Distinguishing West Nile virus infection using a recombinant envelope protein with mutations in the conserved fusion-loop

Abstract

BackgroundWest Nile Virus (WNV) is an emerging mosquito-transmitted flavivirus that continues to spread and cause disease throughout several parts of the world, including Europe and the Americas. Specific diagnosis of WNV infections using current serological testing is complicated by the high degree of cross-reactivity between antibodies against other clinically relevant flaviviruses, including dengue, tick-borne encephalitis (TBEV), Japanese encephalitis (JEV), and yellow fever (YFV) viruses. Cross-reactivity is particularly problematic in areas where different flaviviruses co-circulate or in populations that have been immunized with vaccines against TBEV, JEV, or YFV. The majority of cross-reactive antibodies against the immunodominant flavivirus envelope (E) protein target a conserved epitope in the fusion loop at the distal end of domain II.MethodsWe tested a loss-of-function bacterially expressed recombinant WNV E protein containing mutations in the fusion loop and an adjacent loop domain as a possible diagnostic reagent. By comparing the binding of sera from humans infected with WNV or other flaviviruses to the wild type and the mutant E proteins, we analyzed the potential of this technology to specifically detect WNV antibodies.ResultsUsing this system, we could reliably determine WNV infections. Antibodies from WNV-infected individuals bound equally well to the wild type and the mutant protein. In contrast, sera from persons infected with other flaviviruses showed significantly decreased binding to the mutant protein. By calculating the mean differences between antibody signals detected using the wild type and the mutant proteins, a value could be assigned for each of the flaviviruses, which distinguished their pattern of reactivity.ConclusionsRecombinant mutant E proteins can be used to discriminate infections with WNV from those with other flaviviruses. The data have important implications for the development of improved, specific serological assays for the detection of WNV antibodies in regions where other flaviviruses co-circulate or in populations that are immunized with other flavivirus vaccines.

Neutralizing antibodies against West Nile virus identified directly from human B cells by single-cell analysis and next generation sequencing

“…These results have limited power due to the relatively small sample size of four post-convalescent subjects and seven recently infected subjects. However, they are supported by previous studies where WNV-specific serum antibodies were detected in subjects up to 8 years post-infection 26,31,32,36,38 . Persistence of serum antibodies specific to other viral infections and vaccines (e.g.…”

“…In contrast, only one NAb was also positive in the immunoblot assay. These results may indicate that three of the discovered NAbs recognize conformation dependent epitopes on the WNV E protein that could have been denatured during the immunoblot assay 36 . Similarly, antibodies to the related Flavivirus family member dengue virus, a quaternary epitope was required for neutralization of the virus 9 , indicating that denaturing of the higher order antibody protein structure could result in a loss of function.…”

Modified mRNA Vaccines Protect against Zika Virus Infection

“…Because of the versatility of the platform, we engineered additional mRNA LNP vaccines with mutations in the conserved E-DII-FL, which abolish reactivity of monoclonal and polyclonal antibodies targeting this region (Chabierski et al, 2014;Crill et al, 2012;Hughes et al, 2012;Vogt et al, 2011). The FL mutant mRNA vaccines induced neutralizing antibody responses in non-pregnant female BALB/c mice that protected against virus dissemination to the uterus and brain.…”

“…As such, infection with ZIKV or vaccination with ZIKV structural proteins could induce cross-reactive antibodies, which might enhance DENV infection and disease through ADE (Morens, 1994;Stettler et al, 2016). To minimize this possibility, we generated modified mRNA vaccines by engineering four mutations (T76R, Q77E, W101R, and L107R) in or near the E-DII-FL (prM-E-FL) that abolish antibody reactivity of FL-specific antibodies (Chabierski et al, 2014;Crill et al, 2012;Oliphant et al, 2007). We also generated a separate series of mRNA LNPs by replacing the IgE leader sequence with one from Japanese encephalitis virus (JEV sig ), a feature included in other flavivirus prM-E DNA vaccines to increase the efficiency of host signalase cleavage (Davis et al, 2001;Dowd et al, 2016b), and by further optimizing codon usage ( Fig 3A).…”

West Nile Virus Seroprevalence, Connecticut, USA, 2000–2014

“…For all participants, we assessed previous exposure to WNV by immunoblot for WNV envelope protein ( 6 ). Seroconversion to WNV was distinguished from cross-reactivity to other flaviviruses by rescreening all positive serum against a recombinant WNV-specific mutant envelope protein that lacks the conserved cross-reactive fusion loop epitope ( 7 ). …”