Epitope Dampening Monotypic Measles Virus Hemagglutinin Glycoprotein Results in Resistance to Cocktail of Monoclonal Antibodies

Abstract

The measles virus (MV) is serologically monotypic. Life-long immunity is conferred by a single attack of measles or following vaccination with the MV vaccine. This is contrary to viruses such as influenza, which readily develop resistance to the immune system and recur. A better understanding of factors that restrain MV to one serotype may allow us to predict if MV will remain monotypic in the future and influence the design of novel MV vaccines and therapeutics. MV hemagglutinin (H) glycoprotein, binds to cellular receptors and subsequently triggers the fusion (F) glycoprotein to fuse the virus into the cell. H is also the major target for neutralizing antibodies. To explore if MV remains monotypic due to a lack of plasticity of the H glycoprotein, we used the technology of Immune Dampening to generate viruses with rationally designed N-linked glycosylation sites and mutations in different epitopes and screened for viruses that escaped monoclonal antibodies (mAbs). We then combined rationally designed mutations with naturally selected mutations to generate a virus resistant to a cocktail of neutralizing mAbs targeting four different epitopes simultaneously. Two epitopes were protected by engineered N-linked glycosylations and two epitopes acquired escape mutations via two consecutive rounds of artificial selection in the presence of mAbs. Three of these epitopes were targeted by mAbs known to interfere with receptor binding. Results demonstrate that, within the epitopes analyzed, H can tolerate mutations in different residues and additional N-linked glycosylations to escape mAbs. Understanding the degree of change that H can tolerate is important as we follow its evolution in a host whose immunity is vaccine induced by genotype A strains instead of multiple genetically distinct wild-type MVs.

Mutational Analysis of Measles Virus Suggests Constraints on Antigenic Variation of the Glycoproteins

“…However, this antigenic “drift” does not appreciably occur in the MeV F and H glycoproteins. The molecular basis for the lack of emergent antigenically distinct strains of MeV, relative to other related negative sense RNA virus like influenza A virus, is currently unclear (Fayolle et al, 1999; Lech et al, 2013; Xu et al, 2013). Given that MeV does not undergo major antigenic changes, it is possible that the glycoproteins of MeV are under a rigid, but as of yet undefined, constraint that prevents this evolution from occurring.…”

Antibody neutralization of retargeted measles viruses

“…Residue R533 (green) forms part of the 16DE6 epitope and the R533A mutation in H AALS . MAb epitopes for I-41, 16DE6, cl48, I-44, I-29, 16CD11 have been characterized in previous papers [58, 62]. Epitopes for 20H6 and BH97 were identified by selecting MV-Hedm in the presence of 20H6 or BH97 and by rational design (unpublished data).…”

“…The H protein in MV-H82-EGFR.scFv is engineered to escape completely or partially mAbs tested in this study by encoding mAb escape mutations and rationally designed N-linked glycosylations sites within or adjacent to mAb epitopes. A glycosylation at these sites would have the potential to cloak the epitope from antibody recognition [62], however we have not determined if the carbohydrates are shielding the epitopes or the potential N-linked glycosylation site engineered into the virus is destroying the epitope. The oncolytic potential of MV-H82-EGFR.scFv has not been tested.…”

“…MV-H82-EGFR, in addition to escaping mAbs targeting the receptor binding surface, escaped mAbs cl48/I-44 targeting epitope E3 [62], I-29 targeting epitope E4 [62] also known as epitope vi [69] and partially escaped 16CD11 targeting epitope II [69] (figure 4). Out of the human sera tested in figure 5, two of the individuals had a 4-fold lower NT for MV-H82-EGFR.scFv than MV-Hedm (data not shown).…”

Antigenic Drift Defines a New D4 Subgenotype of Measles Virus

“…The following anti-MeV-H protein MAbs used for neutralization were previously described: BH129, BH047, BH059 (69), BH101, BH026 (70), BH141 (71), and BH097 (72). Protein G affinity-purified MAbs were used, except for BH141 and BH059.…”