2 Material and methods

2.1  Protein derivatives for immunisation

2.1.1  Expression and purification of VLPs

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The generation of the expression plasmids for the carboxy-terminally deleted HBc protein (aa 1-144, HBcd) and chimeric HBc protein carrying 120 aa of the N protein of the hantavirus DOBV at aa 78 of HBcd (HBcdDOB120) have been described previously [Borisova, 88; Geldmacher, 04k].

Purified HBcd and HBcdDOB120 were kindly provided by Dr. Galina Borisova (Biomedical Research and Study Centre, Riga, Latvia). The expression in E. coli and purification of the HBcd particles have been described previously [Geldmacher, 04j]. Briefly, cells of E. coli strain K802 were transformed with plasmids encoding the respective core proteins. After sedimentation the cells were lysed and soluble proteins extracted. Core proteins were precipitated and loaded onto a saccharose gradient or a sepharose CL4B column. Fractions containing the core proteins were identified by SDS-PAGE and Western blot analysis (see chapter 2.2.1), concentrated and stored in glycerol at –20°C until further use. Prior to immunisation, the particles were diluted in PBS.

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The formation of particles of HBcd and HBcdDOB120 particles was kindly proven by negative staining electron microscopy by Dr. Hans R. Gelderblom [Robert Koch-Institut, Berlin, Germany, see Geldmacher, 04i].

2.1.2 Expression and purification of full-length rN protein

The vectors for the yeast expression of the rN proteins of the hantaviruses DOBV, strains Slovenia [DOBV-Slo; Avsic-Zupanc, 95b] and Slovakia [DOBV/Esl/862Aa/98; DOBV-Slk; Sibold, 01b], HTNV, strain Fojnica [HTNV-Foj; Sibold, 99b], PUU, strains Vranica/Hällnäs [PUUV-Vra; Reip, 95], Kazan [PUUV-Kaz; Lundkvist, 97a]) and Sotkamo [PUUV-Sot; Vapalahti, 92] have been generated and generously provided by Ausra Razanskiene [Dargeviciute, 02; Razanskiene et al., 2004]. The yeast expression plasmids for the rN proteins of the New World hantaviruses ANDV, strain AH1 [Lopez, 97b] and SNV, strain 3H226 [Hjelle, 94a] were generated and kindly provided by Jonas Schmidt (Schmidt et al., submitted).

The expression and purification of the rN proteins was performed according to protocols previously described [Dargeviciute, 02; Razanskiene et al., 2004]. Briefly, pFX7-derived expression plasmids encoding a fusion of an amino-terminal hexahistidine (His)-tag and the hantavirus N proteins under the control of a galactose inducible yeast promoter were transformed into the yeast S. cerevisiae wild-type strain FH4C. The synthesis of rN proteins was induced by addition of galactose and proteins were purified under denaturing conditions via their His-tag according to the protocol of the manufacturer (Qiagen). Proteins were characterised in SDS polyacrylamid gel and Western blot (chapter 2.2.1). Hamster polyomavirus (HaPyV) VP1 protein expressed in yeast, kindly provided by Dr. Alma Gedvileite [see Gedvilaite, 04], served as a negative control in Western Blot. For immunisations DOBV rN protein was dialysed against phosphate buffered saline (PBS) and subsequently lyophilised.

2.2 Characterisation of the recombinant protein derivatives

2.2.1  SDS-PAGE and Western Blot

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Protein samples were separated by electrophoresis in 12.5 or 15 % SDS polyacrylamide gels. Protein bands were stained by Coomassie blue. For Western blot, proteins were transferred to cellulose nitrate membrane by semi-dry blotting. After transfer, cellulose membranes were blocked with 5 % dry milk / PBS containing0.1 % Tween (PBS/T) for one hour and incubated 16 to 18 h in PBS/T dilutions of the mAbs 1C12, 4C3 [Lundkvist, 91; diluted 1:1,000], mouse anti-DOBV rN serum (1:2,000 in PBS/T) or polyclonal rabbit serum raised against HBc/GFP particles [Kratz, 99; diluted 1:5,000 in PBS/T]. Thereafter, filters were incubated with the respective horse radish peroxidase (HRP)-conjugated anti-mouse IgG (1:3000, Sigma-Aldrich) or anti-rabbit IgG (1:6,000, Sigma-Aldrich) in PBS/T for 2 hours. The peroxidase staining was performed by adding 4-chloro-1-naphthol (Sigma-Aldrich) supplemented with H2O2.

