Rational design of a multi-valent human papillomavirus vaccine by capsomere-hybrid co-assembly of virus-like particles

The capsid of human papillomavirus (HPV) spontaneously arranges into a T = 7 icosahedral particle with 72 L1 pentameric capsomeres associating via disulfide bonds between Cys175 and Cys428. Here, we design a capsomere-hybrid virus-like particle (chVLP) to accommodate multiple types of L1 pentamers by the reciprocal assembly of single C175A and C428A L1 mutants, either of which alone encumbers L1 pentamer particle self-assembly. We show that co-assembly between any pair of C175A and C428A mutants across at least nine HPV genotypes occurs at a preferred equal molar stoichiometry, irrespective of the type or number of L1 sequences. A nine-valent chVLP vaccine—formed through the structural clustering of HPV epitopes—confers neutralization titers that are comparable with that of Gardasil 9 and elicits minor cross-neutralizing antibodies against some heterologous HPV types. These findings may pave the way for a new vaccine design that targets multiple pathogenic variants or cancer cells bearing diverse neoantigens.


Response to Reviewer Comments on the manuscript [NCOMMS-19-13881-T]:
We thank the two reviewers for recognizing the merit of our work and for their suggestions to improve our manuscript. We have fully addressed the comments with appropriate additional experiments and analyses. To facilitate the navigation of this document, we have copied the reviewers' comments verbatim in blue and typed our responses in black.
Summary of the revisions made, with new experimental data: (1) A nine-valent chVLP with the same composition as Gardasil-9 was generated and subjected to an immunogenicity assay in mice, as compared to the immune response of WT VLP mixture, Gardasil 9, Cervarix HPV vaccines. The neutralizing antibody titers against the nine types of homologous HPV (HPV 6, are comparable with that of Gardasil-9, with minor type-cross neutralization against some of other 11 heterologous HPV types. (2) The stability of a bivalent chVLP was characterized by a multi-faceted approach over a long-term incubation of 10 weeks at 4°C, 25°C and 37°C. We show that chVLPs and WT VLPs have comparable stability.

Reviewer #1
Comments to the Author Reviewer: The major claim is that chimeric HPV VLPs can induce neutralizing responses to multiple HPV types. The approach to produce the chimeric HPV VLPs is quite novel and different from the previous designs to produce HPV VLPs that induce neutralizing antibodies to multiple HPV types using HPV peptides from L2.
The chimeric HPV VLPs were well characterized in vitro and the in vivo study demonstrated that the induce neutralizing antibodies to HPV types included in the in the chimeric VLPs. These titers were not significantly different from native VLPs However, the cross neutralizing antibody responses were limited to a few types so that a pan vaccine design with chimeric HPV VLPs would need to include the most prevalent HPV types.
The quality of the writing including the precision of the description about HPV mechanism of infection and inaccurate terminology (eg hetero-type neutralizing antibodies) should be addressed. The value of the scientific finding are difficult to appreciate due to especially the way they are described in the abstract.

Response:
We thank the reviewer for the encouraging comments on the novelty of our approach. In relation to the neutralization titers elicited by the chVLPs-as also suggested by the Reviewer #2-we include the following text explanation and new data: "Finally, we generated a nine-valent chVLP according to the composition and dosage of L1s in the Gardasil-9 formulation, and measured the neutralizing antibody titers with 20 types of HPV PsVs-nine homologous types (HPV 6, and 11 heterologous ones (HPV 26,. Consistent with other multiple-type chVLPs ( Figure 5 and 7), the nine-valent chVLP showed good physiochemical and particle nature ( Figure S14) and elicited a high neutralizing antibody response against the nine homologous types of HPV similar to that of the WT VLP mixture and Gardasil-9, moreover, additionally induced some minor crossneutralizing antibody titers against heterologous HPV26, -35, -39, -53 and -59 in some of the mice ( Figure 8)." (Page 14, line 306-315).
The limited cross-neutralizing antibody response in terms of both the antibody titer and the type-breadth of the chVLP vaccine may indicate the requirement for a formulation with a stronger adjuvant for higher neutralizing antibody elicitation. In this regard, we toned down the significance of the cross neutralization of the chVLP throughout the manuscript, and rephrased the title from "Capsomere-hybrid human papillomavirus virus-like particles elicit heterotype neutralizing antibodies" to "Rational design of a multiple-valent human papillomavirus vaccine by capsomere-hybrid co-assembly of virus-like particles". Furthermore, we propose a possible strategy with regard to the pan-HPV vaccine design, as described in the Discussion, which is based on this study and our findings in previous work where we created a triple-type HPV vaccine candidate by loop-swapping of the immunogenic surface loops (Li Z, et al. Nature Commun 2018:9;5360): "Furthermore, we surmise that a combination of these two strategies might facilitate the development of a pan-HPV vaccine that could theoretically incorporate 216 types of HPV immunodominant epitopes into a single particle (i.e., 72 pentamers, each bearing triple-type immunogenic loop regions). The number of types covered in the chHPV design approximates the number of HPV genotypes identified to date." (Page 18, We believe that the chVLP rationale could pave the way for a new mode of vaccine design targeting multiple pathogenic variants or hyper-varied cancer cells. Please also refer to our response to Reviewer #2 general comment.
As pointed out, "hetero-type" is incorrect terminology and this has been rephrased as "heterologous type" throughout the manuscript. The abstract has been rewritten to reflect the scientific findings in our study:

