3 Results

3.1  HBcdDOB120 and DOBV rN were expressed in E. coli and S. cerevisiae, respectively

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HBcdDOB120 can be expressed in E. coli and purified according to protocols described previously [Borisova, 88; Geldmacher, 04h]. The HBcdDOB120 protein is pure as proven by Coomassie blue staining (Fig. 3A), and was recognised by HBc-specific as well as N-specific antibodies in Western Blot (Fig. 4A,B).

The HBcd protein is only recognised by the HBc-specific antibodies. The purified HBcd and HBcdDOB120 proteins self-assembles into particles, as shown by electron microscopy [data not shown, see Geldmacher, 04g]. The 120 amino-terminal aa is at least partly exposed on the outside of the particles, as shown by immuno electron microscopy [data not shown, see Geldmacher, 04f].

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FIGURE 3 Coomassie blue stained SDS polyacrylamid gels of the antigens HBcdDOB120 (A) and rN protein of Dobrava virus (DOBV) strain Slovenia (Slo) (B) used in the immunisation experiments. In addition to the proteins used for immunisations the following were analysed: HBcd, which was used as a negative control in the immunisations with HBcdDOB120 (A), as well as the proteins that were used for the detection of cross-reactive antibodies against the N protein of the following hantaviruses: DOBV strain Slovakia (Slk), Hantaan (HTNV) strain Fojnica (Foj), Puumala (PUUV) strains Vranica/Hällnäs (Vra), Kazan (Kaz) and Sotkamo (Sot), Andes (ANDV) strain AH1 and Sin Nombre (SNV) strain 3H226.

The entire rN proteins of DOBV-Slo, DOBV-Slk, HTNV-Foj, PUUV-Vra, PUUV-Sot, PUUV-Kaz, AND-AH1 and SNV-3H226 could be expressed to high level in the yeast S. cerevisiae. They were purified by nickel chelate affinity chromatography by means of the His-tag as proven by Coomassie blue staining of SDS polyacrylamid gel (Fig. 3B). All the proteins, but not the negative control, the hamster polyomavirus (HaPyV) VP1 protein were recognised by an N-specific mAb (Fig. 4C).

FIGURE 4 Western Blot analysis of the antigens HBcdDOB120 (A, B) and rN protein of Dobrava virus (DOBV) strain Slovenia (Slo) (C) used in the immunisation experiments. Detection of proteins were conducted using polyclonal HBc-specific serum (A), polyclonal hantavirus N-specific serum (B) or the N-specific mAb 1C12 (C). Additionally to the immunising antigens HBcd (A, B) is shown, which was used as a negative control in the immunisations with HBcdDOB120, as well as the proteins that were used for the detection of cross-reactive antibodies against the N protein (C) of the following hantaviruses: DOBV strain Slovakia (Slk), Hantaan (HTNV) strain Fojnica (Foj), Puumala (PUUV) strains Vranica/Hällnäs (Vra), Kazan (Kaz) and Sotkamo (Sot), Andes (ANDV) strain AH1 and Sin Nombre (SNV) strain 3H26.

3.2 Preexisiting antibodies to HBc did not abrogate the antibody response to DOBV rN protein after immunisation with HBcdDOB120

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To investigate if an earlier acquired immune response to the carrier protein HBc influences the N-specific immune response after subsequent HBcdDOB120 immunisation, mice with a high HBc-specific antibody titre were immunised with HBcdDOB120 six weeks after the last HBc immunisation (scheme 1, Fig. 2A). For comparison naive mice were immunised with HBcdDOB120 according to the same scheme.

FIGURE 5: DOBV N-specific antibody response in BALB/c (above) and C57BL/6 (below) mice with HBc-specific immunity and naive mice after immunisation with HBcdDOB120. In a first set of immunisations groups of four BALB/c and four C57BL/6 mice were immunised with full-length HBc which resulted in a HBc-specific antibody titre of at least 1:100,000 one months after HBc vaccination (scheme 1, Fig. 2A) or 1:1,000 seven month after HBc vaccination (scheme 2, Fig. 2A). In a second set of immunisation a group of these mice as well as a group of naive mice were immunised once with HBcdDOB120 in complete Freund's adjuvant. Groups of mice were immunised with HBcdDOB120 either 1.5 months (A) (scheme 1, Fig. 2A) or seven months (B) (scheme 2, Fig. 2A) after the last immunisation with HBc. Shown are the means of the reciprocal DOBV rN-specific endpoint titres of four mice with the respective standard deviation. The mean titre of the mice immunised according to the seven months scheme (B) is based on only three mice. Titres did not exhibit significant differences when tested by Mann Whittney U Test (p>0.05).

