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1 [Human Vaccines 4:2, ; March/April 2008]; 2008 Landes Bioscience Research Paper Evolution of type-specific immunoassays to evaluate the functional immune response to Gardasil A vaccine for human papillomavirus types 16, 18, 6 and 11 Judith F. Smith, Rose Kowalski, Mark T. Esser, Martha J. Brown and Janine T. Bryan Vaccine & Biologics Research; Merck Research Laboratories; West Point, Pennsylvania USA Abbreviations: ADHS, antibody-depleted human serum; AAHS, amorphous aluminum hydroxyphosphate sulfate; BSA, bovine serum albumin; βlam, β-lactamase; clia, competitive Luminex immunoassay; cria, competitive radioimmunoassay; ELISA, enzymelinked immunosorbent assay; HAI, hemagglutination inhibition assay; HPV, human papillomavirus; mab, monoclonal antibody; mmu, millimerck units; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PsV, pseudovirus; SEAP, secreted alkaline phosphatase; TMB, 3,3,5,5 -tetramethylbenzidine; VLP, virus-like particle Key words: human papillomavirus, monoclonal antibody, neutralizing antibody, competitive immunoassay, vaccine Epidemiological studies and clinical trials of vaccines depend on the accurate measurement of antibodies within the polyclonal response to infection or vaccination. The assay currently used to measure the antibody response to vaccination with GARDASIL [Quadrivalent Human Papillomavirus (Types 6, 11, 16, 18) Recombinant Vaccine] a quadrivalent vaccine used against human papillomavirus (HPV) types 6, 11, 16 and 18 is a competitive Luminex assay (clia) that uses multiplex technology to detect type specific neutralizing antibodies against all four HPV types in a single serum sample. Here we describe how the clia was developed, as well as how the monoclonal antibodies (mabs), used as competitors in the assay, were characterized. An enzyme linked immunosorbent assay (ELISA) was used to screen eight previously identified mabs for their ability to bind to HPV virus like particles (VLPs) in a type specific and conformation dependent manner. Four of these mabs, H6.M48, K11.B2, H16.V5, and H18.J4, met our specifications and were shown to have the potential to neutralize HPV infection in hemagglutination inhibition and pseudovirus neutralization assays. The competitive immunoassay format was able to distinguish type specific antibodies in the sera of nonhuman primates vaccinated with HPV VLPs, whereas a traditional direct bind ELISA could not. In addition, the serum antibodies measured by the competitive assay are known to be neutralizing, whereas the ELISA does not distinguish neutralizing and nonneutralizing antibodies in a serum sample. By detecting antibodies to neutralizing epitopes, the competitive assay both demonstrates sero conversion and provides a potential functional link between sero conversion and protective immunity in response to vaccination with GARDASIL. Introduction Infection with human papillomavirus (HPV) is one of the most common viral infections in the United States. There are over 100 known types of HPV, of which at least 40 infect the anogenital region. 1 The majority of anogenital HPV disease is caused by two oncogenic (or high risk) types, HPV 16 and 18, which cause approximately 70% of cervical cancer cases worldwide, 2 and two nononcogenic (or low risk) HPV types, HPV 6 and 11, which cause approximately 90% of all genital warts 3 and contribute to the incidence of low grade cervical dysplasias. 4 Infectious HPV viral particles consist of two capsid proteins, L1 and L2, surrounding a double stranded DNA genome. When L1, the 55 kda major capsid protein of HPV, is generated as a recombinant protein in yeast, it self assembles into empty virus like particles (VLPs) that are highly immunogenic. 5 GARDASIL [Quadrivalent Human Papillomavirus (Types 6, 11, 16, 18) Recombinant Vaccine], a highly effective prophylactic vaccine against HPV, is composed of VLPs made from yeast expressed recombinant L1 proteins of HPV types 6, 11, 16, and 18. GARDASIL was recently approved by the United States Food and Drug Administration (FDA). For clinical trials of the vaccine, a high throughput assay was needed to measure type specific, biologically relevant antibodies generated in response to vaccination with GARDASIL. The immune response after exposure to the HPV L1 protein, either through natural infection or vaccination with VLPs, elicits an array of B cell populations; each B cell population produces antibody molecules recognizing unique L1 epitopes. 6,7 It is likely that only some of the antibodies produced confer protection against disease. The most commonly used assay, the enzyme linked immunosorbent assay (ELISA), measures the total IgG response to vaccination. The ELISA identifies antibody binding to all surface *Correspondence to: Judith F. Smith; Merck & Co., Inc.; Vaccine & Biologics Research; P.O. Box 4; West Point, Pennsylvania USA 19486; Tel.: ; judith_smith@merck.com Submitted: 08/06/07; Accepted:11/04/07 Previously published online as a Human Vaccines E-publication: Human Vaccines 2008; Vol. 4 Issue 2

