A viral assembly inhibitor blocks SARS-CoV-2 replication in airway epithelial cells.

The ongoing evolution of SARS-CoV-2 to evade vaccines and therapeutics underlines the need for innovative therapies with high genetic barriers to resistance. Therefore, there is pronounced interest in identifying new pharmacological targets in the SARS-CoV-2 viral life cycle. The small molecule PAV-104, identified through a cell-free protein synthesis and assembly screen, was recently shown to target host protein assembly machinery in a manner specific to viral assembly. In this study, we investigate the capacity of PAV-104 to inhibit SARS-CoV-2 replication in human airway epithelial cells (AECs). We show that PAV-104 inhibits >99% of infection with diverse SARS-CoV-2 variants in immortalized AECs, and in primary human AECs cultured at the air-liquid interface (ALI) to represent the lung microenvironment in vivo. Our data demonstrate that PAV-104 inhibits SARS-CoV-2 production without affecting viral entry, mRNA transcription, or protein synthesis. PAV-104 interacts with SARS-CoV-2 nucleocapsid (N) and interferes with its oligomerization, blocking particle assembly. Transcriptomic analysis reveals that PAV-104 reverses SARS-CoV-2 induction of the type-I interferon response and the maturation of nucleoprotein signaling pathway known to support coronavirus replication. Our findings suggest that PAV-104 is a promising therapeutic candidate for COVID-19 with a mechanism of action that is distinct from existing clinical management approaches.

Exposure of epitope residues on the outer face of the chikungunya virus envelope trimer determines antibody neutralizing efficacy.

UNLABELLED: Chikungunya virus (CHIKV) is a reemerging alphavirus that causes a debilitating arthritic disease and infects millions of people and for which no specific treatment is available. Like many alphaviruses, the structural targets on CHIKV that elicit a protective humoral immune response in humans are poorly defined. Here we used phage display against virus-like particles (VLPs) to isolate seven human monoclonal antibodies (MAbs) against the CHIKV envelope glycoproteins E2 and E1. One MAb, IM-CKV063, was highly neutralizing (50% inhibitory concentration, 7.4 ng/ml), demonstrated high-affinity binding (320 pM), and was capable of therapeutic and prophylactic protection in multiple animal models up to 24 h postexposure. Epitope mapping using a comprehensive shotgun mutagenesis library of 910 mutants with E2/E1 alanine mutations demonstrated that IM-CKV063 binds to an intersubunit conformational epitope on domain A, a functionally important region of E2. MAbs against the highly conserved fusion loop have not previously been reported but were also isolated in our studies. Fusion loop MAbs were broadly cross-reactive against diverse alphaviruses but were nonneutralizing. Fusion loop MAb reactivity was affected by temperature and reactivity conditions, suggesting that the fusion loop is hidden in infectious virions. Visualization of the binding sites of 15 different MAbs on the structure of E2/E1 revealed that all epitopes are located at the membrane-distal region of the E2/E1 spike. Interestingly, epitopes on the exposed topmost and outer surfaces of the E2/E1 trimer structure were neutralizing, whereas epitopes facing the interior of the trimer were not, providing a rationale for vaccine design and therapeutic MAb development using the intact CHIKV E2/E1 trimer. IMPORTANCE: CHIKV is the most important alphavirus affecting humans, resulting in a chronic arthritic condition that can persist for months or years. In recent years, millions of people have been infected globally, and the spread of CHIKV to the Americas is now beginning, with over 100,000 cases occurring in the Caribbean within 6 months of its arrival. Our study reports on seven human MAbs against the CHIKV envelope, including a highly protective MAb and rarely isolated fusion loop MAbs. Epitope mapping of these MAbs demonstrates how some E2/E1 epitopes are exposed or hidden from the human immune system and suggests a structural mechanism by which these MAbs protect (or fail to protect) against CHIKV infection. Our results suggest that the membrane-distal end of CHIKV E2/E1 is the primary target for the humoral immune response to CHIKV, and antibodies targeting the exposed topmost and outer surfaces of the E2/E1 trimer determine the neutralizing efficacy of this response.

A neutralizing monoclonal antibody targeting the acid-sensitive region in chikungunya virus E2 protects from disease.

