key: cord-1055823-l0jbe1az
authors: Brandys, Pascal; Montagutelli, Xavier; Merenkova, Irena; Barut, Güliz T.; Thiel, Volker; Schork, Nicholas J.; Trüeb, Bettina; Conquet, Laurine; Deng, Aihua; Antanasijevic, Aleksandar; Lee, Hyun-Ku; Valière, Martine; Sindhu, Anoop; Singh, Gita; Herold, Jens
title: A mRNA vaccine encoding for a RBD 60-mer nanoparticle elicits neutralizing antibodies and protective immunity against the SARS-CoV-2 delta variant in transgenic K18-hACE2 mice
date: 2022-03-22
journal: bioRxiv
DOI: 10.1101/2022.03.21.485224
sha: a1f9a58ea3df78ff76c53acb2b39a4fce5792e87
doc_id: 1055823
cord_uid: l0jbe1az

Two years into the COVID-19 pandemic there is still a need for vaccines to effectively control the spread of novel SARS-CoV-2 variants and associated cases of severe disease. Here we report a messenger RNA vaccine directly encoding for a nanoparticle displaying 60 receptor binding domains (RBDs) of SARS-CoV-2 that acts as a highly effective antigen. A construct encoding the RBD of the delta variant elicits robust neutralizing antibody response with neutralizing titers an order of magnitude above currently approved mRNA vaccines. The construct also provides protective immunity against the delta variant in a widely used transgenic mouse model. We ultimately find that the proposed mRNA RBD nanoparticle-based vaccine provides a flexible platform for rapid development and will likely be of great value in combatting current and future SARS-CoV-2 variants of concern.

A novel zoonotic betacoronavirus that emerged in Wuhan, China at the end of 2019, subsequently named SARS-CoV-2 by the International Committee on Taxonomy of Viruses in January 2020, has resulted in the ongoing Coronavirus Disease 2019 pandemic with a cumulative total of 465 million reported cases and 6 million reported deaths globally (https://covid19.who.int/table). mRNA-based vaccines for SARS-CoV-2 developed during 2020 have proven to be quite effective in preventing severe COVID-19. However, starting from the third quarter of 2020 new SARS-CoV-2 variants have repeatedly appeared and spread worldwide. As a result, the incidence rate of SARS-CoV-2 infection has increased among vaccinated individuals provided the two available mRNA vaccines . From July to November 2021, the delta variant (B.1.617.2) was dominant worldwide representing over 95% of submitted sequences (https://www.epicov.org). Rapidly waning immunity of mRNA vaccines against the delta variant (Goldberg et al., 2021) (Ferdinands et al., 2022) . Two years into this global health crisis there is still a need for the global deployment of vaccines across individuals of all ages that will be effective in limiting infection and disease with current and future variants of concern (VOC).

As a result of the seriousness of the pandemic, and its complex time course, the pathobiology behind SARS-CoV-2 infection and COVID-19 illness has received considerable attention, laying the groundwork for novel diagnostic, treatment and vaccine strategies. Particularly, the SARS-CoV-2 homotrimeric spike (S) glycoprotein mediates virus entry into the host cell and comprises a N-terminal S1

surface subunit which recognizes host cell receptors, and a C-terminal S2 transmembrane subunit which promotes the fusion of the viral and cellular membranes. The receptor binding domain (RBD) of S1 binds to the host cell angiotensin-converting enzyme 2 (ACE2) receptor (Hoffmann et al., 2020; Walls et al., 2020a; Wrapp et al., 2020) . The S glycoprotein is the immunogen encoded by all currently approved mRNA vaccines. The SARS-CoV-2 RBD is the target of 90% of the neutralizing activity present in COVID-19 convalescent sera and is immunodominant with multiple distinct antigenic sites (Piccoli et al., 2020) . RBDtargeted neutralizing antibodies isolated from COVID-19 convalescent patients provide in vivo protection against SARS-CoV-2 challenge in mouse, hamster and nonhuman primates Wu et al., 2020; Zost et al., 2020) . In addition, recurrent potent neutralizing RBD-specific antibodies correlate with the plasma neutralizing activity of COVID-19 patients (Robbiani et al., 2020) . All these findings indicate that the RBD is a prime target of neutralizing antibodies upon SARS-CoV-2 infection and the immunogen of choice for vaccine development.

