Molecular mechanisms of stress-induced reactivation in mumps virus condensates.

Negative-stranded RNA viruses can establish long-term persistent infection in the form of large intracellular inclusions in the human host and cause chronic diseases. Here, we uncover how cellular stress disrupts the metastable host-virus equilibrium in persistent infection and induces viral replication in a culture model of mumps virus. Using a combination of cell biology, whole-cell proteomics, and cryo-electron tomography, we show that persistent viral replication factories are dynamic condensates and identify the largely disordered viral phosphoprotein as a driver of their assembly. Upon stress, increased phosphorylation of the phosphoprotein at its interaction interface with the viral polymerase coincides with the formation of a stable replication complex. By obtaining atomic models for the authentic mumps virus nucleocapsid, we elucidate a concomitant conformational change that exposes the viral genome to its replication machinery. These events constitute a stress-mediated switch within viral condensates that provide an environment to support upregulation of viral replication.

Disentangling the causes of mumps reemergence in the United States.

Over the past two decades, multiple countries with high vaccine coverage have experienced resurgent outbreaks of mumps. Worryingly, in these countries, a high proportion of cases have been among those who have completed the recommended vaccination schedule, raising alarm about the effectiveness of existing vaccines. Two putative mechanisms of vaccine failure have been proposed as driving observed trends: 1) gradual waning of vaccine-derived immunity (necessitating additional booster doses) and 2) the introduction of novel viral genotypes capable of evading vaccinal immunity. Focusing on the United States, we conduct statistical likelihood-based hypothesis testing using a mechanistic transmission model on age-structured epidemiological, demographic, and vaccine uptake time series data. We find that the data are most consistent with the waning hypothesis and estimate that 32.8% (32%, 33.5%) of individuals lose vaccine-derived immunity by age 18 y. Furthermore, we show using our transmission model how waning vaccine immunity reproduces qualitative and quantitatively consistent features of epidemiological data, namely 1) the shift in mumps incidence toward older individuals, 2) the recent recurrence of mumps outbreaks, and 3) the high proportion of mumps cases among previously vaccinated individuals.

A highly efficacious live attenuated mumps virus-based SARS-CoV-2 vaccine candidate expressing a six-proline stabilized prefusion spike.

With the rapid increase in SARS-CoV-2 cases in children, a safe and effective vaccine for this population is urgently needed. The MMR (measles/mumps/rubella) vaccine has been one of the safest and most effective human vaccines used in infants and children since the 1960s. Here, we developed live attenuated recombinant mumps virus (rMuV)-based SARS-CoV-2 vaccine candidates using the MuV Jeryl Lynn (JL2) vaccine strain backbone. The soluble prefusion SARS-CoV-2 spike protein (preS) gene, stablized by two prolines (preS-2P) or six prolines (preS-6P), was inserted into the MuV genome at the P-M or F-SH gene junctions in the MuV genome. preS-6P was more efficiently expressed than preS-2P, and preS-6P expression from the P-M gene junction was more efficient than from the F-SH gene junction. In mice, the rMuV-preS-6P vaccine was more immunogenic than the rMuV-preS-2P vaccine, eliciting stronger neutralizing antibodies and mucosal immunity. Sera raised in response to the rMuV-preS-6P vaccine neutralized SARS-CoV-2 variants of concern, including the Delta variant equivalently. Intranasal and/or subcutaneous immunization of IFNAR1-/- mice and golden Syrian hamsters with the rMuV-preS-6P vaccine induced high levels of neutralizing antibodies, mucosal immunoglobulin A antibody, and T cell immune responses, and were completely protected from challenge by both SARS-CoV-2 USA-WA1/2020 and Delta variants. Therefore, rMuV-preS-6P is a highly promising COVID-19 vaccine candidate, warranting further development as a tetravalent MMR vaccine, which may include protection against SARS-CoV-2.

Novel sialidase inhibitors suppress mumps virus replication and infection.

