A broadly neutralizing human monoclonal antibody generated from transgenic mice immunized with HCMV particles limits virus infection and proliferation.

Human cytomegalovirus (HCMV) is a ß-herpesvirus that poses severe disease risk for immunocompromised patients who experience primary infection or reactivation. Development and optimization of safe and effective anti-HCMV therapeutics is of urgent necessity for the prevention and treatment of HCMV-associated diseases in diverse populations. The use of neutralizing monoclonal antibodies (mAbs) to limit HCMV infection poses a promising therapeutic strategy, as anti-HCMV mAbs largely inhibit infection by targeting virion glycoprotein complexes. In contrast, the small-molecule compounds currently approved for patients (e.g., ganciclovir, letermovir, and maribavir) target later stages of the HCMV life cycle. Here, we present a broadly neutralizing human mAb, designated 1C10, elicited from a VelocImmune mouse immunized with infectious HCMV particles. Clone 1C10 neutralizes infection after virion binding to cells by targeting gH/gL envelope complexes and potently reduces infection of diverse HCMV strains in fibroblast, trophoblast, and epithelial cells. Antibody competition assays found that 1C10 recognizes a region of gH associated with broad neutralization and binds to soluble pentamer in the low nanomolar range. Importantly, 1C10 treatment significantly reduced virus proliferation in both fibroblast and epithelial cells. Further, the combination treatment of mAb 1C10 with ganciclovir reduced HCMV infection and proliferation in a synergistic manner. This work characterizes a neutralizing human mAb for potential use as a HCMV treatment, as well as a possible therapeutic strategy utilizing combination-based treatments targeting disparate steps of the viral life cycle. Collectively, the findings support an antibody-based therapy to effectively treat patients at risk for HCMV-associated diseases. IMPORTANCE: Human cytomegalovirus is a herpesvirus that infects a large proportion of the population and can cause significant disease in diverse patient populations whose immune systems are suppressed or compromised. The development and optimization of safe anti-HCMV therapeutics, especially those that have viral targets and inhibition mechanisms different from current HCMV treatments, are of urgent necessity to better public health. Human monoclonal antibodies (mAbs) that prevent HCMV entry of cells were identified by immunizing transgenic mice and screened for broad and effective neutralization capability. Here, we describe one such mAb, which was found to target gH/gL envelope complexes and effectively limit HCMV infection and dissemination. Further, administration of the antibody in combination with the antiviral drug ganciclovir inhibited HCMV in a synergistic manner, highlighting this approach and the use of anti-HCMV mAbs more broadly, as a potential therapeutic strategy for the treatment of diverse patient populations.

An in vitro experimental pipeline to characterize the epitope of a SARS-CoV-2 neutralizing antibody.

IMPORTANCE: The COVID-19 pandemic remains a significant public health concern for the global population; the development and characterization of therapeutics, especially ones that are broadly effective, will continue to be essential as severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) variants emerge. Neutralizing monoclonal antibodies remain an effective therapeutic strategy to prevent virus infection and spread so long as they recognize and interact with circulating variants. The epitope and binding specificity of a neutralizing anti-SARS-CoV-2 Spike receptor-binding domain antibody clone against many SARS-CoV-2 variants of concern were characterized by generating antibody-resistant virions coupled with cryo-EM structural analysis and VSV-spike neutralization studies. This workflow can serve to predict the efficacy of antibody therapeutics against emerging variants and inform the design of therapeutics and vaccines.

The CD46 ectodomain participates in human cytomegalovirus infection of epithelial cells.

Human cytomegalovirus (HCMV) primary infections are typically asymptomatic in healthy individuals yet can cause increased morbidity and mortality in organ transplant recipients, AIDS patients, neonates, and the elderly. The successful, widespread dissemination of this virus among the population can be attributed in part to its wide cellular tropism and ability to establish life-long latency. HCMV infection is a multi-step process that requires numerous cellular and viral factors. The viral envelope consists of envelope protein complexes that interact with cellular factors; such interactions dictate virus recognition and attachment to different cell types, followed by fusion either at the cell membrane or within an endocytic vesicle. Several HCMV entry factors, including neuropilin-2 (Nrp2), THBD, CD147, OR14I1, and CD46, are characterized as participating in HCMV pentamer-specific entry of non-fibroblast cells such as epithelial, trophoblast, and endothelial cells, respectively. This study focuses on characterizing the structural elements of CD46 that impact HCMV infection. Infectivity studies of wild-type and CD46 knockout epithelial cells demonstrated that levels of CD46 expressed on the cell surface were directly related to HCMV infectivity. Overexpression of CD46 isomers BC1, BC2, and C2 enhanced infection. Further, CD46 knockout epithelial cells expressing CD46 deletion and chimeric molecules identified that the intact ectodomain was critical for rescue of HCMV infection in CD46 knockout cells. Collectively, these data support a model that the extracellular domain of CD46 participates in HCMV infection due to its surface expression.

An in vitro experimental pipeline to characterize the binding specificity of SARS-CoV-2 neutralizing antibodies.