Numerous mutations are observed throughout the genome of recent variants and these variants are highly infectious compared to the wild-type SARS-CoV-2 genome identified early in the pandemic. In most SARS-CoV-2 strain variants mutations are observed in the neutralizing antibody epitopes of the RBD, allowing escape from neutralizing antibodies (Cele et al., 2021; Collier et al., 2021; Madhi et al., 2021; Planas et al., 2021a; Wang et al., 2021) . The SARS-CoV-2 delta variant is highly contagious and was rapidly spreading at the end of 2021 before the emergence of the omicron variant (Callaway, 2021) . The neutralizing activity of sera from convalescent COVID-19 patients as well as sera from vaccinated individuals decreases for the delta variant compared to the wild-type (Liu et al., 2021b; Planas et al., 2021b) . The RBD of the delta variant (RBDdelta) has acquired the mutations L452R and T478K that are also observed in other variants that are less infectious. It is therefore expected that the RBDdelta immunogen will elicit neutralizing antibodies directed against novel epitopes of the delta variant and other variants of interest. This effect will differ from neutralizing antibodies directed against the wild-type RBD.

The delta variant has also acquired several mutations in the N-terminal domain (NTD) of the S protein. It has been demonstrated that antibodies against a specific site on the NTD can enhance the infectivity of SARS-CoV-2 by inducing the open form of the RBD (Li et al., 2021) . Furthermore, the delta variant escapes from anti-NTD neutralizing antibodies while maintaining functionally-enhancing antibody epitopes (Liu et al., 2021c) , and before the emergence of the omicron variant the delta variant was likely to acquire complete resistance to wild-type spike vaccines (Liu et al., 2021d) . Therefore, the RBD immunogen has an advantage in that it can avoid eliciting enhancing antibodies for the delta variant and potentially for other SARS-CoV-2 variants.

Structure-based antigen design is a widely used approach to improve immunogenicity (Graham et al., 2019; Irvine and Read, 2020; Singh, 2021) and

has been applied to the RBD either by creating RBD multimers or displaying the RBD onto protein or synthetic nanoparticles (NPs) (Borriello et al. 2021 , Cohen et al., 2021 Dalvie et al., 2021; He et al., 2021; King et al., 2021; Ma et al., 2020; Peachman et al., 2021; Tan et al., 2021; Walls et al., 2020b; Walsh et al., 2020) .

The main objective of these various designs is to increase antigen trafficking into draining lymph nodes and to promote B cell receptor clustering and B cell activation. Several RBD-NP recombinant proteins are currently evaluated in clinical trials (NCT04742738, NCT04750343, NCT05175950). In addition, many RBD-NPs designs are incompatible with mRNA vaccines, as they require various additional chemical or enzymatic reactions after protein expression, and the only RBD-based mRNA vaccine being tested in a clinical trial encodes a trimerized RBD (NCT04523571). An mRNA RBD-NP design has the advantage of combining improved immunogenicity, flexibility of design against new variants, and can be rapidly developed as a vaccine as demonstrated by first-generation mRNA vaccines.

Here, we report a messenger RNA encoding for a designed nanoparticle in which multimeric RBDdelta is displayed onto a protein scaffold composed of 60 subunits of the self-assembling bacterial protein lumazine synthase (Zhang et al., 2001) .

The RBDdelta nanoparticle (RBDdelta-NP) elicits potent and protective neutralizing antibody responses in mice, with neutralizing titers an order of magnitude higher than current mRNA vaccine-elicited human sera. We further show that the mRNA RBDdelta-NP vaccine encoding for the RBDdelta protects against COVID-19 disease after SARS-CoV-2 delta variant infection in a widely used transgenic mouse model.

