immunogenicity of novel mumps vaccine candidates generated by_看图王
Immunogenicity of Novel Mumps Vaccine Candidates Generated by Genetic Modi?cation
Pei Xu,a,b Zhenhai Chen,a Shannon Phan,a Adrian Pickar,a Biao He a
Department of Infectious Diseases,College of Veterinary Medicine,University of Georgia,Athens,Georgia,USA a;Intercollege Graduate Program in Cell and Developmental Biology,Pennsylvania State University,University Park,Pennsylvania,USA b
Mumps is a highly contagious human disease,characterized by lateral or bilateral nonsuppurative swelling of the parotid glands and neurological complications that can result in aseptic meningitis or encephalitis.A mumps vaccination program imple-mented since the1960s reduced mumps incidence by more than99%and kept the mumps case numbers as low as hundreds of cases per year in the United States before2006.However,a large mumps outbreak occurred in vaccinated populations in2006 and again in2009in the United States,raising concerns about the ef?cacy of the vaccination program.Previously,we have shown that clinical isolate-based recombinant mumps viruses lacking expression of either the V protein(rMuV?V)or the SH protein(rMuV?SH)are attenuated in a neurovirulence test using newborn rat brains(P.Xu et al.,Virology417:126–136,2011, https://www.sodocs.net/doc/c410669022.html,/10.1016/j.virol.2011.05.003;P.Xu et al.,J.Virol.86:1768–1776,2012,https://www.sodocs.net/doc/c410669022.html,/10.1128/JVI.06019-11) and may be good candidates for vaccine development.In this study,we examined immunity induced by rMuV?SH and
rMuV?V in mice.Furthermore,we generated recombinant mumps viruses lacking expression of both the V protein and the SH protein(rMuV?SH?V).Analysis of rMuV?SH?V indicated that it was stable in tissue culture cell lines.Importantly,
rMuV?SH?V was immunogenic in mice,indicating that it is a promising candidate for mumps vaccine development.
M umps is a human infectious disease characterized by lateral or bilateral nonsuppurative swelling of the parotid glands. In severe cases,mumps can lead to orchitis in postpuberty male patients and damage to the central nervous system.In the prevac-cine era,90%of the population turned seropositive for mumps virus(MuV)by14to15years of age,re?ecting its highly conta-gious nature.Mumps virus is neurotropic and was one of the most common causes of aseptic meningitis before the implementation of mass mumps vaccination programs.
At present,the Jeryl Lynn(JL)vaccine is the most commonly used mumps vaccine,administered as lyophilized live virus with measles and rubella vaccine components.The JL vaccine strain originated from an infectious isolate from a mumps patient in1963(1).The virus was attenuated through continuous passages in embryonic hen eggs and chicken embryos/chicken embryo cell cultures(1).The JL vaccine was licensed in the United States in1967and has been used for over40years.This vaccine has been ef?cacious and safe overall (2–6).However,several large mumps outbreaks have occurred re-cently in the United States and worldwide in populations that have been vaccinated with the JL vaccine(7–10).Major mumps outbreaks in the United States include the2006multistate mumps outbreak, reporting6,584suspected cases originating from the state of Iowa(11, 12)and the2009–2010New York and New Jersey mumps outbreaks with a total of2,078suspected cases reported in2010(13).Both of the outbreaks occurred among highly vaccinated populations,raising questions about the ef?cacy of the current vaccination program in the United States.One possible causality is the antigenic differences be-tween the genotype A vaccine strain and the genotype G circulating wild-type mumps viruses.
In this study,we seek to develop a mumps vaccine candidate through genetic modi?cation of a clinically isolated mumps virus.Mumps virus is a member of the family Paramyxoviridae, subfamily Paramyxovirinae,and genus Rubulavirus(6,14).It is an enveloped virus enclosing a negative-sense,single-stranded,non-segmented RNA genome of15,384nucleotides in length which encodes9viral proteins(15–17).Studies of the function of the Paramyxovirus SH protein reveal that it blocks tumor necrosis factor alpha(TNF-?)induction,signaling,caspase activation,and NF-?B nuclear translocation in transfected and virus-infected cells(18–23).The V protein is an accessory protein translated from the authentic transcript of the V/P gene(24,25).Mumps V protein is an antagonist of antiviral innate immunity.It interferes with type I interferon(IFN)induction by disrupting the recogni-tion of intracellular viral double-stranded RNA(dsRNA)by MDA5(26–28).It also blocks IFN signaling by targeting STAT proteins for proteasome-mediated degradation(29–35).Recom-binant mumps viruses with either the V protein deletion (rMuV?V)or the SH protein deletion(rMuV?SH)are attenu-ated in neurotoxicity in intracerebrally(IC)infected rats(21,36). In this study,we tested the immunogenicity of rMuV?V and rMuV?SH in mice.Furthermore,we generated a recombinant MuV lacking expression of both the SH and V proteins (rMuV?SH?V)and examined antibody and cellular immune re-sponses in mice.
MATERIALS AND METHODS
Plasmids,viruses,and cells.The MuV strain was obtained from a pa-tient during the2005–2006Midwest mumps outbreak in the United States.A full-length cDNA clone of the virus(pMuV)was constructed as previously described(21).Recombinant MuV lacking the V protein (rMuV?V),recombinant MuV lacking the SH protein,and recombinant Received
23September2013Accepted9December2013
Published ahead of print18December2013
Editor:D.S.Lyles
Address correspondence to Biao He,bhe@https://www.sodocs.net/doc/c410669022.html,.
