key: cord-1025964-jyzhbjlc
authors: Malladi, Sameer Kumar; Patel, Unnatiben Rajeshbhai; Rajmani, R S; Singh, Randhir; Pandey, Suman; Kumar, Sahil; Khaleeq, Sara; Gayathri, Savitha; Chakraborty, Debajyoti; Kalita, Parismita; Pramanick, Ishika; Agarwal, Nupur; Reddy, Poorvi; Girish, Nidhi; Upadhyaya, Aditya; Khan, Mohammad Suhail; Kanjo, Kawkab; Bhat, Madhuraj; Mani, Shailendra; Bhattacharyya, Sankar; Siddiqui, Samreen; Tyagi, Akansha; Jha, Sujeet; Pandey, Rajesh; Tripathi, Shashank; Dutta, Somnath; Ringe, Rajesh P.; Varadarajan, Raghavan
title: Immunogenicity and protective efficacy of a highly thermotolerant, trimeric SARS-CoV-2 receptor binding domain derivative
date: 2021-05-24
journal: bioRxiv
DOI: 10.1101/2021.01.13.426626
sha: 59ca417346c4a04f7165a6fb52e1f2ef91b3b992
doc_id: 1025964
cord_uid: jyzhbjlc

The Receptor Binding Domain (RBD) of SARS-CoV-2 is the primary target of neutralizing antibodies. We designed a trimeric, highly thermotolerant glycan engineered RBD by fusion to a heterologous, poorly immunogenic disulfide linked trimerization domain derived from cartilage matrix protein. The protein was expressed at a yield of ∼80-100 mg/liter in transiently transfected Expi293 cells, as well as in CHO and HEK293 stable cell lines, and formed homogeneous disulfide-linked trimers. When lyophilized, the trimer possessed remarkable functional stability to transient thermal stress of upto 100 °C and was stable to long term storage of over 4 weeks at 37 °C unlike an alternative RBD-trimer with a different trimerization domain. Two intramuscular immunizations with a human-compatible SWE adjuvanted formulation, elicited antibodies with neutralizing titers in guinea pigs and mice that were 25-250 fold higher than the corresponding values in human convalescent sera. Against the B.1.351 South African variant, neutralization titers for RBD trimer and human convalescent sera were ∼ three-fold and greater than fourteenfold lower respectively. RBD was also displayed on a designed ferritin-like Msdps2 nanoparticle but this showed decreased yield and immunogenicity relative to trimeric RBD. Trimeric RBD immunized hamsters were protected from viral challenge. The excellent immunogenicity, thermotolerance, and high yield of such trimeric RBD immunogens suggest that they are a promising modality to combat COVID-19. Table of Contents (TOC)/Abstract Graphic

The Coronavirus infectious disease 2019 pandemic caused by SARS-CoV-2 1, 2 has led to ~158.6 million infections and ~3.3 million deaths worldwide as on 13 th May, 2021 3 . India is currently in the throes of a debilitating second wave, with the highest daily infection rate in the world. The viral spike glycoprotein is the most abundant protein exposed on the viral surface and the primary target of host elicited humoral immune responses [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] . Thus, there are a large number of COVID-19 vaccine candidates in various stages of development, with ~ 11 candidates already granted emergency use authorisation. However, all of these are required to be stored either refrigerated or frozen. There is thus an unmet need for efficacious vaccines that can be stored for extended periods at room temperature. In addition, there are recent reports of new strains of the virus with enhanced transmissibility and immune evasion 16, 17 . This emphasizes the urgent need to develop vaccine formulations that elicit high titers of neutralizing antibodies to buffer against viral sequence variation [18] [19] [20] . Spike glycoprotein, like various Class I viral surface glycoproteins, assembles as a trimer with each protomer composed of the surface exposed S1 and membrane anchored S2 subunit 21 . The S1 subunit consists of four independently folding domains: N-terminal domain (NTD), Receptor binding domain (RBD), and two short domains (SD1 and SD2) connected by linker regions 4, 5, 22 . The receptor binding domain (RBD) contains the receptor binding motif (residues 438-505) that facilitates interaction with the ACE2 receptor. Subsequent fusion or endocytosis is mediated by the fusion peptide that constitutes the N-terminal stretch of the S2 subunit 21 . It is now well understood that the majority of neutralizing antibodies in both natural infection and vaccination target the RBD 8, 9, 11, 12, [23] [24] [25] [26] [27] [28] . Thus, various groups are involved in designing RBD-based immunogens [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] . We have previously designed a glycan engineered RBD derivative that was highly thermotolerant and induced moderate titers of neutralizing antibodies 37 . Monomeric versions of immunogens elicit lower binding and neutralizing antibodies than multimeric versions 29, 37, 40, 41 . Potential strategies to improve neutralizing antibody titers include fusions containing repetitive antigenic proteins, Fc fusion based dimerization, nanoparticle design and display strategies, and VLP based display platforms 31, 32, [38] [39] [40] [41] [42] [43] . While effective, several display strategies lead to significant antibody titers against the display scaffold or oligomerization motif, such antibodies might either show undesirable side effects in a small fraction of individuals or direct the response away from the intended target after repeated immunizations.

