this is a non - controlled , longitudinal study , carried out in a sample of 150 children 
 12 - 15 months of age in three health units of the municipality of rio de janeiro who 
 received the combined mmr vaccine according to the basic immunisation schedule and nip 
 routine procedures .
healthy male and female children , living in the city of rio de 
 janeiro , with no significant past medical history , were selected for this study .
the 
 criteria for not including into the study were : ( i ) subjects with a history of measles , 
 rubella and/or mumps , ( ii ) subjects who had already received mmr vaccination documented 
 in the vaccination card , ( iii ) subjects with a history of receiving blood transfusion or 
 blood products , including immunoglobulin in the past one year previous to the study , 
 ( iv ) subjects who presented skin lesions at sites of venipuncture and ( v ) subjects who 
 had used corticosteroids ( except topical or aerosol ) during the last six months or 
 reported use of immunosuppressive drugs .
two blood samples were collected : before and 42 
 days after vaccination ( minimum acceptable 30 days and maximum 60 days ) .
volunteers who 
 did not seroconvert from seronegative to seropositive or who had inconclusive results 
 for any of the antigens were vaccinated again and a third blood sample was collected 
 within the same time range recommended for the second blood collection .
medical records 
 were prepared using the teleform workgroup 2008 program , v.10.2 , which allowed capture 
 of the scanned data , without manual typing and creation of a database . 

vaccine used on the study - the mmr received in bulk from gsk , 
 formulated and distributed by bio - manguinhos , oswaldo cruz foundation ( fiocruz ) , single 
 lot ( 072vva007z ) , in 10 dose presentation , february/2007 production date and valid for 
 two years , was used on the study .
each 0.5 ml dose of reconstituted vaccine contained at 
 least 1,000 50% cell culture infectious dose ( ccid50 ) of attenuated measles 
 virus , schwarz strain , at least 1,000 ccid50 of attenuated rubella virus 
 wistar ra27/3 strain and at least 5,000 ccid50 of attenuated mumps virus , rit 
 4385 strain , derived from jeryl lynn strain .
for each component of the vaccine batch 
 used in the study , the potencies at temperatures varying from 2c to 8c were , in 
 log10 , 4.26 ( measles ) , 5.28 ( mumps ) and 4.02 ( rubella ) and , after storage 
 at 37c , 3.96 , 4.99 and 3.85 , respectively .
the vaccine administered into each volunteer 
 was diluted at the time of study enrollment , according to the nip procedures and each 
 volunteer received only the first dose from each vial . 

laboratory methods - blood samples were placed into an insulated box 
 kept between 4 - 10c from time of collection until arrival at the laboratory .
the maximum 
 time between blood collection and arrival at the laboratory was 6 h. igg antibodies against measles , mumps and rubella were determined at the reference 
 laboratory for measles and rubella , oswaldo cruz institute / fiocruz using an enzyme 
 immunoassay ( eia ) with a commercial kit from siemens ( enzygnost igg ) .
the 
 results for optical density were converted to international units or units per 
 millilitre of serum using a table provided by the manufacturer and categorised as 
 negative for rubella , mumps and measles if < 4.0 iu / ml , < 231 u / ml and < 150 
 miu / ml , respectively . these cut - offs were used in all studies referred to in the 
 bibliographical references .
additionally , results were categorised as seronegative , 
 inconclusive and seropositive , according to the kit instructions . at the end of 2010
, 
 samples were sent for retesting for the mumps component at the gsk biologicals 
 laboratory ( rixensart , belgium ) using the same methodology ( eia ) and diagnostic kit used 
 in the reference laboratory at fiocruz .
igm antibodies against mumps were measured after revaccination of children seronegative 
 after the first dose in order to detect primary immune failures . 