2 Table 1 Reactivity of a panel of monoclonal antibodies against inttact and disrupted HPV VLPs Reactivity of 0.1 mg/ml of purified mab to 5 mg/ml of HPV L1 VLP antigen Monoclonal HPV 6 L1 VLP HPV 11 L1 VLP HPV 16 L1 VLP HPV 18 L1 VLP Antibody Isotype *Intact **Disrupted Intact Disrupted Intact Disrupted Intact Disrupted H6.B10.5 IgG 2b H6.M48 IgG H11.B2 IgG 2b K11.B2 IgG H16.J4 IgG 2b H16.V5 IgG 2b H18.R5 IgG 2b H18.J4 IgG 2b *Intact, VLP coating buffer: 50 mm Histidine buffer + 0.5M NaCl, ph 6.2; **Disrupted, VLP coating buffer: 0.2 M sodium Carbonate buffer M DTT, ph 10.6 Table 2 Heamagglutination inhibition by monoclonal antibodies Monoclonal Assay for Hemagglutination Antibody HPV Type Inhibition Titer VLP L1 (mg/ml) H6.B10.5 HPV 6 *NR H6.M48 HPV 6 <0.78 H11.B2 HPV K11.B2 HPV 11 <0.78 H16.J4 HPV 16 *NR H16.V5 HPV H18.R5 HPV H18.J4 HPV *NR, no response VLP epitopes and does not distinguish between protective and nonprotective antibodies. Further, because it measures total IgG, the ELISA will capture any antibodies in the serum that are cross reactive for other HPV and vaccine VLP types, 8 11 making it difficult to separate out the response to the different vaccine components or to identify subjects who had been infected with one or more vaccine HPV types. Therefore, we wanted to develop an assay to measure antibodies that have the potential to be protective (neutralizing), as a functional measure of sero conversion. Here, we summarize the evolution of our strategy to dissect the polyclonal response to vaccination in order to monitor type specific neutralizing antibodies that are likely to mediate protection against disease. When the HPV vaccine program was initiated, preclinical animal studies were performed to assess the vaccine immunogenicity. We used a set of previously characterized type specific, neutralizing monoclonal antibodies (mabs) directed against HPV L1 VLPs of types 6, 11, 16, and 18 to develop improved assays for monitoring the immune response to HPV infection or vaccination. We first confirmed the type and conformation specificity and neutralization potential of four mabs in an ELISA screening assay. Subsequently, the biological effect of the mab binding to HPV VLPs was demonstrated in two separate neutralization assays, the hemagglutination inhibition (HAI) and the pseudovirus (PsV) neutralization assays. Table 3 We next developed a competitive radioimmunoassay (cria) that uses these antibodies as competitors. In addition to detecting the presence of neutralizing antibodies in the sera of nonhuman primates vaccinated with HPV VLPs, the cria showed less cross reactivity between HPV L1 types than a direct bind ELISA. As the HPV vaccine program progressed, the cria was used to monitor the immune response to the vaccine in Phase I and II human clinical trials. Finally, we developed a competitive, multiplexed, Luminex based immunoassay (clia) that is able to specifically and sensitively detect neutralizing antibodies to the four HPV types in GARDASIL simultaneously in a single serum sample. The clia increased assay throughput and specificity, therefore after appropriate bridging studies were conducted, 12 the clia was used to monitor the immune response in the Phase IIB and Phase III clinical trials of GARDASIL. The clia will facilitate future studies to establish the duration of immunity and the minimum antibody titer against each vaccine component that correlates with protection against HPV infection. Results Pseudovirus neutralization by monoclonal antibodies Monoclonal Assay for HPV Type VLP Concentration Marker Antibody L1 + L2 (mcg/ml) producing Enzyme 50% NE H6.B10.5 HPV *blam H6.M48 HPV6 0.2 blam H11.B2 HPV 11 <0.1 blam K11.B2 HPV blam H16.J4 HPV **SEAP H16.V5 HPV 16 <0.16 SEAP H18.R5 HPV SEAP H18.J4 HPV SEAP *blam, beta-lactamase; **SEAP, secreted alkaline phosphatase Screening of mabs for HPV type specific and conformationdependent binding. We tested eight previously identified mabs, 6,12,13 two each for HPV types 6, 11, 16, and 18, in a screening ELISA Human Vaccines 135

3 to determine their specificity for HPV VLP type and conformation (Table 1). Each mab was assayed for its ability to bind to intact type specific VLPs and to the same VLPs after disruption of their 3 dimensional structures. A mab was considered conformation specific if binding to the native antigen of its target type was at least ten fold greater than binding to the disrupted antigen, and type specific if the OD 450 of wells containing type matched VLPs was >1.000 and the OD 450 values of wells containing VLPs of the other types were Supporting previously published results, H6.M48, K11.B2, H16.V5, and H18.J4 all showed strong type specificity, 12 whereas H6.B10.5 and H11.B2 showed some cross reactivity for types 11 and 6, (respectively refs. 12, 23 and 24). H16.J4 bound both conformational and disrupted VLPs, and therefore it is likely that it recognizes a linear epitope. Both H16.J4 and H18.R5 have previously shown cross VLP binding activity within the HPV 16 related and HPV 18 related types. 