In an alternative strategy, we fused our previously described thermotolerant RBD 37 to a trimerization motif, namely a disulphide linked coiled-coil trimerization domain derived from human cartilage matrix protein (hCMP), to the N-terminus of mRBD. This trimerization domain is expected to be less immunogenic in small animals due to its homology with the corresponding ortholog, than other widely used trimerization domains of bacterial or synthetic origin such as foldon or IZ 44 . In order to compare trimeric RBD with nanoparticle displayed RBD, we also displayed RBD on the surface of ferritin like nanoparticles, employing SpyCatcher-SpyTag technology 45, 46 . hCMP-mRBD expressed as homogenous trimers, possessed comparable thermal stability profiles to the corresponding monomer 37 and remained functional after over 4 weeks upon lyophilization and storage at 37 °C. The trimeric RBD is highly immunogenic in mice and guinea pigs when formulated with SWE adjuvant. SWE is equivalent to the widely used, clinically approved, MF59 adjuvant 47 . Oligomerization increased neutralizing antibody titers by ~25-250 fold when compared with the titers in human convalescent sera, providing a proof of principle for the design strategy. Further the hCMP-mRBD protected hamsters from viral challenge, and immunized sera from mice and guinea pigs neutralized the rapidly spreading B.1.351 viral variant with only a three-fold decrease in neutralization titers. Stable CHO and HEK293 cell lines expressing hCMP-mRBD were constructed and the corresponding protein was as immunogenic, as the protein expressed from transient transfection. Nanoparticle displayed RBD was expressed at lower yield and did not confer any apparent advantage in immunogenicity relative to trimeric RBD. The very high thermotolerance, enhanced immunogenicity, and protection from viral challenge suggest that this trimeric mRBD with inter-subunit, stable disulfides, is an attractive vaccine candidate that can be deployed to combat COVID-19 without requirement of a cold-chain, especially in resource limited settings.

We previously designed a monomeric glycan engineered derivative of the receptor binding domain termed mRBD (residues 332-532 possessing an additional glycosylation site at N532) that induced neutralizing antibodies in guinea pig immunizations 37 . It is known that oligomerization of native antigens can induce higher titers of binding and neutralizing antibodies 31, 40, 42, [48] [49] [50] [51] [52] . We therefore fused mRBD to the disulfide linked trimerization domain derived from human cartilage matrix protein (hCMP) (residues 298-340). We have previously used this domain to successfully trimerize derivatives of HIV-1 gp120. These earlier derivatives were used to successfully elicit high titers of broadly reactive anti-gp120 antibodies in guinea pigs, and rabbits. In rhesus macaques when combined with an MVA prime, the formulation conferred protection against heterologous SHIV challenge, without apparent adverse effects [53] [54] [55] . We hypothesized that RBD fused to the hCMP trimerization domain (residues 298-340), would elicit higher neutralizing antibody titers relative to the corresponding monomer. In the closed state structure model of Spike-2P protein (PDB 6VXX, residues 332-532), the three RBDs are in the down conformation. We separated the coaxially aligned hCMP trimerization domain C-terminal residue 340, Cα plane from the RBD Nterminal Cα plane by ~22 Å to eliminate any steric clashes (Figure 1a ). The distance between the hCMP C-terminus residue 340 and RBD N-terminus residue 332 was ~ 39.0 Å in the modeled structure (Figure 1a) . A fourteen amino acid linker L14 will comfortably span this distance. We employed the same trimerization domain-linker combination used in our previously described HIV-1 gp120 trimer design 56 . Thus, the trimeric hCMP-mRBD design consisted of the N-terminal hCMP trimeric coiled coil domain (residues 298-340) fused to the I332 residue of mRBD by the above linker, followed by the cleavable His tag sequence described previously 37 (Figure 1b) . The hCMP trimerization domain leads to formation of covalently stabilized trimers crosslinked by interchain disulfides in the hCMP domain. This design is termed hCMP-mRBD and hCMP pRBD where the "m" and "p" signifies expression in mammalian or Pichia pastoris cells, respectively.

