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Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays
Published on: April 4, 2011
Molecular characterization of epitopes on the measles virus hemagglutinin protein
A Hu1, H Sheshberadaran, E Norrby
1Department of Virology, Karolinska Institute, School of Medicine, Stockholm, Sweden.
Abstract:
The measles virus (MV) hemagglutinin (H) gene nucleotide sequences of the LEC-WI strain and 11 branched sequential neutralization escape variants of the strain derived by selection with five monoclonal antibodies (Mabs) were determined by direct analysis of amplified polymerase chain reaction products. The parental LEC-WI strain isolated from a patient with subacute sclerosing panencephalitis exhibited H gene sequence characteristics similar to other persistent virus strains derived from brain materials. Mostly single-point H gene mutations, coding for single amino acid substitutions in the H protein, were found to provide explanations for the resistance to the individual Mabs. Resistance to Mabs 16-CD11 and I-41 resulted from changes of Gly-491 to Asp (or Val) and Phe-552 to Val, respectively. One variant (B89) selected by Mab 16-CD11 had a mutation introduced by a single nucleotide deletion and subsequent nucleotide insertion, which caused a shift in the open reading frame. The epitope of Mab I-29 was assigned to Ser-313 or Gly-314, which were changed to Leu and Arg, respectively. The variants subjected to the Mab I-44 selection exhibited change of Ser-189 to Pro. Radioimmunoprecipitation assay and endoglycosidase H (Endo H) treatment revealed that this change destroyed a potential N-linked glycosylation site, indicating that the carbohydrate chain participates in formation of the epitope or indirectly influences its properties. Resistance to Mab 16-DE6 involved three specific amino acid changes in three different places, Gly-211 to Ser, Gly-388 to Asp, and Ser-532 to Phe or Arg-533 to Gly, reflecting the occurrence of a conformational epitope. In conclusion, this study identifies the precise positions of several critical sites on the MV H protein which react with neutralizing antibodies.
Insights
This study maps measles virus (MV) hemagglutinin (H) gene mutations conferring resistance to neutralizing antibodies. Identifying specific amino acid changes reveals critical sites on the H protein for antibody binding and neutralization escape.
Area of Science:
- Virology
- Immunology
- Molecular Biology
Background:
- Measles virus (MV) persistence, particularly in subacute sclerosing panencephalitis, is a significant concern.
- Understanding MV hemagglutinin (H) protein interactions with neutralizing antibodies is crucial for vaccine development and therapeutic strategies.
Purpose of the Study:
- To determine the nucleotide sequences of the MV H gene in the LEC-WI strain and its neutralization escape variants.
- To identify specific mutations responsible for resistance to monoclonal antibodies (Mabs).
- To map the epitopes targeted by neutralizing Mabs on the MV H protein.
Main Methods:
- Polymerase chain reaction (PCR) amplification and direct sequencing of MV H gene.
- Selection of neutralization escape variants using five different monoclonal antibodies (Mabs).
- Analysis of nucleotide and amino acid substitutions, including frameshift mutations.
- Radioimmunoprecipitation assay and endoglycosidase H (Endo H) treatment to assess glycosylation impacts.
Main Results:
- Single-point mutations in the H gene were primarily responsible for resistance to individual Mabs.
- Specific amino acid substitutions were identified for resistance to Mabs 16-CD11, I-41, I-29, I-44, and 16-DE6.
- One variant exhibited a frameshift mutation due to nucleotide deletion and insertion.
- A change at Ser-189 to Pro destroyed a potential N-linked glycosylation site, impacting epitope properties.
- Resistance to Mab 16-DE6 involved multiple amino acid changes, suggesting a conformational epitope.
Conclusions:
- Precise locations of critical neutralizing antibody epitopes on the measles virus H protein have been identified.
- Understanding these mutation-driven escape mechanisms is vital for predicting viral evolution and designing effective immunotherapies.
- The study provides a detailed molecular basis for measles virus neutralization escape.
