RotaTeq® (Merck & Co., Whitehouse Station, NJ) is a live oral rotavirus (RV) vaccine consisting of 5 human-bovine reassortant vaccine viruses. In Finland, RotaTeq vaccine was taken into the national immunization program in September 2009, and is administered on a 3 dose schedule at the ages of 2, 3 and 5 months.
Shedding of RotaTeq vaccine viruses was reported low in the prelicensure trials,1 but on a recent reanalysis of the Rotavirus Efficacy and Safety Trial (REST) study material using reverse transcription-polymerase chain reaction (RT-PCR), shedding of RotaTeq vaccine viruses was detected in up to 65% of the vaccinees with gastroenteritis symptoms.2 Thus, the full extent of shedding of RotaTeq vaccine viruses is not yet characterized. We examined the occasional presence of RotaTeq vaccine viruses in young children seen in hospital mainly for respiratory tract infection (RTI) and correlated these findings with history of RV vaccination. This approach yielded new information on the extent, duration, and type of RotaTeq vaccine virus shedding.
MATERIALS AND METHODS
A prospective study approved by the Ethics Committee of Pirkanmaa Hospital District on the etiology of RTI in children was conducted at Tampere University Hospital from September 1, 2009 to August 31, 2011. All children under 15 years of age, who were admitted into pediatric ward with RTI were eligible for the study.
In this study a stool sample was collected during the hospitalization by a study nurse or nurses working in the pediatric ward or, if not successful, the parents were provided with a sample kit to send a stool specimen within 2 weeks from home. We used this material to examine the presence of rotaviruses and specifically RotaTeq vaccine viruses and therefore excluded all children who had not received any dose of RotaTeq. Dates of administration of RV vaccine brand as well as the vaccine used was enquired from the parents and confirmed from the records of the respective well baby clinic by a study nurse.
Stool samples were stored in freezers at −70°C. Viral RNA was extracted using Qiagen QIAamp Viral RNA Mini Kit (Hilden, Germany) according to the manufacturer’s instructions. RV viral proteins VP7, VP4 and VP6 PCRs, enzyme-linked immunosorbent assay (ELISA) test and sequencing of PCR positive samples were performed as previously reported.3
Positive stool samples were propagated in fetal rhesus monkey kidney (MA104) cells as described previously3 with the modification of using minimum essential medium with 0.5 μg/mL of trypsin instead of minimum essential medium containing 100 U/mL penicillin, 100 U/mL L-glutamine and 100 μg/mL streptomycin.4
During the 2-year-study period a total of 944 children with RTI were recruited into the study and 557 (59.0%) children provided a stool sample. A total of these children, 182 (32.7%) had ever been vaccinated with RotaTeq at any time after September 1, 2009, thus forming our study population. The mean age of the study population was 256 days, ranging from 57 to 643 days; 73.1% were males.
Out of the 182 stool samples, 30 (16.5%) were RV positive by RT-PCR specific for VP7. RV positive samples were identified as RotaTeq vaccine type by sequence analysis. VP6 RT-PCR was positive in 29 and negative in 1 of the 30 VP7 positive cases. Bovine vaccine type VP6 was detected in 28 cases and human type VP6 in 1 case, described later. VP4 RT-PCR was positive in 19 and negative in 11 cases.
RV antigen by ELISA was tested from 19 of the 30 VP7 positive cases. ELISA could not be performed from the remaining 11 samples due to an insufficient amount or type of the sample (diaper). Out of those 19 samples, only 4 (16.7%) were ELISA positive. The ELISA positive cases were all detected with RotaTeq G1P double-reassortant; 1 child was also detected with an additional P VP4.
RotaTeq G1 sequence was detected in 28 cases (93.3%) out of 30 VP7 positive cases. The double-reassortant combination G1P was detected in 11 children (36.7%). The original vaccine type G1 reassortant G1P was detected in 4 children (13.3%), and 1 child had RotaTeq G1 with both P and P. Also 11 children (36.7%) were detected with RotaTeq G1 alone (with no VP4 sequence detected). VP4 reassortant G6P was detected in 2 children. (Table 1)
One child was detected with human VP6 reassorted with RotaTeq G1 VP7 and human wild-type VP4 P. The stool sample of this child was obtained 10 days after the second dose of RotaTeq vaccine. The sample was extracted for several times, and RT-PCR and sequencing were done twice for each protein from each extraction, but the RT-PCR and sequencing results remained identical. The sample could not be propagated in MA104 cells.
A total of the 182 RV vaccinated children, 28 had received 1 dose, 38 had received 2 doses and 116 had received all 3 doses of RotaTeq vaccine at the time they were hospitalized. Shedding of RotaTeq vaccine virus was detected in 14/28 (50.0%) stool samples collected after the first and before the second dose. After the second and before the third dose vaccine virus shedding was detected in 10/38 cases (26.3%), and after the third dose in 6 out of 116 cases (5.2%).
