Abstract
BackgroundThe hemagglutinin-neuraminidase (HN) protein is the major antigenic determinant of the Mumps virus (MuV) and plays an important role in the viral infectious cycle through its hemagglutination/hemadsorption (HA/HD) and neuraminidase (NA) activities. Objective: analyze the biological and immunological properties of a polypeptide derived from a highly conserved region of the HN ectodomain. Methods: a highly conserved region of the HN gene among several MuV genotypes was chosen to be cloned in a eukaryotic expression vector. The pcDNAHN176-construct was transfected into Vero cells and RNA expression was detected by RT-PCR, while the corresponding polypeptide was detected by immunofluorescence and immunochemistry techniques. The HD and NA activities were also measured. The immunogenic properties of the construct were evaluated using two systems: rabbit immunization to obtain sera for detection of the HN protein and neutralization of MuV infection, and hamster immunization to evaluate protection against MuV infection.ResultsA 567 nucleotide region from the HN gene was amplified and cloned into the plasmid pcDNA3.1. Vero cells transfected with the construct expressed a polypeptide that was recognized by a MuV-hyperimmune serum. The construct-transfected cells showed HD and NA activities. Sera from immunized rabbits in vitro neutralized two different MuV genotypes and also detected both the HN protein and the HN176 polypeptide by western blot. Hamsters immunized with the pcDNAHN176-construct and challenged with MuV showed a mild viral infection in comparison to non-immunized animals, and Th1 and Th2 cytokines were detected in them.ConclusionsThe pcDNAHN176-construct was capable of expressing a polypeptide in Vero cells that was identified by a hyperimmune serum anti Mumps virus, and these cells showed the HD and NA activities of the complete MuV HN protein. The construct also elicited a specific immune response against MuV infection in hamsters.
Highlights
The hemagglutinin-neuraminidase (HN) protein is the major antigenic determinant of the Mumps virus (MuV) and plays an important role in the viral infectious cycle through its hemagglutination/hemadsorption (HA/HD) and neuraminidase (NA) activities
Characterization of the pcDNAHN176-construct The PCR amplification of the pcDNAHN176-construct using the set of HN primers initially designed produced a 580-bp fragment, which corresponded to the expected size of the HN insert (Figure 1A, lane 3)
Sequencing of the HN gene fragment indicated that the insert could encode for a 176 amino acid polypeptide that shared a 97.3% similarity with the Urabe HN protein
Summary
The hemagglutinin-neuraminidase (HN) protein is the major antigenic determinant of the Mumps virus (MuV) and plays an important role in the viral infectious cycle through its hemagglutination/hemadsorption (HA/HD) and neuraminidase (NA) activities. Objective: analyze the biological and immunological properties of a polypeptide derived from a highly conserved region of the HN ectodomain. Two important mumps outbreaks were recently reported, one in 2005 in the UK, and the other in 2006 in the USA. In both cases, the G MuV genotype was identified, even though both countries have been using the mumps Jeryl Lynn vaccine, which has been identified as an A genotype [5,6,22]. The genome encodes for three nucleocapsid-associated proteins: an RNA binding protein (N), a phosphoprotein (P) and a large polymerase protein (L), four membrane proteins, an unglycosylated inner membrane or matrix protein (M) and three glycosylated envelope proteins, the fusion protein (F), the hemagglutinin-neuraminidase (HN) protein and the small hydrophobic protein (SH) [23]
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