Ultimately, our study demonstrates the utility of a mRNA vaccine directly encoding a highly immunogenic RBD nanoparticle as a flexible platform for the rapid development of effective vaccines against current and future SARS-CoV-2 variants of concern.

Vaccine-induced immune responses can be enhanced by NPs mimicking the size, shape, multivalency, and symmetric surface geometry of many viruses . High density display of glycosylated antigens onto protein NPs of approximately 40 nm in size enhance humoral immunity as they deposit within the B-cell follicles of the lymph nodes and generate a strong immune response (Singh, 2021) . In order to display SARS-CoV-2 RBD onto a protein NP scaffold we used the self-assembling lumazine synthase (LS) from the hyper-thermophile Aquifex aeolicus as a protein NP scaffold (Zhang et al., 2001) . The spherical LS NP scaffold consists of 60 identical subunits with strict icosahedral 532 symmetry and has been used for the display of HIV antigens (Jardine et al., 2013) .

Antigens arranged in a pathogen-associated structural pattern cross-link B-cell receptors and induce the classical pathway of complement activation resulting in durable antibody responses. The optimal immune response is induced by at least 12-16 neutralizing epitopes spaced by 5-10 nm, as between repetitive immunogens on the surface of a typical RNA virus . We fused the RBD (residues 328-531) to the self-assembling LS and confirmed that with a suitable linker length, 60 copies of glycosylated RBD could be sterically accommodated. We found that such nanoparticles presenting glycosylated RBD could be secreted from mammalian (HEK 293) cells and purified by lectin chromatography with structural homogeneity, albeit with a very low yield. We further verified an optimal diameter of the NP sphere of 30 nm and an optimal spacing of 7 nm between two adjacent RBDs by negative-stain electron microscopy ( Fig. 1) . promoter with a Kozak consensus sequence was found to yield higher expression than human alpha globin 5' UTR (Trepotec et al., 2019) . 3' polyadenylation with a poly(A) tail measuring 120 nucleotides compared with a shorter one and an unmasked poly(A) tail with a free 3' end rather than one extended with unrelated nucleotides each independently enhance RNA stability and translational efficiency (Holtkamp et al., 2006) . We elected to encode the poly(A) tail into the template vector rather than enzymatic polyadenylation, resulting in a limitation to about 80 nucleotides for efficient in-vitro transcription (IVT). We designed a mRNA encoding the RBD of the SARS-CoV-2 delta variant (RBDdelta) fused with LS with a minimal 5' UTR, a human beta-globin 3'UTR and a poly(A) tail of 80 nucleotides. The mRNA was not modified, neither with guanosine/cytidine (G/C) content modification nor with chemical modification.

Overhang at the 3' end of the poly(A) tail hampers translational efficiency of IVT RNA (Holtkamp et al., 2006) , therefore we elected to introduce a site for linearization with BsmBI inserted 3' to the poly(A) tail, resulting in a free 3' end after the poly(A) tail. A construct with the 5' minimal untranslated region UTR1, the human IL-2 signal sequence, a nucleotide sequence encoding RBDdelta fused with LS, the human beta-globin 3'UTR, a poly(A) tail of 80 adenosine residues and the BsmBI restriction site was cloned into a pUC19 vector.

The supercoiled pUC19 DNA was upscaled, linearized with BsmBI, and purified.

In vitro transcription was performed with T7 RNA polymerase in a 2mL reaction.

The mRNA was capped with a cap 1 structure on the 5' end by vaccinia 2'-Omethyltransferase enzymatic capping that protects RNA from decapping and degradation (Picard-Jean et al., 2018) . Capped mRNA was purified by reverse phase chromatography followed by tangential flow filtration (TFF). Final yield of purified mRNA was 2.88 g/l of IVT reaction.

