS AND METHODS
123
Human samples
Wang et al
Page 7
124
The studies herein using human sera were reviewed and approved by the Johns
125
Hopkins University Internal Review Board. Human sera were collected previously from
126
women in four published clinical cohorts: 1) a 38% random sample (n=880) of sera
127
collected as part of a population-based (n=2331) surveillance study in India with 10%
128
high risk HPV DNA prevalence (Community Access to Cervical Health (CATCH) Study)
129
(32) (see Tables 1 and 2 for patient demographics), 2) Patients (n=160) with high grade
130
cervical intraepithelial neoplasia (CIN) (33) (see Supplementary Tables 1 and 2 for
131
patient demographics), 3) Patients (n=19) enrolled in a Phase II study with high-grade
132
vulvar intraepithelial neoplasia (VIN) who were treated with imiquimod for 8 weeks
133
followed by photodynamic therapy (34), and 4) high-grade VIN patients (n=19) who were
134
treated with imiquimod for 8 weeks, followed by vaccination three times at monthly
135
intervals with a fusion protein comprising HPV16 L2, E6, and E7 (TA-CIN) (35). On both
136
phase II trials studies, sera were obtained prior to and after imiquimod treatment (weeks
137
0 and 10 respectively) and after PDT/TA-CIN vaccination (week 26).
138 139
Enzyme-linked immunosorbent assays (ELISA) and neutralization assays
140
Immobilon plates (Nunc) were coated with 500ng/well of purified baculovirus-derived
141
HPV-16/18 L1-VLPs (36), HPV-16/31/45/58 pseudovirions (PsVs), or bacterially-
142
expressed and 6His-tagged HPV16 L2 (37), or L2α(11-88)x5 multimer polypeptide (17).
143
The plates were incubated overnight at 4°C. Wells were then blocked with 1% bovine
144
serum albumin (BSA)–PBS for 1h at 37°C, and incubated with human sera at 1:50
145
dilution for 1h at 37°C. Following a wash step with PBS-0.01% (v/v) Tween 20,
146
peroxidase-labeled Sheep anti-human (GE Heathcare) diluted 1:5000 in 1% BSA–PBS
147
was incubated for 1h. The plates were then washed and developed with 2,2′-azino-bis(3-
148
ethylbenzthiazoline-6-sulphonic acid solution (Roche) for 10 min, and the absorbance
Wang et al
Page 8
149
was measured at 405 nm. For JWW-1 and WW-1 ELISA reactivity comparisons to
150
HPV16 L2 and L2α(11-88)x5, a total of 400ng of antibody in 100μL/well (26.7nM) was
151
utilized as the starting concentration. Neutralization assay comparison between JWW-1,
152
JWW-2 and control IgG was done using our recently described furin-cleaved
153
pseudovirion-based neutralization assay (FC-PBNA) (38).
154 155
SDS-PAGE and Western blotting
156
4-15% gradient SDS-PAGE gels were loaded with 40μg of lysate of 293TT cells
157
transfected with expression vectors for either eGFP or HPV16 L2. The gels were run at
158
110V for 1.5 to 2 hours. The proteins were then transferred onto a nitrocellulose
159
membrane for 90 min at 50V. The membranes were blocked for one hour at RT with 5%
160
non-fat dried milk in phosphate-buffered saline containing 0.1% (v/v) Tween 20 (PBST).
161
The membranes were then incubated with sera diluted at 1:300 or higher (highest
162
1:5000 due to limiting sera) in in 5% milk in PBST overnight at 4°C. The membranes
163
were then washed for 10 min three times with PBST. Membranes were incubated with
164
anti-human IgG HRP secondary diluted 1:5000-10,000 in 5% milk in PBST and
165
incubated for 1h at RT. Membranes were then washed three times with PBST for 10 min
166
each time. Chemiluminescence substrate was added to develop the membranes.