data analysis - a database for the study was created using the 
 statistical package for social sciences program v.17 .
the distribution of absolute and 
 relative frequencies of subjects were tabulated by sex , age group , health units , 
 interval between date of vaccination and date of blood sample collection .
immunogenicity 
 of the mmr vaccine was assessed primarily in terms of the percentage of baseline 
 seronegative children who seroconverted for antibodies against measles , mumps and 
 rubella viruses ( that is , developed antibody levels  cut - off for seropositivity after 
 vaccination ) .
criteria for protocol adherence were : children seronegative before 
 vaccination with available serologic test results before and after vaccination and blood 
 collected from 30 - 60 days after vaccination .
we constructed 95% confidence intervals 
 ( ci ) for proportions using winpepi ( abramson 
 2011 ) .
immunogenicity was also evaluated by geometric mean titre ( gmts ) after 
 vaccination and the magnitude of the immune response could be assessed against the 
 minimum antibody levels for seropositivity . 

ethical aspects - the mmr vaccine administered to volunteers was the 
 same used in the nip routine , having already gone through immunogenicity and 
 reactogenicity clinical studies before registration and use on a large scale .
the study 
 protocol was approved by the research ethical committee of the municipal health 
 secretariat of rio de janeiro ( protocol 48/08 ) .
from may - august 2008 , 165 children were enrolled , of which 150 were eligible and 146 
 ( 96.7% ) had blood samples obtained before and after vaccination .
there was a slight male 
 predominance ( 55% ) and most children ( 92.7% ) were from 12 - 15 months of age .
the 
 intervals between vaccination and blood sampling after vaccination ranged from 34 - 73 
 days and of 146 volunteers who had a second blood sample collected , 97.9% had intervals 
 from 30 - 60 days . before vaccination
, there was one child seropositive for measles , one 
 for mumps and one for rubella and 143 children were susceptible to measles , mumps and 
 rubella .
according to the kit instructions and in children with adherence to protocol , 86 ( 60.1% ) 
 were seropositive for mumps , 42 ( 29.4% ) inconclusive and 15 ( 10.5% ) seronegative . for 
 all children , with exception of one child seropositive to mumps before vaccination , 88 
 ( 60.7% )
were seropositive and the number with negative or inconclusive results after 
 vaccination was 57 ( 39.3% ) . considering 231 u / ml as the cut - off , in children with 
 adherence to protocol , seroconversion for mumps after first vaccination was 89.5% ( 95% 
 ci : 83.3 ; 94.0 ) and 130/145 , 89.7% ( 95% ci : 83.5 ; 94.1 ) , for all available children who 
 were seronegative before vaccination ( table 
 i ) . 

table iresults of mumps serology ( enzyme immunoassay ) after the first dose of 
 combined vaccine against measles , mumps and rubellaigg serologycut - off 231 u / ml n ( % ) according to kit n ( % ) negative15 ( 10.5)15 ( 10.5)inconclusive-42 ( 29.4)positive128 ( 89.5)86 ( 60.1 ) 

 total143 ( 100)143 ( 100 ) 
  post - vaccination gmts were 2,234 ( 95% ci 2039.4 ; 2447.7 ) mui / ml for measles , 596.6 ( 95% 
 ci 517.2 ; 688.3 ) u / ml for mumps and 50.1 ( 45.1 ; 55.8 ) iu / ml for rubella .
when contrasted 
 with cut - off values for seropositivity , the gmts indicated considerably larger magnitude 
 of the immune response for rubella ( cut - off : 4.0 iu / ml ) neither health unit nor time of blood collection after vaccination was relevant 
 regarding immunogenicity ( data not shown ) .
of the 58 children seronegative or with inconclusive serology for mumps after 
 vaccination , 57 were eligible for revaccination ( 1 child was excluded due to a 2nd dose 
 of mmr vaccine received during a mmr campaign , registered on the vaccination card ) . 

blood samples were collected after the second vaccine dose in 54 children ( 94.7% ) and 
 were not collected in three children due to parent / tutor refusal .
the interval between 
 vaccination and revaccination ranged from 203 - 249 days , with a mean of 221 days 
 [ standard deviation ( sd ) : 11.6 ] and a median of 220 days .
the interval between 
 revaccination and third blood collection ranged from 31 - 64 days , with a mean of 39 days 
 ( sd : 6.2 ) and a median of 37 days .
after revaccination , all children had high igg titres for mumps , above 1,200 u / ml , with 
 exception of one child , who had a titre of 457 u / ml . measles and rubella antibody titres 
 showed a modest rise ( table ii ) . 