BALB/c and C57BL/6 mice with a mean HBc-specific antibody titre of 1:250,000 had the same DOBV N-specific antibody titre after immunisation with HBcdDOB120 as mice not possessing an HBc-specific antibody titre before HBcdDOB120 immunisation (Fig. 5A). The preexisiting HBc-specific as well as the induced N-specific antibodies were of all IgG subclasses (data not shown). Similarly, BALB/c mice pre-immunised with carboxy-terminally truncated HBcd developed higher DOBV N-specific titres after HBcdDOB120 immunisation than mice that were not preimmunised with HBcd (data not shown, Geldmacher et al. unpublished data).

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In another experiment groups of mice immunised with HBc were left for seven months before immunising them with HBcdDOB120 (scheme 2, Fig. 2A), to see if a presumable memory immune response to HBc would influence the immune response to DOBV rN protein.

BALB/c and C57BL/6 mice with an HBc-specific antibody titre of 1:1,000 seven months after the HBc immunisation developed a one order of magnitude higher DOBV N-specific antibody titre after immunisation with HBcdDOB120 compared to mice that did not have any HBc-specific antibody titre before HBcdDOB120 immunisation (Fig. 5B). However, the DOBV N-specific antibody titres of mice with and without a preexisting HBcd-specific titre were not significantly different from each other (p>0.05). As observed for the sera of mice immunised according to the "short term" scheme (scheme 1, Fig. 2), HBc-specific and N-specific antibodies were of all IgG subclasses (data not shown).

3.3 HBcdDOB120 and DOBV rN induced antibodies that reacted to virus infected cells

Serum pools of mice immunised with HBcdDOB120 particles or DOBV rN protein were tested for their reactivity with the natively folded N protein in hantavirus-infected cells.

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A serum pool of mice immunised with HBcdDOB120 particles reacted with natively folded N protein in DOBV infected cells, but not with uninfected VeroE6 cells (Fig. 6). In addition, the serum pool also reacted with HTNV and PUUV infected cells (data not shown). Sera of mice immunised with HBcd did not react with DOBV infected cells (data not shown).

A serum pool of mice immunised with DOBV rN also reacted with DOBV infected cells, but not with uninfected VeroE6 cells (Fig. 6). The pool of control sera of mice immunised with PBS did not react with DOBV infected cells (data not shown).

FIGURE 6: Reactivity of serum pools from mice immunised with HBcdDOB120 and DOBV rN protein with DOBV infected VeroE6 cells. Sera from five BALB/c mice taken three weeks after the third immunisation (scheme 3, Fig. 2B) were pooled and diluted 1:1,000. Shown is the immunofluorescence of DOBV infected and non-infected cells incubated with serum pools induced by immunisations with HBcdDOB120 and DOBV rN protein detected by a FITC-conjugated anti-mouse antibody at a 40-fold magnification.

3.4 HBcdDOB120 and DOBV rN induced a strong and long lasting antibody response

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To compare the immunogenicity of HBcdDOB120 and DOBV rN protein by immunising BALB/c and C57BL/6 mice three times with HBcdDOB120 or DOBV rN protein (scheme 3, Fig. 2B). Individual sera taken after each immunisation were tested in ELISA with the homologous rN protein (DOBV-Slo).

Immunisation of BALB/c and C57BL/6 mice with DOBV rN protein resulted in high titres of homologous N-specific antibody titres. The first immunisation induced N-specific antibody titres of 1:8,000 in BALB/c (Fig. 7A) and 1:60,000 in C57BL/6 (Fig. 7B). In BALB/c mice this titre was significantly higher than in mice immunised with HBcdDOB120 (p = 0.032).

BALB/c and C57BL/6 mice immunised with chimeric HBcdDOB120 particles both developed high antibody titres against DOBV rN protein. Three weeks after the first immunisation N-specific antibody titres were as high as 1:20,000 in BALB/c (Fig. 7A) and 1:200,000 in C57BL/6 mice (Fig. 7B). The antibody titre had its peak after the third immunisation with titres of 1:650,000 and 1:250,000, respectively. Eight months after the last immunisation, antibody titres had dropped to 1:55,000 in BALB/c (Fig. 7A) and 1:50,000 in C57BL/6 mice (Fig. 7B). No DOBV rN-specific antibodies were found in the serum pool of control mice immunised with HBcd (data not shown).