167 168
Design and generation of JWW-1 and JWW-2 expression plasmid
169
The rat monoclonal antibody WW-1 is broadly neutralizing and reactive with HPV16 L2
170
17-36 (29). The WW-1 heavy and light chain sequences were obtained by sequencing
171
the hybridoma cDNA (Aldevron, Fargo, USA). Variable region sequences for the mouse
172
monoclonal antibody MAb24B were derived from (39). Both the constant light and heavy
173
chain regions of WW1 and MAb24B were replaced with the constant light and heavy
174
chain regions of human IgG1 sequence and directly synthesized (Biobasic Inc, Ontario,
Wang et al
Page 9
175
Canada). The chimeric heavy and light chains of WW-1 and MAb24B were each cloned
176
into a double expression vector, pVITRO1-neo-mcs (Invivogen, San Diego CA) to
177
generate the JWW-1 and JWW-2 plasmids respectively Plasmids and cDNA sequence
178
information to produce these human chimeric monoclonal antibodies are available from
179
https://www.addgene.org/Richard_Roden/
180 181
Expression and Purification of JWW-1 and JWW-2
182
For initial testing, 5X106 293TT cells were seeded into a 6 well plate the day before
183
transfection. The cells were transfected with either pVITRO1-neo-mcs empty vector
184
template (mock transfection) or JWW-1/JWW-2 using Mirus TransIT 2020 (Mirus Bio,
185
Madison WI) according to the manufacturer’s protocol and maintained in Opti-MEM
186
reduced serum media (Gibco, Life technologies, Grand Island NY). After 72 hours, the
187
supernatant was clarified by centrifugation (1600rpm, 4.5 minutes at room temperature)
188
and passage through a 0.2μm filter (Milipore, Billerica, MA) to remove cellular debris.
189
The filtrate was used for Western blot studies at a 1:100 dilution to determine reactivity.
190
For large scale purification, the cell line Expi293TM was utilized as per the manufacturer’s
191
(Life technologies, Grand Island NY) instructions and purified using HiTrap Protein G HP
192
columns (GE healthcare BioSciences, Pittsburgh, PA). The quantity of antibody was
193
determined using a PierceTM BCA Protein Assay kit, per the manufacturer’s (Pierce)
194
instructions. Purity was assessed by SDS-PAGE and Commassie blue staining.
195 196
Statistics and data analysis
197
For all ELISAs, the mean absorbance and standard deviation (s.d.) of the entire study
198
cohort was first calculated. Subsequently, a positive ELISA value for HPVL2 was
199
designated as an O.D value > mean+3 s.d. Results of ELISA screens were plotted using
200
R package ggplot2. FC-PBNA neutralization titer was defined as the reciprocal of the
Wang et al
Page 10
201
dilution that caused 50% reduction in luciferase activity. FC-PBNA comparisons of RG-1,
202
JWW-1 and JWW-2 were done in triplicate and titrated 2-fold with a starting
203
concentration of 200nM. The values were plotted on Graphpad Prism 6 using the non-
204
linear model Y=Bottom + (Top-Bottom) /(1+10^((LogEC50-X)*HillSlope)).
205 206 207
RESULTS
208
Prevalence of HPV16 L2-specific antibody in sera of an unscreened population at
209
high risk for HPV infection
210
Since the WHO reference reagents for human PV L1-antibodies to HPV16 and HPV18
211
are based on serum pooled from several infected patients, we attempted to screen for
212
L2-reactive sera from patients who are at high-risk for or have active HPV genital
213
disease. We first assessed the prevalence of HPV16 L2-reactive serum antibody
214
responses in a random subset of women enrolled in the ‘CATCH’ (Community Access to
215
Cervical Health) study (Table 1) designed to evaluate the feasibility and impact of
216
introducing cervical screening methods in a previously unscreened population in
217
Medchal Mandal, Andhra Pradesh, India (32). Using ELISA assays, sera from 880
218
women enrolled in the CATCH study were screened for reactivity with full length HPV16
219
L2 protein. When employing a cut-off of mean O.D. + 3 s.d. by our L2 ELISA, eighteen of
220
the 880 sera screened were considered potentially positive (Table 2, Patient 1-18).
221
Since the recombinant HPV16 L2 antigen used in the ELISA was purified from E. coli,
222
there was a possibility that bacterial protein-specific antibody responses might contribute
223
to false positives by reacting with bacterial contaminants in the HPV16 L2 antigen.