14 Evaluation of the mabs in neutralization assays. The humoral immune response to HPV infection or vaccination is polyclonal, and not all antibodies generated are capable of preventing HPV from infecting host cells. We therefore performed two types of assays to test the potential of the selected mabs to neutralize HPV: an HAI assay and a PsV neutralization assay. In the HAI assay, each of the eight mabs tested in the screening ELISA were incubated with the corresponding HPV L1 VLP type. The anti HPV 6, 11, 16, and 18 mabs identified in the screening ELISA as being specific (H6.M48, K11.B2, H16.V5, and H18.J4) strongly inhibited RBC agglutination (Table 2). The mabs that were less type or conformation specific in the screening ELISA required either much higher concentrations to inhibit hemagglutination, or did not inhibit it at all. The in vitro pseudoneutralization assays, provide a biological system where we can evaluate the relative neutralizing potential of antibody samples. The two quantitative assays we used here provide a direct measure of PsV particle entry into the cell with marker DNA for blam or SEAP. Optimum assay performance is at the concentration where the enzymatic activity is reduced to 50% (50% NE, 50% neutralization effect). The assays performed monitored blam enzymatic activity for HPV 6 and 11 PsV and SEAP enzymatic activity for HPV 16 and 18 PsV (see Materials and Methods for details). In single type screening ELISA assays, the mabs that were highly type specific, showed 50% NE of their target PsV at concentrations at or below 0.22 mg/ml for mab H6.M48, H11.B2, K11. B2, H16.V5, H18.R5 and H18.J4 (Fig. 3, Table 3). Where measurement of enzymatic activity resulted in no fluorescence/ luminescence or where all values at any test concentration were below 50% NE, the value is reported as less than the lowest test concentration (i.e., 0.16 mg/ml for H16.V5 with HPV 16 L1+L2 PsV). Thus, all the conformation dependent type specific mabs showed strong neutralization potential. The mabs that were less specific in the screening ELISA showed less neutralization potential. The exception was for H11.B2, which had a slightly greater neutralization potential than K11.B2. Comparison of the direct ELISA and the competitive radioimmunoassay (cria). Direct bind ELISAs are currently the standard measure of immunogenicity. With high amino acid homology between types, concern has been raised about the level of typespecificity that ELISAs can provide. 8,25 Furthermore, ELISAs cannot distinguish between neutralizing and nonneutralizing antibodies. In an attempt to develop a more specific assay that measures serum antibodies that are both type specific and neutralizing, we developed a competitive assay, the cria. To compare the type specificity of our cria with the standard direct bind ELISA, we immunized chimpanzees with monovalent HPV 11, 16, or 18 L1 VLPs or a quadrivalent HPV 6, 11, 16, and 18 vaccine. We tested sera from these animals for the presence of HPV 16 specific antibodies before and after immunization. Both assays successfully detected HPV 16 antibodies in the sera from animals that had received a vaccine containing HPV 16 VLPs, and the response threshold was similar, 6 mmu/ml for the cria and 19 mmu/ml for the ELISA, suggesting similar assay sensitivity (Fig. 4A and B). However, the ELISA also showed substantial cross reactivity for HPV 11 and 18. In contrast, the cria showed lower responses for HPV 11 and 18, although the geometric mean titers (GMTs) for HPV 11 and 18 were still above threshold at some time points. The antibody response generated by the quadrivalent vaccine produced a titer within the same log as the monovalent vaccine, suggesting no apparent immune interference occurred due to immunization with multiple HPV VLP types. Immunogenicity as measured by the detection of neutralizing antibodies in the competitive Luminex Immunoassay (clia). Although the cria is sensitive and specific, the ability to measure antibodies to more than one HPV type in a single serum sample would greatly facilitate the ability to measure the response to all the components of a multivalent vaccine such as GARDASIL. In addition, a high throughput assay that could handle the volume of samples generated in a clinical trial without the use of radioactivity was desirable. To meet these needs, we developed the clia. In a representative experiment, sera from rhesus macaques immunized with the quadrivalent HPV vaccine were tested at multiple time points. Antibodies developed after the first dose declined and then rose again following each booster dose in a pattern that is typical of a prime boost response (Fig. 5). 19 The GMT s remain high through the one year tested. Discussion The ability to measure the titers of type specific, neutralizing antibodies generated in response to natural HPV infection or vaccination with HPV L1 VLPs is important for epidemiologic studies and for clinical trials investigating the magnitude and duration of vaccine immunogenicity. Here, we have described the evolution of our technology for accurately distinguishing type specific, neutralizing antibodies among the many antibodies generated in response to vaccination with monovalent or quadrivalent HPV L1 VLP vaccines. First, to ensure that we selected mabs for use in our competitive assays that were type and conformation specific, we performed a screening ELISA of a panel of eight mabs that had been previously characterized as having varying degrees of specificity (Table 1). We confirmed that a sufficiently specific mab was available for each of the four HPV VLP components of GARDASIL. HPV is species specific, precluding the establishment of an animal model in which to perform a vaccine challenge, and only a few HPV types have been successfully grown in culture. It is therefore critical to establish that the antibody response to a vaccine contains antibodies with the potential to neutralize the virus and prevent it from establishing an infection. To test whether the type specific mabs 136 Human Vaccines 2008; Vol. 4 Issue 2

