Wild-type Mumps Virus Research Articles

Booster doses of an inactivated F genotype mumps vaccine enhance immunogenicity in mice

The mumps virus (MuV) causes a highly contagious human disease characterized by swelling of the parotid glands. Although the administration of an attenuated Jeryl Lynn (JL) MuV vaccine shows efficacy in reducing the incidence of MuV infection, sporadic mumps outbreaks still occur in vaccinated populations. We have previously established that an inactivated F genotype mumps vaccine has a higher neutralizing antibody titer against diverse circulating mumps viruses in mice. Here, we aimed to develop a vaccination strategy to enhance the immune response for MuV and assess the effects of heterologous vaccination compared with homologous approaches. We administered an inactivated F genotype mumps vaccine booster following a homologous prime-boost regime and compared its efficacy with three doses of homologous JL vaccine in mice. We demonstrated robust stimulation of neutralizing antibodies and cellular immune response of interferon-γ-secreting cytotoxic T cells following administration of an inactivated F genotype mumps vaccine booster after a homologous prime-boost regime with JL. Compared with the homologous prime-boost regime, this heterologous prime-boost regime showed protective efficacy against the F genotype of MuV. These findings suggest that the heterologous vaccination strategy based on the administration of an inactivated F genotype mumps vaccine provides more effective cross-protection against circulating wild-type mumps viruses than homologous vaccination.

Genetic Analysis Reveals Differences in CD8+ T Cell Epitope Regions That May Impact Cross-Reactivity of Vaccine-Induced T Cells against Wild-Type Mumps Viruses.

Nowadays, mumps is re-emerging in highly vaccinated populations. Waning of vaccine-induced immunity plays a role, but antigenic differences between vaccine and mumps outbreak strains could also contribute to reduced vaccine effectiveness. CD8+ T cells play a critical role in immunity to viruses. However, limited data are available about sequence variability in CD8+ T cell epitope regions of mumps virus (MuV) proteins. Recently, the first set of naturally presented human leukocyte antigen Class I (HLA-I) epitopes of MuV was identified by us. In the present study, sequences of 40 CD8+ T cell epitope candidates, including previously and newly identified, obtained from Jeryl–Lynn mumps vaccine strains were compared with genomes from 462 circulating MuV strains. In 31 epitope candidates (78%) amino acid differences were detected, and in 17 (43%) of the epitope candidates the corresponding sequences in wild-type strains had reduced predicted HLA-I-binding compared to the vaccine strains. These findings suggest that vaccinated persons may have reduced T cell immunity to circulating mumps viruses due to antigenic differences.

Cross-neutralization between vaccine and circulating wild-type mumps viruses in Korea

Mumps is a contagious disease caused by the mumps virus. It can be prevented using mumps vaccines, administered as a measles-mumps-rubella (MMR) vaccine. For first and second dose immunization, children aged 12–15 months and 4–6 years have been administered this vaccine since 1997 in Korea. Nevertheless, mumps outbreaks still occur in vaccinated populations worldwide. Hence, immunity against these diseases may be attenuated, or there are antigenic differences between currently available vaccine strains and circulating wild-type viruses. After the introduction of national immunization programs in Korea, mumps cases became sporadic. Viral genotypes F, H, and I have emerged since 1998 whereas the vaccine strains belong to genotype A. Here, we compared the amino acid sequences of the haemagglutinin-neuraminidase (HN) gene from wild-type viruses and the mumps vaccine and measured the cross-neutralization titers between them. We selected the F, H, and I wild-type mumps strains circulating in Korea from 1998 to 2016 and analyzed changes in the amino acid sequence of the protein encoded by the HN gene. We measured mumps virus-specific IgG and rapid focus reduction neutralization test (FRNT) titers in Korean isolates and sera obtained from 50 children aged 1–2 years who had been administered a single dose of MMR vaccine. Analysis of the HN protein sequences disclosed no changes in the glycosylation sites but did reveal 4–5 differences between the Korean isolates and the genotype A vaccine strain in terms of the neutralizing epitope sites on their HN proteins. Post-vaccination FRNT titers were significantly lower against genotypes F, H, and I than they were against genotype A. This finding highlights the possibility of a recurrence of mumps outbreaks in vaccinated populations depending on the degree of genetic conservation of the HN gene. Further research into this issue is needed to prevent the resurgence of mumps.