224
Therefore, the 18 sera putatively positive for HPV16 L2-specific antibody by ELISA were
225
re-screened in a Western blot-based assay, in which lysates of 293TT cells transfected
226
with either an expression vector for HPV16 L2, or GFP as a negative control, were
Wang et al
Page 11
227
probed with individual patient sera and a peroxidase-linked secondary antibody specific
228
to human IgG. While the presence of the ~70kDa HPV16 L2 protein in the 293TT lysates
229
was confirmed by Western blot using the mouse monoclonal antibody RG-1 (Figure
230
1E,F), only 6/18 putative positive sera reacted specifically with a band of ~70kDa in the
231
lysates of 293TT cells expressing HPV16 L2 (Figure 1E, summarized in Table 2). This
232
finding suggests that despite a cervical high risk HPV DNA prevalence of 10.8% (as
233
determined by Qiagen hc2 assays) in the CATCH study patients (Table 1), only a minor
234
(0.68%) fraction of their sera was reactive with HPV16 L2 (by both ELISA and Western
235
blot, Figure 1E).
236
HPV-infected individuals frequently mount a type-specific L1 VLP-reactive serum
237
antibody response (30). Therefore the sera of these 18 patients from the CATCH study
238
identified in the HPV16 L2 ELISA screen were also tested for reactivity in two ELISA
239
assays using either baculovirus-derived HPV16 or 18 L1-VLP and previously defined
240
cut-off and standards (36). Importantly, only 1/6 of the verified HPV16 L2-reactive sera
241
was also reactive in the HPV16 L1-VLP ELISA. None of the 6 patients also displayed
242
reactivity against HPV18 L1-VLP (Table 2).
243 244
Given that L2 is highly conserved, it is possible that the negative HPV16 and HPV18 L1-
245
VLP ELISA results were due to infection by other related HPV types. To explore this
246
possibility, we performed HPV-31/45/58 pseudovirion ELISAs with the patient sera.
247
However, none of these 6 HPV16 L2 reactive sera (determined by both ELISA and
248
Western blot) of the CATCH study patients reacted with these additional HPV genotypes
249
either.
250 251
HPV16 L2 seroreactivity of women with HPV16+ CIN
Wang et al
Page 12
252
We next assessed for HPV16 L2-reactive serum antibodies a cohort of patients at the
253
Johns Hopkins Hospital (n=160, mean age 29.5 years) with cervical intraepithelial
254
neoplasia 2/3 (CIN2/3) (Table 3) enrolled in a longitudinal cohort protocol, in which
255
serially collected (≤4) blood samples were obtained. A total of 298 serum samples from
256
this cohort were available for testing. Although the cervical swabs of 99 of the 160
257
patients were HPV16+ (62%), only one of their sera (Table 2, sample 19) was reactive to
258
HPV16 L2 by both ELISA and Western blot assays (Figure 1B, F). The serum was
259
provided by a patient diagnosed with an HPV16+ CIN2 at study entry. Her cone excision
260
at study week 16 was benign suggesting that patient’s lesion underwent spontaneous
261
regression. A year later she was diagnosed with an HPV16-negative CIN3, which also
262
regressed. She had donated serum samples at four visits over this period and each was
263
similarly reactive to HPV16 L2 both via ELISA and Western blot (Table 2, sample 19A-
264
D), but none to either HPV16 or HPV18 L1-VLP by ELISA (Table 2). Together, these
265
data suggest that a systemic antibody response to HPV16 L2 was uncommon even in
266
women with HPV16+ CIN2/3.
267 268
Neither imiquimod nor PDT elicit an HPV16 L2 antibody response in VIN patients
269
The low prevalence of a serum antibody response to HPV16 L2 detectable in the cohort
270
of women with HPV16+ CIN2/3 may reflect HPV infection that is confined to mucosal
271
epithelium. To address the possibility that infection at a different site such as the vulva
272
which might be more immunogenic, sera of 19 patients with high grade vulvar
273
intraepithelial neoplasia 2/3 (VIN2/3), of which 15 were HPV16+, obtained during a
274
phase II study were tested for HPV16 L2-specific antibody (34). None were positive
275
(Figure 1C, 1D).
