Clinical outcomes resulting from SARS-CoV-2 infection vary widely, ranging from asymptomatic cases to the development of mild to severe respiratory illness, and in some instances, chronic lingering disease and mortality. The underlying biological mechanisms driving this wide spectrum of pathogenicity among certain individuals and demographics remain elusive. Autoantibodies have emerged as potential contributors to the severity of COVID-19. Although preliminary reports have suggested the induction of antibodies targeting Angiotensin-Converting Enzyme II (ACE2) post-infection, this assertion lacks confirmation in large-scale studies. In this study, our objective is to comprehensively characterize and quantify the prevalence and expression levels of autoantibodies directed against ACE2 in a sizable cohort (n = 464). Our findings reveal that ACE2-reactive IgM antibodies are the most prevalent, with an overall seroprevalence of 18.8%, followed by IgG at 10.3% and IgA at 6.3%. Longitudinal analysis of individuals with multiple blood draws showed stable ACE2 IgG and IgA levels over time. Upon stratifying individuals based on molecular testing for SARS-CoV-2 or serological evidence of past infection, no significant differences were observed between groups. Functional assessment of ACE2 autoantibodies demonstrated that they are non-neutralizing and failed to inhibit spike-ACE2 interaction or affect the enzymatic activity of ACE2. Our results highlight that ACE2 autoantibodies are prevalent in the general population and were not induced by SARS-CoV-2 infection in our cohort. Notably, we found no substantiated evidence supporting a direct role for ACE2 autoantibodies in SARS-CoV-2 pathogenesis.

For the prevention of SARS-CoV-2 infection, four Fv-antibodies with binding affinity for the ACE2 receptor were screened from an Fv-antibody library. The screened Fv-antibodies were expressed as soluble proteins and estimated to have a high binding affinity, comparable to that between SARS-CoV-2 and the ACE2 receptor. The interaction between the Fv-antibodies and the ACE2 receptor was analyzed using docking simulation, and the significant binding affinity of the screened Fv-antibodies was attributed to the homology in amino acid sequence with the ACE2 receptor. The neutralizing activities of the Fv-antibodies were demonstrated using a cell-based infection assay based on four pseudo-virus types with SARS-CoV-2 variant spike proteins (Wild-type D614, Delta B.1.617.2, and Omicron BA.2, and Omicron BA.4/5).

SARS-CoV-2 has the capacity to evolve mutations that escape vaccine- and infection-acquired immunity and antiviral drugs. A variant-agnostic therapeutic agent that protects against severe disease without putting selective pressure on the virus would thus be a valuable biomedical tool that would maintain its efficacy despite the ongoing emergence of new variants. Here, we challenge male rhesus macaques with SARS-CoV-2 Delta-the most pathogenic variant in a highly susceptible animal model. At the time of challenge, we also treat the macaques with aerosolized RBD-62, a protein developed through multiple rounds of in vitro evolution of SARS-CoV-2 RBD to acquire 1000-fold enhanced ACE2 binding affinity. RBD-62 treatment equivalently suppresses virus replication in both upper and lower airways, a phenomenon not previously observed with clinically approved vaccines. Importantly, RBD-62 does not block the development of virus-specific T- and B-cell responses and does not elicit anti-drug immunity. These data provide proof-of-concept that RBD-62 can prevent severe disease from a highly virulent variant.

Vaccines and antibodies that specifically target or neutralize components of the SARS-CoV-2 virus are effective in prevention and treatment of human patients with SARS-CoV-2 infection. However, vaccines and SARS-CoV-2 neutralization antibodies target a subset of epitopes of viral proteins, and the fast evolution of the SARS-CoV-2 virus and the continuing emergence of SARS-CoV-2 variants confer SARS-CoV-2 immune escape from these therapies. ACE2 is the human cell receptor that serves as the entry point for SARS-CoV-2 into human cells and thus is the gatekeeper for SARS-CoV-2 infection of humans. We report here the development of 4G8C11, an anti-human ACE2 receptor monoclonal antibody that recognizes ACE2 on human cell surfaces. We determined that 4G8C11 blocks SARS-CoV-2 and variant infection of ACE2+ human cells. Furthermore, 4G8C11 has minimal effects on ACE2 receptor activity. 4G8C11 is therefore a monoclonal antibody for ACE2 receptor detection and potentially an effective immunotherapeutic agent for SARS-CoV-2 and variants.

Over the past three years, extensive research has been dedicated to understanding and combating COVID-19. Targeting the interaction between the SARS-CoV-2 Spike protein and the ACE2 receptor has emerged as a promising therapeutic strategy against SARS-CoV-2. This study aimed to develop ACE2-coated virus-like particles (ACE2-VLPs), which can be utilized to prevent viral entry into host cells and efficiently neutralize the virus.

