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Construction of a Vero cell line expression human KREMEN1 for the development of CVA6 vaccines

Virology Journal volume 22, Article number: 12 (2025) Cite this article

Abstract

Coxsackievirus A6 (CVA6) has emerged as a major pathogen causing hand, foot and mouth disease (HFMD) outbreaks worldwide. The CVA6 epidemic poses a new challenge in HFMD control since there is currently no vaccine available against CVA6 infections. The Vero cell line has been widely used in vaccine production, particularly in the preparation of viral vaccines, including poliovirus vaccines and EV71 vaccines. Unfortunately, most CVA6 strains failed to propagate effectively on Vero cells. The expression level of virus-specific receptors on the cell membrane significantly influences viral infection. Here, a Vero cell line with stable over-expressing of KREMEN1 (KRM1), a crucial receptor for CVA6, was constructed using the lentivirus system. The cloned cell line, called Vero-KRM1_#11, could efficiently support most CVA6 infections. The propagation of CVA6-TW00141 strain on Vero-KRM1_#11 was equal to that on RD cells. After four passages, the virus batch was obtained with a titer of about 107 TCID50/mL. Moreover, the purified CVA6 particles produced from Vero-KRM1_#11 or RD cells both could induce high and comparable levels of IgG and neutralizing antibodies. Importantly, passive transfer of the antisera from CVA6-vaccined mice showed 100% preventive efficacy against CVA6 infection in mice. Therefore, KRM1-expressing cells have the potential to serve as a valuable tool for the development and production of CVA6 or polyvalent HFMD vaccines.

Introduction

Hand, foot and mouth disease (HFMD) is a highly contagious infection prevalent in children, caused by over 20 serotypes of Enteroviruses (EVs), primarily affecting infants and children under 5 years of age [1]. The typical symptoms include fever, oral herpes and vesicular rashes on the hands and feet. Although most patients recover spontaneously within two weeks, some may develop severe complications, such as cardiovascular or neurological disorders, even leading to death [2]. Enterovirus A71 (EV71) and Coxsackievirus A16 (CVA16) are the common pathogens of HFMD outbreaks [3, 4]. However, following an outbreak in Finland in 2008 caused by Coxsackievirus A6 (CVA6) [5], this virus has emerged as a significant causative agent of HFMD epidemics across Asia [6, 7], North America [8] and Europe [9]. Additionally, CVA6 infection can manifest in a range of atypical clinical symptoms, including vesiculobullous exanthema [10, 11], onychomadesis [12] and desquamation [13], and has been associated with severe HFMD [6, 14]. Notably, there has been an increase in cases of HFMD among adults linked to CVA6 [15]. The CVA6 epidemic presents new challenges for HFMD control and underscores the urgency of developing effective preventative and management strategies.

Currently, the predominant treatment strategy for HFMD is symptomatic management. Three monovalent inactivated EV71 vaccines, novel and exclusively related to HFMD, have been commercially available in 2016 [16]. These vaccines have demonstrated significant efficacy in protecting against HFMD and associated conditions caused by EV71, but they do not offer protection against other major emerging causes of HFMD, such as CVA6 [17, 18]. Studies have employed serial passaging of viruses in Vero cells to produce adapted viral strains, such as Marek’s disease virus and infectious bursal disease virus. Confluent Vero monolayers were grown in 25 or 75 cm2 cell culture for virus infection, and virus was harvested after the appearance of a maximal CPE by trypsinisation with trypsin/versine, which were harvested and used for subsequent passaging adaptation [19, 20]. This method facilitates the accumulation of beneficial mutations, resulting in viral strains with attenuated pathogenicity suitable for vaccine development. However, progress has been impeded by major challenges, including the low isolation rate of CVA6 strains-potentially less than 1%-and difficulties in cultivating and adapting them on commonly used vaccine production cell lines, such as Vero and MRC-5 [21, 22]. While some studies have shown that formaldehyde-inactivated CVA6 particles exhibit strong immunogenicity in mice, unfortunately, these particles are derived from CVA6-infected RD cell cultures, and these strains are more susceptible to RD cells [23,24,25]. Since RD cells are tumorigenic and unsuitable for vaccine production, constructing cell lines that can efficiently cultivate CVA6 will significantly facilitate vaccine development.

