key: cord-0751187-82b8g111
authors: Mohsen, Mona O.; Balke, Ina; Zinkhan, Simon; Zeltina, Villija; Liu, Xuelan; Chang, Xinyue; Krenger, Pascal S.; Plattner, Kevin; Gharailoo, Zahra; Vogt, Anne‐Cathrine S.; Augusto, Gilles; Zwicker, Marianne; Roongta, Salony; Rothen, Dominik A.; Josi, Romano; da Costa, Joana J.; Sobczak, Jan M.; Nonic, Aleksandra; Brand, Lee‐Anne; Nuss, Katja; Martina, Byron; Speiser, Daniel E.; Kündig, Thomas; Jennings, Gary T.; Walton, Senta M.; Vogel, Monique; Zeltins, Andris; Bachmann, Martin F.
title: A scalable and highly immunogenic virus‐like particle‐based vaccine against SARS‐CoV‐2
date: 2021-09-20
journal: Allergy
DOI: 10.1111/all.15080
sha: 3e24e85d20e3546172b0472fa1dc9c5315f14c40
doc_id: 751187
cord_uid: 82b8g111

BACKGROUND: SARS‐CoV‐2 caused one of the most devastating pandemics in the recent history of mankind. Due to various countermeasures, including lock‐downs, wearing masks, and increased hygiene, the virus has been controlled in some parts of the world. More recently, the availability of vaccines, based on RNA or adenoviruses, has greatly added to our ability to keep the virus at bay; again, however, in some parts of the world only. While available vaccines are effective, it would be desirable to also have more classical vaccines at hand for the future. Key feature of vaccines for long‐term control of SARS‐CoV‐2 would be inexpensive production at large scale, ability to make multiple booster injections, and long‐term stability at 4℃. METHODS: Here, we describe such a vaccine candidate, consisting of the SARS‐CoV‐2 receptor‐binding motif (RBM) grafted genetically onto the surface of the immunologically optimized cucumber mosaic virus, called CuMV(TT)‐RBM. RESULTS: Using bacterial fermentation and continuous flow centrifugation for purification, the yield of the production process is estimated to be >2.5 million doses per 1000‐litre fermenter run. We demonstrate that the candidate vaccine is highly immunogenic in mice and rabbits and induces more high avidity antibodies compared to convalescent human sera. The induced antibodies are more cross‐reactive to mutant RBDs of variants of concern (VoC). Furthermore, antibody responses are neutralizing and long‐lived. In addition, the vaccine candidate was stable for at least 14 months at 4℃. CONCLUSION: Thus, the here presented VLP‐based vaccine may be a good candidate for use as conventional vaccine in the long term.

Since the outbreak of the global pandemic caused by SARS-CoV-2, WHO has reported ~170 million confirmed cases by 3 June 2021 including ~3.5 million deaths (WHO, 3 June 2021). The pandemic has put a heavy toll on public health systems and world's economy.

To limit the damage, efforts have been directed toward vaccine development. On 12th May 2021 around one billion doses of different vaccines have been administered worldwide (WHO, 12th May 2021). Although SARS-CoV-2 causes mostly mild symptoms such as coughing, fever and breathlessness, symptoms may become much more severe, in particular in elderly people and people with chronic diseases which may develop to severe pneumonia and other symptoms including organ failure and death. 1,2 SARS-CoV-2 has a lower fatality rate (2.3%) in comparison with SARS (9.5%) and MERS (34.4%) . 3 However, it transmits much more readily, mostly because non-symptomatic and pre-symptomatic individuals can spread the virus. Thus, while MERS-CoV and SARS-CoV outbreaks have been sporadic and geographically restricted, SARS-CoV-2 has rapidly spread around the world 4, 5 .

The positive-sense ssRNA SARS-CoV-2 virus has a genome of about 29,700 nucleotides with 79.5% identity to SARS-CoV-1. Its genome encodes four main structural proteins; spike protein, membrane protein, nucleocapsid protein, and the envelope protein 6, 7 .