2.2.2 Determination of protein concentration

To determine the concentration of purified proteins, protein samples were mixed with Bradford reagent (0.01 % Coomassie brilliant blue G250, 8.5 % phosphoric acid, 5 % ethanol). After 10 to 30 min the OD595nm values were measured. To estimate the protein concentration, a standard curve with two-fold dilutions of bovine serum albumin (BSA, Sigma-Aldrich) in PBS ranging from 16 - 250 μg/ml was generated. In addition, the estimated protein concentrations of the different rN proteins were compared to each other in SDS polyacrylamid gel by Coomassie blue staining and adjusted accordingly.

2.3 Immunisation of mice

2.3.1  Mice strains

To analyse the immunogenicity of HBcdDOB120 and DOBV rN protein inbred mice strains of two different haplotypes, H-2d (BALB/c) and H2-b (C57BL/6), were used for analysis. Groups of five female mice each were immunised at sex to ten weeks of age. All mice were obtained from the "Bundesinstitut für gesundheitlichen Verbraucherschutz und Veterinärmedizin" in Berlin and held in the Max-Planck-Institute for Infectious Biology, Berlin. Permission of all animal experiments were obtained from the "Landesamt für Arbeitsschutz, Gesundheitsschutz und technische Sicherheit" in accordance to the German laws (§ 8 Abs. 1 des Tierschutzgesetzes).

2.3.2 Immunisation of mice to investigate the influence of HBc-specific preexisting immunity

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The effect of a preexisting immunity against the carrier protein HBc on the development of anti-N antibodies after immunisation with HBcdDOB120 should be addressed. Thus BALB/c and C57BL/6 mice were immunised according to two different protocols, a “short-term” (scheme 1, Fig. 2A) and “long-term“ (scheme 2, Fig. 2A) protocol. In general, mice were first immunised with HBc and then, after animals had developed an antibody titre against HBc as evidenced by ELISA (see chapter 2.4.1), mice were immunised with HBcdDOB120.

In the first set of immunisations, four BALB/c and four C57BL/6 mice per group were immunised subcutaneously (sc) three times with 20 µg each of full length HBc in complete Freund's adjuvants (CFA, Sigma-Aldrich), incomplete Freund's adjuvants (IFA, Sigma-Aldrich) and without adjuvants. Six weeks after the last immunisation the mice had developed a high antibody titre against HBc as evidenced by ELISA (see chapter 2.4.1). In the "short term" experiment the immunisation with HBcdDOB120 (20 μg in CFA) was performed six weeks after the last immunisation with HBc (scheme 1, Fig. 2A). In the "long term" experiments, the immunisation with HBcdDOB120 (20 μg in CFA) was given five months after the last immunisation with HBc (scheme 2, Fig. 2A). Blood was collected three weeks after the final immunisation.

FIGURE 2: Immunisation schemes for BALB/c and C57BL/6 mice. To address the question of the influence of HBc-specific immunity to the response to the DOBV rN protein, mice were immunised with HBc and subsequently with HBcdDOB120 (A, schemes 1 and 2). Immunisations of mice with HBcdDOB120 and DOBV rN protein were done to compare the immune responses of mice after immunisation with the two proteins in antibody response and cellular response (B, scheme 3). Immunisation scheme 3 has previously been used in a hantavirus challenge model in bank voles (Lundkvist et al 1996). Solid arrows illustrate the time points of immunisations, while open arrows show the time points of bleeding of the animals. The grey arrow indicate the time point where animals were sacrificed an proliferation and cytokine assays performed after immunisation with a sub-immunogenic dose (2 μ g) of DOBV rN protein. For details of the immunisations see text (scheme 1 and 2, chapter 2.3.2; scheme 3, chapter 2.3.3) .

