A/Puerto Rico/8/1934 (PR8, H1N1) was preserved by the Laboratory of Preventive Veterinary Medicine, Qingdao Agricultural University. CHO cells (CHO-2H6) lacking the GS gene and MDCK cells (ATCC, CRL-2936) were preserved in the Second Hospital of Nanjing affiliated the University of Chinese Medicine. CHO-2H6 cells were cultured in CHOGrow®302 medium purchased from Yuanpei (Shanghai) Biotechology Co., Ltd. MDCK cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 10 units/mL of penicillin and 10 µg/mL of streptomycin. All cells were maintained at 37 °C and 5% CO₂ atmosphere. All cell lines were validated by short tandem repeats (STR) profiling and tested negative for mycoplasma. 6 to 8 week-old female specific pathogen-free (SPF) BALB/c mice were purchased from Jinan Pengyue (Jinan) Experimental Animal Breeding Co., Ltd., and raised at the Animal Breeding Center of Qingdao Agricultural University with a germ-free environment.

The genes encoding SpyTag-ferritin and SpyCatcher-VHH were synthesized by GenScript (Nanjing) and optimized with CHO preferred codons. Ferritin is adopted from residues 5–174 of Pf ferritin. VHH (R1a-G6) was reported by Simon E. Hufton and colleagues [9]. Three G3S linkers were inserted between SpyTag and Ferritin and between SpyCatcher and VHH. His-tag was added at the carboxy terminus of SpyCatcher-VHH. The amino terminus of the SpyTag-ferritin and SpyCatcher-VHH genes followed after a murine IgG signal peptide sequence. The DNA sequence of the signal peptide is 5^′-GAATTCATGGGTTGGAGTTGCATCATCCTATTTCTAGTGGCCACCGCTACCGGCGTG. Subsequently, the SpyTag–ferritin and SpyCatcher-VHH gene segments were amplified and inserted into the P3 plasmid containing the GS gene between the XbaI and Hind III sites. The successfully cloned P3 plasmid was introduced into GS gene-deficient CHO cells by electroporation. The procedure is set as follows. After being digested, the eukaryotic expression plasmids, 20 µg P3-SpyTag-Ferritin and 20 µg P3-Spy Catcher-VHH respectively, were added into the resuspended CHO-2H6 suspension cell culture and gently mixed, then the cells were incubated at room temperature for 10 minutes. Electroporator (ECM830) parameter (Holliston, MA, USA) is voltage 280 V, time 20 ms. Mixture of plasmids and CHO cells were added into a precooled 4 mm cuvette and electroporation was conducted, then the mixture was transferred into a culture flask preheated to 37 °C, with serum-free CHOGrow®302 medium containing L-alpha-aminoglutamic acid. After 24-hour static culture, the cells were transferred into a 125 mL cell culture flask, and suspended in vertical shaking incubator at 37 °C, 5% CO₂, with speed of 110 rpm. During cell culture, number counting and viability test are performed every 24 hours, then viable cells were frozen for further use.

The CHO cells stably carrying P3-SpyTag-Ferritin or P3-SpyCatcher-VHH were screened by using medium without L-glutamine. After the screening was completed, the CHO cells were continuously cultured, and supernatant was collected for protein purification when the cell viability reached 70%. SpyTag-ferritin were purified using a Superose 6 Increase (GE Healthcare, Wauwatosa, WI, USA) size exclusion column according to the manufacturer’s protocol. SpyCatcher-VHH was purified using a Ni-NTA agarose column (Thermo Fisher Scientific, Shanghai, China) according to the manufacturer’s protocol.

SpyTag-ferritin and SpyCatcher-VHH were mixed at a 1:1 molar ratio and incubated overnight at 4 °C. Ferritin-NP-VHH was then purified using a Superose 6 Increase size exclusion column. Impurities were removed by using a 220 nm filter prior to purify ferritin-NP-VHH.

5 µL purified sample was dropped on a 300-mesh carbon-supported membrane copper grid and stained with 2% (w/v) uranyl acetate. Excessive uranyl acetate was removed until the copper mesh was dry. The sample was then observed under a transmission electron microscope (Hitachi, Tokyo, Japan) at a scale of 100 nm.

