key: cord-0273542-90a6q2z0
authors: Phatarphekar, Abhishek; Vidyadhar Reddy, G. E. C.; Gokhale, Abhiram; Karanam, Gopala; Kuchroo, Pushpa; Shinde, Ketaki; Masand, Girish; Pagare, Shyam; Khadpe, Nilesh; Pai, Sangita S.; Vijayan, Vijita; Ramnath, R. L.; Reddy, K. Pratap; Rao, Praveen; Rao, S. Harinarayana; Ramana, Venkata
title: RelCoVax®, a two antigen subunit protein vaccine candidate against SARS-CoV-2 induces strong immune responses in mice
date: 2022-01-10
journal: bioRxiv
DOI: 10.1101/2022.01.07.475330
sha: d258a6d4e3ad871627e409aeb5293923cac31e07
doc_id: 273542
cord_uid: 90a6q2z0

The COVID-19 pandemic has spurred an unprecedented movement to develop safe and effective vaccines against the SARS-CoV-2 virus to immunize the global population. The first set of vaccine candidates that received emergency use authorization targeted the spike (S) glycoprotein of the SARS-CoV-2 virus that enables virus entry into cells via the receptor binding domain (RBD). Recently, multiple variants of SARS-CoV-2 have emerged with mutations in S protein and the ability to evade neutralizing antibodies in vaccinated individuals. We have developed a dual RBD and nucleocapsid (N) subunit protein vaccine candidate named RelCoVax® through heterologous expression in mammalian cells (RBD) and E. coli (N). The RelCoVax® formulation containing a combination of aluminum hydroxide (alum) and a synthetic CpG oligonucleotide as adjuvants elicited high antibody titers against RBD and N proteins in mice after a prime and boost dose regimen administered 2 weeks apart. The vaccine also stimulated cellular immune responses with a potential Th1 bias as evidenced by increased IFN-γ release by splenocytes from immunized mice upon antigen exposure particularly N protein. Finally, the serum of mice immunized with RelCoVax® demonstrated the ability to neutralize two different SARS-CoV-2 viral strains in vitro including the Delta strain that has become dominant in many regions of the world and can evade vaccine induced neutralizing antibodies. These results warrant further evaluation of RelCoVax® through advanced studies and contribute towards enhancing our understanding of multicomponent subunit vaccine candidates against SARS-CoV-2.

In the past two decades, coronaviruses (CoV) have emerged as a serious threat to humans with 3 major outbreaks being reported starting with severe acute respiratory syndrome (SARS) in late 2002, the Middle Eastern respiratory syndrome (MERS) in 2012 and coronavirus disease in December 2019 [1] [2] [3] . Of these, COVID-19 pandemic caused by the SARS-CoV-2 virus has been the most devastating with greater than 250 million infections and over 5 million deaths reported globally within 2 years of the outbreak first being reported in Wuhan, China [4] . A massive global collaborative effort was needed to develop safe and effective vaccines to protect the world population from the rampaging SARS-CoV-2 virus [5] . Multiple vaccine candidates were developed with unprecedented agility using novel platform technologies such as mRNA and carrier viral vector based vaccines as well as traditional platforms such as the inactivated whole virus and protein subunit vaccines [6] .

Most of the approved vaccines as well as vaccine candidates in development, elicit an immune response against the spike (S) surface glycoprotein of SARS-CoV-2 that recognizes the angiotensin converting enzyme 2 (ACE2) receptor on the target cell via the receptor binding domain (RBD) and enables viral entry [7, 8] . Hence antibodies binding to RBD have the potential to block the RBD-ACE2 interaction and neutralize SARS-CoV-2 viral entry. Indeed, it has been observed that sera from rabbits immunized with RBD had a higher neutralizing antibody titer compared to sera from rabbits immunized with full S protein or S1 domain of S protein [9] and higher anti-RBD antibody titer correlated with higher neutralizing antibody titer in patient sera [10] .