The interaction between a virus and its receptor on the host cell surface is the primary step in viral infection. The cell surface molecule, Kringle containing transmembrane protein 1, KREMEN1 (KRM1) is widely expressed in various human tissues and cells and is present from the early stages of embryonic development through all stages of growth. KRM1 is expressed at high levels in the heart, oesophagus, muscle tissue, among others [26, 27]. KRM1 regulates WNT signaling by binding to DKK and LRP5/6, facilitating the uptake of this complex through clathrin-mediated endocytosis [28]. KRM1 has been identified as an entry receptor for the major group of Enterovirus type A (EV-A) species, including CVA6 and CVA10 [29]. Infection assays showed that blocking KRM1 significantly reduce virus entry into cells, and reintroducing KRM1 expression in the knock-out cells restores the infection, indicating that these viruses critically rely on KRM1 as a host factor [29]. Hence, the insusceptibility of CVA6 in Vero cell lines might be mitigated by constructing cell lines with stable expression of KRM1. In this study, we attempted to construct Vero cell lines with stable KRM1 expression to achieve efficient infection and amplification of CVA6 in diploid cells, thereby providing a potential candidate cell line for CVA6 vaccine research and development.

Materials and methods

Cell lines

Human rhabdomyosarcoma (RD, ATCC# CCL-136), HEK293T/17 (ATCC# CRL-11268) and Verda Reno (Vero, ATCC# CCL-81) cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM, GIBCO) supplemented with 10% fetal bovine serum (FBS, Gibco), 100 μg/mL streptomycin, 100 U/mL penicillin, and 2 mM L-glutamine.

Viruses

The CVA6-TW-00141 strain (GenBank accession no. KR706309, D1 subtype) and the EV71-FJ10 strain were respectively isolated from HFMD clinical specimens in Fujian Province, China. The CVA6-HLJ11 stain (GenBank accession no. MN845762, D2 subtype), CVA6-YN17 strain (GenBank accession no. MN845882, D3 subtype) and CVA6-GD13 strain (GenBank accession no. KF682363, D3 subtype) infectious clones were respectively prepared by transfecting mRNA transcripts, which were synthesized in vitro using a MEGAscript T7 transcription kit (Thermo Fisher Scientific) from linearized full-length cDNA, into RD cells, as previously described [30]. The rescued viruses were propagated in RD cells at 37 ℃, and the supernatants were harvested by freeze-thawing and stored in aliquots at -80 ℃.

Mice

BALB/c mice were obtained from the Beijing Vital River Laboratory Animal Technology Co., Ltd, China. All mice were maintained in a specific-pathogenfree (SPF) facility of Xiamen University. For infection studies, mice were maintained in animal biosafety level 2 (ASBL-2) facility at 23 ℃. All mice were allowed free access to sterilized water and irradiated diet, and provided with a 12 h light-dark cycle. Animal protocols were performed in accordance with the guidelines of the Xiamen University Institutional Committee for the Care and Use of Laboratory Animals, and approved by the Xiamen University Laboratory Animal Center (approval code: XMULAC20160049).

Construction of a KRM1 expression plasmid

The fragments containing the coding sequences of human KRM1 (GenBank accession no. BC063787) were synthesized from Sangon Biotech. The KRM1 gene, flanked by 5’ Xba I and 3’ Pac I sites, was then cloned into pLV-EF1α-IRES-Puro vector, thus creating a pLV-EF1α-KRM1-IRES-Puro plasmid. The constructed plasmid includes the KRM1 gene with a EF1α promoter, as well as a puromycin gene for the selection of transfected cells.

Construction of a transfected Vero cell line expressing KRM1

Vero cells were pre-seeded at 1 × 105 cells per well into 24-well plates and transfected with pLV-EF1α-KRM1-IRES-Puro using Lipofectamine®3000 transfection reagent (Invitrogen) following manufacturer’s instructions. After 48 h post-transfection, The transient expression of KRM1 Vero cells were then infected with CVA6 or EV71. A Vero cell line with stable expressing of KRM1 was constructed using the lentiviral transduction system. In brief, recombinant lentiviruses were packaged by co-transfecting with pLV-EF1α-KRM1-IRES-Puro, psPAX2 (Addgene plasmid #12260) and pMD2.G (Addgene plasmid #12259) into HEK-293T/17 cells using Lipofectamine®3000 transfection reagent. After 8 h post-transfection, the medium was changed to DMEM containing 10% FBS. The supernatants were then harvested at 48 h after transfection and then filtrated by a 0.45 μm pore size filter. After infection of Vero cells with recombinant lentiviruses and incubation for genome integration, stably transformed cells were selected with puromycin (1 μg/mL) and cloned. Two clones were selected for further study, Vero-KRM1_#11 and Vero-KRM1_#26.

RNA isolation and RT-PCR

Total RNA was isolated from 1 × 106 cells using a cultured cell RNA extraction kit (MolPure® Cell RNA Kit, YEASEN) following the manufacturer’s instructions. The levels of KRM1 mRNA were quantified by real time RT-PCR using a one-step RT-PCR kit (GenMag Bio). RT-PCR reaction was performed with the CFX96 Real-Time PCR Detection System (Bio-Rad). Gene-specific primers (forward, 5′-AGCATCCATACAACACTCTGAA-3′; reverse, 5′-CTTCCAGTAGACACCATC.

