Dynamics of the Immune Response in Children to the 23-valent Pneumococcal Capsular Polysaccharide Vaccine (Pneumovax)
Condition: Pneumonia, Pneumococcal
MeSH terms: Pneumonia, Pneumococcal
Study type: Interventional
Phase: Phase 1

Summary: 
      To explore a further dimension of susceptibility to disease, the investigators tested the
      hypothesis that natural variation exists in the rapidity of IgG responses following exposure
      to pneumococcal polysaccharides, and that these differences are sufficiently great to affect
      susceptibility to and outcome of IPD. The study recruited children aged 24-36 months, who had
      recovered from IPD, and age-matched healthy controls and vaccinated them with 1 dose of the
      23-valent PPV to mimic natural exposure. The investigators collected serum samples after
      vaccination and analysed the dynamics of anti-polysaccharide antibody responses to several
      capsular antigens.
    

Detailed description: 
      The details are as described below:

        1. Study site Kilifi District Hospital.

        2. Study population and sampling Cases and controls from the Kilifi Birth Cohort who are
           alive at the end of the cohort observation (at the age of 24 months) and remain resident
           in Kilifi District will be vaccinated with a single 0.5 ml sub-cutaneous dose of 23
           valent pneumococcal polysaccharide vaccine (Pneumovax 23) to compare the dynamics of the
           anti-capsular antibody response to 5 capsular antigens chosen to represent different
           immunological profiles of the pneumococcal capsular polysaccharides (1, 6B, 14, 19F,
           23F).

        3. Recruitment Potential study subjects will be identified and their mothers will be
           approached, informed about the study, including the necessity to undergo HIV testing,
           and invited to take part. A fieldworker will explain the rationale and procedures for
           the study and will give the mother an information sheet/consent form to take away. The
           mother will also be given an appointment slip and a fare to return to the hospital on
           the appointment date. Appointments will be at ward 1. When the mother returns the
           fieldworker will go through the consent form again and will ask the mother to sign. The
           mother will be counselled and her child will be HIV tested. If the participant is
           positive the child will be referred to the Kilifi Family Clinic. If the participant is
           negative, the child will receive the single dose of Pneumovax (23 valent polysaccharide
           vaccine). The mother will be given a schedule of 2 further appointments for blood tests
           and at each visit the fare to and from the hospital will be provided by KEMRI. Study
           subjects who have been vaccinated and subsequently do not attend will be visited by FW
           on a motorcycle to invite them to keep their appointments the very next day. Information
           will be collected concerning age, sex and environmental exposures such as: number of
           elder and younger sibling in the house, cooking smoke exposure, passive cigarette
           smoking, recent admission to hospital.

           Definition of cases:

           Cases of IPD, ARI or radiographic pneumonia and meningitis will be defined as episodes
           of illness, requiring admission to hospital, that occur to cohort members between birth
           and the age of 23 months that also meet at least one of the following syndrome
           definitions: INVASIVE PNEUMOCOCCAL DISEASE is defined by a positive result on EITHER of
           the following criteria: (i) culture of S. pneumoniae from any normally sterile site
           including blood, CSF, lung, synovial fluid but excluding middle ear fluid and external
           eye swabs. OR (ii) Two-fold rise in anti-PsaA antibody concentration measured by ELISA
           in two serum samples obtained at least 5 days apart 21 AND presence in urine of
           pneumococcal capsular polysaccharide detected by latex agglutination assay 46. ARI is
           defined by an acute (<2 weeks) history of cough or difficulty in breathing accompanied
           by a raised respiratory rate (≥50/min in a child <12 months, ≥40/min in a child 12-23
           months). ARI is severe if there is, in addition, chest indrawing, and very severe if
           there is, in addition, central cyanosis and prostration or inability to drink 47.

           RADIOGRAPHIC PNEUMONIA is defined as ARI accompanied by radiographic consolidation. All
           radiographs will be rated both by study physicians and by a consultant radiologist.
           "Radiographic consolidation" will only be considered to be present if documented by both
           observers.

           MENINGITIS: Meningitis is being defined to target the use of additional diagnostic tests
           for IPD. A definition is required which is sensitive but not necessarily highly specific
           for pneumococcal meningitis. Any child that is transferred to the KEMRI high dependency
           unit with a clinical diagnosis of "Meningitis", any child started on the standard
           meningitis antibiotic regime, or any child with an elevated CSF leucocyte count or
           CSF/plasma glucose ratio of <0.5 will be considered as a case of meningitis for the
           purposes of this study.

           A single episode of illness may satisfy one, two, three or all case definitions. The
           definition of IPD is for use in the case-control study. The other definitions are for
           use in the study of incidence and aetiology of severe ARI and radiographic pneumonia and
           to guide the flow of investigations of patients with suspected IPD.

