Foldon fusion of RBD and S1 fragments of SARS‐CoV‐2 to stabilize the structure of subunit protein as a vaccine candidate

The COVID‐19 pandemic threatened public health around the world at the same time as highlighting the urgency of vaccine development. Subunit vaccines are safe and effective vaccine types that utilize parts of viruses to trigger the body’s immune response. Previous research has shown that fusion of the spike protein with the foldon domain (fd) achieved the trimeric form to increase the protein stability of the recombinant subunit protein spike from SARS‐CoV and MERS‐CoV, thus exceeding the immune response in the body. The study aims to observe the expression of RBD‐fd and S1‐fd recombinant proteins from the spike protein of SARS‐CoV‐2 in CHO‐K1 mammalian cells and investigate the binding activity of those proteins with hACE2 receptor, expressed in HEK293T cells using immunofluorescence staining. The plasmids were transiently transfected into the cells, followed by antibiotic selection using G418 as an initial stage to select the positive stable transformants. Protein expression was confirmed by Western blotting and showed an estimated size for monomeric RBD‐fd of 35 kDa and S1‐fd of 55 kDa. However, the trimeric form of the proteins was not observed. In addition, immunofluorescence staining showed the binding activity between the RBD‐fd and S1‐fd proteins and hACE2 expressing cell line, revealing binding and an internalization process.


Introduction
The current pandemic of COVID19 has caused the spreading of human coronavirus which is also known as severe acute respiratory syndrome coronavirus2 (SARS CoV2), and widely menaces public health around the world (Dhama et al. 2020).Compared to the other coronaviruses, MiddleEast respiratory syndrome coro navirus (MERSCoV) and severe acute respiratory syn drome coronavirus (SARSCoV), SARSCoV2 occurs to have a higher infection due to the high rate of human to human transmission (He et al. 2020).According to the World Health Organization (WHO), both MERSCoV and SARSCoV caused outbreaks in several countries.These include some regions in the Middle East, Africa, and South Asia for MERSCoV and China for SARSCoV, in 2012 and 2003, respectively.However, the total infection and the spreading are considered low, therefore, no specific de veloped vaccines and treatments were adjusted to the pa tient's conditions.Consequently, by comparing the case of SARSCoV2 to the other coronavirus cases, the urgency to develop vaccines as the ultimate prevention to the cur rent pandemic has risen (Kashte et al. 2021).
Vaccine is one of the most used biopharmaceutical products, which is crucial to incite the immune system in the body in fighting against infectious diseases to protect the body from diseases in a longterm period.Instead of giving the instant cure to the disease, vaccines are able to reduce the risk and even prevent the disease from happen ing again in the future (Kashte et al. 2021).Therefore, vac cines are an effective approach to treat serious infectious diseases.The current development and improvement of the COVID19 vaccine have been progressively on study all around the world.By investigating the type of available vaccines, the researchers are trying to construct a compe tent and reliable vaccine for the current COVID19 pan demic.One of the types of vaccine that has great potential is the subunit vaccine, which is also known as recombinant protein vaccine.It has a clearer prediction in terms of the response, efficacy, and safety issues associated with the implementation of the vaccine (Yadav et al. 2020).
Previous studies showed that the fusion spike protein from SARSCoV and MERSCoV with foldon domain (fd) was found to achieve the trimeric form to increase the protein stability of subunit vaccines, thus exceeding immune response in the body (Mahmuda et al. 2023).In this study, we developed two subunit vaccines consisting of the SARSCoV2 receptor binding domain (RBD) and S1 fragment of spike protein which are fused with foldon protein to form trimeric structure.The proteins were then expressed in mammalian cells.This labscale research has been performed using one of the subclones of the native Chinese hamster ovary (CHO) cells, CHOK1 cells, as the expression system of the RBDfoldon (RBDfd) and S1 foldon (S1fd) recombinant proteins.According to Kang et al. (2016), CHOK1 is a stable cell line and suitable for therapeutic applications such as recombinant protein in high yield production.Hence, the hypothesis of this study was the RBDfd and S1fd recombinant proteins can be successfully expressed in the CHOK1 cells by show ing the binding activity towards the human angiotensin converting enzyme 2 (hACE2) receptor in the mammalian cells.
We engineered the proteinbased subunit vaccine of SARSCoV2 containing RBD and S1 fragments of spike protein to be expressed in mammalian cells.We introduced signal peptide for recom binant protein secretion into media at the Nterminus.The RBD or S1 fragments was fused with foldon domain using flexible linker containing GS sequence (Figure 1).The in sert genes were ligated into mammalian expression vector (pcDNA3) containing G418 resistance gene sequence to clone the gene of interest (Addgene, #141183).Plasmid preparation and confirmation were carried out by double digestion using restriction enzymes XhoI and NheI (New England Biolabs, #R0146S and #R3131S).The restriction mixture was incubated at 37 °C for 2 h.The confirma tion result can be seen by carrying out gel electrophore sis to visualize the digested plasmid.After the success ful confirmation of digestion, the insert genes (RBDfd and S1Fd) were ligated to the plasmid in separate liga tion reaction using Quick Ligation™ Kit (New England Biolabs, #M2200S) at room temperature for 1 h.For the transformation process, 50 µL of TOP10 competent cells were added each with 2 µL of ligation reaction and incu bated on ice for 15 min, then quickly heatshocked at 42 °C for 50 s, and incubated again on ice for 15 min.Two hundred milliliters of SOC medium were added to the mix ture and incubated in a shaker incubator for 1 h.Then, the mixture was grown overnight in the LB agar medium with 50 µg/mL ampicillin and in the LB agar without ampi cillin as the control by using the spread plate method.The single colonies were observed after overnight incuba tion at 37 °C.The plasmid minipreparation was performed by following manufacturer's instruction using PureLink™ Quick Plasmid Miniprep Kit (Invitrogen, #K210010).The positive transformants were checked using restriction en zymes method, then visualized by gel electrophoresis.

