The results show the value of nanobody technology for identifying novel neutralising epitopes in the S2 region of beta-coronaviruses with potential for the development of new selective anti-viral agents.
Abstract
Entry of coronaviruses into cells is mediated by the viral spike (S) glycoproteins each consisting of S1 receptor binding and S2 membrane fusion subunits. The sequence of the S2 region is very highly conserved amongst variants of SARS-CoV-2 and compared to the S1 unit shares significant sequence identity amongst different beta-coronavirus lineages. By targeting the S2 of SARS-CoV-2 we have identified two selective and potent neutralizing nanobodies (BA.1-C2 and BA.1-D3) that bind to two different quaternary epitopes in the S2 formed by the Heptad Repeat 2 (HR2) trimer at the base of the spike protein. The HR2 sequence is identical in SARS-CoV and SARS-CoV-2 but differs in other beta-coronaviruses explaining the lack of binding to the spike proteins of MERS-CoV or HuCoV-OC43. In combination the anti-SARS-CoV-2 nanobodies prevented viral escape following serial passaging with SARS-CoV-2 (JN.1) and the most potent of these nanobodies reduced viral load in the hamster model of COVID-19, following intranasal administration. Overall, the results show the value of nanobody technology for identifying novel neutralising epitopes in the S2 region of beta-coronaviruses with potential for the development of new selective anti-viral agents.
The continuous evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) highlights the value of broad-spectrum antiviral strategies. Antibody-engineering approaches targeting conserved regions of the Spike protein may enhance neutralizing potency and breadth.
A human antibody (P23) against the Spike protein of SARS-CoV-2 was identified using a human antibody phage display library panning and screening for binding affinity and breadth against multiple coronavirus Spike proteins using surface plasmon resonance (SPR). The epitope of P23 was characterized using the S1 and S2 subunits of SARS-CoV-2 Spike protein and hydrogen–deuterium exchange mass spectrometry (HDX-MS). An IgG–like bispecific fusion protein (Bs-ACE2-P23) was engineered by fusing the extracellular domain (ECD) of human angiotensin-converting enzyme 2 (ACE2) to the N-terminus of the P23 heavy chain (HC). Neutralizing activity was evaluated against both pseudotyped and authentic SARS-CoV-2 variants.
P23 cross-bound Spike proteins from SARS-CoV-2 wild type (WT), D614G and JN.1 variants, Pangolin-CoV, Bat coronavirus RaTG13, and SARS-CoV-1, recognizing an epitope on the S2 subunit adjacent to the fusion peptide (FP). While P23 was ineffective against D614G-containing SARS-CoV-2 variants, Bs-ACE2-P23 exhibited markedly enhanced neutralization potency. This bispecific architecture also improved the neutralizing activity of another FP-targeting antibody.
We developed a bispecific fusion protein with potent broad-spectrum neutralizing activity against SARS-CoV-2 variants. This architecture provides a promising strategy for next-generation coronavirus biologics.
Zhi-Zhong Wei, Ximing Liu, Kailun Wang et al.· Antibody Therapeutics· 0 citations
The phylogenetic relationship of the A222V substitution in the S protein relative to various global isolates is investigated, indicating high mutation rates in the S gene, characterised by diverse point mutations.
Kiky Martha, Ariesaka, M. M. Nuryady et al.· 0 citations
The starting point of this work was a SARS-CoV-2 neutralizing peptide (LW25.13), which binds to the receptor-binding domain of the viral spike protein and inhibits the attachment of the virus to its cellular receptor ACE2. As LW25.13 is unable to neutralize later SARS-CoV-2 variants, such as omicron, we have extended the neutralization breadth of LW25.13 through structural and bioinformatic analysis. This involved the systematic variation of a range of positions and yielded peptides neutralizing SARS-CoV-2 beta and omicron at low nanomolar concentrations, while preserving the strong neutralizing capacity against earlier virus variants (wild-type, alpha, delta), as well as the proteolytic stability and α-helical conformation of the peptide. This gain in neutralizing breadth illustrates the utility of the peptide as a scaffold that can be adapted to different virus variants, which may prove useful for the development of peptides against new coronavirus variants of concern in the future.
