2.1. Human participants, vaccination protocol and blood samples
Five healthy adults 18 – 30 years of age (donors No. 1 – No. 5) were enrolled during the 2008 – 2009 influenza season for the study. In addition, two healthy adults of the same age group (donors No. 6 and No. 7) were enrolled during the 2009 – 2010 influenza season for the flow cytometry sorting experiment (section 3.4). The study protocol was approved by the institutional review board at Stanford University. Informed consent was obtained from each participant. Participants were immunized with one dose of the trivalent inactivated influenza vaccine (TIV) of the current season: 2008 Afluria (CSL, Parkville, Victoria, Australia) for donors No. 1 – No. 5 or 2009 Fluzone (Safoni Pasteur, Swiftwater, PA, USA) for donors No. 6 and No. 7. Both the 2008 and 2009 TIV contained an A/Brisbane/59/2007 (H1N1)-like virus and an A/Brisbane/10/2007 (H3N2)-like virus. In addition, the 2008 TIV contained a B/Florida/4/2006-like virus, while the 2009 TIV contained a B/Brisbane/60/2008-like virus. Blood samples were collected on day 0 before vaccination, as well as day 7 and day 28 post vaccination. Sera were prepared with the day 0 and day 28 blood samples. PBMC were isolated using standard Ficoll gradient centrifugation from heparinized whole blood. B cells were isolated from heparinized whole blood by negative selection with the ResetteSep Human B cell Enrichment cocktail (Stemcell Technologies, Vancouver, BC, Canada), following the manufacturer’s instructions.
2.2. Generation of PPAb
For generation of PPAb from bulk plasmablasts, freshly isolated B cells were resuspended in complete medium (RPMI 1640 supplemented with 10% of heat inactivated fetal calf serum, 100 units/ml of penicillin G and 100 µg/ml of streptomycin) at 3 million cells per ml and cultured in 5% CO2 at 37°C for 4 – 7 days. The conditioned media with secreted PPAb were collected and kept at −20°C until being tested. For generation of PPAb from plasmablast subsets, PBMC and B cells were stained with a cocktail of antibodies against different cell surface markers: PE-Cy7-labeled anti-CD19, Pacific Blue-labeled anti-CD3, PE-labeled anti-CD27, PerCP-Cy5.5-labeled anti-CD38, APC-labeled anti-IgG (all from BD Biosciences, San Jose, CA, USA), and FITC-labeled anti-IgA (Invitorogen, Camarillo, CA, USA). The following cell populations were isolated on a FACS Aria flow cytometer: IgA+ plasmablasts (CD19+CD27highCD38highIgA+) and IgA− plasmablasts (CD19+CD27highCD38highIgA−) from B cells, and CD3+ T cells (CD19−CD3+) from PBMC. Variable numbers of the sorted plasmablast subsets were mixed with sorted T cells as feeder cells and cultured for 7 days in complete RPMI 1640 medium at 3 million total cells (plasmablasts plus T cells) per ml.
2.3. Determination of IgA and IgG concentration in PPAb
The concentration of IgA in the PPAb samples was determined with the IMMUNO-TEK Quantitative Human IgA ELISA kit (Zeptometrix, Buffalo, NY, USA), following the manufacturer’s instructions. The concentration of IgG was determined with a two-antibody sandwitch ELISA. In brief, Ninety-six-well Vinyl Microtiter Microplates (Thermo Fisher Scientific, Waltham, MA, USA) were first coated with goat anti-human IgG (Sigma, Saint Louis, USA) diluted 1:2000 with PBS. Plates were incubated overnight at 4°C, washed with wash buffer (0.1% of Tween 20 in PBS), and blocked with 3% of BSA in PBS for 1 hour at 37°C. PPAb were serially diluted with complete medium, then added to the wells of coated/blocked plates. Serially diluted purified human serum IgG (10 mg/ml, Innovative Research, Novi, MI, USA) was added to wells of the same plate to serve as the standard. The plates was incubated for 1 hour at 37°C, washed, and then incubated for 1 hour at 37°C with peroxidase-conjugated goat anti IgG γ (KPL, Gaithersburg, MD, USA) diluted 1:3000 with complete medium. After washing, the plates were developed with TMB substrate (KPL) and the OD450nm of each well recorded with an ELISA plate reader. The concentration of IgG in the PPAb samples were determined on the standard curve constructed with the OD values of serially diluted human serum IgG standard.
