Arch Virol (2013) 158:193–199 DOI 10.1007/s00705-012-1465-x

BRIEF REPORT

Human papillomavirus type 58 L1 virus-like particles purified by two-step chromatography elicit high levels of long-lasting neutralizing antibodies Xixiu Xie • Yanchun Liu • Ting Zhang • Yanying Xu • Qifeng Bao • Xue Chen • Hongyang Liu • Xuemei Xu

Received: 5 May 2012 / Accepted: 24 July 2012 / Published online: 11 September 2012 Ó Springer-Verlag 2012

Abstract Human papillomavirus (HPV) type 58 is a high-risk type of HPV frequently detected in cervical cancers, especially in Eastern Asia. There are still no commercially available vaccines against HPV 58 infection. High levels of long-lasting neutralizing antibodies are crucial for long-term protection against HPV infection. Here, we have developed a two-step chromatography strategy and have purified highly pure HPV L1 proteins, which form more homogenous and uniform VLPs than those purified by CsCl ultracentrifugation. Low-dosage immunization with HPV 58 L1 VLPs alone or co-administrated with HPV 16 and HPV 18 L1 VLPs is sufficient to induce high levels of long-lasting neutralizing antibodies in mice. Our results suggest that the highly immunogenic HPV 58 L1 VLPs are a good candidate for use in developing effective vaccines against HPV 58 infection.

Cervical cancers represent the most common cancer of women in large parts of the developing world. Approximately 45,689 women are diagnosed with cervical cancer, and 25,561 die from the disease annually in China [1]. High-risk types of human papillomavirus (HPV) have been shown to be the major etiological agents of cervical cancer.

Electronic supplementary material The online version of this article (doi:10.1007/s00705-012-1465-x) contains supplementary material, which is available to authorized users. X. Xie
Y. Liu
T. Zhang
Y. Xu
Q. Bao
X. Chen
H. Liu
X. Xu (&) Department of Biophysics and Structural Biology, Institute of Basic Medical Sciences Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, 5 Dong Dan San Tiao, Beijing 100005, China e-mail: [email protected]

HPV 16 and 18 are the two most prevalent high-risk types all over the world. The prevalence of HPV 16 and 18 in cervical cancer is 53.5 % and 17.2 %, respectively, followed by HPV 45 (6.7 %) and HPV 31 (2.9 %) [2]. HPV 58 (7.2 %) ranks as the third most prevalent HPV type in cervical cancer in China [3] and is the second most prevalent in precancerous lesions in Eastern Asia [1]. Currently, there are two licensed HPV L1 virus-like particle (VLP) vaccines, Cervarix, a bivalent HPV 16/18 vaccine produced in Hi-5 insect cells, and Gardasil, a quadrivalent HPV 16/18/6/11 vaccine produced in Saccharomyces cerevisiae. These two vaccines specifically induce protection against infections and precancerous lesions associated with HPV 16 and 18, but nearly no cross-protection against HPV 58 infection [4, 5]. It has been suggested that the second-generation HPV vaccine should include HPV 58 in order to provide better protection in regions where HPV 58 is prevalent. HPV VLPs can be purified by CsCl gradient ultracentrifugation based on their density, or by various chromatography methods based on the size, shape, net charge or specific ligand-binding properties of the protein. Although CsCl gradient ultracentrifugation has been commonly used to purify small-scale quantities of VLPs for lab research purposes, it has several technical limitations, including long processing time, toxic residue contaminations, limited scalability, and large batch-to-batch variations, which limit its use in large-scale VLP manufacturing and clinical applications. We previously reported that HPV 58 L1 VLPs expressed in Sf9 cells and purified by CsCl ultracentrifugation, could induce neutralizing antibodies in mice [6]. However, it remains unclear whether HPV 58 L1 VLPs can elicit high levels of long-lasting neutralizing antibodies, as HPV 16 and HPV 18 L1 VLPs do. In this study, we developed a two-step chromatography purification strategy

