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.

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Sero-epidemiology of HPV16 L2 and generation of papillomavirus L2-

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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,

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Peter L. Sternf, Patti E. Gravittg, Cornelia L. Trimblea,c,d and Richard B.S. Rodena,c,d*

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Departments of Pathologya, Pediatricsb, Oncologyc, Gynecology and Obstetricsd, The

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Johns Hopkins University, Baltimore, MD, USA; Woman's Cancer Centre , St Mary’s

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Hospital e and Paterson Buildingf Institute of Cancer Sciences, University of Manchester,

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Manchester, UK; Department of Pathologyg, University of New Mexico School of

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Medicine, Albuquerque, NM, USA.

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Running Title: Human/chimeric antibodies to papillomavirus L2

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Key Words: Antibody; serology; HPV; L2; Biological Reference Standard; vaccine;

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papillomavirus

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*Corresponding Author. Department of Pathology, Johns Hopkins University, Room

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308, Cancer Research Building 2, 1550 Orleans St., Baltimore, MD 21231, USA. Tel.: +1

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410 502 5161; fax: +1 443 287 4295; E-mail address: [email protected]

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Funding: The study was funded by Public Health Service grants P50 CA098252 and

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RO1 CA118790 from the National Cancer Institute, and the V foundation

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(http://www.jimmyv.org/). Sai Daayana was funded by Wigan Cancer Research Fund;

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Henry Kitchener and Peter Stern were funded by Cancer Research UK

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(http://www.cancerresearchuk.org/).

22

Competing Interests: Richard Roden and Subhashini Jagu are inventors of L2-related

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patents licensed to Shantha Biotechnics Ltd., GlaxoSmithKline, PaxVax, Inc. and

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Acambis, Inc. Richard Roden has received research funding from Sanofi Pasteur,

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Shantha Biotechnic and GlaxoSmithKline and is a founder of Papivax LLC and a

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scientific advisor to Papivax Biotech Inc.. The terms of these arrangements are

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managed by Johns Hopkins University in accordance with its conflict of interest policies.

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ABSTRACT (225 WORDS)

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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

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comprising HPV16 L2, E7, and E6 (TA-CIN, n=19). Sera were screened sequentially by

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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.

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INTRODUCTION

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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

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derived from HPV6 and HPV11, and provides protection from benign genital warts

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caused by these viruses (4). Recognizing the importance of greater breadth of coverage,

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a nonavalent vaccine targeting 7 of the 15 high risk HPV types (5) and has recently been

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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

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manufacturing costs and complexity. Importantly, >85% of the cervical cancer disease

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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

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develop an affordable vaccine that broadly protects against all hrHPV types. Finally none

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of these vaccines target cutaneous HPV genotypes associated with common warts or

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beta papillomaviruses associated with non-melanoma skin cancer in persons with

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epidermodysplasia verruciformis (EV) or who are immunocompromised.

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Vaccination with the minor capsid protein L2 has potential as an approach to

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comprehensive and inexpensive HPV vaccination as the N-terminal protective epitope

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sequences are well conserved. Preclinical vaccine studies demonstrate that this region

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of L2 can elicit protection against diverse papillomavirus types (12-14). Further, the

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passive transfer of L2-specific neutralizing antibodies into naïve animals is sufficient for

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protection from experimental challenge, providing evidence to support their central role

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in protective immunity. Additionally, because the neutralizing epitopes of L2 are linear

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and conserved, in contrast to the type-restricted conformational epitopes of L1-VLP

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vaccines, cross-reactive L2 vaccines can be produced as a single antigen in bacteria,

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which may reduce manufacturing costs compared to the licensed L1 VLP vaccines.

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However, while L2-specific antibodies are broadly reactive, their ability to neutralize less

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evolutionarily-related types is weaker than for the cognate type. Therefore, we and

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others have sought to enhance cross-protection by either the concatenation of L2

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epitopes derived from several HPV types or displaying L2 epitopes on VLPs. Both

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approaches can broadly protect vaccinated animals against experimental challenge with

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diverse HPV types (15-19).

92 93

To lay the groundwork for L2 vaccine trials, the prevalence of L2-specific neutralizing

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antibodies should be understood in both healthy patients and those with HPV disease.

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Pre-clinical studies suggest that L2 is immunologically subdominant to L1 in the context

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of the capsid suggesting that responses to infection with genital HPV are likely to be rare

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and/or weak when they are detectable. To date, studies addressing L2-specific antibody

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responses to HPV infection are both limited in number and inconsistent with respect to

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sero-prevalence (20-26). Some of these inconsistencies may reflect reliance upon a

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single assay format and/or utilizing only bacterially-expressed L2 antigens (20-26). To

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address this, here we examined the serologic responses to HPV16 L2 in both an

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unscreened healthy population at risk for HPV infection and in patients with high grade

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cervical or vulvar intraepithelial neoplasia (CIN2/3 or VIN2/3 respectively), using HPV16

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L2 ELISA followed by a confirmatory Western blot analysis (i.e. a two-step approach).