Copyright?2014,American Society for Microbiology.All Rights Reserved.
doi:10.1128/JVI.02778-13
https://www.sodocs.net/doc/c410669022.html, Journal of Virology p.2600–2610March2014Volume88Number5
MuV expressing a Renilla luciferase protein have been described before (21).A plasmid containing the MuV genome but lacking both V and SH was constructed by combining the SH open reading frame(ORF)deletion with the plasmid encoding the rMuV?V genome.Primer sequences,de-tailed cloning strategies,and entire cDNA sequences of MuV are available upon request.Jeryl Lynn(JL)vaccine,isolated from the measles,mumps, and rubella(MMR)vaccine,was a gift from Paul Rota at the CDC.
To rescue an infectious virus,plasmid pMuV?SH?V(5?g),along with plasmids pCAGGS-L(1?g),pCAGGS-NP(1.5?g),and pCAGGS-P (200ng),were transfected into BSRT-7cells.Three days later,transfected BSRT-7cells were mixed with Vero cells at a1:1ratio.Ten to14days later, when syncytium formation was observed,supernatants containing puta-tive rMuV?SH?V were collected and plaque puri?ed in Vero cells. Plaques(developing4to7days postinfection[dpi])were ampli?ed in Vero cells once(P0),and their genomes were sequenced.All recombinant viruses used for the following experiments were expanded once in Vero cells from the P0ampli?cation(P1).The rescue procedure was repeated to produce independent stocks of rMuV?SH?V,resulting in6isolates of independently rescued rMuV?SH?V viruses(PX64-1,PX64-4,PX64-61, PX64-67,and PX64-84).
All mumps viruses were grown in Vero cells and harvested at4to7dpi. Virus titers were measured in Vero cells by plaque assay as described previously(37,38).JL virus was grown in Vero cells and concentrated to achieve a working titer.Harvested virus stock was cushioned onto20% sucrose using ultracentrifugation at37,500rpm(Thermo Scienti?c Sor-vall RC6plus centrifuge).Pelleted viruses were resuspended in1%bovine serum albumin(BSA)–Dulbecco’s modi?ed Eagle medium(DMEM)and stored at?80°C.Concentrated JL virus was retitrated in Vero cells by plaque assay.
Vero cells were maintained in DMEM with10%fetal bovine serum (FBS)and1%penicillin-streptomycin(P/S)(Mediatech Inc.,Holu Hill, FL).BSRT-7cells were maintained in DMEM supplemented with10% FBS,1%P/S,10%tryptose phosphate broth(TPB),and400?g/ml Gene-ticin G418antibiotic.Cells were cultured at37°C with5%CO2and passed the day before infection or transfection at appropriate dilution factors to achieve80to90%con?uence the next day.For virus infection,cells were inoculated with viruses in DMEM plus1%BSA at an multiplicity of in-fection(MOI)of0.01,3,or5and incubated for1to2h at37°C with5% CO2.The inocula were then replaced with DMEM supplemented with2% FBS and1%P/S.Cells were transfected with plasmids using Plus and Lipofectamine reagents(Invitrogen,Carlsbad,CA)by following the man-ufacturer-provided protocols.
Sequencing of viruses.Viral RNA was extracted from cell culture su-pernatants using the QIAamp viral RNA extraction minikit(Qiagen Inc., Valencia,CA)by following the manufacturer’s protocol.Isolated viral RNA was reverse transcribed into cDNA using Super Script III reverse transcriptase with random hexamers(Invitrogen).Synthesized cDNA then served as templates for PCR using mumps virus genome-speci?c primers and Taq polymerase(Invitrogen).Fifteen sets of primers,each containing a forward and reverse primer,were designed to divide the genome into15overlapping fragments.The primers were then used for the subsequent sequencing of the PCR products(39).Primer sequences are available upon request.
Immunoblotting.Vero cells in6-well plates at approximately90% con?uence were mock infected or infected with rMuV or rMuV?SH?V at an MOI of0.5.Cells were lysed and collected at different time points postinfection in0.5ml WCEB buffer(50mM Tris-HCl,pH8.0,120mM NaCl,0.5%NP-40,0.00076%EGTA,0.2mM EDTA,10%glycerol)with a mixture of protease inhibitors as described previously(30,31).Cell lysates were brie?y centrifuged to remove cell debris and loaded onto a 10%or17.5%polyacrylamide gel and subjected to SDS-PAGE.Proteins were transferred to an Immobilon-FL transfer membrane(Millipore,Bil-lerica,MA),incubated with primary antibody(anti-MuV V,1:500;anti-MuV NP,1:5,000;anti-MuV P,1:2,000;and anti-MuV SH,1:200)(21) and corresponding secondary antibodies conjugated to horseradish per-oxidase(1:1,000)(KPL,Inc.)and detected using an Amersham ECL West-ern blotting detection kit(GE Healthcare Bioscience,Piscataway,NJ).
Multicycle growth curve in Vero cells.Vero cells in6-cm plates or 6-well plates were mock infected or infected with rMuV,JL,rMuV?V, rMuV?SH,or rMuV?SH?V(multiple isolates)at an MOI of0.01.One ml(6-cm plate)or100?l(6-well plate)of supernatant was collected at1, 2,3,4,5,and6dpi,supplemented with1%BSA,and stored at?80°C. Virus titers were determined by plaque assay in triplicate using Vero cells in6-well plates.After1to2h of incubation with the viruses,the growth medium was changed to DMEM with2%FBS,1%P/S,and1%low-melting-point agarose.Four to7dpi,the Vero cells were stained with Giemsa stain and plaques were counted.
