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Therapeutic Promise of a Human Antibody Against West Nile Virus

 

By: Carol A. Rouzer, VICB Communications
Published: December 6, 2018

 

A new highly potent neutralizing monoclonal antibody protects against West Nile virus infection in vivo.

 

Flaviviruses are single-stranded RNA-containing, enveloped viruses that are primarily transmitted by insects (Figure 1). They include important human pathogens such as dengue virus, Zika virus, yellow fever virus, Japanese encephalitis virus, and west Nile virus (WNV). The most effective means to control these viral illnesses is through the development of vaccines; however, this has only been achieved in the case of yellow fever. An alternative approach is to discover new antiviral therapeutics, including the potential use of neutralizing antibodies that can be administered to exposed individuals or those at risk of exposure. To that end, Vanderbilt Institute of Chemical Biology member James Crowe and his collaborator Theodore Pierson (Viral Pathogenesis Section NIAID) announce the discovery of a highly potent human neutralizing monoclonal antibody (NmAb) against WNV that is protective in murine models of WNV infection [L. Goo, et al. (2018) Nat. Microbiol. published November 19, DOI: 10.1038/s41564-018-0283-7].

 

FIGURE 1. Three dimensional reconstruction (left) and a slice through the center (right) of the Japanese encephalitis virus, a representative flavivirus. Heterodimers of the E and M proteins are shown in a single color. The E and M proteins coat the surface membrane bilayer, which in turn, surrounds the ribonucleoprotein core. Figure reproduced under the Creative Commons Attribution 4.0 International License 4.0 from X. Wang, et al. Nat. Comm., (2017) 8, 14.

 

 

The envelope of flaviviruses comprises a lipid bilayer membrane coated with two proteins, E and M. During viral assembly, the E protein is transported to the membrane surface in complex with prM, which serves as a chaperone. In immature viruses, these proteins form heterotrimeric spikes, but over time, prM is cleaved to yield M, which then combines with E to form E:M:M:E dimeric structures in the membrane (Figures 2 and 3). Composed of DI, DII, and DIII domains, in addition to a helical stem and two transmembrane helices, E is the most surface-exposed of the two proteins. Therefore, it is the primary target of antibodies that neutralize the flaviviruses, including WNV. For example, the previously reported E16 humanized murine NmAb, which has some therapeutic value, recognizes the lateral ridge of the DIII domain. However, for many NmAbs, the epitope is formed from a complex of multiple E proteins, and many recognize immature viruses expressing the prM-containing spikes better than they recognize mature viruses, possibly because the presence of prM helps to expose the targeted epitope.

 


FIGURE 2. Ribbon diagram of an E:M:M:E tetramer. E protein DI, DII, and DIII domains, the E protein transmembrane domain, and the M protein are colored in red, yellow, blue, cyan, and orange, respectively. Figure reproduced under the Creative Commons Attribution 4.0 International License 4.0 from X. Wang, et al. Nat. Comm., (2017) 8, 14.

 

 

     

FIGURE 3. Comparative structures of three flaviviruses, the Japanese encephalitis virus (JEV), the Zika virus (ZIKV), and Dengue virus 2 (DENV2). The parallelogram in each case highlights a single E:M:M:E heterodimer, which is shown enlarged below the respective structure. Colors indicate the distance from the center in nm according to the scale. Figure reproduced under the Creative Commons Attribution 4.0 International License 4.0 from X. Wang, et al. Nat. Comm., (2017) 8, 14.

 

 

In a search for more potent NmAbs, the researchers evaluated serum samples from 13 human donors who had previously been infected with WNV for the presence of potent neutralizing Abs. They found such Abs in 9 of the serum samples using a cell-based assay. They then isolated B cells from the blood of the 3 donors whose serum exhibited the highest neutralizing activity and used these cells to generate Ab-expressing hybridomas. This effort yielded 10 hybridomas secreting mAbs against WNV. Of these, 3 possessed no neutralizing activity, 3 exhibited modest activity, and 4 strongly neutralized the virus. Concentration-dependence studies of the four most potent NmAbs revealed that only one, WNV-86, completely inhibited the virus at the concentrations tested. The concentration of WNV-86 that inhibited the virus by 50% (IC50) was 2 ng/mL, indicating very high potency. Further studies demonstrated that, unlike all the other NmAbs tested, WNV-86 was more active against mature virus (lacking prM) than immature virus. Furthermore, WNV-86 did not potently block binding of viruses to the surface of target cells, indicating that it neutralized WNV at a step subsequent to attachment.
     

The investigators next passaged WNV three times in Vero cells in the presence of WNV-86. At the end of this procedure, the viruses they recovered were resistant to neutralization by the NmAb. Sequencing of the viral genomes indicated that a single mutation in the gene encoding the E protein, resulting in a T64N substitution, was responsible for resistance. This mutation introduced a new glycosylation site, and the researchers confirmed the presence of a glycan at this position in the E protein of the resistant viruses. However, they subsequently found that other mutations at the T64 site could impart resistance against WNV-86 without the addition of a new glycosyl group.
     

Additional serial passages in the presence of WNV-86, starting with a partially resistant T64Q mutant virus, resulted in the recovery of new virus strains that were totally resistant to the NmAb. These viruses encoded an E protein bearing both the T64Q mutation and a T208K mutation. Further work demonstrated that neither of these mutations alone conferred total resistance to WNV-86, but the combination was highly effective.
     

Both T64 and T208 are located in the DII domain of the E protein, suggesting that the epitope for WNV-86 should be located there. The investigators tested this hypothesis by evaluating the sensitivity of 40 viruses bearing other mutations in the E protein to WNV-86. In total, these viruses encoded 56 different mutations, in some cases as double or triple mutations. The results indicated that 29 of the mutations had no effect on WNV-86 sensitivity, while 19 had modest effects, and 6 markedly reduced sensitivity. All 6 of the mutations leading to major reductions in WNV-86 potency were located in the DII region, and most were in the region between T64 and T208 (Figure 4).

 

 

FIGURE 4. Crystal structure of the E protein from WNV with each domain labeled. Gray spheres indicate the locations of residues that were mutated in the search for the WNV-86 epitope. Colored spheres indicate the locations of residues that, when mutated, resulted in a >4-fold loss of neutralization potency for WNV-86. The positions of T64 and T208 are also indicated. Reproduced by permission from Sringer/Nature from L. Goo, et al. (2018) Nat. Microbiol. published November 19, DOI: 10.1038/s41564-018-0283-7. Copyright 2018, Springer Nature.

 

 

To test the ability of WNV-86 to neutralize WNV in vivo, the researchers inoculated mice with the virus and at the same time gave them a single injection of the antibody. WNV-86 provided complete protection of the mice, whereas the second most potent NmAb they isolated, WNV-10, protected only 50% of the infected animals. The latter result was unexpected because WNV-10 was as potent as E16 in vitro, and E16 was fully protective in the in vivo model. In a final study, the researchers also tested the ability of a LALA variant of WNV-86 to protect mice against WNV infection. The LALA variant is unable to interact with Fc receptors, so it cannot stimulate Fc-dependent immune responses in the animal. The LALA variant was only slightly less potent than WNV-86, suggesting that its primary neutralizing potency results from direct interaction with the virus.
     

The results demonstrate the discovery of a highly potent human NmAb against the mature form of WNV. This NmAb is fully protective in a mouse model of viral infection. Further work will better define its epitope and explore its potential therapeutic use in humans.

 

 

 

View Nat. Microbiol. article: A protective human monoclonal antibody targeting the West Nile virus E protein preferentially recognizes mature virions

 

 

 

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