Causes of Rotavirus Vaccine Failure in Zambian Children
Condition: Diarrhoea
MeSH terms: Diarrhea
Study type: Observational

Summary: 
      Zambia recently introduced routine infant immunization against rotavirus - the most important
      cause of severe gastroenteritis and diarrhoea mortality in children. Although vaccines like
      Rotarix are a cost effective tool against infectious diseases, live oral vaccines can be less
      immunogenic and efficacious in developing world settings as compared with industrialized
      countries. Reasons behind this phenomenon are not well understood, but may relate to
      continued maternal antigen exposure and high level maternal immunity that is passed to the
      foetus/newborn transplacentally and/or through breast milk.

      Therefore, three arising hypotheses include: (i) high-level rotavirus-specific maternal
      immunity (in the form of anti-rotavirus breast-milk immunoglobulin A (IgA) and transplacental
      serum IgG) is a major contributor to failed seroconversion following infant vaccination. (ii)
      Malnutrition negatively impacts infant immunity and increases the risk of post-vaccination
      rotavirus gastroenteritis. (iii) Introduction of rotavirus vaccine will alter the molecular
      epidemiology of circulating rotavirus strains detected in vaccinated children presenting with
      severe diarrhea.

      To address these hypotheses, the proposed study will recruit a prospective cohort of 420
      mother-infant pairs. These will be enrolled at the time of vaccination and followed for up to
      four years. Baseline immunological status will be ascertained and seroconversion rates
      determined a month after full immunization. Incident rotavirus gastroenteritis will be
      monitored in the vaccinated infants whenever episodes of diarrhoea occur; through this
      surveillance, the sero-strains of rotaviruses causing disease will be tracked over the four
      year period. Contributions of HIV infection both in mothers and infants, vitamin A and zinc
      deficiency, weight for age Z-scores as well as mid upper arm circumference will also be
      assessed. Knowledge gained from this study will inform future interventional trials on
      strategies to improve rotavirus vaccine effectiveness in the developing world.
    

Detailed description: 
      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.
    

Observational model: Cohort
Time perspective: Prospective
Sampling method: Non-Probability Sample
Study population: 
        Mother-infant pairs presenting for MCH at Kamwala health facility in Lusaka and where the
        infant is 6-15 weeks old, will be recruited into the cohort and followed for a period of
        1-4 years depending on time of enrolment.
      
Biospecimen description: 
      Blood samples for immunogenicity testing will be analyzed in batches.

      Stool samples for rotavirus extraction will also be stored and analyzed in batches.
    
Keywords: Rotavirus; Diarrhoea; Immunisation; Seroconversion; Breastmilk; HIV; Zinc; Vitamin A; Failure