Protamine, a small arginine-rich DNA-binding nuclear protein, condenses sperm DNA in all vertebrates (Balhorn, 2007) and is widely used to deliver different types of RNA in vitro and in vivo. Clinical grade protamine, used to condense and protect RNA from RNase degradation, shows significantly improved cytosolic delivery capacity as compared with other grades (Jarzebska, 2020) . mRNA complexed with the polycationic protein protamine also results in a self-adjuvanted vaccine (Kallen et al., 2013) . The mRNA was complexed by addition of clinical grade protamine to the mRNA at a mass ratio of 1:5. The vaccine was prepared on each injection day with final total mRNA concentration of 840μg/ml. Protamine-condensed RNA was shown to be effective for vaccination using the ear pinna as injection site in mouse (Hoerr et al., 2000) and using intradermal or intramuscular needle-free injection in a human clinical trial (Alberer et al., 2017) .

We assessed the two injection procedures in mice and concluded at the superiority of needle-free injection for the mRNA RBD-NP vaccine ( Fig. 2 A) .

To investigate the prophylactic potential of the mRNA RBDdelta-NP vaccine against the delta variant of SARS-CoV-2 we gave to one group (n=8) of mice a prime/boost regimen of the mRNA RBDdelta-NP vaccine. CB6F1/J female mice 8 weeks old were primed at week 0 and boosted at week 3 with the same dose of 42µg/50µl by intramuscular injection at the caudal thigh with a needle-free injection system (Tropis® injector modified for mouse injection, PharmaJet). Blood was collected and serum prepared on weeks 0 (prior to prime), 3 (prior to boost) and 6.

To determine if the mRNA RBDdelta-NP vaccine elicited SARS-CoV-2 delta neutralizing antibodies we analyzed samples of week 0, 3 and 6 with the cPass™ SARS-CoV-2 neutralization antibody detection kit (GenScript, Cat #L00847). The assay detected any antibodies in serum that neutralize the interaction between the RBDdelta and the ACE2 receptor. The RBDdelta-ACE2 interaction inhibition rate was calculated with the net optical density (OD) of sample and negative control as Inhibition = (1 -OD value of sample/OD value of negative control) x 100%.

Neutralizing antibodies were detected with a cutoff inhibition value of 30% established in a human clinical study.

In all 8 mice receiving the mRNA RBDdelta-NP vaccine for all 8 week 3 samples and all 8 week 6 samples SARS-CoV-2 delta neutralizing antibodies were detected in the mouse sera. For all random week 0 samples no neutralizing antibodies were detected. High inhibition at week 3 (mean 87.0%, standard deviation 10.3%) and

very high inhibition at week 6 (mean 98.0%, standard deviation 0.6%) was observed in all mouse sera. There was a significant difference between week 3

and 6 (Student t-test, p-value= 0.01) (Fig. 3 A) .

The sera of all mRNA RBDdelta-NP-immunized mice collected at week 3 and 6 were also tested for neutralization of cell infection by authentic SARS-CoV-2 delta coronavirus. Two sera of human patients vaccinated with a regular vaccination schedule of BNT162b2 (Pfizer-BioNTech) mRNA vaccine and collected three weeks after the second dose were used as positive controls. The mRNA RBDdelta-NP needle-free intramuscular vaccination schedule induced effective neutralizing antibodies against the SARS-CoV-2 delta variant in 5 mouse sera at week 3 and in all 8 mouse sera at week 6, with median neutralization dose (ND50) ranging from 40 to 5120 at week 6. Mean ND50 was 108 at week 3 and 2095 at week 6, whereas mean ND50 of positive controls was 160. Thus, mean ND50 of sera 3 weeks after the second dose was 13-fold as compared with BNT162b2-elicited human sera. For 3 mouse sera ND50 at week 6 was 32-fold as compared with

BNT162b2-elicited human sera (Fig. 3 B) .

Transgenic mice expressing human angiotensin-converting enzyme 2 under the control of human cytokeratin 18 promoter (K18-hACE2) are highly susceptible to SARS-CoV-2 infection and represent a suitable animal model for the study of viral pathogenesis, and for identification and characterization of prophylactic vaccines for SARS-CoV-2 infection and associated severe COVID-19 disease (Oladunni et al., 2020) .