-CTC-3′; probe, 5′-FAM-AATAGTTGTGCTCACCCAGGCCC-BHQ1-3′) were used for the RT-PCR experiment. RT-PCR was carried out as follows: 15 min at 50 °C and 15 min at 95 °C, followed by 42 cycles of 94 °C for 15 s and 55 °C for 45 s. Each reaction was performed in triplicate. The pLV-EF1α-KRM1-IRES-Puro plasmid was used as a standard sample. A standard curve, generated from serially diluted samples, was used to quantify the number of copies of the gene.

Western blot

1 × 106 RD, Vero, Vero-KRM1_#11, Vero-KRM1_#26 cells were wash in ice-cold PBS and lysed for 10 min in RIPA lysis buffer (BEYOTIME). The cell lysates were centrifuged at 12,000 rpm for 10 min to remove debris. The suspernatant was loaded onto 4–20% Bis-Tris SDS-PAGE gels (SurePAGE™, GenScript) and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking in 5% skim milk in PBS for 1 h, the membranes were incubated with primary human KRM1-specific polyclonal antibody (1 μg/mL, LS-B10086, Lifespan Biosciences) for 1 h. After three washes with PBST (0.05% Tween-20 in PBS), the membranes were further incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit (GAR-HRP) second antibodies (1:5000 dilution) for 30 min. GAPDH (HRP-conjugated GAPDH antibody, Proteintech, 1:10,000 dilution) was used as the internal reference. After three washes with PBST, the membranes were visualized by chemiluminescence (SuperSignalTM West Femto Maximum Sensitivity Substrate, Thermo Fisher Scientific), and the images were obtained by FUSION FX EDGE SPECTRA (Vilber Bio Imaging). All procedures were conducted at room temperature.

Immunofluorescence assay

RD, Vero and Vero-KRM1_#11 cells were pre-seeded at 1 × 105 cells per well into 24-well plates on the 15 mm cover glasses (Nest) and then infected with CVA6 or EV71 at a multiplicity of infection (MOI) of 1. At 12 h post-infection (hpi), cells were fixed with 4% paraformaldehyde for 30 min in the dark and permeabilized with 0.3% Triton X-100 diluted in PBS for 15 min. After blocking with 2% bovine serum albumin (BSA) diluted in PBS at 37℃ for 20 min, cells were incubated with CVA6-specific mAb 4D6 or EV71-specific mAb CT11F9 [31] (4 μg/mL) at 37℃ for 1 h. After three washes with PBST, cells were incubated with Alexa Fluor® 488 goat anti-mouse (GAM-AF488) second antibody (1:1000 dilution) at 37℃ for 30 min in the dark. After three washes with PBST, cells nuclei were stained with 40, 6-diamidino-2-phenylindole (DAPI) for 5 min. Finally, cells were observed and image data were captured using an EVOS M7000 imaging system (Thermo Fisher Scientific).

Viral growth kinetics

RD, Vero or Vero-KRM1_#11 were pre-seeded at 1 × 105 cells per well into 24-well plates and then infected with CVA6-TW-00141 strain at a multiplicity of infection (MOI) of 0.01. Virus propagation was performed at 37℃. At 12, 24, 36, 48, 60, 72, 84, 96 and 108 hpi, one plate was harversted per cell line. Virus was released from the cells by freeze-thawing. After centrifugation, the virus samples were collected and stored at -80℃. Once the final time point was acquired, all samples were titrated using the virus titration assay.

Virus titration assay

Virus titers were determined by the median end-point titration using RD, Vero or Vero-KRM1 cells and expressed as the 50% tissue culture infectious dose (TCID50). In brief, cells were pre-seeded at 5 × 103 cells per well into 96-well plates and then infected with ten-fold serial dilutions of the virus. Each dilution was inoculated with 8 wells for 100 μL per well. After an incubation period of 7 days at 37℃, cells were observed for CPE. The TCID50 values were were calculated according to the Behrens-Kärber method.

Virus production and purification

The virus production and purification experiments were conducted as previously described [32]. In brief, CVA6-TW-00141 strain was grown in RD or Vero-KRM1 cells at a MOI of 0.1 at 37℃, respectively. Virus was harvested 3 days post-infection (dpi), and then centrifuged at 25,000 × g for 30 min to remove the cell debris, and precipitated by using 8% polyethylene glycol (PEG) 8,000 and 0.3 M NaCl in 0.1 M phosphate buffer (PB, pH7.4) at 4℃ for 12 h. After centrifugation, virus was resuspended and then sedimented through a linear 15–45% (w/v) sucrose density gradient at 153,900 g for 4.5 h at 4℃ using a Beckman SW41 Ti rotor. The fractions were collected and independently dialysed against PBS and concentrated by an Ultra-4 centrifugal concentrator (50 kDa, Millipore). The quantity and quality of CVA6 particles were examined by negative-staining electron microscopy (TEM). The protein composition was analyzed by SDS-PAGE.