Immunization of mice.BALB/c mice(female,6to8weeks old)were purchased from Charles River Laboratories(Frederick,MD).Mice were immunized with1?106PFU of rMuV,JL,rMuV?V,rMuV?SH,or rMuV?SH?V in a volume of100?l for intranasal(i.n.)vaccination.For intramuscular(i.m.)vaccination,mice were injected with25?l of inoc-ulum into each side of the caudal thigh bilaterally(106PFU).i.n.-or i.m.-vaccinated mice were boosted with the same amount of virus inocula as the primary vaccination on the21st or22nd day after primary vaccina-tion.Blood samples were obtained from mock or recombinant MuV-vaccinated mice through tail vein puncture.At the termination of each experiment,mice were euthanized with500?l of Avertin(2,2,2-tribro-moethanol)(Sigma-Aldrich)followed by cervical dislocation.Spleens were removed from the mice for splenocyte isolation and in vitro analysis. All mouse immunizations and studies with mumps viruses were per-formed in enhanced biosafety level2facilities with HEPA-?ltered isola-tors and were conducted by following protocols reviewed and approved by the Institutional Animal Care and Use Committee of the University of Georgia.
The ELISPOT assay.Splenocytes were isolated from mouse spleens at the time of euthanasia.Spleens were ground,?ltered through cell strainers (BD Falcon),and washed once with50ml of Hanks’balanced salt solution (Life Technologies)per spleen.Washed splenocytes from each spleen were treated with3ml of Gey’s solution(ammonium chloride,8.29g/li-ter;potassium bicarbonate,1g/liter)for5min at room temperature(RT) to lyse red blood cells.The residual splenocytes were washed once with50 ml Hanks balanced salt solution(HBSS)per spleen and resuspended in10 ml complete tumor medium(CTM)containing0.75g/liter D-glucose (Sigma),7.5ml/liter essential amino acids(50?)(Invitrogen),14ml/liter nonessential amino acids(100?)(Invitrogen),10ml/liter sodium pyru-vate(100?)(Gibco),10ml/liter L-glutamine(100?)(Gibco),0.85g/liter sodium bicarbonate(Sigma),1%gentamicin-penicillin G-streptomycin sulfate(Sigma),and3.4?l/liter2-mercaptoethanol(Fisher)in minimum essential medium,Spinner modi?cation(S-MEM;Sigma).Splenocytes were counted and reconstituted to a concentration of3?106cells/ml and 1.5?106cells/ml in CTM.One hundred?l of splenocytes was plated onto prepared enzyme-linked immunosorbent spot(ELISPOT)plates(Multi-Screen-IP without underdrain;0.45?m,white,sterile;Millipore).The ELISPOT plates were precoated with anti-mouse IFN-?(AN-18; MABTECH)overnight,washed with sterile PBS?ve times,and incu-bated with CTM for1h at RT.One hundred?l of CTM containing either mock-infected or MuV-infected Vero cell lysates at50?g/ml was overlaid onto splenocytes as a stimulant.Vero cell lysates were prepared by rounds of sonication and several freeze-thaw cycles to in-activate any infectious viral particles.The mixture of splenocytes and viral antigens was incubated for40to48h at37°C with5%CO2.The plates were washed after incubation,blotted with biotinylated anti-mouse IFN-?antibody(MAb R4-6A2;MABTECH)and streptavidin-alkaline phosphatase(MABTECH),and developed in5-bromo-4-chloro-3-in-dolylphosphate/nitroblue tetrazolium(KPL).
ELISA.Enzyme-linked immunosorbent assay(ELISA)was performed as previously described(40).Brie?y,immulon2HB96-well microtiter plates(ThermoLab Systems)were coated with MuV proteins at2?g/ml and incubated at4°C overnight.Plates were then washed with KPL wash
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solution (KPL,Inc.),and each well was blocked with 200?l KPL wash solution with 5%nonfat dry milk and 0.5%BSA (Blotto)for 1h at RT.Serum samples were inactivated by heating at 56°C for 0.5h and were serially diluted 2-fold or 4-fold in Blotto.One hundred ?l of diluted serum samples was transferred to the coated plate and incubated for 1h at RT.To detect anti-MuV speci?c antibodies,alkaline phosphatase (AP)-labeled,goat anti-mouse IgG (KPL,Inc.)was diluted in Blotto according to the manufacturer’s instructions,added to each well,and incubated for 1h at RT.Plates were washed and developed by adding 100?l pNPP phosphatase substrate (KPL,Inc.)per well.Optical density (OD)was measured at 405nm on a Bio-Tek Powerwave XS plate reader.