To assess the prophylactic effect of the mRNA RBDdelta-NP vaccine against the delta variant of SARS-CoV-2 we gave to one group (n=16) of K18-hACE2 transgenic mice a prime/boost regimen of the mRNA RBDdelta-NP vaccine (Fig. 4) .

Female K18-hACE2 transgenic mice at 8 weeks old were primed at week 0 and boosted at week 3 with the same dose of 42µg/50µl by intramuscular injection at the caudal thigh with the needle-free injection system. Blood was collected and serum prepared in week 0 (prior to prime), 3 (prior to boost), 6 and 8 (prior to viral challenge). A second control group (n=16) of female K18-hACE2 transgenic mice of same age did not receive any vaccine. Both groups were housed under the same conditions, including a 3-day international shipment during week 4 from California (after immunization) to France (before viral challenge).

To (Fig. 4) . All 8 control mice showed signs of severe disease with rapid body weight loss and reached termination criteria at 7 or 8 dpi ( Fig. 6 and 7). All 8 immunized mice showed body weight gain at 14 dpi with 3 mice experiencing a mild symptomatic episode at 5-6 dpi followed by rapid recovery at 7-8 dpi ( Fig. 6 and 7) .

Eight immunized mice and 8 unvaccinated controls were euthanized at 3 dpi for measurement of viral load and viral titer in lung homogenates (Fig. 4) . Viral load showed a significant reduction in immunized mice (Student t-test, p= 0.0088) ( Fig.   8 A) . Viral titration on Vero-E6 cells showed a more significant reduction in immunized mice (Student t-test, p= 0.0015) and half of the immunized mice showed high protection with mean viral titers 2.6 log10 below the mean of control mice (Fig. 8 B) . (Oladunni et al., 2020) . We also assessed the variation of ten inflammatory cytokine and chemokines in the RNA extracted from total lung homogenates at 3 dpi ( Fig. 9 A) . We found that reduced expression of C-C motif ligand 2 (Ccl2), C-C motif ligand 3 (Ccl3) and C-X-C motif ligand 10 (Cxcl10) contents in immunized mice significantly correlated with reduced lung infection expressed in log10PFU per gram of lung ( Fig. 9 B) . Therefore, the mRNA RBDdelta-NP vaccine also confers protection against lung inflammation mediated by SARS-CoV-2 delta variant infection, as evaluated by titration. We also found a significant increase of the relative expression of subunit p40 of interleukin 12 (Il12p40) and chemokine C-C motif ligand 5 (Ccl5) in lung homogenates of 8 immunized mice versus 8 unvaccinated and challenged mice ( Fig. 9 A). Ccl5 expression is induced during the later stage of inflammation and is an intrinsic property acquired by CD8 + memory T cells (Marçais et al., 2006) and Ccl5 mRNA is translationally silenced in memory phenotype CD8 + T cells until T cell receptors (TCR) stimulation (Swanson et al., 2002) . In mouse viral lung disease Ccl5 mRNA in the lung peaks less than a day after primary viral infection with Ccl5 production by resident epithelial cells and lung macrophages during the innate immune response, and then in a second wave at 7 dpi, primarily with Ccl5 production by infiltrating CD4 + and CD8 + T cells (Culley at al., 2006) . Therefore, elevated Ccl5 expression in immunized mice at 3 dpi was indicative of sustained numbers of memory phenotype T cells in the lung tissue.

Furthermore, interleukin 12 (Il12) expression during infection determines the type and duration of adaptive immune response. Il12 promotes the differentiation of naïve CD4 + T cells into T helper 1 (Th1) cells and aids T-cell expansion and proliferation in cell-mediated immunity (Watford, 2003) . Therefore, the combined increase of Ccl5 and Il12 expression at 3 dpi was indicative of long-term adaptive cell-mediated immune response induced by the mRNA RBDdelta-NP vaccine and warrants further investigation of virus-specific T and B cell response.

