Optimization of an in-house anti-HBs IgG B cell ELISpot
Here, we intermittently used PBMCs from 9 donors, 7 of whom were previously vaccinated with known and detectable anti-HBs antibody titres while 2 were unvaccinated with no previous exposure to Hepatitis B virus as shown in table 2. Samples were assigned unique identification codes expressed as alphabets. All unvaccinated samples had a distinguishing identification of UV code.
Table 2; Characteristics of the optimization samples
Sample ID
|
Sex
|
Age/years
|
Serum anti-HBs titres/ IU/L
|
Vaccinated
S0
|
M
|
34
|
120.2 (˂1yr)
|
U0
|
M
|
28
|
13.78 (~7yrs)
|
B0
|
M
|
37
|
≥500
|
N0
|
F
|
27
|
≥500
|
Un
|
M
|
29
|
≥500
|
Sb
|
M
|
36
|
643
|
M0
|
M
|
25
|
≥500
|
Unvaccinated
|
|
|
|
UV1
|
M
|
38
|
2
|
UV2
|
M
|
32
|
2
|
Greater detection of ASCs with longer IL 2/R848 culture duration.
Two (2) PBMCs samples from vaccinated donors (S0 and U0) were run using a commercially available B cell ELISpot reagent kit (Mabtech). Briefly 1.0x106 thawed PBMCs were stimulated with 10ng/ml of (rh) IL-2 and 1μg/ml of R848 alongside unstimulated controls at 5% CO2 370C for 2 days after which cells were harvested.
There was a trend characterized with a higher frequency of ASCs in unstimulated compared to stimulated cultures with mean frequency of ASCs 25 Ig SCs/106 PBMCs in stimulated [So-45 and Uo-5 Ig SCs/106 PBMCs] and 407 Ig SCs/ 106 PBMCs unstimulated donor cultures [So-507 and Uo-307 Ig SCs/106 PBMCs] (fig 1a). Given the unexpected result, we then run the donor samples (S0 and U0) with a variation of culture time from two (2) to three (3) days (22)
Contrary to the earlier findings but in line with our hypothesis, there was an increase in the frequency of ASCs. For one donor sample, we recorded a frequency of 1450 IgSCs/106 PBMCs in stimulated versus 350 IgSCs/106 in unstimulated wells was recorded for one sample. The other sample (U0) did not demonstrate any difference between stimulated and unstimulated wells (50 IgSCs/1.0X106PBMCs) fig 1b.
Donor U0 was re-run and there was no difference in the result. Together with other three (3) donor PBMCs samples from vaccinated donors B0, N0, and Un, donor U0 was then run with a variation in culture duration to four (4) days.
Stimulated cultures had a substantial higher frequency of IgSCs/106PBMCs (median= 128250) compared to unstimulated donors (median=6500) Figure 2. However donor sample Un was later eliminated from further analysis because of in adequate specimen volume.
Much
Optimization of IL 2 and R848 culture duration
PBMCs samples from HBV vaccinated donor B0 and unvaccinated donor UV1 were cultured and frequencies of ASCs determined at 2, 3, 4, 5 and 6 days of culture.
ASCs continued to proliferate from day 2 to day 4 and a decline was observed from day 4 to day 6 (fig 3). Hence we chose to stimulate and proliferate PBMCs with IL2+R848 for 4days.
Optimized IL 2+R848 4 day protocol did not detect anti HBs-SCs
IgSCs peaked by day 4 of polyclonal stimulation, anti HBs-SCs were however not detectable as shown in fig 4
No significant detection of anti-HBs-SCs irrespective of culture duration or activation method.
Since no anti-HBs-SCS were detected at the optimal 4 day culture, we next assessed the effect of varying culture duration on the frequency of anti-HBs-SCS. The frequency of anti HB-SCs was very low (˂5SFUs/106PBMCs) and infrequent throughout the varying duration times (fig 5).
Additionally we explored an alternative method of activation based on use of CpG+PWM+SAC cocktail (Sebina, et al) (59) and this did not impact on the detection of anti-HBs-SCS either (fig 5a) despite robust induction of IgSCs. In this method, we also observed lower frequencies of IgSCs compared to the IL 2+ R848 based method (fig 5b) and as such, all subsequent stimulation was done using the later.
It was observed that CpG+PWM+SAC stimulation causes an increase in frequency of IgSCs but not anti HBs-SCs, Fig. 6a and IL 2+R848 stimulation was superior to CpG+PWM+SAC fig 6b.
To investigate this further, anti HBs capture antigen used in ELISpot to detect anti-HBs-SCs from a stock concentration of 20 μg/ml by 2 fold dilutions up-to 0.156 μg/ml and this did not impact detection of anti –HBs-SCs was seen fig 7.