4 we had selected had neutralizing potential, we performed two types of neutralization assays. The HAI assay is based on the ability of VLPs to bind to mouse RBCs. A mab that binds to the VLP and prevents it from binding to an RBC would presumably also prevent the corresponding HPV type from binding and infecting an epithelial cell; that is, it would neutralize infection. The mabs that were most specific in our screening ELISA also required very low titers to inhibit hemagglutination, whereas the less specific mabs showed, in general, lower or no neutralizing potential in this assay. One limitation of the HAI assay is that it measures only the ability of an mab to prevent binding of the VLP to the cell surface, but papillomaviruses can also be neutralized after they have bound by preventing their internalization. 16,26 As a result, the neutralization in the HAI assay is likely an underestimation of the neutralization potential of a given antibody. We therefore also performed PsV neutralization assays, in which the mabs were assessed for their ability to interfere with PsV cell surface binding and internalization into a cell. The results from the PsV assays correlated well with those from the HAI assay and further supported our selection of mabs to monitor vaccine titers. It is important to note, however, that although the type specific, conformation dependent H6.M48, K11. B2, H16.V5, and H18.J4 mabs performed the best in these assays, the other antibodies did have titratable neutralizing effects at higher concentrations (Tables 2 and 3). Historically, HPV L1 immunogenicity has been evaluated using direct bind ELISAs. The HPV ELISA can be used to quantify total IgG binding to the L1 VLP, and as such is an excellent tool for identifying sero conversion. However, because of the significant homology between the L1 proteins of different HPV types (60 92%), 9 11,27,28 direct bind ELISAs are unable to provide a definitive measure of type specific antibodies. This is particularly true immediately after vaccination when titers are high. Furthermore, the ELISA does not distinguish between neutralizing and nonneutralizing antibodies. Neutralization assays, such as the HAI assay, although not as sensitive as ELISAs, correlate better with protection from infection. 16 To improve our ability to monitor the type specific neutralizing humoral response to vaccination with HPV VLPs, we developed a competitive assay, the cria. This was developed first for HPV 16, using the well characterized mab H16.V5, which is known to be conformation dependent, type specific, and neutralizing (Tables 1 and 3). 14,17 An HPV 16 cria yielded a higher degree of type specificity for anti HPV 16 antibodies in sera from vaccinated chimpanzees than an HPV 16 ELISA (Fig. 4). The HPV 16cRIA assay was used in Proof of Concept human clinical trials, to evaluate a response to the monovalent HPV 16 L1 VLP vaccine candidate and the quadrivalent HPV vaccine. These trials resulted in 100% sero conversion and the extremely high efficacy supported the rational for monitoring a neutralizing epitope. Indeed, the competitive assay format has proven to be a robust, reproducible, and precise method for monitoring type specific antibody titers in multiple human clinical studies, 29,30-33 where the subject may have circulating antibodies to one or multiple types of HPV. In addition, the results of crias have been correlated with true virus neutralization in the athymic mouse xenograft model. 34 Figure 1. The hemagglutination inhibition assay (HAI) (A, left) VLPs bind to mouse RBCs, causing agglutination and the formation of a floating raft of cells. (A, right) Neutralizing antibodies to the VLPs prevent binding of the VLPs to the RBCs. The RBCs fall to the bottom of the well and form a loose pellet. (B) The pellet formed in the presence of neutralizing antibodies is readily apparent. Here, the type-specific neutralizing mab H6.M48 inhibits agglutination by type 6 VLPs but not type 11 VLPs. Figure 2. Schematic of the competitive Luminex immunoassay (clia) Serum antibodies elicited by immunization compete with phycoerythrin-labeled mabs for binding to HPV type-specific neutralizing epitopes on the VLP. After washes to remove unbound antibodies, the fluorescence is measured. The intensity of the fluorescence is inversely proportional to the titer of anti-hpv neutralizing antibodies in the serum. Each VLP is bound to a microsphere that fluoresces in a different part of the spectrum, enabling the signal from each antibody type to be distinguished. We further refined the competitive assay using multiplex technology to develop a sensitive, nonradioactive, high throughput assay that can measure multiple type specific neutralizing antibodies to all four GARDASIL VLP components in a single serum sample (Fig. 2). 12,22 During the assay development it was demonstrated that the H6.M48 and K11.B2 mabs had greater HPV type specificity and therefore were incorporated. 12 This assay has been used to test the vaccine immune responses in clinical trials of GARDASIL. 30 Sera from vaccinated rhesus macaques showed a typical prime boost response to sequential doses of vaccine and a high titer was maintained for at least one year as illustrated in both assay formats (Fig. 5A clia 19 and B cria). A similar anamnestic response has been seen in women vaccinated with GARDASIL. 30 As the assay measures the displacement of a type specific neutralizing mab, these data provide evidence of type specific sero conversion to all four vaccine Human Vaccines 137