SARS-CoV-2 binds to angiotensin-converting enzyme 2 (ACE2) via the (receptor-binding domain) RBD of its spike protein that protrudes from the viral envelope. Interaction of RBD with ACE2 is the first step in a cascade of events leading to viral entry and ultimately replication 8 . Neutralizing antibodies against SARS-CoV-2 are mostly targeting the RBD of the spike protein. Within RBD, the receptorbinding motif (RBM) is of particular importance as it directly interacts with ACE2 9 . Interestingly, RBM shows no glycosylation or other post-translational modifications and therefore is well suited for production in bacterial expression systems 11 .

Vaccines are the most reliable, cost-effective, and efficient strategy to prevent infectious diseases. Vaccine candidates must induce sufficient quantities of high-affinity antibodies to neutralize the invading virus. Since the initiation of the pandemic, a full spectrum of vaccine types has been tested in preclinical and clinical trials.

Vaccine platforms employed mRNA, DNA, viral vectors, inactivated Switzerland. Email: mona.mohsen@dbmr.unibe.ch

This work was supported by Saiba AG and Inselspital Bern.

Conclusion: Thus, the here presented VLP-based vaccine may be a good candidate for use as conventional vaccine in the long term.

In this study, we describe a novel conventional COVID-19 vaccine that consists of the RBM of SARS-CoV-2 genetically grafted onto the surface of our optimized cucumber-mosaic virus-like particles. We demonstrate that the vaccine candidate (mCuMV TT -RBM) is highly immunogenic in mice and rabbits, can efficiently neutralize SARS-CoV-2, and is stable and highly scalable. The induced antibodies show cross-reactivity with VoC. or live-attenuated virus 10, 12 , and recombinant proteins. The full length of spike protein, RBD, S1 subunit, fusion peptide (FP), and the N-terminal domain (NTD) of the spike protein have been targeted by vaccines that are licensed or undergoing development 4, 13 .

Virus-like particles (VLPs) represent one of the conventional vaccine platforms in the sense that there are globally marketed VLPbased products, for example, hepatitis B virus (HBV) and human papilloma virus (HPV) vaccines have demonstrated the clinical usefulness of this modality. VLPs consist of viral structural proteins that upon recombinant expression, self-assemble into icosahedral or rarely helical particles 14 . Recently, we have developed an immunologically optimized VLP platform based on the cucumber mosaic virus (CuMV TT -VLPs) 15 . CuMV TT -VLPs incorporate a universal T-cell epitope derived from tetanus toxin (TT) 16 . The newly developed platform enhances the interaction between T helper (T H ) cells and B cells, and is expected to improve responses in elderly individuals who are often less reactive to vaccines. This is supported by the fact that pre-existing immunity to the chosen TT epitope is very broad in humans (and animals) as the peptide binds to essentially all HLA-DR molecules and most people have been immunized many times against TT. In addition, CuMV TT -VLPs are packaged with bacterial RNA which is a ligand for toll-like receptor (TLR) 7 and 8 and serves as a potent natural adjuvant 16, 17 . By displaying antigens on CuMV TT -VLPs, it was possible to induce high levels of antigen-specific antibodies in mice, rats, cats, dogs, and horses and treat diseases such as atopic dermatitis in dogs or insect bite hypersensitivity in horses 18, 19 .

In the current study, we have designed and developed a scalable and immunogenic VLP-based COVID-19 vaccine by genetically fusing the RBM of the spike protein from SARS-CoV-2 into CuMV TT -VLPs. The data show that this vaccine is highly immunogenic and capable of inducing both RBD-specific IgG and IgA antibodies as well as a strong viral neutralizing antibody response. Furthermore, the vaccine production process is highly scalable, potentially allowing the production of millions of doses in a single 1000L bacterial fermenter run. After selection of clones with the highest expression level of target proteins, E. coli culture was grown in 100 ml of 2TY medium (1.6% trypton, 1% yeast extract, 0.5% NaCl, 0.1% glucose) containing ampicillin (100 mg/l) on an orbital shaker at 30℃ to the OD 600 value of 0.8-1.0. Then, the cells were induced with 0.2mM IPTG, and the medium was supplemented with 5mM MgCl 2 . Incubation was continued on the rotary shaker (200 rpm, 20℃, 18h). The resulting biomass was collected by low-speed centrifugation and was frozen at −20℃.

