SARS‑CoV-2 RBD219-N1C1: A yeast-expressed SARS-CoV-2 recombinant receptor-binding domain candidate vaccine stimulates virus neutralizing antibodies and T-cell immunity in mice

The COVID-19 pandemic caused by the SARS-CoV-2 virus is posing a major global challenge due to its rapid infectivity and lethality. Despite a global effort towards creating a vaccine, no viable vaccine currently exists. While multiple bioinformatic studies have attempted to predict epitopes, they have focused on the whole spike protein without considering antibody mediated enhancement or Th-2 immunopathology and have missed some important but less antigenic epitopes in the receptor binding domain. Therefore, this study used in silico methods to design and evaluate a potential multiepitope vaccine that specifically targets the receptor binding domain due to its critical function in viral entry. Immunoinformatic tools were used to specifically examine the receptor binding domain of the surface glycoprotein for suitable T cell and B cell epitopes. The selected 5 B cell and 8 T cell epitopes were then constructed into a subunit vaccine and appropriate adjuvants along with the universal immunogenic PADRE sequence were added to boost efficacy. The structure of the vaccine construct was predicted through a de novo approach and molecular docking simulations were performed which demonstrated high affinity binding to TLR 5 receptor and appropriate HLA proteins. Finally, the vaccine candidate was cloned into an expression vector for use as a recombinant vaccine. Similarities to some recent epitope mapping studies suggest a high potential for eliciting neutralizing antibodies and generating a favorable overall immune response.

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Comparative Immunogenicity of the Recombinant Receptor-Binding Domain of Protein S SARS-CoV-2 Obtained in Prokaryotic and Mammalian Expression Systems

Vaccines ◽

10.3390/vaccines10010096 ◽

2022 ◽

Vol 10 (1) ◽

pp. 96

Author(s):

Iuliia A. Merkuleva ◽

Dmitry N. Shcherbakov ◽

Mariya B. Borgoyakova ◽

Daniil V. Shanshin ◽

Andrey P. Rudometov ◽

...

Keyword(s):

Receptor Binding ◽

Neutralizing Antibodies ◽

Expression System ◽

Protein S ◽

Binding Domain ◽

Receptor Binding Domain ◽

Expression Systems ◽

Mice Model ◽

Mammalian Expression ◽

Recombinant Receptor

The receptor-binding domain (RBD) of the protein S SARS-CoV-2 is considered to be one of the appealing targets for developing a vaccine against COVID-19. The choice of an expression system is essential when developing subunit vaccines, as it ensures the effective synthesis of the correctly folded target protein, and maintains its antigenic and immunogenic properties. Here, we describe the production of a recombinant RBD protein using prokaryotic (pRBD) and mammalian (mRBD) expression systems, and compare the immunogenicity of prokaryotic and mammalian-expressed RBD using a BALB/c mice model. An analysis of the sera from mice immunized with both variants of the protein revealed that the mRBD expressed in CHO cells provides a significantly stronger humoral immune response compared with the RBD expressed in E.coli cells. A specific antibody titer of sera from mice immunized with mRBD was ten-fold higher than the sera from the mice that received pRBD in ELISA, and about 100-fold higher in a neutralization test. The data obtained suggests that mRBD is capable of inducing neutralizing antibodies against SARS-CoV-2.

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A novel bivalent DNA vaccine encoding both spike protein receptor-binding domain and nucleocapsid protein of SARS-CoV-2 to elicit T cell and neutralising antibody responses that cross react with variants

10.1101/2021.06.18.448932 ◽

2021 ◽

Author(s):

Victoria A Brentville ◽

Mireille Vankemmelbeke ◽

Rachael L Methingham ◽

Peter Symonds ◽

Katherine W Cook ◽

...

Keyword(s):

T Cell ◽

Dna Vaccine ◽

Receptor Binding ◽

Binding Domain ◽

Antibody Responses ◽

Receptor Binding Domain ◽

Vaccine Platform ◽

Live Virus ◽

Cell Immunity ◽

Protein Receptor

The efficacy of vaccines targeting SARS-CoV-2 is becoming apparent now that the mRNA and adenovirus vector vaccines that have been approved for emergency use are showing promise. However, the longevity of the protective immune response and its efficacy against emerging variants remains to be determined. To improve longevity and future protection against variants, we have designed a DNA vaccine encoding both the SARS-CoV-2 spike (S) protein receptor-binding domain (RBD) and its nucleocapsid (N) protein, the latter of which is highly conserved amongst beta coronaviruses. The vaccine elicits strong pro-inflammatory CD4 Th1 and CD8 T-cell responses to both proteins, with these responses being significantly enhanced by fusing the nucleocapsid sequence to a modified Fc domain. We have shown that the vaccine also stimulates high titre antibody responses to RBD which efficiently neutralise in both a pseudotype and live virus neutralisation assay and show cross reactivity with S proteins from the emerging variants Alpha (B.1.1.7) and Beta (B.1.351). This DNA platform can be easily adapted to target variant RBD and N proteins and we show that a vaccine variant encoding the B.1.351 RBD sequence stimulates cross-reactive humoral and T-cell immunity. These data support the translation of this DNA vaccine platform into the clinic, thereby offering a particular advantage for targeting emerging SARS-CoV-2 variants.