Further, we also designed trimeric RBD constructs (residues 332-532) by fusing hCMP and glycosylated IZ 44 The design utilized the RBD (residues 332-532) from the closed state of the Spike-2P (PDB 6VXX) aligned coaxially with the hCMP trimerization domain, coordinates taken from the homolog CCMP (PDB:1AQ5, Chain 1.1). The N termini of mRBD are labelled as I332 and the hCMP trimerization domain C-termini are labelled as V340. The N, C termini Cα's form vertices of equilateral triangles. The N -terminal plane of RBD (I332) is separated from the C-terminal plane (V340) of the hCMP trimerization domain by ~22.1 Å to avoid steric clashes. The I332 terminus and V340 terminus are ~39 Å apart in the modelled structure and are connected by a 14-residue long linker. b. hCMP-mRBD consists of N-terminal hCMP trimerization domain fused to I332 of RBD by a linker (L14). mRBD-hCMP consists of the C-terminal hCMP trimerization domain fused to N532 of RBD by a linker (L5). mRBD-GlyIZ consists of a C-terminal GlyIZ trimerization domain fused to N532 of RBD by a linker (L5). MsDPS2-mRBD consists of the MsDPS2 nanoparticle fused to SpyTag covalently linked with mRBD-SpyCatcher. c. SEC elution profile of trimeric hCMP-mRBD. d. SDS-PAGE of purified mRBD and hCMP-mRBD in reducing and non-reducing conditions demonstrating formation of disulfide-linked trimers. e. SEC-MALS of purified hCMP-mRBD (MW: 110 ±10 kDa). The red, black and blue profiles are of the molar mass fit, molar mass and refractive index (RI) respectively. f. nanoDSF equilibrium thermal unfolding of hCMP-mRBD. g. SDS-PAGE of purified mRBD-GlyIZ and mRBD-hCMP in reducing conditions. h, i. SEC elution profiles of mRBD-hCMP (H) and mRBD-GlyIZ (I). j. SDS-PAGE of purified MsDpS2-SpyTag, mRBD-SpyCatcher and the resulting MsDPS2-SpyTag-mRBD-SpyCatcher conjugate abbreviated MsDPS2-mRBD for simplicity. The black solid line, triangle without fill and red triangle correspond to MsDPS2-SpyTag nanoparticle, mRDS-SpyCatcher and MsDPS2-mRBD conjugate respectively. k. SPR binding of hCMP-mRBD, mRBD-hCMP, mRBD-GlyIZ and SEC purified complex MsDPS2-mRBD to immobilized ACE2. The curves from highest to lowest correspond to concentrations100 nM, 50 nM, 25 nM, 12.5 nM and 6.25 nM respectively for hCMP-mRBD, mRBD-hCMP and mRBD-GlyIZ. The curves for MsDPS2-mRBD correspond from highest to lowest concentrations of 10 nM, 5 nM, 2.5 nM and 1.25 nM respectively. ND*-No dissociation.

hCMP-mRBD was first expressed by transient transfection in Expi293F suspension cells, followed by single step metal affinity chromatography (Ni-NTA) and tag cleavage. The 

We assessed the immunogenicity of the previously described 37 monomeric mRBD and trimeric hCMP-mRBD adjuvanted with SWE, an AddaVax™ and MF59 equivalent adjuvant, in BALB/c mice. Animals were immunized intramuscularly at day 0, followed by a boost at day 21 37 . Two weeks post boost, sera were assayed for binding and neutralizing antibodies.

Trimeric hCMP-mRBD adjuvanted with SWE elicited 16-fold higher mRBD binding titers scores including f. Lung pathology score g. Inflammation score h. Immune cell influx score i. Edema score. j. Histology of lung sections at varying magnifications (4X, 10X and 40X). Lung pathologies of Unchallenged control (UC), virus challenged and immunized animals. The virus challenged lung histology marked to identify 1. Bronchiolitis and bronchopneumonia, 2. Severe alveolar inflammation (leukocytic alveolitis), alveolar edema and congestion of parenchyma, severe blood hemorrhage and leakage of alveolar sacs, 3. perivascular inflammation and vascular congestion, 4. bronchial infiltration of immune cells with marked edema.