table iiigg ( elisa ) geometric mean titres ( gmt ) after vaccination and revaccination 
 and revaccination / vaccination ratios , for measles , mumps and rubellaigg titresnafter 
 vaccination gmt ( 95% ci)after 
 revaccination gmt ( 95% ci)gmt 
 ratio revaccination / vaccination ( 95% ci)measles ( miu / ml ) 
 542,155.5 ( 1,828.0 ; 
 2,541.6)5,692.1 ( 4,815.5 ; 
 6,728.4)2.6 ( 2.3 ; 
 3.1)mumps ( u / ml )  54247.6 ( 214.3 ; 
 286.0)3,157.0 ( 2,684.9 ; 
 3,712.0)12.8 ( 10.3 ; 
 15.8)rubella ( ui / ml ) 
 5441.3 ( 33.8 ; 
 50.4)151.0 ( 134.2 ; 
 169.9)3.7 ( 3.0 ; 
 4.5 ) 
  moreover , after revaccination , igm for mumps was negative in 52/54 ( 96.3% ) children and 
 in two children results were inconclusive ; for measles , 50/54 ( 92.6% ) were igm negative 
 and four were inconclusive ; for rubella , 53/54 ( 98.1% ) were igm negative and one was 
 inconclusive . due to the high percentage of negative and inconclusive results for mumps after the 
 first mmr vaccination , according to the kit instructions , the pre and post - vaccination 
 samples were sent for blind retesting for mumps at the gsk in rixensart , belgium . 

mumps 
 immunogenicity has been highly variable across mmr studies with features similar to the 
 current study , that is , with the same jeryl - lynn based vaccine from gsk , after the first 
 dose ( mmr used alone or with simultaneous administration of varicella vaccines ) , with 
 similar age at vaccination , using the same enzygnostkit and  231 u / ml 
 cut - off for seropositivity . of note ,
all assumed  231 u / ml for mumps indicated seropositive 
 results ( usonis et al .
studies from 1999 - 2002 presented the highest immunogenicity . a study in 
 2011 in germany showed a gmt for mumps of 523.7 u / ml , with 71.3% seroconversion ( rmke et al .
efficacy trials represent the  best scenarios  of vaccine performance under controlled 
 conditions and are commonly required before a new vaccine is licensed .
they are measured 
 usually as immune responses and when there are correlates of protection ( that is , a 
 cut - off of antibodies above which there will be protection against the disease ) , it is 
 possible to estimate vaccine effectiveness from immunogenicity data .
this is the case 
 for measles and rubella . in the case of mumps , there are no correlates of protection , so 
 it is not possible to estimate effectiveness - that is , the magnitude of reduction of 
 disease rates attributable to vaccination under real life conditions .
many studies 
 evaluated effectiveness of the mumps component in populations vaccinated with jeryl - lynn 
 based vaccines .
the recent mumps outbreak in new york and new jersey reported by the 
 centers for disease control and prevention ( cdc 
 2010 ) estimated a variation on mumps vaccine effectiveness from 73 - 91% after 
 one dose and from 79 - 95% after two doses .
even so , overall vaccine effectiveness is not 
 questioned , as the annual number of mumps cases in the united states of america 
 decreased from 186,000 in 1967 , when the vaccine was introduced , to less than 500 , in 
 the early 2000s .
waning immunity , that is , decrease on seroprotection since time of last 
 vaccination , seems to be an important factor for vaccination failure , both for one and 
 two - dose vaccinees .
waning may also partly explain why vaccination effectiveness has 
 been in general lower than efficacy , which is usually assessed after shorter 
 follow - up .
there is a trade - off between mumps vaccine immunogenicity and reactogenicity , mainly 
 regarding aseptic meningitis . clearly , the jeryl lynn based vaccines are the safest , 
 although probably not the most immunogenic .
this evaluation should be done by each 
 country , according to epidemiological considerations and degree of tolerance for adverse 
 events .
it should be noted that brazil has had a negative experience regarding adverse 
 events with mmr campaigns using mumps strains other than jeryl - lynn ( dourado et al .
the strong response to mumps revaccination on the current study , with a very high after 
 revaccination / after vaccination gmt ratio , suggests insufficient power of this vaccine 
 to induce strong mumps immune response after one dose , but the booster response is 
 reassuring concerning seroconversion after two doses .
moreover , after revaccination , 
 96.3% of children were igm negative for mumps , again suggesting a secondary immune 
 response , with the caveat that the blood collection was taken from one - two months after 
 revaccination , when igm levels are expected to be on the decrease .
however , high igm 
 levels after disease in unvaccinated subjects are maintained for several weeks or months 
 ( cdc 2012 ) .
assessment of igm levels after mmr 
 first dose was found in only one bibliographical reference , using the hoshino mumps 
 strain , the elisa ibl kit , with blood collected four - seven weeks after vaccination and 
 igm seropositivity was found in 71% of children ( tabatabaei 2013 ) .
limitations in the accuracy of the mumps laboratory test , particularly its sensitivity 
 ( 81% ) , may have contributed to the suboptimal mumps immunogenicity results ( backhouse et al .
these considerations assumed no relevant virus circulation , which seemed reasonable , 
 even though reporting of mumps cases is not mandatory in brazil , except outbreaks , which 
 are usually perceived in health care units and are reported on the national notification 
 system , which was not the case .
the results of this study are in agreement with two previous studies done by our group , 
 the first already referred ( silva et al .
2011 ) , 
 which included 1,769 children , and a second not yet published , but with final report 
 approved , which included 183 children 12 - 18 months of age , using the same methodology 
 and cut - offs . in the absence of accepted correlates of protection for mumps ,
no definite statements 
 regarding protection against disease can be derived from the results of this study . 