Within the past year, SARS-CoV-2 variants with mutations in S protein especially in RBD have emerged with the potential to evade neutralizing antibodies in vaccinated individuals as highlighted in certain in vitro neutralization studies [11] . One particular SARS-CoV-2 variant known as the Delta variant (B.1.617.2) has been of particular concern spreading across multiple geographies and demonstrating the potential to escape neutralization by antibodies elicited through immunization with multiple vaccines targeting the S protein [12, 13] . Hence there is a need for next generation vaccines that are able to protect against multiple variants and encompass additional antigens that have a lower potential for mutation thereby providing broader protection.

One such candidate is the nucleocapsid (N), a structural protein required for packaging the viral RNA [14] . The amino acid sequence of N protein is well conserved with 90% homology between SARS-CoV and SARS-CoV-2 [15] . Convalescent patient serum samples have shown significant N reactive T-cells indicating the potential for stimulating strong cell mediated immunity and long term immunity [16] .

Often, protein subunit vaccine candidates are weak immunogens and require formulation with adjuvants to boost immune response. Aluminum compounds (aluminum hydroxide or alum and aluminum phosphate) are commonly used as adjuvants and boost humoral immune responses but do not stimulate cell mediated immunity adequately [17, 18] . Toll like receptor 9 (TLR9) agonists such as CpG oligonucleotides are known to stimulate a Th1 biased T-cell response [19] and can be used in combination with aluminum based adjuvants to stimulate both humoral and cell mediated immune responses.

We developed RelCoVax®, a RBD and N protein two antigen SARS-CoV-2 vaccine candidate.

The vaccine was designed to provide broader protection against SARS-CoV-2 variants and in this study we demonstrate that the vaccine is strongly immunogenic in mice and produces antibodies that can neutralize two strains of SARS-CoV-2 including the Delta variant.

The published sequence of the SARS-CoV-2 virus Wuhan strain (NCBI Accession MN908947.3) [20] was used as a reference for protein sequences of S (residues 1-1273) and N (residues 1-419) proteins. DNA sequences for S and N proteins were codon optimized for expression in Cricetulus griseus (Chinese hamster) ovary or CHO cells and E. coli respectively and synthesized by GeneArt (Thermofisher Scientific). The DNA sequence for RBD (Arg 319 to Phe 541) was amplified from the synthetic S gene and cloned into the EcoRI and HindIII digested pXC17.4 CHO expression vector with a secretion signal sequence using NEBuilder HiFi DNA assembly kit (New England Biolabs, E5520). Similarly, the DNA sequence for the synthetic N gene (Met1 to Ala 419) was amplified and cloned into the NdeI and XhoI digested pET24a(+) vector (Novagen).

The pXC17.4-RBD plasmid was transfected into CHOK1SV GS-KO cells (Lonza) by electroporation and stably transfected clones were selected by addition of methionine sulphoxamine (Sigma Aldrich) to the CD-CHO media (Gibco). The supernatants of individual clones were screened for expression of RBD qualitatively by ELISA using the GENLISA™ Human SARS-CoV-2 (COVID-19) spike protein S1 antigen ELISA kit. (Krishgen Biosystems, KBVH015-10). The highest RBD expressing clone was transferred to the bioreactor for upstream process development.

The pET24a(+)-N plasmid was transformed into chemically competent BL21DE3 pLysS E. coli cells (Novagen) and the transformants were isolated on kanamycin containing LB plates. Single colonies were inoculated and protein expression was induced using isopropyl-β-D-thiogalactopyranoside (IPTG, Sigma Aldrich). Cell lysates were screened for protein expression using SDS-PAGE followed by Coomassie blue staining. A single N expressing clone was transferred to the fermenter for upstream process development.

SARS-CoV-2 RBD was expressed during fermentation by the stably transfected CHOK1SV-GS KO cell line and secreted extracellularly. The cell free supernatant derived during harvest of the fermented broth was processed using Blue Sepharose™ 6 Fast Flow column (GE Healthcare, Cat# 17-0948-01) in flow-through mode followed by a hydrophobic interaction chromatography (HIC) purification step. The protein was then dia-filtered into a low salt buffer and further processed by anion exchange chromatography. The eluted protein was dia-filtered into the storage buffer, processed for a final endotoxin removal step, sterile filtered and stored at -20 (±5) °C.