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by which we obtained highly pure and uniform HPV 16, 18, and 58 L1 VLPs. We further demonstrated that lowdose HPV 58 L1 VLP immunization is sufficient to elicit long-lasting high titers of HPV-58-specific neutralizing antibodies in mice when immunized either alone or with HPV 16 and HPV 18 L1 VLPs. The HPV 16, 18, and 58 L1 genes were modified to optimize codon usage in Sf9 cells as well as the mRNA secondary structure, and they were then expressed in Sf9 cells using a baculovirus system [6]. The GenBank accession numbers of the three genes are GU556964, GU556965, and GU556966. Ninety-six hours after infection, Sf9 cells were harvested and lysed by sonication, and the HPV L1 proteins were purified by size-exclusion and cation-exchange chromatography. The purity of the HPV L1 protein preparations was analyzed by SDS-PAGE with Coomassie blue staining as described previously [6] and by Sf9 host cell protein (HCP) ELISA. Briefly, purified HPV L1 proteins from three batches were diluted separately to 500 ng/ml in PBS. Fifty microliters of each sample was added in duplicate to the wells. The ELISA was then performed using the standard protocol recommended by the manufacturer (Cygnus Technologies). The concentrations of HCPs in the test samples were interpolated from a calibration curve obtained using HCP standards. To determine the physical characteristics of the purified HPV L1 proteins, transmission electron microscopy (TEM) was performed as described previously [6], and dynamic light scattering (DLS) was carried out according to the manufacturer’s instructions (Malvern). Conformation-specific monoclonal antibodies (mAbs) H16.V5, H18.J4 and XM58-07 (made in our lab) were used to investigate by ELISA whether dominant neutralizing epitopes are well retained in HPV L1 VLPs [7]. Briefly, intact or denatured HPV L1 VLPs (100 ng/well) were incubated overnight in 96-well microtiter plates at 4 °C. HPV L1 VLPs were denatured in PBS containing 10 mM DTT for 10 min at 75 °C. The plates were then blocked and incubated with H16.V5 (diluted 1:1000), H18.J4 (diluted 1:700), or XM58-07 (diluted 1:500). ED50 values of HPV 16, 18 and 58 L1 VLPs were examined to evaluate the immunogenic potency of each type of VLP. Three fourfold serial dilutions were prepared for HPV 16 L1 VLPs, whose doses ranged from 0.0125 to 0.200 lg, and four twofold serial dilutions were prepared for HPV 18 and 58 L1 VLPs, whose doses ranged from 0.0125 to 0.100 lg and 0.050 to 0.400 lg, respectively. The samples were diluted in PBS containing 300 mM NaCl and 0.03 % Tween-80, and 100 ll of each dilution was injected subcutaneously into 10 mice. After 21 days, sera were collected and serially diluted twofold for pseudovirus neutralization assays as described previously [6]. A mouse

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was considered to have seroconverted if the corresponding serum neutralizing antibody titer was C100 [8]. The ED50 was defined as the dose required to elicit seroconversion in 50 % of the mice according to the Reed-Muench method [9]. The immunogenicity of HPV L1 VLPs was further investigated in mice. Groups of four- to six-week-old female BALB/c mice (n = 4) were immunized twice intramuscularly with 0.1 lg of L1 VLPs of each type at 2-week interval, either alone or in combination. The groups tested were HPV 16 L1 VLPs (mono 16), HPV 18 L1 VLPs (mono 18), HPV 58 L1 VLPs (mono 58), and HPV 16/18/ 58 L1 VLPs (trivalent). Sera were collected at week 0 (preimmune) and week 4 and analyzed by pseudovirus neutralization assays. All statistical tests were performed using Student’s t-test for independent samples using SPSS software (version 16.0, SPSS). P-values less than 0.05 were considered to be statistically significant. To investigate whether HPV 58 L1 VLPs are able to induce long-lasting neutralizing antibodies when injected alone or together with HPV 16 and 18 L1 VLPs, mice were injected three times intramuscularly with 0.1 lg of L1 VLPs of each type at 2-week intervals with trivalent and mono 58, respectively. Sera were collected at weeks 0 (preimmune), 6, 32 and 48 after the first injection and analyzed by pseudovirus neutralization assays. To investigate whether HPV 58 L1 VLPs induced type-specific or crossreactive neutralizing antibodies, sera from members of the mono 58 immunization group at week 6 were analyzed by HPV 18 or HPV 52 pseudovirus neutralization assays. By applying our recently developed two-step chromatography purification strategy, we obtained approximately 11.6 mg of HPV 16 L1 VLPs, 10 mg of HPV 18 L1 VLPs, and 11.2 mg of HPV 58 L1 VLPs from a one-liter fermentation suspension. As demonstrated by SDS-PAGE with Coomassie blue staining, these HPV 16, 18, and 58 L1 proteins were highly pure (Fig. 1a). A single band was observed, with an expected molecular weight corresponding to that of the L1 protein monomer when 10-12 lg of purified L1 proteins were loaded in each lane. These L1 proteins were further confirmed by Western blot with antiHPV L1 mAb CamVir-1 (Fig. 1a). The purity of the HPV 16, 18, and 58 L1 proteins was further demonstrated by the fact that Sf9 host cell protein contamination was barely detectable using an HCP ELISA kit. As shown in Table 1, HCP contamination was \0.5 % (w/w) in the HPV 16 L1 preparation, 2.2 % (w/w) in the HPV 18 L1 preparation, and \1.4 % (w/w) in the HPV 58 L1 preparation. We next determined the physical characteristics of purified HPV L1 proteins by two independent and wellaccepted methods. By TEM analysis, we found that HPV 16, 18, and 58 L1 proteins were properly folded and uniformly assembled into VLPs with the diameters of

