18 L1 Bi VLPs in Vitro - ACS

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Controlled Hybrid-Assembly of HPV16/18 L1 Bi VLPs in Vitro Shi Jin,† Dong-Dong Zheng,† Bo Sun,‡ Xianghui Yu,‡ Xiao Zha,§ Yongjiang Liu,∥ Shuming Wu,∥ and Yuqing Wu*,† †

State Key Laboratory of Supramolecular Structure and Materials, and ‡The State Engineering Laboratory of AIDS Vaccine, Jilin University, No. 2699, Qianjin Street, Changchun 130012, China § Sichuan Tumor Hospital & Institute, Chengdu 610041, China ∥ Beijing Health Guard Inc., Beijing 100176, China S Supporting Information *

ABSTRACT: Based on the helix4-exchanged HPV16 L1 and HPV18 L1, HPV16 L1 Bi and HPV18 L1 Bi, we have successfully realized the controlled hybrid-assembly of HPV16/18 L1 Bi VLPs (bihybrid-VLPs) in vitro. The bihybrid-VLPs were further confirmed by fluorescence resonance energy transfer (FRET) and complex-immunoprecipitation (Co-IP) assays. The ratio of 16 L1 Bi and 18 L1 Bi in bihybrid-VLPs was verified to be 3:5 based on a modified magnetic Co-IP procedure, when mixing 1 equiv pentamer in assembly buffer solution, but it changed with conditions. In addition, the bihybrid-VLPs showed identical thermal stability as that of normal VLPs, suggesting high potential in practical applications. The present study is significant because it modified one of the vital steps of virus life cycle at the stage of virus assembly, supplying a new approach not only to deepen structural insights but also a possibility to prepare stable, low-cost, bivalent antivirus vaccine. Furthermore, the controlled hybrid-assembly of bihybrid-VLPs in vitro provides suggestions for the design of effective multivalent hybrid-VLPs, being a potential to develop broad-spectrum vaccines for the prevention of infection with multiple types of HPV. KEYWORDS: human papillomavirus (HPV), capsid protein L1, virus like particles (VLPs), controlled hybrid-assembly, bihybrid-VLPs, in vitro

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INTRODUCTION Cervical cancer is the second most common cause of death among women worldwide.1 Almost all cases of cervical cancer result from persistent infection of high-risk types of human papillomavirus (HPV),2−5 especially HPV16 and HPV18 predominate, accounting for about 50% and 20% of cervical cancer, respectively.6,7 HPV are small, nonenveloped viruses whose approximate 8 kb circular genome encodes two structural proteins, L1 and L2, plus several nonstructural proteins that are very important for the virus life cycles.8 The major capsid protein L1 alone, or together with the minor capsid protein L2, can self-assemble in vitro into empty virus like particles (VLPs) when being expressed in a recombinant expression system.9,10 The VLPs are structurally and immunologically similar to the native virions although lacking the potentially oncogenic viral genome, and they are able to induce high titers of neutralizing antibodies (IgG and IgA) and effectively protect animals11−13 and humans 14−18 from papillomavirus infections. Specifically, the licensed quadrivalent (HPV6/11/16/18 L1) VLP vaccine “Gardasil” (Merck) from yeast and the bivalent (HPV16/18 L1) VLP vaccine “Cervarix” (GlaxoSmithKline) from insect cells are both highly effective in protecting women against incident and persistent HPV infection. 19−22 In addition, the U.S. Food and Drug © 2016 American Chemical Society

Administration (FDA) has approved a nine-valent (HPV6/ 11/16/18/31/33/45/52/58 L1) VLP vaccine “Gardasil 9” (Merck) recently, which offers the potential to increase overall prevention of cervical cancer from approximately 70% to 90%.5,23−25 However, the recombinant protein vaccines are largely unavailable in developing countries as the costs of production, distribution, and storage of them are still too high. Furthermore, the incomplete type coverage is particularly problematic for developing countries because most do not have an effective screening program as an alternative to reduce cervical cancer risk from minor oncogenic types. Therefore, the development of an economically advantageous, broad-spectrum vaccine would be a prior and urgent need for the wide introduction of HPV vaccines into the developing countries. The native HPV virions and full-sized VLPs are essentially supramolecular structures; they consist of 72 L1 pentamers (also called capsomeres) that can be purified as a stable unit. The VLP is an icosahedral particle with T = 7 lattice in a diameter of approximately 55−60 nm.26,27 Cryo-electron microscopic analysis reveals that pentamers of T = 7 VLP Received: September 30, 2016 Accepted: November 25, 2016 Published: November 25, 2016 34244