Notably, we detected a robust and specific to PPD peptides (Bo 23 and UV1 7 SFU/1.0x106) in 2 donor samples (B0 and UV1) using IL 2+R848 4day culture method but not Hepatitis B (Bo 0 and UV1 0 SFU/1.0x106). fig 8
HBV vaccine specific stimulation does not activate Memory B cells
Because polyclonal stimulation did not lead to detection of anti HBs-SCs in vaccinated donor PBMCs samples, we then opted for specific antigen stimulation using the rh.HBsAg vaccine. Here we used 4 donor PBMCs; 2 vaccinated donor PBMCs (S0 and U0) were run using a 4 day culture. 2 other donor PBMC samples from vaccinated and unvaccinated (B0 and UV1 respectively) were run with variation in stimulation duration ranging from 2 to 6 days.
Irrespective of the culture duration, we were unable to detect any anti HBs-SCs. Notably, the percentage of recoverable cells in PBMCs stimulated with the vaccine was comparable to the unstimulated cultures (Fig.9).
Optimization of an in-house anti-HBs ELISA
Given that we had used rhHBsAg vaccine as the capture antigen and did not detect anti HBs-SCs, we sought to test the ability of the vaccine to bind anti HBs antibodies in serum of vaccinated individuals using an in house anti-IgG ELISA. A total of 6 serum sample; 5 from vaccinated donors with anti HBs titres ˃500IU/l and 1 sample from unvaccinated donors with no previous exposure to Hepatitis B virus and detectable anti HBs titres , fig 10.
Higher ODs using Horse radish peroxidase compared to alkaline phosphatase s
We initially used one day ELISA protocol with alkaline phosphatase in a biotin-streptavidin linkage which had been successfully used in our previous ELISpot experiments. Briefly 100ng/ml of HBsAg vaccine was coated onto high binding 96well immunlonR 4HBX microtiter ELISA plates alongside a 1in 100 sample dilution.
The mean OD in vaccinated serum samples was 0.009125 not very different from the OD=0 recorded in unvaccinated donors fig 11a. This result prompted us to try another method of detection using unlinked horse radish peroxidase (59). Here, a mean OD of 0.5303 in vaccinated samples was observed and we therefore chose to move forward with this method and all the positive samples showing ODs higher than zero fig11b
Optimal antigen concentration for ELISA coating
Two old serial dilutions of the recombinant HBsAg vaccine were made from 1600ng/ml to 25ng/ml and skimmed milk was used as a background control. An antigen concentration of 100ng/ml with OD=1 was chosen as optimal and was not significantly different from higher concentrations.
Optimal serum dilution
In order to limit serum protein interference with ELISA results, we assessed the effect of serum dilutions on OD. We made 2 fold sample dilution in 1% skimmed milk from 1 in 25 to 1 in 400 and a 1 in 50 serum dilution was deemed optimal based on the comparability with OD from the background (skimmed milk).
Utility of in house ELISA
The ELISA was then used to measure anti-HBs titres in serum of 9 donors shown in table 3 using a plate derived standard. The antibody titres in IU/l produced by the assay were consistently lower less sensitive compared to the gold standard (minividas) table 2.
This implied that the vaccine antigen can only discriminate a small proportion of anti HBs antibodies but could theoretically still be used in the ELISpot to detect anti HBs-SCs.
Table 3; Serum antibody titres
Sample ID
|
Sex
|
Serum anti-HBs titres/ IU/L
[Minividas]
|
Serum anti-HBs titres/ IU/L
[In-house ELISA]
|
S0
|
M
|
120.2
|
18.0
|
U0
|
M
|
76.6
|
13.78
|
B0
|
M
|
>500
|
42.28
|
N0
|
F
|
>500
|
29.385
|
Sb
|
M
|
643
|
84.9
|
M0
|
M
|
>500
|
84.7
|
Culture anti HBs antibody
Until then we were unsure if failure to detect anti HBs-SCs was due to failure of the vaccine to capture anti-HBs produced from anti HBs-SCs or failure of the stimulation conditions to activate hepatitis B specific memory B cells.
To investigate this further, culture supernatant from two (2) donors; vaccinated (Donor B0) and unvaccinated (UV1) was tested for anti HBs titres after 4, 5 and 6 day cultures. No anti HBs anti body was detected at the optimal 4 day time point irrespective of the vaccination status or culture conditions (stimulated or unstimulated supernatants). Small titres of anti HBs (23.68IU/l) were however detected in a 6 day IL 2/R848 stimulated cultures.
Measurement of memory B cell responses among hepatitis B vaccine recipients using the in-house anti-HBs IgG ELISPOT
To test the utility of the optimized B cell ELISpot assay, 30 participants who were initially enrolled into SiVET study were to be randomly selected. This objective was however not achieved due to failure to achieve optimal assay conditions
Determination of the correlation between frequencies of antibody secreting cells in ELISpot with vaccine specific antibody titres in the serum of the participants
To determine if absence of antibody titres is an implication of memory loss/absence, serum antibody titres of 60 participant samples from the SiVET study were to be compared with the memory B cell frequency achieved in the ELISpot assay. This objective was not achieved either due to failure to achieve objective one