Cell cultures
The cryopreserved CHOK1 for RBDfd and S1fd ex pression and HEK293T cells for ACE2 expression were thawed in a 37 °C water bath for 30 s. Several reasons of the utilization of CHOK1 cells were the clear regu lation and standardization on its biosafety and manufac turing practices.CHOK1 also has high tolerance and easily adapts to the environmental changes.Aside from that, its capability to develop in serumfree media and high productivity yield is also intriguing for further vac cine development (Omasa et al. 2010).While HEK293 T cells was used due to its common usage as viral vec tors, high transfection efficiency and good protein pro cessing capability (Tan et al. 2021).One milliliter of each cell suspension was transferred into a tube contain ing 4 mL of F12 complete media for CHOK1 cells, and 4 mL of DMEM (Sigma, #D7777) complete medium for HEK293T cells, respectively.Both of the complete me dia were supplemented with 10% fetal bovine serum al bumin (Sigma, #12103C) and 1% penicillinstreptomycin (Sigma, #P4333).The tube was centrifuged at 3000 rpm for 3 min and the supernatant was discarded.Ten milliliters of complete fresh complete media were added to the tube to resuspend the pellet and transferred to a 10 cm cell culture dish.The culture was incubated at 37 °C with 5% CO 2 concentration for 3 days to obtain approxi mately 3×10 6 cell density.The cell was observed to ensure 8090% confluency prior to the subculture.The growth media was decanted and the cell was washed with 3 mL phosphatebuffered saline (PBS) three times.Five hun dred microliters of trypsin was added and incubated at 37 °C for 3 min.The cells were passage into 2 different 10 cm cell culture dishes and were incubated at 37 °C with 5% CO 2 for 23 days.

Transient transfection
The transfection method of CHOK1 cells in a 6well plate was modified from the method developed by Che ung et al. ( 2018) and Saifudin et al. (2011).The cells that achieved the desired seeding density and confluency were washed using 1× PBS and a new fresh complete medium was added into the plate.After overnight culture, RBD fd or S1fd were transfected into the cells using Lipofec tamine™ LTX (Invitrogen, #A12621).Both plasmid DNA and Lipopectamine were diluted in OptiMEM™ I Re duced Serum Medium (Gibco, #31985062) and were pre pared according to the manufacturer's instruction.Mock transfection as the negative control was done in a different well by adding 1× PBS instead of protein at the same vol ume as samples.Then, the complete transfection media was added into the CHOK1 cells in the 6 wellplate and incubated at 37 °C and 5% CO 2 for 2448 h.The medium was collected one day and two days after the transfec tion for observation.After 72 h of transfection, 0.05% G418 antibiotic (SigmaAldrich, #G8168) was added as the transfection selection approach and was incubated at 37 °C with 5% CO 2 concentration to grow until its maxi mum confluency.

