Shedding Of Rotavirus After First Administration Of Lanzhou Lamb Rotavirus (LLR) Vaccine

BackgroundRotavirus (RV) is the most common identifiable cause of acute gastroenteritis in children shortly after its discovery in1973. Children younger than5years old are mostly infected RV. It is responsible for an estimated39%of childhood diarrhea requiring hospitalization (range25-55%) and approximately611,000(range454,000-705,000) rotavirus-related deaths worldwide annually. Most of death happens in developing countries. China has the second largest birth cohort in the world and the second highest number of deaths due to RV infection. RV was detected in41%of stool specimens of children hospitalized with severe diarrhea in China. About17,000,000infants present with rotavirus gastroenteritis in China each year, resulting in38,405deaths (8%of world deaths).There is still no effective treatment specifically for rotavirus gastroenteritis. So it is important to develop RV vaccine. Lanzhou lamb rotavirus (LLR) vaccine (Lanzhou Institute of Biological Products, Lanzhou, China) isolated from a local lamb with diarrhea in1985, grown in primary calf kidney cells. After37generations, the LLR vaccine has proved to consist of monovalent serotype of G10P[15], group A. This RV vaccine was licensed formally for gastroenteritis prevention (group A rotavirus) among children in China in2000year. Since then nearly5,000,000children younger than5years old have been immunized. The vaccine is reported to induce neutralizing antibody responses in60%of patients but its vaccine efficacy (VE) is unknown since it was not tested against placebo in a controlled phase Ⅲ trial. A matched case-control study of838pair children conducted in2002-2004shows the VE against RV gastroenteritis is73.3%(95%CI, range61.2-81.6%).Oral attenuation vaccine or inactivation vaccine can have vaccine strains in the stools of inoculator after vaccined, this phenomenon calls vaccine deprived shedding. Rotavirus vaccines are all attenuation vaccine or inactivation vaccine, so they can also develop rotavirus vaccine shedding. The shedding rate, volume and time are different between rotavirus vaccines. Generally shedding occurres in day1-10after after vaccination, and shedding volume is about104-107PFU, so suggest that immunocompromi-sed and immunodeficiency people should not contact with rotavirus vaccined children in the first14days. The infectivity of old RRV-TV is higher than other rotavirus vaccines, children and the aged are more danger than adult.However, little data is available on the vaccine’s vaccine deprived shedding. We conducted this study is to detect the rate of rotavirus shedding in stools of immunized infants after the first dose of LLR following2weeks, and to provide data regarding transmission to household contacts (HHC).Subjects and methods1. Study populationThe study was a prospective trial conducted from September2011until October2012at Fengtai district Maternal and Child Health hospital (Beijing). With the parents or guardians informed consent, Infants between2-26months after the first dose of LLR were enrolled. Exclusion criteria were immunocompromised, receiving other vaccines within the past week, getting the symptom of fever or diarrhea within the past week and had rotavirus gastroenteritis before. The study was approved by the Administrative Panel on Human Subjects in Institute of Virus Control and Prevention(Chinese Center for Disease Control, Beijing).2. Vaccines and administrationThe LLR consists of monovalent serotype of G10P[15], group A. there were3.0ml pink color liquid each dose, contains lamb rotavirus>5.51gPFU/ml or1gCCID50. Infants aged2-36month should immunized LLR for3doses, in the same way as one dose one year. The immunization program was included with the normal childhood immunization schedule.3. study designPotentially eligible subjects were infants administrated the first dose of LLR at Fengtai district Maternal and Child Health hospital (Beijing). We got the information of the inclusion and exclusion criteria from parents or guardian face to face. After informed consent was obtained from the parents, they should fulfill a questionnaire at the guidance of a trained investigator. The questionnaire contains the information of infants, HHC numbers, address, telephone