A Novel Double Mosaic Virus-like Particle-Based Vaccine against SARS-CoV-2 Incorporates Both Receptor Binding Motif (RBM) and Fusion Domain

COVID-19 has emerged, and has rapidly become a major health problem worldwide, causing millions of mortalities. Vaccination against COVID-19 is the most efficient way to stop the pandemic. The goal of vaccines is to induce neutralizing antibodies against SARS-CoV-2 virus. Here, we present a novel double mosaic virus-like particle (VLP) displaying two independent neutralizing epitopes, namely the receptor binding motif (RBM) located in S1 and the fusion peptide (AA 817–855) located in S2. CuMVTT virus-like particles were used as VLP scaffold and both domains were genetically fused in the middle of CuMVTT subunits, which co-assembled into double mosaic particles (CuMVTT-DF). A single fusion mosaic particle (CuMVTT-FP) containing the fusion peptide only was used for comparison. The vaccines were produced in E. coli, and electron microscopy and dynamic light scattering confirmed their integrity and homogeneity. In addition, the CuMVTT-DF vaccine was well recognized by ACE2 receptor, indicating that the RBM was in native conformation. Both CuMVTT-FP and CuMVTT-DF vaccines induced high levels of high avidity IgG antibodies as well as IgA recognizing spike and RBD in the case of CuMVTT-DF. Both vaccine candidates induced virus-neutralizing antibodies indicating that the fusion peptide can independently induce virus-neutralizing antibodies. In contrast, CuMVTT-DF containing both RBM and fusion peptide induced a higher level of neutralizing antibodies suggesting that the new double mosaic vaccine candidate CuMVTT-DF consisting of two antigens in one VLP maybe an attractive candidate for scale-up in a bacterial fermentation process for clinical development.

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Development of SARS-CoV-2 Specific IgG and Virus-Neutralizing Antibodies after Infection with Variants of Concern or Vaccination

Vaccines ◽

10.3390/vaccines9070700 ◽

2021 ◽

Vol 9 (7) ◽

pp. 700

Author(s):

Franziska Neumann ◽

Ruben Rose ◽

Janine Römpke ◽

Olaf Grobe ◽

Thomas Lorentz ◽

...

Keyword(s):

Immune Response ◽

Receptor Binding ◽

Neutralizing Antibodies ◽

Neutralization Test ◽

Vaccine Dose ◽

High Avidity ◽

Receptor Binding Domain ◽

Robust Immune Response ◽

Specific Igg ◽

Nucleoprotein N

The humoral immunity after SARS-CoV-2 infection or vaccination was examined. Convalescent sera after infection with variants of concern (VOCs: B.1.1.7, n = 10; B.1.351, n = 1) and sera from 100 vaccinees (Pfizer/BioNTech, BNT162b2, n = 33; Moderna, mRNA-1273, n = 11; AstraZeneca, ChAdOx1 nCoV-19/AZD1222, n = 56) were tested for the presence of immunoglobulin G (IgG) directed against the viral spike (S)-protein, its receptor-binding domain (RBD), the nucleoprotein (N) and for virus-neutralizing antibodies (VNA). For the latter, surrogate assays (sVNT) and a Vero-cell based neutralization test (cVNT) were used. Maturity of IgG was determined by measuring the avidity in an immunoblot (IB). Past VOC infection resulted in a broad reactivity of anti-S IgG (100%), anti-RBD IgG (100%), and anti-N IgG (91%), while latter were absent in 99% of vaccinees. Starting approximately two weeks after the first vaccine dose, anti-S IgG (75–100%) and particularly anti-RBD IgG (98–100%) were detectable. After the second dose, their titers increased and were higher than in the convalescents. The sVNT showed evidence of VNA in 91% of convalescents and in 80–100%/100% after first/second vaccine dose, respectively. After the second dose, an increase in VNA titer and IgGs of high avidity were demonstrated by cVNT and IB, respectively. Re-vaccination contributes to a more robust immune response.

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Virus-Like Particle Based Vaccines Elicit Neutralizing Antibodies against the HIV-1 Fusion Peptide

Vaccines ◽

10.3390/vaccines8040765 ◽

2020 ◽

Vol 8 (4) ◽

pp. 765

Author(s):

Alemu Tekewe Mogus ◽

Lihong Liu ◽

Manxue Jia ◽

Diane T. Ajayi ◽

Kai Xu ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Vaccination Strategy ◽

