Pharmaceutical and immunological evaluation of human papillomavirus viruslike particle as an antigen carrier

Pharmaceutical and Immunological Evaluation of Human Papillomavirus Viruslike Particle as an Antigen Carrier ROXANA M. IONESCU,1 CRAIG T. PRZYSIECKI,2 XIAOPING LIANG,2 VICTOR M. GARSKY,3 JIANG FAN,2 BEI WANG,1 ROBERT TROUTMAN,1 YVETTE RIPPEON,1 ELIZABETH FLANAGAN,2 JOHN SHIVER,2 LI SHI1 1

Biologics and Vaccines PR&D, Merck Research Laboratories, Merck & Co., Inc., P.O Box 4, West Point, Pennsylvania 19486-0004 2

Vaccines and Biologics Research, Merck Research Laboratories, Merck & Co., Inc., P.O Box 4, West Point, Pennsylvania 19486-0004

3

Medicinal Chemistry, Merck Research Laboratories, Merck & Co., Inc., P.O Box 4, West Point, Pennsylvania 19486-0004

Received 28 April 2005; accepted 13 July 2005 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.20493

ABSTRACT: We report the preparation and the immunogenicity of a conjugate vaccine obtained by chemically conjugating a variant of the extracellular peptide fragment of influenza type A M2 protein to the human papillomavirus (HPV) virus-like particle (VLP). Conjugates comprised of approximately 4000 copies of the antigenic peptide per VLP are obtained as the result of the reaction between a C-terminal cysteine residue on the peptide and the maleimide-activated HPV VLP. The resulting conjugates have an average particle size slightly larger than the carrier and present enhanced overall stability against chemical and thermal-induced denaturation. The M2-HPV VLP conjugates lost the binding affinity for anti-HPV conformational antibodies but retained reactivity to a M2-specific monoclonal antibody. The conjugate vaccine formulated with aluminum adjuvant and delivered in two doses of 30-ng peptide was found to be highly immunogenic and conferred good protection against lethal challenge of influenza virus in mice. These results suggest that HPV VLP can be used as a carrier for synthetic or small antigens for the development of subunit vaccines. ß 2005 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 95:70–79, 2006

Keywords: peptide delivery; protein carrier; protein structure; stability; analytical ultracentrifugation; light scattering (dynamic)

INTRODUCTION Conventional vaccines have achieved tremendous success in eradicating or diminishing infectious diseases in the last several decades. However, traditional vaccines are associated with significant drawbacks and challenges in vaccine preparation due to the lack of widely useful, universal template technologies. New approaches of vaccine preparation have been considered for subunit vaccine development.1 One approach is Correspondence to: Li Shi (Telephone: 215-652-8856; Fax: 215-652-5299; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 95, 70–79 (2006) ß 2005 Wiley-Liss, Inc. and the American Pharmacists Association

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the use of minimal antigenic structures such as antigenic peptides to produce safer, well-characterized subunit vaccines. The success of this approach relies not only on the adequate selection of the antigenic sequence, but also on how this sequence is presented to the immune system. The immunogenicity of isolated minimal antigenic peptides alone was found to be very low in general, and an enhancement of antigen presentation or delivery is necessary for developing an efficacious vaccine. Several strategies have been tested for improving the antigen delivery.2–4 One of the most common routes of peptide presentation is to have the antigenic sequence either inserted or chemically coupled via a linker to an antigen