The prevalent human pathogen, mumps virus (MuV; orthorubulavirus parotitidis) causes various complications and serious sequelae, such as meningitis, encephalitis, deafness, and impaired fertility. Direct-acting antivirals (DAAs) targeting MuV which can prevent mumps and mumps-associated complications and sequelae are yet to be developed. Paramyxoviridae family members, such as MuV, possess viral surface hemagglutinin-neuraminidase (HN) protein with sialidase activity which facilitates efficient viral replication. Therefore, to develop DAAs targeting MuV we synthesized MuV sialidase inhibitors. It is proposed that the viral HN has a single functional site for N-acetylneuraminic acid (Neu5Ac) binding and sialidase activity. Further, the known MuV sialidase inhibitor is an analog of Neu5Ac-2,3-didehydro-2-deoxy-N-acetylneuraminic acid (DANA)-which lacks potency. DANA derivatives with higher MuV sialidase inhibitory potency are lacking. The MuV-HN-Neu5Ac binding site has a hydrophobic cavity adjacent to the C4 position of Neu5Ac. Exploiting this, here, we synthesized DANA derivatives with increasing hydrophobicity at its C4 position and created 3 novel sialidase inhibitors (Compounds 1, 2, and 3) with higher specificity for MuV-HN than DANA; they inhibited MuV replication step to greater extent than DANA. Furthermore, they also inhibited hemagglutination and the MuV infection step. The insight-that these 3 novel DANA derivatives possess linear hydrocarbon groups at the C4-hydroxyl group of DANA-could help develop highly potent sialidase inhibitors with high specificity for MuV sialidase, which may function as direct-acting MuV-specific antivirals.

Evaluation of the Interactions between Mumps Virus and Guinea Pig.

Mumps is a highly contagious viral disease that can be prevented by vaccination. In the last decade, we have encountered repeated outbreaks of mumps in highly vaccinated populations, which call into question the effectiveness of available vaccines. Animal models are crucial for understanding virus-host interactions, and viruses such as mumps virus (MuV), whose only natural host is the human, pose a particular challenge. In our study, we examined the interaction between MuV and the guinea pig. Our results present the first evidence that guinea pigs of the Hartley strain can be infected in vivo after intranasal and intratesticular inoculation. We observed a significant viral replication in infected tissues up to 5 days following infection and induction of cellular and humoral immune responses as well as histopathological changes in infected lungs and testicles, without clinical signs of disease. Transmission of the infection through direct contact between animals was not possible. Our results demonstrate that guinea pigs and guinea pig primary cell cultures represent a promising model for immunological and pathogenetic studies of the complex MuV infection. IMPORTANCE Understanding of mumps virus (MuV) pathogenesis and the immune responses against MuV infection is limited. One of the reasons is the lack of relevant animal models. This study explores the interaction between MuV and the guinea pig. We demonstrated that all tested guinea pig tissue homogenates and primary cell cultures are highly susceptible to MuV infection and that α2,3-sialylated glycans (MuV cellular receptors) are being abundantly expressed at their surface. The virus remains in the guinea pig lungs and trachea for up to 4 days following intranasal infection. Although asymptomatic, MuV infection strongly activates both humoral and cellular immune response in infected animals and provides protection against virus challenge. Infection of the lungs and testicles after intranasal and intratesticular inoculation, respectively, is also supported by histopathological changes in these organs. Our findings give perspective for application of guinea pigs in research on MuV pathogenesis, antiviral response, and vaccine development and testing.

Cytotoxic and viricidal effects of human semen on mumps virus-infected lymphocytes: In vitro studies.

Viruses in human semen may be sexually transmitted via free and cell-mediated viral infection. The potential effects of semen on the infection and sexual transmission of most viruses in semen remain largely unclear. The present study elucidated the inhibitory effects of human seminal plasma (SP) on Jurkat cell (JC)-mediated mumps virus (MuV) infection. We demonstrated that MuV efficiently infected JCs and that the JCs infected by MuV (JC-MuV) mediated MuV infection of HeLa cells. Remarkably, SP was highly cytotoxic to JCs and inhibited JC-MuV infection of HeLa cells. The cytotoxic factor possessed a molecular weight of less than 3 kDa, whereas that of the viricidal factor was over 100 kDa. The cooperation of cytotoxic and viricidal factors was required for the SP inhibition of JC-MuV infection, and prostatic fluid (PF) was responsible for both the cytotoxic and viricidal effects of SP. The cytotoxic effects we observed were resistant to the treatment of PF with boiling water, proteinase K, RNase A, and DNase I. Our results provide novel insights into the antiviral properties of SP, which may limit cell-mediated sexual viral transmission.

Epidemiological characteristics and serological survey of mumps 15 years after MMR vaccine was included in the immunization program.

