1-s2.0-S2589004225017389-main-html.html

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Multi-epitope transplantation enables cross-type neutralization of phylogenetically

distant HPV11/6/53 in mice

Ciying Qian, Yujie Xu, Yang Huang, Jie Chen, Yanan Jiang, Jingjia Shen, Fei Gao,

Tianyu Ren, Yihan Lu, Shuyue Zhang, Chengzong Zhang, Daning Wang, Lizhi Zhou,

Tingting Li, Zhibo Kong, Qingbing Zheng, Hai Yu, Ying Gu, Ningshao Xia, Shaowei Li
PII:

S2589-0042(25)01738-9

DOI:

https://doi.org/10.1016/j.isci.2025.113477

Reference:

ISCI 113477

To appear in:

iScience

Received Date: 4 March 2025
Revised Date: 29 June 2025
Accepted Date: 28 August 2025

Please cite this article as: Qian, C., Xu, Y., Huang, Y., Chen, J., Jiang, Y., Shen, J., Gao, F., Ren, T., Lu,

Y., Zhang, S., Zhang, C., Wang, D., Zhou, L., Li, T., Kong, Z., Zheng, Q., Yu, H., Gu, Y., Xia, N., Li, S.,

Multi-epitope transplantation enables cross-type neutralization of phylogenetically distant HPV11/6/53 in

mice, (2025), doi:

https://doi.org/10.1016/j.isci.2025.113477

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Multi-epitope transplantation enables cross-type neutralization of

phylogenetically distant HPV11/6/53 in mice

Ciying Qian

1,2, #

, Yujie Xu

1,2, #

, Yang Huang

1,2, #

, Jie Chen

1,2,

, Yanan Jiang

1,2

Jingjia Shen

1,2

, Fei Gao

1,2

, Tianyu Ren

1,2

, Yihan Lu

1,2

, Shuyue Zhang

1,2

Chengzong Zhang

1,2

, Daning Wang

1,2

, Lizhi Zhou

1,2

, Tingting Li

1,2

, Zhibo

Kong

1,2

, Qingbing Zheng

1,2

, Hai Yu

1,2

, Ying Gu

1,2,

*, Ningshao Xia

1,2,

*, Shaowei

1,2,

1. State Key Laboratory of Vaccines for Infectious Diseases, Xiang An

Biomedicine Laboratory, Discipline of Intelligent Instrument and Equipment,

Department of Experimental Medicine, School of Life Sciences, School of

Public Health, Xiamen University, Xiamen, China.

2. National Institute of Diagnostics and Vaccine Development in Infectious

Diseases, National Innovation Platform for Industry-Education Integration in

Vaccine Research, Xiamen University, Xiamen, China.

**Lead Contact: S.L. (Email: [shaowei@xmu.edu.cn]) will serve as the Lead

Contact for this study. All requests for materials, data, or further information

should be directed to S.L.

# C.Q., Y.X., and Y.H. contributed equally to this work.

* Corresponding Authors: S.L., N.X., and Y. G. are all corresponding authors.

E-mail: (S.L.) shaowei@xmu.edu.cn, (N.X.) nsxia@xmu.edu.cn, (Y. G.)

guying@xmu.edu.cn

Running title:

A chimeric VLP vaccine against HPV types 11, 6 and 53

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Summary

In the post-vaccine era, non-vaccine HPV types necessitate broader-spectrum

HPV vaccines. This study explores epitope transplantation to bridge

phylogenetic gaps between distant types. Using the H11-6HI molecule,

originally designed to cross-neutralize HPV11 and HPV6, we replaced its DE

and FG loops with critical neutralizing epitopes from HPV53, yielding the

chimeric VLP H11-6HI-53DE-FG. Cryo-electron microscopy confirmed a well-

assembled T=7 virus-like structure. Pseudovirus-based neutralization assays

revealed balanced neutralizing antibody titers against HPV11, HPV6, and

HPV53, with ED

values of 0.528 µg, 0.544 µg, and 0.374 µg, respectively, in

mice. This approach demonstrates that multi-epitope transplantation can

overcome phylogenetic barriers between α10 and α6 HPV types, enabling

cross-neutralization between high-risk and low-risk viruses. These results offer

a promising foundation for designing next-generation HPV vaccines with

expanded coverage.

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Introduction

Human papillomavirus (HPV) is a non-enveloped, double-stranded DNA virus

encompassing over 230 identified genotypes, classified into low-risk (LR-HPV)

and high-risk (HR-HPV) categories based on their oncogenic potential

1-5

. These

viruses infect basal epithelial cells through minor abrasions in the skin or

mucosa, with subsequent internalization mediated by capsid proteins

interacting with host receptors. Following entry, the viral genome, assisted by

the L2 capsid protein, translocates to the nucleus

6,7

. HR-HPVs, such as HPV16

and HPV18, are the primary etiological agents of cervical cancer, anogenital

malignancies, and oropharyngeal squamous cell carcinomas

8-11

. Conversely,

LR-HPVs like HPV6 and HPV11 are predominantly associated with benign

conditions such as genital warts and recurrent respiratory papillomatosis

Globally, cervical cancer remains a major public health concern, with over

662,000 new cases and 348,000 deaths reported annually

13,14

. This highlights

the critical need for effective prevention strategies against HPV-associated

diseases. The advent of prophylactic HPV vaccines has revolutionized efforts

to reduce the burden of HPV-related cancers and benign lesions

15-17

. These

vaccines utilize the self-assembling properties of the L1 capsid protein to form

virus-like particles (VLPs), which mimic the natural virion and elicit strong

immune responses

. Among the currently approved vaccines, Gardasil 9

provides the broadest coverage, targeting seven HR types (HPV16, −18, −31,

−33, −45, −52, and −58) and two LR types (HPV6 and −11), collectively

accounting for 90% of HPV-associated cancers and 70% of genital warts

16,19

However, the type-specific nature of existing vaccines limits their coverage,

leaving significant gaps

20-23

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The phylogenetic diversity and structural variability among HPV types present

significant challenges to vaccine development. L1, the major structural protein

of HPV, forms the primary immunogenic component of VLP-based vaccines

This protein contains five surface-exposed loop regions (BC, DE, EF, FG, and

HI), which house most neutralizing antibody epitopes

25-27

. Notably, the

structural disparities among these loops between phylogenetically distant HPV

types underscore the challenges in achieving cross-type neutralization

Existing vaccines elicit predominantly type-specific immunity, with limited cross-

protection, necessitating innovative strategies to bridge these antigenic gaps.

Recent advances in structural vaccinology and epitope transplantation have

highlighted promising approaches for addressing these challenges. By

transplanting neutralizing epitopes from one HPV type into the L1 scaffold of

another, researchers have demonstrated the potential to induce cross-

neutralizing immune responses, particularly within closely related HPV

types

28,29

. Our previous work validated this approach by designing HPV11-6HI

VLPs, wherein immunodominant epitopes from HPV6 were strategically

transplanted onto the L1 scaffold of HPV11. This construct successfully induced

cross-neutralizing responses against both HPV6 and HPV11 in preclinical

models

. However, when applied to phylogenetically distant HPV types, such

as HPV33/59 and HPV58/59, single-loop transplantation strategies yielded

limited success, emphasizing the need for more comprehensive designs to

overcome genetic and structural barriers

28,31

A critical gap in current HPV vaccine research lies in the limited exploration of

cross-type strategies that bridge LR and oncogenic or potentially oncogenic

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types. To address this, we selected HPV6 and HPV11

—

well-characterized LR

types within the α10 species that are widely known for causing benign diseases

such as genital warts and recurrent respiratory papillomatosis

—

as the

structural scaffolds for chimeric VLP design.