Ferritin-NP-VHH and monovalent VHH were tested for their avidity with H1N1 influenza virus by indirect ELISA. ELISA plates were coated with H1N1 influenza virus at 200 ng/well and incubated overnight at 4 °C. Afterwards, the plates were washed three times with 300 µL PBST and blocked in 300 µL of 2.5% nonfat milk at 37 °C for 2 hours. After the plate was washed three times with 300 µL PBST, 400 ng of ferritin-NP-VHH or monovalent VHH was added into each well, and the plate was incubated at 37 °C for 1 hour. After three-times wash with 300 µL PBST in each well, the plates were incubated at 37 °C for 1 hour and 100 µL of mouse anti-His polyclonal antibody (66005-1-lg, Proteintech, Wuhan, China) diluted 1:2000 was added into each well. After three-times wash, 100 µL of 1:5000 diluted goat anti-mouse-HRP-labelled antibody (SA00001-1, Proteintech) was added into each well, and the plate was incubated at 37 °C for 1 hour. After being washed, the plate was incubated with 100 µL of chromogenic solution per well for 15 minutes at room temperature in the dark. Finally, the reaction was terminated with 50 µL of 2 M H₂SO₄ per well, and the OD_450nm was read in an automatic microplate reader (BioTek, Winooski, VT, USA).

For comparing the affinity of ferritin-NP-VHH or monovalent VHH with H1N1 influenza virions, plates were coated with 800 ng of ferritin-NP-VHH or monovalent VHH per well and incubated at 4 °C overnight. After being washed with 300 µL PBST, plates were blocked with 300 µL/well of 2.5% non-fat milk at 37 °C for 2 hours, the plates were washed three times with 300 µL PBST again. Then, 100 µL of H1N1 influenza virions diluted in gradient dilutions was added into each well, and the plates were incubated at 37 °C for 2 hours. After the plate was washed three times, 100 µL of 1:2000 diluted H1N1 HA mAb (11085-MM01, SinoBiological, Beijing, China) was added into each well, and the plates were incubated at 37 °C for 1 hour. After the plate was washed three times, 100 µL of 1:5000 diluted goat anti-mouse-HRP was added into each well, and the plate was incubated at 37 °C for 1 hour. After the plate was washed three times with 100 µL of chromogenic solution per well, the plates were incubated at room temperature for 15 minutes in the dark. Finally, the reaction was stopped with 50 µL of 2 M H₂SO₄ in each well, and the plates were read at an OD_450nm in a plate reader. The binding constant Kd of the binding reaction was accurately calculated by using GraphPad Prism software (GraphPad Software, Boston, MA, USA, version 5.0).

5 $\times$ 10⁴ cells MDCK cells were seeded into 24-well plates per well. Ferritin-NP-VHH or monovalent VHH (0.1 µg, 1 µg, 10 µg, or 50 µg) was mixed with 5 $\times$ 10⁴ PFU of influenza virus and allowed to stand at room temperature for 15 minutes. The mixture was added to each well, with 5 replicates in each group. After being incubated for 48 hours at 37 °C, the supernatant in each well was collected. A standard plaque assay was used to determine the viral titres and plaque size in each well.

Ferritin-NP-VHH and monovalent VHH were placed in dialysis bags containing carbonate buffer (CBS) and dialyzed at 4 °C for 24 hours. Then, 200 nM FITC and 50 nM Ferritin-NP-VHH or monovalent VHH were incubated overnight at 4 °C with rotation to label Ferritin-NP-VHH and VHH, then the mixture was dialyzed in CBS at 4 °C with stirring for 24 hours to remove the surplus FITC. The dialysate was transferred from the dialysis bag into an ultrafiltration tube and centrifuged at 4000 rpm at 4 °C. Subsequently, equal amounts (50 µg) of FITC-labelled ferritin-NP-VHH or monovalent VHH were injected into female BALB/c mice via the tail vein. Blood was collected from the submandibular vein at different time points, and the fluorescence value of the FITC-labelled protein in the blood was measured at an excitation wavelength of 485 nm and an emission wavelength of 535 nm on a VICTOR™ X series multi-label microplate reader (Waltham, MA, USA).