Mumps is a vaccine-preventable acute viral infectious disease. To understand the incidence of mumps and population immunity in Quzhou City after measles mumps rubella vaccine (MMR) was included in the immunization program, we analyzed the epidemiological characteristics of mumps cases from 2009 to 2023 and a cross-sectional serosurvey of IgG antibodies to mumps conducted in 2024. We found that 15 years after the MMR vaccine was included in the immunization program, the incidence of mumps was significantly reduced in all populations, but the incidence remained highest in vaccinated children aged 0-12 years. Vaccine escape may explain the high incidence of mumps in highly vaccinated populations. Updating vaccines or developing a new vaccine that targets multiple viral genotypes may be necessary to improve the effectiveness of the vaccine against infection and fully control infections and outbreaks. The positive rate and concentration of mumps IgG antibody were inconsistent with the incidence data. mumps IgG antibody is not an ideal substitute for immunity and cannot be used to accurately predict whether a target population is susceptible or protected. Natural infections may provide longer-lasting immunity than vaccination.

Long-Lived Plasma Cells Are Contained within the CD19(-)CD38(hi)CD138(+) Subset in Human Bone Marrow.

Antibody responses to viral infections are sustained for decades by long-lived plasma cells (LLPCs). However, LLPCs have yet to be characterized in humans. Here we used CD19, CD38, and CD138 to identify four PC subsets in human bone marrow (BM). We found that the CD19(-)CD38(hi)CD138(+) subset was morphologically distinct, differentially expressed PC-associated genes, and exclusively contained PCs specific for viral antigens to which the subjects had not been exposed for more than 40 years. Protein sequences of measles- and mumps-specific circulating antibodies were encoded for by CD19(-)CD38(hi)CD138(+) PCs in the BM. Finally, we found that CD19(-)CD38(hi)CD138(+) PCs had a distinct RNA transcriptome signature and human immunoglobulin heavy chain (VH) repertoire that was relatively uncoupled from other BM PC subsets and probably represents the B cell response's "historical record" of antigenic exposure. Thus, our studies define human LLPCs and provide a mechanism for the life-long maintenance of anti-viral antibodies in the serum.

Genetic characteristic of mumps virus from 2012 to 2016 and its serum antibody level among general healthy population during 2018-2020 in Jiangsu Province, China.

Mumps is a vaccine-preventable disease with high contagious capability. Its incidence declined rapidly since one dose of mumps vaccine was introduced into Expanded Program of Immunization (EPI) in 2008 in China. Nonetheless, the outbreaks of mumps remain frequent in China. Here we aim to assess herd immunity level followed by one-dose mumps ingredient vaccine and to elucidate the genetic characteristics of mumps viruses circulating in the post vaccine era in Jiangsu province of China. The complete sequences of mumps virus small hydrophobic(SH) gene were amplified and sequenced; coalescent-based Bayesian method was used to perform phylogenetic analysis with BEAST 1.84 software. Commercially available indirect enzyme-linked immune-sorbent IgG assay was used for the quantitative detection of IgG antibody against mumps virus. Our results show that genotype F was the predominant mumps viruses and belonged to indigenous spread, and most of Jiangsu sequences clustered together and formed a monophyly. The prevalence of mumps reached a peak in 2012 and subsequently declined, which presented an obvious different trajectory with virus circulating in other regions of China. The gene diversity of viruses circulating in Jiangsu province was far less than those in China. The antibody prevalence reached 70.42% in the general population during 2018 to 2020. The rising trend of antibody level was also observed. Although mumps antibody prevalence does not reach expected level, mumps virus faces higher pressure in Jiangsu province than the whole of China. To reduce further the prevalence of mumps viruses, two doses of mumps vaccine should be involved into EPI.

Activation of PTH1R alleviates epididymitis and orchitis through Gq and ß-arrestin-1 pathways.

Inflammation in the epididymis and testis contributes significantly to male infertility. Alternative therapeutic avenues treating epididymitis and orchitis are expected since current therapies using antibiotics have limitations associated to side effects and are commonly ineffective for inflammation due to nonbacterial causes. Here, we demonstrated that type 1 parathyroid hormone receptor (PTH1R) and its endogenous agonists, parathyroid hormone (PTH) and PTH-related protein (PTHrP), were mainly expressed in the Leydig cells of testis as well as epididymal epithelial cells. Screening the secretin family G protein-coupled receptor identified that PTH1R in the epididymis and testis was down-regulated in mumps virus (MuV)- or lipopolysaccharide (LPS)-induced inflammation. Remarkably, activation of PTH1R by abaloparatide (ABL), a Food and Drug Administration-approved treatment for postmenopausal osteoporosis, alleviated MuV- or LPS-induced inflammatory responses in both testis and epididymis and significantly improved sperm functions in both mouse model and human samples. The anti-inflammatory effects of ABL were shown to be regulated mainly through the Gq and ß-arrestin-1 pathway downstream of PTH1R as supported by the application of ABL in Gnaq± and Arrb1-/- mouse models. Taken together, our results identified an important immunoregulatory role for PTH1R signaling in the epididymis and testis. Targeting to PTH1R might have a therapeutic effect for the treatment of epididymitis and orchitis or other inflammatory disease in the male reproductive system.