Fusion Peptide ◽

Linear Epitope ◽

Broadly Neutralizing Antibodies ◽

Virus Like Particle ◽

Boost Immunization ◽

Potential Vaccine ◽

Hiv Envelope ◽

Hiv 1

Broadly neutralizing antibodies (bnAbs) isolated from HIV-infected individuals delineate vulnerable sites on the HIV envelope glycoprotein that are potential vaccine targets. A linear epitope within the N-terminal region of the HIV-1 fusion peptide (FP8) is the primary target of VRC34.01, a bnAb that neutralizes ~50% of primary HIV isolates. FP8 has attracted attention as a potential HIV vaccine target because it is a simple linear epitope. Here, platform technologies based on RNA bacteriophage virus-like particles (VLPs) were used to develop multivalent vaccines targeting the FP8 epitope. Both recombinant MS2 VLPs displaying the FP8 peptide and Qβ VLPs displaying chemically conjugated FP8 peptide induced high titers of FP8-specific antibodies in mice. Moreover, a heterologous prime-boost-boost regimen employing the two FP8-VLP vaccines and native envelope trimer was the most effective approach for eliciting HIV-1 neutralizing antibodies. Given the potent immunogenicity of VLP-based vaccines, this vaccination strategy—inspired by bnAb-guided epitope mapping, VLP bioengineering, and prime-boost immunization approaches—may be a useful strategy for eliciting bnAb responses against HIV.

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Structural basis for SARS-CoV-2 neutralizing antibodies with novel binding epitopes

PLoS Biology ◽

10.1371/journal.pbio.3001209 ◽

2021 ◽

Vol 19 (5) ◽

pp. e3001209

Author(s):

Dan Fu ◽

Guangshun Zhang ◽

Yuhui Wang ◽

Zheng Zhang ◽

Hengrui Hu ◽

...

Keyword(s):

Receptor Binding ◽

Neutralizing Antibodies ◽

Therapeutic Interventions ◽

Structural Basis ◽

Binding Motif ◽

Global Public Health ◽

Variable Regions ◽

Treatment Groups ◽

X Ray Crystallography ◽

Antibody Discovery

The ongoing Coronavirus Disease 2019 (COVID-19) pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) threatens global public health and economy unprecedentedly, requiring accelerating development of prophylactic and therapeutic interventions. Molecular understanding of neutralizing antibodies (NAbs) would greatly help advance the development of monoclonal antibody (mAb) therapy, as well as the design of next generation recombinant vaccines. Here, we applied H2L2 transgenic mice encoding the human immunoglobulin variable regions, together with a state-of-the-art antibody discovery platform to immunize and isolate NAbs. From a large panel of isolated antibodies, 25 antibodies showed potent neutralizing activities at sub-nanomolar levels by engaging the spike receptor-binding domain (RBD). Importantly, one human NAb, termed PR1077, from the H2L2 platform and 2 humanized NAb, including PR953 and PR961, were further characterized and subjected for subsequent structural analysis. High-resolution X-ray crystallography structures unveiled novel epitopes on the receptor-binding motif (RBM) for PR1077 and PR953, which directly compete with human angiotensin-converting enzyme 2 (hACE2) for binding, and a novel non-blocking epitope on the neighboring site near RBM for PR961. Moreover, we further tested the antiviral efficiency of PR1077 in the Ad5-hACE2 transduction mouse model of COVID-19. A single injection provided potent protection against SARS-CoV-2 infection in either prophylactic or treatment groups. Taken together, these results shed light on the development of mAb-related therapeutic interventions for COVID-19.

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Engineered receptor binding domain immunogens elicit pan-coronavirus neutralizing antibodies

10.1101/2020.12.07.415216 ◽

2020 ◽

Author(s):

Blake M. Hauser ◽

Maya Sangesland ◽

Evan C. Lam ◽

Jared Feldman ◽

Ashraf S. Yousif ◽

...

Keyword(s):

Receptor Binding ◽

Neutralizing Antibodies ◽

Neutralizing Antibody ◽

Binding Domain ◽

Surface Glycoprotein ◽

Binding Motif ◽

Receptor Binding Domain ◽

Broadly Neutralizing Antibodies ◽

Major Surface Glycoprotein ◽

Major Surface

AbstractEffective countermeasures are needed against emerging coronaviruses of pandemic potential, similar to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Designing immunogens that elicit broadly neutralizing antibodies to conserved viral epitopes on the major surface glycoprotein, spike, such as the receptor binding domain (RBD) is one potential approach. Here, we report the generation of homotrimeric RBD immunogens from different sarbecoviruses using a stabilized, immune-silent trimerization tag. We find that that a cocktail of homotrimeric sarbecovirus RBDs can elicit a neutralizing response to all components even in context of prior SARS-CoV-2 imprinting. Importantly, the cross-neutralizing antibody responses are focused towards conserved RBD epitopes outside of the ACE-2 receptor-binding motif. This may be an effective strategy for eliciting broadly neutralizing responses leading to a pan-sarbecovirus vaccine.