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(a)

(e)

Z-Average (d.nm) : 59.66

PDI: 0.198

(f)

(d)

(c)

Intensity (%)

Intensity (%)

(b)

Z-Average (d.nm) : 74. 74

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0 0.1

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Z-Average (d.nm) : 66.16

(h)

PDI: 0.221

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Z-Average (d.nm) : 151.1

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10000

Size (d.nm)

Size (d.nm)

(g)

PDI: 0.123

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15

PDI: 0.390

(i)

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Z-Average (r.nm) : 55.91

PDI: 0.489

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Intensity (%)

Intensity (%)

Intensity (%)

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5 4 3 2 1

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0 0.1

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Size (d.nm)

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Size (d.nm)

Fig. 1 Purity, transmission electron micrograph and dynamic light scattering analysis of HPV 16, 18, and 58 L1 VLPs. (a) SDS-PAGE with Coomassie blue staining and Western blot assays. The purities of HPV L1 proteins were examined by SDS-PAGE with Coomassie blue staining, and the L1 proteins were confirmed by Western blot. Lane 1, pre-stained protein marker; lane 2, HPV 58 (12 lg); lane 3, HPV 18 (10 lg); lane 4, HPV 16 (12 lg). (b to d) TEM assay. Purified HPV 16 L1 VLPs (b), HPV 18 L1 VLPs (c) and HPV 58 L1 VLPs (d) were loaded onto carbon-coated copper grids, stained with 1 % uranyl

10

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Size (r.nm)

acetate, and examined using a JEM1010 transmission electron microscope at 80 kV with a final magnification of 980,000. Bar = 100 nm. (e to i) DLS assay. Chromatography-purified HPV 58 L1 VLPs (e), HPV 16 L1 VLPs (f), HPV 18 L1 VLPs (g), CsClgradient-purified HPV 58 L1 VLPs (h), and two batches of HPV 18 L1 VLPs (i) were analyzed by DLS. Measurements were performed by Zetasizer Nano ZS at 25 °C. Pdl, polydispersity index; Intensity %, particle percentage calculated in intensity; Z-Average, the mean hydrodynamic diameter based on the intensity of scattered light

Table 1 Detection of Sf9 HCP contamination in purified HPV 16, 18, and 58 L1 VLPs by ELISA L1 VLP type

Percentage (%)b

HCP concentration (ng/ml) Batch 1

Batch 2

Batch 3

Mean

Batch 1

Batch 2

Batch 3

Mean \0.5

HPV 16

\1.4a

5.0

\1.4

\2.6

\0.3

1.0

\0.3

HPV 18

8.9

10.0

14.7

11.2

1.8

2.0

2.9

2.2

HPV 58

9.9

10.0

\1.4

\7.1

2.0

2.0

\0.3

\1.4

a

The detection limit of the Sf9 HCP ELISA kit was 1.4 ng/ml

b

The concentration of total protein in each sample was 500 ng/ml. HCP levels were expressed as a percentage of HCPs within the total proteins (w/w)

