Development of a humanized monoclonal antibody with therapeutic potential against West Nile virus.
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ABSTRACT:
SUBMITTER: Oliphant T
PROVIDER: S-EPMC1458527 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Defining the precise cellular mechanisms of neutralization by potently inhibitory antibodies is important for understanding how the immune system successfully limits viral infections. We recently described a potently inhibitory monoclonal antibody (MAb E16) against the envelope (E) protein of West Nile virus (WNV) that neutralizes infection even after virus has spread to the central nervous system. Herein, we define its mechanism of inhibition. E16 blocks infection primarily at a post-attachment step as antibody-opsonized WNV enters permissive cells but cannot escape from endocytic compartments. These cellular experiments suggest that E16 blocks the acid-catalyzed fusion step that is required for nucleocapsid entry into the cytoplasm. Indeed, E16 directly inhibits fusion of WNV with liposomes. Additionally, low-pH exposure of E16-WNV complexes in the absence of target membranes did not fully inactivate infectious virus, further suggesting that E16 prevents a structural transition required for fusion. Thus, a strongly neutralizing anti-WNV MAb with therapeutic potential is potently inhibitory because it blocks viral fusion and thereby promotes clearance by delivering virus to the lysosome for destruction.
| S-EPMC2679195 | biostudies-other
Project description:BACKGROUND: Dengue virus (DENV) is a significant public health threat in tropical and subtropical regions of the world. A therapeutic antibody against the viral envelope (E) protein represents a promising immunotherapy for disease control. METHODOLOGY/PRINCIPAL FINDINGS: We generated seventeen novel mouse monoclonal antibodies (mAbs) with high reactivity against E protein of dengue virus type 2 (DENV-2). The mAbs were further dissected using recombinant E protein domain I-II (E-DI-II) and III (E-DIII) of DENV-2. Using plaque reduction neutralization test (PRNT) and mouse protection assay with lethal doses of DENV-2, we identified four serotype-specific mAbs that had high neutralizing activity against DENV-2 infection. Of the four, E-DIII targeting mAb DB32-6 was the strongest neutralizing mAb against diverse DENV-2 strains. Using phage display and virus-like particles (VLPs) we found that residue K310 in the E-DIII A-strand was key to mAb DB32-6 binding E-DIII. We successfully converted DB32-6 to a humanized version that retained potency for the neutralization of DENV-2 and did not enhance the viral infection. The DB32-6 showed therapeutic efficacy against mortality induced by different strains of DENV-2 in two mouse models even in post-exposure trials. CONCLUSIONS/SIGNIFICANCE: We used novel epitope mapping strategies, by combining phage display with VLPs, to identify the important A-strand epitopes with strong neutralizing activity. This study introduced potential therapeutic antibodies that might be capable of providing broad protection against diverse DENV-2 infections without enhancing activity in humans.
| S-EPMC3341331 | biostudies-literature
Project description:Gene-based delivery of recombinant antibody genes is a promising therapeutic strategy offering numerous advantages including sustained antibody levels, better safety profile and lower production cost. Here we describe generation of a recombinant antibody Fc-9E2 comprising a fusion protein between human Fc of IgG1 and a single-chain Fv derived from a hybridoma 9E2 secreting a mAb neutralizing West Nile virus (WNV). Fc-9E2 was shown to retain parental mAb's specificity and WNV-neutralizing capacity. Adenovirus-mediated in vivo delivery of the antibody gene resulted in sustained Fc-9E2 serum levels leading to abrogation of lethal WNV infection in an animal model.
| S-EPMC2267767 | biostudies-literature
Project description:A potent anti-West Nile virus (anti-WNV)-neutralizing humanized monoclonal antibody, hE16, was previously shown to improve the survival of WNV-infected hamsters when it was administered intraperitoneally (i.p.), even after the virus had infected neurons in the brain. In this study, we evaluated the therapeutic limit of hE16 for the treatment of WNV infection in hamsters by comparing single-dose peripheral (i.p.) therapy with direct administration into the pons through a convection-enhanced delivery (CED) system. At day 5 after infection, treatments with hE16 by the peripheral and the CED routes were equally effective at reducing morbidity and mortality. In contrast, at day 6 only the treatment by the CED route protected the hamsters from lethal infection. These experiments suggest that hE16 can directly control WNV infection in the central nervous system. In support of this, hE16 administered i.p. was detected in a time-dependent manner in the serum, cerebrospinal fluid (CSF), cerebral cortex, brain stem, and spinal cord in CSF. A linear relationship between the hE16 dose and the concentration in serum was observed, and maximal therapeutic activity occurred at doses of 0.32 mg/kg of body weight or higher, which produced serum hE16 concentrations of 1.3 microg/ml or higher. Overall, these data suggest that in hamsters hE16 can ameliorate neurological disease after significant viral replication has occurred, although there is a time window that limits therapeutic efficacy.