DOI: 10.1021/acsami.6b12456 ACS Appl. Mater. Interfaces 2016, 8, 34244−34251

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ACS Applied Materials & Interfaces

Therefore, the controlled assembly of bihybrid-VLPs in this study provides suggestion for the design of effective multivalent hybrid-VLPs, being potential broad-spectrum vaccines to prevent the infection of multiple types of HPV.

may exist in two states, one forms contact with 6 neighbors as observed in the 60 hexavalent pentamers while the other with 5 as found in the 12 pentavalent pentamers.28 However, an Nterminally truncated L1 protein lacking 10 amino acids (aa) has been shown to assemble into particles consisting of 12 L1 pentamers with a T = 1 lattice referred to as a small VLP.29,30 Crystallographic analysis of T = 1 particles reveals that the interpentameric contacts are established by strong hydrophobic interactions between the α-helices 2 (h2) and 3 (h3) of one pentamer and the α-helix 4 (h4) of a neighboring pentamer.30 The deletion of h2 and h3 impedes even pentamer formation as a large fraction of the L1 protein is found to be insoluble in this case, which suggests an essential role for these regions in L1 folding and soluble expression of pentamers.29−31 Furthermore, the deletion of h4 in L1 forms homogeneous capsomeres but fails in T = 1 or T = 7 particles assembly,29,31,32 demonstrating that the h4 is indispensable for the VLPs formation. To date, L1 pentamers can be produced in large amounts from Escherichia coli (E. coli) and are considered to be very stable at room temperature.29,33,34 Therefore, to reduce the costs of production and storage of vaccine we chose E. coli as the expression system for the reconstructed HPV L1 proteins. In addition, numerous modifications of the L1 protein leading to the formation of pentamers but preventing capsid assembly have been described, such as the replacement of the cysteine residues that form capsid-stabilizing disulfide bonds35,36 or the deletion of h4.29,31,32 However, the influence on supramolecular structure of VLPs assembly has so far not been studied in depth. In the present study, we proposed to realize the controlled assembly of bihybrid-HPV16/18 L1 Bi VLPs (bihybrid-VLPs) by using the pentamers of the reconstructed HPV16 L1 and HPV18 L1 from E. coli. For that, the h4 as well as the related turn sequences in the wild types of HPV16 L1 and HPV18 L1 (16Wt and 18Wt) was exchanged for each other to produce HPV16 L1 Bi and HPV18 L1 Bi (Figure 1).

