Human papillomavirus 16L1-58L2 chimeric virus-like particles elicit durable neutralizing antibody responses against a broad-spectrum of human papillomavirus types

The neutralizing antibodies elicited by human papillomavirus (HPV) major capsid protein L1 virus-like particle (VLP)-based vaccines are largely type-specific. An HPV vaccine inducing cross-neutralizing antibodies broadly will be cost-effective and of great value. To this end, we constructed HPV16L1-58L2 chimeric VLP (cVLP) by displaying HPV58 L2 aa.16-37 on the DE surface region of HPV16 L1. We found that vaccination with the HPV16L1-58L2 cVLP formulated with alum plus monophosphoryl lipid A (Alum-MPL) adjuvant elicited robust neutralizing antibodies in both mice and rabbits against all tested HPV types including HPV16/31/33/35/52/58 (genus α9), HPV18/39/45/59/68 (genus α7), HPV6/11 (genus α10), HPV2/27/57 (genus α4), and HPV5 (genus β1). Importantly, the cross-neutralizing antibody response was maintained at least 82 weeks in mice or 42 weeks in rabbits, and complete protection against HPV58 was observed at week 85 in mice. Our data demonstrate that HPV16L1-58L2 cVLP is an excellent pan-HPV vaccine candidate.

Three available HPV L1 VLP-based prophylactic vaccines, bivalent Cervarix (contains HPV16/18 VLPs), quadrivalent Gardasil (contains HPV16/18/6/11 VLPs), and nonavalent  (contains HPV16/18/31/33/45/52/58/6/11 VLPs), have shown to provide protection against vaccine types [12][13][14][15][16]. There is also evidence of limited cross-protection against HPV31/33/45 with Cervarix or against HPV31 with , but the strength and duration of cross- neutralizing antibody responses are lower and shorter than that of the vaccine types [19][20][21][22]. While the nonavalent vaccine provides broader protection against oncogenic HPV infections and infection-related precancerous lesions, it still does not cover the cutaneous HPV types. Moreover, continuing to add more and more types of VLPs in a vaccine also raises the complexity and cost of production. High cost remains the primary challenge to global implementation of HPV vaccines, especially in the developing countries where nearly 90% of cervical cancer deaths occur [23,24].
An alternative approach to fill in the gap between cross-protection and cost is focused on developing a vaccine using the minor capsid protein L2. Vaccination with the N-terminus of L2 induces cross-neutralizing antibodies, but the antibody titers against the homologous HPVs are lower than that induced by L1 VLPs [25][26][27]. Approaches to boost the immunogenicity of L2 include multimeric display of epitopes on surface region of VLPs from papillomavirus [28][29][30], adeno-associated virus [31], tobacco mosaic virus [32], or on surface region of recombinant bacteriophage [33,34], and delivery of epitopes with FcγR-targeting scaffolds [35,36], bacterial thioredoxin or flagellin scaffold [37,38]. Immunization of KLH-conjugated HPV16 L2 aa.17-36 peptide elicited cross-neutralizing antibodies [39]. The homologous region of L2 aa.17-36 peptide (usually termed RG1 epitope) derived from HPV16, 33 or 45 has been inserted into the DE loop of HPV16 or HPV18 L1VLP to create L1-L2 chimeric VLP vaccines, which have been demonstrated to induce various titers of cross-neutralizing antibodies with different spectrum [29,30,40].
HPV16 is the most common oncogenic type worldwide and accounts for approximately 60% of invasive cervical cancer (ICC) [41,42]. However, the prevalence of other types varies in different regions. For example, HPV58 and 52 are more prevalent oncogenic types in Eastern Asia (ranked third and fourth) than other regions [41][42][43][44][45].
Because HPV58 is highly prevalent in China, we have examined the potential of HPV58 L2 aa.15-37 ( Figure 1, 100% identity with HPV52) to induce cross- Figure 1: Homologous analysis of HPV58 L2 aa.15-37 peptide and comparable sequences from different HPV types. www.impactjournals.com/oncotarget neutralizing antibodies and have generated HPV16L1-58L2 cVLP vaccine by inserting HPV58 L2 aa.16-37 into the DE surface loop of HPV16 L1 VLP. We have found that the chimeric VLP vaccine could induce long-term cross-neutralizing immune responses against a broadspectrum of HPV types.

HPV58 L2 aa.15-37 peptide induces broadly neutralizing antibodies in rabbits
The success of HPV16 RG1 peptide in inducing cross-neutralizing antibody responses lead us to test if HPV58 (the third most prevalent high-risk mucosal type in Eastern Asia) and HPV6 (the most prevalent low-risk mucosal type worldwide) RG1 peptides are also able to do so. HPV58 and HPV6 RG1 peptides were synthesized and conjugated with KLH (termed KLH-58RG1 and KLH-6RG1, respectively). Two rabbits per group were immunized four times with KLH-58RG1 or KLH-6RG1 and sera were collected two weeks after the fourth immunization to determine the neutralizing antibody titers against 13 types of HPV pseudoviruses (PsVs).