2.3.3 Immunisation of mice with HBcdDOB120 and DOBV rN protein

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To investigate the immunogenicity of HBcdDOB120 and DOBV rN protein, groups of five BALB/c and five C57BL/6 mice were immunised according to a scheme which was previously used in a PUUV challenge model [Lundkvist, 96]. Briefly, mice were immunised sc three times with 50 µg of HBcdDOB120 or DOBV-Slo rN protein at intervals of three weeks (scheme 3, Fig. 2B). Mice were immunised with the proteins in PBS with CFA, IFA and without adjuvant, respectively. For negative control, mice were immunised three times with HBcd or PBS in the same adjuvants, respectively.

To assess the N-specific cellular immune response, BALB/c and C57BL/6 mice used for the characterisation of the antibody response were injected with 2 µg of DOBV rN protein seven to eight months after the last immunisation with HBcdDOB120 or DOBV rN protein, respectively (scheme 3, Fig 2B). Four days after immunisation, mice were sacrificed and single cell suspensions were prepared from a pool of inguinal, axial and brachial lymph nodes (see chapter 2.4.3).

2.3.4 Bleeding and storage of blood

Blood was collected by bleeding of the tail vein. After the blood clotting it was centrifuged and sera were aliquoted and stored at –20 °C. Once sera were thawed for use in ELISA, they were subsequently stored at 4 °C.

2.4 Characterisation of the immune response of mice

2.4.1  ELISA

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Elisa was performed as described previously [Geldmacher, 04a]. Briefly, rN proteins [10 µg/ml] in coating buffer (40 mM Na2CO3, 60 mM NaHCO3, pH 9.8) were coated onto Maxisorb Plates (Nunc) overnight at 4°C. Coated plates were blocked with 150 µl / well of blockingbuffer (1 % BSA, 0.01 % Tween-20, PBS) for 30 min at 37 °C. The sera were then diluted in 100 μl / well at a minimum dilution of 1:50 in ELISA-buffer (0.5 % BSA, 0.01 % Tween-20, PBS), titrated three-fold and incubated for 1 h at 37°C. Specific antibody binding was detected by incubation with HRP-labelled rabbit anti-mouse-IgG in ELISA-buffer (1:8,000, Sigma-Aldrich) for 1 h at 37°C. Assays were developed with o-phenylenediamine (Sigma-Aldrich) in 0.05 M phospho-citrate buffer (Sigma-Aldrich) supplemented with 1.5 o/oo H2O2 and stopped with 50 µl 0.6 M H2SO4 after 20 min at room temperature. The optical density (OD) was read at 492 nm with a reference wavelength of 620 nm.

The titres of antibodies of the different IgG subclasses were determined similarly to total IgG as follows. Coating, blocking and serum dilution were done as described above for the IgG ELISA. After incubation of sera, goat anti-IgG1, anti-IgG2a, anti-IgG2b and anti-IgG3 antibodies (1:1,000, Sigma-Aldrich) were added to the plates for one hour. Finally, binding of anti-IgG subclass antibodies was detected by HRP-labelled anti-goat IgG and immuno-staining as described above.

The endpoint titre was defined as the serum dilution where the OD is three times the background OD. The background OD is the OD that is measured in highly diluted sera and does not decrease with further dilution. In our experiments the background OD varied between 0.02 and 0.1. The OD due to unspecific binding was subtracted from each OD of the respective serum dilution. Unspecific binding is the binding to N of antibodies in the negative control sera of the respective dilution.

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To rule out that the IgG subclass titres differed due to a difference in affinity of IgG subclass specific antibodies, ELISAs with coating of plates with threefold dilutions of recombinant IgG1, IgG2a, IgG2b and IgG3 were performed (Sigma Aldrich), starting with concentrations of 100 – 300 ng/ml. After blocking, the adding of anti-IgG1, -IgG2a, -IgG2b or -IgG3 was done according to the IgG subclass ELISA described above. It turned out that the anti-IgG1 antibody gave a three times lower OD as the anti-IgG subclass antibody that gave the highest OD (anti-IgG3) at the same IgG-subclass concentration. Thus the mistake in the IgG subclass distribution in the mouse sera due to the difference in affinity of the anti-IgG subclass antibodies used in the ELISA is maximal three fold and – as log scale is used in the figure – does not have a big impact on the ELISA results.