        4. Laboratory Methods ANTI-CAPSULAR IGG ELISA. The method of Quataert et al. will be used
           with minor modifications 13,14. Flat bottom microtitre plates (Nunc Maxisorb, Nunc,
           Finland) are coated with 20 μg/ml purified capsular polysaccharide (American Type
           Culture Collection, Manassas, Virginia, USA) in 0.01M phosphate buffered saline (PBS),
           pH 7.2, for 5 hours at 37oC and then stored at 4oC. Plates are washed five times with
           PBS, pH7.2, with 0.05% Tween-20 after each stage. Test sera are diluted 1:50 in 1%
           bovine serum albumin, 0.05% Tween-20 in PBS containing 10 μg/ml C-polysaccharide
           (Statens Seruminstitut, Copenhagen, Denmark) and 20 μg/ml polysaccharide of serotypes
           22F (American Type Culture Collection) incubated for 30 mins at room temperature. Test
           sera are aliquoted onto the plate in duplicate in 8 three-fold dilutions and incubated
           at room temperature for 1 hour. A standard reference serum (89SF, Dr Carl Frasch, FDA,
           USA) and a control (post-vaccination serum from an individual with good responses to all
           vaccine serotypes) are assayed on every plate in seven 3-fold dilutions. Bound antibody
           is labelled with alkaline phosphataseconjugated goat anti-human IgG in a 2 hour
           incubation at room temperature. Bound enzyme is visualised with p-nitrophenyl phosphate
           in diethanolamine substrate (Sigma) for 2 hours at room temperature and the reaction is
           stopped with 3M NaOH. Plates are read by an ELISA reader at a wavelength of 450 nm with
           a reference filter of 620nm. Results are analysed by a 4-parameter logistic-log curve
           fit program (ELISA v1.11, Centers for Disease Control and Prevention, Atlanta, GA) and
           expressed in relation to the known concentrations of the reference serum.

           ELISA FOR ANTI-PSAA: The method of Tharpe et al. will be used on pairs or triplicates of
           sera. Flat bottom microtitre plates (Immunlon II HB, Dynatech Corp, Chantilly, VA) are
           coated with 2.5 μg/ml purified PsaA in 0.01M phosphate buffered saline (PBS), pH 7.2,
           for 16 hours at 4oC. PsaA is produced by recombination and expression in Escherichia
           coli of the gene encoding PsaA. Plates are washed four times with PBS, pH7.2, with 0.05%
           Tween-20 after each stage except blocking, when the plates are simply emptied. Plates
           are blocked for 1 hour at 37oC with 1% bovine serum albumin (EIA Grade, Sigma Chemical
           Co., St Louise, MO) 10mg/L in PBS. Test sera are diluted in 1% bovine serum albumin,
           0.05% Tween-20 in PBS and incubated for 1 hour at 37oC. Test sera are assayed in
           duplicate in 8 two-fold dilutions and all sera from one patient are assayed on the same
           plate. A reference serum of high anti-PsaA concentration and a control (Sandoglobulin,
           Sandoz, Switzerland) are assayed on every plate in seven 2-fold dilutions. Bound
           antibody is labelled with mouse monoclonal anti-human IgG-Fc conjugated to horseradish
           peroxidase (clone PH6043, Hybridoma Reagent laboratories, Baltimore, MD) in a 2 hour
           incubation at 37oC. Bound enzyme is visualised with tetramethlybenzidine (TMB
           1-component microwell peroxidase substrate, Kirkegaard and Perry Laboratories Inc,
           Gaithersburg, MD) for 30 minutes at room temperature and the reaction is stopped with
           0.18M Sulphuric Acid. Plates are read by an ELISA reader at a wavelength of 450 nm with
           a reference filter of 620nm. Results are analysed by a 4-parameter logistic-log curve
           fit program (ELISA v1.11, Centers for Disease Control and Prevention, Atlanta, GA) and
           expressed in ELISA units as a percentage of the concentration of the reference serum.

        5. Data storage Recruitment and follow-up forms will be stored in a locked study office.
           Information from the forms will be entered into a computer using Filemaker Pro 5.5 and
           will be linked on study number and visit with the laboratory data.
    

Intervention name(s): Pneumovax
Intervention type(s): Biological
Intervention description(s): 23-valent pneumococcal polysaccharide vaccine
Intervention MeSH terms: Heptavalent Pneumococcal Conjugate Vaccine
Intervention model: Single Group Assignment
Masking: None (Open Label)
Primary purpose: Basic Science
Keywords: Invasive Pneumococcal Disease