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) has led to over 760 million cases and >6.8 million deaths worldwide. We developed a panel of human neutralizing monoclonal antibodies (mAbs) targeting the SARS-CoV-2 Spike protein using Harbour H2L2 transgenic mice immunized with Spike receptor binding domain (RBD) (1). Representative antibodies from genetically-distinct families were evaluated for inhibition of replication-competent VSV expressing SARS-CoV-2 Spike (rcVSV-S) in place of VSV-G. One mAb (denoted FG-10A3) inhibited infection of all rcVSV-S variants; its therapeutically-modified version, STI-9167, inhibited infection of all tested SARS-CoV-2 variants, including Omicron BA.1 and BA.2, and limited virus proliferation in vivo (1). To characterize the binding specificity and epitope of FG-10A3, we generated mAb-resistant rcVSV-S virions and performed structural analysis of the antibody/antigen complex using cryo-EM. FG-10A3/STI-9167 is a Class 1 antibody that prevents Spike-ACE2 binding by engaging a region within the Spike receptor binding motif (RBM). Sequencing of mAb-resistant rcVSV-S virions identified F486 as a critical residue for mAb neutralization, with structural analysis revealing that both the variable heavy and light chains of STI-9167 bound the disulfide-stabilized 470-490 loop at the Spike RBD tip. Interestingly, substitutions at position 486 were later observed in emerging variants of concern BA.2.75.2 and XBB. This work provides a predictive modeling strategy to define the neutralizing capacity and limitations of mAb therapeutics against emerging SARS-CoV-2 variants. Importance: The COVID-19 pandemic remains a significant public health concern for the global population; development and characterization of therapeutics, especially ones that are broadly effective, will continue to be essential as SARS-CoV-2 variants emerge. Neutralizing monoclonal antibodies remain an effective therapeutic strategy to prevent virus infection and spread with the caveat that they interact with the circulating variants. The epitope and binding specificity of a broadly neutralizing anti-SARS-CoV-2 Spike RBD antibody clone against many SARS-CoV-2 VOC was characterized by generating antibody-resistant virions coupled with cryo-EM structural analysis. This workflow can serve to predict the efficacy of antibody therapeutics against emerging variants and inform the design of therapeutics and vaccines.

Investigating N-arylpyrimidinamine (NAPA) compounds as early-stage inhibitors against human cytomegalovirus.

Human cytomegalovirus (CMV) is a ubiquitous ß-herpesvirus that establishes latent asymptomatic infections in healthy individuals but can cause serious infections in immunocompromised people, resulting in increased risk of morbidity and mortality. The current FDA-approved CMV drugs target late stages of the CMV life-cycle. While these drugs are effective in most cases, they have serious drawbacks, including poor oral bioavailability, dose-limiting toxicity, and a low barrier to resistance. Given the clinical relevance of CMV-associated diseases, novel therapies are needed. Thus, a novel class of compounds that inhibits the early stages of the CMV life-cycle was identified and found to block infection of different strains in physiologically relevant cell types. This class of compounds, N-arylpyrimidinamine (NAPA), demonstrated potent anti-CMV activity against ganciclovir-sensitive and -resistant strains in in vitro replication assays, a selectivity index >30, and favorable in vitro ADME properties. Mechanism of action studies demonstrated that NAPA compounds inhibit an early step of virus infection. NAPA compounds are specific inhibitors of cytomegaloviruses and exhibited limited anti-viral activity against other herpesviruses. Collectively, we have identified a novel class of CMV inhibitor that effectively limits viral infection and proliferation.

Discovery and intranasal administration of a SARS-CoV-2 broadly acting neutralizing antibody with activity against multiple Omicron subvariants.

BACKGROUND: The continual emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern, in particular the newly emerged Omicron (B.1.1.529) variant and its BA.X lineages, has rendered ineffective a number of previously FDA emergency use authorized SARS-CoV-2 neutralizing antibody therapies. Furthermore, those approved antibodies with neutralizing activity against Omicron BA.1 are reportedly ineffective against the subset of Omicron subvariants that contain a R346K substitution, BA.1.1, and the more recently emergent BA.2, demonstrating the continued need for discovery and characterization of candidate therapeutic antibodies with the breadth and potency of neutralizing activity required to treat newly diagnosed COVID-19 linked to recently emerged variants of concern. METHODS: Following a campaign of antibody discovery based on the vaccination of Harbor H2L2 mice with defined SARS-CoV-2 spike domains, we have characterized the activity of a large collection of spike-binding antibodies and identified a lead neutralizing human IgG1 LALA antibody, STI-9167. FINDINGS: STI-9167 has potent, broad-spectrum neutralizing activity against the current SARS-COV-2 variants of concern and retained activity against each of the tested Omicron subvariants in both pseudotype and live virus neutralization assays. Furthermore, STI-9167 nAb administered intranasally or intravenously provided protection against weight loss and reduced virus lung titers to levels below the limit of quantitation in Omicron-infected K18-hACE2 transgenic mice. CONCLUSIONS: With this established activity profile, a cGMP cell line has been developed and used to produce cGMP drug product intended for intravenous or intranasal use in human clinical trials. FUNDING: Funded by CRIPT (no. 75N93021R00014), DARPA (HR0011-19-2-0020), and NCI Seronet (U54CA260560).