Selection of transfected cells
Transformants selection was performed by scaling up and down the cell culture into different sizes of the well plates, initiated by 6well plate, followed by 24well plate, 6well plate, 6 cmcell culture dish, and 10 cmcell culture dish, respectively.Cell culture in every stage of scaling was incubated at 37 °C with 5% CO 2 concentration and the ad dition of 0.05% G418 antibiotic.The survived and more stable cells were managed and maintained every two days.The medium from each stage of cell culture scaling was obtained and stored at 20 °C for further analysis and ob servation.The culture medium was further concentrated by adding 500 μL of cell culture medium to Amicon® Ul tra 0.5 mL Centrifugal Filters (MWCO: 10 kDa, Millipore) and centrifuged at 14,000 ×g for 30 min at room temper ature.The filtrate was collected and stored at 20 °C for further usage.

Western blot
The expression of RBDfd and S1fd recombinant pro teins in the transfected cells were observed by using West ern blot.Thirty microliters of each protein sample in re ducing sample buffer were boiled for 5 min.The protein samples were then loaded into 415% precast polyacry lamide gel (Biorad, #4561084) with a running buffer 1× Tris/glycine/SDS (Biorad, #1610732) at 100 V for 1 h.The SDSPAGE gel was removed from the tank assembled into the sandwich Turbo Mini 0.2 PVDF Transfer Packs (Biorad, #1704156) and run with TransBlot Turbo Trans fer System apparatus (Biorad) at 25 V, 1.3 A for 7 min.Af terward, the membrane was incubated with blocking solu tion (10% skim milk in 1× trisbuffered saline (TBS) con taining 0.1% Tween 20 (TBST)) for 1 h.The primary anti body, SARSCoV2 Spike RBD Antibody (R&D Systems, #MAB10540) was added into the blocking solution with a 1:3000 ratio and incubated for 2 h in the gentle shaker.The secondary antibody, mouse alkaline phosphatase antibody (R&D Systems, #AF2910) was added into the blocking solution at 1:2000 ratio and incubated for 1 h.In between the steps of blocking, primary antibody and secondary an tibody, the membrane was washed three times by using 1× TBST.At the end, the membrane was visualized by adding the One Step NBTBCIP substrate (Thermo Scien tific Pierce, #34042).

Binding and internalization into ACE-2 receptor
The binding activity of RBDfd and S1fd recombinant proteins and ACE2 receptor were observed using im munofluorescence staining.HEK293T cells were trans fected with the hACE2 gene, pcDNA3.1hACE2(Ad dgene, #145033) containing plasmid.A half microgram (0.5 μg) purified recombinant plasmid was diluted in 50 μl of OptiMEM I Reduced Serum Medium (Gibco, #31985062).The diluted DNA solution was further added with 1 μL of polyethylenimine (PEI) salt (Sigma, #408727) in 50 μL of OptiMEM I Reduced Serum Medium, mixed gently and incubated overnight at 37 °C with 5% CO 2 concentration.The growth medium was re placed with a new 500 μL of DMEM.These ACE2 trans fected HEK293T cells were grown in the chamber slides and added with 20 µL of each concentrated supernatant from the RBDfd and S1fd transfected CHOK1 cell cul ture.Over expressed of ACE2 receptor were then con firmed by immunofluorescence assay using anti hACE2 antibody as described in section 2.7 below.Both samples were then incubated at 37 °C and 5% CO2 for 23 h (Shang et al. 2021).