SARS-CoV-2 Nucleocapsid was expressed as an intracellular protein in E.coli. The cells were lysed by homogenization and the lysate was centrifuged to obtain the cell free supernatant containing the protein of interest. The supernatant was subjected to depth filtration and processed by anion exchange chromatography in flow through mode. After dia-filtration, the protein was further processed by cation exchange chromatography and the fractions were pooled after analysis of purity by reverse phase (RP) HPLC. The pooled fractions were buffer exchanged into the storage buffer, processed for a final endotoxin removal step, sterile filtered and stored at -70 (±5) °C.

In western blots, RBD was detected using SARS-CoV-2 Spike RBD antibody (Prosci # 9087) as primary antibody and goat anti-rabbit alkaline phosphatase (Jackson Immunoresearch # 111-055-003) as secondary antibody. N protein was detected using SARS-CoV-2 nucleocapsid antibody (Prosci # 35-579) as primary antibody and goat anti-mouse IgG whole molecule (Sigma # A4416) as secondary antibody.

Aluminum hydroxide gel (Alhydrogel®) or alum adjuvant was purchased from Croda, Denmark (Cat # AJV3012) and the CpG deoxy oligonucleotide with a phosphorothioate backbone was custom synthesized by Synbio Technologies, NJ, USA.

To evaluate the binding of RBD to ACE2 receptor, SPR based assays were performed using the Biacore™ T200 (GE Healthcare) system and the kinetic rate constants (ka and kd) and the affinity constant (KD) were determined for 3 individual batches of purified RBD protein. Human ACE2 with Fc tag (Acro Biosystems, AC2-H5257) was captured on the CM5 chip using a running buffer comprising of 10 mM HEPES pH 7.2, 150 mM NaCl and 0.05% Tween-20. The RBD samples were serially diluted from 50 nM to 3.125 nM in running buffer and passed over the chip surface. Single-cycle kinetics was performed using 1:1 kinetic binding model and the sensograms were evaluated using the BIA evaluation software.

Adult male and female BALB/c mice (inbred) were housed at Laboratory Animal Research Services, Reliance Life Sciences, Navi Mumbai. Animals were certified by the veterinarian to be free of any illness. Animals were acclimatized for at least 5 days before initiation of the study. The temperature was maintained at 22 ± 3°C, relative humidity at 50 ± 20% with a light / dark cycle of grown to about 90% confluency prior to starting the assay. Test serum samples along with controls were diluted in MEM + 2% FBS and heat inactivated at 56 °C for 30 mins. Serial dilution of serum samples was performed in a 96 well plate. The viral stocks were diluted in media, added to the wells containing serum dilutions (1:1 ratio at 60 to 100 pfu viral titer per well) and incubated for 1 hr at 37 °C and 5% CO2. After incubation, these were transferred in duplicate to the 24 well plate containing Vero cells after removal of growth media and incubated for 1 hr at 37°C and 5% CO2 with mixing every 15 mins for viral adsorption. The final viral titer per well was between 30-50 pfu.

One mL of overlay media containing carboxy methyl cellulose was added to the wells and the plates were incubated for 5 days at 37 °C and 5% CO2. Wells were washed once with PBS and fixed with formaldehyde solution. After 3 washes, staining was performed by addition of 1% crystal violet solution to the wells. Plates were washed thrice with distilled water, dried and the plaques were counted. The mean number of plaques from the virus only control wells was used to determine the cutoff for desired level of infection reduction. PRNT50 was reported as the highest dilution of test serum for which virus infectivity was reduced by 50%. A standard logical regression model was followed for determination of PRNT50 and cut-off values for determination of valid data points were calculated using the NIH LID Statistical web tool (https://bioinformatics.niaid.nih.gov/plaquereduction/).

To 

All data was analyzed and graphically represented by using Graphpad prism (9.2.0). Statistical significance was determined using unpaired two-tailed T-test and p-values < 0.05 were considered statistically significant (* p-value < 0.05, ** p-value < 0.005, *** p-value < 0.0005, NS -not significant). All preliminary data was tabulated and basic calculations were performed using Microsoft Excel.