Development of Improved Mumps Vaccine Candidates by Mutating Viral mRNA Cap Methyltransferase Sites in the Large Polymerase Protein.

Although a live attenuated vaccine is available for controlling mumps virus (MuV), mumps still outbreaks frequently worldwide. The attenuated MuV vaccine strain S79 is widely used in mumps vaccination in China, but still with many shortcomings, among which the most prominent are the side effects and decreased immunity. Therefore, there is a need to further improve the safety and efficacy of the current MuV vaccine. In the present study, we further attenuated MuV S79 vaccine strain by inhibiting viral mRNA methyltransferase (MTase). We generated a panel of eight recombinant MuVs (rMuVs) carrying mutations in the MTase catalytic site or S-adenosylmethionine (SAM) binding site in the large (L) polymerase protein. These rMuVs are genetically stable and seven rMuVs are more attenuated in replication in cell culture and five rMuVs are more attenuated in replication in lungs of cotton rats compared with the parental vaccine strain S79. Importantly, cotton rats vaccinated with these seven rMuV mutants produced high levels of serum neutralizing antibodies and were completely protected against challenge with a wild-type MuV strain (genotype F). Therefore, our results demonstrate that alteration in the MTase catalytic site or SAM binding site in MuV L protein improves the safety or the immunogenicity of the MuV vaccine and thus mRNA cap MTase may be an effective target for the development of new vaccine candidates for MuV.Electronic supplementary materialThe online version of this article (10.1007/s12250-020-00326-y) contains supplementary material, which is available to authorized users.

A Single Point Mutation in the Mumps V Protein Alters Targeting of the Cellular STAT Pathways Resulting in Virus Attenuation.

Mumps virus (MuV) is a neurotropic non-segmented, negative-stranded, enveloped RNA virus in the Paramyxovirus family. The 15.4 kb genome encodes seven genes, including the V/P, which encodes, among other proteins, the V protein. The MuV V protein has been shown to target the cellular signal transducer and activator of transcription proteins STAT1 and STAT3 for proteasome-mediated degradation. While MuV V protein targeting of STAT1 is generally accepted as a means of limiting innate antiviral responses, the consequence of V protein targeting of STAT3 is less clear. Further, since the MuV V protein targets both STAT1 and STAT3, specifically investigating viral antagonism of STAT3 targeting is challenging. However, a previous study reported that a single amino acid substitution in the MuV V protein (E95D) inhibits targeting of STAT3, but not STAT1. This provided us with a unique opportunity to examine the specific role of STAT 3 in MuV virulence in an in vivo model. Here, using a clone of a wild type MuV strain expressing the E95D mutant V protein, we present data linking inhibition of STAT3 targeting with the accelerated clearance of the virus and reduced neurovirulence in vivo, suggesting its role in promoting antiviral responses. These data suggest a rational approach to virus attenuation that could be exploited for future vaccine development.

Mumps-specific cross-neutralization by MMR vaccine-induced antibodies predicts protection against mumps virus infection

BackgroundSimilar to other recent mumps genotype G outbreaks worldwide, most mumps patients during the recent mumps genotype G outbreaks in the Netherlands had received 2 doses of measles, mumps and rubella (MMR) vaccine during childhood. Here, we investigate the capacity of vaccine-induced antibodies to neutralize wild type mumps virus strains, including mumps virus genotype G. MethodsIn this study, we tested 105 pre-outbreak serum samples from students who had received 2 MMR vaccine doses and who had no mumps virus infection (n=76), symptomatic mumps virus infection (n=10) or asymptomatic mumps virus infection (n=19) during the mumps outbreaks. In all samples, mumps-specific IgG concentrations were measured by multiplex immunoassay and neutralization titers were measured against the Jeryl Lynn vaccine strain and against wild type genotype G and genotype D mumps virus strains. ResultsThe correlation between mumps-specific IgG concentrations and neutralization titers against Jeryl Lynn was poor, which suggests that IgG concentrations do not adequately represent immunological protection against mumps virus infection by antibody neutralization. Pre-outbreak neutralization titers in infected persons were significantly lower against genotype G than against the vaccine strain. Furthermore, antibody neutralization of wild type mumps virus genotype G and genotype D was significantly reduced in pre-outbreak samples from infected persons as compared with non-infected persons. No statistically significant difference was found for the vaccine strain. The sensitivity/specificity ratio was largest for neutralization of the genotype G strain as compared with the genotype D strain and the vaccine strain. ConclusionsThe reduced neutralization of wild type mumps virus strains in MMR vaccinated persons prior to infection indicates that pre-outbreak mumps virus neutralization is partly strain-specific and that neutralization differs between infected and non-infected persons. Therefore, we recommend the use of wild type mumps virus neutralization assays as preferred tool for surveillance of protection against mumps virus infection.