5 HPV VLP antigen types and indicate that the vaccine induced antibodies have neutralization potential. The humoral immune response to HPV L1 VLPs is complex, and the minimum anti HPV antibody concentration that is required for effective protection is not yet known. In clinical trials, GARDASIL vaccination induced an immune response that is at least as great as that observed following natural infection, as measured with the clia 30 or cria. 35 Importantly, the use of the H6.M48, K11.B2, H16.V5 and H18.J4 mabs, which are known to be neutralizing, as competitors in the clia, allows us to identify serum antibodies with the potential to neutralize HPV infection, and therefore provides a possible serological link between sero conversion and protective immunity. Materials and Methods Characterization of monoclonal antibodies. Monoclonal Antibodies (mabs). We examined a total of eight mabs against L1 VLPs of HPV 6 (H6.B10.5, H6.M48), 11 (H11.B2, K11.B2), 16 (H16. J4, H16.V5), and 18 (H18.J4, H18.R5). K11.B2 was developed at Merck Research Laboratories 12 as described below; the others were developed in Professor Neil Christensen s Laboratory at Penn State University and have been previously described (refs. 6, 13 and 14). Generation of mab K11.B2. Female BALB/c mice, four to five weeks old, were purchased from Taconic (Germantown, N.Y.). Mice were immunized intraperitoneally on days 0, 7 and 21 with 20 mg yeast expressed HPV 11 VLPs formulated with the proprietary Merck amorphous aluminum hydroxyphosphate sulfate (AAHS) adjuvant. A final intravenous injection of 20 mg of protein in phosphate buffered saline (PBS) was given to the mice three days prior to cell fusion. The mice were then sacrificed and the spleens removed for cell fusion. Lymphocytes prepared from the spleens 15 were fused with the mouse myeloma partner SP2/0 Ag14 (ATCC 1581, American Type Culture Collection [ATCC], Manassas, VA) using polyethylene glycol 1500 (Boehringer Mannheim, Indianapolis, IN), at a ratio of three parts lymphocytes to one part myeloma cells. HAT selection was used to obtain growing hybridomas. Supernatants were screened by ELISA for reactivity to native HPV 11 VLPs as described below. Positive wells were cloned by limiting dilution, and the supernatants were retested for ELISA HPV 11 VLP reactivity. Screening ELISA for HPV type specific and conformation dependent binding. Immulon 4B microtiter plates were coated overnight at 2 8 C with 5 mg/well of intact L1 VLPs of HPV types 6, 11, 16 or 18 in a buffer of 50 mm histidine, 0.5 M NaCl, ph 6.2, or L1 VLPs that had been disrupted by placing them in a buffer of 0.2 M sodium carbonate (ph 10.6), 0.01 M dithiothreitol at 37 C. The plates were washed three times with PBS containing 0.05% Tween 20 (PBST), and blocked for at least one hour with 1% bovine serum albumin (BSA) in PBST. After three washes, purified antibodies were added in limiting dilution and allowed to incubate for two hours at room temperature (RT). The plates were washed and a goat anti mouse IgG (H+L) HRP conjugate (Zymed, San Francisco, CA; 1:8000 in assay diluent) was added. Assay plates were developed with 3,3,5,5 tetramethylbenzidine (TMB) substrate, and the reaction was stopped with 2 N H 2 SO 4. The optical densities read at 450 nm (OD 450 ) setting were background corrected against control wells that only received the enzyme conjugated antibody and the substrate results were read in a plate reader as the optical density at 450 nm Wells were considered positive if the OD 450 was above Conformation specific antibodies were defined as those whose binding to the intact VLPs was at least 10 fold greater than their binding to the disrupted antigen. Further, binding to any other VLP antigen, either intact or disrupted, was considered significant cross reactivity if the OD 450 was above Hemagglutination inhibition assay. Attachment of HPV to a host cell is the first step in infection. 16 The HAI assay evaluates the ability of a mab to interfere with the binding of a virion or VLP to the cell surface, thereby acting as a surrogate of cell surface neutralization. VLPs incubated with red blood cells (RBCs) agglutinate the RBCs, creating a cross linked raft. In the presence of neutralizing antibodies, the VLPs are not able to agglutinate the RBCs, which then settle into a pellet in the test well (Fig. 1A). RBCs separated from the plasma of Swiss Webster mice were washed and resuspended at 1% (vol/vol) in PBS containing 1 mg/ml BSA. Test antibodies and purified HPV L1 VLPs of types 6, 11, 16 and 18 were diluted in PBS containing 1 mg/ml BSA and incubated at RT for one hour under gentle agitation. The amount of HPV L1 VLP used was optimized for each HPV type. An equal volume of a 1% RBC solution was added to each well containing the antibody/vlp mixture. The plate was incubated, undisturbed, for three hours at 4 C, and