Purification and immunogenicity study of human papillomavirus type 16 L1 protein in Saccharomyces cerevisiae

Journal of Virological Methods 139 (2007) 24–30

Purification and immunogenicity study of human papillomavirus type 16 L1 protein in Saccharomyces cerevisiae Se Na Kim a , Hye Sung Jeong a , Sue Nie Park b , Hong-Jin Kim a,∗ a

b

College of Pharmacy, Chung Ang University, 221 Huksuk-Dong, Dongjak-Ku, Seoul 156-756, Republic of Korea Department of Biologics Evaluation, Korea Food and Drug Administration, 231 Jinheungno, Eunpyeong-Gu, Seoul 122-704, Republic of Korea Received 15 February 2006; received in revised form 1 September 2006; accepted 7 September 2006 Available online 10 October 2006

Abstract Human papillomavirus 16 virus-like particle (HPV16 VLP) vaccines expressed in Saccharomyces cerevisiae are under Phase III trial and are expected to be on the market in the near future. We have established a convenient and economical system for the prophylactic study of vaccines derived from HPV16 VLPs, and neutralization tests to standardize HPV serological methodology as a measure of validation. To purify HPV16 VLPs, yeast cells expressing HPV16 L1 protein were cultured and purified on a small scale by ultracentrifugation and size-exclusion and cationexchange chromatography using open columns. The highly purified HPV16 L1 protein was identified by SDS-PAGE and Western blotting, and electron microscopic analysis confirmed that they self-assembled into VLPs. To test the efficacy of the purified VLPs as a vaccine and their ability to induce humoral immunity, we performed ELISA assays and observed a significant increase in the titer of anti-HPV16 VLPs antibodies in the sera of immunized mice. High anti-HPV16 neutralizing titers were found in the sera of vaccinated mice, as measured by a SEAP-based pseudovirus neutralization assay. These results would be useful in the evaluation of the immunogenicity of HPV vaccine candidates, and provide an international reference standard for HPV serological methods. © 2006 Elsevier B.V. All rights reserved. Keywords: Human papillomavirus (HPV); Virus-like particle (VLP); Neutralization assay

1. Introduction Human papillomavirus (HPV) is the second leading cause of cancer deaths in women worldwide, as it is a major risk factor for cervical cancer (Pisani et al., 1993). HPV is a small double-stranded DNA virus containing a circular genome of approximately 8000 base pairs (Pfister and Fuchs, 1994). Native virions of HPV are non-enveloped, 50–60 nm diameter icosahedral structures composed of 72 capsomers, each composed of five L1 molecules (Baker et al., 1991; Trus et al., 1997). The major capsid protein (L1, ∼55 kDa) can self-assembly into virus-like particles (VLPs), which are structurally similar to native HPV virions (Hagensee et al., 1993; Kirnbauer et al., 1992; Rose et al., 1993; Sasagawa et al., 1995; Volpers et al., 1994). VLPs have been shown to induce high-titer virus neutralizing antibody in animal models (Breitburd et al., 1995;

∗

Corresponding author. Tel.: +82 2 820 5613; fax: +82 2 816 7338. E-mail address: [email protected] (H.-J. Kim).

0166-0934/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2006.09.004

Christensen et al., 1996; Kirnbauer et al., 1996; Suzich et al., 1995). Recombinant VLPs have been expressed in animal, insect, yeast and bacterial cells (Kirnbauer et al., 1992; Lowe et al., 1997; Rose et al., 1993; Zhou et al., 1991), and represent the leading candidate vaccines for preventing cervical cancer (Mandic and Vujkov, 2004). However, the yeast expression system offers the advantage of vaccine development that is cost-effective and easy to adapt to large-scale growth in fermenters; in addition, its potential for contamination by toxins or infectious viruses is small compared with bacterial or mammalian expression systems (Cook et al., 1999; Joyce et al., 1999; Neeper et al., 1996). Currently, the development of VLP vaccines targeted against high-risk HPV types (HPV16 and 18) and low-risk HPV types (HPV6 and 11) is in progress, and quadrivalent HPV L1 VLP vaccines expressed in the yeast system are in Phase III trial and are expected to be available on the market in the near future (Koutsky et al., 2002; Lowy and Frazer, 2003). Hence, standardization of the assays and reference regents for assessing the efficacy of HPV vaccines is greatly needed. Neutralization