To disrupt the cells, the biomass was resuspended 10 ml of buffer (20mM Tris, 5mM EDTA, 5mM Et-SH, 5% glycerol, 10% sucrose, pH 8.0) and further treated with ultrasound (Hielscher 200, power 70%, pulse 50%, 16min) on ice. Then, 0.5% Triton X-100 was added and the solution was rotated at 10 rpm ON at 4℃ without centrifugation.

The solution was then clarified for 10min at 10000 rpm (rotor: F-34-6-38 Eppendorf), and the pellet was discarded. The soluble fraction was loaded on the top of the sucrose gradient (20-60%; in buffer containing 20mM Tris, 2mM EDTA, 5% glycerol, 0.5% TX-100, pH 8.0) and centrifugated in Beckman SW32 rotor for 6h at 25500 rpm at 18℃. The gradient fractions 6m were then removed from the bottom of the 38 ml tube. The CuMV VLP containing fraction (40 and 50% sucrose, pooled) was diluted 1:1 with buffer (20mM Tris, 2mM EDTA, 5% glycerol, pH 8.0). The VLPs were sedimented using Type 70 rotor (Beckman, 50000 rpm, 4h, 4℃). Then, the pellet was dissolved ON in 4 ml of 20mM Tris, 2mM EDTA at 4℃. The solution was clarified by centrifugation (5min, 14000 rpm), the clarified solution overlaid on top of the 30% sucrose "cushion" solution in 20mM Tris, 2mM EDTA, 0.5% TX-100, pH 8.0 The VLPs were sedimented using Beckman TLA100.3 rotor (72000 rpm, 60min, 4℃).

The pellet was solubilized in 2 ml of 20mM Tris, 2mM EDTA, and clarified again by centrifugation (5min, 14000 rpm). Endotoxin measurement in the produced vaccine showed ~50 EU/mg. mCuMV TT -RBM vaccine candidate was next characterized using SDS-PAGE, agarose gel, electron microscopy, and dynamic light scattering (DLS).

Protein concentration was determined using BCA test.

Sample VLP solution (1 mg/ml) was analyzed on a Zetasizer Nano ZS instrument (Malvern Instruments Ltd, UK). The results of three measurements were analyzed by DTS software (Malvern, version 6.32). 16 

Physical stability and integrity of the mosaic CuMV TT -RBM were visualized by transmission electron microscopy (Philips CM12 EM).

For imaging, sample grids were glow discharged and 2μl of purified CuMV TT -RBM (3mg/ml) was added for 30s. Grids were washed 3x with ddH 2 O and negatively stained with 5 μl of 5% uranyl acetate for 30s. Excess uranyl acetate was removed by pipetting and the grids were air dried for 10min. Images were taken with 84,000X and 110,000X magnification.

To test if the vaccine can bind the relevant human receptor ACE2, the plates were coated with 1µg/ml of ACE2 in PBS at a volume of 50μl/well. The plate was incubated at 4℃ overnight. The plate was washed with PBS, Tween 0.01%. Added 50µl/well of Superblock solution (Thermo Fisher, 37518) and incubated for 1h at RT on a shaker.

The blocking solution was flicked off, and 50µl of the CuMV TT -RBM or CuMV TT -VLPs at 1μg/ml was added to the first row of the plate followed by 1:3 dilution. The plate was incubated for 1h at RT, washed with PBS+Tween 0.01%. 50μl of mouse anti-CuMV TT monoclonal antibody (clone 1-1A8/ batch 2) at a concentration of 1μg/ml was added to each well as a secondary antibody and incubated for 1h at RT on a shaker. The plate was washed and 50μl of the detection antibody; HRP labeled goat anti-mouse IgG Fc gamma at a dilution of 1:1000 in PBS-Casein 0.15% was added to each well. The plate was incubated for 1h at RT. The plate was developed, and OD 450 reading was performed (BioTek, USA).