Nucleolar Protein Treacle Is Important for the Efficient Growth of Mumps Virus.

The nucleolus is the largest structure in the nucleus, and it plays roles in mediating cellular stress responses and regulating cell proliferation, as well as in ribosome biosynthesis. The nucleolus is composed of a variety of nucleolar factors that interact with each other in a complex manner to enable its function. Many viral proteins interact with nucleolar factors as well, affecting cellular morphology and function. Here, to investigate the association between mumps virus (MuV) infection and the nucleolus, we evaluated the necessity of nucleolar factors for MuV proliferation by performing a knockdown of these factors with small interfering (si)RNAs. Our results reveal that suppressing the expression of Treacle, which is required for ribosome biosynthesis, reduced the proliferative potential of MuV. Additionally, the one-step growth kinetics results indicate that Treacle knockdown did not affect the viral RNA and protein synthesis of MuV, but it did impair the production of infectious virus particles. Viral matrix protein (M) was considered a candidate Treacle interaction partner because it functions in the process of particle formation in the viral life cycle and is partially localized in the nucleolus. Our data confirm that MuV M can interact with Treacle and colocalize with it in the nucleolus. Furthermore, we found that viral infection induces relocalization of Treacle in the nucleus. Together, these findings suggest that interaction with Treacle in the nucleolus is important for the M protein to exert its functions late in the MuV life cycle. IMPORTANCE The nucleolus, which is the site of ribosome biosynthesis, is a target organelle for many viruses. It is increasingly evident that viruses can favor their own replication and multiplication by interacting with various nucleolar factors. In this study, we found that the nucleolar protein Treacle, known to function in the transcription and processing of pre-rRNA, is required for the efficient propagation of mumps virus (MuV). Specifically, our data indicate that Treacle is not involved in viral RNA or protein synthesis but is important in the processes leading to viral particle production in MuV infection. Additionally, we determined that MuV matrix protein (M), which functions mainly in viral particle assembly and budding, colocalized and interacted with Treacle. Furthermore, we found that Treacle is distributed throughout the nucleus in MuV-infected cells. Our research shows that the interaction between M and Treacle supports efficient viral growth in the late stage of MuV infection.

Immunogenicity of Mumps Virus Genotype G Vaccine Candidates in Jeryl Lynn-Immunized Mice.

Mumps virus (MuV) causes a highly contagious human disease characterized by the enlargement of the parotid glands. In severe cases, mumps can lead to neurological complications such as aseptic meningitis and encephalitis. Vaccination with the attenuated Jeryl Lynn (JL) MuV vaccine has dramatically reduced the incidence of MuV infection. Recently, large outbreaks have occurred in vaccinated populations. The vaccine strain JL was generated from genotype A, while most current circulating strains belong to genotype G. In this study, we examined the immunogenicity and longevity of genotype G-based vaccines. We found that our recombinant genotype G-based vaccines provide robust neutralizing titers toward genotype G for up to 1 year in mice. In addition, we demonstrated that a third dose of a genotype G-based vaccine following two doses of JL immunization significantly increases neutralizing titers toward the genotype G strain. Our data suggest that after two doses of JL vaccination, which most people have received, a third dose of a genotype G-based vaccine can generate immunity against a genotype G strain. IMPORTANCE At present, most individuals have received two doses of the measles, mumps, and rubella (MMR) vaccine, which contains genotype A mumps vaccine. One hurdle in developing a new mumps vaccine against circulating genotype G virus is whether the new genotype G vaccine can generate immunity in humans that are immunized against genotype A virus. This work demonstrates that a novel genotype G-based vaccine can be effective in animals which received two doses of genotype A-based vaccine, suggesting that the lead genotype G vaccine may induce anti-G immunity in humans who have received two doses of the current vaccine, providing support for testing this vaccine in humans.

Viral detection from negative mumps cases with respiratory symptoms in Gwangju, South Korea in 2021.

