Design and characterization of protective pan-ebolavirus and pan-filovirus bispecific antibodies.

Monoclonal antibodies (mAbs) are an important class of antiviral therapeutics. MAbs are highly selective, well tolerated, and have long in vivo half-life as well as the capacity to induce immune-mediated virus clearance. Their activities can be further enhanced by integration of their variable fragments (Fvs) into bispecific antibodies (bsAbs), affording simultaneous targeting of multiple epitopes to improve potency and breadth and/or to mitigate against viral escape by a single mutation. Here, we explore a bsAb strategy for generation of pan-ebolavirus and pan-filovirus immunotherapeutics. Filoviruses, including Ebola virus (EBOV), Sudan virus (SUDV), and Marburg virus (MARV), cause severe hemorrhagic fever. Although there are two FDA-approved mAb therapies for EBOV infection, these do not extend to other filoviruses. Here, we combine Fvs from broad ebolavirus mAbs to generate novel pan-ebolavirus bsAbs that are potently neutralizing, confer protection in mice, and are resistant to viral escape. Moreover, we combine Fvs from pan-ebolavirus mAbs with those of protective MARV mAbs to generate pan-filovirus protective bsAbs. These results provide guidelines for broad antiviral bsAb design and generate new immunotherapeutic candidates.

Monovalent SARS-COV-2 mRNA vaccine using optimal UTRs and LNPs is highly immunogenic and broadly protective against Omicron variants.

The emergence of highly transmissible severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) that are resistant to the current COVID-19 vaccines highlights the need for continued development of broadly protective vaccines for the future. Here, we developed two messenger RNA (mRNA)-lipid nanoparticle (LNP) vaccines, TU88mCSA and ALCmCSA, using the ancestral SARS-CoV-2 spike sequence, optimized 5' and 3' untranslated regions (UTRs), and LNP combinations. Our data showed that these nanocomplexes effectively activate CD4+ and CD8+ T cell responses and humoral immune response and provide complete protection against WA1/2020, Omicron BA.1 and BQ.1 infection in hamsters. Critically, in Omicron BQ.1 challenge hamster models, TU88mCSA and ALCmCSA not only induced robust control of virus load in the lungs but also enhanced protective efficacy in the upper respiratory airways. Antigen-specific immune analysis in mice revealed that the observed cross-protection is associated with superior UTRs [Carboxylesterase 1d (Ces1d)/adaptor protein-3ß (AP3B1)] and LNP formulations that elicit robust lung tissue-resident memory T cells. Strong protective effects of TU88mCSA or ALCmCSA against both WA1/2020 and VOCs suggest that this mRNA-LNP combination can be a broadly protective vaccine platform in which mRNA cargo uses the ancestral antigen sequence regardless of the antigenic drift. This approach could be rapidly adapted for clinical use and timely deployment of vaccines against emerging and reemerging VOCs.

Germline-encoded amino acid-binding motifs drive immunodominant public antibody responses.

Despite the vast diversity of the antibody repertoire, infected individuals often mount antibody responses to precisely the same epitopes within antigens. The immunological mechanisms underpinning this phenomenon remain unknown. By mapping 376 immunodominant "public epitopes" at high resolution and characterizing several of their cognate antibodies, we concluded that germline-encoded sequences in antibodies drive recurrent recognition. Systematic analysis of antibody-antigen structures uncovered 18 human and 21 partially overlapping mouse germline-encoded amino acid-binding (GRAB) motifs within heavy and light V gene segments that in case studies proved critical for public epitope recognition. GRAB motifs represent a fundamental component of the immune system's architecture that promotes recognition of pathogens and leads to species-specific public antibody responses that can exert selective pressure on pathogens.

A pan-variant mRNA-LNP T cell vaccine protects HLA transgenic mice from mortality after infection with SARS-CoV-2 Beta.