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RESULTS AND DISCUSSION 1. Design and Construction of HPV16 L1 Bi and HPV18 L1 Bi Used for Bihybrid-VLPs Assembly. The detailed inspection on the crystal structure of HPV16 L1 VLPs30 shows that the h2 and h3 of one pentamer in the projection domain form a V-shaped groove, into which fits the amino-terminal half of a longer helix (h4) from a neighboring pentamer. The contact areas are strongly hydrophobic, where residues L414, Y418, and V421 in h4 and F404 in pre-h4 form interpentamer packing through nonpolar interactions with the residues of V387, Y390 in h2 and I398, L399 in h3 (Figure 1A). Such hydrophobic interactions are crucially important for the correct folding and conformation keeping of each L1 monomer and pentamer because the tight packing expels water from the pentamer interface, which finally favors the oriented interaction of h2, h3, and h4. Especially, within a pentamer such hydrophobic bonds are repeated five times between monomers with the surrounding ones, together providing significant weak interaction to maintain the correct folding of L1 and VLP formation. In addition, the L1 mutant with h4-deletion formed homogeneous pentamers but failed to assemble into either T = 1 or T = 7 VLPs.29,31,32 These results demonstrated that h4 was indispensable for the VLPs formation. In order to achieve higher (at least not less) binding affinity between the pentamers of HPV16 L1 and HPV18 L1 than the individual ones, we exchanged their h4 and the related turn sequences (in total 40 amino acids) to keep the correct folding and extending orientation of h4 even after exchange, and to keep its interaction with h2 and h3 of the same subtype finally to assemble into hybrid HPV16/18 L1 Bi VLPs. In addition, with C-terminal deletion of 29 amino acids (aa) was generated, as preliminary data suggested a positive effect of this deletion on protein solubility, and truncations of the C-terminus of L1 were tolerated for up to 34 aa without affecting pentamer formation;41 the residues 406-445 of HPV16 L1 were exchanged with those of 407-446 in HPV18 L1 (Figure 1B, C). The reconstructed HPV L1 proteins were referred to as HPV16 L1 Bi (HPV16 L1 BiΔN4h418ΔC29, 16 Bi) and HPV18 L1 Bi (HPV18 L1 BiΔN4h4 16 ΔC29, 18 Bi), respectively. In parallel, the wild-type proteins were referred to as HPV16 L1 (HPV16 L1ΔN4ΔC29, 16Wt) and HPV18 L1 (HPV18 L1ΔN4ΔC29, 18Wt), respectively, for comparison. 2. Expression and Purification of the Reconstructed HPV16 L1 Bi and HPV18 L1 Bi. The HPV L1 mutants of 16 and 18 Bi were constructed, expressed, and purified as GST fusion protein, GST-L1. The purification of the L1 from the cell lysates was performed using GST affinity chromatography and size-exclusion chromatography (SEC). The proteins from the supernatant of the lysates were loaded onto a glutathionesepharose 4B column. After washing with 10× bed volumes of buffer A (3.2 mM NaH2PO4, 16.8 mM Na2HPO4, 200 mM NaCl, 1 mM DTT, 1 mM EDTA, 10 mM PMSF, pH 7.5), large quantities of GST-L1 were retained on the column, later showing a 79 kDa band in SDS-PAGE for GST-L1 (Figure S1). The PreScission Protease (PPase) digestion of the resinbinding GST-L1 released L1 from the GST column, and the soluble 16 and 18 Bi were pinpointed with arrow at the bands of 53 kDa (Figure S1, lanes 6 and 13, respectively),

Figure 1. Structure-based exchange for the h4 and its related turn sequences between HPV16 L1 and HPV18 L1, to realize the hybridassembly of bi-HPV16/18 L1 Bi VLPs. (A) Helix−helix interactions between two pentamers in the T = 1 HPV16 L1 VLPs. The h4 of one pentamer (in yellow) interacts with h2 and h3 of another pentamer (in cyan). (B) Diagram showing the design of the h4-exchanged between HPV16 L1 and HPV18 L1 to produce HPV16 L1 Bi and HPV18 L1 Bi. (C) Sequence alignment of HPV16 L1 and HPV18 L1; those in square frame were exchanged between them.

The results demonstrated that the controlled assembly of bihybrid-VLPs has been successfully realized, being further confirmed by fluorescence resonance energy transfer (FRET) and complex-immunoprecipitation (Co-IP) assays and supplying a new approach to the reports on revealing the protein-cage structures.37−40 In addition, the thermal stability of the bihybrid-VLPs was proven to be similar to that of individual VLPs, suggesting high potential in practical applications. 34245