Importance of reporting sequence & serological data from mumps outbreaks in unvaccinated populations.

Importance of reporting sequence & serological data from mumps outbreaks in unvaccinated populations.

Dynamics of the serologic response in vaccinated and unvaccinated mumps cases during an epidemic

In the last decade, several mumps outbreaks were reported in various countries despite high vaccination coverage. In most cases, young adults were affected who have acquired immunity against mumps solely by vaccination and not by previous wild-type mumps virus infection. To investigate mumps-specific antibody levels, functionality and dynamics during a mumps epidemic, blood samples were obtained longitudinally from 23 clinical mumps cases, with or without a prior history of vaccination, and from 20 healthy persons with no serological evidence of recent mumps virus infection. Blood samples from mumps cases were taken 1–2 months and 7–10 months after onset of disease. Both vaccinated and unvaccinated mumps cases had significantly higher geomean concentrations of mumps-specific IgG (resp. 13,617 RU/ml (95% CI of 9,574–19,367 RU/ml) vs. 1,552 (445–5412) RU/ml at 1–2 months; and 6,514 (5,247–8,088) RU/ml vs. 1,143 (480–2,725) RU/ml at 7–10 months) than healthy controls (169 (135–210) RU/ml) (p = 0.001). Patterns in virus-neutralizing (VN) antibody responses against the mumps vaccine virus were similar, vaccinated and unvaccinated mumps cases had significantly higher ND50 values at both time points of sampling (resp 4,695 (3,779–5,832) RU/ml vs. 1,533 (832–2,825) RU/ml at 1–2 months; 2,478 (1,968–3,122) RU/ml vs. 1,221 (1,029–1,449) RU/ml at 7–10 months) compared with (previously vaccinated) healthy controls (122 (196–76)) RU/ml) (p = 0.001) The unvaccinated mumps cases had significantly lower mumps-specific IgG and VN antibody concentrations at both sampling points compared with previously vaccinated cases, but their antibody concentrations did not differ significantly at the 2 time points. In contrast, the mumps-specific IgG and VN antibody concentrations of the previously vaccinated mumps cases were significantly higher within the first 2 months after onset of mumps and declined thereafter, characteristic for a secondary response. A moderate correlation was found between the level of mumps-specific IgG serum antibodies and VN antibodies for the mumps cases (r = 0.64; p<0.001).

Accumulation of defective interfering viral particles in only a few passages in Vero cells attenuates mumps virus neurovirulence

Immunization programs have implemented live attenuated mumps vaccines which reduced mumps incidence ≥97%. Some of the vaccine strains were abandoned due to unwanted side effects and the genetic marker of attenuation has not been identified so far. Our hypothesis was that non-infectious viral particles, in particular defective interfering particles (DIPs), contribute to neuroattenuation. We showed that non-infectious particles of the mumps vaccine L-Zagreb attenuated neurovirulence of wild type mumps virus 9218/Zg98. Then, we attenuated recent wild type mumps virus MuVi/Zagreb.HRV/28.12 in Vero cells through 16 passages but already the fifth passage (p5) showed accumulation of DIPs and attenuated neurovirulence in a newborn rat model when compared to the second passage (p2). Sequence analysis of the p2 and p5 revealed a single mutation in the 5′ untranslated region of the HN gene. Analysis of the expression level of the HN protein showed that this mutation does not affect the expression of the protein. We conclude that the passages of MuVi/Zagreb.HRV/28.12 in Vero cells for only three passages accumulated DIPs which attenuate neurovirulence.These findings reveal DIPs as a very promising and general neuroattenuating factor which should be considered in the rational design of the new mumps vaccine.

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.