In contrast, we engineered

epitopes from HPV53, a less widely recognized but epidemiologically relevant

type, to test cross-type epitope transplantation and neutralization potential.

Although HPV53 is classified by the International Agency for Research on

Cancer (IARC) as a possibly carcinogenic type (Group 2B), its clinical and

epidemiological significance should not be overlooked, particularly in East

Asian populations

32-34

. Several large-scale studies conducted in China have

consistently identified HPV53 among the top four most prevalent high-risk-

associated genotypes. For example, in a cervical screening study of 105,679

women in Wuhan (2015

–

2022), HPV53 ranked fourth among high-risk types,

with a prevalence of 1.87%. Among patients diagnosed with high-grade

100

squamous intraepithelial lesions (HSIL+) by ThinPrep cytologic test (TCT),

101

HPV53 ranked fifth

. Similar findings were observed in Liaocheng (2.77%

102

prevalence in 34,076 women)

and in Jilin Province, where HPV53 accounted

103

for 3.4% of infections in a cohort of 21,282 women and was found in 8.9% of

104

cervical cancer cases

. In South Korea, HPV53 was the most frequently

105

detected genotype among HPV-positive women, with a prevalence of 9.69% in

106

a cohort of 18,170 individuals

. Globally, HPV53 is detected in 1.2% of women

107

with normal cytology, but its prevalence rises to 7.3% in low-grade lesions and

108

3.5% in high-grade lesions

. Despite its exclusion from current vaccine

109

formulations, the regional burden and phylogenetic divergence of HPV53 from

110

vaccine-included LR types like HPV6 and HPV11 make it a structurally and

111

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immunologically meaningful candidate for testing cross-type neutralization.

112

Therefore, in this study, we used the HPV11 L1 scaffold incorporating the HI

113

loop of HPV6 (H11-6HI) as a foundation, and introduced additional neutralizing

114

loops from HPV53 (DE and FG) to construct a chimeric VLP. This design allows

115

us to assess the feasibility of multi-epitope transplantation in bridging

116

phylogenetic gaps and expanding the coverage of next-generation HPV

117

vaccines.

118

In this study, we address the critical challenge of bridging the phylogenetic gap

119

between possibly carcinogenic type and LR HPV types by designing a chimeric

120

VLP, H11-6HI-53DE-FG. Building on the original design of H11-6HI, which

121

incorporates the immunodominant HI loop of HPV6 onto the L1 scaffold of

122

HPV11, we strategically introduced two key neutralizing epitopes from HPV53

123

(DE and FG loops) into this established framework. This multi-epitope

124

transplantation approach resulted in a chimeric VLP capable of inducing robust

125

cross-neutralization across HPV6, HPV11, and HPV53. This study not only

126

demonstrates the feasibility of using multi-epitope transplantation to overcome

127

phylogenetic barriers but also establishes a framework for developing next-

128

generation HPV vaccines with expanded coverage. By addressing the unmet

129

need for protection against both LR and

possibly carcinogenic

types, our

130

findings lay the groundwork for inclusive and broad-spectrum HPV prevention

131

strategies.

132

Results

133

Phylogenetic analysis of HPV L1 sequences

134

According to the carcinogenic classification by the International Agency for

135

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Research on Cancer (IARC), 25 HPV types have been identified as potentially

136

oncogenic. Among them, HPV6 and HPV11 are classified as LR types, while

137

several other HPV types have uncertain carcinogenic potential

3,4

. In this study,

138

we obtained 46 representative L1 gene sequences from the International HPV

139

Reference Center, comprising all 25 oncogenic HPV types, two low-risk types

140

(HPV6 and HPV11), and several types with undefined carcinogenicity, to

141

conduct a comprehensive phylogenetic analysis. As shown in Supplementary

142

Fig. 1, phylogenetic analysis revealed that HPV6 and HPV11, both belonging

143

to the α10 species, exhibit close evolutionary relatedness. In contrast, they are

144

evolutionarily distant from HPV53, a possibly carcinogenic type classified within

145

the α6 species. Further sequence identity analysis using Clustal Omega

146

software, showed an overall amino acid sequence similarity of 61.8% among

147

HPV11, HPV6, and HPV53 for the L1 protein. The highest similarity was

148

observed between HPV11 and HPV6 (91.45%), whereas HPV53 shared 64.41%

149

and 63.42% sequence identity with HPV11 and HPV6, respectively. These

150

findings further corroborate the closer evolutionary relationship between HPV11

151

and HPV6, while their divergence from HPV53 is substantially greater than that

152

observed between HPV56/66 and HPV53. To gain deeper insights into

153

sequence variation, we compared the loop regions of HPV11 and HPV53 in the

154

L1 protein (Supplementary Fig. 2). Significant differences were observed in the

155

number of amino acid residues across the BC, DE, EF, and FG loop regions,

156

with variations of 7, 19, 9, and 15 residues, respectively. Given that the loop

157

regions of L1 play a critical role in the immunogenicity of VLPs, we hypothesize

158

that a homologous loop replacement strategy could be a potential approach for

159

designing broad-spectrum, cross-protective VLP-based vaccines. However, the

160

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feasibility of cross-protection between distantly related types, such as HPV53

161

and HPV6/11, remains to be experimentally validated.

162

Expression and purification of H11-6HI and HPV53 single- and double-

163

loop swapped chimeric molecules in E. coli

164

We engineered a series of chimeric molecules by replacing the loop sequences

165

encoding HPV53 BC, DE, EF, and FG with the homologous loop regions of the

166

H11-6HI molecule. These modifications included both single-loop and double-

167

loop exchanges, yielding a total of 10 HPV11/6/53 chimeric molecules. The

168

coding sequences of these chimeras were subsequently cloned into the pTO-

169

T7 vector for expression in Escherichia coli (Figure 1A). All chimeric constructs

170

were expressed as soluble proteins and isolated from the supernatant of

171

bacterial lysates using a two-step chromatography protocol. This approach is

172

designed to facilitate their assessment and potential development as vaccine

173

candidates. Initially, SP-FF strong cation exchange chromatography was

174

employed to purify HPV pentamers. As illustrated in Figure 1B, with the

175

exception of H11-6HI-53BC-DE, the target proteins were eluted at high salt

176

concentrations, each with a molecular weight of approximately 55 kDa and

177

achieving satisfactory purity levels. To promote the assembly of HPV pentamers

178

into VLPs, the protein-containing fractions were subjected to dialysis to remove

179

dithiothreitol. This was followed by Superdex 200 purification to obtain high-

180

purity HPV VLPs. The SDS-PAGE analysis demonstrated that the protein purity

181

significantly improved after the second purification step (Figure 1C). Additionally,

182

these high-purity VLPs exhibited strong reactivity with the HPV L1 specific

183

antibody 4B3, as shown in Figure 1D.

184

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Characterization of the in vitro assembled H11-6HI and HPV53 chimeric

185

VLPs

186

To comprehensively characterize the physical and chemical properties of the

187

chimeric VLPs, we employed a variety of analytical techniques, including

188

transmission electron microscopy (TEM), high-performance size exclusion

189

chromatography (HPSEC), and dynamic light scattering (DLS). The H11-6HI

190

and HPV53 VLPs served as controls for these analyses. As depicted in Figure

191

2A, exchanging single-loop regions between H11-6HI and HPV53 did not

192

impact their self-assembly in vitro. All four single-loop swap constructs

193

successfully assembled into uniform and regular VLPs, with retention times of

194

approximately 13 minutes (± 0.2 minutes) observed in HPSEC. DLS

195

measurements indicated particle radii around 30 nm for the H11-6HI-53 single-

196

loop chimeric molecules, closely mirroring the attributes of the H11-6HI control.