We expressed a part of the S protein extending from residue 319 to 541 (223 amino acids) encompassing the RBD and the full length N protein (419 amino acids) (Fig. 1A ). Both RBD and N protein were purified to homogeneity through multistep purification processes. Under nonreducing conditions, the RBD protein band was observed between 24 and 31 KDa (Fig. 1B) indicating monomeric form of the protein (MW = 25 KDa). The N protein sequence lacks cysteine residues and was observed at the expected size below 52 KDa (MW = 45.6 KDa). To determine whether the expressed RBD protein has folded correctly and is able to bind to its target, its interaction with the ACE2 receptor was probed. We observed that RBD bound to ACE2 with high affinity in the low nM range (Table 1) well within the reported KD range of 5 nM to 70 nM for RBD-ACE2 interaction [7, 21] [22] .

No mortality or morbidity was observed in any of the animals due to treatment in all studies. 

Initially we studied the effect of adjuvants alum and CpG oligonucleotide on anti-RBD and anti-N antibody titers in mice sera. The mice were immunized and serum collected as per schematic in Fig. 2A . With no adjuvants (G1), RBD was poorly immunogenic and did not produce significant antibody titers even after 2 doses compared to the placebo group (P) (Fig. 2B) . When alum was included as an adjuvant (G2 and G4), a strong stimulation of anti-RBD antibody titer was observed compared to other groups. The highest anti-RBD antibody geometric mean titer (GMT) was observed for G4 when both alum and CpG oligonucleotide were added as adjuvants. The difference between the anti-RBD titer for G2 and G4 was statistically significant after 1 st dose but not after the 2 nd dose. Unlike RBD, mice in group G1 displayed significantly higher anti-N antibody titer compared to mice in group P indicating that N protein is immunogenic even in absence of adjuvants (Fig. 2C ). Similar to RBD, mice in the groups G2 and G4 displayed a sharp increase in the anti-N antibody titers compared to other groups indicating strong stimulation with alum.

Overall, G4 mice displayed the highest anti-N antibody titers with strong statistical difference compared to G2 mice after 1 st dose but not after 2 nd dose.

We also evaluated the anti-RBD and anti-N antibody titers in mice sera with a 10-fold difference 3B ). For anti-N antibody titers, statistical significance was observed between the 2 dosing regimens on day 14 as well as on day 28 due to an approximately 3-fold decrease in anti-N GMT between day 21 and day 28 for the 1 µg dose group (Fig. 3C) .

We optimized the formulation of our vaccine candidate to include a preservative and had to increase the minimum formulation volume to 200 µL per dose while maintaining the same amount of each antigen (10 µg) and adjuvants per dose. We also formulated RelCoVax® in a volume of 500 µL per dose envisaged for humans. In subsequent studies, the anti-RBD and anti-N antibody response induced by both these formulations was studied following 2 doses each per mouse (Fig.   4A ). For the 500 µL formulation only 40% of the volume could be injected into each mouse Serum dilutions for PRNT50 ranged from 10 fold to 2560 fold hence these were the limits of quantification for the titer values. Relative to placebo, significantly stronger virus neutralization was observed in test serum pools indicating presence of neutralizing antibodies against both strains of the virus (Fig. 4C) . Immunization with the 200 µL formulation produced higher overall neutralizing antibody titer (statistical significance was observed between the 200 µL formulation and the 500 µL formulation against the Delta strain).

Cellular responses to RBD and N proteins in immunized animals as well as control group dosed as per schematic in Fig. 5A were determined by stimulating splenocytes with both antigens individually as well as in combination. Media only was used as negative control and Con A was used as a positive control (data not shown). We observed higher overall IFN-γ positive cells per million splenocytes in test mice compared to placebo mice across all treatment groups (Fig. 5B ).

In the test group, significantly higher stimulation was seen in N protein and RBD plus N protein treated group compared to media treated group (p-values of 0.0097 and 0.0098 respectively). We did not see much IFN-γ response in media treated and RBD treated placebo mice splenocytes but N protein and RBD plus N protein treatment resulted in high IFN-γ response even in placebo group. However, the test mice splenocytes showed significantly higher IFN-γ response to N protein and RBD plus N protein compared to placebo group (p-values of 0.015 and 0.0023 respectively).