Immunofluorescence (IF) assay
The IF process covered fixing, permeabilization, block ing, primary and secondary antibody incubation, and vi sualization.The chamber slide containing cells were fixed by adding 100 µL of 3,7% formaldehyde solution on top of the cells and incubated for 10 min at room tempera ture.The formaldehyde solution was discarded and the cells were washed three times using 500 µL PBS.One hundred microliters (150 µL) 0.2% TritonX was added for permeable process and incubated for 10 min at room temperature.The cells were washed two times using 500 µL followed by the adding of 150 uL 1% BSA (Thermo Scientific, #B14) to the cells and incubated for 30 min at room temperature for blocking nonspecific binding.The primary antibodies (antiRBDmouse and antihuman ACE2rabbit antibodies) (R&D Systems, #MAB105808 and #MAB10823) to detect RBDfd or S1Fd, and human ACE2 respectively, were diluted in blocking solution at a ratio of 1:250.The diluted antibodies were added into each chamber slide and incubated for 1 h at room temperature.The secondary antibodies (AF488conjugated antimouse and AF594conjugated antirabbit) (Abcam, #ab150113 and #ab150080) at ratio of 1:2000 and 1:1000 in blocking solution, respectively were added to chamber slide and in cubated overnight at 4 °C.After the primary and secondary antibodies incubation, the cells were washed three times using 500 µL PBS.The mounting of the slides was done by using Fluoroshield™ with DAPI (Sigma, #F6057) and dried overnight.The cells in each chamber slide were ob served under the fluorescence microscope (Olympus) with magnification of 20×.

Engineering and production of subunit vaccines
The recombinant plasmids were confirmed before tran siently transfected into CHOK1 cells.The plasmid ob tained from miniprep was tested by double digestion us ing NheI and XhoI restriction enzymes and visualized us ing gel electrophoresis.The result of double digestion can easily be observed by comparing bands between undi gested and digested plasmids (Table 1).The successfully digested plasmid showed favorable results by showing two bands representing insert gene either RBDfd or S1fd (lower) and vector pcDNA3 (higher) as shown in Figure 2. The purity and concentration of plasmids were mea sured before transiently transfected into the cells.We used LipopectamineLTX to enhance the efficiency of transfec tion.The expressed protein was observed 24 h and 48 h af ter transfection by slot blotting using antibody specific into RBD spike glycoprotein SARSCoV2 (data not shown).
On day 3, the transfected cells were selected by using G418 treatment.The treated cultures were scaled down to a 24well dish and then gradually scaled up again un til in a 10 cmdish to increase the protein expression.The protein expression in every single dish were confirmed by slot blotting (data not shown).Western blot analysis was performed only in the medium of 10 cmdish, and thick bands at approximately 35 kDa for RBDfd and 55 kDa for S1fd.As a comparison, the negative control (mock transfection) showed no band as expected (Figure 3).

In vitro binding analysis of recombinant subunit vaccines into ACE2 receptor
To investigate the biological effect of the subunit vaccines, we utilized immunofluorescence staining for hACE2 re ceptors and recombinant protein RBDfd or S1fd was per formed.The HEK 293T was used to over expressed the ACE2 protein using transient transfection technique.Be fore adding the concentrated supernatant containing RBD fd or S1fd into ACEoverexpressed HEK 293T, it was confirmed that the HEK 293T cells has already expressed ACE2 protein, which can be shown on Figure 4a.The red color showed the ACE2 protein expressed in membrane HEK 293T cells.Furthermore, the proteins binding to the ACE2expressing HEK 293T cells was confirmed.The RBDfd or S1fd protein from concentrated supernatant of the 10 cmdish was added into transiently transfected ACE2expressing HEK 293 cells.The cells were incu bated with mouse antiRBD primary antibody, followed by secondary antibodies (AF488conjugated antimouse).
The successful binding showed a green color in membrane cells area, which represents the interaction of the recom binant RBDfd or S1fd with the hACE2 receptor in the cell membrane (Figure 4b, c).Finally, we investigated the binding and internalization of RBDfd and S1fd to ACE 2expressing cells by using two primary antibodies, which are rabbit antiACE2 and mouse antiRBD spike SARS CoV2 antibodies, followed by two secondary antibod ies (AF594conjugated antirabbit and AF488conjugated antimouse).The result showed that both RBDfd and S1fd protein successfully bound and internalized into the hACE2 protein expressing cells, which were shown by yellowishorange color inside the cells.Therefore, the RBDfd and S1fd proteins were determined to be able to bind with the hACE2 protein (Figure 4d, e).