197

Building upon the single-loop exchanges, we introduced additional loop

198

regions from HPV53 to produce double-loop swap constructs. Refer to Figure

199

2B for a visual depiction of the described VLP assembly and relevant

200

chromatographic profiles, apart from the H11-6HI-53BC-DE construct, which

201

failed to assemble completely and could not form VLPs separable from

202

pentamers, all other double-loop swap constructs retained the particle

203

properties characteristic of the H11-6HI scaffold. Notably, these constructs did

204

not adopt the irregular rod-shaped morphology commonly associated with

205

HPV53 assemblies, as reported in previous studies

. Instead, the double-loop

206

chimeric VLPs displayed a single-peak distribution in HPSEC, indicative of

207

good uniformity, and their particle sizes remained consistent with the H11-6HI

208

scaffold.

209

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Our findings suggest that homologous loop region exchanges between the

210

phylogenetically distant HPV11 and HPV53 types exert minimal influence on

211

VLP assembly. This preservation of structural integrity and particle uniformity

212

highlights the robustness of the VLP scaffold despite significant genomic

213

alterations. These insights are critical for optimizing the design and

214

development of VLP-based vaccines, where cross-type chimeric constructs can

215

be beneficial.

216

Immunogenicity analysis of H11-6HI and HPV53 chimeric VLPs in mice

217

To investigate the immunogenic properties of the well-assembled H11-6HI-53

218

chimeric VLPs—including their ability to maintain neutralizing antibody titers

219

against HPV11 and 6, as well as to induce neutralizing antibodies against

220

HPV53—we conducted a series of immunizations in 6-week-old SPF BALB/c

221

female mice. The mice were randomly divided into groups (n = 5) and

222

immunized via intraperitoneal injection using aluminum adjuvant at doses of 5.0

223

μg, 1.0 μg, and 0.2 μg per mouse. The immunization schedule included time

224

points at 0, 2, and 4 weeks, with blood samples collected from the orbital vein

225

at 2, 3, 5, and 6 weeks. Neutralizing antibody titers were assessed using a

226

PBNA.

227

As illustrated in Figure 3A, regardless of whether the HPV11/6/53 wild-type (WT)

228

or chimeric molecules (excluding chimeric VLPs that completely lost

229

immunogenicity) were administered, both induced neutralizing antibody titers

230

that increased with the number of immunizations. After a single immunization,

231

the chimeric molecules predominantly induced limited neutralizing titers against

232

HPV11, surpassing those generated by the WT HPV11. Remarkably, most

233

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experimental groups showed a substantial elevation in neutralizing antibody

234

titers following the third immunization, exhibiting an immunization frequency-

235

dependent effect.

236

Detailed characterization of sera collected two weeks post the third

237

immunization was conducted. This included evaluations of type-specific

238

antibody levels, cross-type neutralizing antibody levels, and the dose-

239

dependency of neutralizing antibody titers (Figure 3B). Consistent with previous

240

findings

, HPV11 and HPV6 demonstrated cross-neutralization due to their

241

close phylogenetic relationship, while no cross-neutralization was observed for

242

the more distantly related HPV53. Upon replacing a single loop region with

243

HPV53, the H11-6HI chimeric VLP failed to induce HPV53-specific neutralizing

244

antibodies. However, the introduction of a second loop region corresponded

245

with varying degrees of enhancement in HPV53-specific neutralizing antibody

246

levels. The chimeric constructs H11-6HI-53BC-EF and H11-6HI-53DE-FG

247

exhibited the highest overall immunogenicity, significantly outperforming the

248

control H11-6HI. At the 5 μg dose, H11-6HI-53BC-EF induced the strongest

249

neutralizing antibody responses against all three HPV types. However, its

250

cross-type neutralization potency declined with lower antigen doses and was

251

ultimately surpassed by H11-6HI-53DE-FG. Notably, H11-6HI-53DE-FG

252

maintained neutralizing titers of 10^2 even at the 0.2 μg dose. It is also worth

253

noting that replacing additional loop regions with those from HPV53 reduced

254

neutralization activity against HPV11.

255

To further evaluate the immunogenic performance of the chimeric molecules

256

H11-6HI-53BC-EF and H11-6HI-53DE-FG, we assessed both type-specific and

257

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cross-type binding antibody titers. The results, presented in Figure 4,

258

demonstrate that these chimeric constructs exhibited superior capacity to

259

induce binding antibodies. Across all groups, the levels of binding antibodies

260

were markedly higher than those of neutralizing antibodies. Notably, the cross-

261

binding titers between WT HPV11 and HPV6 exceeded 10^4, even at the lowest

262

dose of 0.2 μg, while virtually no cross-binding was observed with the more

263

distantly related HPV53. The H11-6HI chimeric molecule elicited balanced

264

binding titers for HPV11 and HPV6, comparable to those induced by their

265

respective WT VLPs, and displayed minimal cross-binding to HPV53. In

266

contrast, the H11-6HI-53 chimeric VLP significantly enhanced binding antibody

267

titers against HPV53 compared to WT HPV11, likely due to the incorporation of

268

HPV53-specific epitopes. For the H11-6HI-53DE-FG construct in particular, the

269

binding antibody titers against HPV53 were significantly higher than those

270

induced by WT HPV11 at all tested doses (

< 0.0001 at 5.0 μg;

< 0.001 at

271

1.0 μg;

< 0.01 at 0.2 μg). Consistent with the trend observed in neutralizing

272

antibody titers, H11-6HI-53BC-EF outperformed H11-6HI-53DE-FG at higher

273

doses. However, at lower doses, it elicited lower levels of binding antibodies

274

than H11-6HI-53DE-FG. Overall, the trends in binding antibody titers closely

275

mirrored those observed for neutralizing antibody levels. These results highlight

276

the potential of incorporating multiple key neutralizing epitopes to overcome

277

phylogenetic barriers, thereby enabling cross-neutralization between distantly

278

related HPV types such as HPV11 and HPV53.

279

Evaluation of ED

in vaccine candidates

280

The half-maximal effective dose (ED

) is a critical metric in vaccine evaluation,

281

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quantifying the dose required to achieve 50% of the desired response (typically

282

seroconversion). Lower ED

values indicate higher vaccine potency and

283

efficacy. In this study, the ED

evaluation confirmed the ability of the H11-6HI-

284

53DE-FG VLP to maintain cross-neutralizing activity across HPV11, HPV6, and

285

HPV53 at comparable doses (Table 1). For the WT HPV11, HPV6, and HPV53

286

VLPs, the ED

values were 0.054 μg, 0.031 μg, and 0.023 μg, respectively,

287

indicating strong type-specific seroconversion. However, these WT VLPs failed

288

to induce half-maximal seroconversion against other HPV types within the

289

measured dose range, highlighting their limited cross-neutralizing potential. In

290

contrast, the chimeric H11-6HI-53DE-FG VLP exhibited balanced cross-

291

neutralizing capabilities across the three HPV types, with ED

values of 0.528

292

μg, 0.544 μg, and 0.374 μg for HPV11, HPV6, and HPV53, respectively.

293

Remarkably, the chimeric VLP achieved half-maximal seroconversion against

294

all three components, demonstrating its capacity to bridge phylogenetic gaps.

