Maja Šantak

Antigenic differences between vaccine and circulating wild-type mumps viruses decreases neutralization capacity of vaccine-induced antibodies

A recent resurgence of mumps in doubly vaccinated cohorts has been observed, identifying genotype G as the current predominant genotype. In this study, the neutralization efficacy of guinea pig sera immunized with three vaccine viruses: L-Zagreb, Urabe AM9 and JL5, was tested against seven mumps viruses: three vaccine strains and four wild-type strains (two of genotype G, one of genotype C, one of genotype D) isolated during 1998-2011. All sera neutralized all viruses although at different levels. The neutralization efficiency of sera decreases several fold by temporal order of virus isolation. Therefore, we concluded that gradual evolution of mumps viruses, rather than belonging to a certain genotype, results in an antigenic divergence from the vaccine strains that decrease the neutralization capacity of vaccine-induced antibodies. Moreover, the amino-acid sequence alignment revealed three new potentially relevant regions for escape from neutralization, i.e. 113-130, 375-403 and 440-443.

Concentration and purification of rubella virus using monolithic chromatographic support.

The production of economically acceptable viral vaccines of high quality requires simple and efficient methods for purification and concentration of viral particles. Ion-exchange chromatography (IEC) has become one of commonly used methods for large-scale downstream purification of viruses. Viruses possess different biological and/or biochemical properties and therefore IEC conditions must be established specifically for each virus. Live attenuated rubella virus vaccines have been manufactured and successfully used widely to protect people from rubella and congenital rubella syndrome for almost 40 years. The aim of this study was to search for an efficient method for concentration and purification of rubella virus using IEC. The selected operating conditions using quaternary amine monolithic supports enabled highly efficient binding, purification and concentration of rubella virus from complex biological suspension without additional procedures. Eluted viral particles maintained their infectivity and viral recovery was almost 100%. At the same time, viral preparation was successfully depleted from host cell protein and DNA. This work indicates the possibility of using monoliths to improve the rubella virus yields in productions where high virus titers during cultivation can hardly be achieved.

Comparisons of mumps virus potency estimates obtained by 50% cell culture infective dose assay and plaque assay

The two most commonly used methods for the determination of a virus potency are plaque assay and 50% cell culture infective dose (CCID(50)) assay, both based on cytopathic effect observation. We compared the potency estimates obtained by plaque and CCID(50) assays for nine mumps virus strains that produce different cytopathic effects in Vero cells. The ratios of CCID(50) and plaque assay quantification results differed for different strains and were in a range of 0.66-10, indicating that quantification results for some mumps virus strains are almost identical regardless of whether CCID(50) or plaque method is used, while the potency estimates of other strains strongly depend on the choice of the assay.

A comparison of complete untranslated regions of measles virus genomes derived from wild-type viruses and SSPE brain tissues

We compared complete untranslated regions (UTRs) of two subacute sclerosing panencephalitis (SSPE) measles virus (MV) strains and two wild-type (wt) MV strains, all belonging to the same genotype (D6). In comparison to wt MVs of the same genotype, base changes were identified in the two SSPE measles virus strains at 27 and 33 noncoding positions, respectively. Majority of these residues are unique for each of the SSPE virus sequences in comparison to all other reported measles virus strain sequences. The location of some of these changes indicates that they may modify cis-acting regulatory sequences including gene-end signal of the P gene, H/L gene junction and Kozak consensus element of the L gene. Further, within the long UTR between M and F genes, deletions and insertions were identified. Thus, our study could be significant for additional investigation using reverse genetics and recombinant viruses, of possible influence of mutations in UTRs on establishment and maintenance of chronic progressive CNS disease caused by MV persistence.

Clinical and molecular characterization of a parechovirus type 1 outbreak in neonates in Croatia.

During July 2009 an outbreak in neonates represented with gastrointestinal and respiratory symptoms was observed at the Neonatal Postintensive Care Unit, Clinical Hospital Center, Zagreb. Human parechovirus type 1 (HPeV1) was isolated from seven patients, one of whom was asymptomatic. All but one were premature neonates with serious underlying conditions, and all recovered fully after several days. In order to characterize the HPeV1s, sequencing of the VP1/2A region was conducted on six isolates from the outbreak and four isolates detected in Croatia in 2008 and 2007. The analysis of sequence similarity showed that the nucleotide identity between the prototype strain (Harris) and HPeV1 isolated in Croatia was 76.5–77.5%. Croatian strains from 2007 and 2009 clustered together with strains from the Netherlands and Germany detected in 2003 and 2006, respectively, while strains from 2008 clustered with the strain from Finland detected in 2000. Change of the dominant strains each year may suggest antigenic variation as a result of viral response to specific immunity of the target population. J. Med. Virol. 83:137–141, 2011.

Variability of hemagglutinin-neuraminidase and nucleocapsid protein of vaccine and wild-type mumps virus strains.