S.N. Kim et al. / Journal of Virological Methods 139 (2007) 24–30

assays have often been used to evaluate the efficacy of viral vaccine candidates. However, obtaining infectious HPV virions in cell culture systems is difficult. Therefore, an alternative type of in vitro assay of HPV neutralization has been developed (Pastrana et al., 2004) that involves the use of pseudovirions consisting of capsids that are formed in vitro by expressing the viral L1/L2 protein. However, conflicting results have been obtained in trials using this assay in different laboratories around the world. Therefore, standardization of the assay and the criteria used to evaluate the immune response to this vaccine should make it possible to make comparisons of the outcomes of different trials. In the present study, we performed an immunogenicity study of a vaccine that was derived from HPV16 VLPs, and neutralization tests to standardize HPV serological methods as a measure of validation. HPV16 VLPs that are made in the yeast expression system were purified by a simple, small-scale three-step purification process, and we assessed their immunogenicity by ELISAs and by SEAP-pseudovirus neutralization assays. 2. Materials and methods

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plus 1 M NaCl), and fractions were analyzed using SDS-PAGE and Western blotting. 2.3. Western blotting analysis Samples containing HPV16 L1 protein were electrophoresed on 12.5% acrylamide gels under reducing and denaturing conditions and transferred onto PVDF membranes (Q-Biogene, USA). The protein was detected using mouse anti-HPV16 L1 (Camvir-1; Chemicon International Inc., USA) as primary antibody and goat anti-mouse IgG–HRP conjugate (Sigma, USA) as secondary antibody. The membrane was visualized using Western blotting luminol reagent (Santa Cruz Biotechnology, USA). 2.4. Electron microscopy Purified HPV16 L1 was dialyzed against PBS at 4 ◦ C for 3 h, absorbed to carbon-coated grids and negatively stained with 2% phosphotungstic acid. TEM was performed using a TEM200CX transmission electron microscope at final magnifications of 41,000×.

2.1. Expression of HPV16 L1 protein in yeast

2.5. Immunization of BALB/c mice with HPV16 VLPs

Recombinant HPV16 L1 protein was expressed as previously described (Park et al., 2002). Saccharomyces cerevisiae strain EGY48 not transformed with the HPV16 L1 gene was used as a negative control. EGY48 was grown with shaking at 30 ◦ C for 24 h in YPD medium, which consists of 1% yeast extract, 2% peptone and 2% glucose (DIFCO Laboratories, USA).

The immunogenicity of the purified HPV16 VLPs was tested in BALB/c mice. Twenty female 6-week-old BALB/c mice, purchased from ORIENT Co., Korea, were maintained in an air-conditioned room and supplied with sterile chow and water, and used when they reached 7 weeks of age. They were divided into four groups; 15 were immunized by subcutaneous injection of 5 ␮g purified HPV16 VLPs adsorbed to Freund’s complete adjuvant (Sigma, USA), and five controls were inoculated with adjuvant only. After 3 weeks, all the mice were boosted twice with the same amount of VLPs adsorbed to Freund’s incomplete adjuvant (Sigma, USA). Ten days later, they were bled from their tails, and sera were collected and stored at −20 ◦ C.