"Abstract
The capsid of human papilloma virus (HPV) spontaneously arranges into a T=7 icosahedral particle with 72 L1 pentameric capsomeres associating via disulfide bonds between Cys175 and Cys428. Here, we designed a capsomere-hybrid virus-like particle (chVLP) to accommodate multiple types of L1 pentamers by the reciprocal assembly of single C175A and C428A L1 mutants, either of which alone encumbers L1 pentamer particle self-assembly. We show that coassembly between any pair of C175A and C428A mutants across at least nine homologous HPV genotypes occurs at a preferred equal molar stoichiometry, irrespective of the type or number of L1 sequences. A nine-valent chVLP vaccine-formed through the structural clustering of homologous HPV epitopes-confers neutralization titers that are comparable with that of Gardasil-9 and elicits minor cross-neutralizing antibodies against some heterologous HPV types. These findings may pave the way for a new vaccine design that targets multiple pathogenic variants or hyper-varied cancer cells." (Page 2, line 21-34).
Additional comments Comment 1: Line 45 in introduction incorrectly states that vaccines only induced type specific protection.

Response:
We apologize for this incorrect description. We have rephrased the sentence as: "…they induce mostly type-restricted neutralizing antibodies and have limited cross-protection against a few of the non-vaccine types." (Page 4, line 65-67). Please also refer to our responses to Reviewer #2, Comment 1.

Comment 2:
In vitro antigenicity work demonstrated the loss of some type specific mAb binding, but is not clear how the authors claim that "some new type-cross neutralization" (line 325).
Response: As suggested, we have revised the text as follows: "Moreover, few mAbs, such as chVLP-specific mAb 10C3 and anti-HPV59 mAb 13A6, only reacted with HPV16/52 bivalent chVLPs instead of their parentaltype VLPs ( Figure 6C), which suggests …"(Page 17, line 365-367) Comment 3: Despite the extensive in vitro evaluation there is no data on the stability of the chimeric VLPs, given ½ the disulfide bonds, vs non-chimeric VLPs.
Response: Indeed, stability is a key point of concern for translating chimeric VLPs into vaccines. We examined multiple attributes of HPV16L1-C175A-HPV52L1-C428A chVLPs during storage at 4°C, 25°C, and 37°C for up to 10 weeks, specifically assessing sequence integrity with SDS-PAGE and WB, thermal stability with DSC, and size distribution with HPSEC and AUC. We further confirmed morphology with TEM and the hydrodynamic radius with DLS. The WT HPV16 and HPV52 VLPs were stored and examined in parallel.
"Despite of ½ disulfide bonds formed, the chVLPs exhibited comparable stability with that of WT VLPs during storage at 4°C, 25°C, and 37°C for up to 10 weeks, as assessed via protein integrity with SDS-PAGE and western blotting ( Figure S4), thermal stability with DSC ( Figure S5 "To formulate the multiple-type HPV VLP vaccine, each VLP type was first individually absorbed to an aluminum hydroxide adjuvant, and then mixed together, resulting in a final amount of 0.42 mg aluminum hydroxide suspended in 0.5 or 1.0 mL solutions. For the multi-valent chVLPs, each chVLP was formulated with aluminum hydroxide adjuvant with an equivalent amount of L1 and aluminum hydroxide as that in the multiple-type HPV VLPs in 1.0 mL solutions. The commercial HPV vaccines, Gardasil-9 (Lot no. N023354) and Cevarix (Lot no. S007151) were purchased (Hong Kong) and diluted with aluminum hydroxide adjuvant solution, according to intended antigen amount, to serve as controls." (Page 26, line 585-593)