Luciferase activity-based neutralization assay.Serum samples were serially diluted 2-fold starting from 1:10or 1:40up to 1:20,480.Recom-binant virus expressing a Renilla luciferase protein (rMuV-Luc)was di-luted to 2,000PFU/ml in 1%BSA-DMEM.One portion of serum (40?l)was mixed with an equal volume of rMuV-Luc virus (80PFU/40?l)into each well of a 96-well plate and incubated at 37°C with 5%CO 2.Each 96-well plate contained ?ve serum samples and one standard in duplicate.After 1h of incubation,trypsinized Vero cells in 4%FBS,2%P/S in DMEM were added to each well of the 96-well plates.At 48to 72h postin-fection,infected Vero cells were lysed and analyzed for total luciferase activity per well using the Renilla Luciferase assay system (Promega)and a Veritas microplate luminometer (Promega).The neutralizing titer was calculated as the highest dilution level with luciferase readings exceeding that produced by 40PFU of rMuV-Luc virus in standard control wells.Statistics.P values were calculated using Student’s t test (two-tailed,type 2).Correlations of titers determined by luciferase activity-based neu-tralization assay to that determined by plaque reduction neutralization assay were calculated by R 2.
RESULTS
Immunogenicity of recombinant mumps viruses lacking either the V protein or the SH protein in mice.To analyze the immu-nogenicity of the current mumps vaccine,JL,and the clinical MuV isolate from the 2006outbreak (referred to as MuV),mice were vaccinated with JL or MuV via the intranasal (i.n.)or intramus-cular (i.m.)route and boosted at 22days postprimary vaccination with the same virus,dose,and route as the primary vaccination.
Serum samples were collected at 14days postboost (dpb).As ex-pected,JL generated higher neutralizing antibody titers against JL than MuV,and MuV generated higher anti-MuV titers than JL,regardless of the route of immunization (Fig.1).This result is consistent with a previous report that sera from JL-vaccinated humans had higher anti-JL neutralizing titers than anti-MuV neu-tralizing titers (41).
Previous studies have shown that rMuV ?V (lacking V protein expression)or rMuV ?SH (lacking SH protein expression)are atten-uated in a neurovirulency potency test in rat brains (21,36),suggest-ing these viruses are good candidates for vaccine development.To investigate the immunogenicity and vaccine potential of rMuV ?V and rMuV ?SH in mice,BALB/c mice were mock vaccinated (PBS)or vaccinated with rMuV,JL,rMuV ?SH,or rMuV ?V and boosted at 22days postprimary vaccination through i.m.injection with the same virus and dose as the primary vaccination.We chose the i.m.route because the trivalent MMR vaccine is usually administered by intra-muscular (i.m.)or deep subcutaneous injection (42),but mostly via i.m.administration.Serum samples were collected at 14dpb.The total IgG antibody titer against MuV was measured by ELISA using plates coated with puri?ed MuV (Fig.2).We found that all groups generated robust anti-MuV antibody responses.
Mumps virus is a human respiratory virus transmitted via re-spiratory secretions such as saliva and nose and throat discharge (6).i.n.vaccination induces both local immunity in the respira-tory tract and systemic immunity.Mucosal immunity provides direct and rapid protection against virus challenge.To examine the immunogenicity of rMuV ?V and rMuV ?SH compared to that of rMuV and JL,BALB/c mice were mock vaccinated (PBS)or vaccinated with rMuV,JL,rMuV ?SH,or rMuV ?V intranasally and boosted at 22days postprimary vaccination with the same virus type and dose as the primary i.n.vaccination.Serum samples were collected at 14dpb.Total IgG antibody titers against MuV were measured by ELISA (Fig.3).All groups generated robust anti-MuV antibody responses.
Rescue of recombinant viruses lacking both V and SH pro-teins.To further enhance the safety of vaccine candidates,we con-structed a recombinant virus lacking expression of both the V and SH proteins.The genome length of the newly synthesized cDNA (pMuV ?SH ?V)complied with the rule of six for Paramyxovirus
FIG 1Cross-reactivity of JL and MuV.BALB/c mice were i.n.or i.m.immu-
nized with PBS,MuV,or JL at 106PFU/mouse and boosted at 22days post-vaccination with the same virus at 106PFU/mouse.Serum samples were col-lected at 14days postboost.Heat-inactivated serum samples of individual mice from the same group were pooled to perform the plaque reduction neutraliza-tion test (PRNT).Serum samples were 2-fold serial diluted from 1:30to 1:3,840.A volume of 120?l diluted serum was mixed with 120?l diluted virus containing 80PFU of either JL or rMuV virus and incubated at 37°C for 1h.The count of residual unneutralized PFU per 100?l was determined by plaque assay in 6-well plates of Vero cells.PRNT titer is determined as the ?rst dilution level with residual PFU of more than half of the input per 100?
l.
FIG 2i.m.immunization with rMuV ?SH or rMuV ?V induced antibody responses in mice.BALB/c mice were i.m.vaccinated with PBS,rMuV,JL,rMuV ?SH,and rMuV ?V at 106PFU and boosted 22days postvaccination with 106PFU.Serum samples were collected at 14dpb,and total antibody titers in these samples were measured by ELISA coated with MuV viral pro-teins.
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(43).Infectious recombinant viruses (rMuV ?SH ?V)were rescued from BSRT-7cells transfected with pMuV ?SH ?V and helper plas-mids as described before (21).To con?rm rescue of the virus,viral RNA was extracted from cell culture medium containing rescued viruses (Fig.4B ).The SH gene and the V/P gene region were ampli-?ed using reverse transcription-PCR and sequenced.As shown in Fig.4,the SH ORF truncation as well as the V deletion was con?rmed (Fig.4C and D ).
