Up-Regulation of CD40 Ligand and Induction of a Th2 Response in Children Immunized with Pneumococcal Polysaccharide Vaccines

Study population.We evaluated 15 patients (2 to 13 years of age) referred to our pediatric allergy-immunology clinic for evaluation of recurrent respiratory infections. None of these patients had immunoglobulin, IgG subclass, or other known primary or secondary immunodeficiencies. They had all received routine immunizations, including DPT, required for their ages (by history). As part of their evaluation, patients received one does of a 23-valent pneumococcal polysaccharide vaccine (Pnu-Immune; Wyeth-Lederle, Pearl River, N.Y.). One patient who failed to respond to 23-PV received a single dose of an experimental 7-valent pneumococcal conjugate vaccine within 6 months of immunization with 23-PV after parental consent (28). Four unimmunized healthy adults were also included as controls. Serum concentrations of antipneumococcal polysaccharide antibodies and mRNAs for CD40L, IL-4, IL-12p40, and IFN-γ extracted from in vitro-stimulated peripheral blood mononuclear cells (PBMC) were measured before and 4 to 6 weeks after pneumococcal vaccination for patients and 4 weeks apart for the unimmunized controls.

Experimental heptavalent pneumococcal conjugate vaccine.The experimental pneumococcal conjugate vaccine (supplied by Wyeth-Lederle) contains polysaccharide conjugates of serotypes 4, 6B, 9V, 14, 19F, and 23F and an oligosaccharide conjugate of serotype 18C produced by reactive amination. The carrier protein for all conjugates is CRM197, a nontoxic variant of diphtheria toxin. This vaccine has recently been approved for use by the Food and Drug Administration (FDA).

Cell preparations and cultures.PBMC from 2 ml of blood were isolated by Ficoll-Hypaque (density, 1.077g/cm³; Sigma, St. Louis, Mo.) gradient centrifugation. After an overnight incubation in RPMI 1640 at 37°C in humidified air containing 5% CO₂, PBMC (5 × 10⁵) were cultured for 5 h in the absence or presence of concanavalin A (ConA) (20 mg/ml; Miles Scientific, Naperville Ill.), tetanus toxoid (TT) adsorbed USP (1:500; Connaught Laboratories Inc., Swiftwater, Pa.), 23-PV (1:500; Pnu-Immune, Lederle-Praxis Biologicals), and pneumococcal polysaccharide serotype 3, 14, or 18C (20 μg/ml; American Type Culture Collection, Manassas, Va.). Preliminary kinetic studies revealed that optimal mRNA expression of CD40L, IL-4, IL-12p40, and IFN-γ was observed at 4 to 6 after stimulation. For comparison purposes, equal numbers of cells were used for both pre- and postimmunization samples; all cultures were incubated for 5 h. After incubation, cells were washed twice with ice-cold phosphate-buffered saline and mRNA was extracted.

mRNA capture.mRNA was extracted from stimulated cells using the commercial mRNA Capture Kit (Roche, Indianapolis, Ind.) according to the manufacturer's instructions. Briefly, the cell pellet was lysed with 200 μl of cell lysis buffer; DNA was sheared mechanically by passing the lysate through a 21-gauge needle six times. Four microliters of biotin-labeled oligo(dT₂₀) (1:20 dilution from stock solution) was added to the cell lysate and incubated at 37°C for 5 min. After hybridization of the mRNA with biotin-oligo(dT₂₀), 50 μl of the solution was added to streptavidin-coated PCR tubes and incubated at 37°C for 3 min. The tubes were then washed three times with 250 μl of washing buffer.