Vaccine preparation and immunization of mice

The purified CVA6 particles were inactivated by adding β-propiolactone and mixed with an equal volume of aluminum adjuvant. Female BALB/c mice aged 6–8 weeks old (n = 5 per group) were immunized intraperitoneally (i.p.) at a dosage of 1.5 μg of inactivated CVA6 vaccine in a volume of 500 μL. Each mice was immunized at weeks 0 and 2, and subsequently bled at weeks 0, 4, 6, 8 and 10. The blood samples were centrifuged at 12,000 rpm at 4 ℃ for 15 min. The sera were heat-inactivated at 56 ℃ for 30 min, and then stored at -20 °C for ELISA and neutralization assays.

ELISA

The 96-well ELISA plates were coated with 50 ng per well of purified CVA6 particles in PBS. After incubation at 4℃ overnight, the plates were washed once with PBST and saturated with the saturation buffer (0.25% casein and 1% gelatin in PBS buffer) at 37℃ for 2 h. Serum samples were diluted in PBS and added to the plates, followed by incubation at 37℃ for 1 h. After five washes with PBST, the plates were added with GAM-HRP (1:5000 dilution) and incubated at 37℃ for 30 min. For color development, after five washes with PBST, the plates were incubated with a solution of 3,3′,5,5′-tetramethylbenzidine (TMB) at 37℃ for 15 min, and the reaction was terminated with 2 M H2SO4. Finally, the absorbance was measured at 450/620 nm with a plate reader.

In vitro neutralization assay

RD cells were pre-seeded at 1 × 104 cells per well into 96-well plates. Serum samples from mice were two-fold serially diluted ranging from 1:32 to 1:2,048 and mixed with equal volume of CVA6-TW-00141 strain (100 TCID50) in 96-well plates at 37℃ for 2 h. Virus-serum mixtures were added into the cell well and then incubated at 35℃ for 7 days. Each well was observed by microscope. The neutralization titers were the averages of the triplicate readings calculated based on the highest dilution in over 50% cytopathic effect (CPE). The neutralizing titer of sera with values ≥ 32 was considered the threshold for positivity.

In vivo protection test

One-day-old BALB/c mice (n = 6 per group) were firstly administered i.p. with 100 μL diluted sera 6 h before challenged i.p. with 100 μL of CVA6-TW-00141 strain (5 × 105 TCID50 per mouse). The mice in the control group were treated with PBS via the same route. All mice were monitored daily for survival, clinical illness and weight until 20 dpi. The grade of clinical disease was scored as follows: 0, healthy; 1, lethargy and inactivity; 2, wasting; 3, limb weakness; 4, hind-limb paralysis; and 5, morbidity and death.

Statistics

Statistical analysis was performed using GraphPad Prism version 8.0. Anti-CVA6 IgG titers and neutralizing titers were presented as the geometric mean titer (GMT) and converted to the logarithmic scale. The results are expressed in terms of the means ± standard deviations (SD) and analyzed by using the unpaired Student’s t-test. The health scores were shown as means. The survival curves were evaluated by the Log-rank (Mantel-Cox) test. A P value < 0.05 was considered statistically significant. Statistical details of the experiments can be found in the Results and Figure legends.

Results

Transient expression of KRM1 renders Vero cells susceptible to infection of CVA6

Vero cells are commonly used as stromal cells for vaccine production, particularly in the preparation of viral vaccines, including poliovirus and EV71 vaccines. The interaction of virus and receptor is the key step in viral infection. To determine whether Vero cells can support replication of CVA6 when KRM1 is overexpressed, a KRM1-encoding plasmid, pLV-EF1α-KRM1-IRES-Puro, was transiently transfected into Vero cells. Two days post-transfection with the KRM1 gene, the Vero cells were infected with several CVA6 strains or one EV71 strain at an MOI of 1, and the CPE was monitored for five days. We found that transiently KRM1-transfected Vero supported CVA6 infection and observed CPE similarly to EV71 infection, while no development CPE on wild-type Vero cells (Fig. 1A). Additionally, EV71 or CVA6 viruses were inoculated into pre-seeded cells at an MOI of 1. After 12 h of infection, the cells were fixed and subjected to immunofluorescence detection, using EV71 or CVA6 specific antibodies as the primary antibody and GAM-AF488 as the secondary antibody. The results showed that positive signals were detected in Vero-KRM1 cells infected with CVA6, but not on wild-type Vero cells, while positive signals were detected in both Vero-KRM1 and Vero cells infected with EV71 (Fig. 1B). These results confirm that increased overexpression of the KRM1 receptor protein did not significantly affect EV71 infection but did promotes the susceptibility of Vero to CVA6 virus.