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carrier. Carriers that have been used in animal studies or commercialized vaccines are tetanus toxoid (TT)5, keyhole limpet hemocyanin (KLH)6, outer-membrane-protein-complex (OMPC) from N.meningitidis6, protein rP40 from the outermembrane-protein-A5, and pyruvate dehydrogenase.7 A special category of carriers is represented by virus capsid proteins that have the capability to self-assemble into virus-like particles (VLPs). Examples of VLPs used as peptide carriers are hepatitis B virus surface antigen (HBsAg) and core antigen (HBcAg)8, hepatitis E virus particles9, polyoma virus10, and bovine papilloma virus.11 More recently, antigen presenting artificial VLPs were constructed to mimic the molecular weight and size of real virus particles.12 The advantage of using papillomavirus VLPs as peptide antigen carrier is that it allows the presentation of antigenic sequence in an ordered array that ensures optimal response from the immune system. Moreover, exposure of the antigenic sequence in a matrix that mimics an icosahedral virion was found to abrogate the ability of the humoral immune system to distinguish between self and foreign.13 By linking mouse self-peptide TNF-a to papilloma virus VLPs high-titers, long-lasting auto antibodies were induced in mice. One of the challenges in using VLPs as minimal antigen carriers is to avoid the decrease in immunogenicity of the developed conjugate vaccine due to the presence of anti-carrier antibodies induced by preexposure to the VLP carrier. The human papillomavirus (HPV) VLPs possess a typical icosahedral lattice structure about 60 nm in size and each VLP is formed by the assembly of 72 L1 protein pentamers (called capsomeres).14,15 One of the advantages of using HPV VLP as antigen carrier is that the VLP has a well-defined quaternary structure amenable for extensive characterization of the final product. Bovine papillomavirus VLPs have been used successfully to carry an antigenic sequence either inserted into the L1,13 or L216 proteins of the VLPs or fused to streptavidin which then is bound to biotinylated VLPs.11 In this study we describe the HPV VLP system as an antigen carrier for developing conjugate vaccines in which the antigenic sequence is chemically coupled to the capsid surface of HPV VLPs. The chemical coupling avoids the potential problems of peptide insertion into the L1 sequence that can interfere with the proper assembly of the VLPs and is much simpler than the fusion/

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biotinylation procedure. Moreover, chemical coupling allows much higher peptide loads per VLP compared to previous reported procedures.11 Our data indicate that the peptide conjugation process does not induce significant alteration in the morphology of HPV VLPs. The antigenic sequence, M2 peptide, selected to test the carrier abilities of HPV VLPs corresponds to the extracellular segment of the M2 protein in influenza virus A. This particular sequence is of interest because it is very well conserved and an influenza vaccine based on it would avoid the current seasonal adaptations. It was previously shown that this M2 peptide either fused into HBcAg sequence17 or conjugated to OMPC6 induced a strong antibody mediated protection in mice against lethal virus challenge. As shown in this report, similar immune responses were observed with M2-HPV VLP conjugate vaccine formulated with aluminum adjuvant. The results support the use of the HPV VLP system as an effective antigen carrier for subunit vaccine development.

MATERIALS AND METHODS HPV VLPs and M2-Peptide HPV type 16 VLPs were expressed and purified from Saccharomyces cerevisiae as described in the literature.18 The antigen used in this study is a synthetic 25-residue M2-peptide prepared by standard t-Boc solid phase synthesis. The sequence of the peptide is similar to the extracellular segment of the M2 protein in Influenza virus strain A/Aichi/470/68 (H3N1)6 and contains an unnatural amino acid, 6-aminohexanoic acid (Aha).

Antigen-Carrier Conjugation HPV VLPs in 50 mM NaHCO3 pH 8.4 at 14 mM in L1 protein concentration were mixed with a commercial heterobifunctional cross-linker 4(N-maleimidomethyl)-cyclohexane-1-carboxylate (sSMCC) (Pierce Endogen, Rockfort, IL) to a final sSMCC/L1 protein (mol/mol) ratio of
100. The reaction proceeded for 1 h at 2–88C and was then desalted by dialysis against a pH 6.2 buffer containing 10 mM Histidine, 0.5 M NaCl, 0.015% polysorbate 80 to generate sSMCC activated HPV VLPs. The maleimide equivalents were determined by the DTNB assay.6 The M2-peptide JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 1, JANUARY 2006