To con?rm that genomic changes in rMuV ?SH ?V abolish V and SH expression,Vero cells were mock infected or infected with rMuV or rescued rMuV ?SH ?V (PX64-67strain).Expression lev-els of MuV NP,P,V,and SH proteins were examined using West-ern blotting.While NP and P were detected in both rMuV-and rMuV ?SH ?V-infected cells,expression of V or SH protein was only detected in rMuV-infected Vero cells (Fig.4E ).
Analysis of rMuV ?SH ?V in tissue culture cells.To select an rMuV ?SH ?V virus that replicates well for vaccine production pur-poses,the replication capability of rMuV ?SH ?V viruses from 6in-
FIG 3i.n.immunization with rMuV ?SH or rMuV ?V induced antibody re-sponses in mice.BALB/c mice were i.n.vaccinated with PBS,rMuV,JL,rMuV ?SH,and rMuV ?V with 106PFU and boosted 22days postvaccination with 106PFU.Serum samples were collected at 14dpb,and total antibody titers of these samples were measured by ELISA coated with MuV viral pro-
teins.
FIG 4Generation of recombinant MuV lacking V and SH proteins (rMuV ?SH ?V).(A)Schematics of pMuV ?SH ?V.A 156-bp section was removed from the SH gene of pMuV ?V,a cDNA genome of mumps virus lacking expression of V protein.(B)Reverse transcription-PCR con?rmed the mutation in the SH ORF in rescued rMuV ?SH ?V.Recombinant viruses (rMuV ?SH ?V)were rescued from pMuV ?SH ?V through transfection of BSRT-7cells with pMuV ?SH ?V,together with the helper plasmids (pCAGGS-L,pCAGGS-NP,and pCAGGS-P).RNA was extracted from rMuV ?SH ?V-infected Vero cells.Two primers,PX47F and PX48R (sequences are available upon request),were used to amplify the SH gene region.(C and D)Sequence con?rmation of the mutated regions in the SH ORF and the V/P editing site.The reverse transcription-PCR product of the SH gene was sent for sequencing.Sequencing results con?rmed the mutation was successfully introduced into rMuV ?SH ?V.(E)Western blot con?rmation of the deletion of V and SH proteins in rMuV ?SH ?V viruses.Vero cells were mock infected or infected with rMuV or rMuV ?SH ?V at an MOI of 0.5.Cell lysates were collected at 48hpi and were blotted against MuV NP,P,V,and SH proteins.
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dividual rescues,designated PX64-N,were compared by multicycle growth kinetics in Vero cells.While most rMuV?SH?V viruses reached the peak virus titer within the?rst72h postinfection(hpi), PX64-4grew slower than the others and peaked at96hpi.Three strains(PX64-4,PX64-61,and PX64-67)grew to a titer close to107 PFU/ml,and the other three strains(PX64-1,PX64-2,and PX64-81) had titers ranging from5.5?105to6?105,1to1.5logs lower than the former strains(Fig.5A).PX64-67had a growth pattern similar to that of full-length rMuV(virus titer peaking during?rst48hpi)and the highest virus titer among rMuV?SH?V viruses.Therefore,it was chosen and designated rMuV?SH?V for the following studies.Pre-viously,whenweobtainedrMuV?V,mutationsinregionsotherthan the designed V/P editing site always arose.The entire genome of the rescued rMuV?SH?V viruses were sequenced to determine whether genome-wide mutations occurred during virus rescue.PX64-67con-tained an additional single-nucleotide change(C-T)in genomic po-sition1913(termed1913C-T)(NP gene end)and7894T-A silent (HN ORF)mutations compared to rMuV.To compare the growth of rMuV?SH?V to rMuV and parental viruses(rMuV?SH and rMuV?V),Vero cells were infected with rMuV,rMuV?SH, rMuV?V,and rMuV?SH?V at an MOI of0.01.While rMuV?SH showed growth kinetics comparable to those of rMuV,consistent with previously published data(21),rMuV?V and rMuV?SH?V were about a half log lower in virus titer.The virus titer of rMuV?SH?V decreased after48hpi,remaining about a half log lower than that of rMuV?V and one log lower than that of rMuV or rMuV?SH(Fig.5B).
Intracellular viral protein expression of rMuV?SH?V was compared to that of rMuV.NP,P,and V protein expression levels were examined(Fig.5C).Comparable NP and P protein levels were detected in Vero cells infected with rMuV or rMuV?SH?V. However,secretion of infectious viral particles of rMuV?SH?V-infected Vero cells was less than that of rMuV-infected Vero cells at all time points postinfection(Fig.5C).At48hpi,a more intense P protein band was observed in rMuV?SH?V-infected cells than in rMuV-infected cells,consistent with a previous report of higher P expression at early time points in rMuV?V viruses,a likely result of increased P transcription from the V/P gene due to deletion of the V mRNA transcript(36).Expression of the V protein was only detected in rMuV-infected cells.
Maintenance of V and SH protein deletion in rMuV?SH?V through10passages in Vero cells.To examine the stability of rMuV?SH?V,it was passed in Vero cells continuously for10 passages at a low MOI.At passage10(rMuV?SH?V P10),the culture medium from infected Vero cells was used for viral RNA extraction,followed by whole-genome sequencing to determine the consensus genome sequence.Sequencing results revealed3 additional mutations:1silent mutation in the HN ORF,an R154K mutation in P,and an N2063H mutation in L.Interestingly,the G-A nucleotide mutation at position2445in the P ORF is the?rst nucleotide of the6-guanine editing site(GGGGGG)of the V/P gene,which has been altered to GAGGAGGG in rMuV?SH?V and rMuV?V viruses(36).Importantly,none of these mutations affected deletion of V or SH.