2.4.2 Immunofluorecence assay (IFA)

VeroE6 cells infected with DOBV (strain Slovenia), HTNV (strain 76-118) or PUUV (strain Sotkamo) were seeded in 8 – 12-well coverslips and grown overnight. The next day, cells were washed with PBS, fixed with acetone/methanol (1:2) and a 1:1,000, 1:5,000 or 1:10,000 dilution of serum pools of mice immunised three times with HBcdDOB120 or DOBV rN protein (scheme 3, Fig. 2) in PBS/10% rabbit serum was added. For visualisation, rabbit anti–mouse IgG fluorescein isothiocyanate (FITC)-conjugated secondary antibody was used.

2.4.3 Preparation of single cell suspensions from lymph nodes

.For the investigation of the lymphocyte proliferation, inguinal, axial and brachial lymph nodes of single mice were pooled and mashed through a 70 µm cell strainer (FALCON). Cells were pelleted by centrifugation (530 x g, 5 min, 4 °C) and taken up in RPMI medium (Biochrome), 5 % heat-inactivated foetal calf serum (FCS, Biochrome), 2 mM L-glutamine, 100U/ml penicillin, 100 µg/ml streptomycin, 50 µM 2-mercaptoethanol and 25 mM HEPES. The concentration of living cells was determined by trypan blue dye staining. A minimum of 100 live cells were counted with a Bürker chamber and cells were adjusted to the concentration needed (see chapter 2.4.4).

2.4.4 Proliferation and cytokine assays for the determination of N-specific lymphocytes

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To analyse the proliferation of N-specific lymphocytes, lymph node cells of mice immunised with HBcdDOB120 or DOBV rN proteins (scheme 3, Fig 2B) were restimulated in vitro with DOBV rN protein. Doublets of 600,000 cells were seeded in 200 µl medium / well of a 96-well round bottom plate. Cells were restimulated at 37 °C, 5 % CO2 for 3 days with concentrations of 0, 0.03, 0.16, 0.80, 4.00 and 20.00 µg/ml DOBV-Slo rN protein. As a positive control cells were treated with 4 µg/ml concanavalin A (Con A, Sigma-Aldrich). In addition, cells from animals immunised with DOBV rN protein were restimulated with rG2 to assess the impact that the His tag or potential yeast contamination might have on the proliferation.

After 24 hours of restimulation 30 µl per well supernatant was removed for IL-2 testing and BrdU was added to the cells according to the BrdU proliferation test protocol (Roche). After 48 hours of restimulation 50 µl per well supernatant was removed for the IL-4 and IFN-γ testing and 44,000 of the 600,000 cells per well were transferred to a flat-bottom microtitre plate. After centrifugation (530 x g, 5 min, 4 °C) of the plate the medium was removed. The rest of the cells were restored in the incubator. After another 72 hours, 44,000 cells were removed for the proliferation test and the supernatant was saved for IL-4 and IFN-γ tests The BrdU ELISA was performed according to the BrdU Proliferation ELISA protocol (Roche). The stimulation indices (SI) was calculated as the ratio of the OD value obtained by the respective antigen concentration to the OD value obtained by medium only.

As outlined above supernatants were collected from lymph node cells after 24 h restimulation for IL-2 testing and after 48 h and 72 h restimulation for IL-4 and IFN-γ testing. IL-2, IL-4 and IFN-γ was quantified in the supernatant according to the manufacturers protocols (R&D) for a sandwich ELISA. Detection limits of the tests were between 7 and 30 pg/ml.

2.5 Data analysis

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Antibody endpoint titres of each mouse as determined by ELISA was transformed to 10log and average and standard deviation determined for each immunisation group (HBcdDOB120 and DOBV rN protein). By means of the proliferation assay, average SI values (see 2.4.4) and standard deviations were determined for each mouse and each restimulation antigen concentration from duplets. This average SI was then averaged again to calculate the average of each of the immunisation groups for each restimulating antigen concentration. Similarily, cytokine concentrations in the supernatant were determined for each mouse and each restimulating antigen concentration and averages and standard deviations were calculated for each immunisation group. All data analysis were done for BALB/c and C57BL/6 mice separately. Where appropriate, data were analysed for significant differences using a Mann-Whittney U Test (see respective results paragraphs).

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