key: cord-0910796-j1ywcj2d
authors: Mi, Taotao; Wang, Tiantian; Xu, Huifang; Sun, Peng; Hou, Xuchen; Zhang, Xinwei; Ke, Qian; Liu, Jiawen; Hu, Shengwei; Wu, Jun; Liu, Bo
title: Kappa-RBD produced by glycoengineered Pichia pastoris elicited high neutralizing antibody titers against pseudoviruses of SARS-CoV-2 variants
date: 2022-03-06
journal: Virology
DOI: 10.1016/j.virol.2022.03.001
sha: 9f286d55e516f7b548b093468593e9685e86e19c
doc_id: 910796
cord_uid: j1ywcj2d

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) kappa (B.1.617.1) variant represented the main variant of concern (VOC) for the epidemic in India in May 2021. We have previously established a technology platform for rapidly preparing SARS-CoV-2 receptor-binding domain (RBD) candidate vaccines based on glycoengineered Pichia pastoris. Our previous study revealed that the wild-type RBD (WT-RBD) formulated with aluminum hydroxide and CpG 2006 adjuvant effectively induces neutralizing antibodies in BALB/c mice. In the present study, a glycoengineered P. pastoris expression system was used to prepare recombinant kappa-RBD candidate vaccine. Kappa-RBD formulated with CpG and alum induced BALB/c mice to produce a potent antigen-specific antibody response and neutralizing antibody titers against pseudoviruses of SARS-CoV-2 kappa, delta, lambda, beta, and omicron variants and WT. Therefore, the recombinant kappa-RBD vaccine has sufficient potency to be a promising COVID-19 vaccine candidate.

glycoengineered P. pastoris expression system was used to prepare recombinant 23 kappa-RBD candidate vaccine. Kappa-RBD formulated with CpG and alum induced 24 BALB/c mice to produce a potent antigen-specific antibody response and neutralizing 25 antibody titers against pseudoviruses of SARS-CoV-2 kappa, delta, lambda, beta, and 26 omicron variants and WT. Therefore, the recombinant kappa-RBD vaccine has 27 sufficient potency to be a promising COVID-19 vaccine candidate.

Introduction 32 SARS-CoV-2 is responsible for the global COVID-19 pandemic. As of 33 February 2022, more than 423 million cases of COVID-19 infections have been 34 reported worldwide, with 5.8 million mortalities caused by COVID-19 infections [1] . 35 SARS-CoV-2 infects the respiratory system with symptoms including cough and 36 fever and acute respiratory distress in severe cases [2] . The rapid spread of COVID-19, 37 the severe clinical symptoms, and the emergence of virus variants require urgent 38 development of new vaccines and treatments. 39 SARS-CoV-2 is an enveloped, single-stranded, positive-sense RNA virus [3] . 40 The spike (S) glycoprotein, which induces neutralizing antibodies against viral 41 infection, comprises a receptor-binding subunit S1 and a membrane fusion subunit S2.

S is an essential part of the virus mechanism for binding, fusion, and entry into 43 mammalian cells. The S1 subunit contains an N-terminal domain (NTD), a 44 receptor-binding domain (RBD), and two small subdomains (Fig. 1a) . The RBD In the present study, we designed a system to express a kappa mutant RBD 71 vaccine in yeast and evaluated its effectiveness against SARS-CoV-2 pseudoviruses, 72 facilitating the future development of SARS-CoV-2 preventive vaccines. which cultured in the BMGY with a concentration of 1% methanol, expressed the 100 target protein, as previously described [13] .

The RBD was collected from the fermentation supernatant and purified chromatographies [13] .

Protein structural assessment. The purified RBD was treated with 107 peptide-N-asparagine (PNGase F), and the resulting protein was analyzed by 108 SDS-PAGE and western blotting using anti-SARS-CoV spike S1 (rabbit) and HRP 109 goat anti-rabbit IgG antibodies (dilution ratio of 1:2500).

The relative molecular mass, RBD reduction, and deglycosylation were The RBD purity was analyzed by high-performance liquid chromatography 117 (HPLC) using a ZORBAX 300SB-C8 column (5 μm, 4.6 mm × 15 cm) (Agilent). The protein) 216 amino acids in length with E484Q, and L452R mutations (Fig. 1a) .

Mutations in the SARS-CoV-2 RBD for each variant are listed in Fig. 1b .

The RBD was expressed in a glycoengineered P. pastoris expression system 190 (Fig. 1c) . The proteins expressed in yeast were purified by cationic exchange, 191 hydrophobic, anion exchange, and gel exclusion chromatographies (Fig. 1d ) [13] . The (Fig. S1 ).

After SEC-HPLC (TSKgel G3000sw, TOSOH) and RP-HPLC (Agilent,

ZORBAX 300SB-C8) analysis, the RBD sample yielded a single peak (Fig. S2) , 199 indicating a protein purity of 100 %.