295

These findings underscore the potential of H11-6HI-53DE-FG as a vaccine

296

candidate, capable of inducing cross-neutralizing responses against

297

phylogenetically distant HPV types. Although H11-6HI-53BC-EF elicited

298

stronger neutralizing and binding antibody titers against all three component

299

types at higher doses, it achieved only 50% seroconversion against HPV11

300

under the low-dose gradient used in the ED

assay and failed to reach this

301

threshold for HPV6 and HPV53. This limited performance at lower doses further

302

supports the superior cross-type immunogenicity of the DE and FG loop-based

303

construct, in line with previous studies identifying the DE and FG loops as

304

dominant immunogenic regions of HPV53

305

Thermal Stability and Structural Integrity of the Chimeric Vaccine

306

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Candidate H11-6HI-53DE-FG

307

Thermal stability is a critical attribute for vaccines, significantly influencing their

308

shelf life, distribution logistics, accessibility, cost-effectiveness, and overall

309

public health impact. Utilizing a MicroCal VP-Capillary DSC system, we

310

determined the melting temperatures (Tm values) of WT HPV11, HPV6, the

311

scaffold molecules H11-6HI and the chimeric H11-6HI-53. As illustrated in

312

Figure 5, the Tm values were 77.87

C for HPV11 and 79.31

C for HPV6. The

313

double-type chimeric VLP H11-6HI exhibited a Tm of 76.89

C, slightly lower

314

than those of WT HPV11 and HPV6. However, upon incorporation of HPV53

315

epitopes, the chimeric VLP H11-6HI-53BC-EF showed a pronounced decrease

316

in thermal stability, characterized by a bimodal melting profile. This profile likely

317

reflects the presence of two distinct VLP populations with different assembly

318

stabilities: poorly assembled particles with a lower Tm of 55.18

C and well-

319

assembled particles with a higher Tm. Such heterogeneity is commonly

320

observed in VLPs assembled from pentamers derived from different HPV types.

321

In contrast, the chimeric VLP H11-6HI-53DE-FG demonstrated enhanced

322

thermal stability, with a Tm of 81.32

—

exceeding all other tested constructs

—

323

indicating superior particle thermal stability.

324

Further evaluation via cryo-electron microscopy (Cryo-EM) provided insights

325

into the structural integrity of the H11-6HI-53DE-FG VLP (EMD-63835),

326

attaining a resolution of 19.48 Å as assessed by the Fourier Shell Correlation

327

(FSC) criterion at 0.143 (Figure 5B). Remarkably, H11-6HI-53DE-FG displayed

328

structural characteristics comparable to those of H11-6HI VLPs, as detailed in

329

a previous study

, underscoring its potential applicability as a viable vaccine

330

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candidate. Therefore, H11-6HI-53DE-FG emerges as a promising chimeric VLP

331

for further investigations focusing on cross-immunity.

332

Discussion

333

Over the past few decades, the rational design of HPV vaccines has made

334

substantial progress, with current strategies focusing on broadening antigenic

335

coverage, enhancing immune responses, and addressing the limitations of

336

type-specific immunity. Although prophylactic HPV vaccines have significantly

337

reduced the global burden of HPV-related diseases, the continued circulation

338

of non-vaccine HPV types and the phylogenetic divergence between

possibly

339

carcinogenic types

and LR-types underscore the need for innovative vaccine

340

designs.

341

Current mainstream approaches to broaden vaccine protection include

342

increasing the number of VLP types in multivalent formulations and exploring

343

L2-based vaccines. The former strategy has yielded successive advances, from

344

bivalent to quadrivalent and currently nonavalent HPV vaccines, with even

345

more extensive formulations (e.g., 11-valent [CTR20201029] and 15-valent

346

[CTR20241041]) undergoing clinical evaluation. While these multivalent

347

formulations effectively expand type coverage, they inevitably increase the total

348

protein dose (e.g., the nonavalent vaccine contains 270 μg per dose) and

349

require the establishment of additional, type-specific production lines to

350

manufacture each distinct VLP component. Moreover, further increases in

351

antigenic components may exacerbate immune interference, necessitating

352

dose adjustments that could raise the risk of adverse events and elevate

353

production costs

. L2-based vaccines, targeting the more conserved minor

354

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capsid protein, have long been considered a promising alternative. In principle,

355

they could provide broader protection across genotypes and be more cost-

356

effective. However, L2 vaccines face major development hurdles: the full-length

357

L2 protein is difficult to express and purify, structural data remain limited, and

358

immunogenicity of key epitopes (such as the RG1 epitope) is relatively weak.

359

Consequently, many L2 strategies rely on multimeric epitope display platforms

360

such as nanoparticles or tandem repeats

. Thus, despite their theoretical

361

advantages, L2-based vaccines are still in early-stage development and have

362

not reached the manufacturing maturity required for widespread deployment.

363

In contrast, our L1 epitope transplantation strategy represents a pragmatic and

364

scalable design framework. By targeting immunodominant surface loops from

365

epidemiologically relevant genotypes and integrating them into established L1

366

scaffolds, we created chimeric VLPs capable of cross-neutralizing multiple HPV

367

types. In this study, the H11-6HI-53DE-FG construct achieved cross-

368

neutralization against HPV11, HPV6, and HPV53 using a single immunogen.

369

The required protein dose was only ~1/3 of the wild-type VLP mixtures

370

necessary for the same type coverage. Our approach has been successfully

371

extended to multiple clusters of genotypes (e.g., HPV16/35/31, 18/45/59,

372

58/33/52, and others), supporting the feasibility of designing 7

–

8 chimeric

373

constructs to collectively cover 20-21 clinically significant HPV types

374

From a production standpoint, all our chimeric VLPs are expressed in

E. coli

375

consistent with the manufacturing platforms used for licensed vaccines such as

376

Cecolin and Cecolin-9. The production of 7

–

8 chimeric VLPs would require a

377

limited number of upstream production lines, and constructs based on existing

378

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vaccine components (e.g., HPV6, HPV11, HPV16) could leverage current

379

infrastructure with minimal adjustments. This contrasts with multivalent

380

strategies that require independent expression and purification of each VLP

381

component. Therefore, although the de novo formulation of chimeric VLPs

382

entails initial development work, it does not introduce greater long-term

383

manufacturing complexity compared to scaling up multivalent formulations.

384

Taken together, our approach offers a rational, potentially more dose-sparing,

385

and production-efficient pathway to develop broad-spectrum HPV vaccines.

386

Unlike conventional multivalent vaccines that scale type coverage linearly with

387

component number, our strategy enables nonlinear expansion of protection

388

through multi-epitope transplantation while maintaining manageable

389

manufacturing demands.

390

In prior studies, single-loop transplantation strategies showed limited success

391

in inducing cross-neutralization between phylogenetically distant HPV types

392

Similarly, in this study, chimeric constructs incorporating single loops from

393

HPV53 (H11-6HI-53BC/DE/EF/FG) failed to elicit cross-neutralization between

394

possibly carcinogenic

-HPV and LR-HPV types. However, when two loops from

395

HPV53 were transplanted, cross-neutralization improved significantly. The H11-

396

6HI-53DE-FG chimeric construct achieved a notable breakthrough by inducing

397

balanced cross-neutralization across HPV11, HPV6, and HPV53. This multi-

398

epitope transplantation approach not only retained part of the immunogenicity

399

of the H11-6HI scaffold but also, demonstrated effective cross-neutralization

400

between

possibly carcinogenic

and LR HPV types. These findings validate the

401

feasibility of designing chimeric VLPs to bridge phylogenetic gaps and provide

402

theoretical and practical pathways for developing broad-spectrum HPV

403

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vaccines.