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dissolved in N2-sparged buffer was mixed with sSMCC-activated HPV VLPs to a thiol/maleimide (mol/mol) ratio of
3. Alternatively, activated/ quenched HPV VLP (A/Q HPV VLP) was prepared by mixing sSMCC-activated HPV VLPs with N-acetylcysteine at a thiol/maleimide (mol/ mol) ratio
10. The reactions proceeded for
15 h at 2–88C. Both samples were then treated with bmercaptoethanol to quench any excess maleimide. Finally, the samples were dialyzed (Dispodialyser MWCO 300000 Spectrum Industries Inc., Rancho Dominguez, CA) against 0.5 M NaCl and 0.015% polysorbate 80. Similar results were obtained when the free thiols in HPV VLPs were quenched with iodoacetamide prior to conjugation. Determination of Protein Concentration and Peptide Load per VLP The concentration of protein in solution was determined by a colorimetric bicinchoninic acid (BCA) assay. The peptide load per VLP was determined by amino acid analysis. Samples were hydrolyzed for 70 h in 6 N HCl at 1108C and then quantitated after cation-exchange chromatography treatment (AAA Services, Inc., Boring, OR). The amount of peptide was determined by either referencing to the Aha content or conducting an analysis based on the procedure described in the literature.19 Both methods gave similar results. SDS–PAGE Analysis of Conjugates SDS–Page was performed on NuPage 10% BisTris gels (Invitrogen, Carlsbad, CA) under reducing conditions. The conjugates were mixed with NuPAGE LDS sample buffer (2% final detergent concentration) and NuPAGE sample reducing agent and kept at 708C for 10 min prior to loading into the gel. The running buffer was NuPAGE MES SDS containing NuPAGE antioxidant. Colloidal Blue Staining kit was used for gel staining. SeeBlue Plus2 prestained standard (Invitrogen) was used as molecular weight marker. Detection of Conjugate Interactions with Anti-HPV and Anti-M2 Antibodies The binding of HPV type 16 VLPs and M2-HPV VLP conjugates to antibodies specific to HPV type 16 or M2 protein was evaluated using the surface plasmon resonance technique on a Biacore 2000 instrument. The anti-HPV type 16 antibodies used in the testing (conformational antibodies JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 1, JANUARY 2006

H16.V5, H16.E70, and linear epitope binding antibody H16.J4) were purchased from Dr. N.D. Christensen at Pennsylvania State University. The anti-M2 antibody L18.H12 was provided by M. Horton. Immobilization of the rat anti-mouse Fcg antibody on the chip was done in three steps: (a) Activating the carboxymethyl groups on the sensor chip surface, (b) coupling the rat antimouse Fcg antibody with reactive esters, (c) blocking the unreacted NHS-esters with of ethanolamine-HCl. The mouse neutralizing monoclonal antibodies were diluted in the Biacore running buffer prior to binding to the immobilized rat anti-mouse antibody. Finally, the antigens were bound to the captured anti-HPV or anti-M2 antibodies. The relative antigenicity of an antigen is defined as the relative Biacore response calculated based on the binding intensity (RU) of sample and reference to a specific antibody. For anti-HPV antibodies, the original HPV VLP lot was used as reference. For anti-M2 antibody, the M2-HPV VLP lot was used as reference. ðR Uantigen =R Uantibody Þsample Reference Conc  100%  Sample Conc ðR Uantigen =R Uantibody Þreference

Conjugate and Carrier Particle Size Measurements Dynamic light scattering (DLS) measurements were performed on a Malvern 4700 instrument with detection at 908 and room temperature. The output power was at 0.25 W, aperture of 100 and total protein concentration of 0.1 mg/mL. The size reported represents the Z-average hydrodynamic diameter as resulted from monomodal analysis of data obtained in five consecutive measurements on the same sample. SEC-HPLC was performed on a HP 1100 System equipped with a Shodex OHpak SB-805 column and an elution buffer containing 25 mM phosphate, 0.75 M NaCl pH 7.0. Sedimentation velocity experiments were performed on an analytical ultracentrifuge Beckman XL-I using a rotor An6Ti and a double-sector cell. The rotor speed was 10000 rpm and the boundary movement was observed by absorption at 280 nm. Data was analyzed using the program DCDTþ (http://www.jphilo.mailway.com). Electron microscopy measurements were performed by Electron Microscopy BioServices (Monrovia, MD) using a JEOL 1200 EX Transmission Electron Microscope at high magnification. Airdried samples were stained with 2% phosphotungstic acid.

EVALUATION OF VIRUSLIKE PARTICLE AS AN ANTIGEN CARRIER

Turbidity Measurements The heat-induced increase in the turbidity of HPV VLP or M2-HPV VLP conjugate solutions was monitored on a spectrophotometer HP 8453 equipped with a thermal controller type 89090A. The variation in optical density at 350 nm was recorded as the temperature increased from 248C to 748C at rate of
1.58C/min.