276
Wang et al
Page 13
277
The lack of an L2 antibody response might also reflect the localized and non-lytic
278
replication of HPV and thus minimal inflammation associated with genital HPV16
279
infection. Therefore we reasoned that topical application of the TLR7 agonist imiquimod
280
at the lesion site might act as an adjuvant that would promote a serologic response to
281
L2. The patients were treated subsequently with imiquimod applied to the lesion once in
282
the first week, twice in the second week, and three times weekly for six subsequent
283
weeks. At week 10, one week after completing the imiquimod regimen, a peripheral
284
blood sample was obtained. No L2-specific serologic response was detected in any of
285
the subjects. Following imiquimod treatment, the patients received photodynamic
286
therapy (using methylaminolevulinate cream and red light via an Aktilite 128 (Photocure
287
ASA) set to deliver a dose of 50 J/cm2) at weeks 12 and 16 of the study (34). Peripheral
288
blood was obtained at week 26. No significant differences in L2 ELISA reactivity were
289
identified in within-subject comparisons before versus after treatment (Figure 1C and
290
Figure 1D). This finding suggests that any potential adjuvant effect caused by local
291
innate stimulation with imiquimod, even upon lesion damage with photodynamic therapy
292
was insufficient to elicit detectable systemic L2 responses in the clinical setting of
293
persistent HPV16+ VIN2/3.
294 295
Vaccination elicits an HPV16 L2 antibody response in VIN patients
296
The inability to detect a serologic response against HPV16 L2 in patients with HPV16+
297
high grade anogenital intraepithelial neoplasia (AGIN) may reflect some technical issue
298
with the assay or that the AGIN patients have a genetic background that is incompatible
299
with the induction of an L2 antibody response. To further investigate, HPV16 L2-specific
300
serologic responses in high grade VIN patients (n=19) treated with imiquimod for 10
301
weeks followed by three vaccinations with 125μg of a fusion protein comprised of HPV16
302
L2, E7, and E6 (TA-CIN) were assessed. Consistent with an earlier VIN cohort study, no
Wang et al
Page 14
303
HPV L2 responses were induced following topical imiquimod application (Figure 1D).
304
However, after vaccination with TA-CIN, 63% of the patient group (12/19) developed an
305
HPV16 L2 response (Figure 1D) which was also detectable by Western blot (Data not
306
shown). Thus the failure to detect an L2 antibody response in HPV16+ VIN patients prior
307
to L2 vaccination does not reflect either a technical issue with the assay or the inability of
308
most VIN patients to mount an L2 antibody response.
309 310
L2 antibody responses induced by HPV infection are not detectably neutralizing
311
Our screening efforts yielded 7 patients (see Table 2) with HPV16 L2 antibody verified
312
by both ELISA and Western blot. As 6/7 were also HPV16 L1-VLP ELISA negative, we
313
next assessed if their L2 responses had neutralizing potential. Using the standard in vitro
314
HPV neutralization assay against HPV16 pseudovirions, none were detectably
315
neutralizing (all EC50200 [N/A]
HPV 16 (Luciferase Reporter)
HPV 26 (Luciferase Reporter)
HPV 45 (Luciferase Reporter)
HPV 58 (Luciferase Reporter)
602 603 604
JWW-1
6.6 [2.3-19]
JWW-2
71 [5.2-980]
human IgG control
>200 [N/A]
RG-1
>200 [N/A]
JWW-1
67 [10-510]
JWW-2
75 [12-480]
Rat IgG control
>200 [N/A]
Wang et al
605
Page 31
REFERENCES
606 607
1.
608 609
zur Hausen, H. 2002. Papillomaviruses and cancer: From basic studies to clinical application. Nature Reviews Cancer 2:342-350.
2.
Walboomers, J. M. M., M. V. Jacobs, M. M. Manos, F. X. Bosch, J. A. Kummer, K. V.
610
Shah, P. J. F. Snijders, J. Peto, C. J. L. M. Meijer, and N. Munoz. 1999. Human
611
papillomavirus is a necessary cause of invasive cervical cancer worldwide. Journal of
612
Pathology 189:12-19.
613
3.
614 615
Human papillomavirus and cervical cancer. Lancet 370:890-907. 4.
616 617
Schiffman, M., P. E. Castle, J. Jeronimo, A. C. Rodriguez, and S. Wacholder. 2007.
Schiller, J. T., X. Castellsague, and S. M. Garland. 2012. A review of clinical trials of human papillomavirus prophylactic vaccines. Vaccine 30 Suppl 5:F123-138.
5.
Joura, E. A., A. R. Giuliano, O. E. Iversen, C. Bouchard, C. Mao, J. Mehlsen, E. D.
618
Moreira, Jr., Y. Ngan, L. K. Petersen, E. Lazcano-Ponce, P. Pitisuttithum, J. A.