2.2. Purification of HPV16 L1 protein To purify intracellular HPV16 L1 protein, cells were harvested and lysed (Park et al., 2002). Cleared lysates were layered onto cushions of 45% sucrose in breakage buffer (20 mM sodium phosphate, pH 7.2, 100 mM NaCl, 1.7 mM EDTA) and pelleted by ultracentrifugation at 25,000 rpm for 10 h. The pellets were resuspended in 1 ml breakage buffer with 0.01% Tween-80 (Shi et al., 2005) and fractionated at room temperature by size-exclusion chromatography on a 1.0 cm × 100 cm Glass Econo-column (Bio-Rad Laboratories Inc., USA) that was loaded with SephacrylTM S-1000 resin (Amersham Pharmacia Biotech, Sweden). The running buffer for this column was 10 mM sodium phosphate, pH 7.2, 150 mM NaCl, 0.01% Tween80. Fractions were collected into glass tubes and analyzed by SDS-PAGE and Western blotting for the presence of HPV16 L1 protein. The fractions of highest purity were pooled, equilibrated against binding buffer (20 mM Tris, pH 7.2, 100 mM NaCl, 0.1 mM EDTA, 5% glycerol, 15 mM 2-mercaptoethanol) and applied to P-11 cationic phosphocellulose (Whatman, UK) in a 0.8 cm × 4 cm Poly-Prep chromatography column (BIO-RAD Laboratories Inc., USA) that had previously been equilibrated with binding buffer at 4 ◦ C. Following a wash with a one-column volume of washing buffer (binding buffer plus 0.25 M NaCl), HPV16 L1 protein was eluted with elution buffer (binding buffer

2.6. Enzyme-linked immunosorbent assays (ELISAs) HPV16 VLP-specific antibody was assayed by ELISA. The ELISA plates were coated overnight with 100 ng/well of purified VLPs in PBS at 4 ◦ C, washed three times with washing buffer (PBS-T; 0.05% Tween-20 in PBS), and blocked with 2% BSA in PBS-T for 1 h at room temperature. Unabsorbed proteins were removed by washing, and serially diluted mouse sera were added in diluent buffer (0.3% BSA in PBS-T). The plates were then incubated at 37 ◦ C for 1 h. Thereafter, they were washed with PBS-T, goat anti-mouse IgG–HRP conjugate was added to the wells, and incubation continued for a further hour at 37 ◦ C. Unbound secondary antibody was removed by washing and bound antibody was stained with substrate—that is, one tablet of o-phenylendiamine (Sigma, USA) in 25 ml of phosphate–citrate buffer, dissolved with a capsule of sodium perborate (Sigma, USA) in 100 ml of dH2 O. The color reaction was stopped by adding 3 M H2 SO4 and absorbance was recorded at 492 nm.

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Fig. 1. Analysis of the expression of HPV16 L1 protein in recombinant Y2805 cells by SDS-PAGE (A) and Western blotting (B). Cell were lysed and electrophoresed (lanes 1 and 2), and separated into 45% sucrose supernatant (lanes 3 and 4) and pellet (lanes 5 and 6) fractions by ultracentrifugation. The negative control (EGY48) is shown in (A), lanes 1, 3 and 5 and (B), lanes 1, 3 and 5. Molecular weight reference markers (lane 1) are displayed at the left; the arrowhead at the right indicates the position of recombinant L1 (∼55 kDa).