Furthermore,10single plaques(designated SP-1to SP-10) were obtained from rMuV?SH?V P10,and the V/P and SH gene regions were sequenced(Table1).The V protein and the SH ORF deletion were maintained in all10progeny strains from passage 10,including the1913C-T mutation in the NP gene end region. While9out of10strains contained the2445G-A mutation in the V/P gene editing site,1strain lost/failed to retain this mutation.To con?rm that the2445G-A mutation had no effect on the V pro-tein deletion,expression of the V protein in SP-1-to SP-10-in-fected Vero cells was examined by Western blotting(Fig.6).No expression of V was detected,indicating that the mutation had no effect on V protein deletion.
Intramuscular immunization of BALB/c mice with rMuV?SH?V generated an antibody response against MuV.BALB/c mice were i.m.vaccinated with rMuV,JL,rMuV?SH,rMuV?V,or
FIG5Analysis of rMuV?SH?V in tissue culture cells.(A)Multicycle growth
rate of rMuV?SH?V.Vero cells were mock infected or infected with
rMuV?SH?V viruses from different rescues(PX64-1,PX64-2,PX64-4,PX64-
61,or PX64-67)at an MOI of0.1.Supernatants collected from culture me-
dium of infected cells at1,2,3,4,5,and6dpi were plated onto Vero cells for
plaque assay,from which virus titer was calculated.(B)Multicycle growth
curve of rMuV?SH?V virus compared to parent viruses.PX64-67was se-
lected for subsequent animal experiments.In vitro growth of rMuV?SH?V
(PX64-67)was compared to rMuV,JL,rMuV?V,and rMuV?SH in Vero cells
as described for panel A.(C)Viral protein expression levels of rMuV?SH?V at
different time points postinfection.NP,P,and V protein expression levels in
rMuV?SH?V-infected Vero cells(MOI of0.01)were examined from1to4
dpi and compared to those of rMuV infection.
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rMuV?SH?V as described above.Serum samples were collected as described previously.Serum IgG antibody titers against MuV were measured using ELISA with plates coated with MuV viral proteins(lysed virions).Neutralizing antibody titers against MuV were measured by an rMuV-Luc-based neutralization assay as de-scribed in Materials and Methods(linear correlation of rMuV-Luc-based neutralization assay to traditional plaque reduction neutralization assay was con?rmed by an R2value of0.9317using ferret serum samples[data not shown]).Similar approaches have been used for adenovirus,measles virus,and respiratory syncytial virus(RSV)to substitute for the traditional plaque reduction neu-tralization test(PRNT)(44–47).We used rMuV-Luc,which was constructed based on the genetic background of MuV,as the tar-geting virus to compare the potentials of humoral responses in-duced by the vaccine candidates as well as the JL strain in mice to protect against the circulating mumps virus in the United States. No signi?cant differences were detected among the groups for
total antibody titers(Fig.7A).However,the neutralizing antibody titers of serum samples showed some differences.The average neutralizing titer of JL-inoculated mice was signi?cantly lower than that of rMuV-infected mice,which had the highest neutral-izing titer.Mice inoculated with rMuV?SH,rMuV?V,or rMuV?SH?V had similar average titers(Fig.7B).
Intranasal immunization of BALB/c mice with rMuV?SH?V generated an antibody response against MuV.BALB/c mice were i.n.inoculated with rMuV,JL,rMuV?SH,rMuV?V,or rMuV?SH?V at106PFU,and serum samples were collected for measurement of both total antibody titer and neutralizing titer against MuV.rMuV?SH-inoculated mice developed the highest total antibody titer,and the rMuV group had a higher titer than the JL group.No signi?cant differences were detected among the JL group,rMuV?V group,and rMuV?SH?V groups(Fig.8A). The rMuV group had the highest neutralizing titer,the JL group had the lowest neutralizing titer,and the other three groups (rMuV?SH,rMuV?V,and rMuV?SH?V)ranked between them (Fig.8B).Although i.n.-immunized mice exhibited neutralizing antibody titer patterns like those observed in the i.m.groups,sta-tistically signi?cant differences were found between the rMuV and JL groups(P?0.001),JL and rMuV?SH groups(P?0.001),JL and rMuV?SH?V groups(P?0.038),and rMuV?V and rMuV?SH groups(P?0.034).
Adaptive T cell responses were induced in mice vaccinated with rMuV?SH?V.To investigate the cellular immune re-sponses induced by rMuV?SH?V,i.m.-or i.n.-inoculated mice were euthanized at28dpb and splenocytes were isolated for ELISPOT assay.In i.m.-inoculated mice,the JL group had the highest T cell response levels and rMuV?SH?V had the lowest T cell response levels,with no distinguishable differences among the rMuV,rMuV?SH,and rMuV?V groups(Fig.9A).Differences were signi?cant between JL and rMuV?SH,JL and rMuV?SH?V, rMuV?SH and rMuV?SH?V,and rMuV?V and rMuV?SH?V. In i.n.-inoculated mouse groups,rMuV-and rMuV?V-immu-
TABLE1V/P gene and SH gene sequences of rMuV?SH?V P10single plaque-puri?ed viruses a
Virus V protein
deletion
NP GE or V/P GS
mutation
V/P editing site
mutation
SH ORF
deletion
SP-1Yes1913C-T2445G-A Yes
SP-2Yes1913C-T2445G-A Yes
SP-3Yes1913C-T2445G-A Yes
SP-4Yes1913C-T2445G-A Yes
SP-5Yes1913C-T 1578A-C Yes
SP-6Yes1913C-T2445G-A Yes
SP-7Yes1913C-T2445G-A Yes
SP-8Yes1913C-T2445G-A Yes
SP-9Yes1913C-T2445G-A Yes
SP-10Yes1913C-T2445G-A Yes
a rMuV?SH?V was passed continuously in Vero cells for10passages.Ten plaques were randomly obtained from rMuV?SH?V at passage10and grown in Vero cells(SP-1to SP-10).The V/P and SH gene regions of SP-1to SP-10were sequenced.Mutations found in these regions are shown.