Response:
We have now included the statistical analyses, as follows: "The repeated neutralization titers were averaged to generate mean values and corresponding standard deviations (SDs). Non-neutralization samples were assigned a value of half of the limit of detection for visualization. Statistical significance was assessed by one-way ANOVA: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001." (Page 38, line 964-967) Comment 6: Not clear of the added value of Figure 6C: generation of mAb to ChVLPs ?
Response: To explore any potential variation in antigenicity upon chVLP assembly, we generated mAbs from mice immunized with chVLPs, and that found most neutralizing mAbs were type-specific, similar to their parental types. However, a few mAbs showed binding activities that varied between chVLPs and WT VLPs; for example, mAb 10C3 recognized only chVLPs. These findings suggest some variation in antigenicity in response to the reciprocal assembly of chVLPs. The sentences were rephrased as "Moreover, few mAbs, such as chVLP-specific mAb 10C3 and anti-HPV59 mAb 13A6, only reacted with HPV16/52 bivalent chVLPs instead of their parental-type VLPs ( Figure 6C), which suggests that the hybrid assembly may slightly alter the original antigenicity that would stem from the prototypic pentamers or VLPs. Hybridassembly might also create some type-cross neutralization sites on the interpentameric interface, given that the interface is constituted by the two stretches of L1 sequences from the two parental types via the reciprocal linkage of the unmutated cysteine residues. Interestingly, although the antigenicity of various chVLPs was altered, potent antibody elicitation for the prototypical neutralization was still maintained and generated some type-cross protection." (Page 17, line 365-374) Comment 7: Methodology for Figure S3 not very clear?

Response:
A detailed protocol for disulfide bond analysis has now been added to the Materials & Methods section: "The total or free sulfhydryl (SH) content of HPV16L1-C175A-HPV52L1-C428A chVLPs and WT VLPs was determined according to the method of Yongsawatdigul and Park. For the total SH content, samples were serially diluted to a final L1 concentration of 0.2, 0.4, 0.6, or 0.8 mg/mL with solubilizing buffer (0.086 M Tris-HCl, 0.09 M glycine, 0.04 M EDTA, 8 M urea, pH 8.0). For the free SH content, samples were diluted in the same manner but with urea-free solubilizing buffer (0.086 M Tris-HCl, 0.09 M glycine, 0.04 M EDTA, pH 8.0). Each diluted sample (5 mL) was then mixed with 50 μL Ellman's reagent (2 mM DTNB in 0.2 M Tris-HCl, pH 8.0), and the mixtures were incubated at 25°C for 40 min. The SH content was calculated from the absorbance measured at 412 nm using a molar extinction coefficient of 13,600 M −1 ·cm −1 . A simple linear regression model was applied to optimally fit the correlation between the concentration of VLPs and the absorbance values. The disulfide bond concentrations for chVLPs and WT VLPs were calculated using the equations Csh=73.53*(A/C) and Cdb=(Csht-Cshf)/2, where Csh is the concentration (μmol/g) of the SH content, A is the absorbance value at 412 nm, C is the concentration of the samples, Csht and Cshf are the concentration (μmol/g) of the total SH content and free SH content, respectively, and Cdb is the concentration (μmol/g) of the disulfide bond content of the samples. Each sample was analyzed in triplicate." (Page 23 , line 519-536)

Reviewer: 2
Comments to the Author Reviewer: This is an interesting study using a novel strategy generating capsomere-hybrid HPV L1 VLP to develop HPV vaccine candidates that confer broad-spectrum protection.
The authors rationally design single C175A or C428A L1 mutants that assemble into pentamers, but are deficient for VLP assembly. Mixing C175A and C428A L1 mutants of different HPV types in equimolar ratios hybrid VLP are generated that mimic wild-type L1 VLP morphologically and immunologically. Up to seven types are shown to co-assemble, and the authors propose a maximum of 72 types could co-assemble.
The hybrid VLP were extensively characterized by biophysical and biochemical means for particulate, structural characteristics, thermal stability. Although the HPV16-containing hybrid VLP retained the immunodominant neutralization epitope recognized by mAb V5, some hybrid VLP lost high-affinity binding sites to mAb.
In contrast to a mixture of VLP that induced antisera with neutralization of the included (homologous) VLP as expected, the hybrid VLP containing the same mixture of capsomers of HPV types were also highly immunogenic and in addition induced cross-neutralization to heterologous types (e.g. the heptatype hybrid VLP induced neutralization against 10 HPV types).
The authors claim to be able to develop an improved HPV vaccine with better cross-protection than licensed multivalent HPV vaccines. However, it remains unclear, if a multivalent combination of capsomers into hybrid VLP (as in this study) has a competitive advantage over a combination of wild-type L1 VLP (e.g. Gardasil-9). Also, Cervarix and Gardasil vaccines induce some crossneutralization and cross-protection.