then visually observed and digitally scanned to generate a permanent image. Inhibition of hemagglutination was determined by visual inspection. A uniform RBC color distribution across the well indicated hemagglutination, whereas a tight pellet of cells forming a focal red dot centrally located within the well indicated HAI (see Fig. 1B). In vitro pseudovirus neutralization assays. HPV cannot be propagated in routine cell culture; therefore, PsV infection assays have been used as surrogate tests for virus neutralization. We used two different in vitro PsV neutralization assays to test the ability of the mabs to prevent PsV infection of cells grown in culture. Overall, the advances in PsV neutralization assays for both low risk and high risk HPV types are relatively similar; however, the assays available for the low risk HPV types 6 and 11 have used L1+L2 PsV externally coupled to a reporter plasmid containing b lactamase (blam), whereas the assays for the high risk HPV types 16 and 18 have advanced to encapsulate a reporter plasmid containing the secreted alkaline phosphatase (SEAP) gene within the PsV. As these kinds of assays are technically challenging, for each HPV type, we used the available assay that was most reliable and reproducible in our hands. We used HPV 6 and HPV 11 PsV neutralization assays developed by Merck 17 to evaluate the neutralization potential of the monoclonal antibodies H6.B10.5, H6.M48, H11.B2, and K11.B2. First, HPV (L1+L2) PsV were generated and chemically coupled to blam DNA (blam:hpv PsV) using the linker molecule sulfosuccinimidyl 4 (N maleimidomethyl)cyclohexane 1 carboxylate (Pierce Chemical Co., Rockford, IL). Serial monoclonal antibody dilutions were added to wells containing a monolayer of cells of the human cervical carcinoma cell line C33A (HTB 31, ATCC), followed by the addition of blam:hpv 6 PsV or blam:hpv 11 PsV in OptiMEM (Invitrogen, Carlsbad, CA). C33A cells are susceptible to infection with blam:hpv PsV. Upon entry of the blam:hpv PsV into the C33A cells, blam is produced in the cytoplasm. Assay plates were cultured for 48 hours, washed, and incubated with a fluorescent cephalosporin derivative, CCF4 (Aurora Biosciences, 138 Human Vaccines 2008; Vol. 4 Issue 2

6 Figure 3. Type-specific mabs show neutralization potential in a pseudovirion assay. Type-specific mabs were tested for their ability to prevent entry of HPV L1+L2 PsVs into C33A cells (A and B) or 293TT cells (C and D). (A) HPV 6, (B) HPV 11, (C) HPV 16, (D) HPV 18. Neutralization in (A and B) was measured by the fluorescence (AFU, arbitrary fluorescence units) of the substrate CCF4, which is cleaved by βlam. Neutralization in (C and D) was reported as chemiluminescence (RLU, relative light units) of the substrate CSPD, which is dephosphorylated by SEAP (C and D). Each point is the mean ± standard deviation of 3 (A and B) or 2 (C and D) measurements. The level of 50% Neutralizing Effect (50% NE) is indicated by the green lines. Values below the 50% NE threshold was considered to reflect neutralizing potential. Note that each assay is normalized to its own controls; therefore the scales differ between panels βlam. Neutralization in (C and D) was reported as chemiluminescence (RLU, relative light units) of the substrate CSPD, which is dephosphorylated by SEAP (C and D). Each point is the mean ± standard deviation of 3 (A and B) or 2 (C and D) measurements. The level of 50% Neutralizing Effect (50% NE) is indicated by the green lines. Values below the 50% NE threshold was considered to reflect neutralizing potential. Note that each assay is normalized to its own controls; therefore the scales differ between panels.{bb}± San Diego, CA). CCF4 is a membrane permeable substrate for blam and, once hydrolyzed by the enzyme in the cytoplasm, fluoresces blue. Fluorescence was measured on a CytoFluor 4000 (Perkin Elmer). A neutralizing mab will prevent the PsV from entering the cell, resulting in lower fluorescence values. If the mab is not neutralizing, the PsV will enter the cells, delivering the blam gene and resulting in increased blue fluorescence. Positive control wells contained no mab, which defined the maximum florescence attainable, whereas negative control wells containing no PsV or mab defined the Figure 4. Comparison of type specificity in an ELISA and a competitive radioimmunoassay. Monovalent and Quadivalent immunized chimpanzee sera were evaluated in two assay systems: direct bind HPV 16 ELISA and HPV 16 Competitive Radioimmunoassay (cria). The solid line indicates the GMT results and open symbols indicate the titer of the individual animals. While the same standard sera was used in both assays, the quantitative readout for the two assays is different. The Y axis on the ELISA represents the total IgG bound to the HPV 16 VLP expressed in mmu/ml. The Y axis on the cria plots the serum s ability to displace the binding of H16.V5 expressed in mmu/ml relative to the immunized serum standard. Dotted lines indicate the thresholds at which samples were considered positive for anti-hpv 16 antibodies. Human Vaccines 139