The mosquito-borne alphavirus, chikungunya virus (CHIKV), has recently reemerged, producing the largest epidemic ever recorded for this virus, with up to 6.5 million cases of acute and chronic rheumatic disease. There are currently no licensed vaccines for CHIKV and current anti-inflammatory drug treatment is often inadequate. Here we describe the isolation and characterization of two human monoclonal antibodies, C9 and E8, from CHIKV infected and recovered individuals. C9 was determined to be a potent virus neutralizing antibody and a biosensor antibody binding study demonstrated it recognized residues on intact CHIKV VLPs. Shotgun mutagenesis alanine scanning of 98 percent of the residues in the E1 and E2 glycoproteins of CHIKV envelope showed that the epitope bound by C9 included amino-acid 162 in the acid-sensitive region (ASR) of the CHIKV E2 glycoprotein. The ASR is critical for the rearrangement of CHIKV E2 during fusion and viral entry into host cells, and we predict that C9 prevents these events from occurring. When used prophylactically in a CHIKV mouse model, C9 completely protected against CHIKV viremia and arthritis. We also observed that when administered therapeutically at 8 or 18 hours post-CHIKV challenge, C9 gave 100% protection in a pathogenic mouse model. Given that targeting this novel neutralizing epitope in E2 can potently protect both in vitro and in vivo, it is likely to be an important region both for future antibody and vaccine-based interventions against CHIKV.

Detection of host immune responses in acute phase sera of spontaneous resolution versus persistent hepatitis C virus infection.

Prior to the identification of hepatitis C virus (HCV), transfusion-transmission was common. Viral transmission in subjects with a known date of infection allows the study of the immune responses to acute HCV infection. We analysed 39 soluble immune factors in serum samples from subjects with transfusion-transmitted HCV. Dynamic expression kinetics of interferon gamma-induced protein 10 (IP-10), tumour necrosis factor-alpha and interleukin (IL)-10 were observed during acute HCV infection. Serum IP-10 was the only analyte that was significantly elevated in HCV resolvers compared with uninfected controls. In individuals who progressed to chronic HCV elevated levels of IP-10 and IL-10 coincided with first significant alanine aminotransferase elevation and remained elevated during the first year of acute HCV infection. In addition to monitoring lack of reduction in viral load, serum levels of IP-10 and IL-10 expression during acute HCV infection may be useful biomarkers to predict the progress to chronic HCV.

Transfusion transmission of HCV, a long but successful road map to safety.

Improved blood banking practices and the development and implementation of increasingly sensitive serological and nucleic acid amplification technology assays for screening donors for HCV over the past few decades have helped minimize the residual risk from transfusion transmitted HCV in the developed world. Furthermore, studies of transfusion transmitted infections and of donors identified as infected by routine screening have provided significant insights into HCV transmission, epidemiology and pathogenesis. However, transfusion transmission of HCV is still a significant route of infection in the developing world. Key preventive mechanisms to ensure safe blood include elimination of paid donors and development of national donor pools comprising volunteer repeat blood donors, combined with implementation of standardized and maximally sensitive screening assays for HCV. There is also a need to develop up-to-date data on HCV disease burden on a global scale, in part, derived from systematic screening of donors for HCV infection. We suggest the creation of blood donor databases and specimen repositories, both at national and international levels, to facilitate epidemiological surveillance and pathogenesis and treatment studies in the future.

Hepatitis C virus NS5A anchor peptide disrupts human immunodeficiency virus.

In the absence of an effective vaccine, there is an urgent need for safe and effective antiviral agents to prevent transmission of HIV. Here, we report that an amphipathic alpha-helical peptide derived from the hepatitis C virus NS5A anchor domain (designated C5A in this article) that has been shown to be virocidal for the hepatitis C virus (HCV) also has potent antiviral activity against HIV. C5A exhibits a broad range of antiviral activity against HIV isolates, and it prevents infection of the three in vivo targets of HIV: CD4(+) T lymphocytes, macrophages, and dendritic cells by disrupting the integrity of the viral membrane and capsid core while preserving the integrity of host membranes. C5A can interrupt an ongoing T cell infection, and it can prevent transmigration of HIV through primary genital epithelial cells, infection of mucosal target cells and transfer from dendritic cells to T cells ex vivo, justifying future experiments to determine whether C5A can prevent HIV transmission in vivo.

Focused dampening of antibody response to the immunodominant variable loops by engineered soluble gp140.