In vivo experiments were performed using 8-to 12-week-old female, BALB/cOlaHsd wild-type (wt) mice purchased from Harlan 

Wild-type BALB/cOlaHsd mice were vaccinated subcutaneously (s.c.)

using different regimens and doses as summarized in Table 1 

SARS-CoV-2 RBD wildtype , RBD K417N , RBD E484K , RBD N501Y , RBD K417N/E484K

To determine the total IgG antibodies against the candidate vaccine mCuMV TT -RBM in sera of vaccinated mice, ELISA plates were coated with SARS-CoV-2 RBD wildtype or with SARS-CoV-2 Spike IgG subclasses were measured from sera collected on day 42

following the same described ELISA protocol. The following secondary antibodies were used: goat anti-mouse IgG1-HRP and goat anti- 

To test IgG antibody avidity against SARS-CoV-2 full spike protein and RBD protein, threefold serial dilutions of 1/20 diluted mice sera, were added to duplicate ELISA plates coated over night with 1μg/ml RBD or spike protein. After incubation at RT for 1h, the plates were washed once in PBS-0.01% Tween, and then washed 3x with 7 M urea in PBS-0.05%Tween or with PBS-0.05% Tween for 5min every time.

After washing with PBS-0.05%Tween, goat anti-mouse IgG conjugated to horseradish peroxidase (HRP) (Jackson ImmunoResearch, West Grove, Pennsylvania) was added 1/2000 and incubated for 1h at RT. Plates were developed and read at OD 450 .

To determine the neutralizing ability and capacity of vaccine-induced antibodies, a CPE assay was performed using wildtype SARS-CoV-2 (SARS-CoV-2/ABS/NL20). Serum samples were heat-inactivated for 30min at 56℃. Two fold serial dilutions were prepared starting at 1:20 up to 1:160. 100 TCID 50 of the virus was added to each well and incubated for 37℃ for 1h. The mixture has been added on a monolayer of Vero cells and incubated again for 37℃ for 4 days.

Four days later, the cells were inspected for cytopathic effect. The titer was expressed as the highest dilution that fully inhibits formation of CPE.

Data were analyzed and presented as (mean ±SEM) using Student's t test, one-way ANOVA or Area Under Curve (AUC) as mentioned in the figure legend, with GraphPad PRISM 9. The value of p<0.05 was considered statistically significant (*p<0.01, **p<0.001, ***p<0.0001).

Bacterial lysates were generated as described in 2.1 and applied to a sucrose gradient in a continuous flow centrifugation (AW Promatix 1000™). The fractions obtained were analyzed by SDS-PAGE, native agarose gel electrophoresis, and electron microscopy.

Our first attempt to generate a COVID-19 vaccine using CuMV TT VLPs platform, utilized eukaryotically expressed recombinant RBD which was chemically coupled to the VLPs using Succinimidyl 6-((beta-maleimidopropionamido)hexanoate) SMPH cross-linker 21 .

This method resulted in a vaccine candidate that induced high levels of RBD-specific antibodies which were able to strongly inhibit RBD binding to ACE2 and neutralize SARS-CoV-2/ABS/NL20 virus 22 .

In an attempt to produce a more readily scalable vaccine candidate with better yields, we genetically fused RBM into CuMV TT to produce a mosaic vaccine as illustrated in Figure 1A ( Figure 1F ). The observed larger size than seen by electron microscopy is due to water molecules surrounding the VLPs. Figure 1G ).

Next, we tested the stability of mCuMV TT -RBM after storage at +4℃ for 14 months. The stability of the vaccine was assessed and compared to a freshly produced vaccine by performing SDS-PAGE ( Figure 2A and E), agarose gel ( Figure 2B and F), DLS analysis ( Figure 2C and G), and electron microscopy ( Figure 2D and H) . The results indicated that the vaccine is highly stable for 14 months at +4℃ with no signs of degradation.

We investigated the immunogenicity of the developed vaccine mCuMV TT -RBM in BALB/c mice using the s.c. vaccination route.

We tested different doses and vaccination regimens as summarized in Table 1 

Previous studies have shown that the different IgG subclasses play an essential role against viruses by complement fixation, enhancing opsonization, and immune effector function 24 . Accordingly, it is of interest to assess the IgG subclasses induced by the developed vaccine candidate. We therefore tested the vaccine for its ability to induce IgG1, IgG2a, IgG2b, and IgG3. The results indicated that mCuMV TT -RBM induces the production of all four subclasses when binding is assessed on RBD protein as shown in Figure 4A and B.

Analysis of Log 10 OD 50 showed a predominance of IgG2a. These results were confirmed when assessing the binding to the spike protein ( Figure 4C and D).