7 minimum florescence level. The mid point between the maximum and minimum florescence levels in each assay was defined as the 50% neutralization effect for that assay. The PsV neutralization assays developed by Pastrana and Buck at the National Cancer Institute 8 were used to evaluate the HPV 16 and HPV 18 antibodies H16.V5, H16.J4, H18.R5 and H18.J4. Briefly, HPV 16 and HPV 18 L1+L2 PsV were produced that encapsulated a target reporter plasmid containing the SEAP gene (PsV:SEAP). These PsV:SEAP were generated in the modified human embryonic kidney cell line, 293TT. Serially diluted mabs were combined with the specific HPV type PsV:SEAP at a concentration determined to be optimal in previous dilution infection experiments. The combined mab and PsV were then added to a monolayer of 293TT cells. When PsV enter 293TT cells, SEAP is produced and secreted into the supernatant of the well. The addition of a chemiluminescent substrate (1.25 mm CSPD, BD Biosciences, Clontech, Palo Alto, CA) enables the SEAP enzyme activity to be measured. Supernatants were sampled from the culture plates at 72 hours and were evaluated for SEAP enzymatic activity as a marker of PsV internalization. Antibody neutralization of the PsV would prevent PsV entry into cells, and SEAP production would therefore not be expected in the presence of neutralizing antibodies. Plates were read on a Dynex Luminometer 15 to 20 minutes after the addition of the substrate solution, using a setting of 0.2 second read/well. Development of competitive immunoassays to measure serum antibodies. Generation of Anti HPV VLP Nonhuman Primate Sera. Assay controls and test sera were prepared by immunization of chimpanzees. 18 HPV L1 VLPs adsorbed to Merck amorphous aluminum hydroxyphosphate sulfate (AAHS) adjuvant were delivered by intramuscular (IM) injection on day 0 and at weeks 8 and 24. Ten micrograms of HPV 16 L1 VLP or HPV 11 L1 VLP were delivered per dose; two animals received HPV 16 VLPs and two animals received HPV 11 VLPs. HPV 18 L1 VLP and the quadrivalent (HPV 6, 11, 16 and 18) vaccine were given to two additional animals each. The quadrivalent vaccine contained 10 mg of each component. Serum samples were collected at day 0 and every four weeks thereafter. The data set presented here includes test results from day 0 and weeks 8, 12, 24 and 28. Test sera was generated in four rhesus macaques, these were immunized with yeast derived HPV 6, 11, 16 and 18 VLPs at (2, 4, 4 and 2 mg/dose, respectively). The VLPs were formulated on AAHS and delivered by IM injection at weeks 0, 8 and 24. Sera were collected at weeks 0, 2, 4, 8, 10, 12, 16, 20, 24, 26, 28 and HPV 16 antibody ELISA. The HPV 16 antibody ELISA was used to evaluate total IgG in the immunized serum against the HPV L1 VLPs. Polystyrene 96 well plates (Nunc International, MaxiSorp F96, Denmark) were coated with baculovirus derived HPV 16 L1 VLP, produced in insect cells 20 in PBS, and incubated overnight at 4 C. Wells without VLP were used to control for background. Plates were washed with PBS plus 0.05% Tween 20 and blocked in PBS containing 1% BSA for 2 hours at RT. Sera were diluted with serial two fold dilutions from 1:100 to 1:204,800 in sample diluent (PBS containing 1% BSA and 0.05% Tween 20). The assay standard was serum from an HPV 16 hyperimmunized chimpanzee. The dilution for the standard curve was a serial three fold dilution from 10.1 to millimerck Units/ml (mmu/ml). The amount of bound IgG was detected with alkaline phosphatase conjugated goat anti human IgG (Kirkegaard and Perry Laboratories, Gaithersburg, MD). Color development of 100 ml of the substrate [1 mg/ml pnpp (Sigma, St. Louis, MO) in a solution of 1 M diethanolamine and 1 mm MgCl 2, ph 9.8] was stopped after 1 hour by the addition of 100 ml of 3 N NaOH. The OD 405 of each sample was read on a Titertek Multiskan ELISA Plate Reader. The background value was established as the average of the serum ODs of wells without VLP. Samples were normalized by subtracting the background from the average of the VLP coated wells. Binding of IgG was calculated (in ELISA mmu/ml) by comparison to the standard curve using a 4 parameter logistic curve fit. 21 The cutoff value for serological response was determined relative to the standard curve using multiple runs of normal human serum from individuals negative for HPV by polymerase chain reaction (PCR) and having low risk of HPV exposure. The cutoff value was determined to be 19 mmu/ml. HPV 16 competitive radioimmunoassay (H16 cria). A limiting amount of the conformation specific monoclonal antibody H16. V5 was used to test for the presence of anti HPV 16 antibodies in serum based on competition for a limited number of antigen epitopes. Polystyrene beads (1/4 inch with specular finish, Precision Plastic Ball Co., Franklin Park, IL) were precoated overnight at 4 C using normal goat serum at a dilution of 1:1000 in PBS, 400 ml at 7.5 beads/ml. The beads were washed with deionized water and permitted to air dry on a stainless steel tray. Antigen coating, bead washing, and bead storage were all performed in a buffer containing 50 mm MOPS M NaCl, ph 7.0. Yeast derived HPV 16 L1 VLPs were added to 600 ml MOPS buffer to a concentration of 50 ng/ml, and 3000 precoated beads were added to produce 5 beads/ ml. Coating was performed for 1 hour at RT with mild agitation. The beads were washed, and sufficient buffer was added to keep the VLP coated beads submerged at 4 C. Equal volumes (100 ml) of serum sample and diluted H16.V5 (1:800,000 dilution in PBST, containing 1% BSA) were