2.7. Production of secreted alkaline phosphatase (SEAP)-based pseudovirus HPV16 pseudoviruses were produced as previously described (Buck et al., 2004; Pastrana et al., 2004) with minor modifications. Briefly, 293TT cells were co-transfected with p16L1h, p16L2h and pYSEAP. Some 44 h later, they were resuspended at 108 cells/ml in DPBS supplemented with 0.25% Brij 58, 9.5 mM MgCl2 , 0.1% Benzonase (Sigma, USA) and 0.1% Plasmid-Safe ATP-Dependent DNase (Epicentre, USA). The resuspended cells were matured by incubation at 37 ◦ C for 16 h (Buck et al., 2004), and 0.17 vol. 5 M NaCl was added. The mixtures were placed at 4 ◦ C for 10 min, and clarified by centrifugation at 1500 × g for 10 min at 4 ◦ C. To obtain crude pseudovirus, the supernatants were aliquoted into siliconized microcentrifuge tubes (Sigma, USA) and frozen at −80 ◦ C. For Optiprep-purified pseudovirus, the supernatants were purified on a pre-formed 27, 33, 39% Optiprep (Sigma, USA) step gradient, and fractions were collected by bottom puncture of the tube. The fractions were inspected for purity by 10% SDS-PAGE, then pooled and frozen at −80 ◦ C. 2.8. Neutralization assay (SEAP-based pseudovirus neutralization assay) Neutralization assays were performed as previously described (Pastrana et al., 2004) with minor modifications. Briefly, 293TT cells were plated 3–4 h in advance in 96-well tissue-culture-treated flat-bottomed plates (Nalge Nunc International, USA) at 30,000 cells/well in 100 ␮l neutralization buffer. Crude pseudovirus preparations were diluted 2000-fold, and Optiprep-purified preparations were diluted 400-fold, and incubated with diluted sera at 4 ◦ C for 1 h. The pseudovirus–antibody mixtures were then added to the previously plated cells and incubation continued. After 72 h, the SEAP content of the supernatants was analyzed using the colorimetric SEAP assay (Buck et al., 2005). A total of 40-␮l aliquots of the supernatant were transferred to 96-well ELISA plates (Nalge Nunc International, USA) and 20 ␮l of 0.05% CHAPS was added in PBS (Sigma, USA). The mixtures were incubated at 65 ◦ C for 30 min and 200 ␮l of coloring substrate was added—that is, one tablet of

p-nitrophenyl phosphate (PNP; Sigma, USA) in 20 ml of 2 M diethanolamine (Sigma, USA) plus 1 mM MgCl2 and 0.5 mM ZnCl2 . The reaction mixtures were incubated at room temperature in the dark for 1–4 h and absorbances were recorded at 405 nm. Serum neutralization titers are defined as the reciprocal of the highest dilution that causes at least a 50% reduction in SEAP activity (Pastrana et al., 2004). 3. Results 3.1. Expression and purification of HPV16 L1 protein The production of HPV16 L1 protein by recombinant S. cerevisiae Y2805 cells was analyzed by SDS-PAGE and Western blotting. As shown in Fig. 1, L1 bands of approximately 55 kDa were detected only in extracts derived from Y2805 cells transformed with the HPV16 L1 expression plasmid, not in control EGY48 extracts. To purify the protein, the cell extracts were ultracentrifuged over a 45% sucrose cushion and the pellet and supernatant were analyzed by SDS-PAGE and Western blotting. L1 was detected in both the pellet and supernatant of recombinant Y2805 cells. However, more was present in the pellet than in the supernatant (Fig. 1). The pellet was subjected

Fig. 2. Size-exclusion chromatography of the pellet fraction. The elution profile from the size-exclusion column is shown. The horizontal bar indicates the fractions pooled and purified by cation-exchange chromatography.

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Fig. 3. SDS-PAGE (A) and Western blotting (B) of HPV16 L1 protein in the fractions collected from the size-exclusion column. Lane 1, pellet fraction after ultracentrifugation; lanes 2–17, fractions number 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62 and 64. Molecular weight markers are shown in lane M.

to size-exclusion chromatography using a SephacrylTM S-1000 column (Fig. 2). Fractions (1 ml) were collected and analyzed for HPV16 L1 by SDS-PAGE and Western blotting. As shown in Fig. 3, L1 was detected in fractions 40–54, which had higher purities than the other fractions. The bar in Fig. 2 indicates the fractions that were pooled and submitted to cation-exchange chromatography using a P-11 cation column. The HPV16 L1, which has a predicted pI (isoelectric point) of 8.27 (predicted by ProtParam) (Wilkins et al., 1999), bound to the P-11 cationic column under weakly acidic conditions (pH 7.2). No HPV16 L1 protein was eluted in the 0.25 M NaCl wash step that was designed to remove weakly bound proteins (Fig. 4), and pure HPV16 L1 was eluted with elution buffer containing 1 M NaCl and detected by SDS-PAGE and Western blotting (Fig. 4).