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size obtained independently from DLS might be a little bit different from the crystal size as the effects of conformation and/or hydration of protein, etc.44 Then the TEM images (Figure S3A, B) confirmed the sizes of pentamers observed in DLS with uniform size and morphology (10 nm donutlike structure), being very close to the previously reported L1 pentamers.29,34 These results indicated that the h4-exchange between 16Wt and 18Wt did not affect the pentamers formation. Afterward, in order to confirm the correct folding of the reconstructed 16 and 18 Bi after expression, as well as to compare the secondary structural changes between them and 16Wt-P and 18Wt-P, respectively, the far-UV CD spectrum was measured (Figure S4). The quantitative analysis on the spectra revealed that the contents of α-helix, β-sheet, β-turn were 13.20%, 36.60%, 22.20% for 16Wt-P and 12.70%, 37.80%, 22.80% for 18Wt-P, respectively, being close to the revealed structural features of L1-P in crystal.32 In addition, the contents of α-helix, β-sheet, β-turn were evaluated to be 12.10%, 38.60%, 23.60% for 16 Bi-P and 11.90%, 39.40%, 24.50% for 18 Bi-P, respectively. Albeit they were somewhat different from those of 16Wt-P and 18Wt-P, which truly confirmed both 16 Bi-P and 18 Bi-P folded correctly even after h4-exchange. In the following performed UV CP-temp analyses (Figure 3 and

corresponding to the molecular weight of the target L1 monomer. The L1 proteins eluted from the GST column were further purified and analyzed by using SEC on Superdex-200 (16/60 column) in buffer B (3.2 mM NaH2PO4, 16.8 mM Na2HPO4, 200 mM NaCl, pH 7.5), and the obtained FPLC elution profile is presented in Figure 2. Similar as in an earlier report,42 three

Figure 2. FPLC elution profiles of HPV16 L1 Bi and HPV18 L1 Bi. Western-blot (top) of 16 Bi-P and 18 Bi-P probed with the typespecific HPV16 L1 and HPV18 L1 polyclonal antibody (16PAb and 18PAb), respectively. Lanes 1−3 :16PAb; lanes 4−6: 18PAb. Lanes 1 and 4: 16 Bi-P; lanes 2 and 5: standard proteins; lanes 3 and 6: 18 BiP.

proteins such as ferritin (440 kDa), bovine serum albumin (66 kDa), and ovalbumin (45 kDa) were chosen as standard molecules. Both 16 and 18 Bi L1 showed two peaks as the target protein with elution positions at ∼265 and ∼53 kDa, corresponding to the molecular weight of a L1 pentamer (Bi− P, major peak) and a monomer (Bi-M, minor peak), respectively. The lower monomer production for HPV18 L1 Bi than that of HPV16 L1 Bi was exactly in parallel to those obtained between HPV18 L1Wt and HPV16 L1Wt43 because of an unknown reason. The L1 obtained from the peak positions of pentamer (referred as 16 Bi-P and 18 Bi-P, respectively) were detected by western-blot with the polyclonal antibody of HPV16 L1 (16PAb) and HPV18 L1 (18PAb), respectively (top panel of Figure 2). The 18 Bi-P protein did not show a band in lane 3 (Figure 2, left-hand top panel) and neither did the 16 Bi-P protein in lane 4 (Figure 2, right-hand top panel), indicating the 16PAb only specifically recognized 16L1 but no cross-reactivity with 18L1, and vice versa. In addition, part of the protein was eluded in void as large aggregates because the molecular weight was beyond the resolution of this size-exclusion column. A recent study showed that the proteins from the monomer peak could not assemble into pentamer further, possibly because they existed as partially misfolded, metastable states.43 3. Characterization of the Pentamers of HPV16 L1 Bi and HPV18 L1 Bi. The results of dynamic light scattering (DLS) provided the size distribution of pentamers, confirming the correct quaternary structure of them. The column bar of the hydrodynamic diameter distributions (Figure S2A, B) indicated the average size of 13.65 and 12.32 nm for 16 Bi-P and 18 Bi-P, respectively, being close to the diameter expected for pentamers.28,29,31 It was worth noting that the hydrodynamic

Figure 3. Thermal stability of 16Wt-P, 18Wt-P, 16 Bi-P, and 18 Bi-P and the corresponding VLPs, as determined by UV cloud point with temperature ramping.