RotaTeq G1P double-reassortant was commonly shed after each dose of the vaccine. After the first dose 8 children shed RotaTeq vaccine-derived G1P, and 3 children shed after the second and 1 child after the third dose. None of these children had diarrhea. The vaccine strains and combinations in relation to the latest vaccine dose are shown in Table 1.
The duration of shedding, as counted from the latest vaccination date, was over 14 days (prolonged) in 16 cases (53.3%) and over 30 days in 9 cases (30.0%). The proportion of long-time (over 14 days) shedders became larger after each dose of vaccine received; after the first dose prolonged shedding was detected in 4/14 (28.6%) children, respectively after the second 7/10 (70.0%) and after the third dose 5/6 (83.3%).
The longest duration of shedding was 84 days counted from the third immunization in a child detected with RotaTeq G1 VP7, while VP4 and VP6 RT-PCRs were negative. The other 5 cases after the third dose were detected 9, 22, 39, 52 or 53 days after the third dose, and the respective genotypes were RotaTeq G1 alone, G1P, G1 alone, G6P and G1 alone.
In this study we used children hospitalized for respiratory infection without gastroenteritis symptoms as proxies for healthy children to follow the shedding of RotaTeq vaccine viruses after routine vaccination. In these children under the age of 2 years who had received RotaTeq vaccine we detected the presence of RotaTeq vaccine viruses in 16.5%. This is higher than detected in the prelicensure studies using the plaque assay method on samples collected within a week after each dose,1,5,6 but largely in line with more recent studies, which have shown shedding rates of around 20% using RT-PCR or enzyme immunoassay as detection methods.7–9
The previous studies have not detected prolonged shedding on such a scale, although Hsieh et al10 detected RotaTeq strains up to 28 days after inoculation. Very long shedding, like our finding up to the age of 8 months, has not been described previously in healthy infants, but Patel et al.11 detected shedding over 200 days in immunocompromised children. We detected 6 children with prolonged shedding 9–84 days after the third dose of RotaTeq vaccine. However, it is not certain after which dose the shedding started in such cases. It is possible that our occasional sampling detected children who became long time shedders already after the first dose, in which case the longest duration of shedding might have been 6 months.
Dominance of RotaTeq G1 genotype in shedding was a new finding, as G1 was detected in 93.3% of the cases. Still, also in prelicensure studies of RotaTeq G1 and P were actually the most common genotypes shed after vaccination, and also the G1P combination was detected already in the early studies.12,13 In the composition of RotaTeq vaccine, the titer of G1 is nearly the same as that of G3 and G4, and lower than G2; therefore the higher shedding rate of RotaTeq G1 cannot be explained by a higher inoculum.14 In fact we did not detect any other vaccine-derived human G-types but G1 among the shedders. The only unifying factor with prolonged shedding was RotaTeq G1 VP7, with the exception of 2 cases with bovine–human G6P combination. The properties that make G1 such a predominant genotype in prolonged infection, and consequently shedding, remain unknown and need further study.
The major limitation of our study was the design of the original study, which was not planned to determine the rate or duration of shedding of RotaTeq vaccine, but only provided stool samples at random time points. However, even with this less than optimal study design we could determine that long term shedding of vaccine viruses is not uncommon. While we could not determine after which dose the long-term shedding started, it is reasonable to speculate that already the first dose may select those individuals who eventually become long-term shedders.
Shedding of RV vaccine strains in asymptomatic children is usually not regarded as clinically significant, with the possible exception of transmission to susceptible or immunocompromised contacts. The use of sensitive RT-PCR in detection of vaccine viruses has been criticized, and the plaque assay in cell culture defended, on the grounds that RT-PCR may not detect live infectious viruses but only parts (RNA) of the virus.2 However, a prolonged presence of the vaccine viruses even as detected by RT-PCR only is an indication of prolonged infection in the intestinal cells and might be associated with clinical consequences, although these are unknown as yet.
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2. Matson DO, Vesikari T, Dennehy P, et al. Analysis by rotavirus gene 6 reverse transcriptase-polymerase chain reaction assay of rotavirus-positive gastroenteritis cases observed during the vaccination phase of the Rotavirus Efficacy and Safety Trial (REST). Hum Vaccin Immunother. 2014;10:2267–2275
3. Hemming M, Räsänen S, Huhti L, et al. Major reduction of rotavirus, but not norovirus, gastroenteritis in children seen in hospital after the introduction of RotaTeq vaccine into the National Immunization Programme in Finland. Eur J Pediatr. 2013;172:739–746
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9. Donato CM, Ch’ng LS, Boniface KF, et al. Identification of strains of RotaTeq rotavirus vaccine in infants with gastroenteritis following routine vaccination. J Infect Dis. 2012;206:377–383
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14. Merck. RotaTeq (prescription information). 2007 Whitehouse Station, NJ Merck & Co Inc.