619
Restrepo, G. Stuart, L. Woelber, Y. C. Yang, J. Cuzick, S. M. Garland, W. Huh, S. K.
620
Kjaer, O. M. Bautista, I. S. Chan, J. Chen, R. Gesser, E. Moeller, M. Ritter, S.
621
Vuocolo, and A. Luxembourg. 2015. A 9-valent HPV vaccine against infection and
622
intraepithelial neoplasia in women. N Engl J Med 372:711-723.
623
6.
Schabath, M. B., L. L. Villa, H. Y. Lin, W. J. Fulp, G. O. Akogbe, M. E. Abrahamsen,
624
M. R. Papenfuss, E. Lazcano-Ponce, J. Salmeron, M. Quiterio, and A. R. Giuliano.
625
2013. Racial differences in the incidence and clearance of human papilloma virus (HPV):
626
the HPV in men (HIM) study. Cancer Epidemiol Biomarkers Prev 22:1762-1770.
627
7.
Hariri, S., E. R. Unger, S. E. Powell, H. M. Bauer, N. M. Bennett, K. C. Bloch, L. M.
628
Niccolai, S. Schafer, M. Steinau, and L. E. Markowitz. 2012. Human papillomavirus
629
genotypes in high-grade cervical lesions in the United States. J Infect Dis 206:1878-
630
1886.
Wang et al
631
8.
Page 32
Niccolai, L. M., C. Russ, P. J. Julian, S. Hariri, J. Sinard, J. I. Meek, V. McBride, L.
632
E. Markowitz, E. R. Unger, J. L. Hadler, and L. E. Sosa. 2013. Individual and
633
geographic disparities in human papillomavirus types 16/18 in high-grade cervical
634
lesions: Associations with race, ethnicity, and poverty. Cancer 119:3052-3058.
635
9.
636 637
Agosti, J. M., and S. J. Goldie. 2007. Introducing HPV vaccine in developing countries - Key challenges and issues. New England Journal of Medicine 356:1908-1910.
10.
Wheeler, C. M., X. Castellsague, S. M. Garland, A. Szarewski, J. Paavonen, P.
638
Naud, J. Salmeron, S. N. Chow, D. Apter, H. Kitchener, J. C. Teixeira, S. R. Skinner,
639
U. Jaisamrarn, G. Limson, B. Romanowski, F. Y. Aoki, T. F. Schwarz, W. A. Poppe,
640
F. X. Bosch, D. M. Harper, W. Huh, K. Hardt, T. Zahaf, D. Descamps, F. Struyf, G.
641
Dubin, M. Lehtinen, and H. P. S. Group. 2012. Cross-protective efficacy of HPV-16/18
642
AS04-adjuvanted vaccine against cervical infection and precancer caused by non-
643
vaccine oncogenic HPV types: 4-year end-of-study analysis of the randomised, double-
644
blind PATRICIA trial. Lancet Oncol 13:100-110.
645
11.
Malagon, T., M. Drolet, M. C. Boily, E. L. Franco, M. Jit, J. Brisson, and M. Brisson.
646
2012. Cross-protective efficacy of two human papillomavirus vaccines: a systematic
647
review and meta-analysis. Lancet Infect Dis 12:781-789.
648
12.
Christensen, N. D., J. W. Kreider, N. C. Kan, and S. L. DiAngelo. 1991. The open
649
reading frame L2 of cottontail rabbit papillomavirus contains antibody-inducing
650
neutralizing epitopes. Virology 181:572-579.
651
13.
Gambhira, R., S. Jagu, B. Karanam, P. E. Gravitt, T. D. Culp, N. D. Christensen, and
652
R. B. Roden. 2007. Protection of rabbits against challenge with rabbit papillomaviruses
653
by immunization with the N terminus of human papillomavirus type 16 minor capsid
654
antigen L2. J Virol 81:11585-11592.
Wang et al
655
14.
Page 33
Gambhira, R., B. Karanam, S. Jagu, J. N. Roberts, C. B. Buck, I. Bossis, H. Alphs,
656
T. Culp, N. D. Christensen, and R. B. Roden. 2007. A protective and broadly cross-
657
neutralizing epitope of human papillomavirus L2. J Virol 81:13927-13931.
658
15.
Jagu, S., B. Karanam, R. Gambhira, S. V. Chivukula, R. J. Chaganti, D. R. Lowy, J.