Mumps is the second-most reported infectious disease in South Korea; however, due to the low pathogen confirmation rate in laboratory diagnoses, we proposed a method for reevaluating the high incidence rate via the laboratory verification of other viral diseases. In 2021, 63 cases of pharyngeal or cheek mucosal swabs of suspected mumps cases in Gwangju, South Korea, were assessed for causative pathogens using massive simultaneous pathogen testing. More than one respiratory virus was detected in 60 cases (95.2%), 44 (73.3%) of which were codetected. Human rhinovirus was detected in 47 cases, followed by human herpesvirus (HHV)6 in 30; HHV4 (17), human bocavirus (17), HHV5 (10), and human parainfluenza virus 3 (6) were also detected. Our findings suggest the need for further investigations on the pathogenesis of diseases mimicking mumps, which are considered to aid with appropriate public health responses, treatment, and the prevention of infectious disease outbreaks.

Combining genomics and epidemiology to track mumps virus transmission in the United States.

Unusually large outbreaks of mumps across the United States in 2016 and 2017 raised questions about the extent of mumps circulation and the relationship between these and prior outbreaks. We paired epidemiological data from public health investigations with analysis of mumps virus whole genome sequences from 201 infected individuals, focusing on Massachusetts university communities. Our analysis suggests continuous, undetected circulation of mumps locally and nationally, including multiple independent introductions into Massachusetts and into individual communities. Despite the presence of these multiple mumps virus lineages, the genomic data show that one lineage has dominated in the US since at least 2006. Widespread transmission was surprising given high vaccination rates, but we found no genetic evidence that variants arising during this outbreak contributed to vaccine escape. Viral genomic data allowed us to reconstruct mumps transmission links not evident from epidemiological data or standard single-gene surveillance efforts and also revealed connections between apparently unrelated mumps outbreaks.

Demonstration of bacterium-free very rapid reverse genetics system using mumps virus.

The reverse genetics system is a very powerful tool for analyzing the molecular mechanisms of viral propagation and pathogenesis. However, full-length genome plasmid construction is highly time-consuming and laborious, and undesired mutations may be introduced by Escherichia coli. This study shows a very rapid E. coli-free method of full-genome construction using the mumps virus as an example. This method was able to reduce dramatically the time for full-genome construction, which was used very efficiently for virus rescue, from several days or more to ~2 days, with a similar accuracy and yield to the conventional method using E. coli/plasmid.

Co-circulation of two Mumps virus genotypes in Assam, India.

Mumps is a vaccine-preventable disease, and research on the vaccine's efficacy has recently indicated declining efficacy that has failed to protect against primary infections or reinfections, leading to a global resurgence in nations that use mumps vaccine in their national immunization programmes (NIPs). Lack of reports on its infection, documentation and published studies prevents it from being recognized as a public health issue in India. The waning of immunity is ascribed to the changes between the circulating and vaccine strains. The goal of the current study was to describe the circulating MuV strains in the Dibrugarh district of Assam, India, from 2016 to 2019. Blood samples were examined for IgM antibodies, and throat swab samples were put through Taqman assay for molecular detection. The small hydrophobic (SH) gene was targeted for genotyping through sequencing, and its genetic variations and phylogenetic analysis were carried out. Mumps RNA was found in 42 cases, and Mumps IgM in 14, of which 60% (25/42) of the cases were male and 40% (17/42) were female mostly affecting children between the ages of 6 and 12. Sequence and phylogeny analyses of SH gene revealed Genotypes C (83%) and G (17%) were simultaneously circulating during the study period. The study offers crucial genetic baseline information for the creation of Mumps prevention and control measures. Therefore, based on the research, it is clear that developing an effective vaccination strategy should take into account all currently prevalent genotypes in order to provide better protection against the disease's comeback.

Increase of Diversity of Mumps Virus Genotype G SH Variants Circulating Among a Highly Immunized Population: Spain, 2007-2019.

MuV caused three epidemic waves in Spain since genotype G emerged in 2005, despite high vaccination coverage. SH gene sequencing according to WHO protocols allowed the identification of seven relevant variants and 88 haplotypes. While the originally imported MuVi/Sheffield.GBR/1.05/-variant prevailed during the first two waves, it was subsequently replaced by other variants originated by either local evolution or importation, according to the additional analysis of hypervariable NCRs. The time of emergence of the MRCA of each MuV variant clade was concordant with the data of the earliest sequence. The analysis of Shannon entropy showed an accumulation of variability on six particular positions as the cause of the increase on the number of circulating SH variants. Consequently, SH gene sequencing needs to be complemented with other more variable markers for mumps surveillance immediately after the emergence of a new genotype, but the subsequent emergence of new SH variants turns it unnecessary.