Fig. 1
figure 1

Transient expression of KRM1 renders vero cells susceptible to CVA6 infection. (A) Representative cytopathic effect images of CVA6 and EV71 infections in wide-type Vero cells and transfected Vero cells, and control cells without infection. Scale bar, 100 μm. (B) Representative fluorescence confocal images of CVA6 and EV71 infections in wide-type Vero cells and transfected Vero cells, and control cells without infection. The primary antibodies were CVA6-specific mAb 4D6 or EV71-specific mAb CT11F9 and the second antibody (green) was Alexa Fluor 488-conjugated goat anti-mouse. The nuclei were stained with DAPI (blue). Scale bar, 100 μm

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Construction of a Vero cell line stably expressing KRM1

Given the effective CVA6 infection observed in Vero cells with transient overexpression of KRM1, we attempted to construct a stable Vero cell line overexpressing KRM1 using a lentiviral system, employing puromycin selection and limiting dilution methods to select and isolate positive cells (Fig. 2A). After multiple rounds of screening, we obtained two Vero cell clones, named Vero-KRM1_#11 and Vero-KRM1_#26. Western blot analysis revealed that these clones had relatively higher levels of KRM1 expression compared to untransfected Vero cells and RD cells (Fig. 2B). Further, real-time PCR analysis using gene-specific primers for KRM1 showed that the KRM1 mRNA expression levels in Vero-KRM1_#11 and Vero-KRM1_#26 were 1.12 × 108 and 9.3 × 107 copies/million cells, respectively, which are about 100 times higher than those in wild-type Vero cells and RD cells (Fig. 2C). These results indicate that the Vero-KRM1_#11 and Vero-KRM1_#26 cell lines have been successfully modified and can stably express high levels of KRM1. We monitored the cell counts daily over a 10-day period after initially seeding 10,000 cells per well in a 24-well plate to compare the growth rates between parental Vero and Vero-KRM1_#11. The growth rates of the two types of cells are similar (Supplementary Fig. 1). Initially, the cells grow slowly during a period known as the lag phase. After 3–7 days, they enter the logarithmic growth phase, where the growth rate peaks and cell viability is at its best. Eventually, the cells transition into the stationary growth phase. We further evaluated the stability of genetic modifications during cell culture passaging. The cells from 10, 12, 14 and 16 passages were chosen to analyze the mRNA and expression levels of KRM1 protein. The results showed the levels of KRM1_#11 mRNA were about 1 × 108 copies/million cells (Supplementary Fig. 2). These reveals that after 16 consecutive passages, the expression level of KRM1 in the cells remained essentially constant. Given the higher KRM1 expression in Vero-KRM1_#11, this clone was selected for subsequent virus infection verification.

Fig. 2
figure 2

Construction of a transfected Vero cell line with stable expressing of KRM1. (A) Schematic of the experimental workflow for constructing a transfected Vero cell line with stable expressing of KRM1. (B) Western blot analysis of RD, Vero, Vero-KRM1_#11 and Vero-KRM1_#26 cells with anti-KRM1 polyclonal antibody. GAPDH was used as the internal reference. (C) The levels of KRM1 mRNA as measured by quantitative real-time RT-PCR. The experiments were performed independently in triplicate. The values are expressed as the mean ± SD. Statistical significance was determined using unpaired Student’s t test. ****p < 0.0001, n.s., not significant

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Vero-KRM1 susceptible to infection and replication of CVA6

To confirm the susceptibility and replication ability of the modified Vero-KRM1_#11 cells to CVA6, infection validation was performed using four representative CVA6 strains of different genotypes, TW-00141, GD13, YN17 and HLJ11. Following infection for 24 h at an MOI of 1, all tested CVA6 strains induced noticeable CPE in RD and Vero-KRM1_#11 cells (Fig. 3A). Immunofluorescence assay results showed significant virus-specific green fluorescence signals in CVA6-infected cells, whereas uninfected cells did not display a positive signal (Fig. 3B). To further assess and compare the infection and replication efficiency of CVA6 in Vero-KRM1_#11, RD and Vero cells, cells were preseeded at 1 × 105 cells per well and then infected with CVA6-TW-00141 at an MOI of 0.01 for five days. Viral lysates from infected cells were collected every 12 h and titrated to analyze the viral growth kinetics. The results indicated that CVA6-TW-00141 could effectively replicate in Vero-KRM1_#11 and RD cells, with comparable propagation efficiency in both cell types. As the infection time increased, the viral titer gradually increased. However, CVA6-TW-00141 could not effectively replicate in wide-type Vero cells (Fig. 3C). Subsequently, the CVA6-TW-00141 strain, generated through reverse genetics technology, was used as the initial P0 generation for serial propagation in Vero-KRM1_#11 cells. The viral titer was measured for each generation harvested. The results demonstrated that after continuous passage, the viral titers reached about 107 TCID50/mL from P4 onwards, and the viral titer for 4–20 passages indicates that the titer fluctuates with the number of passages but generally stabilizes within the relatively high titer from 107 to 108 TCID50/mL, meeting the inoculation needs for large-scale virus production (Fig. 3D). These findings indicate that overexpression of KRM1 increases the susceptibility of Vero cells to CVA6, and the Vero-KRM1_#11 cell line is expected to become one of the cellular substrates for the production of CVA6 vaccine.