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mouse adapted viruses A/Puerto Rico/8/34 (PR8; H1N1) and X-31(H3N2), a reassortant between PR8 and A/Aichi/68 (H3N2), were propagated in allantoic fluid of 10 day-old embryonated eggs. The mice were anesthetized with ketamine/xylazine. Twenty microliters of virus with 1 LD90 was instilled into nostrils. After challenge, the mouse survival rates were recorded daily. The mortality rate was calculated as: (number of mice at the day specified/number of mice at day 0) 100%.

Immunogenicity of Conjugate Four- to ten-week-old female Balb/c mice were obtained from Charles River Laboratories (Wilmington, MA). All animal studies described in this report were approved by the Merck Research Laboratories Institutional Animal Care and Use Committee. M2-HPV VLP adsorbed on Merck aluminum adjuvant (MAA) at different peptide doses was delivered by 0.1 mL I.M. in two injections, four weeks apart. The control group was represented by naı¨ve mice. The mice were challenged 3 weeks after the second injection. The peptide doses of 3, 30, and 300 ng correspond to about 5, 50, and 500 ng of HPV VLP. The dose of MAA delivered at each injection was 45 mcg. AntiM2 geometric mean titers were determined at 2 weeks after each injection. ELISA measurements, viral challenge, and postchallenge monitoring were described in detail elsewhere.6 Briefly, for M2 antibody ELISA, 96-well plates were coated with 50 mL per well of M2 peptide at a concentration of 4 mg per mL in 50 mM bicarbonate buffer, pH 9.6 at 48C over night. Plates were washed with phosphate buffered saline (PBS) and blocked with 3% skim milk in PBS containing 0.05% Tween-20 (milk-PBST). Testing samples were diluted in a fourfold series in PBST. One hundred microliters of a diluted sample was added to each well, and the plates were incubated at 248C for 2 h and then washed with PBST. Fifty microliters of predetermined dilutions of HRP-conjugated secondary antibodies in milk-PBST was added per well and the plates were incubated at 248C for 1 h. Plates were washed and 100 mL of 1 mg/mL o-phenylenediamine dihydrochloride in 100 mM sodium citrate, pH 4.5 was added per well. After 30 min incubation at 248C, the reaction was stopped by adding 100 ml of 1N H2SO4 per well, and the plates were read at 490 nm using an ELISA plate reader. The antibody titer was defined as the reciprocal of the highest dilution that gave an OD490 nm value above the mean plus two standard deviations of the conjugate control wells. For viral challenge,

RESULTS AND DISCUSSIONS Antigenic Peptide Loading on the Virus-Like Particle The peptide load on the HPV VLP was determined after acid hydrolysis using amino acid analysis by either quantitating the unnatural amino acid (Aha, 6-aminohexanoic acid) in the peptide or by multiple regression least-square analysis of data.19 Both methods indicated a peptide loading of about 11 peptides per L1 protein. There are 360 copies of L1 protein in a HPV VLP (a VLP contains 72 L1 protein pentamers or capsomers) thus resulting in a total load of about 4000 peptide copies per VLP. This number is significantly larger than the previously reported total number of peptides carried on a bovine papillomavirus particle.11 In the bovine papillomavirus case, an antigenic peptide was fused to streptavidin (SA) and the fusion construct interacted with biotinylated VLPs. The L1 protein of the VLPs was found to accommodate
1.5 SA tetramers resulting in a ratio of
6 peptides per L1 monomer. This load is about half of that found with our conjugation of M2 peptide to HPV VLP. It is possible that the bulkiness of the SA tetramer precludes a higher antigen loading in the reported case. The conjugation efficiency can be evaluated by determining how many of the initial sites activated by sSMCC resulted in a peptide coupling. Amino acid analysis can provide a quantitative estimation of tranexamic acid (TXA) which is the product of sSMCC cross-linker in the hydrolysis process. The measured average amount of TXA indicated
19 activated sites per L1 protein, suggesting that only 58% (or 11/19) of the activated sites were involved in peptide coupling. It is proposed that some of the activated sites may interact with proximal side chains of Cys, Lys, or His resulting in significant cross-linking of the protein. In Figure 1 are shown the bands observed in SDS–PAGE analysis of the HPV VLP with/without exposure JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 1, JANUARY 2006

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Figure 1. SDS–PAGE under reducing conditions on HPV VLPs with/without conjugation. Lane 1: M2-HPV VLP with iodoacetamide treatment, lane 2: M2-HPV VLP without iodoacetamide treatment, lane 3: molecular weight markers, lane 4: HPV VLP.