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FIGURE 7: Kinetics of DOBV N-specific antibody response. BALB/c (A) and C57BL/6 (B) mice were immunised according to scheme 3 (Fig. 2B) three times with 50 µg of HBcdDOB120 or DOBV rN protein in complete Freund's adjuvant (week 0), incomplete Freund's adjuvant (week 3) or without adjuvant (week 6), respectively. N-specific antibody endpoint titres (three times the background) were determined in ELISA for each mouse separately. Shown are the means of reciprocal endpoint titres of 5 mice and the respective standard deviations. Stars indicate significant differences in log 10 titres between mice immunised with HBcdDOB120 compared to mice immunised with DOBV rN protein as determined by Mann Whittney U Test (p < 0.05).

Like in HBcdDOB120 immunised mice the highest antibody titre was found after the third immunisation with titres of 1:800,000 and 1:1,000,000, respectively. At this time point significantly higher titres were found in mice immunised with DOBV rN protein compared to HBcdDOB120. Eight months after the last DOBV rN protein immunisation, antibody titres had dropped to 1:150,000 in BALB/c (Fig. 7A) and 1:100,000 in C57BL/6 mice (Fig. 7B). Serum pools from sera of animals immunised with DOBV-Slo rN protein did not develop any titre against yeast-expressed DOBV-Slo G2, used as a negative control (data not shown). In addition, no DOBV rN-specific antibodies were found in the serum pool of control mice immunised with PBS (data not shown).

3.5 HBcdDOB120 and DOBV rN protein induced antibodies are highly cross-reactive to the rN proteins of other hantaviruses

The reactivity of sera from mice immunised three times with HBcdDOB120 or DOBV-Slo rN protein to the rN proteins of other hantaviruses was analysed. Therefore, individual sera taken two weeks after the final immunisation with HBcdDOB120 or DOBV rN protein (both based on the N protein of DOBV-Slo) were tested for antibodies reacting with the rN proteins of DOBV-Slk, HTNV-Foj, PUUV-Vra, PUUV-Kaz and PUUV-Sot, SNV-3H226 and ANDV-AH1.

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Sera of mice immunised with HBcdDOB120 were all reactive to the rN proteins of DOBV-Slk, HTNV-Foj and PUUV-Vra, PUUV-Kaz, and PUUV-Sot, ANDV-AH1 and SNV-3H226 when tested in ELISA (Fig. 8A,B). The pattern of cross-reactivity did not differ between BALB/c and C57BL/6 mice. The cross-reactivity was highest to the heterologous rN proteins of the more closely related HTNV. However, reactivities were significantly lower to the more distantly related PUUV-Kaz (only in BALB/c mice, p=0.032) and PUUV-Vra (p=0.008 BALB/c; p=0.016 C57BL/6). Reactivities to PUUV-Sot, SNV and ANDV were not found to be significantly different to the reactivity to DOBV (Fig. 8A,B).

In immunofluorescence assay, a highly diluted (1:10,000) serum pool from HBcdDOB120 vaccinated BALB/c mice showed highest fluorescence intensity and more cells immunofluorescence positive on HTNV infected cells. Sera showed similar intensity on DOBV infected VeroE6 cells but much lower intensity of fluorescence and fewer immunofluorescence positive PUUV infected cells (data not shown). Interestingly, the level of cross-reactivity was lower when a serum pool was tested in Western Blot. Serum of mice immunised with HBcdDOB120 reacted to a low extent to the rN proteins of PUUV-Kaz and PUUV-Sot and did not react at all to rN protein of PUUV-Vra (data not shown). However, both tests are not quantitative as the intensity of immunofluorescence and the intensity of the bands in Western Blot could only be estimated.