Fig. 3
figure 3

Vero-KRM1_#11 cells susceptible to replication of CVA6. (A) Representative cytopathic effect images of CVA6-infected Vero-KRM1_#11 cells or uninfected cells. Scale bar, 100 μm. (B) Representative fluorescence confocal images of CVA6-infected Vero-KRM1_#11 cells or uninfected cells. The primary antibody was CVA6-specific mAb 4D6 and the second antibody (green) was Alexa Fluor 488-conjugated goat anti-mouse IgG. The nuclei were stained with DAPI (blue). Scale bar, 50 μm. (C) Growth kinetics of CVA6-TW-00141 stain infection on RD, Vero and Vero-KRM1_#11 cells after infection at an MOI of 0.01. The virus samples were collected at the indicated time points and then titrated. The experiments were independently performed in triplicate. The values are expressed as the mean ± SD. Statistical significance was determined using unpaired Student’s t test. n.s., not significant. (D) Virus titers of CVA6-TW-00141 strain grown on Vero-KRM1_#11 cells from P3 to P20 generation of serial passages. The experiments were performed independently in triplicate. The values are expressed as the mean ± SD

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CVA6 prepared on Vero-KRM1 induced neutralizing antibodies in mice

To evaluate the potential of Vero-KRM1_#11 as a stromal cell line for CVA6 vaccine production, we conducted an experiment to compare the production of the CVA6 particle antigens in Vero-KRM1_#11 cells and RD cells. Cells were infected with CVA6-TW-00141 at an MOI of 1 for three days, and we observed similar CPE in both cell types. To determine the ability of Vero-KRM1_#11 cells to produce viral particles, we collected virus culture supernatant for purification. We used ultracentrifugation with sucrose density gradient and a 50 KDa centrifugal concentrator to purify and enrich the CVA6 particles. We successfully observed CVA6 particles prepared on Vero-KRM1_#11 cells, which had a diameter of approximately 30 nm, similar to those observed in RD cells (Fig. 4A). To determine the protein composition, CVA6 particles produced on Vero-KRM1_#11 and RD cells were subjected to further analysis using SDS-PAGE and western blot. The viral particles produced on both types of cells have similar proteomic compositions, including the capsid proteins VP0, VP1, VP2 and VP3. Of these, VP0, VP1 and VP3 can assemble into procapsid, while VP1, VP2, and VP3 can assemble into A particles (Fig. 4B, Supplementary Fig. 3). These results suggest that the Vero-KRM1_#11 cell line could be a suitable candidate for producing intact CVA6 particles.

Fig. 4
figure 4

Characterization of the purified CVA6-TW-00141 particles. (A) Representative negative-staining TEM images of the purified CVA6-TW-00141 particles prepared on either Vero-KRM1_#11 or RD cells. Scale bars, 200 nm in left pannels and 100 nm in right pannels. (B) SDS-PAGE analysis of the purified CVA6-TW-00141 particles prepared on either Vero-KRM1_#11 or RD cells

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To further evaluate the immunogenicity of viral particles produced by the two types of cells, female BALB/c mice aged six weeks were immunized with a dose of 1.5 μg per injection. Each group of mice was boosted once with the same dose and route two weeks after the initial immunization. As a control, mice were immunized with adjuvant (Fig. 5A). The levels of IgG antibodies that were directed against the purified CVA6 antigens in the immunized mouse serum were assayed by ELISA. Both groups (CVA6 antigens prepared on Vero-KRM1_#11 or RD cells) exhibited potent reactivity to the coated antigens, achieving a total IgG antibody titer of approximately 103 in the serum after the second immunization, whereas sera from the adjuvant control group showed no reactivity (Fig. 5B). The antisera were subsequently analyzed using an in vitro neutralization assay to evaluate their capacity to neutralize CVA6. Post-second immunization, the geometric mean titer (GMT) against CVA6-TW-00141 of serum from mice immunized with CVA6 antigens prepared on Vero-KRM1_#11 cells was from 256 to 1024 from week 4 onwards, and the GMT against other CVA6 strains were from 64 to 1024, a result comparable to that of antigens prepared on RD cells (Fig. 5C, Supplementary Fig. 4). The results indicated good immunogenicity and cross-neutralization of CVA6-TW-00141 prepared on Vero-KRM1_#11.