FIG6Lack of V expression in isolates from the10th passage of rMuV?SH?V. Vero cells were mock infected or infected with rMuV or SP-1to SP-10.One hundred?l of infected Vero cell lysates was subjected to Western blotting to detect NP and V protein expression levels.FIG7Evaluation of antibody responses in mice i.m.vaccinated with rMuV?SH?V.BALB/c mice were i.m.immunized with PBS,rMuV,JL, rMuV?SH,rMuV?V,or rMuV?SH?V at106PFU/mouse and boosted at22 days postvaccination with the same virus at106PFU/mouse.(A)ELISA results measuring total antibody titer at14dpb.Serum samples were collected at14 dpb.The total antibody titer against MuV was measured through ELISA.(B) Neutralizing antibody titer at14dpb.Neutralizing antibody titers in serum samples collected at14dpb were measured through an rMuV-Luc-based neu-tralization assay.P values of?0.05are shown.
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nized mice had the lowest responding cell counts (Fig.9B ).Signif-icant differences were observed between JL and rMuV ?V,rMuV and rMuV ?SH,rMuV and rMuV ?SH ?V,rMuV ?SH and rMuV ?V,and rMuV ?V and rMuV ?SH ?V groups.
DISCUSSION
The JL vaccine is one of the most successful vaccines developed during the third quarter of the last century.It was produced by the propagation of mumps virus in embryonated hen’s eggs that re-sulted in attenuation (48–51).Introduction of the in vitro tissue/cell culture technique into vaccinology facilitated the develop-ment and production of the majority of currently licensed live-attenuated vaccines in the United States against viral infections (52–56).For mumps vaccine candidates,JL is the great success,but unfortunately there were many failures.Different passages of attenuated viruses were tested in animal models or in ?eld trials in order to select a vaccine seed with the most reduced virulence and greatest immunogenicity (1).Selected vaccine candidates need to be biologically characterized in order to be distinguished from
virulent strains.There are currently no standardized attenuation markers for mumps vaccines,partially due to the semirational and semiempirical nature of the traditional attenuation method (1,57–60).The rate of aseptic meningitis following vaccination with JL (estimated one case per 1.8million doses)is below background levels (61).However,other live attenuated mumps virus vaccines have had much higher incidences of vaccine-associated meningi-tis.The Urabe vaccine,which was widely distributed in Japan,Europe,and Canada,is estimated to cause one case of meningitis in every 1,000to 11,000doses distributed in the United Kingdom and one case of meningitis in every 62,000doses distributed in Canada.The Urabe vaccine has been withdrawn due to safety concerns.
In this study,based on the establishment of reverse genetic technology of negative-sensed,nonsegmented RNA
viruses
FIG 8Evaluation of antibody responses in mice i.n.vaccinated with rMuV ?SH ?V.BALB/c mice were i.n.immunized with PBS,rMuV,JL,rMuV ?SH,rMuV ?V,or rMuV ?SH ?V at 106PFU and boosted at 22days postvaccination with the same virus at 106PFU.(A)ELISA results measuring total antibody titer at 14dpb.Serum samples were collected at 14dpb.Total antibody titers against MuV were measured through ELISA.(B)Neutralizing antibody titer at 14dpb.Neutralizing antibody titers in serum samples col-lected at 14dpb were measured through an rMuV-Luc-based neutralization assay.P values for signi?cantly different groups were found for rMuV and JL groups (0.001),rMuV and rMuV ?V groups (0.016),JL and rMuV ?SH groups (0.001),JL and rMuV ?SH ?V groups (0.038),and rMuV ?V and rMuV ?SH groups (0.034).For simplicity,P values comparing the rMuV and JL groups and JL and rMuV ?SH ?V groups are shown.
FIG 9Cellular immune responses induced by rMuV ?SH ?V vaccination in mice.(A)Memory T cell responses in mumps virus i.m.-immunized mice.BALB/c mice were i.m.immunized with PBS,rMuV,JL,rMuV ?SH,rMuV ?V,or rMuV ?SH ?V at 106PFU and boosted at 22days postvaccination with the same virus at 106PFU.Splenocytes were extracted from mouse spleens and used for ELISPOT assay.Splenocytes were stimulated with MuV-infected Vero cell lysates or with mock-infected Vero cell lysates at 50?g/ml.P values of ?0.05are shown.(B)Memory T cell responses in mumps virus i.n.-immu-nized mice.BALB/c mice were i.m.immunized with PBS,rMuV,JL,rMuV ?SH,rMuV ?V,or rMuV ?SH ?V at 106PFU and boosted at 22days postvaccination with the same virus at 106PFU.Splenocytes were extracted from mouse spleens and used for ELISPOT assay.Splenocytes were stimulated with MuV-infected Vero cell lysates or with mock-infected Vero cell lysates at 50?g/ml.P values of ?0.05are shown.