To determine the prophylactic effect of immune response against the RBDdelta-NP antigen we assessed the correlation between clinical results and serology results in vaccinated mice. RBDdelta-ACE2 inhibition ratio in mouse sera showed significant correlation, as measured by linear regression analysis and ranked by decreasing p-value, with body weight variation (Fig. 10 A) and clinical score (Fig.   10 B) . Thus, anti-RBDdelta antibodies elicited by the mRNA RBDdelta-NP vaccine are an important correlate of protection against COVID-19 disease in K18-ACE2

transgenic mice.

We also assessed the correlation between infection in the lungs and serology results. The correlation with RBDdelta-ACE2 inhibition ratio (Fig. 11 A) was not significant. However, viral titer in lungs after infection correlated significantly with serum neutralization of cell infection with SARS-CoV-2 delta variant (p-value= 0.025 and p-value= 0.006 in non-parametric Spearman correlation) (Fig. 11 B) .

In this study we demonstrated that mRNA RBDdelta-NP vaccine elicits a robust 

PB and JH are the founders and owners of Phylex BioSciences, Inc., the company that holds IP related to RBD-NP. PB is a named inventor on several RBD-NP vaccine patents. The other authors declare no commercial or financial conflict of interest.

A sample of glycosylated RBD-NP protein was applied to a carbon coated copper grid with uranyl-formate staining. Grids were prepared in duplicates and EM imaging was performed. (A) One of the 186 micrographs that were acquired.

(B) 3271 particles were picked up manually and 2D-aligned into 20 classes.

Nanoparticles are homogeneous in size, look as expected by the design with scattered RBD densities around the LS core and a diameter D ~30 nm. Distance between adjacent RBDs on spherical NP surface is calculated as (π D 2 /60) 1/2 ~7 nm. 

Sera samples analyzed with the SARS-CoV-2 neutralization antibody detection assay.

RBDdelta-ACE2 interaction inhibition rate at week 3 and 6 in CB6F1/J female mice (n= 8).

Week 3 and 6 were significantly different in two-tailed paired t-test (p-value= 0.016).

Sera samples analyzed with the SARS-CoV-2 delta variant neutralization test.

Median neutralization dose (ND50) at week 3 and 6 in CB6F1/J female mice (n= 8). BNT162b2-elicited human sera as reference. Week 3 and 6 were not significantly different in two-tailed paired t-test (p-value= 0.063). 

Correlation between SARS-CoV-2 neutralization antibody assay results and clinical results was assessed in infected K18-hACE2 transgenic mice of immunized Group 1 followed until 14 dpi (n= 8 with individual color code).

week 8 and body weight variation between day 0 and mean of body weights during acute disease period (5-8 dpi). Correlation is significant (p-value= 0.0014, R= 0.92).

week 8 and highest reached clinical score. Correlation is significant (p-value= 0.0038, R= -0.88). Correlation is significant (p-value= 0.025, R= -0.77) and non-parametric Spearman correlation is more significant (p-value= 0.006, R= -0.89).

Animal welfare for the mouse studies in the U.S. was conducted in compliance with the U.S. Department of Agriculture (USDA) Animal Welfare Act ( 

A 15μl sample of expressed protein at 1mg/ml concentration was diluted in TBS at 20μg/ml and applied to a carbon coated copper grid (440-mesh) with uranylformate staining. Grids were prepared in duplicates and EM imaging was performed with a TF20 microscope (200kV) and Tietz 4k/4k camera. 186 micrographs were acquired and 3271 particles were picked up manually and 2Daligned into 20 classes for analysis.

mRNA encoding RBD-NP mRNA encoding the RBDdelta fused with LS was designed with a minimal 5' UTR of 14 nucleotides, a human beta-globin 3'UTR, 3' polyadenylation with a poly(A) tail of 80 nucleotides, and unmodified nucleosides.

A construct with the 5' minimal untranslated region, the human IL-2 signal sequence, a nucleotide sequence encoding RBDdelta fused with LS, the human beta-globin 3'UTR, a poly(A) tail of 80 adenosine residues and the BsmBI restriction site was cloned into a pUC19 vector. The supercoiled pUC19 DNA was upscaled, linearized with BsmBI, and purified. In vitro transcription was performed with T7 RNA polymerase in a 2mL reaction. The mRNA was capped with a cap 1 structure on the 5' end by vaccinia 2'-O-methyltransferase enzymatic capping.