Table S1), 16Wt-P, 18Wt-P, 16 Bi-P, and 18 Bi-P exhibited Tm values at 53.2, 53.9, 51.9, and 52.5 °C, respectively, only slight weaker thermal stability of mutants being shown than the wildtypes. Then the integrity and stability of L1 pentamers were checked by trypsin digestion (Figure S5A, B), being very close to those previously reported for wild-type pentamers.29,45 Combining the results of DLS, TEM, CD spectra, UV CPtemp, and trypsin digestion together, it indicated that both 16 Bi-P and 18 Bi-P could form pentamer well with proper size and stability, albeit very slight secondary structural variations were revealed from the wild-types. 4. Hybrid-Assembly and Characterization of BihybridVLPs in Vitro. 4.1. Fluorescence Resonance Energy Transfer (FRET) between Cy3−16 Bi-P and Cy5−18 Bi-P. Fluorescence resonance energy transfer (FRET) is a distance-dependent spectroscopic techanique.46−49 To assay the hybrid-assembly of Cy3−16 Bi-P and Cy5−18 Bi-P (1:1), FRET between them were detected by time-dependent fluorescence (Figure S6B). The gradual quenching of Cy3 at 566 nm was accompanied by a band appearance and gradual increase at 666 nm for Cy5, 34246


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suggesting more 18 Bi-P might be involved in a 16/18 Bi VLP than 16 Bi-P. In addition, the Tm value of 16 Bi VLPs was relatively lower than those of 16/18 Bi VLPs and 18 Bi VLPs, and from another aspect it indicated 16/18 Bi VLPs had closer structural properties to 18 Bi VLPs. Furthermore, the differences of Tm values (Figure 3) reasonably indicated that all the capsid structure of VLPs were much more stable than those of pentamers, in showing approximately 10 °C higher Tm. Meanwhile, the integrity and stability of HPV VLPs were further proved by trypsin digestion. After 2 h incubation with trypsin the VLPs appeared intact (Figure S5C), indicating they were stable enough to resist the trypsin digestion. 4.3. SLS Monitoring on the Assembly Processes of VLPs in Vitro. The process of the hybrid-assembling of 16 Bi-P and 18 Bi-P into VLPs was monitored by time-dependent static light scattering (SLS) under constant stirring (black line in Figure S9), which increased slowly for the first 10 h but then was followed by a quick increase where the capsid-like structures should be largely formed. The fluorescence scattering intensity continuously increased with time and reached a plateau after 23 h. When performing the assembly for each Bi-P alone at identical conditions, the fluorescence scattering intensities of 16 Bi-P increased at almost the same rate as the mixture for the first 10 h, but it slowed down in the following, different from the intensity of 18 Bi-P which increased fast with time and reached plateau quickly. The differences in SLS plots indicated all of them could assemble into VLPs but in different kinetic ways. Especially, the large difference in kinetics plots between them reflected the intrinsic structure-dependence of assembly. For the first 10 h, the assembly rate of the mixture of 16 Bi-P and 18 Bi-P (1:1) was similar to that of 16 Bi-P, indicating they had similar assembly kinetics where the interpentamers interactions of 16 Bi might be predominant; Subsequently, the assembly rate of the mixture of 16 Bi-P and 18 Bi-P (1:1) fell in between those of 16 Bi-P and sole 18 Bi-P, reflecting that the different assembly kinetics and the interactions between 16 Bi-P and 18 Bi-P might be predominated in the 16/18 Bi VLPs. These observations again suggested that 16 Bi-P and 18 Bi-P might be assembled into a hybrid 16/18 Bi VLP; however, we could not eliminate the possibility to produce the mixture of 16 Bi VLPs and 18 Bi VLPs. That is, the mixture of 16 Bi-P and 18 Bi-P could assemble into three kinds of VLPs, 16 Bi VLPs, 18 Bi VLPs, and 16/18 Bi VLPs. 5. Purification of Bihybrid-HPV16/18 L1 Bi VLPs in Vitro. 5.1. Separation of VLPs by Sucrose Gradient Sedimentation. After VLPs assembly and sucrose gradient sedimentation assays, each fraction was analyzed by westernblot (Figure 4A, B). All L1 proteins were found to appear mainly in two groups separated in fractions 3−6 and 10−12, corresponding to a large number of VLPs and a small amount of pentamers, respectively. Further analysis by TEM images (Figure 4C, D) revealed the L1 in fractions 3−6 were essentially VLPs, which showed uniform size and morphology as spherical particles with diameters between 40 and 60 nm, while the L1 in fractions 10−12 were homogeneous pentamers with the diameter of approximate 10 nm. The proteins corresponding to fractions 3−6 were collected, eliminating the possibility and/or interference of the aggregates and pentamers. 5.2. Classic Magnetic Complex-Immunoprecipitation (CoIP) Assay To Purify Bihybrid-HPV16/18 L1 Bi VLPs. After the monitoring of 16 Bi-P and 18 Bi-P assembly in vitro, to substantiate the formation of 16/18 Bi VLPs and eliminate the

