Causes of Rotavirus Vaccine Failure in Zambian Children

The study will be conducted at Kamwala health facility in Lusaka where the Maternal Child Health (MCH) and antiretroviral therapy (ART) clinics as well as the Centre for Infectious Disease Research Zambia (CIDRZ) Kamwala Research Unit are co-located. Kamwala has a catchment population of over 10,000 with approximately 30 new infants presenting to MCH each month with 6-14 week (DPT-HiB-Hep) immunisation rates above 95%. Mother-infant pairs will be approached during the initial visit to MCH by the study clinic assistant or peer counsellor with information about this study in the local language of choice. Those generally interested will be invited to the research clinic which is close by, for more detailed information during which motivated mothers will be recruited and taken through the written informed consent process by the study nurse. For illiterate participants, an independent, literate individual will witness and validate the informed consent process. A specific log will be maintained to show information on dates and numbers of those invited to the study, reasons for refusal documented.

A senior research nurse, study nurse, and clinical research assistant will be responsible for informed consent and enrolment procedures under the supervision of the investigator. A paediatrician will be available for hands-on consultation when needed.

Cohort Follow-up. Enrolled mothers will be asked to complete demographic and behavioural questionnaires, a participant locator form, and undergo phlebotomy and mechanical expression of breast-milk at the enrolment visit for determination of anti-rotavirus IgG and IgA (in serum and breast-milk). Infants will have a baseline anti-rotavirus IgA and IgG antibodies determined to ascertain baseline exposure to wild-type and transplacental immunoglobulin followed by a second serum IgA determination one month following completion of rotavirus vaccination (at approximately 14-20 weeks).

Infants will then be followed quarterly in-clinic for the remainder of the study and will also make interim clinic visits if the child has diarrhoea. At each visit, the child will be weighed, height measured, and anthropometrics taken, including skin fold for subcutaneous body fat and mid upper arm circumference. During any symptomatic interim visits, assessments will be made of disease severity (by Vesikari scale) and a sample of stool will be collected for determination of rotavirus antigen and genotype if rotavirus positive. The patient will also be sent home with a diarrhoea diary to be completed over the following two weeks. Following identification of an child with severe gastroenteritis, an age-matched control from within the cohort (without recent gastroenteritis) will be brought in for measurement of Vitamin A and serum zinc levels. Children with gastroenteritis will be treated according to national guidelines (per World Health Organization (WHO) Integrated Management of Childhood Illness). If referral is required, the child will be referred to outpatient clinic, or in serious cases the University Teaching Hospital.

Study Procedures. Same as above.

Laboratory Methods.

1. Detection of Rotavirus-specific IgA and IgG will occur by ELISA. Detection of rotavirus-specific IgA and IgG will occur by ELISA. Briefly, the procedure is as follows. Microplate wells (e.g., Nunc Immuno I) are incubated with purified viral antigen preparations, then washed with phosphate buffered saline with Tween detergent (PBS-T). Serum and breast-milk samples are diluted serially (in PBS-T containing 1% bovine serum albumin) and incubated in the coated microplates for 2 hours at 37oC then overnight at 4oC. Microplates are then washed and peroxidase-conjugated anti-human IgA or IgG goat antibodies (Sigma Immunochemicals) is added. The reaction is then terminated with H2SO4, and optical density is measured by EIA plate reader. All samples are assayed in duplicate against each viral antigen and against a control antigen. A test well was considered positive by its optical density at 450 nm, e.g., if it is greater than or equal to two times that of its own control. Specificity is controlled by including wells containing control antigen incubated in the absence of the sample, and sensitivity is checked by including wells containing viral antigen incubated in the presence of samples known to contain high titre of a particular rotavirus-specific immunoglobulin class.

2. Detection of Rotavirus Antigen will occur by Rotaclone ELISA. Stool samples will be tested for rotavirus by ELISA (Rotaclone®, Meridian Biosciences) according to manufacturer's specifications. Briefly, sample will be brought to room temperature and diluted with buffered saline with 0.02% thimerosol. Diluted sample and enzyme conjugate are then incubated with a monoclonal antibody to the VP6 protein within the Rotaclone microplate. Substrate buffers containing urea peroxide and tetramethylbenzidine are then added, and the reaction is terminated with H2SO4. Spectrophotometric determination of result is then made by measuring absorbance at 450 nm against an air blank. Specimens with absorbance units (A450) greater than 0.150 are considered positive. Those with absorbance equal to or below 0.150 are considered negative.

3 Determination of Rotavirus VP7 Genotype. Viral RNA is extracted from a 10% suspension of rotavirus antigen-positive faecal material in PBS (pH 7.0) using TRIzolTM Reagent (GIBCO Life Technologies) according to the manufacturer's protocol. Viral RNA is then taken through RT-PCR as described earlier.