Fig. 5
figure 5

Immunogenicity andin vivo protection of the purified CVA6-TW-00141 particles. (A) The animal experiment was performed following the procedures explained in the schematic diagram. Groups of 6–8 weeks old BALB/c mice were immunized i.p. with the inactivated CVA6 particles prepared on either Vero-KRM1_#11 or RD cells. A control group was inoculated with adjuvant alone. Each mice was immunized at weeks 0 and 2 with a dosage of 1.5 μg (n = 5 per group), and subsequently bled at weeks 0, 4, 6, 8 and 10. The green arrows represent immunization, and the red arrows represent blood collection. (B and C) Antibody response induced by CVA6 particles in mice was measured. Serum anti-CVA6 IgG titer (B) and neutralizing titer (C) were presented as the geometric mean titre (GMT) and converted to the logarithmic scale. The values are expressed as the mean ± SD. Significance was determined using unpaired Student’s t test. n.s., not significant. (D-F)In vivo animal protective efficacy of antisera collected from mice immunized with the CVA6 particles prepared on Vero-KRM1_#1. One-day-old BALB/c mice were firstly administered with diluted antisera 6 h before challenged with CVA6-TW-00141 strain. Mice were monitored daily for survival (D), clinical illness (E) and weight (F) until 20 dpi. Experiments were repeated independently twice, and one representative result is shown. Survival curves were compared by the log-rank (Mantel-Cox) test. (n = 6; ***p < 0.001)

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In addition, the in vivo protective efficacy of the antisera was assessed using a neonatal mouse model infected with CVA6. The antisera were collected two weeks after the final immunization. One-day-old BALB/c mice were firstly administered i.p. with 1:10 diluted sera, and each mouse was challenged with 5 × 105 TCID50 of CVA6 after 6 h. Upon continuously monitoring of weight and health index of the mice for 20 days, it was observed that antiserum provided 100% protection against the pathogen, with no significant fluctuations in health index or deviations from normal weight growth. In contrast, all mice in the control group died within 6 dpi (Fig. 5D-F). Thus, the CVA6 antigens prepared on Vero-KRM1_#11 cells exhibit strong antigenicity and immunogencity, which is comparable to those prepared on RD cells. These findings demonstrate that Vero-KRM1_#11 cells have great potential as a production cell line for the CVA6 vaccines.

Discussion

In 2008, the first outbreak of HFMD caused by CVA6 occurred in Finland [5, 33]. Since then, CVA6 has continued to circulate globally, emerging as a significant pathogen responsible for HFMD due to its increased pathogenicity, infectivity, and variations in lesion sites and severity resulting from CVA6 gene recombination. In recent years, HFMD caused by CVA6 has rapidly spread across numerous countries and regions, leading to multiple outbreaks. Although the EV71 vaccine has been implemented and both the CVA16 monovalent vaccine and recombinant EV71-CVA16 bivalent vaccine have entered clinical trials, these vaccines do not provide cross-protection against CVA6 [34, 35]. As a new pathogen associated with HFMD outbreaks, CVA6 presents considerable challenges for the prevention and control of HFMD epidemics in China.

When evaluating cells for vaccine production, it is essential to consider factors such as permissiveness to viruses, viral growth, and scalability. If a virus cannot effectively adapt and replicate within the cells, the purpose of cell culture is undermined; similarly, without the capability for mass production, the prospects for product development are not promising. The key to developing a vaccine for human use lies in the availability and security of a cell line with high production capacity, and only a limited number of cell lines have been approved by regulators for this purpose. Vero cells are among the vaccine production cell lines recognized by the World Health Organization and the Chinese Pharmacopoeia, commonly used as viral culture substrates in production. Vero cells can support the proliferation of various viruses, including Japanese encephalitis, poliomyelitis, rabies, and others; notably, they have also been approved for the production of human virus vaccines against the new coronavirus [36]. Furthermore, researchers at the Institute of Biological Products in Beijing have demonstrated that Vero cells exhibit no tumorigenicity after passing through 250 generations. However, it has been observed that most CVA6 strains are difficult to infect and replicate efficiently in Vero cells, which has led to a slow development of CVA6 vaccines.