404

However, inducing cross-neutralization between phylogenetically distant HPV

405

types remains a significant challenge. Although the H11-6HI-53DE-FG chimeric

406

VLP elicited neutralizing antibody titers against HPV53 in the range of 2

–

3 log

407

units, the overall potency remained moderate, suggesting that the

408

immunogenicity of the transplanted epitopes is still limited. Moreover, when

409

multiple key loop regions

—

especially the two major neutralizing loops of

410

HPV11

—

were replaced within the H11-6HI scaffold, we observed a noticeable

411

reduction in neutralization titers against both HPV11 and HPV6, with

412

performance inferior to that of the WT HPV11/6 VLPs. Notably, H11-6HI-53DE-

413

FG exhibited a greater reduction in HPV11 and HPV6 neutralization titers at the

414

5 µg dose compared to other dual-loop chimeras. Nonetheless, this construct

415

showed a more balanced neutralizing response across all three target types at

416

low antigen doses, comparable to or even exceeding that of single- or dual-loop

417

replacements, suggesting that while multi-epitope transplantation can broaden

418

type coverage, it may also compromise the immunogenicity of the original

419

scaffold type.

420

Further insights were revealed when we constructed a chimeric VLP containing

421

only the DE loop of HPV53 on the H11-6HI scaffold. This construct completely

422

lost neutralizing activity against all three target types, indicating that the DE loop

423

is likely a critical neutralization epitope for HPV11. The partial recovery of

424

neutralization observed in the dual-loop chimeras may stem from structural

425

remodeling of the functional epitopes on the VLP surface induced by multiple

426

epitope substitutions. This hypothesis, however, requires validation through

427

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high-resolution structural analysis to determine the precise conformational

428

changes caused by heterologous epitope grafting. Future vaccine optimization

429

should focus on the accurate identification of neutralizing epitopes for each

430

HPV type and ensuring their faithful conformational presentation on

431

heterologous scaffolds to maximize immunogenic efficacy.

432

Interestingly, despite limited neutralizing activity, some chimeric VLPs induced

433

markedly high binding antibody titers. For example, H11-6HI-53BC-EF elicited

434

binding titers against HPV53 exceeding 10^6, while the corresponding

435

neutralization titers remained around 10^3 at the 5 µg dose. Similar trend was

436

observed in wild-type VLPs derived from closely related HPV genotypes, such

437

as HPV11 and HPV6—both of which are covered by licensed vaccines like

438

Cecolin-9 produced using a

E. coli

expression system. These VLPs exhibited

439

strong cross-binding reactive without compromising their potent neutralizing

440

capacity as vaccine immunogens. Thus, the observed dissociation between

441

binding and neutralization titers suggests that fine-tuning this balance may not

442

be the primary challenge in chimeric VLP design. Instead, the key lies in

443

accurately identifying the most immunodominant neutralizing epitopes of each

444

genotype and transplanting them onto a structurally compatible scaffold that

445

preserves their native antigenic conformation.

446

Therefore, in the practical development of third-generation HPV vaccines based

447

on chimeric VLPs (e.g., eight constructs covering 21 HPV types), we prioritize

448

homologous epitope transplantation between phylogenetically related

449

genotypes to maximize cross-type immunogenicity. However, when this

450

approach fails to preserve or transfer immunogenicity effectively, our findings

451

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demonstrate that multi-epitope transplantation across more distantly related

452

types provides a viable alternative, offering a complementary strategy to

453

broaden vaccine coverage.

454

For vaccine efficacy evaluation, we employed a PBNA instead of an in vivo

455

mouse vaginal challenge model. While the mouse vaginal model is a sensitive

456

approach for assessing protective antibody responses, it lacks scalability for

457

high-throughput antibody detection

42,43

. PBNA, a WHO-recommended assay,

458

offers robust, scalable, and reliable evaluation of L1-based HPV vaccines

44,45

459

This assay has played a critical role in the development and evaluation of

460

licensed HPV vaccines produced using

E. coli

expression systems, including

461

Cecolin and Cecolin-9. Head-to-head immunogenicity comparison of one dose

462

Cecolin and Gardasil in Chinese girls aged 9

–

14 years have demonstrated that

463

HPV vaccines derived from

E. coli

systems exhibit immunogenicity comparable

464

to those produced via eukaryotic expression platforms

. Given the well-

465

established correlation between PBNA-derived antibody titers and in vivo

466

vaccine efficacy, we believe that the immunogenicity results demonstrated by

467

PBNA in this study are representative of protective efficacy across different

468

animal models and are closely aligned with actual in vivo protection. Given the

469

well-established correlation between PBNA-derived antibody titers and in vivo

470

vaccine efficacy, we are confident that the vaccine efficacy demonstrated

471

through PBNA in this study is comparable across different animal models and

472

strongly correlates with actual in vivo protection.

473

In summary, this study demonstrates the potential of multi-epitope

474

transplantation to overcome the phylogenetic barriers between HR and LR HPV

475

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types. By strategically introducing two immunodominant epitopes from HPV53

476

into the H11-6HI scaffold, we successfully designed a chimeric VLP, H11-6HI-

477

53DE-FG, capable of inducing balanced cross-neutralization across HPV11,

478

HPV6, and HPV53. These findings highlight the feasibility of leveraging epitope

479

transplantation to design broad-spectrum HPV vaccines, offering an alternative

480

pathway for addressing gaps in current vaccine strategies. While challenges

481

remain in optimizing immunogenicity and maintaining scaffold stability, this

482

approach provides a promising framework for the rational design of next-

483

generation HPV vaccines. Future efforts should focus on further refining epitope

484

selection, scaffold engineering, and evaluating these constructs in clinically

485

relevant models to advance their development as effective and inclusive HPV

486

vaccines.

487

Limitations of the study

488

This study identified promising multivalent vaccine antigens capable of cross-

489

neutralizing HPV11, HPV6, and HPV53. To ensure clinical translatability, these

490

chimeric VLP constructs require detailed evaluation of their immunogenicity and

491

protective efficacy in preclinical models like rabbits and cynomolgus macaques,

492

which better mimic human immune responses. Assessing these candidates

493

across diverse models will provide critical insights into their broad-spectrum

494

protective potential and capacity to prevent HPV-related diseases. Additionally,

495

advancing these vaccine formulations toward human application necessitates

496

thorough examination of their safety, production scalability, and long-term

497

stability under various storage conditions. Addressing these gaps through

498

rigorous preclinical and translational research will enhance the feasibility of

499

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implementing this vaccine design, offering a pathway for developing more

500

inclusive and effective strategies for HPV prevention.

501

Resource availability

502

Lead contact

503

Further information and requests for resources and reagents should be

504

addressed to and will be fulfilled by the Lead Contact Shaowei Li

505

(shaowei@xmu.edu.cn).

506

Materials availability

507

This study did not generate unique reagents.

508

Data and code availability

509

All data supporting the findings of this study are included in the main article and

510

supplementary materials.

511

The cryo-EM density map generated during the study has been deposited in

512

the Electron Microscopy Data Bank (EMDB) under accession number EMD-

513

63835, which is also listed in the Key Resources Table.

514

No original or custom code was generated or used in this study, and no

515

additional datasets or resources were deposited in external repositories.

516

Any additional information required to reanalyze the data reported in this paper

517

is available from the lead contact upon request.

518

519

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Author contributions

520

S.L., N.X. and Y.G. designed the study. C.Q., Y.X., Y.H., J.C., Y.J., J.S., F.G.,

521

T.R., Y.L., S.Z., C.Z., D.W., L.Z., T.L. and Z.K. performed the experiments. C.Q.,

522

Q.Z., H.Y. and S.L. analyzed the data. C.Q. wrote the original draft. C.Q. and

523

S.L. participated in discussion and interpretation of the results. Funding

524

acquisition: S.L., N.X., Y.G. and L.Z.. All authors contributed to experimental

525

design.