45-70 nm, 40-68 nm, and 40-65 nm, respectively (Fig. 1b, c and d). Furthermore, using DLS assays, we observed that the size distribution of HPV 58 L1 proteins was unimodal, with a mean polydispersity index (PdI) of 0.198 and a mean hydrodynamic diameter (Z-average) of 59.66 nm, suggesting that HPV 58 L1 proteins were highly uniform and assembled into VLPs (Fig. 1e). Similar results were found for HPV 16 and 18 L1 proteins (Fig. 1f, g). In contrast to the homogeneous VLPs purified by two-step chromatography, HPV 58 and HPV 18 L1 proteins purified from Sf9

cells by CsCl ultracentrifugation displayed multiple peaks with a relatively wide size range (Fig. 1h, i), suggesting the heterogeneity of these CsCl ultracentrifugation purified VLPs. Thus, these data demonstrate that HPV 16, 18, and 58 L1 VLPs purified from Sf9 cells by the two-step chromatography purification strategy are pure, uniform, and well characterized, suggesting the potential use of this strategy in large-scale VLP production. To determine the immunogenicity of HPV 16, 18, and 58 L1 VLPs, we first examined whether the dominant

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Fig. 2 (a) The binding activities of HPV L1 VLPs. Type-specific neutralizing mAb H16.V5 (diluted 1:1000), H18.J4 (diluted 1:700), or XM58-7 (diluted 1:500) reacted with intact (I) and denatured (D) HPV 16, 18, and 58 L1 VLPs in ELISA. HPV L1 VLPs were denatured in PBS containing 10 mM DTT for 10 min at 75 °C. Results are expressed as the mean ± SD (n = 4). (b and c) Neutralizing antibody levels of sera from mice immunized with trivalent and corresponding monovalent vaccines. Groups of mice (n = 4) were immunized twice intramuscularly with 0.1 lg L1 VLPs of each type at 2-week intervals, either alone or in combination, and the corresponding groups were HPV 16 L1 VLPs (mono 16), HPV 18 L1 VLPs (mono 18), HPV 58 L1 VLPs (mono 58), and HPV 16/18/58 L1 VLPs (trivalent). Immune sera were detected at week 4 with pre-

immune sera as controls. (b). For long-term experiments, groups of mice (n = 4) were injected three times intramuscularly with 0.1 lg L1 VLPs of each type at 2-week intervals with trivalent and mono 58 vaccines. The anti-HPV 58 neutralizing antibody titers were detected at weeks 6, 32 and 48 after the first immunization (c). (d) Neutralization of HPV pseudovirion types 18 and 52 with HPV 58 L1 VLP anti-sera. Sera from the mono 58 immunization group at two weeks after the third injection were analyzed by HPV 18 or 52 pseudovirus neutralization assays. Serum neutralizing antibody titers were calculated from log10-transformed values. Values between trivalent and corresponding monovalent vaccine groups were compared using Student’s t-test

neutralizing epitopes are well retained in our purified HPV L1 VLPs. H16.V5- and H18.J4-recognized epitopes were confirmed by ELISA to be present on the surface of HPV 16 and HPV 18 L1 VLPs, respectively (Fig. 2a). A typespecific neutralizing mAb, XM58-07, could bind efficiently to intact HPV 58 L1 VLPs (Fig. 2a). We next calculated the ED50 value of each VLP preparation. The ED50 values of HPV 16, 18, and 58 L1 VLPs were 0.0275 lg, 0.0172 lg, and 0.1140 lg, respectively (Supplementary material). Of note, the ED50 of HPV 18 L1 VLPs was lower than that of HPV 16 and HPV 58 L1 VLPs. Finally, we

examined the neutralizing antibody levels induced by these L1 VLPs by the well-established pseudovirus neutralization assay. As shown in Fig. 2b, monovalent vaccines as well as trivalent vaccine were able to induce high levels of neutralizing antibodies against the corresponding immunogen type(s) at week 4, although a relatively low antiHPV 58 neutralizing antibody titer was found in one of the four mice in the trivalent group. There were no significant differences in type-specific neutralizing antibody titers to vaccine components between the trivalent and corresponding monovalent vaccine groups (P[0.05). Consistent