and information of stools collection for the following2weeks. Then parents got14plastic stools collection kits with instructions for stools collection. After infants discharged home, our investigator would go to their address every day in the following2weeks for stools collection, inquire whether vaccine adverse events of fever, irritability, vomiting and/or diarrhea happened in infants and record symptoms of fever, vomiting and diarrhea in HHC. Samples were then delivered to Institute of Virus Control and stored at-20℃.Data were entered into a study database for analysis.4. Analysis of stool specimensAll stools after administration of the first dose of LLR were initially tested for presence of rotavirus antigen sensitive EIA (ProSpecTM Rotavirus Microplate Assay, Oxoid Ltd, UK) at viral diarrhea study lab affiliated Institute of Virus Control and Prevention. The EIA used to detect rotavirus antigen in stool samples in this study had previously been described. This lab was the central lab used for viral diarrhea monitoring and Prevention in china. The EIA checking assay was a sensitive assay in an antigen capture EIA format that uses96-well test plates. EIA detection was following the specification of assay strictly. The absorbance of each stool sample was read using a spectrophotometer set at450nm. And the cut-off value is by adding0.2absorbance units to the negative control value. At least one positive control and one negative control were included on each plate to determine plate validity.5. Genotype by reverse transcription-polymerase chain reaction (RT-PCR)All stool samples of the EIA positive infants, including EIA negative samples of EIA positive infants and some samples of EIA negative infants that were selected randomly, were tested with RT-PCR to detect the genotype of shedding. Rotavirus double-stranded RNA (dsRNA) was extracted using the QuantiTech RNA kit (Qiagen, Valencia, USA). The extracted dsRNA was used as template for amplification. The primers for human rotavirus genotype checking were provided by viral diarrhea study lab affiliated Institute of Virus Control and Prevention. Two primers were designed to be specific for the VP7gene segment and VP4gene segment of strain LLR respectively for lamb rotavirus genotype checking. The forward and reverse primers used for the amplification of the partial VP7(G genotype) and VP4(P genotype) gene segments of the LLR strain were as outlined below. VP7, LLR-VP7F (forward primer),(5’-TGAGTGGACGAGTACATTGT-3’);LLR-VP7R(reverseprimer),(5’-AAT GGTCAAGTCGTGGTAG-3’);amplified fragment451bp.VP4, LLR-VP4F(forward primer),(5’-GAT GCT TCT AGC ACC AAC ATC AG-3’); LLR-VP4R (reverseprimer),(5’-CAG TGA ATG GAG TGA ACG AC-3’); amplified fragment513bp.These oligonucleotides were designed based on the sequences of the LLR VP7(Accession number;HM800948) and LLR VP4(Accession number;JQ013506). The following steps were performed:viral RNA was denatured at97℃for5min. Then, reverse transcription, followed by RT-PCR, were carried out using the Qiagen OneStep RT-PCR kit (Qiagen, Valencia, USA). The RT-PCR reaction was carried out with an initial reverse transcription step at42℃for30min, followed by PCR activation at95℃for15min, and40cycles of amplification with the following cycling conditions using a GeneAmp PCR system9700thermal cycler (AppliedBiosystemsGroup, Foster City, USA):30s at94℃,30s at55℃,60s at72℃, a final extension of7min at72℃, and a4℃cooling step. Amplicons were run in a1%agarose gel. The desired bands were then excised and purified with the QIAquickGel extraction kit (QIAGEN, Inc.,Valencia, USA) according to the manufacturer’s protocol and saved for sequencing. the RT-PCR products were sequenced by ruibiotech co.Ltd. Nucleotide sequence similarity searchesof the GenBank database (release143.0) were performed using the National Center for Biotechnology Information (NCBI, National Institutes of Health, Bethesda, MD, USA) Basic Local Alignment Search Tool (BLAST).6. Statistical analysisCumulative and daily proportions of fecal rotavirus vaccine virus shedding during the14days following vaccination with95%confidence intervals (CI) were calculated. Duration of shedding and peak shedding days within the14-day collection period were determined.Result1. DemographicsA total of121infants were enrolled,1infant was not contacted,5infants were exited with no stool specimen. At least1stool specimen was collected from115infants. Collections were made of1172specimens from these subjects. The male female ratio was1.12:1. 