526

Acknowledgements

527

This work was supported by the National Key Research and Development

528

Program of China (Grant No. 2024YFC2310601), the National Natural Science

529

Foundation of China (Nos. 82271873, 82471864, and 82302062), the Xiamen

530

Industry-University-Research Collaboration Program (No. 2024CXY0113), the

531

Xiang'an Innovation Laboratory Technology Program (No. 2023XAKJ0101019),

532

and the Fundamental Research Funds for the Central Universities (Nos.

533

20720220004 and 20720220006). The funders had no role in the study design,

534

data collection and analysis, decision to publish, or preparation of the

535

manuscript.

We sincerely thank the drawing support provided by the Biorender

536

platform (https://app.biorender.com/).

537

Declaration of interests

538

The authors declare no competing interests.

539

540

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Figure titles and legends

541

Figure 1. Expression, purification, protein purity analysis and in vitro VLP

542

self-assembly of H11-6HI and HPV53 chimeric L1 proteins

543

(A) Schematic overview illustrating the key steps in the manufacturing process

544

to produce high quantities of HPV WT and chimeric VLPs using Escherichia coli

545

cells.

546

(B) SDS-PAGE analysis of purified HPV11-6HI-53 chimeric L1 protein obtained

547

from SP sepharose chromatography.

548

549

Superdex 200 sepharose chromatography.

550

(D) Western blot analysis of purified chimeric VLP proteins separated via

551

reducing SDS-PAGE, detected using a broad-spectrum linear monoclonal

552

antibody (mAb) 4B3.

553

Figure 2: Morphological, size, and homogeneity characterization of H11-

554

6HI-53 chimeric VLPs

555

(A) Characterization of H11-6HI and HPV53 chimeric VLPs with a single

556

homologous loop replacement: TEM images depict the particle morphology with

557

a 100 nm scale bar (top). HPSEC elution profiles illustrate particle size and

558

purity (middle). DLS provides the particle size distribution data (bottom).

559

(B) Characterization of H11-6HI and HPV53 chimeric VLPs with two

560

homologous loop replacements: TEM images show the morphology of particles

561

with a 100 nm scale bar (top). HPSEC elution profiles reveal particle size and

562

purity (middle). DLS particle size distribution analysis is presented (bottom).

563

Figure 3: Induction of Type-Specific and cross-type neutralizing

564

antibodies by H11-6HI-53 chimeric VLP vaccination

565

BALB/c mice (n = 5) were intraperitoneally inoculated with5.0 μg, 1.0 μg, or 0.2

566

Journal Pre-proof

μg doses of WT or chimeric HPV11, HPV6, and HPV53 VLPs at weeks 0, 2,

567

and 4. The elicitation of neutralizing antibodies was quantitatively assessed six

568

weeks following the initial immunization, employing neutralization assays where

569

the assay's sensitivity threshold is delineated by a dotted line. All data were

570

subjected to one-way analysis of variance (ANOVA) and are presented as

571

mean ± standard deviation (SD); *

< 0.05, **

< 0.01, ***

< 0.001, ****

572

0.0001. The error bars represent the SD, and symbols denote individual mice.

573

(A) Dose-dependent immunogenicity of the H11-6HI-53 chimeric VLPs.

574

(B) Type-specific and cross-neutralizing antibody titers induced by H11-6HI-53

575

chimeric VLPs two weeks after three immunizations.

576

Figure 4. Induction of Type-Specific and cross-type IgG titers by

577

HPV11/6/53 chimeric candidate vaccine.

578

BALB/c mice (n = 5) were intraperitoneally inoculated with 5.0 μg, 1.0 μg, or 0.2

579

μg doses of WT or chimeric HPV11, HPV6, and HPV53 VLPs at weeks 0, 2,

580

and 4. Serum binding titers of type-specific and cross-type antibodies against

581

HPV11, HPV6, and HPV53 were detected by indirect ELISA. The sensitivity

582

threshold of the assay is indicated by a dotted line. All data were subjected to

583

two-way analysis of variance (ANOVA) and are presented as mean ± standard

584

deviation (SD); *

< 0.05, **

< 0.01, ***

< 0.001, ****

< 0.0001. The error

585

bars represent the SD, and symbols denote individual mice.

586

Figure 5. Thermal Stability and Cryo-EM Characterization of HPV11-6HI-

587

53 Chimeric Candidate Vaccine.

588

(A) Thermal stability comparison of WT, double-type chimeric VLP, and triple-

589

type chimeric VLP of HPV11, HPV6, and HPV53.

590

(B) Cryo-EM structural visualization of H11-6HI-53DE-FG chimeric VLPs,

591

illustrated with radial color grading from 260 Å to 290 Å, detailing the complex

592

conformational nuances. Resolution validation of the icosahedral architecture

593

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Table:

597

Table 1. Half effective dosage (ED

) of HPV11/6/53 chimeric VLPs with

598

aluminum adjuvant in mice

599

Antigen

(VLP)

Dose

(μg)

PsV11

PsV6

PsV53

Seroconversio

n no./

inoculated no.

(μg)

Seroconversio

n no./

inoculated no.

(μg)

Seroconversio

n no./

inoculated no.

(μg)

HPV11

0.300 8/8

0.054

1/8

>0.300

0/8

>0.300

0.100 5/8

3/8

0/8

0.033 1/8

1/8

0/8

0.011 1/8

0/8

HPV6

0.300 0/8

>0.300

8/8

0.031

0/8

>0.300

0.100 0/8

3/8

0/8

0.033 0/8

7/8

0/8

0.011 0/8

0/8

HPV53

0.300 0/8

>0.300

0/8

>0.300

7/8

0.023

0.100 0/8

0/8

8/8

0.033 0/8

0/8

6/8

0.011 0/8

0/8

HPV11&6&53

0.300 8/8

0.144

8/8

0.144

8/8

0.019

0.100 2/8

2/8

8/8

0.033 0/8

0/8

8/8

0.011 0/8

0/8

H11-6HI-53BC-EF 0.900 7/8

0.142

1/8

>0.900

0/8

>0.900

0.300 1/8

0/8

0.100 4/8

2/8

1/8

0.033 0/8

0/8

0.011

0/8

H11-6HI-53DE-FG 0.900 6/8

0.528

5/8

0.544

5/8

0.374

0.300 1/8

1/8

3/8

0.100 1/8

2/8

0/8

0.033 0/8

0/8

0.011

0/8

3/8

600

601

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STAR

★

Methods

602

Key resources table

603

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Antibodies
4B3

Li et al.

N/A

Goat anti-mouse IgG-HRP

This paper

N/A

Bacterial and virus strains

E.coli

ER Chemically Competent Cell

This paper

N/A

DH5α Chemically Competent Cell

Invitrogen

Cat # 12034013

Biological samples

Serum samples from BALB/c mice

This paper

N/A

Chemicals, peptides, and recombinant proteins
All HPV L1

This paper

N/A

Critical commercial assays
Plasmid Small Extraction Kit

Tiangen

Cat# DP103

EndoFree Maxi Plasmid Kit

TianGen

Cat# DP117

Trypsin

Thermo Fisher

Lot # TG269188

DMEM

Thermo Fisher

Cat #11965092

1x PBS, pH 7.4

Thermo Fisher

Cat # 10010031

Fetal bovine serum (FBS)