Response:
We thank the reviewer for the encouraging summary of our chVLP co-assembly strategy. As suggested, we now include a comparison of our chVLP with the commercial formulations that show equal neutralization as well as minor cross-neutralization: "Finally, we generated a nine-valent chVLP according to the composition and dosage of L1s in the Gardasil-9 formulation, and measured the neutralizing antibody titers with 20 types of HPV PsVs-nine homologous types (HPV 6, and 11 heterologous ones (HPV 26,. Consistent with other multiple-type chVLPs ( Figure 5 and 7), the nine-valent chVLP showed good physiochemical and particle nature ( Figure S14) and elicited a high neutralizing antibody response against the nine homologous types of HPV similar to that of the WT VLP mixture and Gardasil-9, moreover, additionally induced some minor crossneutralizing antibody titers against heterologous HPV 26, in some of the mice (Figure 8)." (Page 14,.
"It is reasonable that the cross-neutralizing antibody response is limited for chVLPs, due to that the immunodominant epitopes of HPV L1 are mainly located on the five surface loops. Furthermore, the "suspended bridge" region, which varies in the capsomere-hybrid co-assembly, may have a lower immunogenicity when considered in the context of the surface loops that constitute most of immunodominant epitopes. Therefore, the type-cross neutralization of chVLPs should be further tested, with the assistance of some stronger adjuvants, such as AS04, in non-human primates." (Page 17,. Please also refer to our comments in response to Reviewer #1 general comment. With this in mind, we toned down the significance of the cross neutralization of chVLPs throughout the manuscript, and rephrased the title as "Rational design of a multiple-valent human papillomavirus vaccine by capsomere-hybrid co-assembly of virus-like particles". In regards to the final comment made by the reviewer, we have removed our previous claim that, "This method provides a new approach that may aid in the development of an improved HPV vaccine with better cross-protection against the various HPV genotypes." Instead, we include the following text in the Discussion: "Nonetheless, the nine-valent chVLP has additional competitive advantages over Gardasil 9 in terms of practical vaccine development, including a more stable pentamer stage in the purification process, a more controllable assembly without the requirement for a reductant (e.g., DTT) during assembly, and a "one-time" formulation." (Page 17, line 381-385). Furthermore, we propose a possible strategy with regard to the pan-HPV vaccine design based on the findings of this study and of our previous study (Li Z, et al. Nature Commun 2018:9;5360), as follows: "Furthermore, we surmise that a combination of these two strategies might facilitate the development of a pan-HPV vaccine that could theoretically incorporate 216 types of HPV immunodominant epitopes into a single particle (i.e., 72 pentamers, each bearing triple-type immunogenic loop regions). The number of types covered in the chHPV design approximates the number of HPV genotypes identified to date." (Page 18, line 392-397).
We believe that the chVLP rationale could pave the way for a new mode of vaccine design, targeting multiple pathogenic variants or hyper-varied cancer cells.
Major point This study would be much stronger if the experimental chVLP vaccine efficacy were tested (using Gardasil-9 in comparison) also in vivo in the mouse vaginal model (or rabbit cutaneous model) against challenge with (homologous and heterologous) HPV pseudovirion types for which neutralization is claimed.

Response:
We acknowledge that a mouse vaginal model using PsV challenge in vivo is a sensitive measure to test for the production of protective antibodies against HPV infection (Longet et al. J Virol. 2011;85.24:13253-13259;Day et al. Clin. Vaccine Immunol. 2012:CVI-00139) and particularly beneficial for testing L2-based vaccine development (Wu et al., PLoS one 2011;6:e27141;Jagu et al., J Virol 2013;87:6127-6136). However, such a model cannot sufficiently meet the demand for the high-throughput detection of serum neutralizing antibody titers (Jagu et al. J Virol 2013;87.11:6127-6136). Therefore, unfortunately, such in vivo animal models were not used for this study, and are beyond the scope of this work. Instead, WHO guidelines indicate that a PsV-based neutralization assay (PBNA) in a cell model is the "gold standard" for assessing the protective potential of antibodies induced by HPV L1 vaccines (Dessy et al. Hum Vaccines 2008:4.6;425-434;World Health Organization Expert Committee on Biological Standardization. Guidelines to assure the quality, safety and efficacy of recombinant human papillomavirus virus-like particle vaccines. WHO, Geneva 2007;World Health Organization. WHO meeting on the standardization of HPV assays and the role of WHO HPV LabNet in supporting vaccine introduction. WHO, Geneva 2008). As such, we previously used the high-throughput and robust PBNA to develop our HPV16/18 bivalent vaccine (launched in China recently), HPV 6/11 bivalent vaccine, HPV 9-valent vaccine, and HPV 20-valent vaccine (Gu et al. Vaccine 2017;35:4637-4645;Pan et al. Vaccine 2017;35:3222-3231;Wei et al. Emerg Microbes Infect. 2018;26;7:160;Li et al. Nature Commun. 2018;9:5360), as well as the 20-type neutralization assay carried out in this study.