All experiments were approved by the Animal Ethics Committee of Qingdao Agricultural University and performed according to the committee’s guidelines. Female BALB/c mice (6–8 week-old) were randomly divided into groups of each group with 8 mice and housed under designated pathogen-free conditions with food and water ad libitum. Before administration and challenge, mice were anaesthetized with isoflurane. All steps are performed in a biosafety hood equipped with a charcoal filter, including (1) prepare mixture of 20%v/v isoflurane in propylene glycol, (2) soak cotton pad in the mixed isoflurane, (3) retrieve mouse nose close to the soaked cotton with isoflurane mixture. The depth of anesthesia can be adjusted by moving the nostrils closer to or farther from the cotton pad, and then intranasally administered 50 µg ferritin-NP-VHH or monovalent VHH at 4 hours before and 24 hours after challenge with 2$\times$LD₅₀ influenza virus A/Puerto Rico/8/1934 (H1N1). A group treated with PBS was used as a native control. Body weight and survival rate were continuously monitored for 14 days. Humane endpoints were applied when weigh loss is greater than 25%. To determine the pathological changes and viral titres in the lung tissue of mice treated with ferritin–NP-VHH or monovalent VHH, another independent experiment was performed. On the 3rd day after infection with influenza virus A/Puerto Rico/8/1934 (H1N1), following deep euthanasia in accordance with institutional animal care guidelines, mouse cervical dislocation was performed, then tissues were dissected under sterile and controlled conditions. For lung sample collection, the thoracic cavity was opened, then the lungs were removed, and peripheral tissue was dissected from the lower lobes while avoiding major airways to ensure comparable sampling across animals. Next, whole lung samples were sent to Servicebio to perform H&E staining for pathological change testing in lung tissue or viral replication by testing the concentration of the N gene segment by RT-PCR.

Lung specimens were collected, fixed in 10% neutral buffered formalin, and embedded in paraffin wax. Five-micrometer-thick sections were stained with hematoxylin and eosin (H&E) and evaluated for histopathological alterations.

Lung samples of mice were collected and total RNA was extracted by using an RNeasy RNA extraction kit (Qiagen, Düsseldorf, Germany) according to the manufacturer’s protocol. The concentration of viral RNAs (vRNAs) was quantified by real-time RT-PCR with primers specific for the NP gene which was conducted in two steps by using a SuperScript III Platinum two-step qRT-PCR kit with SYBR green (Invitrogen, Carlsbad, CA, USA) as per the manufacturer’s instructions in an iCycler iQ-Multicolor real-time PCR detection system (Bio-Rad, Hercules, CA, USA). Specifically, vRNA was reverse-transcribed by using the Uni-12 primer and amplified by PCR using F5^′-GATTGGTGGAATTGGACGAT and B5^′-AGAGCACCATTCTCTCTATT as primers. The concentrations of vRNA were obtained by comparison with serially diluted plasmid carrying NP gene segment. All RNA determinations were assayed in duplicate and repeated three times.

Comparisons between different groups were performed by using nonparametric one-way ANOVA with the Tukey multiple comparison test and Fisher’s exact test. The survival data were analysed by using log-rank test. All analyses were performed by using GraphPad Prism version 5.0 for Windows (GraphPad Software). p values 0.05 were considered to be significant.

In this study, ferritin-NP-VHH was generated using a modified SpyTag/SpyCatcher technique in which VHH was linked at the C-terminal of SpyCatcher convalently, as shown in Fig. 1 [27, 28, 29, 30]. Both SpyTag-ferritin and SpyCatcher-VHH were expressed in glutamine synthetase (GS)-deficient CHO cells [31, 32, 33, 34, 35]. The self-assembled nanoparticles were confirmed by transmission electron microscopy (TEM), and the particle size of Ferritin–NP-VHH was slightly larger than that of SpyTag-ferritin, as shown in Fig. 2.

Fig. 1.

Design of the multimerization of nanobodies. Using modified SpyTag/SpyCatcher technology, the VHH was exposed to the outer surface of the ferritin shell. Each ferritin is composed of 24 ferritin monomers, and 24 VHHs can be displayed on the surface of each ferritin particle to form anti-influenza virus nanoparticles, thereby realizing the multimerization of nanobodies. VHH, variable domains of heavy-chain antibody.

Fig. 2.

Images of ferritin particle (A) and ferritin-NP-VHH (B) were taken by transmission electron microscopy. Under the same magnification conditions, the particle size of ferritin-NP-VHH is significantly larger than that of ferritin particle. NP, nanoparticle. Scale bar = 100 nm.

To evaluate the specific binding activity of ferritin-NP-VHH, monovalent VHH with influenza virus, ELISA plates were coated with H1N1 influenza virus overnight, after which equivalent amounts (0.03 µM each) of ferritin-NP-VHH or monovalent VHH were added to each well. The plates were read in a plate reader at an absorbance of 450 nm. Finally, the data were analysed by using GraphPad Prism software (version 5.0), and the binding constant Kd was calculated. The results showed that both ferritin-NP-VHH and monovalent VHH are efficiently bound to the influenza virus. However, compared with monovalent VHH, ferritin-NP-VHH was more sensitive to influenza virus capture by 7.18-fold, as shown in Fig. 3.

Fig. 3.