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with the lowest ED50 of HPV 18 L1 VLPs, anti-HPV 18 neutralizing antibody titers induced by trivalent and mono 18 vaccines were higher than those of anti-HPV 16 and anti-HPV 58. These results indicated that HPV 16, 18, and 58 L1 VLPs purified by two-step chromatography are highly immunogenic and elicit effective neutralizing antibody responses. High levels of long-lasting neutralizing antibodies are crucial for long-term protection against HPV infection. To evaluate the long-term effect of HPV 58 L1 VLP immunization, we examined the long-lasting neutralizing antibody levels in mice by the pseudovirus neutralization assay. Anti-HPV 58 neutralizing antibody titers in both trivalent and mono-58-immunized mice were maintained at high levels (above 103) for at least 48 weeks (Fig. 2c). No significant differences were found in the levels of longlasting HPV-58-specific neutralizing antibodies between the trivalent and mono 58 vaccine groups (P[0.05). Longlasting serum HPV-16- and HPV-18-specific neutralizing antibodies presented the same profile (data not shown). It has been reported that HPV 58 L1 VLPs induce no or marginal cross-neutralization against high-risk HPV types 16, 31, 33, 39, 45, and 59 [10], and therefore the neutralization of other common high-risk HPV types, 18 and 52, by HPV 58 L1 VLP antisera was investigated. No crossneutralization was observed between HPV 58 and HPV 18 or HPV 52 (Fig. 2d). The results showed that neutralization induced by HPV 58 L1 VLPs is type-specific. Taken together, we conclude that HPV 16, 18, and 58 L1 VLPs purified by two-step chromatography from Sf9 cells are highly immunogenic and induce high levels of long-lasting neutralizing antibodies in mice. In this report, we produced HPV 16, 18 and 58 L1 VLPs in Sf9 cells. The Sf9 cell line is a clonal isolate of Sf21, which was derived from the pupal ovarian tissue of a fall army worm, Spodoptera frugiperda. Their small, regular size makes Sf9 cells exceptional for the formation of monolayers and plaques. In the production of the HPV 16/18 L1 VLP vaccine Cervarix, Sf9 cells were used for recombinant virus amplification, and Hi-5 cells, which originated from the ovarian cells of a cabbage looper, Trichoplusia ni, were used for the expression of L1 VLPs. We compared the expression levels of the optimized HPV 16 L1 gene in Hi-5, Sf9 and Sf21 cells and found that L1 expression levels in Sf9 and Sf21 cells were comparable, whereas in Hi-5 cells, the L1 expression level was too low to be detected (data not shown). It would be more practical to use only one cell line rather than two for large-scale VLP production. Our results showed that Sf9 cells are an appropriate tool to produce well-characterized HPV L1 VLPs. HPV L1 VLPs, expressed in Sf9 cells and purified by CsCl ultracentrifugation, have been demonstrated for some

