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

Abstract

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.

Keywords: Jeryl Lynn; mumps vaccine.

FIG 1

Schematic of recombinant viruses. N, nucleoprotein; V, V protein; P, phosphoprotein; M, matrix protein; F, fusion protein; SH, small hydrophobic protein; HN, hemagglutinin-neuraminidase protein; L, large protein/RNA-dependent polymerase. Vaccine construct sequences are based on the JL vaccine (genotype A) and the IA/06 isolate (genotype G). rMuV(ΔVΔSH) was generated by mutating the editing site of the V/P gene to only transcribe P and deleting the SH ORF. The recombinant JL (rJL) vaccines were generated by deleting the SH ORF and changing the JL F and/or HN to IA F and/or HN.

FIG 2

Generation of an infectious recombinant JL. (A) Confirmation of rJL virus rescue. After plaque purification, viral RNA was extracted from the medium and subjected to RT-PCR. Primers flanking the NP gene and M gene region were used to amplify a 3.1-kb region of the viral genome. The JL vaccine and rJL viruses are shown along with a mock. The recovered virus matched the JL major component. (B) Syncytium formation after rJL infection. Vero cells were infected at MOIs of 0.01 and 5 with rJL. After 48 h postinfection (hpi), syncytium formation was observed by microscopy.

FIG 3

Characterization of recombinant viruses. (A) Confirmation of recombinant virus rescues. After plaque purification, a stock was propagated, and viral RNA was extracted from the medium and subjected to RT-PCR. Primers in the NP gene and P gene region were used to amplify a 1.6-kb region of the viral genome. (B) Viral protein expression of recombinant viruses. Vero cells were infected at a low MOI. Cells were lysed at 48 hpi and subjected to immunoblotting with an anti-MuV-F and anti-actin antibody. (C) Growth kinetics at a low MOI. Vero cells were infected with the recombinant viruses at an MOI of 0.01. Medium was collected at 24-h increments and titered by plaque assay. (D) Growth kinetics at a high MOI. Vero cells were infected at an MOI of 5 with the recombinant viruses, and medium was collected at 12-h increments. Titers were determined by plaque assay. JL and IA were used as controls. The growth rate of each vaccine was compared to that of the JL vaccine strain. n = 3; two-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test with JL; color corresponds to the vaccine indicated in the key; *, P < 0.05. (E) Plaque formation in Vero cells. The recombinant viruses were plaqued in Vero cells, grown for 8 days, and then stained with crystal violet.

FIG 4

Neutralization titers in mice immunized with vaccine candidates. Neutralization titers in mice postimmunization. Plaque reduction neutralization titers against genotype A (JL) virus (A) and genotype G (IA) virus (B) are shown. The mean 50% plaque reduction neutralization titer and SEM are reported. Bars represent the mean plaque reduction neutralization titer, and individual samples are plotted as points within the bars to show the SEM. The lower limit of detection (LOD) is defined as the starting dilution of serum that was plaqued in this assay, and negative samples are reported as below the detection level. n = 5 to 7; one-way ANOVA with comparison to PBS; *, P < 0.05, **, P < 0.01, ***, P < 0.001.

FIG 5

Longevity of antibodies in mice immunized with vaccine candidates. (A) Timeline of BALB/c study. BALB/c mice were intranasally immunized with 10⁵ PFU and then boosted with the same dose 21 days later. Mice were bled at 35, 56, and 364 dpv. ELISAs and/or plaque reduction neutralization assays were performed. The study concluded 1 year post-prime immunization. (B) ELISA titers in mice postvaccination. Mice were bled at days 56 and 364 post-prime immunization. Serum antibody titers were determined by ELISA. Endpoint titers were determined using serum from day 56 and day 364. Plates were coated with either genotype A (left) or genotype G (right) antigens. The reciprocal of the highest positive dilution and SEM of the average titer are reported. n = 5; one-way ANOVA with comparison to the JL vaccine candidate was performed; *, P < 0.05. (C) Longevity of neutralization titers in mice postvaccination. Plaque reduction assays were performed with day 56 and day 364 serum samples to compare NAb levels over time. The mean 50% plaque reduction neutralization titer and SEM are reported. The bars represent the average plaque reduction neutralization titer. The LOD is defined as the starting dilution of serum that was plaqued in this assay, and negative samples are reported as below the detection level. Neutralization titers against genotype A (JL) virus (left) and against genotype G (IA) virus (right) are shown. n = 5; multiple t tests compared results at 56 and 364 DPV; *, P < 0.05.

FIG 6

Neutralization titer in mice after a third dose of a genotype G-based vaccine. (A) Timeline of BALB/c study. BALB/c mice were intramuscularly immunized with 10⁶ PFU of JL and then boosted with the same dose at 1 month postprime. At 3 months postprime, mice were boosted intramuscularly or intranasally with 10⁶ PFU of JL, rMuV(ΔVΔSH), or rJL(ΔSH, IA-F/HN). Mice were bled at 2.5 and 3.5 months and then terminated at 6 months postprime. Serum was obtained for neutralization. (B to D) Neutralization titers in mice postvaccination. Plaque reduction assays were performed on sera collected from mice. The mean 50% plaque reduction neutralization titer and SEM are reported (n = 6). Bars represent the average plaque reduction neutralization titer, and individual samples are plotted as points within the bars to show the SEM. A dotted line represents the LOD, and negative samples were plotted below the detection level. (B) Neutralization titers after prime-boost of JL given intramuscularly (2.5 months after prime). (C) Neutralization titers 3.5 months postprime and 2 weeks post-IA-based immunization. (D) Neutralization 6 months postprime and 3 months post-genotype G-based immunization. One-way ANOVA with comparison to PBS; *, P < 0.05, **, P < 0.01, ***, P < 0.001.

FIG 7

Neurotoxicity of vaccine candidates. The severity of hydrocephalus in rats inoculated with MuV (IA/06), rMuV(ΔVΔSH), and rJL(ΔSH, IA-F/HN) was measured as described in Materials and Methods. Number of neonate rats per group: WT Iowa (IA/06), 10; rMuV(ΔVΔSH), 26; rJL(ΔSH, IA-F/HN), 18. The mean score of neurotoxicity and standard deviation (SD) are shown for each virus. The dotted line represents the neurotoxicity score of the JL vaccine that was previously established.