FIGURE 8: Analysis of the cross-reactivity of antibodies of BALB/c (A) and C57BL/6 (B) mice two weeks after the third immunisation with 50 µg HBcdDOB120 or DOBV rN protein (scheme 3, Fig. 2B). Antibodies induced by either of those two constructs based on the rN protein of hantavirus strain Dobrava Slovenia (DOBV-Slo) were tested on the reactivity with the rN proteins of the hantaviruses DOBV strain Slovakia (Slk), Hantaan virus (HTNV) strain Fojnica (Foj), Puumala viruses (PUUV) strains Kazan (Kaz), Vranica/Hällnäs (Vra), Sotkamo (Sot), Sin Nombre virus (SNV) strain 3H226 and Andes virus (ANDV) strain AH1. Endpoint titres (three times the background) of each animal was determined by ELISA. Shown are the arithmetic mean values and standard deviations of the reciprocal endpoint titres of five animals. Stars indicate significant differences in log 10 titres between DOBV-Slo-specific titres compared to antibody titres reacting to the N protein of other hantaviruses as determined by Mann Whittney U Test (p < 0.05).

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In general, the sera of mice immunised with DOBV-Slo rN protein reacted in ELISA strongly with all rN proteins (Fig. 8A,B). The pattern of cross-reactivity did not differ between BALB/c (Fig. 8A) and C57BL/6 (Fig. 8B) mice. The reactivity of the sera from both BALB/c and C57BL/6 mice immunised with DOBV-Slo rN protein to the rN protein of the more closely related DOBV-Slk and HTNV-Foj was slightly stronger when compared with the reactivity to the rN proteins of the PUUV strains (Fig. 8A,B). In both mouse strains, the reactivity was significantly lower to PUUV-Vra (p=0.008), PUUV-Sot (p=0.008 BALB/c; p=0.032 C57BL/6). Only sera from C57BL/6, but not BALB/c mice immunised with DOBV rN reacted to significantly lower level to ANDV-AH1 rN protein (p=0.032).

In immunofluorescence assay highly diluted (1:10,000) pool of sera from DOBV rN protein immunised BALB/c mice reacted strongly to HTNV and DOBV infected VeroE6 cells but weaker to PUUV infected cells (data not shown). In Western Blot, the serum pool of mice immunised with DOBV-Slo rN protein reacted with the rN proteins of both DOBV, HTN and all three PUUV strains (data not shown).

3.6 HBcdDOB120 and DOBV rN induced N-specific antibodies of all IgG subclasses

To get a first idea which kind of immune response is induced by HBcdDOB120 particles and DOBV rN protein in mice, we determined titres of N-specific antibodies of the IgG subclasses IgG1, IgG2a, IgG2b and IgG3. In addition, titre ratios of IgG1/IgG2a as well as titre ratios of all four IgG subclasses to total IgG were calculated to characterise the (mostly T cell secreted) cytokine environment when B cells were activated.

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HBcdDOB120 induced in BALB/c and C57BL/6 mice N-specific antibodies of all IgG subclasses, IgG1, IgG2a, IgG2b and IgG3 to a similar extend (Figs 9A,B). The ratio of IgG1/IgG2a was between 1.0 and 1.2 in BALB/c and between 1.1 and 1.5 in C57BL/6 mice at four time points after the first immunisation (Tab. 4).

Besides IgG1 and IgG2a antibodies, IgG2b and IgG3 were measured in the sera of mice immunised with HBcdDOB120. In the sera taken the mean IgG2b/IgG ratios lay between 0.70 and 1.02 while the IgG3/IgG ratios were between 0.67 and 0.82 (Tab. 4).

FIGURE 9: IgG subclass distribution of DOBV N-specific antibodies in sera of mice two weeks after the last of three immunisations with HBcdDOB120 or DOBV rN protein. Shown are the arithmetic means and standard deviations of the reciprocal endpoint titres (three times the background) of five animals. Each titre was determined separately by ELISA for each animal. Comparability of the anti-IgG1, anti-IgG2a, anti-IgG2b and anti-IgG3 antibodies used in the ELISA was confirmed in a special ELISA (see chapter 2.4.1).

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In mice sera after immunisation with DOBV rN protein N-specific IgG of all subclasses were found to beinduced (Figs 9C,D). However, we observeda slight dominance of IgG1 over IgG2a, IgG2b and IgG3 in the sera of BALB/c mice (Fig. 9C). This dominance of IgG1 over the other IgG subclasses was detected in sera after each immunisation step (Tab. 4). In the sera of C57BL/6 mice IgG1 dominated over IgG2b, followed by IgG2a and IgG3 (Fig. 9D). As observed in BALB/c mice, this dominance could be seen in sera of C57BL/6 mice after each immunisation (Tab. 4).