Licensed COVID-19 vaccines ameliorate viral infection by inducing production of neutralizing antibodies that bind the SARS-CoV-2 Spike protein and inhibit viral cellular entry. However, the clinical effectiveness of these vaccines is transitory as viral variants escape antibody neutralization. Effective vaccines that solely rely upon a T cell response to combat SARS-CoV-2 infection could be transformational because they can utilize highly conserved short pan-variant peptide epitopes, but a mRNA-LNP T cell vaccine has not been shown to provide effective anti-SARS-CoV-2 prophylaxis. Here we show a mRNA-LNP vaccine (MIT-T-COVID) based on highly conserved short peptide epitopes activates CD8+ and CD4+ T cell responses that attenuate morbidity and prevent mortality in HLA-A*02:01 transgenic mice infected with SARS-CoV-2 Beta (B.1.351). We found CD8+ T cells in mice immunized with MIT-T-COVID vaccine significantly increased from 1.1% to 24.0% of total pulmonary nucleated cells prior to and at 7 days post infection (dpi), respectively, indicating dynamic recruitment of circulating specific T cells into the infected lungs. Mice immunized with MIT-T-COVID had 2.8 (2 dpi) and 3.3 (7 dpi) times more lung infiltrating CD8+ T cells than unimmunized mice. Mice immunized with MIT-T-COVID had 17.4 times more lung infiltrating CD4+ T cells than unimmunized mice (7 dpi). The undetectable specific antibody response in MIT-T-COVID-immunized mice demonstrates specific T cell responses alone can effectively attenuate the pathogenesis of SARS-CoV-2 infection. Our results suggest further study is merited for pan-variant T cell vaccines, including for individuals that cannot produce neutralizing antibodies or to help mitigate Long COVID.

IMM-BCP-01, a patient-derived anti-SARS-CoV-2 antibody cocktail, is active across variants of concern including Omicron BA.1 and BA.2.

Monoclonal antibodies are an efficacious therapy against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, rapid viral mutagenesis led to escape from most of these therapies, outlining the need for an antibody cocktail with a broad neutralizing potency. Using an unbiased interrogation of the memory B cell repertoire of patients with convalescent COVID-19, we identified human antibodies with broad antiviral activity in vitro and efficacy in vivo against all tested SARS-CoV-2 variants of concern, including Delta and Omicron BA.1 and BA.2. Here, we describe an antibody cocktail, IMM-BCP-01, that consists of three patient-derived broadly neutralizing antibodies directed at nonoverlapping surfaces on the SARS-CoV-2 Spike protein. Two antibodies, IMM20184 and IMM20190, directly blocked Spike binding to the ACE2 receptor. Binding of the third antibody, IMM20253, to its cryptic epitope on the outer surface of RBD altered the conformation of the Spike Trimer, promoting the release of Spike monomers. These antibodies decreased Omicron SARS-CoV-2 infection in the lungs of Syrian golden hamsters in vivo and potently induced antiviral effector response in vitro, including phagocytosis, ADCC, and complement pathway activation. Our preclinical data demonstrated that the three-antibody cocktail IMM-BCP-01 could be a promising means for preventing or treating infection of SARS-CoV-2 variants of concern, including Omicron BA.1 and BA.2, in susceptible individuals.

Antibodies induced by an ancestral SARS-CoV-2 strain that cross-neutralize variants from Alpha to Omicron BA.1.

Neutralizing antibodies that recognize the SARS-CoV-2 spike glycoprotein are the principal host defense against viral invasion. Variants of SARS-CoV-2 bear mutations that allow escape from neutralization by many human antibodies, especially those in widely distributed ("public") classes. Identifying antibodies that neutralize these variants of concern and determining their prevalence are important goals for understanding immune protection. To determine the Delta and Omicron BA.1 variant specificity of B cell repertoires established by an initial Wuhan strain infection, we measured neutralization potencies of 73 antibodies from an unbiased survey of the early memory B cell response. Antibodies recognizing each of three previously defined epitopic regions on the spike receptor binding domain (RBD) varied in neutralization potency and variant-escape resistance. The ACE2 binding surface ("RBD-2") harbored the binding sites of neutralizing antibodies with the highest potency but with the greatest sensitivity to viral escape; two other epitopic regions on the RBD ("RBD-1" and "RBD-3") bound antibodies of more modest potency but greater breadth. The structures of several Fab:spike complexes that neutralized all five variants of concern tested, including one Fab each from the RBD-1, -2, and -3 clusters, illustrated the determinants of broad neutralization and showed that B cell repertoires can have specificities that avoid immune escape driven by public antibodies. The structure of the RBD-2 binding, broad neutralizer shows why it retains neutralizing activity for Omicron BA.1, unlike most others in the same public class. Our results correlate with real-world data on vaccine efficacy, which indicate mitigation of disease caused by Omicron BA.1.

The HCV Envelope Glycoprotein Down-Modulates NF-κB Signalling and Associates With Stimulation of the Host Endoplasmic Reticulum Stress Pathway.