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We also examine whether topical application of a Toll-like receptor 7 (TLR7) agonist,

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imiquimod, at the lesion site and either ablation of the lesion with photodynamic therapy

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or intramuscular vaccination with HPV16 L2E7E6 fusion protein (TA-CIN) elicit serum

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antibody responses to L2.

109 110

In addition to sero-prevalence, validated serologic assessment methods that are both

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functionally sensitive as well as high-throughput will be required to assess neutralizing

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antibody responses of patients. Recently, several new in vitro assays with enhanced

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sensitivity to L2-specific neutralizing antibodies have been developed (27-29). A key

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requirement for their routine validation is a reproducible positive control (30). Indeed, the

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need for standardization of assays for assessing responses to HPV L1-VLP vaccination

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led the World Health Organization (WHO) to develop international serologic standards

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for HPV16 and HPV18 L1-VLP reactive antibodies respectively, using serum pooled

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from HPV-infected women (30, 31). Here, we sought to follow a similar approach in

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generating a standard for future L2-based vaccination validation studies, by identifying

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patient sera with L2-reactive neutralizing antibody.

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MATERIALS AND METHODS

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Human samples

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The studies herein using human sera were reviewed and approved by the Johns

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Hopkins University Internal Review Board. Human sera were collected previously from

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women in four published clinical cohorts: 1) a 38% random sample (n=880) of sera

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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)

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(32) (see Tables 1 and 2 for patient demographics), 2) Patients (n=160) with high grade

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cervical intraepithelial neoplasia (CIN) (33) (see Supplementary Tables 1 and 2 for

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patient demographics), 3) Patients (n=19) enrolled in a Phase II study with high-grade

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vulvar intraepithelial neoplasia (VIN) who were treated with imiquimod for 8 weeks

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followed by photodynamic therapy (34), and 4) high-grade VIN patients (n=19) who were

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treated with imiquimod for 8 weeks, followed by vaccination three times at monthly

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intervals with a fusion protein comprising HPV16 L2, E6, and E7 (TA-CIN) (35). On both

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phase II trials studies, sera were obtained prior to and after imiquimod treatment (weeks

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0 and 10 respectively) and after PDT/TA-CIN vaccination (week 26).

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Enzyme-linked immunosorbent assays (ELISA) and neutralization assays

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Immobilon plates (Nunc) were coated with 500ng/well of purified baculovirus-derived

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HPV-16/18 L1-VLPs (36), HPV-16/31/45/58 pseudovirions (PsVs), or bacterially-

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expressed and 6His-tagged HPV16 L2 (37), or L2α(11-88)x5 multimer polypeptide (17).

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The plates were incubated overnight at 4°C. Wells were then blocked with 1% bovine

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serum albumin (BSA)–PBS for 1h at 37°C, and incubated with human sera at 1:50

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dilution for 1h at 37°C. Following a wash step with PBS-0.01% (v/v) Tween 20,

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peroxidase-labeled Sheep anti-human (GE Heathcare) diluted 1:5000 in 1% BSA–PBS

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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

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was measured at 405 nm. For JWW-1 and WW-1 ELISA reactivity comparisons to

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HPV16 L2 and L2α(11-88)x5, a total of 400ng of antibody in 100μL/well (26.7nM) was

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utilized as the starting concentration. Neutralization assay comparison between JWW-1,

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JWW-2 and control IgG was done using our recently described furin-cleaved

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pseudovirion-based neutralization assay (FC-PBNA) (38).

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SDS-PAGE and Western blotting

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4-15% gradient SDS-PAGE gels were loaded with 40μg of lysate of 293TT cells

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transfected with expression vectors for either eGFP or HPV16 L2. The gels were run at

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110V for 1.5 to 2 hours. The proteins were then transferred onto a nitrocellulose

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membrane for 90 min at 50V. The membranes were blocked for one hour at RT with 5%

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non-fat dried milk in phosphate-buffered saline containing 0.1% (v/v) Tween 20 (PBST).

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The membranes were then incubated with sera diluted at 1:300 or higher (highest

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1:5000 due to limiting sera) in in 5% milk in PBST overnight at 4°C. The membranes

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were then washed for 10 min three times with PBST. Membranes were incubated with

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anti-human IgG HRP secondary diluted 1:5000-10,000 in 5% milk in PBST and

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incubated for 1h at RT. Membranes were then washed three times with PBST for 10 min

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each time. Chemiluminescence substrate was added to develop the membranes.

