Proteins were expressed in transiently transfected (HEK) 293T cells (ATCC CRL-1573)
Proteins were expressed in transiently transfected (HEK) 293T cells (ATCC CRL-1573). Gn-Gc lattice. Genome sequence analysis confirmed that this region of the RVFV Gn-Gc assembly was under selective pressure and constituted a site of vulnerability around the virion surface. These data provide a blueprint for the rational design of immunotherapeutics and vaccines capable of preventing RVFV contamination and a model for understanding Ab-mediated neutralization of bunyaviruses more generally. Keywords: phlebovirus, Rift Valley fever computer virus, antibody, Oleanolic Acid (Caryophyllin) structure, bunyavirus, virus-host interactions, immune response, vaccine, antiviral, neutralization Graphical Abstract Open in a separate window Highlights ? The Gn glycoprotein of Rift Valley fever computer virus elicits potent neutralizing antibodies ? Derived a class of monoclonal antibodies that protects in an animal model ? A distinct region on RVFV Gn constitutes a key site of vulnerability ? Antibodies are predicted to prevent exposure of viral fusion loops Allen et?al. reveal a molecular basis of antibody-mediated neutralization of Rift Valley fever computer virus, an important human and animal pathogen. They isolate and demonstrate the protective efficacy of a monoclonal antibody in a murine model of computer virus infection, providing a blueprint for rational therapeutic and vaccine design. Introduction First reported in 1931 (Daubney et?al., 1931), Rift Valley fever computer virus (RVFV) is an arbovirus endemic to Africa and the Arabian peninsula that causes recurrent epidemics and epizootics. RVFV is usually of both agricultural and biomedical importance, as contamination of livestock results in high incidences of neonatal mortality and zoonosis; human disease ranges from moderate self-limiting febrile illness to severe disease characterized by hemorrhagic diatheses, encephalitis, and ocular pathologies (Al-Hazmi et?al., 2003, Mohamed et?al., 2010, Sow et?al., 2014). Human populations throughout East Africa are at high risk for RVFV contamination, with seroprevalence reported to exceed 8% in communities located near water reservoirs that support mosquito populations (Pourrut et?al., 2010). No licensed antivirals or vaccines for RVFV are currently available, although a number of vaccine candidates are in development (Dungu et?al., 2018, Faburay et?al., 2017). The genetically diverse group of viruses within the genus protective efficacy of our mAb, RV-Gn1, in animals highlights the potential power of RVFV-specific nAbs as prophylactics. By analogy with mAb cocktails developed against EBOV GP (Qiu et?al., 2012), we anticipate that this identification of nAbs specific to other spatially distinct epitopes on the surface of the RVFV Gn-Gc complex assembly will be an important consideration for IL9 antibody the development of synergetic, non-competing combinations of anti-RVFV mAbs. STARMethods Key Resources Table (National Research Council, 2011). Four male 8C10?week aged New Zealand White rabbits were used in immunization studies. Mice All murine procedures with animals were undertaken according to the United Kingdom Animals (Scientific Procedures) Act 1986. These studies were approved by the ethical review process of Public Health England, Porton Down, UK, and by the Home Office, UK via Establishment License 70/1707 and project license P82D9CB4B. A set of humane end points based on clinical manifestation of disease were defined in the protocol of the project license. Female BALB/c 6-8?week aged mice were used in these experiments. Cell lines HEK293F female embryonic kidney cells were cultured in Freestyle 293F expression media (GIBCO, Thermofisher). HEK293T female human embryonic kidney cells were cultured in DMEM supplemented with 10% FCS, non-essential amino acids and L-glutamine. (GIBCO, Thermofisher). Female vero cells were cultured Oleanolic Acid (Caryophyllin) in DMEM with 10% FCS. Hybridoma cell lines were generated commercially by Epitomics. Hybridoma cell cultures were produced in hybridoma SF media (Life Technologies). Cell lines were maintained in a humidified incubator at 37C, supplied with 5%C8% CO2. HEK293F cells were agitated at 135?rpm. Cell lines were not authenticated following purchase. Method Details Immunization of rabbits with recombinant RVFV Gn Four male 8C10?week aged New Zealand White rabbits were primed (intramuscularly) with the full-length RVFV Gn ectodomain (120?g) adjuvanted with Adjuplex? (Sigma Aldrich) (Wegmann et?al., 2015) at a ratio of 1 1:5 adjuvant to immunogen in sterile PBS (1?mL total volume). Following immunization, a further two boosts were conducted at four week intervals. The final boost for rabbit 8315 was at week 16 and was performed intravenously seven days before Ab Oleanolic Acid (Caryophyllin) isolation. Sera were prepared from blood collected prior to immunization and seven days following each immunization/boost. Murine RVFV contamination model Female BALB/c 6-8?week aged mice were housed in groups of three. Groups of six mice (randomly assigned, two boxes per group) were treated intravenously with RV-Gn1 (200?g.