| S-EPMC1913249 | biostudies-literature
Project description:The early diagnosis of West Nile virus (WNV) infection is important for successful clinical management and epidemiological control. The non-structural protein 1 (NS1) of flavivirus, a highly conserved and secreted glycoprotein, is abundant in the serum of flavivirus-infected patients and represents a useful early diagnostic marker. We developed a WNV-specific NS1 antigen-capture ELISA using two mouse monoclonal antibodies (MAbs) that recognised distinct epitopes of the NS1 protein of WNV as capture and detection antibodies. The antigen-capture ELISA displayed exclusive specificity to WNV without cross-reaction with other related members of the flavivirus family, including the dengue virus, yellow fever virus, Japanese encephalitis virus, and tick-borne encephalitis virus. Additionally, the specificity was presented as no false positive in normal (0/1003) and DENV-infected (0/107) human serum specimens. The detection limit of the antigen-capture ELISA was as low as 15 pg/ml of recombinant WNV NS1 protein (rWNV-NS1) and 6.1 plaque-forming units (PFU)/0.1 ml of WNV-infected culture supernatant. In mice infected with WNV, the NS1 protein was readily detected in serum as early as one day after WNV infection, prior to the development of clinical signs of the disease. The sensitivity of the NS1 capture ELISA (93.7%) was significantly higher (79.4%) than that of real-time reverse transcription polymerase chain reaction in 63 serum samples from WNV-infected mice (p = 0.035). This newly developed NS1 antigen-capture ELISA with high sensitivity and specificity could be used as an efficient method for the early diagnosis of WNV infection in animals or humans.
| S-EPMC4193763 | biostudies-literature
Project description:Over the past decade, West Nile virus (WNV) has spread to all 48 of the lower United States as well as to parts of Canada, Mexico, the Caribbean, and South America, with outbreaks of neuroinvasive disease occurring annually. At present, no therapeutic or vaccine is available for human use. Epidemics of WNV and other emerging infectious disease threats demand cost-efficient and scalable production technologies that can rapidly transfer effective therapeutics into the clinical setting. We have previously reported that Hu-E16, a humanized anti-WNV mAb, binds to a highly conserved epitope on the envelope protein, blocks viral fusion, and shows promising postexposure therapeutic activity. Herein, we generated a plant-derived Hu-E16 mAb that can be rapidly scaled up for commercial production. Plant Hu-E16 was expressed at high levels within 8 days of infiltration in Nicotiana benthamiana plants and retained high-affinity binding and potent neutralizing activity in vitro against WNV. A single dose of plant Hu-E16 protected mice against WNV-induced mortality even 4 days after infection at rates that were indistinguishable from mammalian-cell-produced Hu-E16. This study demonstrates the efficacy of a plant-produced mAb against a potentially lethal infection several days after exposure in an animal challenge model and provides a proof of principle for the development of plant-derived mAbs as therapy against emerging infectious diseases.