Thermo Fisher

Cat # 10099

Polyethylenimine Linear (PEI) MW40000

（

rapid lysis

）

Yeasen

Cat # 40816ES03

SurePAGEᵀᴹ, Bis-Tris, 10x8, 4-12%, 10 wells

GenScript

Cat# M00652

Deposited data
H11-6HI-53DE-FG VLP

This paper

EMD-63835

Experimental models: Cell lines
HEK293FT

ATCC

RRID: CVCL_6911

Experimental models: Organisms/strains
BALB/c mice

Slac Laboratiry Animal
Co.,shanghai, China

N/A

Oligonucleotides

Recombinant DNA
HPV11/6/53 chimeric L1

This paper

N/A

Software and algorithms
PyMOL

PyMOL

https://pymol.org/

GraphPad Prism (version 8.0.1)

Graphpad

https://www.graphpa
d.com/

ChimeraX software

Goddard, T. D. et al.

https://www.cgl.ucsf.
edu/chimerax/

EPU

Thermo

Fisher

Scientific

N/A

SPSS Statistics software

IBM

https://www.ibm.com
/products/spss-
statistics

Clustal Omega software

EMBL-EBI

https://www.ebi.ac.u
k/Tools/msa/clustalo/

Journal Pre-proof

MicroCal Origin 8.0 software

OriginLab Corporation

https://www.originlab
.com/

cisTEM software

Grant Lab

https://cistem.org/

Other

Experimental model and study participant details

604

Mice

605

All experiments were conducted using age-matched (6-8 weeks) female

606

BALB/c mice, purchased from SLAC Laboratory Animal Co., Shanghai, China,

607

and maintained under specific-pathogen-free conditions. The sex of the mice

608

was female only; no comparison of sex/gender effects was performed, which is

609

acknowledged as a limitation of this study. The experimental protocols were

610

approved by the Xiamen University Laboratory Animal Management Ethics

611

Committee (Protocol No. XMULA20220301), and all procedures were

612

performed in strict compliance with animal ethics guidelines and approved

613

protocols.

614

Cell lines

615

HEK293FT cell line was purchased from Thermo Fisher (Cat# R70007), and

616

cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with

617

10% Pansera ES, Fetal bovine serum (PAN Biotech, Cat# ST30-2620), 1%

618

MEM Non-Essential Amino Acids Solution (Thermo Fisher Scientific, Cat#

619

11140-050) and 1% penicillin–streptomycin (BasalMedia, Cat# S110JV) at

620

37 °C and 5% CO2. The HEK293FT cell line has been authenticated by the

621

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supplier. Mycoplasma contamination testing was not performed, which is a

622

limitation of the study.

623

Method details

624

Phylogenetic relationships analysis of the HPV L1 sequences

625

L1 DNA sequences of 46 HPV types were retrieved from the International

626

Human Papillomavirus Reference Center

. Sequence alignment was

627

performed using Clustal Omega

, and phylogenetic relationships were

628

analyzed with the Neighbor-Joining method in MEGA 12

. Phylogenetic trees

629

were visualized using the Interactive Tree of Life (iTOL)

630

HPV virus-like particle preparation

631

The construction and assembly validation of H11-6HI and HPV53 VLPs were

632

carried out as previously described

30,39

. In this study, chimeric L1 genes

633

incorporating sequences from both H11-6HI and HPV53 were designed based

634

on those earlier constructs, synthesized using Gibson assembly, and cloned

635

into the pTO-T7 expression vector for expression in

E. coli

strain ER2566. The

636

transformed

E. coli

cells were cultured in LB liquid medium at 37°C until the

637

optical density at 600 nm (OD

600

) reached 0.6–0.7. Protein expression was then

638

induced with 5 μM isopropyl-β-d-thiogalactopyranoside (IPTG) at 25°C for 10

639

hours. Harvested cells were resuspended in lysis buffer (50 mM Tris-Base [pH

640

7.2], 10 mM EDTA, and 0.3 M NaCl) and lysed by sonication. The lysate was

641

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clarified by centrifugation, and the supernatant was purified using SP or XS

642

sepharose chromatography (GE Healthcare). To promote VLP self-assembly,

643

the purified L1 proteins were dialyzed against a neutral buffer containing 10 mM

644

phosphate buffer (pH 6.5) and 0.5 M NaCl without dithiothreitol. The VLPs were

645

further purified using Superdex 200 gel filtration chromatography.

646

SDS-PAGE and Western Blotting

647

SDS-PAGE was performed according to the modified method of Laemmli

648

Samples were diluted to a final protein concentration of 0.3-0.5 mg/mL and

649

mixed with an equal volume of Laemmli sample buffer (125 mM Tris-HCl, pH

650

6.8, 4% w/v SDS, 20% w/v glycerol, 200 mM dithiothreitol, and 0.002% w/v

651

bromophenol blue). The mixtures were heated at 100°C for 10 min, and 10

652

microliters of each sample were loaded onto a 10% polyacrylamide separating

653

gel under reducing conditions. Following electrophoresis, the gels were stained

654

with Coomassie Brilliant Blue and destained with a solution of 33% methanol,

655

10% acetic acid, and 57% distilled water until the protein bands became visible.

656

Separated proteins were then transferred to nitrocellulose membranes using

657

Trans-Blot® Turbo™ 5x Transfer Buffer (Bio-Rad) at 80 volts for 1.5 hours (Mini

658

Trans-Blot Cell, Bio-Rad). The membranes were blocked with 5% skimmed milk

659

for 1 hour at room temperature, then incubated with HPV broad-spectrum linear

660

monoclonal antibody 4B3 at a 1:1000 dilution for 1 hour. Membranes were

661

washed five times with PBS containing 0.1% v/v Tween-20 (PBST) (WANTAI

662

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BioPharm), followed by incubation with goat anti-mouse alkaline phosphatase-

663

conjugated antibodies (Abcam; Cambridge, UK) at a 1:5000 dilution for 1 hour.

664

The color development was performed using NBT/BCIP reagent (Pierce

665

Biotechnology; Rockford, IL) for 5 min.

666

Negative-stain electron microscopy

667

Purified WT and chimeric VLPs were diluted to 0.2 mg/mL with phosphate buffer

668

(pH 6.5, containing 0.5 M NaCl) and adsorbed onto 200-mesh carbon-coated

669

copper grids (Quantifoil Micro Tools) at room temperature for 2 min. Following

670

adsorption, the grids were stained with 2% phosphotungstic acid (pH 6.4) for 1

671

min. Specimens were examined and imaged using an FEI Tecnai T12

672

transmission electron microscope (TEM) operating at 120 kV and a

673

magnification of 15,000×.

674

High-performance size exclusion chromatography (HPSEC)

675

The homogeneity analysis of WT and chimeric VLPs was conducted using a

676

high-performance size exclusion chromatography (HPSEC) system (Waters

677

Corporation) equipped with a TSK G5000 PWXL column (7.5 mm × 300 mm;

678

TOSOH, Tokyo, Japan). The mobile phase consisted of a phosphate buffer

679

containing 0.5 M NaCl at pH 6.5, with a flow rate maintained at 0.5 mL/min.

680

Detection was performed at a wavelength of 280 nm.

681

Dynamic light scattering (DLS)

682

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For DLS experiments, VLPs samples with a final concentration of 0.5 mg/mL

683

were centrifuged at 13,000 rpm for 10 min to remove large particulate matter.

684

The clarified samples were then carefully transferred into clean, plastic cuvettes

685

with a 10-mm path length, ensuring overfilling of the 50-μL sample cells.

686

Measurements were conducted using a NanoBrook Omni DLS system

687

(Brookhaven Instruments). Data collection and analysis followed the standard

688

procedures provided by the instrument manufacturer. The hydrodynamic radius

689

(Rh) of the particles was calculated using the Stokes-Einstein equation. The

690

mean hydrodynamic particle size distribution of WT and chimeric VLPs was

691

determined from the average of three separate acquisitions.