RT-PCR.Captured mRNA was used as substrate for the synthesis of cDNA by reverse transcription (RT). The RT reaction was performed using avian myeloblastis virus reverse transcriptase (Roche). The reaction was performed at 42°C for 120 min. The cDNA was then amplified using commercially available sequence specific primers for IL-4 (456 bp), IL-12p40 (373 bp), IFN-γ (501 bp), CD40L (293 bp), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (600 bp) (Stratagene, La Jolla, Calif.) by PCR. Direct incorporation of digoxigenin (DIG)-labeled nucleotides in the PCR mixture containing 0.2 mM dATP, dCTP, dGTP, 0.19 mM dTTP, and 0.01 mM DIG 11-dUTP (Roche) was used to measure the PCR product by enzyme-linked immunosorbent assay (ELISA). Cycling parameters were as follows: a 5-min denaturation at 95°C and 5-min annealing at 60°C, followed by the appropriate number of cycles of 1 min at 72°C, 45 s at 95°C, 45 s at 60°C, and a final 7 min at 72°C (Twin Block System, Ericomp, San Diego, Calif.). All PCR products were analyzed in preliminary studies within the linear range of amplification as follows: IL-4, 34 cycles; IL-12p40, 30 cycles; CD40L, 26 cycles; IFN-γ, 22 cycles, and GAPDH, 18 cycles. In all cases, the resultant PCR products of 600 bp (GAPDH), 501 bp (IFN-γ), 456 bp (IL-4), 373 bp (IL-12p40), and 293 bp (CD40L) were visualized by ethidium bromide staining on 2% agarose gels under UV light.

Quantification of PCR amplicons by capillary electrophoresis.Capillary electrophoresis using the Gold-P/ACE System 5000 with laser-induced fluorescence detection (Beckman, Fullerton, Calif.) was performed as previously described (33). To quantify mRNA, the amount of each different amplicon in each PCR sample was measured by integrating the fluorescence peak areas.

Quantification of PCR amplicons by ELISA-PCR.A commercial ELISA-PCR system (Roche) was used for the semiquantitative detection of DIG-labeled PCR products by a hybridization-based microtiter plate assay. Wells of a streptavidin-coated microtiter plate were filled with 10 μl of PCR product, followed by denaturation with 25 μl of 0.2 N NaOH for 5 min and hybridization with 2 pmol of the respective 3′-biotinylated capture probe (Integrated DNA Technologies, Coralville, Iowa). The capture probes were specific for the following amplified target sequences: 5′-GAC AAC TTT GGT ATC GTG GAA GGA-3′ for GAPDH, 5′GGC ATT TTG AAG AAT TGG AAA GAG GAG-3′ for IFN-γ, 5′-TGC GTT CAG GTC CAG GGC AAG AGC-3′ for IL-12p40, 5′-TTC ACA GGA CAG GAA TTC AAG CCC-3′ for IL-4, and 5′-AAT CCA TTC ACT TGG GAG GAG-3′ for CD40L. Capture was allowed to proceed for 3 h at 45°C. Afterwards, the wells were washed three times with washing solution (Roche). To each well was added 200 μl of anti-DIG–peroxidase (10 U/ml; Roche) diluted 1:1,000 in a buffer containing 100 mM Tris-HCl and 150 mM NaCl (pH 7.5). The plates were incubated at 37°C for 30 min and then washed as before. The reaction was visualized by adding 200 μl of ABTS [2,2′-azinobis(3-ethylbenzthiazdinesulfonic acid)] substrate solution (Roche) to each well and incubating the plates at 37°C for 30 min in the dark. The optical density (OD) was read at 405 nm with a reference at 490 nm using a Bio-Rad (Hercules, Calif.) reader. The run was considered valid if all negative control values were less than 0.2 OD unit and the positive control value was greater than 1.0 OD unit. ODs were converted into logs of copy numbers of initial cDNAs in the patient samples. Quantification of PCR products was performed by comparison to a standard curve. The standard curve for each amplicon was created using serial dilutions of known concentrations (logs of copy numbers of initial cDNAs) of plasmids coding for GAPDH, IFN-γ, IL-12p40, IL-4, or CD40L (American Type Culture Collection). Plasmids were isolated, serially diluted, PCR amplified, and detected as described above. OD units for patient samples were then converted to copy numbers based on their respective standard curves.

ELISA for antipneumococcal IgG.IgG antipneumococcal antibody levels against serotypes 1, 3, 4, 6B, 9V, 14, 18C, 19F, and 23F were determined by an ELISA protocol calibrated against the FDA 89-SF reference sample (CBER; FDA, Washington, D.C.) as previously described (29).

Statistical analysis.Comparisons between pre- and postimmunization paired data were performed using the Wilcoxon test (one tailed). A P value of <0.05 was considered significant.