659
T. Schiller, and R. B. S. Roden. 2009. Concatenated Multitype L2 Fusion Proteins as
660
Candidate Prophylactic Pan-Human Papillomavirus Vaccines. J Natl Cancer Inst
661
101:782-792.
662
16.
Jagu, S., K. Kwak, B. Karanam, W. K. Huh, V. Damotharan, S. V. Chivukula, and R.
663
B. Roden. 2013. Optimization of multimeric human papillomavirus L2 vaccines. PLoS
664
One 8:e55538.
665
17.
Jagu, S., K. Kwak, J. T. Schiller, D. R. Lowy, H. Kleanthous, K. Kalnin, C. Wang, H.
666
K. Wang, L. T. Chow, W. K. Huh, K. S. Jaganathan, S. V. Chivukula, and R. B.
667
Roden. 2013. Phylogenetic considerations in designing a broadly protective multimeric
668
L2 vaccine. J Virol 87:6127-6136.
669
18.
Schellenbacher, C., K. Kwak, D. Fink, S. Shafti-Keramat, B. Huber, C. Jindra, H.
670
Faust, J. Dillner, R. B. S. Roden, and R. Kirnbauer. 2013. Efficacy of RG1-VLP
671
Vaccination against Infections with Genital and Cutaneous Human Papillomaviruses. J
672
Invest Dermatol 133:2706-2713.
673
19.
Schellenbacher, C., R. Roden, and R. Kirnbauer. 2009. Chimeric L1-L2 Virus-Like
674
Particles as Potential Broad-Spectrum Human Papillomavirus Vaccines. Journal of
675
virology 83:10085-10095.
676
20.
Lehtinen, M., J. Niemela, J. Dillner, P. Parkkonen, T. Nummi, E. Liski, P. Nieminen,
677
T. Reunala, and J. Paavonen. 1993. Evaluation of serum antibody response to a newly
678
identified B-cell epitope in the minor nucleocapsid protein L2 of human papillomavirus
679
type 16. Clin Diagn Virol 1:153-165.
Wang et al
680
21.
Page 34
Jenison, S. A., X. P. Yu, J. M. Valentine, and D. A. Galloway. 1991. Characterization
681
of human antibody-reactive epitopes encoded by human papillomavirus types 16 and 18.
682
J Virol 65:1208-1218.
683
22.
Jenison, S. A., J. M. Firzlaff, A. Langenberg, and D. A. Galloway. 1988. Identification
684
of Immunoreactive Antigens of Human Papillomavirus Type-6b by Using Escherichia-
685
Coli-Expressed Fusion Proteins. J Virol 62:2115-2123.
686
23.
Yaegashi, N., S. A. Jenison, M. Batra, and D. A. Galloway. 1992. Human-Antibodies
687
Recognize Multiple Distinct Type-Specific and Cross-Reactive Regions of the Minor
688
Capsid Proteins of Human Papillomavirus Type-6 and Type-11. J Virol 66:2008-2019.
689
24.
Kanda, T., H. Teshima, K. Katase, S. Umezawa, H. Watabe, H. Takahashi, T. Onda,
690
and K. Yoshiike. 1995. Occurrence of the antibody against human papillomavirus type
691
16 virion protein L2 in patients with cervical cancer and dysplasia. Intervirology 38:187-
692
191.
693
25.
Suchankova, A., O. Ritter, I. Hirsch, V. Krchnak, Z. Kalos, E. Hamsikova, B.
694
Brichacek, and V. Vonka. 1990. Presence of Antibody Reactive with Synthetic Peptide
695
Derived from L2 Open Reading Frame of Human Papillomavirus Types-6b and Type-11
696
in Human Sera. Acta Virologica 34:433-442.
697
26.
Paez, C. G., N. Yaegashi, S. Sato, and A. Yajima. 1993. Prevalence of Serum Igg
698
Antibodies for the E7 and L2 Proteins of Human Papillomavirus Type-16 in Cervical-
699
Cancer Patients and Controls. Tohoku Journal of Experimental Medicine 170:113-121.
700
27.
Day, P. M., Y. Y. Pang, R. C. Kines, C. D. Thompson, D. R. Lowy, and J. T. Schiller.