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Engineered Receptor Binding Domain Immunogens Elicit Pan-Coronavirus Neutralizing Antibodies Outside the Receptor Binding Motif

SSRN Electronic Journal ◽

10.2139/ssrn.3773801 ◽

2021 ◽

Author(s):

Blake M. Hauser ◽

Maya Sangesland ◽

Evan Christopher Lam ◽

Jared Feldman ◽

Ashraf S. Yousif ◽

...

Keyword(s):

Receptor Binding ◽

Neutralizing Antibodies ◽

Binding Domain ◽

Binding Motif ◽

Receptor Binding Domain

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L18F substrain of SARS-CoV-2 VOC-202012/01 is rapidly spreading in England

10.1101/2021.02.07.21251262 ◽

2021 ◽

Cited By ~ 3

Author(s):

Frederic Grabowski ◽

Marek Kochańczyk ◽

Tomasz Lipniacki

Keyword(s):

Molecular Evolution ◽

South African ◽

Receptor Binding ◽

Growth Rates ◽

Neutralizing Antibodies ◽

Spike Protein ◽

Binding Motif ◽

Viral Genomes ◽

African Strain ◽

Escape Mutants

AbstractThe Variant of Concern (VOC)-202012/01 (also known as B.1.1.7) is a rapidly growing lineage of SARS-CoV-2. In January 2021, VOC-202012/01 constituted about 80% of SARS-CoV-2 genomes sequenced in England and was present in 27 out of 29 countries that reported at least 50 viral genomes. As this strain will likely spread globally towards fixation, it is important to monitor its molecular evolution. Based on GISAID data we systematically estimated growth rates of mutations acquired by the VOC lineage to find that L18F substitution in viral spike protein has initiated a substrain characterized by replicative advantage of 1.70 [95% CI: 1.56–1.96] in relation to the remaining VOC-202012/01 substrains. The L18F mutation is of significance because when recently analyzed in the context of the South African strain 501Y.V2 it has been found to compromise binding of neutralizing antibodies. We additionally indicate three mutations that were acquired by VOC-202012/01 in the receptor binding motif of spike, specifically E484K, F490S, and S494P, that may also give rise to escape mutants. Such mutants may hinder efficiency of existing vaccines and expand in response to the increasing after-infection or vaccine-induced seroprevalence.

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Deamidation drives molecular aging of the SARS-CoV-2 spike receptor-binding motif

10.1101/2021.05.20.445042 ◽

2021 ◽

Author(s):

Ramiro Lorenzo ◽

Lucas A. Defelipe ◽

Lucio Aliperti ◽

Stephan Niebling ◽

Tânia F. Custódio ◽

...

Keyword(s):

Receptor Binding ◽

Neutralizing Antibodies ◽

Vaccine Development ◽

Strong Dependence ◽

Computational Prediction ◽

Binding Motif ◽

Virus Infectivity ◽

Asparagine Deamidation ◽

Molecular Aging ◽

Virion Surface

The spike is the main protein component of the SARS-CoV-2 virion surface. The spike receptor binding motif mediates recognition of the hACE2 receptor, a critical infection step, and is the preferential target for spike-neutralizing antibodies. Post-translational modifications of the spike receptor binding motif can modulate viral infectivity and immune response. We studied the spike protein in search for asparagine deamidation, a spontaneous event that leads to the appearance of aspartic and isoaspartic residues, affecting both the protein backbone and its charge. We used computational prediction and biochemical experiments to identify five deamidation hotspots in the SARS-CoV-2 spike. Similar deamidation hotspots are frequently found at the spike receptor-binding motifs of related sarbecoviruses, at positions that are mutated in emerging variants and in escape mutants from neutralizing antibodies. Asparagine residues 481 and 501 from the receptor-binding motif deamidate with a half-time of 16.5 and 123 days at 37 °C, respectively. This process is significantly slowed down at 4 °C, pointing at a strong dependence of spike molecular aging on the environmental conditions. Deamidation of the spike receptor-binding motif decreases the equilibrium constant for binding to the hACE2 receptor more than 3.5-fold. A model for deamidation of the full SARS-CoV-2 virion illustrates that deamidation of the spike receptor-binding motif leads to the accumulation in the virion surface of a chemically diverse spike population in a timescale of days. Our findings provide a mechanism for molecular aging of the spike, with significant consequences for understanding virus infectivity and vaccine development.

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A high-affinity RBD-targeting nanobody improves fusion partner’s potency against SARS-CoV-2

PLoS Pathogens ◽

10.1371/journal.ppat.1009328 ◽

2021 ◽

Vol 17 (3) ◽

pp. e1009328

Author(s):

Hebang Yao ◽

Hongmin Cai ◽

Tingting Li ◽

Bingjie Zhou ◽

Wenming Qin ◽

...