167 168

Design and generation of JWW-1 and JWW-2 expression plasmid

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The rat monoclonal antibody WW-1 is broadly neutralizing and reactive with HPV16 L2

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17-36 (29). The WW-1 heavy and light chain sequences were obtained by sequencing

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the hybridoma cDNA (Aldevron, Fargo, USA). Variable region sequences for the mouse

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monoclonal antibody MAb24B were derived from (39). Both the constant light and heavy

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chain regions of WW1 and MAb24B were replaced with the constant light and heavy

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chain regions of human IgG1 sequence and directly synthesized (Biobasic Inc, Ontario,

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Canada). The chimeric heavy and light chains of WW-1 and MAb24B were each cloned

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into a double expression vector, pVITRO1-neo-mcs (Invivogen, San Diego CA) to

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generate the JWW-1 and JWW-2 plasmids respectively Plasmids and cDNA sequence

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information to produce these human chimeric monoclonal antibodies are available from

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https://www.addgene.org/Richard_Roden/

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Expression and Purification of JWW-1 and JWW-2

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For initial testing, 5X106 293TT cells were seeded into a 6 well plate the day before

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transfection. The cells were transfected with either pVITRO1-neo-mcs empty vector

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template (mock transfection) or JWW-1/JWW-2 using Mirus TransIT 2020 (Mirus Bio,

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Madison WI) according to the manufacturer’s protocol and maintained in Opti-MEM

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reduced serum media (Gibco, Life technologies, Grand Island NY). After 72 hours, the

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supernatant was clarified by centrifugation (1600rpm, 4.5 minutes at room temperature)

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and passage through a 0.2μm filter (Milipore, Billerica, MA) to remove cellular debris.

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The filtrate was used for Western blot studies at a 1:100 dilution to determine reactivity.

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For large scale purification, the cell line Expi293TM was utilized as per the manufacturer’s

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(Life technologies, Grand Island NY) instructions and purified using HiTrap Protein G HP

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columns (GE healthcare BioSciences, Pittsburgh, PA). The quantity of antibody was

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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.

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Statistics and data analysis

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For all ELISAs, the mean absorbance and standard deviation (s.d.) of the entire study

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cohort was first calculated. Subsequently, a positive ELISA value for HPVL2 was

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designated as an O.D value > mean+3 s.d. Results of ELISA screens were plotted using

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R package ggplot2. FC-PBNA neutralization titer was defined as the reciprocal of the

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dilution that caused 50% reduction in luciferase activity. FC-PBNA comparisons of RG-1,

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JWW-1 and JWW-2 were done in triplicate and titrated 2-fold with a starting

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concentration of 200nM. The values were plotted on Graphpad Prism 6 using the non-

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linear model Y=Bottom + (Top-Bottom) /(1+10^((LogEC50-X)*HillSlope)).

205 206 207

RESULTS

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Prevalence of HPV16 L2-specific antibody in sera of an unscreened population at

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high risk for HPV infection

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Since the WHO reference reagents for human PV L1-antibodies to HPV16 and HPV18

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are based on serum pooled from several infected patients, we attempted to screen for

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L2-reactive sera from patients who are at high-risk for or have active HPV genital

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disease. We first assessed the prevalence of HPV16 L2-reactive serum antibody

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responses in a random subset of women enrolled in the ‘CATCH’ (Community Access to

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Cervical Health) study (Table 1) designed to evaluate the feasibility and impact of

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introducing cervical screening methods in a previously unscreened population in

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Medchal Mandal, Andhra Pradesh, India (32). Using ELISA assays, sera from 880

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women enrolled in the CATCH study were screened for reactivity with full length HPV16

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L2 protein. When employing a cut-off of mean O.D. + 3 s.d. by our L2 ELISA, eighteen of

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the 880 sera screened were considered potentially positive (Table 2, Patient 1-18).

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Since the recombinant HPV16 L2 antigen used in the ELISA was purified from E. coli,

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there was a possibility that bacterial protein-specific antibody responses might contribute

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to false positives by reacting with bacterial contaminants in the HPV16 L2 antigen.

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Therefore, the 18 sera putatively positive for HPV16 L2-specific antibody by ELISA were

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re-screened in a Western blot-based assay, in which lysates of 293TT cells transfected

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with either an expression vector for HPV16 L2, or GFP as a negative control, were

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probed with individual patient sera and a peroxidase-linked secondary antibody specific

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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.

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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

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We next assessed for HPV16 L2-reactive serum antibodies a cohort of patients at the

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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

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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

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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

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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]

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Seroepidemiology of Human Papillomavirus 16 (HPV16) L2 and Generation of L2-Specific Human Chimeric Monoclonal Antibodies.

Presently, the seroprevalence of human papillomavirus (HPV) minor capsid antigen L2-reactive antibody is not well understood, and no serologic standar...
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