Immunization studies with modified gp120 monomers using a hyperglycosylation strategy, in which undesired epitopes are masked by the selective incorporation of N-linked glycans, were described in a previous paper (Selvarajah S, et al., J Virol 2000;79:12148-12163). In this report, we applied the hyperglycosylation strategy to soluble uncleaved gp140 trimers to improve the antigenic and immunogenic profile in the context of a trimeric conformation of the immunogen. The JR-FL gp140 gene was added upstream of a soluble trimerization domain of chicken cartilage matrix (CART) protein and expressed predominantly as a trimer and called gp140-CART wild-type. In the hyperglycosylated gp140-CART mCHO(V) mutant, four extra sugar attachment motifs on the variable loops helped mask epitope recognition by monoclonal antibodies specific to the variable loops. The gp140-CART mCHO(V) mutant and gp140-CART wild-type soluble trimer protein were used to immunize rabbits. The gp140-CART mCHO(V) immune sera had reduced antibody response to the variable loops compared to gp140-CART wild-type immune sera as shown by peptide reactivity, competition assays, and the reduced ability of sera to neutralize SF162 virus (a variable loop neutralization-sensitive virus). The antibody response to the CD4 binding site was retained in the gp140-CART mCHO(V) mutant immune sera similar to gp140-CART wild-type immune sera. The results demonstrate that the strategy of hyperglycosylation is clearly useful in the context of a compact form of Env immunogen such as the soluble gp140 trimer in dampening responses to variable loops while maintaining responses to an important epitope, the CD4 binding site. However, the results also show that in order to elicit broadly neutralizing antibodies that target conserved epitopes, the soluble gp140 trimer immunogen template will require further modifications.

A virocidal amphipathic {alpha}-helical peptide that inhibits hepatitis C virus infection in vitro.

An amphipathic alpha-helical peptide (C5A) derived from the membrane anchor domain of the hepatitis C virus (HCV) NS5A protein is virocidal for HCV at submicromolar concentrations in vitro. C5A prevents de novo HCV infection and suppresses ongoing infection by inactivating both extra- and intracellular infectious particles, and it is nontoxic in vitro and in vivo at doses at least 100-fold higher than required for antiviral activity. Mutational analysis indicates that C5A's amphipathic alpha-helical structure is necessary but not sufficient for its virocidal activity, which depends on its amino acid composition but not its primary sequence or chirality. In addition to HCV, C5A inhibits infection by selected flaviviruses, paramyxoviruses, and HIV. These results suggest a model in which C5A destabilizes viral membranes based on their lipid composition, offering a unique therapeutic approach to HCV and other viral infections.

Cell-free human immunodeficiency virus type 1 transcytosis through primary genital epithelial cells.

Although the transport of human immunodeficiency virus type 1 (HIV-1) through the epithelium is critical for HIV-1 colonization, the mechanisms controlling this process remain obscure. In the present study, we investigated the transcellular migration of HIV-1 as a cell-free virus through primary genital epithelial cells (PGECs). The absence of CD4 on PGECs implicates an unusual entry pathway for HIV-1. We found that syndecans are abundantly expressed on PGECs and promote the initial attachment and subsequent entry of HIV-1 through PGECs. Although CXCR4 and CCR5 do not contribute to HIV-1 attachment, they enhance viral entry and transcytosis through PGECs. Importantly, HIV-1 exploits both syndecans and chemokine receptors to ensure successful cell-free transport through the genital epithelium. HIV-1-syndecan interactions rely on specific residues in the V3 of gp120 and specific sulfations within syndecans. We found no obvious correlation between coreceptor usage and the capacity of the virus to transcytose. Since viruses isolated after sexual transmission are mainly R5 viruses, this suggests that the properties conferring virus replication after transmission are distinct from those conferring cell-free virus transcytosis through the genital epithelium. Although we found that cell-free HIV-1 crosses PGECs as infectious particles, the efficiency of transcytosis is extremely poor (less than 0.02% of the initial inoculum). This demonstrates that the genital epithelium serves as a major barrier against HIV-1. Although one cannot exclude the possibility that limited passage of cell-free HIV-1 transcytosis through an intact genital epithelium occurs in vivo, it is likely that the establishment of infection via cell-free HIV-1 transmigration is a rare event.