701
2012. A human papillomavirus (HPV) in vitro neutralization assay that recapitulates the
702
in vitro process of infection provides a sensitive measure of HPV L2 infection-inhibiting
703
antibodies. Clin Vaccine Immunol 19:1075-1082.
Wang et al
704
28.
Page 35
Sehr, P., I. Rubio, H. Seitz, K. Putzker, L. Ribeiro-Muller, M. Pawlita, and M. Muller.
705
2013. High-throughput pseudovirion-based neutralization assay for analysis of natural
706
and vaccine-induced antibodies against human papillomaviruses. PLoS One 8:e75677.
707
29.
Wang, J. W., S. Jagu, K. Kwak, C. Wang, S. Peng, R. Kirnbauer, and R. B. Roden.
708
2014. Preparation and properties of a papillomavirus infectious intermediate and its
709
utility for neutralization studies. Virology 449:304-316.
710
30.
Ferguson, M., D. E. Wilkinson, and T. Zhou. 2009. WHO meeting on the
711
standardization of HPV assays and the role of the WHO HPV Laboratory Network in
712
supporting vaccine introduction held on 24-25 January 2008, Geneva, Switzerland.
713
Vaccine 27:337-347.
714
31.
Wilkinson, D. E. H., Alan B; Faust, Helena ;Panicker, Gitika ;Unger, Elizabeth R.;
715
Dillner, Joakim; Ngamkham, Jarunya and WHO Expert Committee on Biological
716
Standardization 2012. Collaborative study to evaluate the proposed 1st WHO
717
international standard for antibodies to human papillomavirus type 18. World Health
718
Organization. WHO/BS/2012.2191 [http://www.who.int/iris/handle/10665/96608]
719
32.
Gravitt, P. E., P. Paul, H. A. Katki, H. Vendantham, G. Ramakrishna, M. Sudula, B.
720
Kalpana, B. M. Ronnett, K. Vijayaraghavan, and K. V. Shah. 2010. Effectiveness of
721
VIA, Pap, and HPV DNA testing in a cervical cancer screening program in a peri-urban
722
community in Andhra Pradesh, India. PLoS One 5:e13711.
723
33.
Trimble, C. L., S. Piantadosi, P. Gravitt, B. Ronnett, E. Pizer, A. Elko, B. Wilgus, W.
724
Yutzy, R. Daniel, K. Shah, S. Peng, C. Hung, R. Roden, T. C. Wu, and D. Pardoll.
725
2005. Spontaneous regression of high-grade cervical dysplasia: effects of human
726
papillomavirus type and HLA phenotype. Clin Cancer Res 11:4717-4723.
727 728
34.
Winters, U., S. Daayana, J. T. Lear, A. E. Tomlinson, E. Elkord, P. L. Stern, and H. C. Kitchener. 2008. Clinical and immunologic results of a phase II trial of sequential
Wang et al
Page 36
729
imiquimod and photodynamic therapy for vulval intraepithelial neoplasia. Clin Cancer
730
Res 14:5292-5299.
731
35.
Daayana, S., E. Elkord, U. Winters, M. Pawlita, R. Roden, P. L. Stern, and H. C.
732
Kitchener. 2010. Phase II trial of imiquimod and HPV therapeutic vaccination in patients
733
with vulval intraepithelial neoplasia. Br J Cancer 102:1129-1136.
734
36.
Viscidi, R. P., B. Snyder, S. Cu-Uvin, J. W. Hogan, B. Clayman, R. S. Klein, J.
735
Sobel, and K. V. Shah. 2005. Human papillomavirus capsid antibody response to
736
natural infection and risk of subsequent HPV infection in HIV-positive and HIV-negative
737
women. Cancer Epidemiol Biomarkers Prev 14:283-288.
738
37.
Roden, R. B., W. H. t. Yutzy, R. Fallon, S. Inglis, D. R. Lowy, and J. T. Schiller. 2000.
739
Minor capsid protein of human genital papillomaviruses contains subdominant, cross-
740
neutralizing epitopes. Virology 270:254-257.
741
38.
Wang, J. W., S. Jagu, C. Wang, H. C. Kitchener, S. Daayana, P. L. Stern, S. Pang, P.
742
M. Day, W. K. Huh, and R. B. Roden. 2014. Measurement of neutralizing serum
743
antibodies of patients vaccinated with human papillomavirus L1 or L2-based
744
immunogens using furin-cleaved HPV Pseudovirions. PloS one 9:e101576.