In sera taken at four time points the mean IgG2b/IgG ratios laid between 0.48 and 0.61 for BALB/c and 0.84 and 0.93 for C57BL/6 without showing a time-dependent pattern (Tab. 4). The IgG3/IgG ratios were between 0.65 and 0.73, decreasing with time after immunisation (Tab. 4).

TABLE 4: Ratios of N-specific antibody titre of IgG subclasses to total IgG and IgG1 to IgG2a ratios at various time points after immunisation of BALB/c and C57BL/6 mice with HBcdDOB120 particles or DOBV rN protein. Animals were immunised three times (weeks 0, 3 and 6) with 50 μ g in adjuvants according to scheme 3 (Fig. 2B) and bled at various time points. Shown are the means (in bold) and the standard deviation (s) of five animals of the ratios of antibody titres that as determined by ELISA.

time pointa

mouse

strain

immunisation antigen

IgG1/IgGb

mean d s

IgG2a/IgGb

mean d s

IgG2b/IgGb

mean d s

IgG3/IgGb

mean d s

IgG1/IgG2ac

mean d s

3

6

8

35

BALB/c

HBcdDOB120

0.97

0.98

0.82

0.90

0.11

0.08

0.06

0.07

0.81

0.90

0.82

0.84

0.09

0.07

0.06

0.07

0.86

0.83

0.72

0.70

0.11

0.08

0.08

0.07

0.81

0.81

0.74

0.72

0.10

0.03

0.05

0.04

1.20

1.10

1.01

1.08

0.12

0.15

0.13

0.15

3

6

8

35

DOBV rN

1.08

0.95

1.02

1.04

0.07

0.04

0.03

0.08

0.68

0.73

0.77

0.81

0.05

0.05

0.03

0.05

0.60

0.50

0.48

0.60

0.05

0.10

0.13

0.11

0.73

0.70

0.72

0.70

0.05

0.04

0.04

0.02

1.58

1.30

1.31

1.28

0.09

0.08

0.00

0.06

3

6

8

35

C57BL/6

HBcdDOB120

0.93

0.92

0.89

0.77

0.04

0.10

0.03

0.08

0.64

0.68

0.79

0.57

0.08

0.03

0.04

0.02

0.93

0.90

1.02

0.98

0.07

0.06

0.07

0.04

0.82

0.79

0.73

0.67

0.01

0.04

0.07

0.05

1.47

1.35

1.14

1.36

0.17

0.16

0.05

0.13

3

6

8

35

DOBV rN

1.00

0.98

0.99

0.87

0.07

0.03

0.03

0.04

0.64

0.67

0.69

0.59

0.04

0.03

0.05

0.04

0.92

0.93

0.92

0.84

0.04

0.03

0.00

0.09

0.72

0.72

0.67

0.65

0.03

0.03

0.04

0.04

1.58

1.47

1.44

1.48

0.13

0.08

0.13

0.04

a weeks after first immunisation
b ratios of the N-specific log10 reciprocal antibody titre of IgG subclass to IgG total
c ratios of the N-specific log10 reciprocal antibody titre of IgG1 to IgG2a
dThe HBcdDOB120 induced ratios that significantly differ from DOBV rN induced ratios are in italic
and underlined (p<0.05). Analysis of significant differences by Mann-Whittney U Test, done
separately for BALB/c and C57BL/6 mice.

3.7 Proliferation of N-specific lymphocytes was low after immunisation with HBcdDOB120 or DOBV rN protein

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To investigate the cellular immune response, mice immunised three times with HBcdDOB120 or DOBV rN protein were subsequently immunised with a low dose of DOBV rN protein in order to bring the N-specific lymphocytes back to the draining lymph nodes.

Mice previously immunised with HBcd and PBS, respectively, were also immunised with the same dose of DOBV rN to guarantee the "sub-immunogenic" nature of this low dose. Proliferation of cells was determined using 6x105 (BALB/c) or 4x105 (C57BL/6) cells per per well.

In mice immunised with HBcdDOB120 only small (BALB/c) or no (C57BL/6) proliferative response after restimulation with DOBV rN could be detected (Fig. 10). The highest proliferative response, a stimulation index (SI) of 2.2 developed after a restimulation with 4 µg/ml DOBV rN protein. These results are representative for three other experiments conducted on cells from BALB/c mice treated the same way as in the experiment shown here (data not shown). Restimulation of cells with 4 µg/ml ConA resulted in strong proliferation with SIs of 4 to 14 (BALB/c) or 40 (C57/BL6) (data not shown).