There are currently multiple COVID-19 vaccines that have been given emergency use approval and others with encouraging Phase I data 66 are in advanced clinical trials. There remains a need for cheap, efficacious, COVID-19 vaccines that do not require a cold chain and elicit antibodies capable of neutralizing emerging variants of concern (VOC). Despite the extraordinarily rapid pace of vaccine development, there are currently many countries where not even a single dose has been administered. This will prolong the pandemic and promote viral evolution and escape 73 . It has also become clear that minimizing the extent of non SARS-CoV-2 derived immunogenic sequence in the vaccine is highly desirable. We previously designed a thermotolerant, monomeric, glycan engineered RBD (residues 332-532) that elicited neutralizing antibodies. In the present study we sought to improve the immunogenicity without negatively altering biophysical and antigenic characteristics of the designed immunogen by designing trimeric and nanoparticulate RBDs. Overall, trimeric hCMP-mRBD elicited higher binding and neutralizing antibodies in both mice and guinea pigs compared to monomeric mRBD. The best trimeric mRBD involved fusion with the hCMP trimerization domain at the N-terminus of mRBD. Relative to other trimerization domains such as foldon and GCN4 derivatives 32,65 , this forms a trimer that is stabilized by intermolecular disulfides and hence will not dissociate, even at high dilutions. A fusion of hCMP with HIV-1 gp120 has been extensively tested in guinea pigs, rabbits and non-human primates as an HIV-1 vaccine candidate and showed promising immunogenicity without any apparent adverse effects 48, 54, 55 .

This trimerization sequence has sequence identities with the corresponding ortholog of 81, 91 and 51% in mice, guinea pigs and hamsters, consistent with the low hCMP directed titers in guinea pigs. Thus, hCMP titers in humans are expected to be negligible, given 100% sequence identity with the host protein. Immunization studies in non-human primates with hCMP-mRBD will be shortly initiated to confirm this. In addition, fusion of the short disulfide forming stretch to the other trimerization domains is being carried out to examine the modularity of this motif.

Like our previously described monomeric mRBD, hCMP-mRBD shows remarkable thermotolerance. Lyophilized hCMP-mRBD was stable to extended storage at 37 °C for over four weeks and to transient 90-minute thermal stress of upto 100 °C. In contrast, to the alternative GlyIZ trimerization sequence, the disulfide linked hCMP-mRBD was more homogeneous and thermotolerant, demonstrating that the latter feature is not a given for any multimeric RBD formulation. Mice were immunized with various trimeric and nanoparticle displayed RBD constructs ( Figure 1 65 . In contrast to recently described, highly immunogenic multicomponent nanoparticle systems 39, 40 , the present single component, trimeric RBD might be easier to purify and manufacture and in our hands, nanoparticle display did not confer any significant benefit in immunogenicity over trimerization, whilst the former elicited considerably higher titers of scaffold directed antibodies. However, multicomponent as well as Spy-tagged nanoparticles do have the potential advantage of modularity and being able to display two or more antigens simultaneously. Trimeric RBD elicited sera neutralized B·1·351 pseudovirus with only a relatively small drop in neutralization titer compared to that seen in convalescent sera (Figure 3f-3j) . This large drop in HCS has also been observed in other studies, for example it was observed 75 

We describe a thermotolerant, homogenous, intermolecular disulfide-linked, trimeric RBD that is highly expressed, immunogenic, and elicits sera which neutralize both wildtype and B.1.351

virus. This is an excellent candidate for future clinical development and deployment, is easily manufacturable at a large scale, and eliminates the requirement of a cold-chain.

The present trimeric mRBD construct consists of an N-terminal trimerization domain of human cartilage matrix protein (hCMP) (hCMP residues 298-340) (accession number AAA63904) linked by a 14-residue flexible linker (ASSEGTMMRGELKN) derived from the V1 loop of HIV-1 JR-FL gp120 linked to RBD residues 332-532 (accession number YP_009724390.1) with an engineered glycosylation site (NGS) at N532 fused to an HRV-3C precision protease cleavage site linked to a 10x Histidine tag by a GS linker. The hCMP-mRBD construct reincorporated a glycosylation motif "NIT" at the N-terminal of the mRBD recapitulating the native glycosylation site at N331 in SARS-CoV-2 RBD.

The C-terminal fusion of hCMP trimerization domain was obtained by fusing mRBD (residues 332-532) to hCMP (residues 298-340) by a five-residue linker (GSAGS). This construct is termed mRBD-hCMP. Additionally, the C-terminal fusion of Glycosylated IZ trimerization domain was obtained by fusing mRBD (residues 332-532) to Glycosylated IZ (residues "NGTGRMKQIEDKIENITSKIYNITNEIARIKKLIGNRTAS") by a five residue linker (GSAGS). This construct is termed mRBD-GlyIZ. mRBD (residues 332-532) was fused to SpyCatcher (residues 440-549) and the construct was 

mRBD, hCMP-mRBD, mRBD-hCMP, mRBD-GlyIZ, mRBD-SpyCatcher, mSpyCatcher protein was purified from transiently transfected Expi293F cells following manufacturer's guidelines (Gibco, Thermofisher) as described previously 37 . A minimum of three independent batches of purifications were performed for all the constructs.