mixed in each well of a 20 well Abbott assay plate. A single HPV 16 antigen coated bead was then added to each assay well. Samples were assayed in duplicate. Assay plates were sealed with adhesive sheets, incubated at RT overnight, and washed with deionized water. An 125 I labeled goat anti mouse IgG (NEN Life Sciences, Boston, MA) was used as a secondary antibody to detect bound H16.V5. The plate was sealed with a fresh adhesive sheet and incubated at 37 C for 2 to 2.5 hours. The amount of H16.V5 that bound to the HPV 16 VLP was determined using a gamma counter to measure the radioactive counts emitted by the 125 I (Wallac Wizard, PerkinElmer). The relative inhibition of H16. V5 binding by serum antibodies was compared to a standard curve, constructed by dilution of hyperimmunized chimpanzee sera, using a four parameter logistic curve fit. The immune serum used for the standard curve was assigned an arbitrary value in cria mmu/ml. The cutoff value for a serological response was determined relative to the standard curve, using multiple runs of preimmune chimpanzee serum and normal human serum from individuals negative for HPV by PCR and having low risk of HPV exposure. The cutoff value was determined to be 6 mmu/ml. HPV competitive Luminex Immunoassay (clia). The clia uses fluorescent tag based multiplexing technology to measure antibodies to all four GARDASIL antigens simultaneously. VLPs of each type are covalently bound to 5 mm diameter microspheres that fluoresce at a specific wavelength; different VLP types are bound to microspheres that fluoresce at unique wavelengths, allowing them to be differentiated when mixed. Test serum diluted in ADHS and phycoerythrin 140 Human Vaccines 2008; Vol. 4 Issue 2

8 Figure 5. Comparison of the performance of the multiplexed HPV quadrivalent competitive Luminex immunoassay (clia) and the competitive Radioimmunoassays (cria). Rhesus macaques were immunized with quadrivalent HPV vaccine on AAHS adjuvant at weeks 0, 8, and 24. At the indicated time points, the GMTs of neutralizing mabs to each vaccine component were measured using a clia and the competitor antibodies H6.M48, K11.B2, H16.V5, and H18.J4 (A). Adapted from ref. 19. The same samples were also tested in each of the single type cria assays (Panel B). In the HPV 6 cria the competitive mab used was H6.B10.5. In the HPV 11 cria the competitive mab was H11.B2. The competitor monoclonal antibodies used for the HPV 16 and 18 cria assays were the same as those used in the clia. (PE) conjugated, type specific, neutralizing competitor mabs were added to the well. The micropheres of all four types were added to the test well where antibodies could compete for limited VLP binding sites on the beads (Fig. 2). Type specific neutralizing antibodies in serum will successfully displace the PE conjugated competitor mabs, decreasing the amount of fluorescence. Microspheres are analyzed individually using two lasers, one to detect the microsphere type and the other to measure the amount of PE fluorescence, generating separate values for each HPV type. The assay was conducted (as previously described by Opalka et al. and Dias et al. refs. 12 and 22). In brief, a quadriplex reference standard was prepared for each HPV type in a prescreened assay matrix of antibody depleted human serum (ADHS). The reference standard was a pool of serum samples from African Human Vaccines 141

9 green monkeys hyperimmunized with monovalent HPV L1 VLP types 6, 11, 16 or 18. The quadriplex reference standard was diluted in a serial two fold dilution in ADHS to create a 12 point standard curve with final well concentrations ranging from 0.25 to 500 mmu/ml. The assay plate setup contained four controls: ADHS diluent and high, low, and negative titer controls. All standards, controls, and samples were tested in duplicate. Baseline testing of serum samples was performed at a 1:4 dilution; final assay conditions consisted of 25 ml of each of the following: serum, ADHS, mab PE quadriplex (0.1 mg/ml for each mab), and VLP microspheres (5,000 VLP microspheres per well per type). The plate was covered with a foil seal and placed on a shaker (600 to 800 rpm) at RT for 16 to 25 hours. The contents of the assay plate were transferred to a 1.2 mm hydrophilic low protein binding filter plate (Millipore, Billerica, MA) and washed three times. The microspheres were suspended in 125 ml of assay wash buffer for analysis on a Bio Plex Suspension Array System (Bio Rad, Hercules, CA). Fluorescent units were read and duplicate wells averaged. Dilution corrected serum values (in mmu/ml) were computed based on a four parameter logistics fit of the standard curve on each assay plate. Samples that exceeded the limits of quantitation for the standard curve were retested at higher dilutions. Acknowledgments We acknowledge the writing assistance of Dr. Naomi L. Ruff, who received funding from Merck & Co., Inc. References 1. Schiffman M, Castle PE. Human papillomavirus: Epidemiology and public health. Arch Pathol Lab Med 2003; 127: Clifford GM, Smith JS, Aguado T, Franceschi S. Comparison of HPV type distribution in high grade cervical lesions and cervical cancer: A meta analysis. Br J Cancer 2003; 89: Gissmann L, Wolnik L, Ikenberg H, Koldovsky U, Schnürch HG, zur Hausen H. 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