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SARS-CoV-2 RBD219-N1C1: A Yeast-Expressed SARS-CoV-2 Recombinant Receptor-Binding Domain Candidate Vaccine Stimulates Virus Neutralizing Antibodies and T-cell Immunity in Mice

10.1101/2020.11.04.367359 ◽

2020 ◽

Cited By ~ 1

Author(s):

Jeroen Pollet ◽

Wen-Hsiang Chen ◽

Leroy Versteeg ◽

Brian Keegan ◽

Bin Zhan ◽

...

Keyword(s):

Recombinant Protein ◽

Receptor Binding ◽

Neutralizing Antibodies ◽

Low Cost ◽

Neutralizing Antibody ◽

Binding Domain ◽

Receptor Binding Domain ◽

Wild Type ◽

Cell Immunity

AbstractThere is an urgent need for an accessible and low-cost COVID-19 vaccine suitable for low- and middle-income countries. Here we report on the development of a SARS-CoV-2 receptor-binding domain (RBD) protein, expressed at high levels in yeast (Pichia pastoris), as a suitable vaccine candidate against COVID-19. After introducing two modifications into the wild-type RBD gene to reduce yeast-derived hyperglycosylation and improve stability during protein expression, we show that the recombinant protein, RBD219-N1C1, is equivalent to the wild-type RBD recombinant protein (RBD219-WT) in an in vitro ACE-2 binding assay. Immunogenicity studies of RBD219-N1C1 and RBD219-WT proteins formulated with Alhydrogel® were conducted in mice, and, after two doses, both the RBD219-WT and RBD219-N1C1 vaccines induced high levels of binding IgG antibodies. Using a SARS-CoV-2 pseudovirus, we further showed that sera obtained after a two-dose immunization schedule of the vaccines were sufficient to elicit strong neutralizing antibody titers in the 1:1,000 to 1:10,000 range, for both antigens tested. The vaccines induced IFN-γ, IL-6, and IL-10 secretion, among other cytokines. Overall, these data suggest that the RBD219-N1C1 recombinant protein, produced in yeast, is suitable for further evaluation as a human COVID-19 vaccine, in particular, in an Alhydrogel® containing formulation and possibly in combination with other immunostimulants.

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Yeast-Expressed SARS-CoV Recombinant Receptor-Binding Domain (RBD219-N1) Formulated with Aluminum Hydroxide Induces Protective Immunity and Reduces Immune Enhancement

10.1101/2020.05.15.098079 ◽

2020 ◽

Cited By ~ 15

Author(s):

Wen-Hsiang Chen ◽

Xinrong Tao ◽

Anurodh Agrawal ◽

Abdullah Algaissi ◽

Bi-Hung Peng ◽

...

Keyword(s):

Receptor Binding ◽

Neutralizing Antibodies ◽

Neutralizing Antibody ◽

Binding Domain ◽

Pseudotyped Virus ◽

Receptor Binding Domain ◽

Protein Vaccine ◽

S Protein ◽

Recombinant Receptor ◽

High Level

AbstractWe developed a severe acute respiratory syndrome (SARS) subunit recombinant protein vaccine candidate based on a high-yielding, yeast-engineered, receptor-binding domain (RBD219-N1) of the SARS beta-coronavirus (SARS-CoV) spike (S) protein. When formulated with Alhydrogel®, RBD219-N1 induced high-level neutralizing antibodies against both pseudotyped virus and a clinical (mouse-adapted) isolate of SARS-CoV. Here, we report that mice immunized with RBD219-N1/Alhydrogel® were fully protected from lethal SARS-CoV challenge (0% mortality), compared to ∼ 30% mortality in mice when immunized with the SARS S protein formulated with Alhydrogel®, and 100% mortality in negative controls. An RBD219-N1 formulation Alhydrogel® was also superior to the S protein, unadjuvanted RBD, and AddaVax (MF59-like adjuvant)-formulated RBD in inducing specific antibodies and preventing cellular infiltrates in the lungs upon SARS-CoV challenge. Specifically, a formulation with a 1:25 ratio of RBD219-N1 to Alhydrogel® provided high neutralizing antibody titers, 100% protection with non-detectable viral loads with minimal or no eosinophilic pulmonary infiltrates. As a result, this vaccine formulation is under consideration for further development against SARS-CoV and potentially other emerging and re-emerging beta-CoVs such as SARS-CoV-2.

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A recombinant receptor-binding domain in trimeric form generates completely protective immunity against SARS-CoV-2 infection in nonhuman primates

10.1101/2021.03.30.437647 ◽

2021 ◽

Author(s):

Limin Yang ◽

Deyu Tian ◽

Jian-bao Han ◽

Wenhui Fan ◽

Yuan Zhang ◽

...