Keyword(s):

Receptor Binding ◽

Neutralizing Antibodies ◽

Binding Motif ◽

Fusion Partner ◽

Receptor Binding Domain ◽

Single Chain ◽

Neutralization Activity ◽

Effective Strategies ◽

High Affinity ◽

Considerable Research

A key step to the SARS-CoV-2 infection is the attachment of its Spike receptor-binding domain (S RBD) to the host receptor ACE2. Considerable research has been devoted to the development of neutralizing antibodies, including llama-derived single-chain nanobodies, to target the receptor-binding motif (RBM) and to block ACE2-RBD binding. Simple and effective strategies to increase potency are desirable for such studies when antibodies are only modestly effective. Here, we identify and characterize a high-affinity synthetic nanobody (sybody, SR31) as a fusion partner to improve the potency of RBM-antibodies. Crystallographic studies reveal that SR31 binds to RBD at a conserved and ‘greasy’ site distal to RBM. Although SR31 distorts RBD at the interface, it does not perturb the RBM conformation, hence displaying no neutralizing activities itself. However, fusing SR31 to two modestly neutralizing sybodies dramatically increases their affinity for RBD and neutralization activity against SARS-CoV-2 pseudovirus. Our work presents a tool protein and an efficient strategy to improve nanobody potency.

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Structural basis for potent neutralization of SARS-CoV-2 and role of antibody affinity maturation

Nature Communications ◽

10.1038/s41467-020-19231-9 ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 4

Author(s):

Nicholas K. Hurlburt ◽

Emilie Seydoux ◽

Yu-Hsin Wan ◽

Venkata Viswanadh Edara ◽

Andrew B. Stuart ◽

...

Keyword(s):

Receptor Binding ◽

Neutralizing Antibodies ◽

Substantial Reduction ◽

Affinity Maturation ◽

Structural Basis ◽

Binding Motif ◽

Neutralization Potential ◽

Neutralizing Monoclonal Antibody ◽

Potent Neutralization

Abstract SARS-CoV-2 is a betacoronavirus virus responsible for the COVID-19 pandemic. Here, we determine the X-ray crystal structure of a potent neutralizing monoclonal antibody, CV30, isolated from a patient infected with SARS-CoV-2, in complex with the receptor binding domain. The structure reveals that CV30 binds to an epitope that overlaps with the human ACE2 receptor binding motif providing a structural basis for its neutralization. CV30 also induces shedding of the S1 subunit, indicating an additional mechanism of neutralization. A germline reversion of CV30 results in a substantial reduction in both binding affinity and neutralization potential indicating the minimal somatic mutation is needed for potently neutralizing antibodies against SARS-CoV-2.

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AP205 VLPs Based on Dimerized Capsid Proteins Accommodate RBM Domain of SARS-CoV-2 and Serve as an Attractive Vaccine Candidate

Vaccines ◽

10.3390/vaccines9040403 ◽

2021 ◽

Vol 9 (4) ◽

pp. 403

Author(s):

Xuelan Liu ◽

Xinyue Chang ◽

Dominik Rothen ◽

Mariliza Derveni ◽

Pascal Krenger ◽

...

Keyword(s):

Receptor Binding ◽

Neutralizing Antibodies ◽

Large Scale ◽

Vaccine Candidate ◽

Herd Immunity ◽

Capsid Proteins ◽

Binding Motif ◽

C Terminus ◽

The World ◽

Electron Microscopy Analysis

COVID-19 is a novel disease caused by SARS-CoV-2 which has conquered the world rapidly resulting in a pandemic that massively impacts our health, social activities, and economy. It is likely that vaccination is the only way to form “herd immunity” and restore the world to normal. Here we developed a vaccine candidate for COVID-19 based on the virus-like particle AP205 displaying the spike receptor binding motif (RBM), which is the major target of neutralizing antibodies in convalescent patients. To this end, we genetically fused the RBM domain of SARS-CoV-2 to the C terminus of AP205 of dimerized capsid proteins. The fused VLPs were expressed in E. coli, which resulted in insoluble aggregates. These aggregates were denatured in 8 M urea followed by refolding, which reconstituted VLP formation as confirmed by electron microscopy analysis. Importantly, immunized mice were able to generate high levels of IgG antibodies recognizing eukaryotically expressed receptor binding domain (RBD) as well as spike protein of SARS-CoV-2. Furthermore, induced antibodies were able to neutralize SARS-CoV-2/ABS/NL20. Additionally, this vaccine candidate has the potential to be produced at large scale for immunization programs.

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