745
39.
Nakao, S., S. Mori, K. Kondo, K. Matsumoto, H. Yoshikawa, and T. Kanda. 2012.
746
Monoclonal antibodies recognizing cross-neutralization epitopes in human
747
papillomavirus 16 minor capsid protein L2. Virology 434:110-117.
748
40.
Pastrana, D. V., R. Gambhira, C. B. Buck, Y. Y. Pang, C. D. Thompson, T. D. Culp,
749
N. D. Christensen, D. R. Lowy, J. T. Schiller, and R. B. Roden. 2005. Cross-
750
neutralization of cutaneous and mucosal Papillomavirus types with anti-sera to the
751
amino terminus of L2. Virology 337:365-372.
752
41.
Kondo, K., H. Ochi, T. Matsumoto, H. Yoshikawa, and T. Kanda. 2008. Modification
753
of human papillomavirus-like particle vaccine by insertion of the cross-reactive L2-
754
epitopes. Journal of medical virology 80:841-846.
Wang et al
755
42.
Page 37
Rubio, I., A. Bolchi, N. Moretto, E. Canali, L. Gissmann, M. Torrimasino, M. Muller,
756
and S. Ottonello. 2009. Potent anti-HPV immune responses induced by tandem repeats
757
of the HPV16 L2 (20-38) peptide displayed on bacterial thioredoxin. Vaccine 27:1949-
758
1956.
759
43.
Tumban, E., J. Peabody, D. S. Peabody, and B. Chackerian. 2011. A pan-HPV
760
vaccine based on bacteriophage PP7 VLPs displaying broadly cross-neutralizing
761
epitopes from the HPV minor capsid protein, L2. PLoS One 6:e23310.
762
44.
Embers, M. E., L. R. Budgeon, M. Pickel, and N. D. Christensen. 2002. Protective
763
immunity to rabbit oral and cutaneous papillomaviruses by immunization with short
764
peptides of L2, the minor capsid protein. J Virol 76:9798-9805.
765
45.
Campo, M. S., G. J. Grindlay, B. W. O'Neil, L. M. Chandrachud, G. M. McGarvie, and
766
W. F. Jarrett. 1993. Prophylactic and therapeutic vaccination against a mucosal
767
papillomavirus. J Gen Virol 74 ( Pt 6):945-953.
768
46.
Gambhira, R., P. E. Gravitt, I. Bossis, P. L. Stern, R. P. Viscidi, and R. B. S. Roden.
769
2006. Vaccination of healthy volunteers with human papillomavirus type 16 L2E7E6
770
fusion protein induces serum antibody that neutralizes across papillomavirus species.
771
Cancer Res 66:11120-11124.
772
47.
Kawana, K., T. Yasugi, T. Kanda, N. Kino, K. Oda, S. Okada, Y. Kawana, T. Nei, T.
773
Takada, S. Toyoshima, A. Tsuchiya, K. Kondo, H. Yoshikawa, O. Tsutsumi, and Y.
774
Taketani. 2003. Safety and immunogenicity of a peptide containing the cross-
775
neutralization epitope of HPV16 L2 administered nasally in healthy volunteers. Vaccine
776
21:4256-4260.
777
48.
Wang, J. W., S. Jagu, C. G. Wang, H. C. Kitchener, S. Daayana, P. L. Stern, S. Pang,
778
P. M. Day, W. K. Huh, and R. B. S. Roden. 2014. Measurement of Neutralizing Serum
779
Antibodies of Patients Vaccinated with Human Papillomavirus L1 or L2-Based
780
Immunogens Using Furin-Cleaved HPV Pseudovirions. PloS one 9.
Wang et al
781
49.
Page 38
Buck, C. B., N. Cheng, C. D. Thompson, D. R. Lowy, A. C. Steven, J. T. Schiller,
782
and B. L. Trus. 2008. Arrangement of L2 within the papillomavirus capsid. Journal of
783
virology 82:5190-5197.
784
50.
World Health Organization. 2006. Guidelines to assure the quality, safety and efficacy
785
of recombinant human papillomavirus-like particle vaccines. WHO Press.
786
WHO/BS/06.2050
787
[http://www.who.int/immunization/hpv/learn/guidelines_to_assure_the_quality_safety_an
788
d_efficacity_ecbs_2006.pdf]
789 790