Discussion
Subunit vaccines are the vaccine using some parts of the germs or viruses in the structure construction to incite the immune response in the body, without causing any infec tion risks (Chong et al. 2015).Therefore, the improvement can be made for constructing COVID19 vaccine by uti lizing the essential remnant of the virus, such as the spike protein, or even smaller remnants such as RBD that lo cated in the spike protein of the virus.Several previous studies also used RBDbased or spikebased recombinant protein as the vaccine development in other coronaviruses (MERSCoV and SARSCoV), which showed great im munogenicity effects (Li et al. 2020).However, in the native state, spike protein formed in prefusion state as a trimeric glycoprotein mediating both binding to host cell receptors and fusion of virus and host cell membranes.Therefore, some improvements in developing structure based design of recombinant subunit vaccine to form a trimeric prefusion state of spike protein can be promoted by fusing with other protein fragments.Most approved COVID19 vaccines use prefusion stabilized structure of spike protein, including mRNA, viral vector and subunit vaccines (Creech et al. 2021).In this study, we combined the RBD or S1 fragment with foldon domain using GS flexible linker without any protein tags.Foldon protein consists of 27 amino acids sequences that can help to in crease the length of the RBD in the recombinant protein and achieve the trimeric form (Tai et al. 2016).A sig nal peptide utilizing hemagglutinin sequence was used as stated in the registered patent (P00202211076) for protein secretion.
Mammalian cells are widely used as the expression system of recombinant proteins.About 85% of the re combinant proteins that are available in the market are expressed in mammalian cells, due to the capability to express the complex recombinant proteins (Tripathi and Shrivastava 2019).One of the most commonly used mam malian cells is CHO cells that well known for its stabil ity, and the available cell lines is CHOK1.In addition, the stable cell lines are able to produce a high yield re combinant protein product, flexible to grow in a serum free medium, and less affected by the infections of human viruses (O'Flaherty et al. 2020).Therefore, this cell has a great advantage in developing biopharmaceutical products such as a vaccine.In labscale research, the expression of recombinant protein is essential for proceeding to larger scale production.
In this study, we used transient transfection for protein production, followed by selection of G418 as the initial stage for stable cell line production.The protein expressed were confirmed by Western blot analyses using antibody specific into RBD spike glycoprotein of SARSCoV2.The proteins were analyzed in reduced SDSPAGE.There fore, the size of both proteins shown as a monomeric form.The two bands of S1fd protein were observed in the blot, but not for the RBDfd.There are several causes that makes S1fd sample had two bands in the Western blot.First, it may be caused by protease degradation (Mahmood and Yang 2012), that cleaved the peptide in S1 peptide but not at RBD sequence.Second, it might be due to a glyco sylation difference (Semaan et al. 2012).The S1 subunit has 13 putative Nglycosites, and 3 of them in RBD sites (Gong et al. 2021).Therefore, the hyperglycosylated S1 protein expressed in CHO cells may be produced in het erogeneity glycoforms, resulting different size of protein.
The binding activity of expressed proteins were also con firmed using in vitro assay using ACE2 expressing trans genic cells.The proteins were bound and internalized into hACE2 receptor showing the biological effect of those re combinant proteins.However, we have not managed to determine the trimeric form of both foldon fusion pro teins.For the next study, the trimeric form might be in vestigated using non reducing protein sample buffer be fore SDSPAGE or using native PAGE and size exclusion chromatography.Further study, the sophisticated instru ment such as cryoelectron microscopy can be used to in vestigate the trimeric structure of these antigens design.The confirmation of trimeric form is essential to ensure the antigens will have a great immunogenicity as vaccine candidates (Hsieh et al. 2020).

Conclusions
In summary, both RBDfd and S1fd have been success fully expressed in CHOK1 adherent cells and character ized using specific antibody.Moreover, both proteins have a binding activity with receptor hACE2 based on in vitro studies.However, further studies must be performed to purify the proteins and characterize trimeric structure of these protein design.

FIGURE 1
FIGURE 1 Gene constructs representing RBD or S1 fusion foldon (a) and the illustration of the plasmid DNA mapping (b).

FIGURE 4
FIGURE 4 Binding and internalization of RBD-fd and S1-fd into hACE2 expressing cells.(a) hACE2 protein was transiently transfected into HEK 293T cells, expressed in membrane cells exerting red color.Blue color indicated the nucleus which was stained with DAPI.(b, c) The binding of RBD-fd and S1-fd proteins in hACE2 expressing cells, respectively.(d, e) The binding and internalization of RBD-fd and S1-fd proteins in hACE2 expressing cells, resulting the yellowish colors.

TABLE 1
Results of the P. aeruginosa protease clear zone test.
Cut S1-fd with Restriction Enzyme 2,000Cut RBD-fd with Restriction Enzyme 800