Extracted viral RNA is then taken through nested PCR utilizing primers which are complimentary to the 3' ends of both viral RNA strands within gene segment 9, which encodes viral protein 7 (VP7). These primers are used for first strand synthesis. Primer RVG9 and six serotype-specific primers (aBT1, aCT2, aET3, aDT4, aAT8, and aFT9) which are located in six variable regions on the gene 9 and correspond to G-serotypes 1, 2, 3, 4, 8 and 9, are used in second amplification. Briefly, 5 µl of RNA with 25 pmol of each primer Beg9 and End9 is denatured for 5 min at 97°C and cooled on ice. Thereafter, 8 µl of RT reaction mixture containing 1.5 µl of 10X PCR buffer (500 mM KCl, 100 mM Tris-HCl (pH 8.3), 2 µl of 25 mM MgCl2, 2 µl of 2.5 mM of each dATP, dCTP, dGTP and dTTP, 10 U AMV reverse transcriptase (Promega) and 20 U Rnasin ribonuclease inhibitor (Promega), is added to the sample-primer mixture and incubated at 42°C for 60 min. PCR buffer mixture (35 µl), containing 3.5 µl of 10X PCR buffer, 2 µl of 2.5 mM peach dATP, dCTP, dGTP and dTTP, 2.5 U AmpliTaq DNA polymerase (Perkin Elmer) and 27 µl of water are added to RT reaction. The reaction mixture is denatured at 94°C for 3 min, followed by 30 cycles (1 min at 94°C, 2 min at 42°C, 3 min at 72°C) and a final extension of 5 min at 72°C.

First-round PCR products (2 µl) are transferred to 48 µl of second PCR reaction mixture, containing 5 µl of 10X PCR buffer, 25 pmol each primers aBT1, aCT2, aET3, aDT4, aAT8, aFT9 and RVG9, 0.2 mM each dATP, dGTP, dCTP and dTTP, 2mM MgCl2, 2.5 U of Taq polymerase. Second-round cycling conditions are identical to first round, except that only 25 cycles are run. The PCR products are analyzed by gel electrophoresis to identify expected lengths of amplified VP7, allowing determination of genotype. Other primer sets, PCR conditions and/or sequencing is used to distinguish genotype when/if the initial primers fail.

4 Determination of VP4 Genotype. The strategy for PCR typing of gene 4 is identical, in principle, to that used for VP7.64 First strand synthesis (reverse transcription) takes advantage of highly conserved areas of VP4 utilizing primers Con2 and Con3 (Box 3), which are complimentary to the 3' ends of both viral RNA strands within gene segment 4. 1 to 5 µL of viral RNA is added to 0.5 ml microcentrifuge tubes containing 3.5 µl of dimethylsulfoxide (Sigma) in a final volume of 8.5 µl, and the samples are mixed and denatured at 97°C for 5 min. The samples are then cooled on ice for 5 min and collected by brief centrifugation. 13.5 µL of H20, 16 µL of deoxynucleoside triphosphates (1.25 mM each dATP, dGTP, dCTP, and dTTP), 5 µL 1OX buffer (100 mM Tris-hydrochloride [pH 8.3], 500 mM KCl) (Perkin-Elmer Cetus), 3.5 µL of 25 mM MgCl2, 2 µl of primer (containing 25 µM of each primer, Con 2 and Con 3), and 9 U reverse transcriptase is then added to each denatured dsRNA sample tube (to give a final reaction volume of 49 µ1) and incubated at 42oC for 60 minutes.

Following addition of 1 µL (1.9U) of Taq polymerase and 100 µL mineral oil, first-round PCR is carried out for 30 cycles (1 min 94oC, 2 min at 50oC, and 2 min at 72oC. A cooling cycle is used to bring the samples to 17OC at the completion of the experiment. The second-round typing reaction if performed using 0.5 to 5 µL of the first-round product (5 µL if no visible product; 0.5 µL if visible product) is mixed with 45 µL reaction mixture in a final volume of 50 µL. If less than 5 µL of first round product is added, the remaining volume, to complete 50 µL, is 10mM TrisHCl (pH 8.3)-2.5mM MgCl¬2. The reaction mixture is 19.5 µL water, 16 µL dNTPs, 5 µL 10X Buffer II, 3 µL 25 mM MgCl2, 1 µL of the primer cocktail (containing 20 µM each of Con3, 1T-1, 2T-1, 3T-1, and 4T-1), and 0.5 µL (2.5U) Taq. The samples are overlaid with mineral oil and taken through 25 rounds of PCR (same cycles as first round). The samples are then analyzed by gel electrophoresis and product size enables determination of VP4 genotype. Other primer sets and/or sequencing is used to distinguish genotype when/if the initial primers fail.

5 Determination of Vitamin A and Zinc levels: Serum samples will be evaluated for Vitamin A level by ELISA (e.g., Human Vitamin A, EIAAB) according to the manufacturer's instructions. Briefly, the microtitre plate provided in this kit has been pre-coated with an antibody specific to Vitamin A. Standards or samples are then added to the appropriate microtitre plate wells with a biotin-conjugated polyclonal antibody preparation specific for VA and Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a tetramethylbenzidine substrate solution is added to each well. Only those wells that contain Vitamin A, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in colour. The enzyme-substrate reaction is terminated by the addition of a H2SO4 and the color change is measured spectrophotometrically at a wavelength of 450 nm. The concentration of VA in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Zinc levels will be measured from blood using assays such as Lampugnani's simple colorimetric method which uses a spectrophotometric method with chromogen 4-(2-pyridylazo) resorcinol sodium salt to measure serum zinc concentrations.