Next, we tested the ability of mCuMV TT -RBM to induce immunoglobulin class-switching to IgA using the two regimens D0/D14 and D0/D28. D0/D28 regimen induced higher RBD-specific IgA titers in comparison with D0/D14 regimen ( Figure 4E ). Determining the Log 10 OD 50 of IgA titers in both regimens confirmed the difference in antibody titers ( Figure 4F ).

We have previously produced a number of RBD variants, including the UK, Brazilian, and Indian VoC 25, 26 , and have shown, as others, that mutations at position E484K strongly reduce recognition by convalescent sera 26, 27 . It is therefore of significant interest that our vaccine induced immune sera recognized all VoC equally well as the wild type ( Figure 5A and B) . Hence, this vaccine candidate may have the potential to protect equally well against all VoC that have occurred up to now.

F I G U R E 3 mCuMV TT -RBM induces high levels of specific antibodies with high avidity against RBD and spike protein of SARS-CoV-2. A, Vaccination regimen (Prime/Boost) D0/D14 or D0/D28, bleeding schedule, and groups. B, Log 10 OD 50 of RBD-specific IgG titers for the groups vaccinated with CuMV TT as a control or mCuMV TT -RBM on days 7, 14, 21, 28, 35, and 42 using D0/D14 regimen. C, Log 10 OD 50 of spike-specific IgG titers for the groups vaccinated with CuMV TT or mCuMV TT -RBM on days 7, 14, 21, 28,3 5, and 42 using D0/D14 regimen. D, Log 10 OD 50 of RBD-specific IgG titers for the groups vaccinated with CuMV TT or mCuMV TT -RBM on days 7, 14, 21, 28, 35, and 42 using D0/D28 regimen. E, Log 10 OD 50 of spike-specific IgG titers for the groups vaccinated with CuMV TT or mCuMV TT -RBM on days 7, 14, 21, 28,3 5, and 42 using D0/D28 regimen. F, Comparison between Log 10 OD 50 of RBD-specific IgG titers on days 35 and 42 using D0/D14 or D0/D28 regimens. G, Comparison between Log 10 OD 50 of spike-specific IgG titers on days 35 and 42 using D0/D14 or D0/D28 regimens. H, Avidity of RBD-specific IgG titers in mice vaccinated with mCuMV TT -RBM using D0/D14 regimen, sera were treated with PBST or 7 M Urea. I, Avidity of spike-specific IgG titers in mice vaccinated with mCuMV TT -RBM using D0/D14 regimen, sera were treated with PBST or 7 M Urea. J, Avidity Index of RBD-specific IgG in mice vaccinated with mCuMV TT -RBM using D0/D14 or D0/D28 regimens, sera were treated with PBST or 7 M Urea. K, RBD-specific IgG titer for the group vaccinated with mCuMV TT -RBM on days 42 and 134. L, spike-specific IgG titer for the group vaccinated with mCuMV TT -RBM on days 42 and 134. Statistical analysis (mean ±SEM) using one-way ANOVA in B-G or Student's t test in J, n=10 or 5. One representative of 3 similar experiments is shown. The value of p<0.05 was considered statistically significant (*p<0.01, **p<0.001, ***p<0.0001)

In comparison with infections with other viruses, patients infected with COVID-19 produce neutralizing antibodies at relatively low levels 28, 29 . Hence, we compared the total RBDspecific IgG titers induced in mouse sera after vaccination with mCuMV TT -RBM to SARS-CoV-2 convalescent sera (total of 5 different sera). The results showed that the vaccine induces higher RBD-specific antibody titers even after a single priming dose compared to natural infection with SARS-CoV-2 and the booster injection further increased the RBD-specific antibody responses ( Figure S3A and B) . Analysis of Area Under Curve (AUC) indicated a strong difference (p<0.0001) between mouse sera collected on day 14 (after priming) or day 42 (after boost) to convalescent sera ( Figure S3C and D).

In a next step, we compared the efficacy of vaccinating mice with a total of 2 doses of mCuMV TT -RBM (Prime/Boost) versus 3 doses (Prime/1 st Boost/2 nd Boost) as illustrated in Figure 6A . The results showed no significant difference in the induced RBD-specific antibody titers following the 1 st boost in comparison with sera following the 2 nd boost ( Figure 6B and C) . Furthermore, the avidity of the induced antibodies after the 2 nd boost shows a slightly higher value when compared to the ones induced following the 1 st boost ( Figure 6D and E) even though the difference did not reach statistical significance.