Rescue of wild-type mumps virus from a strain associated with recent outbreaks helps to define the role of the SH ORF in the pathogenesis of mumps virus

Mumps virus (MuV) causes acute infections in humans. In recent years, MuV has caused epidemics among highly vaccinated populations. The largest outbreak in the U.S. in the past 20 years occurred in 2005–2006 with over 5000 reported cases in which the majority of the cases was in vaccinated young adults. We sequenced the complete genome of a representative strain from the epidemic (MuV-IA). MuV-IA is a member of genotype G, the same genotype of MuV that was associated with the outbreak in the UK in 2004–2005. We constructed a reverse genetics system for MuV-IA (rMuV-IA), and rescued a virus lacking the open reading frame (ORF) of the SH gene (rMuV∆SH). rMuV∆SH infection in L929 cells induced increased NF-κB activation, TNF-α production and apoptosis compared to rMuV-IA. rMuV∆SH was attenuated in an animal model. These results indicated that the SH ORF of MuV plays a significant role in interfering with TNF-α signaling and viral pathogenesis during virus infection.

Erratum to: Evidence for recombination between vaccine and wild-type mumps virus strains

In a paper published last year in Archives of Virology recombination occurring in mumps virus has been reported (Zhang et al., Arch Virol. 2010 Sep;155(9):1493–1496. Epub 2010 Jun 25.). This conclusion was based on the complete genome sequence of mumps virus strain 9218/Zg98 deposited in GenBank in January 2008 by Dubravko Forcic, Zagreb, Croatia (dforcic@imz.hr). Since this sequence proved to be erroneous, the paper has been retracted.

RETRACTED ARTICLE: Evidence for recombination between vaccine and wild-type mumps virus strains

Recombination of mumps virus (MuV) has rarely been reported. In this study, phylogenetic and recombination analyses were performed on 30 complete MuV genomes, including 17 vaccine and 13 wild-type strains. One potentially significant recombination event was found to have occurred between the lineage represented by the vaccine strain L3/Russia/Vector (AY508995) as the minor parent and wild MuV strain Drag94 (AY669145) as the major parent, and this led to a recombinant, 9218/Zg98 (EU370206), a wild-type MuV strain isolated from a 3-year-old boy with parotitis. In summary, we found a recombinant of MuV derived from vaccine and wild-type MuV strains.

Leucine at Position 383 of Fusion Protein Is Responsible for Fusogenicity of Wild-Type Mumps Virus in B95a Cells

Objective: Mumps virus is isolated in Vero cells and, recently, B95a cells have been reported to be susceptible to it. Currently circulating wild-type mumps virus strains (genotypes B, G, J and L) induced cytopathic effects in both Vero and B95a cells. On the other hand, the Hoshino vaccine strain (KO3) did not induce cytopathic effects in B95a cells. In this study, differences in fusion inducibility were investigated. Methods: Nucleotide sequences of the fusion (F) and hemagglutinin-neuraminidase (HN) protein regions were compared. The F and HN expression plasmids were constructed and fusion analysis was conducted, using recombinant F expression plasmids under the control of T7 RNA polymerase. Results: Extensive cell fusion was observed when B95a cells were transfected with the wild-type F expression plasmid as the F expression partner; 13–16 amino acid differences were observed in the F protein region between the KO3 and the wild types. F expression plasmids with leucine at position 383 of the F protein induced large cell fusion in B95a cells. Conclusion: Leucine at position 383 of the F protein of the wild types was the critical amino acid for fusion inducibility in B95a cells.

A Point Mutation, E95D, in the Mumps Virus V Protein Disengages STAT3 Targeting from STAT1 Targeting

Mumps virus, like other paramyxoviruses in the Rubulavirus genus, encodes a V protein that can assemble a ubiquitin ligase complex from cellular components, leading to the destruction of cellular signal transducer and activator of transcription (STAT) proteins. While many V proteins target the interferon-activated STAT1 or STAT2 protein, mumps virus V protein is unique in its ability to also target STAT3 for ubiquitin modification and proteasome-mediated degradation. Here we report that a single amino acid substitution in the mumps virus V protein, E95D, results in defective STAT3 targeting while maintaining the ability to target STAT1. Results indicate that the E95D mutation disrupts the ability of the V protein to associate with STAT3. A recombinant mumps virus carrying the E95D mutation in its P and V proteins replicates normally in cultured cells but fails to induce targeting of STAT3. Infection with the recombinant virus results in the differential regulation of a number of cellular genes compared to wild-type mumps virus and increases cell death in infected cells, producing a large-plaque phenotype.

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.