to the conjugation procedure. Nonactivated HPV VLPs present protein bands of the expected mobility,15 with a dominant species representing the monomeric L1 protein and two faint bands for a fragment and oligomeric species. However, M2HPV VLP with or without iodacetamide pretreatment could not penetrate the gel despite the strong chemical (2% detergent, reducing conditions) and thermal (10 min at 708C) pretreatment prior to loading into the wells. No penetration in the gels was observed for (A/Q) HPV VLP as well. Therefore it was concluded that significant intra-VLP cross-linking occurs after maleimide activation. As it will be shown below, VLP size measurements indicate that the impact of inter-VLP cross-linking on the particle size distribution of VLPs is negligible. In regard to the spatial distribution of the antigenic peptide on the surface of HPV VLPs, we consider the primary amine of the Lys side chain as the most likely site of sSMCC activation. There are 34 Lys in the L1 protein of HPV type 16 and nine of these lysines are located in the Cterminus. The molecular picture shown in Figure 2 reveals that the putative activation sites on HPV type 16 VLPs are evenly spread on the VLP surface. The NZ atoms of Lys residues presented in Figure 2 are oriented towards the exterior of the VLP. Except for Lys 230, all Lys residues have more than 25% of the surface exposed to the solvent. The C-terminus region is very flexible and accessible to proteases, so it is very probable that the side chains of Lys situated in this region are available for activation. Unfortunately, the CJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 1, JANUARY 2006

Figure 2. Ribbon diagram of the L1 protein as determined by X-ray in a 12-capsomere VLP.14 The individual medium gray spheres represent the NZ atoms of 19 Lys chains that are on the exterior surface of the VLP. The dark gray cluster shows Phe 50 that is part of the epitope for both H16.V5 and H16.E70 antibodies. The light gray cluster represents the binding loop for H16.J4 antibody. The figure was generated using the program MolMol.26

terminus region was not resolved in the X-ray structure.14 Pharmaceutical Characterization of M2-HPV VLP Conjugates DLS measurements indicate a slight increase in the average particle size of the carrier (HPV VLP) upon conjugation from
60 nm for the untreated carrier (HPV VLP) to
80 nm for the conjugate (M2-HPV VLP). The A/Q HPV VLPs reveal an average hydrodynamic size of
65 nm, a value that is very close to the size of the untreated carrier. SEC-HPLC results (Fig. 3A) present the main peak of M2-HPV VLP conjugate eluting at

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The M2-HPV VLP conjugates observed by EM (Fig. 4) present a size distribution between 40 to 95 nm, with a mean at approximately 65 nm. This value is very close to that of the untreated HPV VLPs. However, in contrast with the unconjugated carrier, the conjugates were found to have a ‘‘fuzzy appearance’’ in M2-HPV VLP, which may be due to the presence of conjugated peptide. The multi-VLP clusters shown in EM images are observed for HPV VLP as well; therefore they may be the result of sample preparation for EM measurement and are not representative for the sample in solution. In conclusion, EM results support that the morphology of HPV VLPs was preserved and that no major disruption of HPV VLP scaffold occurred during the chemical conjugation process. The profiles of heat-induced aggregation determined by a solution turbidity assay for treated and untreated HPV VLPs or the conjugates are shown in Figure 5. For untreated HPV VLPs, the heatinduced aggregation (as revealed by the increase in optical density due to light scattering) becomes detectable at 608C and increases in an abrupt manner if the temperature is further increased. For the A/Q VLPs or M2-HPV VLP conjugates, the turbidity of solution does not present detectable

Figure 3. Particle size distribution for HPV VLP type 16 (solid line), activated/quenched HPV VLP (dashed line), and conjugate M2-HPV VLP (solid line with circles) as determined by (A) SEC-HPLC and (B) Analytical Ultracentrifugation.

shorter retention time compared to A/Q or untreated HPV VLPs; that corresponds to a particle size of the conjugate larger than that of A/Q or untreated HPV VLPs. The small shoulders in the chromatograms reveal the presence of a small fraction of aggregated material before and after the conjugation. Finally, sedimentation velocity data (Fig. 3B) presents a distribution of sedimentation coefficients for the M2-HPV VLP centered at s* values larger than that of the untreated or A/ Q HPV VLPs. The slight increase of the sedimentation coefficient of conjugate compared to carrier alone is consistent with a small size increase upon conjugation as revealed by DLS and chromatographic measurements. The overall results also suggest that no significant inter-VLP cross-linking (and implicit, aggregation) occurs during the conjugation process.