With the COVID-19 pandemic caused by the SARS-CoV-2 virus continuing to spread across the world including in some countries where a substantial population has been vaccinated, there is a need for development of vaccines targeting multiple viral antigens. In this paper we have described the development of RelCoVax®, a two antigen adjuvanted subunit vaccine candidate comprising of the RBD and N proteins of SARS-CoV-2 virus and its immunogenicity in mice.

Vaccine treatment did not affect the body weight or feed and water consumption and no treatment related clinical signs of toxicity were observed during experimental period in any of the animals barring transient local injection site reactions indicating that the vaccine was generally safe for the treated animals.

Previous studies with the SARS-CoV virus have indicated that RBD produced through heterologous expression and adjuvanted with alum can neutralize viral entry into cells [23] . In addition to alum, we also included a CpG oligonucleotide, an adjuvant known to stimulate a Th1 biased immune response with stronger stimulation of cell mediated immunity. This combination of adjuvants has also been tested with SARS-CoV-2 subunit vaccine candidates based on the full length S protein [24, 25] . Our vaccine candidate produced a robust antibody response to both RBD and N antigens in immunized mice with endpoint titers well over 1000 fold higher compared to placebo treated mice after a 2 dose regimen. Elevated anti-RBD and anti-N antibody levels could be detected nearly 2 weeks after first dose and the second dose led to a strong increase in titer to peak levels within a week after administration. A dose of 10 µg of each antigen produced higher titers than 1 µg of each antigen. However, we did observe a dose sparing effect when formulation volumes were readjusted to include a preservative. Here, a dose of 40% of each antigen and adjuvant produced nearly the same levels of anti-RBD and anti-N antibody titers (200 µL Vs 500 µL formulation).

There is a strong rationale for increasing the coverage of SARS-CoV-2 antigens beyond the S protein with new mutations being identified in the S protein including the RBD in emerging variants, enabling them to escape neutralizing antibodies in individuals immunized with the 1 st generation of spike protein based vaccines [26] . Recent reports have emerged wherein N protein has been explored as a vaccine candidate and has displayed promising results. Immunization with N protein alone was shown to protect both mice and hamsters against SARS-CoV-2 viral challenge and a positive correlation was established between N peptide reactive T-cell response and the level of protection [27] . Inclusion of N protein along with S protein for immunization has also been shown to aid viral clearance from distal organs like the brain compared to S protein alone [28] . We observed that N protein is highly immunogenic and capable of inducing anti-N antibodies without addition of adjuvants. The splenocytes of RelCoVax® immunized animals showed a strong IFN-γ response to stimulation with N protein and RBD plus N protein compared to unstimulated controls. Stimulation with RBD alone did produce a slightly higher mean IFN-γ response but a high mouse to mouse variation was seen and overall it was not significant compared to unstimulated control. Here we have demonstrated the ability of N protein to strongly stimulate T cell responses with a Th1 bias. Other studies have also reported Th1 biased T-cell responses in NHP and mice models upon immunization with S protein or a modified RBD protein in combination with N protein with no evidence of vaccine associated enhanced respiratory disease (VAERD) [29, 30] .

With the emerging threat of new SARS-CoV-2 variants, a vaccine capable of providing broad protection against multiple strains is desired. We tested the ability of sera from immunized mice to neutralize two SARS-CoV-2 virus strains including the Delta variant of concern that is currently to Phe 541 and includes the receptor binding motif (RBM) [7] . Affinity constants for interaction of RBD and ACE2 as measured by SPR. Three independent batches of purified RBD were evaluated for ACE2 binding. 

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We would like to acknowledge Dr Ruta Kulkarni and her team at NIBEC, Pune for performing the pRNT50 tests with immunized mice sera provided by Reliance Life Sciences Pvt. Ltd.

VR conceptualized the vaccine and provided overall guidance, AP, GECVR, GK and KS designed the constructs, performed the cloning experiments and expression studies, GM, SP, NK, SSP, and VV developed the upstream and downstream processes for protein production as well as characterization, PK performed the SPR experiments, GECVR, AP, AG and GK performed formulation studies, SHR provided guidance for animal testing, RRL and KPR performed animal experiments, GECVR, AP, GK and AG developed and performed ELISA assays, PR provided technical guidance and designed the ELISpot experiments that were performed by PR, AP and AG. AP wrote the manuscript.