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FIGURE 10: Analysis of N-specific proliferation of lymph node cells. One Mouse was immunised three times sc with HBcdDOB120 or DOBV rN protein (scheme 3, Fig 2B). Seven months after the last immunisation these mice were injected with a sub-immunogenic dose of 2 µg DOBV rN protein to induce the N-specific lymphocytes to home to the draining lymph nodes. Control mice previously immunised with HBcd or PBS were immunised the same way. Four days after the injection of the sub-immunogenic dose doublets of 6 x 10 5 (BALB/c) or 4 x 10 5 (C57BL/6) cells from the pooled inguinal, axial and brachial lymph nodes were restimulated for 72 h in vitro with different concentrations of DOBV rN protein or left untreated in complete RPMI medium. In addition, cells from animals immunised with DOBV-Slo rN were restimulated with rG2 protein to assess if the His tag or potential yeast contaminations have an impact on the proliferation. Level of proliferation was determined by means of brom-desoxyuridine incorporation (Roche). The stimulation index was calculated as the proliferation induced by the respective antigen concentration divided by the proliferation induced by medium alone. Shown are means of doublets with the respective standard deviations.

Immunisation of mice with DOBV rN protein resulted in proliferative responses in BALB/c and C57BL/6 mice after restimulation with DOBV rN protein (Fig. 10). The strongest N-specific responses, with SIs of over 5 (BALB/c) and over 3 (C57BL/6), were found after restimulation with 4 µg/ml and 20 µg/ml DOBV rN protein.

In BALB/c mice restimulation with His-tagged rG2 protein purified in a similar way as DOBV rN protein also induced cells that were immunised with DOBV rN protein to proliferate (Fig. 10). The proliferation of cells from BALB/c mice immunised with DOBV rN protein caused by rG2 protein was variable and in some antigen concentrations higher than the proliferation caused by DOBV rN protein (Fig. 10). The same was seen in the other three experiments (data not shown).

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In contrast, lymph node cells of C57BL/6 mice immunised with DOBV rN protein did not proliferate at all when restimulated with rG2 protein. The same was the case for the lymph node cells from C57BL/6 and BALB/c mice immunised with PBS after restimulation with DOBV rN protein or rG2 protein (Fig. 10 and data not shown). Cells from mice immunised with HBcdDOB120 did also not proliferate after restimulation with 20 μg/ml rG2 protein (data not shown). Restimulation of cells with 4 µg/ml Con A resulted in strong proliferation (see 3.4.1.1).

3.8 Higher cytokine levels were secreted after immunisation with DOBV rN protein than after immunisation with HBcdDOB120

Cytokines IL-2, IL-4 and IFN-γ were measured in the supernatant of mouse cells used in the proliferation assay (see above). As in the proliferation assay, cytokine concentrations in the supernatant of BALB/c cells were not comparable to the cytokine concentrations in the supernatant of C57BL/6 cells because less cells of the latter per well were cultured (see above).

The cells of BALB/c mice (Figs 11, 13) and a C57BL/6 (Fig. 12) mouse immunised with HBcdDOB120 secreted only very little amounts of IL-2 after restimulation with DOBV rN protein. The highest levels of IL-2, 80 pg/ml in BALB/c (Fig. 11) and 7 pg/ml in C57BL/6 (Fig. 12) cell supernatants, were detected after restimulation with 20 µg/ml DOBV rN protein.

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Cells of mice immunised with HBcdDOB120 secreted very little (BALB/c) or no (C57BL/6) IL-4 and IFN-γ after restimulation with DOBV rN protein. Like IL-2, BALB/c cells secreted the highest amount of IFN-γ (230 pg/ml) after restimulation with 20 µg/ml DOBV rN (Fig. 11) At this concentration lymphocytes secreted the most IL-4 and IFN-γ after 48 h (Fig. 13) and 72 h (Fig. 11) of restimulation. However, in one experiment highest IL-4 concentrations (80 pg/ml) were found after restimulation of lymphocytes with 5 μg/ml DOBV rN (data not shown).