To test the capacity of the induced antibodies to neutralize the real virus, we have assessed reduction of cytopathic effect (CPE) using 100 TCID 50 of SARS-CoV-2/ABS/NL20. Titers are expressed as the highest dilution that inhibits formation of CPE by 100%.

A significant difference in neutralization capacity was seen when comparing sera of day 42 of vaccinated mice vs the control group (p= 0.0014). In addition, sera from mice after the 2 nd boost (day 63) further enhanced the neutralization titer when compared to the control group (p. <0.00001) or the group received only 1 boost (p. 0.0021) ( Figure 6F ). The obtained results showed the capacity of the vaccine candidate to completely block the cytopathic effect of the virus.

Manufacturability, in particular scalability and production yields are a critical attribute in selecting vaccines candidates to address a global pandemic. For this reason, we focused here on a VLP-based vaccine that can be efficiently produced in bacteria. Indeed, as the RBM of SARS-CoV-2 is not glycosylated and has no other post-translational modifications, it may be an optimal candidate for a VLP-based vaccine candidate produced in E. coli. Some studies have shown that neutralizing antibodies against SARS-CoV-2 are low in general and wane relatively rapidly 44 ; some patients may even completely lack long-lasting SARS-CoV-2 antibodies 28, 29 . This may be explained by coronaviruses morphological structure as they are large particles with long spike proteins resulting in RBD trimers spaced by 25 nm. Other viruses and VLPs are capable of inducing optimal and long-lived neutralizing antibodies thanks to the 180 monomers forming a repetitive surface structure with epitopes spaced by 5-10 nm 28 . The induced antibodies using mCuMV TT -RBM vaccine could be detected at similar levels 3 months following the booster immunization, indicating longevity of the induced response.

The most important goal of any anti-viral vaccine is the induction of neutralizing antibodies that can inhibit SARS-CoV-2 infection. Our test sera were probed for their ability to inhibit a cytopathic effect of wildtype SARS-CoV-2 isolate on Vero cells. The induced antibodies have shown good neutralizing capability of the virus in particular following the 2 nd booster dose.

We have not directly measured the induction of T H cell responses by our vaccine candidate, mostly because the expected effector mechanism is induction of neutralizing antibodies. In addition, the size of the RBM may be too small to reliably induce a T H cell response in inbred mice. However, we have previously shown that VLP-specific T H cell responses mediate isotype-switch for B cells specific for antigens displayed on the VLPs. Furthermore, the bacterial RNA packaged in VLPs, such as CuMV TT , drive CD8, and T H 1 responses 47,48 .

We and others have shown recently that N501Y mutation enhanced the binding affinity to ACE2 but did not significantly affect the recognition of RBD by convalescent sera. On the other hand, E484K mutation resulted in abolished recognition by convalescent sera 45 . mCuMV TT -RBM is shown here to induce antibodies of much higher affinity/avidity than SARS-CoV-2 typically does in humans. This increased affinity/avidity translates to increased crossreactivity with SARS-CoV-2 VOC. Indeed, antibodies induced by the here presented vaccine candidate recognizes VoCs from Brazil, UK, and India with equal efficiency suggesting that our vaccine can protect against the new variants.

In addition to stability and high immunogenicity, production yields of mCuMV TT -RBM are of key importance. Indeed, we were able to show in 2-litre bioreactor and continuous flow centrifugation that millions of doses may be produced in a single 1000-litre fermenter run. This is particularly important for less affluent countries, where affordability of vaccines is an important aspect.

Collectively, we have shown in this study that our novel mosaic VLP-based vaccine candidate can efficiently induce high levels of specific anti-RBD and spike antibodies that effectively neutralize SARS-CoV-2 and highly cross-recognize all emerging viral VoC tested. As COVID-19 continues to represents a global threat to human health, it seems rational to further develop this vaccine candidate.

This work was supported by Saiba AG and Inselspital Bern. We thank PD Dr. AlexanderEggel and Dr. Daniel Brigger for providing wildtype RBD protein. 

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