Generation of HPV16L1-58L2 cVLPs
To potentially enhance and broaden crossneutralizing responses induced by the HPV58 RG1 peptide, we engineered an HPV16L1-58L2 chimeric protein by displaying HPV58 L2 aa.16-37 on the DE loop of HPV16 L1 and expressed it in baculovirus expression system. The HPV16L1-58L2 protein was highly expressed similar to HPV16L1 protein as analyzed by SDS-PAGE with coomassie blue staining ( Figure 2A). The HPV16L1-58L2 protein was confirmed by Western blot with Camvir-1 monoclonal antibody or polyclonal rabbit serum raised against HPV58 L2 aa.15-37 showing a strong migrating band at 50-55 kDa ( Figure 2B, 2C). After purification by ultracentrifugation, HPV16L1-58L2 protein was tested by SDS-PAGE with coomassie blue staining and a strong band was observed when 5 μg of each protein was loaded ( Figure 2D). The purity was about 90% as tested by HCP ELISA. Dynamic light scattering (DLS) analyses of purified HPV16L1-58L2 protein showed the uniform hydrodynamic diameter distribution with a mean polydispersity index of 0.211 and an average hydrodynamic diameter of 107.8 nm ( Figure 2E). Further transmission electron microscopy (TEM) analysis showed that HPV16L1-58L2 protein was assembled into fullsize VLPs (average diameter, 49 nm) with the unimodal distribution of particle diameter ( Figure 2F, 2G).

HPV16L1-58L2 cVLPs induced long-term protection in mice
As shown in Figure 5, the cross-neutralizing antibody response induced by HPV16L1-58L2 cVLPs was maintained at least 42 weeks in rabbits and 82 weeks in mice. The antibody titer threshold for protection has not been clearly identified, but antibody titers are more than 100-fold lower than the minimum titer detectable by standard PBNA are sufficient to confer protection against PsV genital infections in mouse model [46]. To evaluate the long-term protective response induced by HPV16L1-58L2 cVLPs, the immunized mice were vaginal challenged with HPV58 PsV at week 85. Although 2 of 5 neutralizing antibody titers against HPV58 were not detectable at week 82, complete protection was still conferred at week 85 ( Figure 6).

DISCUSSION
HPV L1 VLPs are highly immunogenic but induce mainly type-specific neutralizing antibodies. The recently approved Gardasil-9 contains HPV L1 VLPs from 7 types of high-risk HPVs and 2 types of low-risk HPVs. Thus, an HPV vaccine that can be produced cost-effectively and covers a broader spectrum of HPV types than Gardasil-9 is desired. In this study, we used HPV16 L1 VLP as the backbone to display HPV58 L2 aa.16-37 into HPV16 L1 DE loop. We found that the chimeric VLPs induced broadly neutralizing antibodies against over 17 HPV types. Thus, we believe that it is a promising candidate for a new generation of HPV vaccine.
Although RG1 sequences from different HPVs are relatively conserved (Figure 1), it remains unclear whether the RG1 peptide from other HPVs (except for HPV16) can induce strong and broad-spectrum neutralizing antibodies. Jagu et al. [26] showed that sera from mice immunized with a synthetic tandem repeat of L2 aa.17-36 derived from HPV58 and other 21 HPV types neutralized HPV16/18/45, but not HPV58/6. Tumban et al. [33] showed that mice vaccinated with a mixture containing 8 recombinant PP7 bacteriophages (a singlestrand RNA bacteriophage of Pseudomonas aeroginosa) each displaying one type of HPV L2 aa.17-31 (including HPV58), provided only partial in vivo protection against HPV58, and the neutralizing activity of the antisera was not known. To our knowledge, the immunogenicity of HPV58 RG1 peptide to elicit cross-neutralizing antibody responses is not well studied. Here we found that immunization of KLH-58RG1 peptide could induce cross-neutralizing antibody responses against all 13 tested HPV types with relatively high titers whereas KLH-6RG1 antisera neutralized only 10 HPVs with very low titers. These data indicated that the immunogenicity of any HPV type RG1 peptide is difficult to be empirically predicted.
As the immunogenicity of HPV58 RG1 is relatively high, we inserted HPV58 L2 aa.16-37 into the DE surface loop of HPV16 L1 VLP to generate HPV16L1-58L2 chimeric protein. The protein was highly expressed with good stability and efficiently self-assembled into VLPs (Figure 2), and induced potent broad-spectrum neutralizing antibody responses (Figures 3, 4). Contrarily, the HPV16L1-6L2 chimeric protein, which constructed by inserting HPV6 L2 aa.17-38 into the DE surface loop of HPV16 L1 VLP, was shown to be unstable, and induced much lower levels of antibody response even against HPV6 (not shown). There are three amino acid differences (86% homology) between HPV58 L2 aa.16-37 and HPV6 L2 aa.17-38, which may influence the interaction between the residues of inserted peptide and L1 backbone, and result in the differences in stability and immunogenicity of the two chimeric proteins. Therefore, it is important to optimize L2 peptides with good immunogenicity and select L1-L2 cVLPs with high stability for screening vaccine candidates.
The long lasting cross-neutralizing antibody responses are essential for HPV protection since HPV vaccines are mostly given to preadolescents, the protection should sustain a decade, at least from preadolescence through midlife. Previous studies have indicated that in vivo protection induced by either L1 VLP-or L2-based vaccine is mediated by neutralizing antibodies [48][49][50][51][52]. Schellenbacher et al. [40] determined the duration of neutralizing antibodies induced by 16L1-16RG1 VLP to 8 HPV PsVs and found that they could detect neutralizing antibodies to HPV16/18/31, but not to other 5 types, at week 52. Our data showed that HPV16L1-58L2 cVLP induced long lasting neutralizing antibody response, the neutralizing antibodies against 15 HPVs that were maintained at relatively high levels at week 42 in rabbits ( Figure 5A). It also induced durable neutralizing antibody response in mice up to week 82 ( Figure 5B), the longest neutralizing antibody response among the L2-based vaccines reported so far to our best knowledge.
Although increasing L1 VLP type in multivalent VLP vaccines can broaden the vaccine protection spectrum, it is not cost-effective since adding one VLP type mainly increases one protection type. We believe that formulating multivalent VLP vaccines with the HPV16L1-58L2 cVLP will induce much broader protection spectrum and similar level of HPV16 neutralizing antibody responses comparing to the vaccines formulated with HPV16 L1 VLP, since the HPV16L1-58L2 cVLP induce similar high titers of HPV16 neutralizing antibodies as HPV16 L1 VLP, and various titers of cross-neutralizing antibodies against a broad spectrum of HPVs.
In summary, our study demonstrate that the HPV16L1-58L2 cVLP is an attractive pan-HPV vaccine candidate to induce strong and durable cross-neutralizing antibodies against a broad-spectrum HPV types, representing a novel candidate for HPV vaccines.