Nina Raasch, L. Weissenborn, Elie Richel et al.· Journal of Medicinal Chemist...· 0 citations
As the subunit of the SARS-CoV-2 spike protein (SARS-CoV-2 SP), the receptor-binding domain (RBD) can specifically bind to human angiotensin-converting enzyme 2 (ACE2), enabling viral infection of host cells. Therefore, it is of utmost significance to explore probes that can bind to the conserved epitope of the SARS-CoV-2 RBD with good affinity, so as to recognize various SARS-CoV-2 variants for diagnosis, vaccination, and the development of new detection methods. Herein, we biopanned peptide RN3 (sequence: YSIDWVFHHPML) by phage display, which can bind to the SARS-CoV-2 RBD with excellent affinity and selectivity. Molecular dynamics simulation and molecular docking verified that the His8 and Met11 residues in peptide RN3 are bound to the Asn487 and Tyr489 residues of the conserved epitope of the SARS-CoV-2 RBD subunit on the SARS-CoV-2 SP (His8-Asn487 and Met11-Tyr489, respectively) by hydrogen bonding. Alanine scanning confirmed that Met11 of peptide RN3 is the key amino acid for binding to the SARS-CoV-2 SP. The median inhibition concentration for peptide RN3 inhibiting the SARS-CoV-2 SP binding to ACE2 was 37 nM. Subsequently, phage RN3 was used as a capture probe, the SARS-CoV-2 SP-specific binding peptide Pn (sequence: WNLDLSQWLPPMGGGSKKKC) as a detection probe, and Au@Pd NP-based peroxidase-mimicking nanozyme for signal amplification. A phage RN3/antigen/peptide Pn sandwich ELISA was established for the selective detection of SARS-CoV-2 SP with a linear range of 5-1000 pg/mL and a limit of detection of 2.93 pg/mL. This method was applied to double-blind testing of 13 randomized clinical samples, which could distinguish between positive and negative samples. The detected results are well consistent with those of the gold-standard RT-PCR method. Thus, this study proposed phage-displayed dual peptides to construct a reliable and inexpensive ELISA for screening SARS-CoV-2 infection. A similar strategy can be extended to study other pathogens.
Mingyang Wang, Haipeng Yu, Wanjian Liu et al.· Analytical Chemistry· 1 citation
The emergence of the SARS-CoV-2 pandemic led to the spread of highly transmissible variants, such as the Delta variant, which originated in India, underscoring the urgent need to develop new antivirals, therapeutics, and vaccines. In our previous study, we showed that Membrane-Envelope Virus-like Particles exhibit antigenicity and neutralization activity. Hence, our present study was conducted to evaluate whether the M protein alone can form VLPs that elicit an immune response. Using computational methods, we identified key interacting residues in M-protein that contribute to VLP formation and interact with other structural proteins, including Spike (S), Nucleocapsid (N), and Envelope (E). The SARS-CoV-2-M protein was expressed in Sf-21 insect cells, and the resulting VLPs were purified, analyzed for shape and size, and characterized using DLS, FESEM, and TEM. The purified VLPs were injected into BALB/c mice to evaluate their immune response compared with uninfected controls. The biophysical analysis confirms that the particles are round and have a size of ~ 180–200 nm. The serum levels of IgG, IgM, and IgA were found to be higher in immunized mice than in uninfected mice. Further qRT-PCR analysis demonstrated the levels of IFN-γ, IL-2, and IL-12, indicating a TH1-biased immune response against the M protein. Our study demonstrates that the highly conserved M protein can self-assemble into VLPs and elicit humoral and cellular immune response. Furthermore, our study indicates that while M-protein VLPs elicit significant antibodies and cytokine responses, they do not induce detectable neutralizing activity when given alone.
Akash Kumar, K. K. Inampudi, Vikas Kumar et al.· Virology Journal· 0 citations
The ongoing threat of zoonotic coronavirus spillover into humans, exemplified by severe acute respiratory syndrome virus 2 (SARS-CoV-2), underscores the urgent need for pan-coronavirus therapeutics that can be deployed to mitigate future pandemics.
Here, we identified monoclonal antibodies from COVID-19 convalescent donors that target two conserved epitopes in the S2 domain of the coronavirus Spike glycoprotein: the central helix (CH) and a membrane-proximal epitope in the heptad repeat 2 (HR2), which we designate the lower stalk (LS).
CH-directed antibodies exhibited broad cross-reactivity across betacoronaviruses, whereas LS-directed antibodies demonstrated reactivity primarily within sarbecoviruses. Using cryogenic electron microscopy (cryo-EM), we determined sub-4 Å structures of three cross-reactive CH antibodies–ch.005, ch.007, and ch.010–bound to the prefusion-stabilized SARS-CoV-2 S2 protein, revealing distinct binding poses and contact residues relative to previously described CH antibodies. In parallel, X-ray crystallography studies yielded a sub-2 Å structure of the ls.019 Fab in complex with the LS peptide, providing, to our knowledge, the first structural visualization of a human monoclonal antibody engaging this epitope.
Together, these findings advance our knowledge of two conserved and structurally vulnerable sites within the coronavirus S2 subunit—the central helix and the lower stalk—that can guide the development of broad-spectrum antibody therapeutics and vaccines against current and emerging coronaviruses.
Howard Hughes Medical Institute
Vaccines and Immunotherapy (VAC)
Adonis A. Rubio, M. Abernathy, Davide F. Robbiani et al.· Journal of Immunology· 0 citations
