The ongoing evolution of SARS-CoV-2, with the emergence of variants that evade vaccine-induced and infection-acquired immunity, poses a significant challenge to global pandemic control. Existing vaccines and antiviral therapies, while effective against some variants, demonstrate waning efficacy against newly emerging strains. The development of new variants necessitates a continuous cycle of vaccine updates and the search for new antiviral agents. This adaptability necessitates a shift towards therapeutic strategies that are not directly dependent on the specific viral sequence. Previous studies explored the development of therapeutic agents that target conserved host components of viral entry, as this approach offers a potential solution to circumventing the challenges presented by the virus's mutation rate. This study investigated the potential of a variant-agnostic therapeutic agent to reduce severe COVID-19 disease, without inducing selective pressure that could lead to the evolution of further immune-evasive strains. The research focuses on the development and efficacy of RBD-62, a protein engineered to have greatly enhanced binding affinity to ACE2, thereby outcompeting the SARS-CoV-2 spike protein for receptor binding. This host-targeted approach offers the theoretical advantage of maintaining efficacy against numerous variants without the need for continuous adaptation.

The literature review section details the limitations of existing COVID-19 therapies and vaccines in the face of emerging variants. Studies highlighting the reduced efficacy of vaccines like mRNA-1273 and BNT162b2 against Omicron are cited, alongside research on the waning neutralization capacity of monoclonal antibodies against new subvariants. The review also notes that while antiviral agents such as nirmatrelvir/ritonavir (Paxlovid) have shown efficacy, concerns remain about the potential for drug-resistant mutations. The research highlights the need for variant-agnostic therapeutic agents that act independently of specific viral mutations, either targeting the host or acting on conserved viral components. Earlier studies on the development of ACE2-binding inhibitors are discussed, laying the foundation for the development and testing of RBD-62. These studies demonstrated the principle of utilizing a high-affinity decoy receptor to competitively block viral entry and the potential for engineering proteins with vastly improved binding affinities.

This study employed a challenge model using rhesus macaques, a highly susceptible animal model known for effectively reproducing SARS-CoV-2 infection. Male rhesus macaques were randomly assigned to either a treatment group (receiving aerosolized RBD-62) or a control group (receiving PBS). RBD-62 was administered via aerosolization to target both the upper and lower airways, immediately prior to challenge with SARS-CoV-2 Delta variant. Treatment continued every 24 hours for five days. Viral replication was assessed by measuring subgenomic RNA (sgRNA) copies in bronchoalveolar lavage (BAL) fluid and nasal swabs. Cultivable virus titers were also determined via tissue culture infectious dose 50% (TCID50) assays. Immune responses were evaluated through measuring serum and mucosal IgG and IgA binding titers to different variant RBDs (wild-type, Delta, and Omicron BA.1). T-cell responses were assessed by intracellular cytokine staining (ICS) of peripheral blood mononuclear cells (PBMCs) and BAL cells after stimulation with SARS-CoV-2 spike and nucleocapsid peptides. Memory B-cell responses were measured using flow cytometry with fluorescently labeled variant-specific probes. In vitro assays determined RBD-62's ability to block ACE2 binding and SARS-CoV-2 infection across a panel of variants, including WA1, Delta, BA.1, BA.5, XBB.1.5, and XN.1. Statistical analysis was performed using Wilcoxon rank-sum test with Holm's adjustment for multiple comparisons. All animal procedures were conducted according to NIH standards and approved by relevant Animal Care and Use Committees.

The in vitro assays showed that RBD-62 potently inhibited ACE2 binding and SARS-CoV-2 infection across multiple variants, exhibiting 100-fold greater potency compared to wild-type RBD. In the in vivo challenge study with the Delta variant, RBD-62 treatment significantly reduced viral RNA copies and cultivable virus in both the upper and lower airways on days 2 and 4 post-challenge, indicating prevention of infection at both sites. The effect was observed in both the lungs and the nose. The study also demonstrated a lack of significant impact on the development of virus-specific T- and B-cell responses. RBD-62 treatment did not prevent the development of antibodies against the virus or inhibit the formation of memory B cells capable of binding to multiple variants, including Omicron BA.1 and Delta. While the kinetics of immune responses were slightly delayed in the RBD-62 group compared to the control group, indicating a muted primary response, RBD-62 administration did not significantly impact the overall magnitude of the immune response. The frequency of memory B cells binding to Delta and/or BA.1 was similar in the treated and untreated groups, suggesting no substantial impairment in the development of long-term immunity.

The findings demonstrate that RBD-62, a high-affinity ACE2 antagonist, can effectively suppress SARS-CoV-2 replication in both upper and lower airways in a rhesus macaque model, even with a highly pathogenic variant like Delta. Importantly, this suppression occurred without impairing the development of robust virus-specific T and B cell responses. This indicates a potential approach for therapeutic intervention that does not rely on targeting the rapidly mutating viral components, therefore holding promise for efficacy against currently circulating and future SARS-CoV-2 variants. The muted primary immune response observed in the treated group might be a consequence of reduced viral antigen load due to effective viral suppression, highlighting a potential need to explore strategies for balancing viral control with robust immune stimulation. The observed equivalent protection in both the upper and lower airways, a significant advantage over some current vaccines, highlights the potential for RBD-62's application not just for treatment but also for pre-exposure prophylaxis. The aerosolization method used allows for efficient delivery to both sites.

This study provides strong evidence for the potential of RBD-62 as a variant-proof therapeutic agent for COVID-19. Its ability to suppress viral replication in both upper and lower airways, without inhibiting immune responses, suggests a valuable approach for preventing severe disease. Future research should focus on optimizing RBD-62 dosage and duration of treatment to balance antiviral activity with immune response, explore combination therapies (e.g., with vaccination), and investigate the long-term effects on immune memory and protection against reinfection.

The study used only male rhesus macaques, and the results may not be fully generalizable to other primate species or humans. The study focused on the Delta variant; however, the in vitro data suggest broad efficacy against different variants. The observation of a muted primary response in the treated group suggests that further investigations are needed to fully assess the long-term impact on immunity and the risk of potential virus rebound following cessation of RBD-62 treatment. Furthermore, the efficacy of RBD-62 as a post-exposure treatment was not fully assessed within the timeframe of this study.