Capped mRNA was purified by reverse phase chromatography using POROS™ resin (Thermo Fisher) followed by tangential flow filtration (TFF). Final yield of purified mRNA was 2.88 g/l of IVT reaction. RNA integrity was analyzed by denaturing PAGE. mRNA sample and ladder were denatured at 70°C for 2 minutes and kept on ice, then 200ng of mRNA mixed with diluent marker was loaded on gel.

The mRNA was complexed by addition of clinical grade protamine to the mRNA at a mass ratio of 1:5. The vaccine was prepared on each injection day with final total mRNA concentration of 840mg/l. mRNA integrity prior to and after formulation was analyzed on 1% agarose gel with SYBR safe stain (Thermo Fisher).

All groups of immunized mice were primed at week 0 and boosted at week 3 with the same dose of 42µg/50µl by intramuscular injection at the caudal thigh under 1-5% isoflurane anesthesia with a needle-free injection system (Tropis® injector prototype modified for mouse injection, PharmaJet). Blood was collected by retroorbital sinus or submandibular vein puncture and serum samples were prepared in week 0 (prior to prime), 3 (prior to boost), 6, and additionally in week 8 (prior to viral challenge) for the transgenic mice. As an exception for the injection route study (Fig. 2 A) boost was at week 2 and blood collection at week 4. For plaque assay, 10-fold serial dilutions of samples in DMEM were added onto VeroE6 monolayers in 24 well plates. After one-hour incubation at 37°C, the inoculum was replaced with equivalent volume of 5% FBS DMEM and 2%

carboxymethylcellulose. Three days later, cells were fixed with 4% formaldehyde, followed by staining with 1% crystal violet to visualize the plaques.

Total RNA was prepared from one lung lobe collected at 3 dpi using lysing matrix (Table XX) .

The following thermal profile was used: a single cycle of polymerase activation for 2 min at 50°C and 10 s at 95°c, followed by 40 amplification cycles of 15 s at 95°C and 1 min 60°C (annealing-extension step). Mouse Gapdh was used as an endogenous reference control to normalize differences in the amount of input nucleic acid. The average CT values were calculated from the technical replicates for relative quantification of target cytokines/chemokines. The differences in the CT cytokines/chemokines amplicons and the CT of the endogenous reference control, termed dCT, were calculated to normalize for differences in the quantity of nucleic acid. Relative expression was calculated as 2 dCT .

Sera samples of week 0, 3, 6 and 8 were analyzed with the cPass™ SARS-CoV-2 neutralization antibody detection kit (GenScript, Cat #L00847) to detect any antibodies that neutralize the interaction between the RBDdelta and the ACE2 receptor. The kit contains two key components: the horseradish peroxidase (HRP) conjugated recombinant SARS-CoV-2 RBD (HRP-RBD), and a capture plate coated with human ACE2 receptor protein (hACE2). The protein-protein interaction between HRP-RBD and hACE2 is blocked by neutralizing antibodies against the RBD. The assay was performed as per the manufacturer's protocol, except the HRP-RBD component was replaced with HRP conjugated RBDdelta. First, the samples and controls were pre-incubated with the HRP-RBDdelta for 30 min at 37°C

to allow the binding of the circulating neutralization antibodies to HRP-RBDdelta.

The mixture was then added to the capture plate and incubated for 15 min at 37°C.