Binding of ferritin-NP-VHH or monomer VHH with influenza virus. Both ferritin-NP-VHH and VHH can specifically bind to influenza virus, but the capture affinity of ferritin-NP-VHH (Kd = 0.4684 $\pm$ 0.01893 HAU/mL) for the virus is 7.18 times greater than that of the monomer VHH (3.361 $\pm$ 0.07851 HAU/mL) (Error bars represent SD).

Next, we determined the specific neutralizing activity of ferritin-NP-VHH or monovalent VHH with influenza virus by using the standard plaque assay method. The neutralization and plaque size results show that ferritin-NP-VHH conferred higher neutralizing activity than monovalent VHH against the influenza virus, as shown in Fig. 4A,B.

Fig. 4.

Neutralization of ferritin-NP-VHH or monomer VHH with influenza virus. Both ferritin-NP-VHH and VHH showed potent neutralizing activity against influenza virus. However, neutralization (A) and plaque size (B) suggested ferritin-NP-VHH conferred higher neutralizing activity against influenza virus than monomeric VHH (SD was specified as error bars and ****p value 0.0001).

To compare the half-life of ferritin-NP-VHH with that of monovalent VHH in vivo, the fluorescence values in the blood of BABL/c mice injected with ferritin-NP-VHH-FITC or monovalent VHH-FITC by tail vein were measured. The results show that the half-life of ferritin-NP-VHH in the blood is 2.44 times longer than that of monovalent VHH, as shown in Fig. 5.

Fig. 5.

Ferritin-NP-VHH had a longer half-life than mononer VHH did in mice. To compare the half-lives of ferritin-NP-VHH and VHH, the fluorescence values in the blood of BABL/c mice injected with ferritin-NP-VHH/VHH-FITC were measured. The results show that the half-life of ferritin-NP-VHH in the blood is approximately 2.44 times longer than that of VHH. FITC, fluorescin isothiocyanate.

We subsequently investigated the protective potential of ferritin-NP-VHH against lethal infection with influenza A virus in vivo. Eight-week-old female BALB/c mice were inhaled intranasally with 50 µg ferritin-NP-VHH or 50 µg monovalent VHH at 4 hours before and 24 hours after challenge with the influenza A/Puerto Rico/8/1934 (H1N1) virus at a dose of 2$\times$LD₅₀. In the control group, the mice were treated with PBS. The results revealed that mice treated with ferritin-NP-VHH had significantly less weight loss and a higher survival rate than mice treated with PBS control or monovalent VHH did, as shown in Fig. 6. Moreover, significantly greater body weight loss after challenge with influenza virus was observed in the monovalent VHH group than in the ferritin-NP-VHH group on Day 6 (p 0.01). These findings suggest that ferritin-NP-VHH provides potential protection against lethal infection with influenza viruses in mice.

Fig. 6.

Ferritin-NP-VHH and monomeric VHH provided protection for mice against challenge with lethal influenza virus. Compared with PBS control mice, mice treated with ferritin-NP-VHH were significantly better protected, as indicated by weight loss and lethality caused by influenza virus infection. Furthermore, compared with mice in the VHH group, mice in the ferritin-NP-VHH group had significantly less body weights after the challenge (n = 8 SD was specified as error bars, **p 0.01). (A) Flowchart illustrating the schedule of animal experiments. (B) Changes in the body weight of mice following A/PR/8/34 (H1N1) challenge. (C) The survival rate of mice post-H1N1 challenge. PBS, phosphate buffer saline.

Additionally, pathological changes were detected in the lungs of mice challenged with the influenza A/Puerto Rico/8/1934 (H1N1) virus. Significant histological changes, such as diffuse inflammation of the interalveolar septa accompanied by intra-alveolar oedema, were observed in the lung sections of mice in the PBS group; otherwise, few pathological changes and less viral replication were observed in the lung tissues of mice in the ferritin-NP-VHH group (Fig. 7A–C). The NP gene segment was presented in a very low level (Fig. 7D). These findings indicated that ferritin-NP-VHH protected mice from infection by the influenza A/Puerto Rico/8/1934 (H1N1) virus.

Fig. 7.

Pathological alterations in the lungs of mice. Pathological alterations in each tissue section were examined via microscopy at an original magnification of 40$\times$. (A) PBS group, (B) VHH group, (C) ferritin-NP-VHH group. The results showed that ferritin-NP-VHH and VHH reduced the infiltration of inflammatory cells, improved the widening of alveolar septa, and reduced serous exudate, and ferritin-NP-VHH conferred slightly better protection than VHH did. (D) Concentrations of the NP gene segment (n = 8 and SD was specified as error bars, ****p value 0.0001). Scale bar = 20 µm.