692

Differential scanning calorimetry (DSC)

693

The thermal stability of the WT and chimeric VLPs was evaluated using a

694

MicroCal VP-Capillary DSC system (GE Healthcare, MicroCal Products Group,

695

Northampton, MA). Samples were diluted to a concentration of 0.5 mg/mL in a

696

phosphate buffer (pH 6.5, containing 0.5 M NaCl). DSC was performed across

697

a temperature range of 10°C to 90°C at a scanning rate of 100°C per hour. Data,

698

including DSC curves and melting temperatures (Tm), were analyzed using

699

MicroCal Origin 8.0 software (OriginLab Corp., Northampton, MA).

700

Cryo-electron microscopy (Cryo-EM)

701

Aliquots of 3 μL of purified H11-6HI-53DE-FG virus-like particles (VLPs) (2.0

702

mg/mL) were applied to glow-discharged Quantifoil grids (R1.2/1.3, 200 mesh;

703

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Quantifoil Micro Tools) using a Vitrobot Mark IV (Thermo Fisher Scientific) at

704

100% humidity and 4°C. Cryo-electron microscopy (cryo-EM) images were

705

captured on a FEI Tecnai F30 transmission electron microscope (Thermo

706

Fisher Scientific) at 300 kV, equipped with a Falcon III direct electron detector.

707

The electron acceleration voltage was set to 300 kV with 93,000× magnification,

708

resulting in a pixel size of 1.120 Å. Data were automatically collected with

709

Thermo Fisher EPU software. Icosahedral 3D reconstructions were performed

710

using AUTO3DEM

or cisTEM

software. Resolution of final 3D maps was

711

determined by the gold-standard Fourier shell correlation (FSC) curve at a

712

cutoff of 0.143

. Visualizations were generated using ChimeraX software

713

nzyme-linked immunosorbent assay (ELISA)

714

Purified WT or chimeric VLPs were coated onto 96-well microplates at a

715

concentration of 300 ng per well and incubated for 2 h at 37°C. Following a

716

single wash with PBST buffer (20 mmol/L PBS [pH 7.4], 150 mmol/L NaCl, and

717

0.05% Tween-20), the plates were blocked with 200 μL of enzyme dilution buffer

718

(PBS containing 0.25% casein, 1% gelatin, and 0.05% proclin-300),

719

supplemented with 10% sucrose. Subsequently, the wells were incubated with

720

100 μL of 3-fold serially diluted vaccinated mouse serum, starting at a dilution

721

of 1:2000, for 45 min at 37°C. Wells were then washed five times with PBST

722

buffer and incubated with HRP-conjugated anti-mouse IgG for 45 min at 37°C,

723

followed by another five washes. Finally, 100 μL of substrate solution (WANTAI

724

Journal Pre-proof

BioPharm) was added to each well and incubated for 15 min at 37°C. The

725

reaction was then quenched with the addition of 50 μL of 2 mol/L H

. Optical

726

density (OD) values were measured at 450 nm, with a reference wavelength of

727

630 nm. Serum binding titers were calculated using GraphPad Prism software

728

(GraphPad Software, San Diego, CA).

729

HPV pseudovirus preparation

730

The pvitro-L1-L2 co-expression vectors and pcDNA3.1-EGFP used in the

731

experiment were kindly provided by Dr. J. T. Schiller. Plasmids carrying codon-

732

optimized HPV L1 and L2 genes were co-transfected into 293FT cells (Thermo

733

Fisher, Cat# R70007) in a 1:1 mass ratio with the pcDNA3.1-EGFP plasmid.

734

Transfections were performed using Polyethylenimine Linear (PEI) MW40000

735

(rapid lysis; Yeasen, Cat# 40816ES03). The cells were harvested 72 hours

736

post-transfection, after a single wash with DPBS. Subsequently, the collected

737

cells were lysed in a lysis buffer comprising 0.5% Brij58 (Sigma-Aldrich), 0.2%

738

Benzonase (Merck Millipore, Darmstadt, Germany), 0.2% PlasmidSafe ATP-

739

Dependent DNase (Epicenter Biotechnologies, Madison, WI), and DPBS-Mg

740

solution. The lysates were then incubated at 37°C for 16-18 hours. After

741

incubation, the lysates were treated with ice-water for 15 minutes at 4°C.

742

Following this, 5 M NaCl was added to the lysates at a volume equal to 0.19

743

times the lysate volume. The resultant pseudovirus was harvested for use in

744

subsequent neutralization assays. The 50% Tissue Culture Infective Dose

745

Journal Pre-proof

(TCID

) of the pseudovirus was determined using the Reed-Muench method

746

, as previously described

The pseudoviruses were then diluted to 3 × 10^5

747

TCID

/μL for the Pseudovirus-based Neutralization Assay (PBNA).

748

Pseudovirus-based neutralization assay (PBNA)

749

To detect serum neutralizing antibody titers, mouse sera were initially diluted in

750

ES serum-containing DMEM to various starting concentrations (ranging from

751

2.5 to 1000). These initial dilutions were then subjected to 2-fold serial dilutions,

752

maintaining a total volume of 60 μL for each dilution. The diluted sera (60 μL)

753

were subsequently mixed with an equal volume (60 μL) of HPV PsV diluent,

754

which contained a PsV concentration of 3 × 10^5 TCID

/μL. The sera-PsV

755

mixtures were then incubated at 37°C for 1 h to allow interaction. Following this

756

incubation, 100 μL of each mixture was added to HEK293FT cells that had been

757

pre-seeded in 96-well plates at a density of 1.5 × 10^4 cells per well and

758

incubated for 4-6 h at 37°C to allow adhesion. The cells were then incubated

759

with the mixtures for an additional 72 h at 37°C. The extent of PsV infection was

760

quantified using the ELISPOT method to count the number of green fluorescent

761

spots in each well. Wells with spot counts lower than 50% of the average value

762

of the negative control wells were considered to indicate effective inhibition of

763

PsV infection due to the presence of serum neutralizing antibodies. The

764

neutralization titer of the antibody was calculated as the log10 of the highest

765

serum dilution that achieved greater than 50% inhibition of infection.

766

Journal Pre-proof

Vaccine Immunogenicity Evaluation

767

Female BALB/c mice (n = 5), aged 6-8 weeks, were immunized intraperitoneally

768

with 1 mL of vaccine following a prime-boost-boost regimen on days 0, 14, and

769

28. Each immunization consisted of either chimeric or WT VLPs formulated with

770

aluminum adjuvant at doses of 5.0, 1.0, or 0.2 μg per dose. The final endotoxin

771

concentration of all antigen preparations was validated by the Limulus

772

Amebocyte Lysate (LAL) assay (Lonza), <1 EU/μg

. Serum samples were

773

collected at week 6, and neutralizing titers were assessed using the PBNA.

774

Analysis

775

Female BALB/c mice (n = 8) were administered a single intraperitoneal

776

vaccination with varying doses (0.011, 0.033, 0.100, 0.300, or 0.900 μg) of

777

either WT or chimeric VLPs mixed with aluminum adjuvant. Serum samples

778

were collected five weeks post-immunization. Neutralizing antibody responses

779

were assessed using pseudovirus-based cell models for HPV11, HPV6, and

780

HPV53. The results were analyzed using the Reed and Muench method

781

Quantification and statistical analysis

782

Statistical analyses were performed using SPSS Statistics software (IBM Corp.,

783

Armonk, NY, USA). Data were subjected to one-way or two-way analysis of

784

variance (ANOVA) and are presented as mean ± standard deviation (SD).

785

Statistical significance was denoted as follows: *

< 0.05, **

< 0.01, ***

786

Journal Pre-proof

References:

788

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