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(62–66),we examined the possibility of generating a new mumps vaccine candidate through deletion of V and SH protein expres-sion from a clinical isolate from a2006Iowa mumps epidemic (MuV,genotype G).Deletion of either of the proteins has been previously reported to reduce mumps neurotoxicity in IC-in-fected rats.Importantly,since deletion of the V protein alone (from the MuV strain)is suf?cient to reduce the neurovirulence potential of the recombinant virus to a level comparable to that of the JL vaccine(21,36),the lack of V protein expression can be used as an attenuation marker for our vaccine candidates.
Attenuation based on targeted genetic modi?cation has several advantages.The most commonly used mumps vaccine,JL,is a mixture of at least two well-distinguished components(67–69). Surveillance of the compositional balance between the two com-ponents during vaccine preparation and propagation has been proposed(68).However,cDNA-derived recombinant viruses have de?ned consensus sequences and higher homogeneity.They are plaque puri?ed,whole genome sequenced,and passed in Vero cells only for the purpose of ampli?cation.Vaccine candidates can be continuously rescued from the cDNA plasmids with a de?ned consensus sequence and clear genetic markers for attenuation.All processes are cell culture based,bypassing the necessity of high-quality pathogen-free chickens,chicken eggs,or any other animals used for in vivo adaption(70–72).Omission of the serial passages saves time for vaccine development and avoids potential adap-tion-induced antigen shifts of the vaccine strains;therefore,it re-tains the maximum amount of immunogenic epitopes.
Mumps viruses are classi?ed into12genotypes based on ge-netic variability of the SH gene(73,74).Different subtypes of mumps viruses exhibit distinguished geographic distribution worldwide.Although the driving force of such distribution re-mains unclear,emergence of new subclusters of circulating mumps viruses within a genotype(75,76)indicates evolution of wild-type mumps viruses under various selection forces.Failure to detect genotype A wild-type mumps viruses in countries/re-gions immunized with genotype A vaccine in recent studies may be due to a vaccine-based selection pressure.This pressure may select for genotypes with increased virulence and heterogeneity compared to current vaccines(77–82).Decreased neutralization capabilities against heterogenotypes among subtypes of mumps viruses(83–86)and lack of cross protection between different subtypes(genotype D against genotype A)in human natural in-fection have been reported(87).It would be ideal to use a geno-type-matched vaccine candidate(genotype G),which elicits more speci?c immune responses that effectively protect against the cir-culating mumps viruses in the United States(genotype G)(11,13, 78,79).
Although additional mutations occurred during rMuV?SH?V virus rescue and during passages of rMuV?SH?V in Vero cells,no regaining of the V protein or the SH protein was observed in any rMuV?SH?V viruses analyzed,indicating that rMuV?SH?V is stable in tissue culture cells.Interestingly,besides two silent mu-tations in the HN and L ORFs,rMuV?SH?V(PX64-67)pos-sessed one nucleotide change(C-T)in genomic position1913in the V/P GS region,which has been previously seen in the course of rMuV?V virus rescue(36).This mutation is believed to be impor-tant in regulating the transcription/translation level of P protein, emphasizing the signi?cance of a proper ratio between NP and P protein during virus growth.
One challenge of developing a new mumps vaccine is the lack of correlation between protection and immune responses.While a neutralization titer is thought to be essential in protection against mumps infection(79),investigations of serum samples of patients versus nonpatients during recent mumps outbreaks revealed no de?ned cutoff neutralizing antibody titer against mumps virus, indicating a potential role for cellular immunity in effective pro-tection against mumps challenge(41,86).In this study,the inves-tigation of immunogenicity of rMuV?SH?V in i.n.-and i.m.-vaccinated mice showed that rMuV?SH?V was able to induce a neutralizing titer comparable to those induced by rMuV?SH and rMuV?V and a higher titer than that induced by JL vaccine.Fur-thermore,rMuV?SH?V vaccination also stimulated T cell re-sponses in mice,although the role of cell-mediated immunity in mumps disease protection remains to be demonstrated.We also observed that rMuV?SH induced slightly higher total antibody titers than those induced by rMuV,and rMuV?SH?V induced higher antibody titers than those induced by rMuV?V,suggesting that deletion of SH leads to better antigen presentation.Similar results have been reported for a closely related virus,parain?uenza virus5(PIV5),in which PIV5lacking SH is more immunogenic than PIV5(88).The mouse models have been widely used to test vaccine ef?cacy for various human viruses(89–92).However,it is not a good model for mumps virus infection.The ef?cacy of rMuV?SH?V in nonhuman primates,which is a good model for mumps virus infection(40),should be examined before testing this candidate in humans.In summary,rMuV?SH?V was able to elicit both antibody and cellular responses against MuV in i.n.-and i.m.-vaccinated mice,providing a safe and immunogenic mumps vaccine candidate.
ACKNOWLEDGMENTS
We appreciate the comments,suggestions,and technical help from mem-bers of the He laboratory.We thank Paul Rota for providing the JL vaccine strain.We are grateful to Kaori Sakamoto for carefully reading the man-uscript prior to submission.
This work has been supported by a grant from the NIH(AI097368to B.H.).
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