As we know, KRM1 serves as a host entry receptor for CVA6 [29]. Interestingly, our study revealed that CVA6 strains could induce CPE in RD cells (data not shown), while Vero cells exhibited resistance to CVA6 infection (Fig. 1), despite comparable KRM1 receptor expression levels in both cell types (Fig. 2B-C, Supplementary Fig. 2). This raises the question of whether RD cells possess an additional helper receptor that facilitates viral infection, a hypothesis that warrants further investigation. We would further explore the helper receptor of CVA6 to deepen and enrich our understanding of the molecular mechanisms involved in virus entry. Due to the increased expression of virus-specific receptors, which can enhance the susceptibility of resistant cells to the virus, our study successfully constructed a Vero cell line with stable over-expressing of KRM1 that was found to effectively support the infection by multiple representative strains of CVA6. In Vero-KRM1 cells, CVA6 was able to replicate continuously through multiple passages, and the virus titer was maintained at a stable level of approximately 107 TCID50(Fig. 3D). Furthermore, we prepared CVA6 antigens using Vero-KRM1 cells and observed that the types of CVA6 particles were similar to those prepared in RD cells. Immunization of mice with the purified particle antigens induced antibody levels comparable to those elicited by antigens prepared on RD cells. Therefore, Vero-KRM1 cells represent a promising candidate for CVA6 virus vaccine production.

While the parent Vero cell line has been approved for viral vaccine production, genetic modifications that lead to the overexpression of receptors may have regulatory implications. Consequently, the modified Vero cell line cannot be directly utilized for the clinical production of viral vaccines, as the impact on tumorigenicity remains uncertain. Fortunately, a fully characterized Vero cell line is available as starting material, which could expedite the registration process. For instance, certain virus safety tests may not need to be repeated, and it is feasible to insert transgenes into specific regions of the genomic DNA to mitigate the risk of oncogenic insertion. Vero cell lines that have been registered for vaccine production have been successfully modified to express KRM1 on their surface, and these cell lines have demonstrated stability in KRM1 expression. Many strains of the CVA6 virus have been successfully cultured on Vero-KRM1 cells, which are unable to grow on wild-type Vero cells, indicating that modified Vero cells could serve as a valuable tool for CVA6 vaccine production. The application of receptor overexpressing modified cell lines in human vaccine production necessitates further validation in terms of safety, stability, and other factors. Although the results showed the expression level of KRM1 in the cells remained essentially constant after 16 consecutive passages (Supplementary Fig. 2), the further research should be conducted to answer whether the genetic modification remains stable over extended cell culture passages and whether KRM1 expression or unintended mutations occur over time.

Data availability

No datasets were generated or analysed during the current study.

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Acknowledgements

Not applicable.

Funding

This study was supported by grants from the National Natural Science Foundation of China (32470996, 82101918, 82272310, and 82172248), the Fundamental Research Funds for the Central Universities (20720220006), and the Xiamen Science and Technology Program (2022CXY0102). The funders had no role in the study design, data collection and analysis, decision to publish, or manuscript preparation.

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  1. Dongqing Zhang, Yuxiang Zou and Jiaying Wu contributed equally to this work.

Authors and Affiliations

  1. State Key Laboratory of Vaccines for Infectious Diseases, Xiang An Biomedicine Laboratory, School of Life Sciences, School of Public Health, Xiamen University, Xiamen, 361102, PR China

    Dongqing Zhang, Yuxiang Zou, Jiaying Wu, Longfa Xu, Zhifeng Ke, Yuanyuan Wu, Zhenhong Zhou, Mujin Fang, Ningshao Xia, Rui Zhu & Tong Cheng

  2. National Institute of Diagnostics and Vaccine Development in Infectious Diseases, Collaborative Innovation Center of Biologic Products, National Innovation Platform for Industry-Education Integration in Vaccine Research, Xiamen University, Xiamen, 361102, PR China

    Dongqing Zhang, Yuxiang Zou, Jiaying Wu, Longfa Xu, Zhifeng Ke, Yuanyuan Wu, Zhenhong Zhou, Mujin Fang, Ningshao Xia, Rui Zhu & Tong Cheng

  3. Department of Pediatrics, Zhongshan Hospital, Xiamen University, Xiamen, 361004, PR China

    Ling Chen

  4. Beijing Wantai Biological Pharmacy Enterprise Co., Ltd, Beijing, 102206, PR China

    Henggang Xu & Jianping Chu

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Contributions

R.Z., L.X., D.Z., T.C. and N.X. contributed to the experimental design. D.Z., R.Z., Y.Z., L.C. and T.C. contributed to the manuscript preparation. R.Z., Y.W., D.Z., Z.Z, M.F., Z.K., Y.W., J.C. and H.X. contributed to the virus preparation and characteristic analysis. Y.W., Y.Z., D.Z., J.C. and H.X. contributed to the preparation and in vitro characterization of antibody. D.Z., Y.Z. and J.W. performed the animal experiments. All authors approved the final version. All authors discussed the results and commented on the manuscript.

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Correspondence to Rui Zhu or Tong Cheng.

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Zhang, D., Zou, Y., Wu, J. et al. Construction of a Vero cell line expression human KREMEN1 for the development of CVA6 vaccines. Virol J 22, 12 (2025). https://doi.org/10.1186/s12985-024-02618-1

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