Virus-like particles were generated through the utilization of a packaging plasmid in conjunction with a plasmid containing the ACE2 envelope sequence. Subsequently, ACE2-VLPs and ACE2-EVs were purified via ultracentrifugation. The quantification of VLPs was validated through multiple methods, including Nanosight 3000, TEM imaging, and Western blot analysis. Various packaging systems were explored to optimize the ACE2-VLP configuration for enhanced neutralization capabilities. The evaluation of neutralization effectiveness was conducted using pseudoviruses bearing different spike protein variants. Furthermore, the study assessed the neutralization potential against the Omicron BA.1 variant in Vero E6 cells.

ACE2-VLPs showed a high neutralization capacity even at low doses and demonstrated superior efficacy in in vitro pseudoviral assays compared to extracellular vesicles carrying ACE2. ACE2-VLPs remained stable under various environmental temperatures and effectively blocked all tested variants of concern in vitro. Notably, they exhibited significant neutralization against Omicron BA.1 variant in Vero E6 cells. Given their superior efficacy compared to extracellular vesicles and proven success against live virus, ACE2-VLPs stand out as crucial candidates for treating SARS-CoV-2 infections.

This novel therapeutic approach of coating VLPs with receptor particles provides a proof-of-concept for designing effective neutralization strategies for other viral diseases in the future.

Since the SARS-CoV-2 Omicron virus has gained dominance worldwide, its continual evolution with unpredictable mutations and patterns has revoked all authorized immunotherapeutics. Rapid viral evolution has also necessitated several rounds of vaccine updates in order to provide adequate immune protection. It remains imperative to understand how Omicron evolves into different subvariants and causes immune escape as this could help reevaluate the current intervention strategies mostly implemented in the clinics as emergency measures to counter the pandemic and, importantly, develop new solutions. Here, we provide a review focusing on the major events of Omicron viral evolution, including the features of spike mutation that lead to immune evasion against monoclonal antibody (mAb) therapy and vaccination, and suggest alternative durable options such as the ACE2-based experimental therapies superior to mAbs to address this unprecedented evolution of Omicron virus. In addition, this type of unique ACE2-based virus-trapping molecules can counter all zoonotic SARS coronaviruses, either from unknown animal hosts or from established wild-life reservoirs of SARS-CoV-2, and even seasonal alpha coronavirus NL63 that depends on human ACE2 for infection.

The development of effective antiviral compounds is essential for mitigating the effects of the COVID-19 pandemic. Entry of SARS-CoV-2 virions into host cells is mediated by the interaction between the viral spike (S) protein and membrane-bound angiotensin-converting enzyme 2 (ACE2) on the surface of epithelial cells. Inhibition of this viral protein-host protein interaction is an attractive avenue for the development of antiviral molecules with numerous spike-binding molecules generated to date. Herein, we describe an alternative approach to inhibit the spike-ACE2 interaction by targeting the spike-binding interface of human ACE2 via mRNA display. Two consecutive display selections were performed to direct cyclic peptide ligand binding toward the spike binding interface of ACE2. Through this process, potent cyclic peptide binders of human ACE2 (with affinities in the picomolar to nanomolar range) were identified, two of which neutralized SARS-CoV-2 entry. This work demonstrates the potential of targeting ACE2 for the generation of anti-SARS-CoV-2 therapeutics as well as broad spectrum antivirals for the treatment of SARS-like betacoronavirus infection.

Immunologically impaired individuals respond poorly to vaccines, highlighting the need for additional strategies to protect these vulnerable populations from COVID-19. While monoclonal antibodies (mAbs) have emerged as promising tools to manage infectious diseases, the transient lifespan of neutralizing mAbs in patients limits their ability to confer lasting, passive prophylaxis from SARS-CoV-2. Here, we attempted to solve this problem by combining cell and mAb engineering in a way that provides durable immune protection against viral infection using safe and universal cell therapy.

Mouse embryonic stem cells equipped with our FailSafe™ and induced allogeneic cell tolerance technologies were engineered to express factors that potently neutralize SARS-CoV-2, which we call 'neutralizing biologics' (nBios). We subcutaneously transplanted the transgenic cells into mice and longitudinally assessed the ability of the cells to deliver nBios into circulation. To do so, we quantified plasma nBio concentrations and SARS-CoV-2 neutralizing activity over time in transplant recipients. Finally, using similar cell engineering strategies, we genetically modified FailSafe™ human-induced pluripotent stem cells to express SARS-CoV-2 nBios.

Transgenic mouse embryonic stem cells engineered for safety and allogeneic-acceptance can secrete functional and potent SARS-CoV-2 nBios. As a dormant, subcutaneous tissue, the transgenic cells and their differentiated derivatives long-term deliver a supply of protective nBio titers in vivo. Moving toward clinical relevance, we also show that human-induced pluripotent stem cells, similarly engineered for safety, can secrete highly potent nBios.

Together, these findings show the promise and potential of using 'off-the-shelf' cell products that secrete neutralizing antibodies for sustained protective immunity against current and future viral pathogens of public health significance.