Functional consequences of attenuating mutations in the haemagglutinin–neuraminidase, fusion and polymerase proteins of a wild-type mumps virus strain

Wild-type mumps viruses (MuVs) are highly neurotropic and, prior to widespread vaccination programmes, were a major cause of viral meningitis and encephalitis in most developed countries. At present, there are no markers for virus attenuation, apart from the failure of a passaged isolate to produce clinical symptoms in vaccinees. Indeed, some MuV vaccines have retained residual neurovirulence properties and have caused aseptic meningitis in vaccinees. Three amino acid changes associated with the neuroattenuation of a wild-type MuV strain were identified previously. This study evaluated the impact of these changes on the function of the respective proteins. The data demonstrated that the Ser-->Asp amino acid substitution at position 466 in the haemagglutinin-neuraminidase protein resulted in decreased receptor binding and neuraminidase activity, the Ala/Thr-->Thr selection in the fusion protein resulted in decreased fusion activity, and the Ile-->Val substitution in the polymerase resulted in increased replicative/transcriptional activity. These data suggest a polygenic component (i.e. specific and inter-related roles of these amino acid changes) to MuV neuroattenuation.

P.3.a.010 Risperidone long-acting injection: a one year observational study in a naturalistic setting

Immunization programs have implemented live attenuated mumps vaccines which reduced mumps incidence ≥97%. Some of the vaccine strains were abandoned due to unwanted side effects and the genetic marker of attenuation has not been identified so far. Our hypothesis was that non-infectious viral particles, in particular defective interfering particles (DIPs), contribute to neuroattenuation. We showed that non-infectious particles of the mumps vaccine L-Zagreb attenuated neurovirulence of wild type mumps virus 9218/Zg98. Then, we attenuated recent wild type mumps virus MuVi/Zagreb.HRV/28.12 in Vero cells through 16 passages but already the fifth passage (p5) showed accumulation of DIPs and attenuated neurovirulence in a newborn rat model when compared to the second passage (p2). Sequence analysis of the p2 and p5 revealed a single mutation in the 5′ untranslated region of the HN gene. Analysis of the expression level of the HN protein showed that this mutation does not affect the expression of the protein. We conclude that the passages of MuVi/Zagreb.HRV/28.12 in Vero cells for only three passages accumulated DIPs which attenuate neurovirulence.These findings reveal DIPs as a very promising and general neuroattenuating factor which should be considered in the rational design of the new mumps vaccine.

Mumps Virus-Specific Antibody Titers from Pre-Vaccine Era Sera: Comparison of the Plaque Reduction Neutralization Assay and Enzyme Immunoassays

Mumps virus-neutralizing antibodies are believed to be the most predictable surrogate marker of protective immunity. However, assays used to detect neutralizing antibodies, such as the plaque reduction neutralization (PRN) assay, are labor- and time-intensive and consequently are often supplanted by the more rapid and inexpensive enzyme immunoassay (EIA) technique. For virus infections for which international antibody standards exist and are bridged to clinical studies of protection (e.g., measles and rubella), the EIA has been successfully used to determine immune surrogate endpoints, yet no such international reference exists for mumps serology. Since both virus-neutralizing and nonneutralizing antibodies are measured in the EIA, in the absence of a mumps serological standard, the EIA may be prone to yielding false-positive results when utilized for assessing surrogate markers of protective immunity. Moreover, since mumps virus-specific antibody titers are generally low in comparison to antibody levels induced by other viruses and EIA procedures often employ relatively high serum dilution factors, the EIA may be prone to yielding false-negative results. To examine these issues, a PRN assay and two commercially available EIA kits were used to evaluate wild-type mumps virus serological responses in human serum samples from the pre-mumps vaccine era. Our results indicate that the PRN assay is a more sensitive and specific method of measuring serological responses to wild-type mumps virus.

The Rat‐Based Neurovirulence Safety Test for the Assessment of Mumps Virus Neurovirulence in Humans: An International Collaborative Study

Because of the highly neurotropic and neurovirulent properties of wild-type mumps viruses, most national regulatory organizations require neurovirulence testing of virus seeds used in the production of mumps vaccines. Such testing has historically been performed in monkeys; however, some data suggest that testing in monkeys does not necessarily discriminate among the relative neurovirulent risks of mumps virus strains. To address this problem, a collaborative study was initiated by the National Institute for Biological Standards and Control in the United Kingdom and the Food and Drug Administration in the United States, to test a novel rat-based mumps virus neurovirulence safety test. Results indicate that the assay correctly assesses the neurovirulence potential of mumps viruses in humans and is robust and reproducible.