Following acute HCV infection, the virus establishes a chronic disease in the majority of patients whilst few individuals clear the infection spontaneously. The precise mechanisms that determine chronic HCV infection or spontaneous clearance are not completely understood but are proposed to be driven by host and viral genetic factors as well as HCV encoded immunomodulatory proteins. Using the HIV-1 LTR as a tool to measure NF-κB activity, we identified that the HCV E1E2 glycoproteins and more so the E2 protein down-modulates HIV-1 LTR activation in 293T, TZM-bl and the more physiologically relevant Huh7 liver derived cell line. We demonstrate this effect is specifically mediated through inhibiting NF-κB binding to the LTR and show that this effect was conserved for all HCV genotypes tested. Transcriptomic analysis of 293T cells expressing the HCV glycoproteins identified E1E2 mediated stimulation of the endoplasmic reticulum (ER) stress response pathway and upregulation of stress response genes such as ATF3. Through shRNA mediated inhibition of ATF3, one of the components, we observed that E1E2 mediated inhibitory effects on HIV-1 LTR activity was alleviated. Our in vitro studies demonstrate that HCV Env glycoprotein activates host ER Stress Pathways known to inhibit NF-κB activity. This has potential implications for understanding HCV induced immune activation as well as oncogenesis.

Structural basis for continued antibody evasion by the SARS-CoV-2 receptor binding domain.

Many studies have examined the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants on neutralizing antibody activity after they have become dominant strains. Here, we evaluate the consequences of further viral evolution. We demonstrate mechanisms through which the SARS-CoV-2 receptor binding domain (RBD) can tolerate large numbers of simultaneous antibody escape mutations and show that pseudotypes containing up to seven mutations, as opposed to the one to three found in previously studied variants of concern, are more resistant to neutralization by therapeutic antibodies and serum from vaccine recipients. We identify an antibody that binds the RBD core to neutralize pseudotypes for all tested variants but show that the RBD can acquire an N-linked glycan to escape neutralization. Our findings portend continued emergence of escape variants as SARS-CoV-2 adapts to humans.

Characterization of an Anti-Ebola Virus Hyperimmune Globulin Derived From Convalescent Plasma.

BACKGROUND: Convalescent plasma has been used to treat many viral diseases including Ebola. The manufacture of a purified anti-Ebola virus (EBOV) intravenous immunoglobulin (IVIG) from pooled convalescent plasma is described in this paper. METHODS: An enzyme-linked immunosorbent assay (ELISA) targeting an EBOV surface glycoprotein antigen was used to determine the immunoglobulin titer of pooled plasma and purified anti-EBOV IVIG. Anti-EBOV IVIG was also tested in neutralization assays using a vesicular stomatitis virus pseudovirion expressing EBOV glycoprotein on its surface and with live EBOV. Finally, the efficacy of the anti-EBOV IVIG was assessed in a mouse model of EBOV infection. RESULTS: In the ELISA, the anti-EBOV IVIG was shown to have a 7-fold increase in immunoglobulin G (IgG) titer over pooled convalescent plasma. In both the pseudovirion and live virus assays, the anti-EBOV IVIG showed approximately 5- to 6-fold increased potency over pooled plasma. Anti-EBOV IVIG also significantly improved survivability in mice infected with the virus when administered concurrently or 2 days after infection. CONCLUSIONS: These data support this purified anti-EBOV IVIG merits additional investigation and clinical trials for treatment and postexposure prophylaxis of Ebola virus disease. The experience gained can be applied to manufacture hyperimmune globulins against other emerging viruses.

Anti-SARS-CoV-2 Activity of Adamantanes In Vitro and in Animal Models of Infection.

Coronavirus disease 2019 (COVID-19) has had devastating effects worldwide, with particularly high morbidity and mortality in outbreaks on residential care facilities. Amantadine, originally licensed as an antiviral agent for therapy and prophylaxis against influenza A virus, has beneficial effects on patients with Parkinson's disease and is used for treatment of Parkinson's disease, multiple sclerosis, acquired brain injury, and various other neurological disorders. Recent observational data suggest an inverse relationship between the use of amantadine and COVID-19. Adamantanes, including amantadine and rimantadine, are reported to have in vitro activity against severe acute respiratory syndrome coronavirus (SARS-CoV) and, more recently, SARS-CoV-2. We hypothesized that adamantanes have antiviral activity against SARS-CoV-2, including variant strains. To assess the activity of adamantanes against SARS-CoV-2, we used in vitro and in vivo models of infection. We established that amantadine, rimantadine, and tromantadine inhibit the growth of SARS-CoV-2 in vitro in cultured human epithelial cells. While neither rimantadine nor amantadine reduces lung viral titers in mice infected with mouse-adapted SARS-CoV-2, rimantadine significantly reduces viral titers in the lungs in golden Syrian hamsters infected with SARS-CoV-2. In summary, rimantadine has antiviral activity against SARS-CoV-2 in human alveolar epithelial cells and in the hamster model of SARS-CoV-2 lung infection. The evaluation of amantadine or rimantadine in human randomized controlled trials can definitively address applications for the treatment or prevention of COVID-19.

