The continued evolution of SARS-CoV-2, particularly the emergence of Omicron variants, necessitates the development of broadly neutralizing antibodies that overcome vaccine and antibody limitations. While passive administration of neutralizing antibodies offers a promising therapeutic strategy, the high mortality associated with SARS-CoV-1 highlights the need for treatments effective against both viruses and their variants. Respiratory viral load correlates with disease severity, suggesting that direct delivery of antivirals to the respiratory tract could improve efficacy at lower doses. Multivalent antibodies are cost-effective and easier to produce than antibody cocktails; nanobodies (Nbs), with their single-domain structure, are particularly amenable to multivalent engineering. Previous research identified aRbd-2, a broad-spectrum nanobody targeting a conserved RBD epitope. This study focuses on a newly identified Nb, aS3, which targets a distinct RBD core epitope and cross-reacts with SARS-CoV-1 and SARS-CoV-2 variants. The study engineers a novel bispecific Nb dimer (2-3-Fc) from aS3 and aRbd-2, aiming for potent neutralization of virtually all VOCs and effective in vivo protection via intranasal administration.

The introduction section mentions the challenges posed by emerging SARS-CoV-2 variants and the limitations of existing vaccines and antibodies. It highlights the need for broadly neutralizing agents that are effective against both SARS-CoV-1 and SARS-CoV-2. The success of multivalent engineering and the advantages of nanobodies (Nbs) over conventional antibodies are also discussed, citing previous work on a broad-spectrum nanobody, aRbd-2. This sets the stage for the introduction of the novel nanobody aS3 and the bispecific dimer 2-3-Fc.

The study involved several key methodologies: 1. **Nanobody Identification and Characterization:** A phage display library was used to identify nanobodies from immunized alpacas. Binding kinetics were measured using Biacore, and neutralizing activity was assessed using pseudovirus and authentic virus neutralization assays (ELISA, micro-neutralization test, plaque reduction neutralization test (PRNT)). 2. **Structural Analysis:** X-ray crystallography was employed to determine the crystal structure of aS3 in complex with SARS-CoV-1 RBD, providing insights into the binding mechanism and cross-reactivity. 3. **Bispecific Nanobody Engineering:** The aS3 nanobody was engineered into a dimeric construct (aS3-Fc) and further fused with aRbd-2 to create the bispecific dimer 2-3-Fc. 4. **In vivo Studies:** The efficacy of the bispecific nanobody dimer was evaluated in a hamster model of Omicron infection, comparing systemic and intranasal administration routes and doses. 5. **Variant Analysis:** The binding and neutralizing activity of the nanobodies were tested against various SARS-CoV-2 variants, including Omicron subvariants. Specific techniques mentioned include competitive ELISA, Surface Plasmon Resonance (SPR), and cryo-electron microscopy (cryo-EM). Detailed interaction analyses were performed to understand the molecular basis of nanobody binding and neutralization.

The key findings of the study include: 1. **aS3 Cross-reactivity:** The nanobody aS3 demonstrated strong cross-reactivity with both SARS-CoV-1 and wild-type SARS-CoV-2, binding tightly to the RBD core epitope. 2. **Enhanced Neutralization by aS3-Fc:** The dimeric construct aS3-Fc showed significantly improved binding affinity and neutralizing potency compared to the monomeric aS3. 3. **Potent Neutralization of Variants by 2-3-Fc:** The bispecific dimer 2-3-Fc exhibited single-digit ng/mL neutralizing potency against all major variants of concern, including BA.5, demonstrating broad neutralization capacity. 4. **In vivo Protection:** A single systemic dose of 2-3-Fc (10 mg/kg) completely protected hamsters from Omicron infection. Even more impressively, a much lower intranasal dose (5 mg/kg) drastically reduced viral loads and eliminated virus titers in the trachea and lungs. 5. **Y296-3-Fc Enhanced Activity:** A variant of 2-3-Fc, Y296-3-Fc, exhibited superior neutralization of the BA.2.75 Omicron subvariant.

The study successfully demonstrates the development of a highly effective bispecific nanobody dimer for broad neutralization of SARS-CoV-1 and SARS-CoV-2 variants. The potent neutralizing activity and successful in vivo protection, especially via the low-dose intranasal route, highlight the therapeutic and prophylactic potential of 2-3-Fc. The findings suggest that intranasal administration could offer a simple and effective preventive approach against Omicron and its subvariants. The identification of a variant, Y296-3-Fc, with enhanced activity against BA.2.75 underscores the potential for further optimization and variant-specific adaptation. The study’s results contribute significantly to the arsenal of tools available to combat SARS-CoV-2 and related viruses.

This study successfully developed a bispecific nanobody dimer (2-3-Fc) with potent and broad neutralizing activity against SARS-CoV-1 and SARS-CoV-2 variants, including Omicron. The efficacy of low-dose intranasal administration offers a promising prophylactic strategy. Future research could focus on further optimization of the nanobody for even broader neutralization and enhanced efficacy, alongside clinical trials to assess its safety and efficacy in humans.

While the study demonstrates promising results in a hamster model, further studies are needed to confirm the findings in larger animal models and ultimately in human clinical trials. The long-term efficacy and potential for immune escape remain to be fully determined. The study's focus on specific Omicron subvariants might not fully represent the complete spectrum of future variants.