however , the data here provided strengthen the need of a second mmr dose to ensure 
 maximum protection against mumps .
serological and epidemiological studies after two 
 doses should be implemented to know if further doses and at which intervals are 
 needed .
data from the current study confirm the high immunogenicity of the mmr measles and 
 rubella components with one dose and suggest lower immunogenicity of the mumps 
 component . after a second dose ,
the main and immediate implication of these findings is the reassurance 
 that two mmr doses are highly immunogenic for all antigens included on the vaccine .
as 
 there is no serological correlate of protection for mumps , the implications of 
 immunogenicity data should be considered cautiously , but they may be useful regarding 
 immunisation practices and guidance , taking into account other variables , such as 
 epidemiological data .
 a non - controlled longitudinal study was conducted to evaluate the combined vaccine 
 against measles , mumps and rubella ( mmr ) immunogenicity in 150 children vaccinated in 
 the routine of three health units in the city of rio de janeiro , brazil , 2008 - 2009 , 
 without other vaccines administered during the period from 30 days before to 30 days 
 after vaccination . 
 a previous study conducted in brazil in 2007 , in 1,769 children 
 ranging from 12 - 15 months of age vaccinated against yellow fever and mmr 
 simultaneously or at intervals of 30 days or more between doses , had shown low 
 seroconversion for mumps regardless of the interval between administration of the two 
 vaccines . 
 the current study showed 89.5% ( 95% confidence interval : 83.3 ; 94.0 ) 
 seroconversion rate for mumps . all children seroconverted for measles and rubella . 
 
 after revaccination , high antibody titres and seroconversion rates were achieved 
 against mumps . 
 the results of this study and others suggest that two mmr doses confer 
 optimal immunoresponses for all three antigens and the possible need for additional 
 doses should be studied taking into account not only serological , but also 
 epidemiological data , as there is no serological correlate of protection for 
 mumps . 