Keyword(s):

Receptor Binding ◽

Neutralizing Antibodies ◽

Nonhuman Primates ◽

Rhesus Macaques ◽

Vaccination Strategy ◽

Binding Domain ◽

Receptor Binding Domain ◽

Recombinant Receptor ◽

High Level ◽

Cellular Immune

Safe and effective vaccination is critical to combatting the COVID-19 pandemic. Here, we developed a trimeric SARS-CoV-2 receptor-binding domain (RBD) subunit vaccine candidate that simulates the natural structure of the spike (S) trimer glycoprotein. Immunization with RBD-trimer induced robust humoral and cellular immune responses and a high level of neutralizing antibodies that were maintained for at least 4 months. Moreover, the antibodies that were produced in response to the vaccine effectively neutralized the SARS-CoV-2 501Y.V2 variant. Of note, when the titers of the antibodies dropped to a sufficiently low level, only one boost quickly activated the anamnestic immune response, resulting in complete protection against the SARS-CoV-2 challenge in rhesus macaques without typical histopathological changes or viral replication in the lungs and other respiratory tissues. Our results indicated that immunization with SARS-CoV-2 RBD-trimer could raise long-term and broad immunity protection in nonhuman primates, thereby offering an optimal vaccination strategy against COVID-19.

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SARS-COV-2 Recombinant Receptor-Binding-Domain (RBD) Induces Neutralizing Antibodies Against Variant Strains of SARS-CoV-2 and SARS-CoV-1

Vaccine ◽

10.1016/j.vaccine.2021.08.081 ◽

2021 ◽

Author(s):

John Lok Man Law ◽

Michael Logan ◽

Michael A. Joyce ◽

Abdolamir Landi ◽

Darren Hockman ◽

...

Keyword(s):

Receptor Binding ◽

Neutralizing Antibodies ◽

Binding Domain ◽

Receptor Binding Domain ◽

Recombinant Receptor

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Stereotypic neutralizing VH antibodies against SARS-CoV-2 spike protein receptor binding domain in COVID-19 patients and healthy individuals

Science Translational Medicine ◽

10.1126/scitranslmed.abd6990 ◽

2021 ◽

pp. eabd6990

Author(s):

Sang Il Kim ◽

Jinsung Noh ◽

Sujeong Kim ◽

Younggeun Choi ◽

Duck Kyun Yoo ◽

...

Keyword(s):

B Cells ◽

Receptor Binding ◽

Neutralizing Antibodies ◽

De Novo ◽

Binding Domain ◽

Host Cells ◽

Spike Protein ◽

Receptor Binding Domain ◽

Healthy Individuals

Stereotypic antibody clonotypes exist in healthy individuals and may provide protective immunity against viral infections by neutralization. We observed that 13 out of 17 patients with COVID-19 had stereotypic variable heavy chain (VH) antibody clonotypes directed against the receptor-binding domain (RBD) of SARS-CoV-2 spike protein. These antibody clonotypes were comprised of immunoglobulin heavy variable (IGHV)3-53 or IGHV3-66 and immunoglobulin heavy joining (IGHJ)6 genes. These clonotypes included IgM, IgG3, IgG1, IgA1, IgG2, and IgA2 subtypes and had minimal somatic mutations, which suggested swift class switching after SARS-CoV-2 infection. The different immunoglobulin heavy variable chains were paired with diverse light chains resulting in binding to the RBD of SARS-CoV-2 spike protein. Human antibodies specific for the RBD can neutralize SARS-CoV-2 by inhibiting entry into host cells. We observed that one of these stereotypic neutralizing antibodies could inhibit viral replication in vitro using a clinical isolate of SARS-CoV-2. We also found that these VH clonotypes existed in six out of 10 healthy individuals, with IgM isotypes predominating. These findings suggest that stereotypic clonotypes can develop de novo from naïve B cells and not from memory B cells established from prior exposure to similar viruses. The expeditious and stereotypic expansion of these clonotypes may have occurred in patients infected with SARS-CoV-2 because they were already present.

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mRNA vaccination boosts cross-variant neutralizing antibodies elicited by SARS-CoV-2 infection

Science ◽

10.1126/science.abg9175 ◽

2021 ◽

pp. eabg9175 ◽

Cited By ~ 6

Author(s):

Leonidas Stamatatos ◽

Julie Czartoski ◽

Yu-Hsin Wan ◽

Leah J. Homad ◽

Vanessa Rubin ◽

...

Keyword(s):

Receptor Binding ◽

Neutralizing Antibodies ◽

Binding Domain ◽

Receptor Binding Domain ◽

Previous Infection

Emerging SARS-CoV-2 variants have raised concerns about resistance to neutralizing antibodies elicited by previous infection or vaccination. We examined whether sera from recovered and naïve donors collected prior to, and following immunizations with existing mRNA vaccines, could neutralize the Wuhan-Hu-1 and B.1.351 variants. Pre-vaccination sera from recovered donors neutralized Wuhan-Hu-1 and sporadically neutralized B.1.351, but a single immunization boosted neutralizing titers against all variants and SARS-CoV-1 by up to 1000-fold. Neutralization was due to antibodies targeting the receptor binding domain and was not boosted by a second immunization. Immunization of naïve donors also elicited cross-neutralizing responses, but at lower titers. Our study highlights the importance of vaccinating both uninfected and previously infected persons to elicit cross-variant neutralizing antibodies.

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