2. Rotavirus antigen screening39(3.3%) of1172stool specimens were rotavirus EIA-positive on1or more post-vaccination days. In the2weeks post-immunization,16/115(13.91%) infants had at least one rotavirus antigen (EIA)-positive stool. Shedding occurred as early as post-vaccination day1and as late as post-vaccination day13. Peak proportions of shedding occurred on post-vaccination days5through10, suggesting that shedding peaked time was Day5-10. Looking specifically at the shedding patterns of those infants who had EIA-positive specimens, the mean duration of shedding within the14days post-vaccination was3days;6(37.5%) infants had positive EIA-positive specimens on1day only,2(12.5%) infants on2days,1(6.25%) infant on3days and4day respectively, and3(18.75%) infants on5days and6days respectively. The OD value was read using a spectrophotometer set at450nm. The mean OD value was0.62, suggesting the virus valume of shedding was about7.8×105/ml according the introduction of EIA assay.3. RT-PCRGenetype was identified in all16EIA-positive subjects (177specimens) and7EIA-negative subjects (61specimens) by RT-PCR with primers specific for the VP7gene segment and VP4gene segment followed by nucleotide sequencing of the PCR amplification. There was no positive semple got in hunman rotavirus RT-PCR cchecking, no infants got human rotavirus infected after LLR vaccination. As for lamb rotavirus, the VP7sequence had a positivity rate of101/177(57.06%) and the VP4sequence had a positivity rate of103/177(58.2%) among the EIA-positive subjects, with111/177(62.71%) specimens being positive for both VP7and VP4sequence. In addition,61EIA-negative specimens selected from7EIA-negative infants were also negative for vaccine-type rotavirus by RT-PCR. In each case, the gene fragment tested was100%identical to the corresponding gene fragment of LLR, the parent strain of the monovalent rotavirus vaccine.4. Safety8/115(7%) infants developed with solicited adverse events of fever, irritability, vomiting and/or diarrhea in the2weeks post-immunization. Parents reported5episode of fever,3episodes of diarrhea and1episode of irritability. There were no episodes of intussusception in the2weeks post-immunization in the infants. Only one infant with solicited adverse events of fever was EIA-positive, suggestion no directly correlation in solicited adverse events and shedding. Both hospitalizations and medical histories were reviewed by the investigators and admitting neonatologist and were not considered to be related to vaccination. Only1/442HHC developed symptom of diarrhea during the2weeks post-immunization of study subjects. We collected7stool specimens of this infant in7days, the EIA and RT-PCR checking turned out negative.Conclusion(1) The shedding rate was13.9%in the14days fllowing the first dose of LLR, the95%confidence was8.1-21.6%. It was posibilty to develop rotavirus vaccine deprived shedding for infants in the14days, and the peak time of shedding was day5-10after vaccination. The durion shedding time was as short as one day, as long as six days, the mean shedding time was3days. The mean OD value was0.62by EIA checking, suggesting the virus valume of shedding was about7.8×105/ml.(2) Age was an important factor for shedding in this study. Shedding rate in Infants aged2-12months was20.5%, aged13-24months was13%, and aged24-36months was0. The shedding rate was decreased apparently as the age increment.(3) The shedding virus was LLR strain according to RT-PCR checking, genestype was G10P[15]. No infants got human rotavirus infected after LLR vaccination.(4) There were7%infants developed with solicited adverse events of fever, irritability, vomiting and/or diarrhea in the2weeks post-immunization. No directly correlation in solicited adverse events and shedding. Only1/442HHC developed symptom of diarrhea during the2weeks post-immunization of study subjects.(5) A new RT-PCR method to detect LLR srtain was created, as two primers were designed for VP7gene and VP4gene of lamb rotavirus. The specificity and sensibility of the new method were validated, the result were satisfactory.