2.4. Enumeration of total and influenza-specific ASC
Total and influenza-specific IgA or IgG ASC were enumerated with ELISPOT. Ninety-six-well MultiScreen HTS plates (Millipore, Billerica, MA, USA) were coated with affinity purified goat antihuman IgA+IgG+IgM (H+L) (KPL) at a concentration of 4 µg/ml in PBS to detect total ASC, or coated with 2008 TIV (Fluzone) at a concentration of 4.5 µg/ml in PBS to detect influenza-specific ASC. Wells coated with PBS served as negative controls. Plates were incubated overnight at 4°C and then blocked for 2 hours at 37°C with complete medium. Freshly isolated B cells were resuspended in complete medium containing phosphatase conjugated goat anti-human IgA antibody (KPL) at 0.2 µg/ml or phosphatase conjugated goat anti-human IgG (H+L) antibody (KPL) at 0.2 µg/ml, dispensed into wells of the coated/blocked plates at 2 fold serial dilutions and incubated for 4 hours or longer at 37°C with 5% CO2. Plates were washed with PBS and developed with a Blue alkaline phosphatase substrate kit (Vector, Burlingame, CA, USA). The number of IgG and IgA ASC in each well was determined by counting the spots. Nonspecific spots detected in the negative control (PBS) wells were subtracted from the counts of influenza-specific and total ASC.
2.5. Influenza-specific binding activity of serum and PPAb samples
Influenza vaccine (TIV)- or individual vaccine virus strain-specific IgG or IgA binding activity was determined with ELISA. Ninety-six-well Vinyl Microtiter Microplates were coated with 2008 TIV at 2.7 µg/ml in PBS, or individual 2008 influenza vaccine strains (kindly provided by Dr. Kanta Subbarao of NIH) at 57 HA unit/ml in PBS (4 HA unit/well). Plates were incubated overnight at 4°C, washed with wash buffer, and blocked with complete medium for 1 hour at 37°C. PPAb or serum samples were serially diluted with complete medium, added to the wells of coated/blocked plates and incubated for 1 hour at 37°C. Wells incubated with complete medium served as negative control. The plates were then washed, incubated for 1 hour at 37°C with peroxidase-conjugated goat anti IgG γ (KPL) diluted 1:4000 with complete medium, or peroxidase-conjugated goat anti IgA α (KPL) diluted 1:2000 dilution with complete medium. After washing, the plates were developed with TMB substrate (KPL) and the OD450nm of each well determined with an ELISA plate reader. The cutoff OD450nm value was set at mean + 2 × SD (standard deviation) of OD450nm value of all negative control wells. Titer of each sample was determined as the highest dilution at which the mean OD450nm value of duplicate wells was greater than the cutoff.
2.6. Microneutralization and HAI assays
Serum samples were pretreated with receptor-destroying enzyme (Denka Seiken, Tokyo, Japan) overnight at 37°C and subsequently heated at 56°C f or 45 minutes. Neutralization antibody titers were determined for 100% inhibition of cytopathic effect (CPE) in Madin-Darby canine kidney cells (European Collection of Animal Cell Cultures, Wiltshire, United Kingdom). Serially diluted serum or PPAb specimens were incubated with 50 TCID50 (50% tissue culture infective doses) of the indicated influenza virus for 1 hour at 33°C and transferred to the Madin-Darby canine kidney cell monolayers in 96-well culture plates. After 6 days of culture at 33°C in a CO2
incubator, CPE in the wells was examined under a light microscope. The neutralizing antibody titer of a given sample was defined as the reciprocal of the last serum or PPAb dilution with no observed CPE (Belshe et al. 2000
). A titer of 10 was assigned to all serum samples in which the first dilution (1:20) was negative. A titer of 5 was assigned to all PPAb samples in which the first dilution (1:10) was negative.
The HAI assay was performed using a standard technique (Centers for Disease Control and Prevention 1998
); serially diluted 25-µl aliquots of serum or PPAb samples in PBS were mixed with 25-µl aliquots of virus, corresponding to four HA units, in V-bottom 96-well plates (Nunc, Rochester, NY, USA) and incubated for 30 minutes at room temperature. At the end of the incubation, 50 µl of 0.5% chicken (for influenza A/H1N1 and B viruses) or turkey (for influenza A/H3N2 virus) red blood cells was added and incubated for a minimum of 45 minutes before reading for HAI activity. The HAI titer of a given sample was defined as the reciprocal of the last serum dilution with no HA activity. A titer of 2 was assigned to all samples in which the first dilution (1:4) was negative.
2.7. Statistical analysis
Paired t-tests were used for comparing different samples (PPAb versus serum). All titer data were logarithm transformed for analysis.