HRV-3C precision protease digestion was performed to remove the C-terminal 10xHis tag (Protein: HRV-3C = 50:1). HRV-3C digestion was performed for 16 hrs at 4 °C in PBS (pH 7·4). Ni Sepharose 6 Fast flow resin (GE Healthcare) affinity exclusion chromatography was performed to obtain the tagless protein (containing the tag C-terminal sequence: LEVLFQ).

The unbound tagless proteins concentration was determined by absorbance (A280) using NanoDrop™2000c with the theoretical molar extinction coefficient calculated using the ProtParam tool (ExPASy).

Protein nanoparticles present an attractive platform for antigenic display and immune stimulation by mimicking natural infection 79 . In this study, we conjugated mammalian purified RBD (mRBD) from SAR-CoV-2 virus to a self-assembling bacterial protein -DNA Binding 

Equilibrium thermal unfolding of hCMP-mRBD (-10xHis tag) protein, before or after thermal stress was carried out using a nanoDSF (Prometheus NT.48) as described previously 37 . Two independent measurements were carried out in duplicate with 2-4 μM of protein in the temperature range of 15-95 °C at 100% LED power and initial discovery scan counts (350nm) ranging between 5000 and 10000. In all cases, when lyophilized protein was used, it was reconstituted in water, prior to DSF.

hCMP-mRBD protein kinetic binding studies to ACE2-hFc and CR3022 antibody were performed on a ProteOn XPR36 Protein Interaction Array V.3.1 (Bio-Rad). The GLM sensor chip was activated with sulfo-NHS and EDC (Sigma) reaction. Protein G (Sigma) was covalently coupled following activation. ~3500-4000 RU of Protein G (10 µg/mL) was coupled in 10mM sodium acetate buffer pH 4·5 at a flow rate of 30 µl/min for 300 seconds in desired channels. Finally, 1M ethanolamine was used to quench the excess sulfo-NHS esters.

Following quenching, ligand immobilization was carried out at a flow rate of 30 µl/min for 100 seconds. ACE2-hFc or CR3022 were immobilized at ~800 RU on desired channels excluding a single blank channel that acts as the reference channel. hCMP-mRBD analyte interaction with 

Left lobes of lung, fixed in 4% of paraformaldehyde were processed, embedded in paraffin, and cut into 4 μm correct symbol, and sectioned by microtome for haematoxylin and eosin staining. The lung sections were microscopically examined and evaluated for different pathological scores by a veterinary immunologist. Four different histopathological scores were assigned as follows 1. Percent of infected part of lung tissues considering the consolidation of lung; 2. Lung inflammation scores, considering the severity of alveolar and bronchial inflammation; 3. Immune cell influx score, considering the infiltration of lung tissue with the numbers of neutrophils, macrophages and lymphocytes; 4. Edema score, considering the alveolar and perivascular edema. The scores and parameters were graded as absent (0), minimal (1), mild (2), moderate (3), or severe (4) 81 .

Three-time freeze-thawed right lower lobe from the lung of each hamster was homogenized in 1ml of RNAiso Plus Reagent (Takara) and total RNA was isolated as per the manufacturer's protocol using chloroform and isopropanol reagents. The quantity and quality (260/280 ratios) of RNA extracted was measured by Nanodrop. The extracted RNA was further diluted to 27 ng/μl in nuclease free water. The viral sub genomic RNA copy number was quantified by using Pseudovirus neutralization titers (ID50) were determined as the serum dilution at which infectivity was blocked by 50%. The three RBD mutations (K417N, E484K, N501Y) were introduced into the parental Spike-Δ19-D614G clone using overlap PCR and Gibson recombination. The assembled full-length Spike containing the B.1.351 RBD mutations was cloned in pcDNA3.4 vector and was confirmed by sequencing and used to generate the corresponding pseudovirus as described above. The investigators performing the neutralization assays were blinded to the group identities.

The P values for ELISA binding titers, neutralization titers, were analysed with a two-tailed 

Additional experimental information methods and figures available as single pdf file.

Rajesh Ringe (rajeshringe@imtech.res.in), Raghavan Varadarajan (varadar@iisc.ac.in). 

All the data are in the manuscript. Additional requests for data will be approved upon reasonable request and upon an approval of signed data access agreement.

A provisional patent application has been filed for the RBD formulations described in this 

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We thank Dr. Neil King for kindly providing the ACE2-hFc fusion protein, and Drs. Lynda Govt. of India PM CARES fund (SP/OPSA-20-0004) to RV. We also acknowledge funding