Ethics statement
All animal work was done in accordance with the guidelines of the Institutional Animal Care and Use Committee of the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences, and all experimental protocols were approved by the Institutional Animal Care and Use Committee.

Generation of HPV16L1-58L2 chimeric protein
Our previous work showed that the optimized HPV16L1 delta C31 gene (accession number: GU556964) expressed HPV16 L1 VLP at high level in baculovirus expression system [53]. In this study HPV58 L2 aa.16-37 gene sequence was genetically engineered into the DE loop of the above HPV16 L1 between nt.408 and nt.412 (aa.136 and aa.138) with three point mutations in the flanking sequence (Y135G, A136P, N138P) by PCR strategy. The HPV16L1-58L2 construct was subcloned into pFastBac1 vector and expressed in the Bac-to-Bac baculovirus expression system according to the manufacturer's instructions (Invitrogen).
HPV16L1-58L2 chimeric proteins were purified by CsCl ultracentrifugation as described previously [53] with minor modifications. Briefly, the supernatant of cell lysate was purified by ultracentrifugation on 27% CsCl (w/w)-PBS density gradients for 20 h, then dialyzed the interest fraction in 0.5 M NaCl PBS at 4°C for 2-3 h and ultracentrifugation on 5% sucrose (w/w)/60% CsCl (w/w)-0.5 M NaCl PBS for 2.5 h and then on 27% CsCl (w/w)-0.5 M NaCl PBS density gradients for 20 h. VLPs were dialyzed against PBS at 4°C for 3 days. Cell lysates or purified proteins were analyzed by SDS-PAGE with coomassie blue staining or Western blot with Camvir-1 (MILLIPORE, MAB885) or polyclonal rabbit serum raised against HPV58 L2 aa.15-37.

Dynamic light scattering (DLS) and transmission electron microscopy (TEM)
DLS measurements were performed on a Malvern Zetasizer Nano ZS (Malvern). Samples were filtered through a 0.45 μm filter followed by equilibration to 25 °C. The hydrodynamic size was recorded as Z-average hydrodynamic diameter. All data reported here are the averages of three measurements of the same sample.
For TEM analysis, purified proteins were adsorbed on a carbon-coated grid for 1 minute, rinsed with distilled water, negatively stained with 1% uranyl acetate for 3 minutes and examined with a TEM-1400 electron microscope operating at 80 kV with a magnification of 80,000×.

Animal immunizations
New Zealand white (NZW) rabbits and BALB/c mice were purchased from the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences, and kept in the animal facility of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences.

Standard PsV-based neutralization assay (PBNA)
Standard PBNA was performed as described previously [35]. Every sample was analyzed in duplicate. Statistical significance was determined by two-tailed, unpaired t-test in Graphpad Prism 5. P values <0.05 were considered to be statistically significant.

CONFLICTS OF INTEREST
The authors declare no competing financial interests.