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types of HPV [6, 11, 12]. However, due to safety concerns, CsCl is not permitted for human vaccine production. It has also been suggested that CsCl ultracentrifugation purification causes aggregation of VLPs during storage and has a negative impact on the function of VLPs [13]. Therefore, we developed a two-step chromatography method to purify HPV 16, 18, 58 L1 VLPs. In contrast to the heterogeneity of CsCl-gradient-purified HPV 58 and 18 VLPs, chromatography-purified HPV 16, 18 and 58 L1 VLPs are highly uniform, as demonstrated by TEM and DLS. We also found that the HPV 18 L1 VLPs purified by two-step chromatography could elicit relatively high levels of neutralizing antibodies at two weeks after the second immunization (Fig. 2b, panel 5) as compared to the same dosage of HPV 18 L1 VLPs purified by CsCl ultracentrifugation at the same time point (ref. 6, Fig. 5A), which indicates that these uniform HPV L1 VLPs purified by two-step chromatography may have relatively higher immunogenicity than those purified by CsCl ultracentrifugation, as shown for other VLPs [13, 14]. In addition, by applying two-step chromatography, we obtained a high yield (10-11.6 mg/ liter of Sf9 cells) of pure HPV 16, 18, and 58 L1 VLPs. Thus, our study demonstrates the advantages of two-step chromatography purification in large-scale HPV L1 VLP production. Studies have shown that immunization with L1 VLPs could protect animals from subsequent challenge with papillomavirus [15]. Moreover, protection can also be achieved by passive transfer of serum antibodies from vaccinated to naive animals [15], indicating that protection is predominantly mediated by serum neutralizing antibodies. Clinical trials also showed that the neutralizing antibody kinetics were associated with the long-term protection efficacy of Cervarix [16], indicating that the potency and persistence of the neutralizing antibodies are crucial for long-term protection against HPV infection. Long-term neutralizing antibody responses induced by HPV 16 and 18 L1 VLPs have been investigated in clinical trials [16, 17], but long-lasting neutralizing antibodies induced by HPV 58 L1 VLPs have not been reported. We previously reported that high-dose immunization with HPV 58 L1 VLPs (5 lg/injection) purified by CsCl ultracentrifugation elicited long-lasting VLP-reactive antibodies by VLP-based ELISA [6]. VLP-based ELISA detects the total VLP-reactive IgG, including reactive neutralizing antibodies and reactive non-neutralizing antibodies. Pastrana et al. found that sera from an HPV 16 L1 VLP vaccinee reacted with HPV 18 L1 VLPs at a relative high titer (2560) by VLP-based ELISA, but they were unable to neutralize HPV 18 pseudovirions [18]. Our preliminary data showed that pre-incubation sera from mice immunized with HPV 16 L1 VLPs with HPV 18 L1 VLPs resulted in a 28.6 % decline in serum IgG titers against HPV 16, while

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pre-incubation with HPV 58 L1 VLPs reduced 35.7 % in serum IgG titers against HPV 16, but the same sera could not neutralize HPV 18 or HPV 58 pseudovirions (submitted for publication). The above results indicated that crossreactive and non-neutralizing antibodies contribute to a substantial portion of the total VLP-reactive IgG antibodies measured by VLP-based ELISA, and the magnitude of these cross-reactive non-neutralizing antibodies will become greater as the constitutive number of L1 VLP types increases. So far, the correlation between the VLP-based ELISA and the pseudovirus neutralizing assay for measuring the protective antibody response induced by trivalent or even highly multivalent L1 VLP vaccine has not been reported [19, 20]. In the present study, we extended these studies and demonstrated that low-dosage immunization with HPV 58 L1 VLPs (0.1 lg/injection) purified from Sf9 cells by two-step chromatography can elicit high levels of long-lasting type-specific neutralizing antibody in mice, as determined by the pseudovirus neutralization assay, ‘the gold standard’ for assessing the protective potential of HPV vaccines as recommended by WHO [19]. Therefore, our study not only provides a good method of large-scale HPV VLPs production but also strongly suggests that the highly immunogenic HPV 58 L1 VLPs are a good candidate for developing effective vaccines against HPV 58 infection, which is frequently detected in cervical cancers in Eastern Asia. Acknowledgments We thank Dr. Yufei Xu (Harvard Medical School, Boston) for critical reading of the manuscript. We thank Prof. John T. Schiller (National Cancer Institute, Maryland) for providing the 293TT cell line, p18sheLL plasmid and p52sheLL plasmid, Prof. Tadahito Kanda (National Institute of Infectious Diseases, Tokyo) for p58sheLL plasmid, Prof. Martin Mu¨ller (Deutsches Krebsforschungszentrum, Heidelberg) for p16sheLL plasmid, and Prof. Neil D. Christensen (Pennsylvania State University College of Medicine, Hershey) for mAbs H16.V5 and H18.J4. This research was supported by the Key Program of National High Technology Research and Development Program of China (863 Program, No. 2007AA215181), Natural Science Foundation of China (No. 31070813), and Natural Science Foundation of Beijing (No. 7102110).

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