3.2. In vitro self-assembly of purified HPV16 L1 We confirmed, by electron microscopy, that the purified HPV16 L1 protein self-assembled into VLPs. As shown in Fig. 5, the completely assembled structures were 51 ± 15 nm in diameter, which is identical to the dimensions of HPV VLPs that were reported previously (Rossi et al., 2000; Schiller and Lowy, 1996). 3.3. Induction of HPV16 VLP-specific antibodies by immunization with purified HPV16 VLPs To determine whether or not immunization with the yeast-derived HPV16 VLPs could induce humoral immunity, mice were injected with purified HPV16 VLPs, and

Fig. 4. SDS-PAGE (A) and Western blotting (B) of HPV16 L1 protein in the fractions collected from cation-exchange column. The fractions collected from the size-exclusion column (lane 1) were applied to a P-11 cation column equilibrated at pH 7.2 and the unbound material was collected (lane 2). Following removal of weakly bound protein (lane 3) by a low-salt wash (0.25 M NaCl), L1 was eluted from the column with buffer containing 1 M NaCl (lane 4). Molecular weight markers are shown in lane M.

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3.4. Purified HPV16 VLPs induce strongly neutralizing antibodies against HPV16 pseudovirus

Fig. 5. Electron micrograph of yeast-expressed HPV16 L1 protein demonstrating in vitro self-assembly. Shown is purified HPV16 L1 protein (Fig. 4) after equilibration in PBS for 3 h. The equilibrated L1 protein was adsorbed to carbon-coated copper grids, stained with phosphotungstic acid and examined by transmission electron microscopy. The magnification is 41,000× (bars, 50 nm).

HPV16 VLPs-specific antibodies were detected by ELISAs in the sera of the immunized mice. As shown in Table 1, high ELISA titers (76,800–115,200) were observed in all the sera from the 15 mice that were injected with HPV16 VLPs.

Table 1 ELISA titers of anti-HPV16 VLPs antibodies in sera from immunized mice with HPV16 VLPs Group namea Group 1 (102,400 ± 35,050c )

Mouse ID no.

ELISA titer for serum (IgG)b

1 2 3 4 5

64,000 128,000 64,000 128,000 128,000

Group 2 (115,200 ± 28,620c )

6 7 8 9 10

128,000 128,000 128,000 128,000 64,000

Group 3 (76,800 ± 28,620c )

11 12 13 14 15

64,000 64,000 64,000 64,000 128,000

Group 4d (<100c )

16 17 18 19 20

a

<100 <100 <100 <100 <100

Group of five mice divided each case. ELISA titer were deduced at an OD 2.0 × Group 4 (control). c Average of 5 mice/group ± S.D. (standard deviation). d Group 4 (mouse ID nos. 16, 17, 18, 19, 20) indicate control group that were inoculated only with the adjuvant. b