The unbound HRP-RBDdelta as well as any HRP-RBDdelta bound to non-neutralizing antibody was captured on the plate, while the circulating neutralization antibodies-HRP-RBDdelta complexes remained in the supernatant and were removed during washing. After washing steps, 3, 3', 5, 5'-tetramethylbenzidine ( 

To introduce the variant specific mutations into the spike gene, we used twelve 50 bp primers containing the desired nucleotide changes in a PCR reaction. These PCR products were used to replace WU-Fragments 9 and 10 (covering the spike region). The WU-Fragments encoding the whole SARS-CoV-2 genome are described in Thao et al. (2020) . The WU-Fg. 1.3-8, 11, 12 and the newly created PCR fragments with 50 bp homologous overlaps, were then used for the in-yeast TAR cloning method as described previously (Thao et al., 2020) to generate infectious cDNA clones. In vitro transcription was performed for EagI-cleaved

YACs and PCR-amplified SARS-CoV-2 N gene using the T7 RiboMAX Large Scale RNA production system (Promega) as described previously. Transcribed capped mRNA was electroporated into baby hamster kidney (BHK-21) cells expressing SARS-CoV-2 N protein. Electroporated cells were co-cultured with susceptible VeroE6 cells expressing TMPRSS2 to produce passage 0 (P.0) of the recombinant SARS-CoV-2 S-delta . Subsequently, progeny virus was used to infect fresh VeroE6-TMPRSS2 cells to generate P.1 stocks for downstream experiments.

Vero-E6 cells were seeded at a density of 1.5x10 4 cells/100μl per well (in DMEM supplemented with 10% FBS, 1% non-essential amino acids (NEAA) and

1% Penicillin-Streptomycin) in 96-well cell culture plates one day before and incubated over night at 37°C, 5% CO2. Two-fold dilution series of control sera and serum samples of week 3, 6 and 8 were prepared in quadruplicates in 96-well cell culture plates using unsupplemented DMEM cell culture medium (50μl /well). To each well, 50μl of DMEM containing 100 plaque forming units (PFU) of SARS-CoV-2 S-delta from P.1 stock described above were added and incubated for 60 min at 37°C. Subsequently 100 μl of serum and virus mixtures were added on confluent Vero E6 cells and 96-well plates were incubated for 72 h at 37°C. The cells were fixed for 10 min at room temperature with 4% buffered formalin solution, then cells were counterstained with a solution containing 1% crystal violet for another 10 min at room temperature. Finally, the microtiter plates were rinsed with deionized water and immune serum-mediated protection from cytopathic effect was visually assessed.

Statistical analyses were performed using Prism v9.3.1 (GraphPad Software) ( Fig.   2 -3, 5, 10-11) or using R Statistical Software v4.1.2 (R Core Team 2021) ( Fig. 6-9 ). Some datasets were analyzed after Log-transformation as indicated in the figure legends. Statistical differences between groups in datasets with one categorical variable were evaluated by two sample t-test (2 groups 

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Reduced sensitivity of SARS-CoV-2 variant Delta to antibody neutralization

Convergent antibody responses to SARS-CoV-2 in convalescent individuals

Eliciting B cell immunity against infectious diseases using nanovaccines

RANTES production by memory phenotype T cells is controlled by a posttranscriptional, TCR dependent process

A COVID-19 vaccine candidate using SpyCatcher multimerization of the SARS-CoV-2 spike protein receptor-binding domain induces potent neutralising antibody responses

Ultrapotent human antibodies protect against SARS-CoV-2 challenge via multiple mechanisms

Rapid reconstruction of SARS-CoV-2 using a synthetic genomics platform

Maximizing the translational yield of mRNA therapeutics by minimizing 5'-UTRs

Virus-like particles are efficient tools for boosting mRNA

Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein

Elicitation of Potent Neutralizing Antibody Responses by Designed Protein Nanoparticle Vaccines for SARS-CoV-2

Safety and Immunogenicity of Two RNA-Based Covid-19 Vaccine Candidates

Antibody resistance of SARS-CoV-2 variants B.1.351 and B.1.1.7

Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation

The biology of IL-12: coordinating innate and adaptive immune responses

A noncompeting pair of human neutralizing antibodies block COVID-19 virus binding to its receptor ACE2

X-ray structure analysis and crystallographic refinement of lumazine synthase from the hyperthermophile Aquifex aeolicus at 1.6 Å resolution: determinants of thermostability revealed from structural comparisons

Potently neutralizing and protective human antibodies against SARS-CoV-2