The affinity of kappa-RBD and WT-RBD toward the His-ACE2 ectodomain 201 was determined by BLI ( Fig. 1f and Table S1 ). The results showed that the affinity of 10 µg RBD-Alum group was 3.9 × 10 2 . A significant difference between the three 211 groups was observed (P < 0.001). Two weeks after the booster immunization, the 212 serum antibody titer of the mice in the 10 µg RBD-Alum-CpG group was 5.12 × 10 6 , the serum antibody titer of the mice in the 2.5 µg RBD-Alum-CpG group was 1.08 × 214 10 6 , and the serum antibody titer of the mice in the 10 µg RBD-Alum group was 1.06 215 × 10 4 , and the difference in the antibody titers between the three groups was highly 216 significant (P < 0.001) (Fig. 2c-e) . The results showed that the 10 µg RBD dose 217 induced higher total anti-RBD IgG titers, indicating that a higher antigen dose 218 increased the levels of antibodies.

The ratio of IGg1/IGg2a reflects the dominant response type in the body, and The RBD vaccine induced a cellular immune response. Interleukin-4 (IL-4) 10 5 cells, and 85-157 spots/4 × 10 5 cells, respectively) (Fig. 3a) . In addition, 245 compared with the 10 µg RBD-Alum-CpG group, the secretion of IFN-γ, IL-2, and 246 IL-4 in the Alum-CpG group under CpG stimulation was increased significantly 247 (142-215 spots/4 × 10 5 cells, 19-91 spots/4 × 10 5 cells, and 6-32 spots/4 × 10 5 cells, 248 respectively) (Fig. 3b) . Therefore, the 10 µg RBD-Alum-CpG group produced a Several studies have shown that candidate SARS-CoV-2 vaccines prepared with an 322 aluminum hydroxide gel can induce a specific Th2-biased response and may induce 323 pulmonary eosinophilic immunopathology in murine models [34] . an independent t-test (*P < 0.05, **P < 0.01, ***P < 0.005). Normal saline: 0.9% (w/v) NaCl. 

World Health Organization. Weekly operational update on Coronavirus disease (COVID-19)

Clinical Characteristics of 138 Hospitalized Patients With 2019 Novel Coronavirus 353 -Infected Pneumonia in Wuhan

SARS-CoV-2 spike produced in insect cells elicits high neutralization titres in 355 non-human primates

Structure of The SARS-CoV-2 spike receptor-binding dom ain bound to the ACE2 82

Structure, function, and evolution of coronavirus spike proteins

Annual Review of Virology

Elucidation of cellular targets and exploitation of the receptor-binding domain 373 of SARS-CoV-2 for vaccine and monoclonal antibody synThesis

A Vaccine Based on The Receptor-Binding Domain of The Spike Protein

Glycoengineered Pichia pastoris Targeting SARS-CoV-2 Stimulates Neutralizing and Protective Antibody Responses

Convergent evolution of SARS-CoV-2 spike mutations, L452R, E484Q 379 and P681R, in The second wave of COVID-19 in Maharashtra

Assessment of infectivity and The impact on neutralizing activity of immune 381 sera of The COVID-19 variant, CAL

The SARS-CoV-2 spike L452R-E484Q variant in the Indian B.1.617 strain 383 showed significant reduction in the neutralization activity of immune sera

Influence of immunoglobulin isotype on therapeutic antibody function

Multiple SARS-CoV-2 variants escape neutralization by 388 vaccineinduced humoral immunity

Evidence of escape of SARS-CoV-2 variant B.1.351 from natural 390 and vaccine-induced sera

Reduced neutralization of SARS-CoV-2 B.1.1.7 variant by 392 convalescent and vaccine sera

A vaccine targeting the RBD of the S protein of SARS-CoV-2 induces 394 protective immunity

Use of combinatorial genetic libraries to humanize N-linked 396 glycosylation in the yeast Pichia pastoris

Optimization of humanized IgGs in glycoengineered Pichia pastoris 398

Use of HDEL-tagged Trichoderma reesei mannosyl oligosaccharide 400 1,2-α-D-mannosidase for N-glycan engineering in Pichia pastoris

The new SARS-CoV-2 strain shows a stronger binding affinity to ACE2 due to 402 N501Y mutant

Early transmissibility assessment of the N501Y mutant strains of

Sequence analysis of the emerging SARS-CoV-2 variant omicron in South Africa

Omicron and Delta variant of SARS-CoV-2: a comparative 416 computational study of spike protein

FDA, Development and Licensure of Vaccines to Prevent COVID-19: Guidance for Industry

Severe acute respiratory syndrome-associated coronavirus 419 vaccines formulated with delta inulin adjuvants provide enhanced protection while ameliorating lung eosinophilic 420 immunopathology

and transmembrane region (TM). (b) Mutations in the RBD of different SARS-CoV-2 variants. (c) SDS-PAGE analysis of various glycoengineered yeast/RBD clones

f) BLI profiles measuring the interaction between recombinant RBD and ACE2. A11, A12: 50 nM RBD