Figure 4. Electron microscopy image of M2-HPV VLP. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 1, JANUARY 2006

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Figure 5. Temperature-induced aggregation monitored by OD at 350 nm for HPV VLP type 16 (solid line), activated/quenched HPV-VLP (dashed line), and conjugate M2-HPV VLP (solid line with circles).

aggregation below 708C. It is very likely that the enhanced stability against heat-induced aggregation is due to the intra-VLP cross-linking induced by sSMCC treatment. The additional intra-VLP bonds formed via sSMCC may prevent L1 protein from partial unfolding and subsequent exposure of hydrophobic surfaces. It is worth noting that the conjugation or sSMCC treatment resulted in the change of the surface properties of the HPV VLPs, which may in part contribute to the stability enhancement of the carrier. Future studies using different antigenic sequences may clarify the contribution of the surface modification and crosslinking to the overall stability enhancement of the conjugates.

Phe 50.20 As shown in Figure 2, there are 6 Lys residues, which flank Phe 50. It is very likely that conjugation of a peptide to any of the Lys residues around Phe 50 will perturb the antibody binding. H16.J4 binds to a loop on the top of L1 protein in VLP. There is only one Lys along this loop, which may not become conjugated with peptide because the binding to H16.J4 is not altered in M2-HPV VLP. One concern is whether the peptide is presented in the correct 3-D configuration on the surface of the carrier. The M2 protein is an integral membrane protein of the Influenza A virus and the antigenic sequence selected represents the extracellular part of M2. The M2 protein is a homotetramer formed by two disulfide-linked dimers21 and, to our knowledge, no detailed 3-D structure was reported in the literature about the extracellular segment of M2. CD and fluorescence measurements suggest that the unconjugated peptide in solution is predominantly in random structural configuration (data not shown). Although these findings disfavor presentation of the peptide in a defined structural configuration on the surface of VLP, preliminary results obtained by surface plasmon resonance indicate that the M2HPV VLP conjugate is recognized by a relevant anti-M2 antibody L18.H12. No binding to anti-M2 antibody was detected under similar conditions with HPV VLPs or (A/Q) HPV VLP (the ‘‘relative antigenicity’’ as defined in Materials and Methods was below 5%). The monoclonal antibody L18.H12 binds to a region comprising the residues in the first half of M2 sequence and has antiviral activity (M. Horton, personal communication). In vivo Immunological Evaluation

In vitro Antigenicity Analysis of M2-HPV Conjugates The spatial distribution of antigen was further investigated by determining the binding of M2HPV VLP and A/Q HPV VLP to linear and conformational anti-HPV mouse antibody (mAB). The binding affinity of M2 HPV VLP for the conformational or neutralization antibodies H16.V5 and H16.E70 was found to be dramatically lower (less than 1%) compared to that of nontreated HPV VLPs, while the binding to linear antibody H16.J4 was not significantly affected by conjugation (relative antigenicity
100%). The relative antigenicity for the binding of (A/Q) HPV VLP to H16.V5 antibody was found also less than 1%. The epitopes involved in the binding of the conformational antibodies H16.V5 and H16.E70 comprise JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 1, JANUARY 2006

Balb/c mice were immunized with two administrations of M2-HPV VLP conjugate vaccine formulation at different M2 peptide doses as described in Materials and Methods. Results of ELISA measurements on blood samples taken 2 weeks after each immunization indicate that the conjugate elicited high anti-M2 antibody response (Fig. 6A). Although the titers increase in a systematic manner as the M2 peptide dose is increased from 3 to 300 ng, the difference in titers between the lowest and highest dose is within one log unit. These results indicate that the antigenic peptide at nanogram doses can induce a significant immune response when presented on a suitable carrier. It is worth noting that similar titers are observed in mice when the M2 peptide is conjugated on a