Lymphocytes from control mice immunised with HBcd secreted also low amounts of cytokines after restimulation with DOBV rN protein (Figs 11-13). Highest concentrations of IL-4 (< 70 pg/ml) and IFN-γ (< 90 pg/ml) were emitted by BALB/c lymphocytes after restimulation with DOBV rN protein for 72 h (Figs 11).

The cells from BALB/c mice (Fig. 11, 13) and a C57BL/6 mouse (Fig. 12) immunised with DOBV rN protein both secreted IL-2 after restimulation with DOBV rN protein. However, level of IL-2 was much higher in the supernatant of BALB/c lymphocytes compared to the supernatant of C57BL/6 lymphocytes. The highest level of secretion was observed after restimulation with 20 µg/ml, 1,500 pg/ml in BALB/c cells and 75 pg/ml in C57BL/6 cells. Restimulation of the lymph node cells from BALB/c, but not the C57BL/6 mouse with rG2 control protein resulted in the detection of IL-2 secretion of more than half of the level after restimulation with rN protein (Figs 11, 12).

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Immunisation of BALB/c and C57BL/6 mice with DOBV rN protein induced lymph node cells to secrete high amounts of IL-4 and IFN-γ after restimulation with DOBV rN protein (Figs 11-13). Highest secretion of IL-4 was 930 pg/ml (BALB/c, Fig. 11) and 540 pg/ml (C57BL/6, Fig. 12) after restimulation of cells with 20 µg/ml DOBV rN protein. Also IFN-γ was secreted most after restimulation of cells with 20 µg/ml, 4,800 pg/ml in BALB/c (Fig. 11) and 1,400 pg/ml in C57BL/6 cells (Fig. 12). In the same line, BALB/c lymphocytes secreted the highest amounts of IL-4 and IFN-γ after 48 h of restimulation with 20 μg/ml rN protein (Figs 13).

FIGURE 11: Analysis of IL-2, IL-4 and IFN-γ secreted by lymphocytes from four BALB/c mice. Mice were immunised with HBcdDOB120 or DOBV rN protein and supernatant from cultured lymphocytes was collected after 24h (IL-2) or 72 h (IL-4, IFN- γ ) of restimulation with different concentrations of DOBV rN protein. Negative control mice were immunised with HBcd and PBS, respectively. Cells from mice immunised with DOBV rN protein were additionally restimulated with rG2 protein to estimate cytokine secretion due to cells reacting to His-tag or potential yeast contaminations. Supernatants were taken from cells used in the proliferation assay (Fig. 10) and cytokine concentrations were determined by sandwich ELISA (chapter 2.4.4).

Restimulation with rG2 protein also caused the cells from BALB/c, but not cells from the C57BL/6 mouse immunised with DOBV rN protein to secrete high amounts of IL-4 and IFN-γ (Figs 11, 12). However, the level of IL-4 and IFN-γ induction was lower at most restimulating antigen concentration when compared to rN protein stimulation (Figs 11, 12).

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FIGURE 12: Analysis of IL-2, IL-4 and IFN-γ secreted by lymphocytes from one C57BL/6 mouse. Mice were immunised with HBcdDOB120 or DOBV rN protein and supernatant from cultured lymphocytes was collected after 24h (IL-2) or 72 h (IL-4, IFN- γ ) of restimulation with different concentrations of DOBV rN protein. Negative control mice were immunised with HBcd and PBS, respectively. Cells from mice immunised with DOBV rN protein were additionally restimulated with rG2 protein to estimate cytokine secretion due to cells reacting to His-tag or potential yeast contaminations. Supernatants were taken from cells used in the proliferation assay (Fig. 10) and cytokine concentrations were determined by sandwich ELISA (chapter 2.4.4).

Cells of mice immunised with PBS did not secrete any IL-2, IL-4 or IFN-γ (Figs 11-13).

FIGURE 13: Analysis of IFN-γ and IL-4 secreted by lymphocytes from four BALB/c mice. Mice were immunised with HBcdDOB120 or DOBV rN protein and supernatant from cultured lymphocytes was collected after 48 h of restimulation with different concentrations of DOBV rN protein. Negative control mice were immunised with HBcd and PBS, respectively. Cells from mice immunised with DOBV rN protein were additionally restimulated with rG2 protein to estimate cytokine secretion due to cells reacting to His-tag or potential yeast contaminations. Supernatants were taken from cells used in the proliferation assay (Fig. 10) and cytokine concentrations were determined by sandwich ELISA (chapter 2.4.4).


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