Biomaterials and Oxygen Join Forces to Shape the Immune Response and Boost COVID-19 Vaccines.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to an unprecedented global health crisis, resulting in a critical need for effective vaccines that generate protective antibodies. Protein subunit vaccines represent a promising approach but often lack the immunogenicity required for strong immune stimulation. To overcome this challenge, it is first demonstrated that advanced biomaterials can be leveraged to boost the effectiveness of SARS-CoV-2 protein subunit vaccines. Additionally, it is reported that oxygen is a powerful immunological co-adjuvant and has an ability to further potentiate vaccine potency. In preclinical studies, mice immunized with an oxygen-generating coronavirus disease 2019 (COVID-19) cryogel-based vaccine (O2-CryogelVAX) exhibit a robust Th1 and Th2 immune response, leading to a sustained production of highly effective neutralizing antibodies against the virus. Even with a single immunization, O2-CryogelVAX achieves high antibody titers within 21 days, and both binding and neutralizing antibody levels are further increased after a second dose. Engineering a potent vaccine system that generates sufficient neutralizing antibodies after one dose is a preferred strategy amid vaccine shortage. The data suggest that this platform is a promising technology to reinforce vaccine-driven immunostimulation and is applicable to current and emerging infectious diseases.

Memory B cell repertoire for recognition of evolving SARS-CoV-2 spike.

Memory B cell reserves can generate protective antibodies against repeated SARS-CoV-2 infections, but with unknown reach from original infection to antigenically drifted variants. We charted memory B cell receptor-encoded antibodies from 19 COVID-19 convalescent subjects against SARS-CoV-2 spike (S) and found seven major antibody competition groups against epitopes recurrently targeted across individuals. Inclusion of published and newly determined structures of antibody-S complexes identified corresponding epitopic regions. Group assignment correlated with cross-CoV-reactivity breadth, neutralization potency, and convergent antibody signatures. Although emerging SARS-CoV-2 variants of concern escaped binding by many members of the groups associated with the most potent neutralizing activity, some antibodies in each of those groups retained affinity-suggesting that otherwise redundant components of a primary immune response are important for durable protection from evolving pathogens. Our results furnish a global atlas of S-specific memory B cell repertoires and illustrate properties driving viral escape and conferring robustness against emerging variants.

SARS-CoV-2 evolution in an immunocompromised host reveals shared neutralization escape mechanisms.

Many individuals mount nearly identical antibody responses to SARS-CoV-2. To gain insight into how the viral spike (S) protein receptor-binding domain (RBD) might evolve in response to common antibody responses, we studied mutations occurring during virus evolution in a persistently infected immunocompromised individual. We use antibody Fab/RBD structures to predict, and pseudotypes to confirm, that mutations found in late-stage evolved S variants confer resistance to a common class of SARS-CoV-2 neutralizing antibodies we isolated from a healthy COVID-19 convalescent donor. Resistance extends to the polyclonal serum immunoglobulins of four out of four healthy convalescent donors we tested and to monoclonal antibodies in clinical use. We further show that affinity maturation is unimportant for wild-type virus neutralization but is critical to neutralization breadth. Because the mutations we studied foreshadowed emerging variants that are now circulating across the globe, our results have implications to the long-term efficacy of S-directed countermeasures.

Memory B cell repertoire for recognition of evolving SARS-CoV-2 spike.

Memory B cell reserves can generate protective antibodies against repeated SARS-CoV-2 infections, but with an unknown reach from original infection to antigenically drifted variants. We charted memory B cell receptor-encoded monoclonal antibodies (mAbs) from 19 COVID-19 convalescent subjects against SARS-CoV-2 spike (S) and found 7 major mAb competition groups against epitopes recurrently targeted across individuals. Inclusion of published and newly determined structures of mAb-S complexes identified corresponding epitopic regions. Group assignment correlated with cross-CoV-reactivity breadth, neutralization potency, and convergent antibody signatures. mAbs that competed for binding the original S isolate bound differentially to S variants, suggesting the protective importance of otherwise-redundant recognition. The results furnish a global atlas of the S-specific memory B cell repertoire and illustrate properties conferring robustness against emerging SARS-CoV-2 variants.