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Figure 6. A: Geometric mean titer of anti-M2 antibody induced by M2-HPV VLP in mice at T ¼ 2 and 6 weeks after immunizations at T ¼ 0 and T ¼ 4 weeks with vaccines containing M2-HPV VLP at different peptide doses. B: Rate of survival against lethal challenge for mice immunized with vaccines containing M2-HPV VLP at different peptide doses.

larger-size carrier, the N. meningitidis outermembrane protein complex (OMPC).6 The survival rates of mice against lethal challenge are shown in Figure 6B. The group receiving the lowest dose of peptide (3 ng) shows only 60% survival, whereas the protection in groups with higher doses of 30 or 300 ng peptide is 100%. It is interesting to note that, while the groups receiving the middle dose (30 ng) and the low dose (3 ng) of the M2-VLP differed in antibody titers by only threefold, the former showed complete protection whereas the latter showed only 60% protection. The antibody titers at the 3 ng dose may suggest a protection threshold: mice with titers above the threshold survive whereas those with the titers below the threshold do not. No survival after challenge was observed for the control group, confirming that the virus challenge and the vaccine protection were both effective. As previously reported,6 some weight loss is observed after challenge even in the groups with 100% survival (data not shown). In conclusion, the

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vaccination of Balb/c mice with M2-HPV VLP conjugate vaccine efficiently protects the animals against live virus challenge. It is generally accepted that small peptides which lack helper T-cell epitopes are not immunogenic, and that various strategies need to be adopted to improve their ability to induce protection.27–29 Although we have not evaluated directly the immunogenicity of a free M2 peptide in this study, previous reports in the literature30 clearly indicate that the epitope density is a critical factor in the magnitude of the humoral and protective response. Upon immunization at constant antigen dose, it was found30 that the survival rate against an Influenza A challenge can vary between 100% to 0% when the number of M2 peptide copies fused to Glutathione-S-transferase varied between 16 and 1, respectively. The carrier-induced epitope-specific suppression has been described in literature.5 Therefore, future experiments should determine how the immunogenicity of the conjugate is affected by the presence of anti-carrier antibodies in vivo. Previous studies with M2-OMPC6 indicate that pre-exposure to carrier did not abolish, but slightly diminished the response to the Influenza conjugate vaccine. However, it was suggested that subsequent boosts could overcome the detrimental effect of preexisting antibodies to the carrier. Despite the overwhelming number of cases in which pre-immunization with a carrier was shown to impair the antibody response, one cannot simply propose a priori that the presence of anti-carrier antibodies has an adverse effect on the immunogenicity of a conjugate vaccine. It was reported that prior immunity to carrier (TT) was beneficial either to anti-hCG (human chorionic gonadotropin22) or to malarial peptide23 response. In a different case describing recombinant flagella as a carrier of influenza peptide epitopes it was found that there was no effect of pre-exposure to carrier.24 It would be interesting to determine in the case of HPV VLPs whether there is any difference in animal models pre-exposed to the carrier in the untreated form (as an anti-HPV vaccine) or the treated form (as a carrier presenting a different antigen). It was found that more than 75% of reactive human sera were completely blocked by H16.V5 antibody.25 The fact that conjugated M2-HPV VLP does not bind to H16.V5 antibody suggests that carrier suppression to vaccines prepared through chemical conjugation between antigen and HPV VLPs would not be a major concern for those who were pre-exposed to HPV. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 1, JANUARY 2006

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Previous experiments with M2-OMPC6 have demonstrated that the protection against influenza virus lethal challenge can be passively transferred by the administration of immunized animal sera, indicating that neutralizing antibodies were sufficient to confer protection. It is very likely that a similar humoral response was triggered by the immunization with M2-HPV VLP conjugate. In regard to the cellular response, previous experiments showed that HPV type 16 VLPs induced a strong Th2 response as measured by CD4þ T cells production of IL-4.18 It was also proposed that nonconformational antigenic sequences presented by HPV VLPs might enhance the cell-mediated immune response.16

ACKNOWLEDGMENTS Chris Culberson kindly determined the solvent exposure of the Lys side-chains in L1. We thank Alex Ni for advice on SEC-HPLC and Analytical Ultracentrifugation assays, Robert Evans, Paul Keller, and Emilio Emini for valuable discussions, Melanie Horton for providing the anti-M2 monoclonal antibody and Carl Burke for useful discussions and critical review of the manuscript.

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