SEAP-based pseudovirus was produced in 293TT cells in which the parent 293 cells had been engineered to express high levels of SV40 large T antigen. The 293TT cells were co-transfected with the codon-modified HPV16 capsid genes, L1 and L2, together with a secreted alkaline phosphatase (SEAP) reporter plasmid containing the SV40 origin of replication. Infection of 293TT cells with SEAP-based pseudovirus was monitored by SEAP activity in the culture supernatant. Antibody-mediated neutralization of the pseudovirus is revealed by a reduction in SEAP activity (Pastrana et al., 2004). To determine whether or not injection of the yeast-derived HPV16 VLPs induced neutralizing antibodies against infectious HPV16pseudovirus, we performed SEAP-based pseudovirus neutralization assays. The 293TT cells were infected with SEAP-based pseudovirus together with serial two-fold dilutions of the sera of the immunized mice. After incubation for 72 h, SEAP activity was measured in the culture supernatants. To assess inter-assay variation, we carried out three separate neutralization assays on different days using the same pseudovirus stock. As shown in Table 2, all the sera of the mice immunized with HPV16 VLPs had high neutralization titers (2560–40,960) and no neutralizing activity (<40) was detected in the sera from the control mice. We performed the neutralization assays with two pseudovirus preparations (crude and Optiprep-purified pseudovirus) and found that the neutralization titers obtained with the Optiprep-purified pseudovirus were on average 1.7-fold higher than those with the crude preparation (Table 2). Because the latter also contained non-infectious empty VLPs (Buck et al., 2004), use of the Optiprep-purified pseudovirus increases the sensitivity of the SEAP neutralization assay. Neutralization titers did not vary by more than four-fold in the triplicate assays. 4. Discussion In this report, we describe the production of the L1 major capsid protein of human papillomavirus (HPV) type 16 in a yeast expression system, and its purification by a three-step process. The purified protein formed capsid-like particles spontaneously and elicited high titers of neutralizing antibodies in immunized mice. The protocol described yields pure HPV16 L1 protein by a simple purification protocol performed on a small scale using open columns without special equipment. Our procedure is easier to use and costs less than that employed by Cook et al. (1999), who reported the expression and purification of large quantities of HPV11 L1 protein from the yeast expression system. In addition, the SEAP-based pseudovirus neutralization assay for testing the efficacy of the HPV vaccine was reproducible and appears to be as sensitive as, and possibly more HPV-genotype-specific, than a standard VLPbased ELISA. Moreover, this assay system has the advantage that sufficient SEAP-pseudovirus can be produced in one flask for a thousand titrations in the 96-well plate format (Pastrana et al., 2004). Thus, we believe that this is a good surrogate assay for detecting the virus-neutralizing activity of sera. The SEAP

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Table 2 SEAP-pseudovirus neutralization assay using sera from immunized mice with HPV16 VLPs Group namea

Mouse ID no.

SEAP-pseudovirus neutralizationb Crude PSV

Group 1 (crude PSV:

16,440 ± 8700c ;

Optiprep-purified PSV

1 2 3 4 5

6,400 25,600 10,240 15,360 25,600

17,067 54,613 10,240 20,480 27,307

Group 2 (crude PSV: 7940 ± 5150c ; Optiprep-purified PSV: 17,070 ± 14,530c )

6 7 8 9 10

10,240 2,560 15,360 7,680 3,840

20,480 5,120 40,960 10,240 8,533

Group 3 (crude PSV: 10,500 ± 3190c ; Optiprep-purified PSV: 18,260 ± 7810c )

11 12 13 14 15

10,240 15,360 6,400 10,240 10,240

20,480 27,307 5,973 17,067 20,480

Group 4d (crude PSV: <40c ; Optiprep-purified PSV: <40c )

16 17 18 19 20

a b c d

Optiprep-purified PSV:

25,940 ± 17,170c )

<40 <40 <40 <40 <40

<40 <40 <40 <40 <40

Group of five mice divided each case. Endpoint-neutralizing titer was defined as 50% inhibition of the amount of SEAP compared to the virus added without antibody. Average of 5 mice/group ± S.D. (standard deviation). Group 4 (mouse ID nos. 16, 17, 18, 19, 20) indicate control group that were inoculated only with the adjuvant.

activity in the SEAP-based pseudovirus neutralization assay was measured by a colorimetric assay that is much less expensive than the chemiluminescence method (Pastrana et al., 2004), and can be performed with a conventional ELISA plate spectrophotometer. However, recombinant HPV VLPs purified from yeast are inherently unstable and tend to aggregate in solution in the absence of stabilizers (Shi et al., 2005). This aggregation problem causes loss of HPV VLPs during purification. In our experiments, the yield of pure VLPs per batch was not high. The aggregation problem will need to be solved using different buffer conditions, such as high salt and non-ionic surfactants, to increase the yield (Shi et al., 2005). In conclusion, our purification system is convenient and economical for preparing HPV16 VLP vaccines, and the results of this study could be applied to validate the efficacy of HPV vaccine candidates. In addition, our purification system could be of use as an international reference standard for HPV serological methods. Acknowledgement This work was supported by the KFDA under grant 05092731. References Baker, T.S., Newcomb, W.W., Olson, N.H., Cowsert, L.M., Olson, C., Brown, J.C., 1991. Structures of bovine and human papillomaviruses. Analysis by

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