which should be attributed to the progressive assembly of VLPs with time. Due to a significant energy loss of Cy3 while only part of it was transferred to Cy5, therefore, the intensity changes between them are obviously different. In addition, when the molar ratio of Cy5−18 Bi-P to Cy3−16 Bi-P was varied from 0.5:1 to 2:1, the peak intensity at 666 nm for Cy5 increased (Figure S6A−C). The intensity ratio of Cy5 to Cy3 (I666/I566) correlated almost linearly with the protein ratio (Figure S7), confirming the composition changes in bihybridVLPs. It suggested the incorporation of different amount of 16 Bi-P and 18 Bi-P to the buffer solution changed the assembly ratio of them in bihybrid-VLPs. Meanwhile, as negative control when fixing the excitation wavelength (λex) at 535 nm for Cy3− 16 Bi-P alone, the fluorescence intensity of it did not change with time, while the fixing of λex at 645 nm for Cy5−18 Bi-P alone or its mixture with Cy3−16 Bi-P (Figure S6D−F) led to no emission change either in a 24 h monitoring. The results indicated the FRET indeed occurred between Cy3−16 Bi-P and Cy5−18 Bi-P in the assembly, which suggested 16 Bi-P and 18 Bi-P were assembled into a VLP. 4.2. Characterization of Bihybrid-HPV16/18 L1 Bi VLPs in Vitro. After the assembly in buffer C (43.8 mM NaH2PO4, 6.2 mM Na2HPO4, 1 M NaCl, 2 mM CaCl2, 2 mM MgCl2, 0.03% Tween 80, pH 6.0), the average sizes of particles were evaluated to be 51.68, 52.90, and 51.25 nm (Figure S2C−F), corresponding to the VLPs of 16/18 Bi, 16 L1 Bi, and 18 L1 Bi, respectively. Such results were in good consistency with the expected size for the native virus and the previously reported ones for HPV (55−60 nm).26−28 The TEM images in Figure S3C−E and the size distribution in Figure S8 clearly showed the presence of assembled VLPs with irregular shape, broad size distributed between 40 and 70 nm in diameter, but no aggregation. Comparing the shape and size distribution of 16 Bi VLPs and 18 Bi VLPs, those of 16/18 Bi VLPs were even more regular. The results indicated the pentamers have been fully assembled into T = 7 capsids with 72 pentamers. Based on the TEM images, the mean diameters of VLPs for 16 Bi, 18 Bi, hybrid 16/18 Bi were evaluated to be 50.86, 49.02, and 49.54 nm, respectively (Figure S8), being reasonably slightly smaller than those obtained by DLS (Figure S2). Such results matched more closely to the expected size of HPV VLPs than the reported ones produced from Saccharomyces cerevisiae, which showed bigger size of 110−120 nm accompanying also with partial aggregation.50 In addition, the TEM images of 16/18 Bi VLPs in Figure S3C showed more uniform size distribution than those of sole 16 Bi VLPs or 18 Bi VLPs (Figure S3D, S3E), indicating most of VLPs there (Figure S3C) were different from those of sole 16 Bi VLPs or 18 Bi VLPs. Therefore, we hypothesized that 16 Bi-P and 18 Bi-P were hybrid-assembled into a VLP, being predominated in the assembled VLPs that possibly contained 16 Bi VLPs, 18 Bi VLPs as well as 16/18 Bi VLPs together. Afterward, the thermal stability of VLPs was revealed by using UV CP-temp analyses (Figure 3 and Table S1), where the 16 Bi VLPs, 18 Bi VLPs, and 16/18 Bi VLPs at three different molar ratios of 16 Bi-P to 18 Bi-P exhibited Tm values at 61.2, 62.6, 62.0, 62.3, and 62.4 °C, respectively. It is interesting to observe that the obtained VLPs from five different conditions showed very similar transition plots. Of note, albeit the different molar ratios of 16 Bi-P to 18 Bi-P in solution changed the composition of bihybrid VLPs, they had only slight influence on the thermal stability of 16/18 Bi VLPs. The 16/18 Bi VLPs showed closer Tm values to 18 Bi VLPs than 16 Bi VLPs, 34247