Mumps virus-specific immune response outcomes and sex-based differences in a cohort of healthy adolescents.

Despite high levels of MMR-II usage in the US, mumps outbreaks continue to occur. Evidence suggests that mumps vaccine-induced humoral immunity wanes over time. Relatively few studies have examined cell-mediated immunity or reported on sex-based differences. To better understand sex-based differences in the immune response to mumps vaccine, we measured neutralizing antibody titers and mumps-specific cytokine/chemokine responses in a cohort of 748 adolescents and young adults after two doses of MMR vaccine. We observed significantly higher neutralizing antibody titers in females than in males (120.8 IU/mL, 98.7 IU/mL, p = 0.038) but significantly higher secretion levels of MIP-1α, MIP-1ß, TNFα, IL-6, IFNγ, and IL-1ß in males compared to females. These data demonstrate that sex influences mumps-specific humoral and cell-mediated immune response outcomes, a phenomenon that should be considered during efforts to improve vaccines and prevent future outbreaks.

Streamlined Whole-Genome Sequencing of Mumps Virus for High-Resolution Outbreak Analysis.

Since 2015, the United States has experienced a resurgence in the number of mumps cases and outbreaks in fully vaccinated populations. These outbreaks have occurred predominantly in close-quarter settings, such as camps, colleges, and detention centers. Phylogenetic analysis of 758 mumps-positive samples from outbreaks across the United States identified 743 (98%) as genotype G based on sequence analysis of the mumps small hydrophobic (SH) gene. Additionally, SH sequences in the genotype G samples showed almost no sequence diversity, with 675 (91%) of them having identical sequences or only one nucleotide difference. This uniformity of circulating genotype and strain created complications for epidemiologic investigations and necessitated the development of a system for rapidly generating mumps whole-genome sequences for more detailed analysis. In this study, we report a novel and streamlined assay for whole-genome sequencing (WGS) of mumps virus genotype G. The WGS procedure successfully generated 318 high-quality WGS sequences on nucleic acid from genotype G-positive respiratory samples collected during several mumps outbreaks in the United States between 2016 and 2019. Sequencing was performed by a rapid and highly sensitive custom Ion AmpliSeq mumps genotype G panel, with sample preparation performed on an Ion Chef and sequencing on an Ion S5. The WGS data generated by the AmpliSeq panel provided enhanced genomic resolution for epidemiological outbreak investigations. Translation and protein sequence analysis also identified several potentially important epitope changes in the circulating mumps genotype G strains compared to the Jeryl-Lynn strain (JL5) used in vaccines in the United States, which could explain the current level of vaccine escapes.

Decreased humoral immunity to mumps in young adults immunized with MMR vaccine in childhood.

In the past decade, multiple mumps outbreaks have occurred in the United States, primarily in close-contact, high-density settings such as colleges, with a high attack rate among young adults, many of whom had the recommended 2 doses of mumps-measles-rubella (MMR) vaccine. Waning humoral immunity and the circulation of divergent wild-type mumps strains have been proposed as contributing factors to mumps resurgence. Blood samples from 71 healthy 18- to 23-year-old college students living in a non-outbreak area were assayed for antibodies and memory B cells (MBCs) to mumps, measles, and rubella. Seroprevalence rates of mumps, measles, and rubella determined by IgG enzyme-linked immunosorbent assay (ELISA) were 93, 93, and 100%, respectively. The index standard ratio indicated that the concentration of IgG was significantly lower for mumps than rubella. High IgG avidity to mumps Enders strain was detected in sera of 59/71 participants who had sufficient IgG levels. The frequency of circulating mumps-specific MBCs was 5 to 10 times lower than measles and rubella, and 10% of the participants had no detectable MBCs to mumps. Geometric mean neutralizing antibody titers (GMTs) by plaque reduction neutralization to the predominant circulating wild-type mumps strain (genotype G) were 6-fold lower than the GMTs against the Jeryl Lynn vaccine strain (genotype A). The majority of the participants (80%) received their second MMR vaccine ≥10 years prior to study participation. Additional efforts are needed to fully characterize B and T cell immune responses to mumps vaccine and to develop strategies to improve the quality and durability of vaccine-induced immunity.