A trimeric human angiotensin-converting enzyme 2 as an anti-SARS-CoV-2 agent.

Effective intervention strategies are urgently needed to control the COVID-19 pandemic. Human angiotensin-converting enzyme 2 (ACE2) is a membrane-bound carboxypeptidase that forms a dimer and serves as the cellular receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). ACE2 is also a key negative regulator of the renin-angiotensin system that modulates vascular functions. We report here the properties of a trimeric ACE2 ectodomain variant, engineered using a structure-based approach. The trimeric ACE2 variant has a binding affinity of ~60 pM for the spike protein of SARS­CoV­2 (compared with 77 nM for monomeric ACE2 and 12-22 nM for dimeric ACE2 constructs), and its peptidase activity and the ability to block activation of angiotensin II receptor type 1 in the renin-angiotensin system are preserved. Moreover, the engineered ACE2 potently inhibits SARS­CoV­2 infection in cell culture. These results suggest that engineered, trimeric ACE2 may be a promising anti-SARS-CoV-2 agent for treating COVID-19.

Rapid and complete inactivation of SARS-CoV-2 by ultraviolet-C irradiation.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has devastated global public health systems and economies, with over 52 million people infected, millions of jobs and businesses lost, and more than 1 million deaths recorded to date. Contact with surfaces contaminated with droplets generated by infected persons through exhaling, talking, coughing and sneezing is a major driver of SARS-CoV-2 transmission, with the virus being able to survive on surfaces for extended periods of time. To interrupt these chains of transmission, there is an urgent need for devices that can be deployed to inactivate the virus on both recently and existing contaminated surfaces. Here, we describe the inactivation of SARS-CoV-2 in both wet and dry format using radiation generated by a commercially available Signify ultraviolet (UV)-C light source at 254 nm. We show that for contaminated surfaces, only seconds of exposure is required for complete inactivation, allowing for easy implementation in decontamination workflows.

Molecular basis for a germline-biased neutralizing antibody response to SARS-CoV-2.

The SARS-CoV-2 viral spike (S) protein mediates attachment and entry into host cells and is a major target of vaccine and drug design. Potent SARS-CoV-2 neutralizing antibodies derived from closely related antibody heavy chain genes (IGHV3-53 or 3-66) have been isolated from multiple COVID-19 convalescent individuals. These usually contain minimal somatic mutations and bind the S receptor-binding domain (RBD) to interfere with attachment to the cellular receptor angiotensin-converting enzyme 2 (ACE2). We used antigen-specific single B cell sorting to isolate S-reactive monoclonal antibodies from the blood of a COVID-19 convalescent individual. The seven most potent neutralizing antibodies were somatic variants of the same IGHV3-53-derived antibody and bind the RBD with varying affinity. We report X-ray crystal structures of four Fab variants bound to the RBD and use the structures to explain the basis for changes in RBD affinity. We show that a germline revertant antibody binds tightly to the SARS-CoV-2 RBD and neutralizes virus, and that gains in affinity for the RBD do not necessarily correlate with increased neutralization potency, suggesting that somatic mutation is not required to exert robust antiviral effect. Our studies clarify the molecular basis for a heavily germline-biased human antibody response to SARS-CoV-2.

A trimeric human angiotensin-converting enzyme 2 as an anti-SARS-CoV-2 agent in vitro.

Effective intervention strategies are urgently needed to control the COVID-19 pandemic. Human angiotensin-converting enzyme 2 (ACE2) is a carboxypeptidase that forms a dimer and serves as the cellular receptor for SARS-CoV-2. It is also a key negative regulator of the renin-angiotensin system (RAS), conserved in mammals, which modulates vascular functions. We report here the properties of a trimeric ACE2 variant, created by a structure-based approach, with binding affinity of ~60 pM for the spike (S) protein of SARS-CoV-2, while preserving the wildtype peptidase activity as well as the ability to block activation of angiotensin II receptor type 1 in the RAS. Moreover, the engineered ACE2 potently inhibits infection of SARS-CoV-2 in cell culture. These results suggest that engineered, trimeric ACE2 may be a promising anti-SARS-CoV-2 agent for treating COVID-19.