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turn, and the proteins obtained finally from the elution were detected by western-blot (Figure 6A, B). The Clean-Blot IP Detection Reagent as the secondary antibody could detect only the native antibody in western-blot, which therefore provided accurate and specific detection of 16/18 Bi VLPs without interference of denatured 16PAb and 18PAb from Co-IP assay. Finally, both HPV16 L1 and HPV18 L1 were verified in the elution, which absolutely confirmed we obtained pure 16/18 Bi VLPs in this way. In addition, the band ratio of 16B-P and 18BP was estimated approximately to be 3:5 based on the gray in western-blot as evaluated by UVP Bioimaging system of labworks software. Furthermore, the size of 16/18 Bi VLPs was validated by DLS (Figure 6C), confirming the capsid-like structure of VLP with the average size of 52.65 nm. When the hybrid assembly was performed at a molar ratio of 2:1 for 16 Bi-P to 18 Bi-P, the similar treatment resulted in a band ratio of 5:7 for 16 Bi-P to 18 Bi-P in bihybrid-VLPs finally (Figure S10). It indicated strong influence of molar ratio between 16 Bi-P to 18 Bi-P on the composition of bihybridVLPs. Such a conclusion was further supported by FRET, which were performed on the hybrid assembly of 16 Bi-P and 18 Bi-P in different molar ratios (Figure S6A, C). The fluorescence intensity ratio of Cy5 to Cy3 (I666/I566) versus the molar ratio of Cy5−18 Bi-P to Cy3−16 Bi-P varied linearly from 0.5:1 to 2:1 (Figure S7), confirming the composition changes of bihybrid-VLPs at different conditions. Finally, both the FRET plots performed on the dye-labeled wild-type protein pentamer, Cy3−16Wt-P and Cy5−18Wt-P (Figure S11) and the western-blot obtained from the classic magnetic Co-IP assay (Figure S12) denied the possibility of coassembly between 16Wt-P and 18Wt-P without h4 exchange. Such results confirmed that the controlled hybrid-assembly of HPV16/18 L1 Bi VLPs in vitro indeed originated from the exchange of key segment between HPV 16 and 18 L1. Comparing the 17-aa sequence in h4 of HPV16 L1 and 18 L1, they are indeed highly conserved to each other. Among them the hydrophobic residues of L414, Y418, and V421 in h4 of HPV16 L1, involving the hydrophobic interactions with h2 and

Figure 4. Analysis on VLPs assembly. The mixture of 16 Bi-P and 18 Bi-P was dialyzed against assembly buffer , and subsequently analyzed by sucrose gradient sedimentation analysis with western-blot (A, B) and TEM images employing negative staining (C, D). Western blot probed with the 16PAb (A) and 18PAb (B), respectively.

effects of 16 Bi VLPs and 18 Bi VLPs involved, we purified and assayed the bihybrid-VLPs by classic magnetic Co-IP by using linear-dependent type-specific HPV16 L1 and HPV18 L1 polyclonal antibody (16PAb and 18PAb). The diagram of classic magnetic Co-IP procedure is shown in Figure 5. The magnetic separation avoided centrifugation which might break weak binding of antibody−antigen and cause partial loss of target protein. Therefore, here we replaced the protein A/G beads resin with protein A/G magnetic beads resin to improve the purification efficiency of 16/18 Bi VLPs. The mixture solution of VLPs, which might include 16 Bi VLPs, 18 Bi VLPs, and 16/18 Bi VLPs, went through 16PAb and 18PAb resin in