| S-EPMC2823901 | biostudies-other
Project description:Envelope protein-targeted vaccines for flaviviruses are limited by concerns of antibody-dependent enhancement (ADE) of infections. Nonstructural protein 1 (NS1) provides an alternative vaccine target that avoids this risk since this protein is absent from the virion. Beyond its intracellular role in virus replication, extracellular forms of NS1 function in immune modulation and are recognized by host-derived antibodies. The rational design of NS1-based vaccines requires an extensive understanding of the antigenic sites on NS1, especially those targeted by protective antibodies. Here, we isolated human monoclonal antibodies (MAbs) from individuals previously naturally infected with WNV, mapped their epitopes using structure-guided mutagenesis, and evaluated their efficacy in vivo against lethal WNV challenge. The most protective epitopes clustered at three antigenic sites that are exposed on cell surface forms of NS1: (i) the wing flexible loop, (ii) the outer, electrostatic surface of the wing, and (iii) the spaghetti loop face of the β-ladder. One additional MAb mapped to the distal tip of the β-ladder and conferred a lower level of protection against WNV despite not binding to NS1 on the surface of infected cells. Our study defines the epitopes and modes of binding of protective anti-NS1 MAb antibodies following WNV infection, which may inform the development of NS1-based countermeasures against flaviviruses. IMPORTANCE Therapeutic antibodies against flaviviruses often promote neutralization by targeting the envelope protein of the virion. However, this approach is hindered by a possible concern for antibody-dependent enhancement of infection and paradoxical worsening of disease. As an alternative strategy, antibodies targeting flavivirus nonstructural protein 1 (NS1), which is absent from the virion, can protect against disease and do not cause enhanced infection. Here, we evaluate the structure-function relationships and protective activity of West Nile virus (WNV) NS1-specific monoclonal antibodies (MAbs) isolated from the memory B cells of a naturally infected human donor. We identify several anti-NS1 MAbs that protect mice against lethal WNV challenge and map their epitopes using charge reversal mutagenesis. Antibodies targeting specific regions in the NS1 structure could serve as the basis for countermeasures that control WNV infection in humans.
| S-EPMC8510529 | biostudies-literature
Project description:West Nile virus is a mosquito-borne flavivirus closely related to the human epidemic-causing dengue, yellow fever and Japanese encephalitis viruses. In establishing infection these icosahedral viruses undergo endosomal membrane fusion catalysed by envelope glycoprotein rearrangement of the putative receptor-binding domain III (DIII) and exposure of the hydrophobic fusion loop. Humoral immunity has an essential protective function early in the course of West Nile virus infection. Here, we investigate the mechanism of neutralization by the E16 monoclonal antibody that specifically binds DIII. Structurally, the E16 antibody Fab fragment engages 16 residues positioned on four loops of DIII, a consensus neutralizing epitope sequence conserved in West Nile virus and distinct in other flaviviruses. The E16 epitope protrudes from the surface of mature virions in three distinct environments, and docking studies predict Fab binding will leave five-fold clustered epitopes exposed. We also show that E16 inhibits infection primarily at a step after viral attachment, potentially by blocking envelope glycoprotein conformational changes. Collectively, our results suggest that a vaccine strategy targeting the dominant DIII epitope may elicit safe and effective immune responses against flaviviral diseases.
| S-EPMC7095628 | biostudies-literature
Project description:Flaviviruses, such as West Nile virus (WNV), are significant human pathogens. The humoral immune response plays an important role in the control of flavivirus infection and disease. The structure of WNV complexed with the Fab fragment of the strongly neutralizing mAb E16 was determined to 14.5-Angstrom resolution with cryo-electron microscopy. E16, an antibody with therapeutic potential, binds to domain III of the WNV envelope glycoprotein. Because of steric hindrance, Fab E16 binds to only 120 of the 180 possible binding sites on the viral surface. Fitting of the previously determined x-ray structure of the Fab-domain III complex into the cryo-electron microscopy density required a change of the elbow angle between the variable and constant domains of the Fab. The structure suggests that the E16 antibody neutralizes WNV by blocking the initial rearrangement of the E glycoprotein before fusion with a cellular membrane.
| S-EPMC1567891 | biostudies-literature
Project description:A decade has passed since the appearance of West Nile virus (WNV) in humans in the Western Hemisphere in New York City. During this interval, WNV spread inexorably throughout North and South America and caused millions of infections ranging from a sub-clinical illness, to a self-limiting febrile syndrome or lethal neuroinvasive disease. Its entry into the United States triggered intensive research into the basic biology of WNV and the elements that comprise a protective host immune response. Although no therapy is currently approved for use in humans, several strategies are being pursued to develop effective prophylaxis and treatments. This review describes the current state of knowledge on epidemiology, clinical presentation, pathogenesis, and immunobiology of WNV infection, and highlights progress toward an effective therapy.
| S-EPMC2759769 | biostudies-literature