Cell culture methods.

2.6.1. Separation and Cryopreservation of peripheral blood lymphocytes (PBMCs).

Blood samples from the Hepagene™ vaccinated individuals were obtained specifically for in-vitro culture analysis in EDTA (Sardstedt, Leicester, UK) at the discretion of the attending physician and the kind agreement of the vaccine trial subject. The blood was transferred into an equal volume of RPMI 1640 medium (ICN Biomedicals Inc., California, USA). The diluted blood was layered onto 10ml of ficol (Lymphoprep; Nycomed, Oslo, Norway) using a disposable Pasteur pipette and centrifuged at 2,400 rpm for 20 minutes. Peripheral blood mononuclear cells (PBMCs) were removed from the interface and transferred to a clean vessel and washed three times in RPMI 1640 with 10 lU/ml penicillin and lOpg/ml streptomycin (ICN Biomedicals Inc., California, USA). After thorough washing the PBMCs were counted using a haemocytometer.

A freezing mix of 90% heat inactivated foetal calf serum (PCS) (Biowhittaker, Wokingham, UK) and 10% dimethylsulphoxide (DMSO) (BDH, Poole, UK) was added to the PBMCs at a concentration of 5x10^ cells/ml. Expeditiously the mixture was aliquoted in 1 ml volumes into 1.8 ml labelled freezing vials (Nunc, Kamstrup, Denmark) then placed in a suitable container before transfer to a -70°C freezer. After 18 hours the vials were put into liquid nitrogen (-192°C) until required.

When required for culture the PBMCs were thawed rapidly by gentle agitation of the vial in a 37°C waterbath briefly, before transfer to a large volume (20 ml) of RPMI 1640 with 10 lU/ml penicillin and 10|Lig/ml streptomycin. The large volume was needed to avoid DMSO cytotoxicity, which reduces cell viability. The PBMCs were washed thoroughly to remove all the DMSO and PCS before use.

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The ability of the cells to proliferate in the presence of particular antigens stimuli was assessed. The PBLs were resuspended in ‘complete medium’, which is RPMI, 10 lU/ml penicillin, 10|ig/ml streptomycin (both from ICN Biomedicals Inc, California, USA) plus 10% normal AB positive human serum (North London Blood Transfusion Centre, London, UK). The PBLs were counted then seeded at 1x10^ viable cells/well in 96 well round bottomed plates (Nunc, Kamstrup, Denmark).

Optimal concentrations were determined for the non-adjuvanted Hepagene™ vaccine antigen and hepatitis B surface antigen and tetanus toxoid (Evans Medical, Speke, UK). Positive controls for proliferation measured by incubation of PBMCs with the T-cell mitogen phytohemagglutanin (PHA; 2|ig/ml, Murex, Hartford, UK) or the PBMCs were cultured with optimal concentrations of a recall antigen tetanus toxoid. To assess the background proliferation in complete medium PBMCs were cultured with medium alone. Optimal duration of cultures was determined at 37°C in a humidified 5% CO^

incubator. Cells were then pulsed with 0.5 |iCi/well of methyl ^H-thymidine

(Amersham, Amersham, UK) 18 hours before harvesting onto glass fibre filters

(Pharmacia LKB, Biotechnology, Uppsala, Sweden). Scintillation counting was

performed using a Wallac betacounter 1450 plus (Wallac, Turku, Finland). To ensure standardisation of conditions all cultures were performed in triplicate. Antigen specific proliferation was expressed as stimulation indices (mean counts per minute of cells with antigen / mean counts per minute of cells alone).

2.6.3. Frequency analysis of Hepagene™ -responsive PBLs.

The Hepagene™ antigen specific T-cell frequency was determined by dilution analysis from Hepagene™ trial subjects at time points 6, 18 and 20 months. Varying numbers of PBMCs were added to individual wells of a 96 well round bottomed plates. The PBMCs were seeded into the plates at 2 x l0 \ lxlO \ 6xlO\ 3x10% 1x10"^, 3x10^ and 1x10^ in replicates of 12 in the presence of 3pg/ml nonadjuvanted Hepagene™ antigen. The positive controls for proliferation were the same as above. Cells were cultured in complete media for 6 days. Those cultures demonstrating ^H-thymidine incorporation higher than the arithmetic mean plus 3 standard deviations (SD) of that shown by

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cultures incubated in the absence of antigen were scored positive. The fraction of wells demonstrating negative cultures was calculated for each concentration of seeded PBMCs (Taswell, 1981).

2.6.4. Establishment of Hepagene^'^-specifie T cells lines.

Blood in heparin was obtained from consenting Hepagene™ trial subjects made available by permission from the Academic Department of Travel Medicine and Vaccines, Royal Free Hospital School of Medicine. The PBMCs were prepared as outlined above and the concentration adjusted to 1x10^ cells per vial for freezing. Sufficient vials of 1x10^ PBMCs are thawed made up to 2 ml then aliquoted into wells of a labelled flat bottomed 24 well plate (Nunc, Kamstrup, Denmark). The cells were either cultured in the presence of Spg/ml nonadjuvanted Hepagene™ vaccine antigen or 2|iig/ml of PHA as a non-specific T-cell stimulator. The cultures were incubated the cells at 37°C in a humidified 5% CO^ incubator for 6 days. Simultaneously cells were seeded into a round-bottomed 96 well plate in the presence of Hepagene™ vaccine antigen to analyse the specificity of the bulk culture.

Following 7 days, 20ng/ml recombinant IL-2 in 3mis of complete medium was added to the bulk cell lines. This promoted the growth and expansion of the T cells activated over the previous 6 days by the immunogenic regions in Hepagene™. The cultures were returned to the incubator for a further 7 days.

During the incubation period of the bulk cell line the APCs within the culture die, therefore, secondary stimulation of the T cells with antigen and autologous feeder cells were required. These were prepared by irradiating autologous PBMCs with a sublethal dose of 4000 rads, which prevented feeder cell growth and division. The bulk cell lines were centrifuged at 1500 rpm for 10 minutes in conical tubes. The T cells were counted, adjusted to 1x10* cells/ml and added to 2x10* autologous feeder cells in a 24 well plate and incubated for a further 7 days. At the same time 2x10^ Hepagene™ - specific T cells were added to 4x10^ autologous feeder cells in the presence of either Hepagene™ vaccine antigen, HBsAg or peptides derived from the vaccine sequence for 6 days to assay cell line specificity.

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The cells were maintained by alternative weekly cycles. In the first week cultures received Hepagene™ or PHA, plus autologous feeder cells and assayed for specificity. In the second week, the cells received recombinant IL-2 alone.