Figure 5. Diagram of the modified magnetic complex-immunoprecipitation (Co-IP) procedure. The Fc region of the HPV L1 polyclonal antibody is bound to Protein A/G magnetic beads. Unbound components were washed away, and then HPV L1 polyclonal antibodies and HPV L1 proteins were eluted with the buffer G that disrupted the binding interactions. 34248


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Figure 6. Western-blot of proteins obtained from the elution of classic magnetic Co-IP as detected with (A) 16PAb and (B) 18PAb, respectively. (C) Hydrodynamic diameter distribution of VLPs as determined by DLS measurements.

Finally, the pentamer ratio of HPV16 L1 Bi and HPV18 L1 Bi was verified to be 3:5 based on a modified magnetic Co-IP procedure when mixing 1 equiv pentamer in assembly buffer solution, and it changed with condition. The present study supplies a new approach to deep structural insights and to prepare stable, low-cost, bivalent antivirus vaccine, which provides suggestion for the design of effective multivalent hybrid-VLPs and/or broad-spectrum vaccines for the prevention of infection with multiple types of HPV.

h3, are highly conserved in HPV18 L1. It demonstrated that even after exchanging the h4 between 16Wt and 18Wt the left five-different aa might not affect much the hydrophobic interaction. Therefore, considering the turn-related sequences (residues 403−413) in pre-h4 acting as a flexible hinge or adapter and bridging the gaps between donor and acceptor pentamer; while the turn-related sequences (residues 430− 446) in post-h4 extend around the circumference of the target pentamer, linking h4 with the C-terminal segment,30 both of them were also considered to be exchanged between 16Wt and 18Wt. Therefore, the results confirmed that both the h4 and the related two turn sequences (in total 40 amino acids) are crucially important in the hybrid-assembly between HPV16 L1 Bi-P and 18 L1 Bi-P. In addition, the current issued multivalent vaccines are essentially the mixture of capsid proteins.5,19−22 The immune interference was often observed when comparing the typespecific neutralizing antibody levels induced by multivalent vaccine to those by corresponding monovalent vaccines. This kind of interference would become more obvious when formulating more subtypes of VLPs into multivalent vaccines. Increasing the concentration of one L1 protein could reduce immune interference on it, but it increases immune interference on other types of coimmunized L1 proteins.51 Meanwhile, with increase of the concentration of each VLP component, the stability of the vaccine would be decreased while the cost of HPV vaccine would also be increased. Furthermore, in comparing with the direct mixing of VLPs from type 16 and 18, bihybrid-VLP induced similar titer antibodies of types 16 and 18 with a relatively low concentration of VLP. That is, hybrid assembly of HPV L16 and L18 decreased both the concentration of VLPs and the immune interference, and consequently would reduce the cost of HPV vaccine in future. Therefore, the controlled assembly of bihybrid-VLPs in vitro provided the possibility to design effective, multivalent, broadspectrum vaccine.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b12456. Experimental details and additional analyses data (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +86-431-85168730. Fax: +86431-85193421. ORCID

Yuqing Wu: 0000-0003-4883-5982 Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding

The projects of the National Natural Science Foundation of China (No. 91027027 and 21373101). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We greatly appreciate the financial support from the projects of the National Natural Science Foundation of China (No. 91027027 and 21373101) and the State Key Laboratory for Supramolecular Structure and Materials, Jilin University.

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CONCLUSION By using the pentamers of reconstructed proteins of HPV16 L1 Bi and HPV18 L1 Bi from E. coli, we have successfully realized the controlled hybrid-assembly of HPV16/18 L1 Bi VLPs in vitro, which is significant because it is the first report on the hybrid-assembly of HPV VLPs. The assembly of bihybrid-VLPs was further confirmed by FRET and Co-IP assays. Both UV CP-temp and trypsin digestion assays together showed very close thermal stability of bihybrid-VLPs to that of each sole VLPs, suggesting high potential in practical applications.

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REFERENCES

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