The mumps virus (MuV) molecular evolution is characterized by the co-circulation of numerous distinct strains. Standardized phylogenetic analyses based on the nucleotide sequences of the SH gene are important for mumps surveillance, but lack the information regarding antigenic properties. So far, the location of antigenic epitopes has been determined for two MuV proteins, the hemagglutinin-neuraminidase (HN) and the nucleocapsid (N) protein. We performed multiple sequence comparisons of putative HN and N protein sequences in order to describe their diversity and plasticity, and to determine the level of similarity between vaccine and wild-type strains. The results of full-length HN or N protein phylogeny showed that MuV strains form a number of differing clades which are in concordance with grouping obtained by standard MuV genotyping. When vaccine strains are compared to all wild-type strains, the highest mean percentage of amino acid differences in both HN and N protein analysis was found for Jeryl Lynn 5 and Jeryl Lynn 2 strains while the lowest value was obtained for Leningrad-3 and L-Zagreb strains. When only 3 antigenic regions of the HN protein, comprising 45 amino acids in total, were investigated, the diversity is considerably diminished: 51.5% of all putative HN proteins show identical sequences (including those of vaccine strains L-Zagreb, Leningrad-3, Hoshino and Urabe). Another 26.5% proteins (including Miyahara vaccine strain) differ in only one amino acid, while the others differ in two to five amino acids from the most common sequence. Jeryl Lynn 2 and Jeryl Lynn 5 strains differ in four amino acids each. N protein antigenic sites have been mapped within its hypervariable C-terminus. Our results indicate that there might be genotype-specific amino acids residing in this antigenic region. The results of our study present the background information for investigations of MuV heterogeneity and antigenic diversity.

Detection of genetic lineages of human metapneumovirus in Croatia during the winter season 2005/2006.

Human metapneumovirus (HMPV) is an important respiratory pathogen, especially among young children. The genetic characteristics of HMPV circulating in Croatia have not been studied so far. The aim of this study was to determine the incidence of HMPV infection in hospitalized children with acute respiratory tract infection (ARTI) in the season 2005/2006 in Croatia, as well as to perform the genotypic analysis of detected HMPV strains. From December 1 to March 31 nasopharyngeal secretions (NPSs) were collected from 402 inpatients up to 5 years of age with ARTI. NPSs were tested by real‐time RT‐PCR assay targeting the nucleoprotein (N) gene of HMPV. HMPV infection was detected in 33 patients (8.2%). To perform the phylogenetic study, partial nucleotide sequences were obtained for HMPV fusion (F) gene of 30 HMPV positive samples. Phylogenetic analysis showed the circulation of two main genetic lineages (A and B), with B lineages being prevalent. It also showed the existence of two sublineages within the group B (B1 and B2) and three subclusters within lineage A (A1, A2a and A2b). Further molecular analysis revealed point mutations in HMPV strains of sublineage B1. J. Med. Virol. 80: 1282–1287, 2008.

Influence of charge ratio of liposome/DNA complexes on their size after extrusion and transfection efficiency

Background Physicochemical characteristics of liposome/DNA complexes influence transfection efficiency and affect each other in a very intricate way. The result of this is discrepancies in conclusions drawn about the individual influence of each one. Methods Aiming to elucidate the influence of liposome/DNA charge ratio and size on transfection efficiency and on each other, we used liposome/DNA complexes with charge ratio (+/−) in the range of 1–50 and extruded through membranes of 400, 200, and 100 nm. Plasmid DNA encoding green fluorescent protein was used to measure transfection efficiency by flow cytometry. Sizes of liposome/DNA complexes were measured by dynamic light scattering. Results Liposome size was reduced after extrusion but this was mainly driven by the charge ratio and not by the size of the membrane pores. Reduction of complex size at each charge ratio positively correlated with transfection efficiency. When the size of the complexes was approximately constant, increasing the charge ratio was found to promote transfection efficiency. Cationic lipid N-(1-(2,3-dioleoyloxy)propyl)N,N,N trimethylammonium chloride was used for modulation of positive charge and a cytotoxicity test showed that increasing its amount increases cytotoxicity. Conclusion It can be concluded that charge ratio dictates the size of the complex whereas overall size reduction and higher charge ratios promote transfection efficiency in vitro.

Genetic heterogeneity of L-Zagreb mumps virus vaccine strain

BackgroundThe most often used mumps vaccine strains Jeryl Lynn (JL), RIT4385, Urabe-AM9, L-Zagreb and L-3 differ in immunogenicity and reactogenicity. Previous analyses showed that JL, Urabe-AM9 and L-3 are genetically heterogeneous.ResultsWe identified the heterogeneity of L-Zagreb throughout the entire genome. Two major variants were defined: variant A being identical to the consensus sequence of viral seeds and vaccine(s) and variant B which differs from variant A in three nucleotide positions. The difference between viral variants in L-Zagreb strain is insufficient for distinct viral strains to be defined. We demonstrated that proportion of variants in L-Zagreb viral population depends on cell substrate used for viral replication in vitro and in vivo.ConclusionL-Zagreb strain should be considered as a single strain composed of at least two variant viral genomes.

Identification of conformational neutralization sites on the fusion protein of mumps virus.

In spite of the success of the mumps vaccination, recent mumps outbreaks have been reported even among individuals with a history of mumps vaccination. For a better understanding of why the vaccination failed in cases of vaccinees who fell ill during recent mumps outbreaks, the immunological events during infection and/or vaccination should be better defined. In the work presented here we sought to identify new neutralization sites on the mumps virus surface glycoproteins. By using anti-mumps mAbs, three amino acid positions at residues 221, 323 and 373 in the F protein of mumps virus were shown to be located in at least two conformational neutralization epitopes. mAbs that specifically target these sites effectively neutralized mumps virus in vitro. The newly acquired glycosylation site at position 373 or loss of the existing one at position 323 was identified as the mechanism behind the escape from the specific mAbs. Based on the findings of this study, we suggest that the influence of the antigenic structure of the F protein should not be ignored in a thorough investigation of the underlying mechanism of the mumps vaccine failure or when making a strategy for development of a new vaccine.