The ongoing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), resulting in the emergence of new variants that are resistant to existing vaccines and therapeutic antibodies, has raised the need for novel strategies to combat the persistent global COVID-19 epidemic. In this study, a monoclonal anti-human angiotensin-converting enzyme 2 (hACE2) antibody, ch2H2, was isolated and humanized to block the viral receptor-binding domain (RBD) binding to hACE2, the major entry receptor of SARS-CoV-2. This antibody targets the RBD-binding site on the N terminus of hACE2 and has a high binding affinity to outcompete the RBD. In vitro, ch2H2 antibody showed potent inhibitory activity against multiple SARS-CoV-2 variants, including the most antigenically drifted and immune-evading variant Omicron. In vivo, adeno-associated virus (AAV)-mediated delivery enabled a sustained expression of monoclonal antibody (mAb) ch2H2, generating a high concentration of antibodies in mice. A single administration of AAV-delivered mAb ch2H2 significantly reduced viral RNA load and infectious virions and mitigated pulmonary pathological changes in mice challenged with SARS-CoV-2 Omicron BA.5 subvariant. Collectively, the results suggest that AAV-delivered hACE2-blocking antibody provides a promising approach for developing broad-spectrum antivirals against SARS-CoV-2 and potentially other hACE2-dependent pathogens that may emerge in the future.

ACE2 and Mpro in the pathology of SARS-CoV-2 show great potential in developing COVID-19 drugs as therapeutic targets, due to their roles as the "gate" of viral entry and viral reproduction. Of the many potential compounds for ACE2 and Mpro inhibition, α-mangostin is a promising candidate. Unfortunately, the potential of α-mangostin as a secondary metabolite with the anti-SARS-CoV-2 activity is hindered due to its low solubility in water. Other xanthone isolates, which also possess the xanthone core structure like α-mangostin, are predicted to be potential alternatives to α-mangostin in COVID-19 treatment, addressing the low drug-likeness of α-mangostin. This study aims to assess the potential of xanthone derivative compounds in the pericarp of mangosteen (Garcinia mangostana L.) through computational study. The study was conducted through screening activity using molecular docking study, drug-likeness prediction using Lipinski's rule of five filtration, pharmacokinetic and toxicity prediction to evaluate the safety profile, and molecular dynamic study to evaluate the stability of formed interactions. The research results showed that there were 11 compounds with high potential to inhibit ACE2 and 12 compounds to inhibit Mpro. However, only garcinone B, in addition to being indicated as active, also possesses a drug-likeness, pharmacokinetic, and toxicity profile that was suitable. The molecular dynamic study exhibited proper stability interaction between garcinone B with ACE2 and Mpro. Therefore, garcinone B, as a xanthone derivative isolate compound, has promising potential for further study as a COVID-19 treatment as an ACE2 and Mpro inhibitor.

SARS-CoV-2 has the capacity to evolve mutations to escape vaccine-and infection-acquired immunity and antiviral drugs. A variant-agnostic therapeutic agent that protects against severe disease without putting selective pressure on the virus would thus be a valuable biomedical tool. Here, we challenged rhesus macaques with SARS-CoV-2 Delta and simultaneously treated them with aerosolized RBD-62, a protein developed through multiple rounds of in vitro evolution of SARS-CoV-2 RBD to acquire 1000-fold enhanced ACE2 binding affinity. RBD-62 treatment gave equivalent protection in upper and lower airways, a phenomenon not previously observed with clinically approved vaccines. Importantly, RBD-62 did not block the development of memory responses to Delta and did not elicit anti-drug immunity. These data provide proof-of-concept that RBD-62 can prevent severe disease from a highly virulent variant.

Entry of SARS-CoV-2 into human respiratory cells, mediated by the spike protein, is absolutely dependent on the cellular receptor ACE2 (angiotensin-converting enzyme-2). This makes ACE2 an attractive target for therapeutic intervention in COVID-19. In this issue, Zuo et al. discover that vitamin C, an essential nutrient and common dietary supplement, can target ACE2 for ubiquitin-dependent degradation, resulting in the inhibition of SARS-CoV-2 infection (Zuo et al, 2023). The study identifies novel mechanisms of cellular ACE2 regulation and may inform the design of therapeutics targeting SARS-2 and related coronaviruses.

CTL-mediated killing of virally infected or malignant cells is orchestrated at the immune synapse (IS). We hypothesized that SARS-CoV-2 may target lytic IS assembly to escape elimination. We show that human CD8+ T cells upregulate the expression of ACE2, the Spike receptor, during differentiation to CTLs. CTL preincubation with the Wuhan or Omicron Spike variants inhibits IS assembly and function, as shown by defective synaptic accumulation of TCRs and tyrosine phosphoproteins as well as defective centrosome and lytic granule polarization to the IS, resulting in impaired target cell killing and cytokine production. These defects were reversed by anti-Spike antibodies interfering with ACE2 binding and reproduced by ACE2 engagement by angiotensin II or anti-ACE2 antibodies, but not by the ACE2 product Ang (1-7). IS defects were also observed ex vivo in CTLs from COVID-19 patients. These results highlight a new strategy of immune evasion by SARS-CoV-2 based on the Spike-dependent, ACE2-mediated targeting of the lytic IS to prevent elimination of infected cells.