Production of a Highly Immunogenic Antigen from SARS-CoV-2 by Covalent Coupling of the Receptor Binding Domain of Spike Protein to a Multimeric Carrier

Abstract

Since the discovery of SARS-CoV-2, several antigens have been proposed to be part of COVID-19 vaccines. The receptor binding domain (RBD) of Spike protein is one of the promising candidates to develop effective vaccines since it can induce potent neutralizing antibodies. We previously reported the production of RBD in Pichia pastoris and showed it is structurally identical to the protein produced in mammalian HEK-293T cells. In this work we designed an RBD multimer construct with the purpose of increasing RBD immunogenicity. We produced multimeric particles by a transpeptidation reaction between the RBD expressed in P. pastoris and Lumazine Synthase from Brucella abortus (BLS), which is a highly immunogenic and very stable decameric protein of 170 kDa. We vaccinated mice with two doses 30 days apart, and then we measured humoral immune response. When the number of RBD copies coupled to BLS was high (6-7 RBD molecules per BLS decamer, in average), the immune response was significantly better than that elicited by RBD alone or even by RBD-BLS comprising low number of RBD copies (1-2 RBD molecules per BLS decamer). Remarkably, the construct with high number of RBD copies induced high IgG titers with high neutralizing capacity. Furthermore, a superior immune response was observed when Al(OH)3 adjuvant was added to this formulation, exhibiting a higher titer of neutralizing antibodies. Altogether our results suggest that RBD covalent coupled to BLS forming a multimer-particle shows an advantageous architecture to the antigen-presentation to the immune system which enhances immune responses. This new antigen should be considered a potent candidate for a protein-based vaccine.

SciScore for 10.1101/2021.04.25.441271: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Ethics	IRB: Immunization Protocols: Mice immunization was carried out by experts from the High Level Technological Service from CONICET (STAN No. 4482), under guidelines from the Institutional Committee for the Care and Use of Laboratory Animals (CICUAL).
Sex as a biological variable	Six groups of five Females mice (6-8 week-old) (n=5) were immunized subcutaneously with the same amount of RBD protein (15 µg) coupled or not to BLS in the presence or absence of 500 µg aluminum hydroxide (Al(OH)3).
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
Half maximal inhibitory concentration (IC50), …

SciScore for 10.1101/2021.04.25.441271: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Ethics	IRB: Immunization Protocols: Mice immunization was carried out by experts from the High Level Technological Service from CONICET (STAN No. 4482), under guidelines from the Institutional Committee for the Care and Use of Laboratory Animals (CICUAL).
Sex as a biological variable	Six groups of five Females mice (6-8 week-old) (n=5) were immunized subcutaneously with the same amount of RBD protein (15 µg) coupled or not to BLS in the presence or absence of 500 µg aluminum hydroxide (Al(OH)3).
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
Half maximal inhibitory concentration (IC50), corresponding to the serum antibody dilution causing a 50% reduction of GFP positive cells compared to control “virus only” treated cells, was determined using the same software according to Ferrara and Temperton44.	Temperton44 suggested: None
Experimental Models: Cell Lines
Sentences	Resources
Briefly, HEK-293T cells (2×107) were seeded in a 150-mm tissue culture dish in DMEM medium containing 10% FBS.	HEK-293T suggested: None
Experimental Models: Organisms/Strains
Sentences	Resources
BALB/c mice were obtained from the animal facility of the Faculty of Veterinary Sciences, University of La Plata (Argentina), and housed at the animal facility of the Instituto de Ciencia y Tecnología Dr. César Milstein, Fundación Pablo Cassará.	BALB/c suggested: RRID:IMSR_ORNL:BALB/cRl)
Recombinant DNA
Sentences	Resources
BLS Expression and Purification: A gene encoding a variant of lumazine synthase of Brucella abortus (BLS) including an N-terminal TEV site followed by a Gly-Gly-Gly sequence and a Gly-Ser-GLy-Ser-GLy spacer (Met-Glu-Asn-Leu-Tyr-Phe-Gln-Gly-Gly-Gly-Gly-Ser-GLy-Ser-Gly-BLS) was assembled and cloned in the pET11a vector (Novagen).	pET11a suggested: RRID:Addgene_105501)
ACE2 Expression and Purification: The expression plasmid pcDNA3-sACE2(WT)-8his, which includes the human ACE2 soluble domain (residues 1-615) coding gene, followed by a Gly-Ser-Gly linker and eight His residues for purification, was transfected in HEK-293T mammalian cells.	pcDNA3-sACE2 suggested: None
ACE2 concentration was determined by UV spectrophotometry using an absorption coefficient of 157220 M−1 cm−1. TEV Protease Expression and Purification: Tobacco Etch Virus protease (TEV) was a gift from Helena Berglund (Addgene plasmid # 125194; http://n2t.net/addgene:125194; RRID:Addgene_125194)40.	detected: RRID:Addgene_125194)
Sortase A Expression and Purification: The Sortase A pentamutant (eSrtA) in pET29 was a gift from David Liu (Addgene plasmid # 75144; http://n2t.net/addgene:75144; RRID:Addgene_75144)41.	pET29 suggested: None detected: RRID:Addgene_75144)
Addgene plasmid #11619; http://n2t.net/addgene:11619; RRID:Addgene_11619), a lentivirus backbone (VRC5602, NIH), and the Spike protein (VRC7475_2019-nCoV-S-WT, NIH).	detected: RRID:Addgene_11619)
The next day, cells were transfected with 10 μg of VRC5602, 5 μg pLB-GFP, and 3 μg of VRC7475_2019-nCoV-S-WT in OptiMEM medium using PEI in a 1:3 DNA:PEI ratio.	pLB-GFP suggested: None
Software and Algorithms
Sentences	Resources
Virus titers were calculated by counting the GFP positive cells using an automated counting tool in ImageJ (NIH), a titer of 200-225 GFP positive cells/field using 100X magnification was used for the assay.	ImageJ suggested: (ImageJ, RRID:SCR_003070)
Sera antibody neutralization titers were calculated by a nonlinear regression curve fit using GraphPad Prism software Inc. (La Jolla, CA).	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

Results from JetFighter: We did not find any issues relating to colormaps.

Results from rtransparent:

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No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

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Production of a Highly Immunogenic Antigen from SARS-CoV-2 by Covalent Coupling of the Receptor Binding Domain of Spike Protein to a Multimeric Carrier

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