4 Discussion

4.1  A preexisiting immunity to the carrier protein rather boosts the immunity to the antigenic insert

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Hepatitis B is a widespread infection resulting in a high prevalence of anti-HBc antibodies in the human population worldwide. These pre-exisiting anti-HBc antibodies might influence the success of a vaccine based on HBc as a carrier protein. In this study data are presented that a preexisting immunity to HBc induced by immunisation with particles formed by entire core protein did not abrogate the DOBV N-specific antibody response after immunisation with HBcdDOB120. Similarly, in mice preexisting antibodies to HBV small surface antigen (HBs) only slightly suppressed the antibody response against part of the hepatitis C virus envelope presented on HBs particles [Netter, 03]. In line, in mice immunised with a Haemophilus influenzae type B (HiB) polysaccharide/BSA vaccine, an enhancing effect of BSA carrier protein-specific preexisting antibodies on the anti-HiB immune response was observed [Schneerson, 80]. In children that were primed with a diphteria toxin also an enhanced immunogenicity of a HiB polysaccharide coupled to diphteria toxin was found [Granoff, 93].

The antigen dose used for priming the anti-carrier immune response has been reported to influence the outcome whether the immune response to the respective antigen presented on the carrier is enhanced or suppressed [Peeters, 91]. Therefore, additional studies are needed to investigate if the titre of preexisting anti-core antibodies might influence the results of the immunisation with chimeric particles. Additionally it should be investigated, if the dose of HBcdDOB120 influences DOBV N-specific titre in mice with anti-core immunity.

These data are in contrast to the negative influence of a preexisting immunity to the carrier when trying to deliver a vaccinia virus construct encoding HTNV N and glycoproteins to vaccinia virus-immune volunteers [McClain, 00]. In the same line, in mice with preexisting immunity to an adenovirus (AdV) carrier, the expression of luciferase provided by an AdV-luciferase gene vector was significantly decreased compared to mice lacking an AdV-specific immunity [Vlachaki, 02]. Preexisting antibodies against human papillomavirus (HPV) capsid proteins L1/L2 also abolished the protection from a tumor challenge in mice immunised with chimeric HPV-L1/L2 VLPs harbouring a tumor protein segment expressed by the tumor [Da Silva, 01c; Da Silva, 03b].

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Likely, the presence of carrier-specific antibodies might result in the opsonisation of most particles, of both viral vectors and VLPs, and their uptake by antigen presenting cells (APCs) via Fc receptors. Therefore, a possible explanation for the different influence of a preexisting immunity on DNA-based vectors versus nucleic acid-free VLPs might be the following. When vectors or particles are opsonised with antibodies, foreign protein-encoding DNA in the vector gets degraded. On the other hand the foreign protein insert of chimeric VLPs will gets presented better on MHC class I and II. This enhanced MHC class II presentation could induce a stronger insert-specific antibody response. As such an enhancement of peptide presentation on MHC class I and II has been shown after covering protein with antibodies [Zheng, 01a; Zheng, 01b; Rafiq, 02a]this could be an explanation for the higher antibody response in mice with preexisting carrier-specific antibody titre. However, this does not explain the lower protectivity against tumors in mice with a preexisting L1/L2-specific immune response after immunisation of mice with chimeric L1/L2/tumor antigen particles [Da Silva, 01b; Da Silva, 03a]. It seems that particles that consist on carrier proteins that confer receptor dependent uptake, like papillomavirus L1/L2 [Da Silva, 01a] induction of insert specific immune response is hampered by preexisiting carrier specific immune response. This is in contrast to particles based on carrier proteins HBc (this study) and small HBs [Netter, 03].

Here, only the influence of a preexisting antibody response to the carrier protein on the insert-specific antibody response was measured. But, as preimmunisation induced HBc-specific IgG of all subclasses, it is likely that the preimmunisation also induced HBc-specific T-cells, as would happen in a natural HBV infection. This suggests that an immunisation with HBc-derived VLPs would be successful in people that once were infected with HBV. However, it remains to be proven if HBc-specific T cells have an abolishing effect on the induction of insert-specific immunity or protection. Additionally it should be proven if the immune response obtained in mice with a preexisting anti-core immunity can be confirmed in the situation of a preexisting immunity due to HBV infection, e.g. in a primate model.

4.2 DOBV N proteins induce a similar immune response as other hantavirus N proteins

In south-east Europe the A. flavicollis-adapted DOBV is the major cause of severe clinical courses of HFRS. In addition, experimental inoculation of this virus was found to kill suckling mice [Klingström, 03]. While the immunogenicity and protectivity of PUUV rN protein in rodents has been investigated in several studies [de Carvalho Nicacio, 01b; Dargeviciute, 02; de Carvalho Nicacio, 02], less is known about the immunogenicity of DOBV rN protein. Therefore, in this study the immunogenicity of two different protein derivatives based on the N protein of an A. flavicollis-adapted DOBV, HBcdDOB120 and DOBV rN protein was investigated.

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In line with initial immunisation studies in rabbits [Razanskiene, 04], high antibody titres were induced in BALB/c and C57BL/6 mice strains by immunisation with HBcdDOB120 and DOBV rN protein. This strong immunogenicity is also reflected by the fact, that already after the primary immunisation a high-titred antibody response was observed. These data are similar to those found for E. coli-expressed PUUV-Kaz and yeast-expressed PUUV-Vra rN proteins in the same mice strains [de Carvalho Nicacio, 01a; A. Zvirbliene et al., unpublished data]. The homologous antibody titres observed by us after immunisation with DOBV rN protein were comparable to homologous antibody titres in bank voles after immunisation with PUUV-Kaz rN protein, but much higher than those found in bank voles after immunisation with DOBV-Slo rN protein [de Carvalho Nicacio, 02]. If this difference is caused by the different expression systems used to produce the rN proteins or by a suboptimal detection of bank vole antibodies by anti-mouse IgG remains to be elucidated.

As previously observed for entire E. coli-expressed rN proteins of different hantaviruses (de Carvalho Nicacio et al., 2002), immunisations of mice with chimeric HBc particles and entire DOBV rN protein resulted in a highly cross-reactive antibody response. As discussed below (chapter 4.4), this confirms again previous data about a highly cross-reactive region located in the amino-terminus of the N protein.

In the same line, rabbit sera raised against yeast-expressed rN proteins of DOBV and HTNV [Razanskiene, 04] and sera from mice immunised with DOBV-Slo rN and HBcdDOB120 protein (this study) showed the same pattern of cross-reactivity. The sera reacted to high extent to the rN proteins of HTNV and DOBV and to slightly lower extent to PUUV, SNV and ANDV. The same lower reactivity to PUUV and ANDV rN protein was found in sera of DOBV-Slo rN protein immunised bank voles [de Carvalho Nicacio, 02]. For the sera of DOBV rN and HBcdDOB120 immunised mice from this study this pattern of cross-reactivity was not only observed in ELISA, but also in immunofluorescence analysis using HTNV- and PUUV-infected Vero E6 cells. When looking at PUUV immunised rabbits [Razanskiene, 04] and PUUV or ANDV immunised bank voles [de Carvalho Nicacio, 02] the reverse cross-reactivity can be seen, with lower reactions to rN proteins of DOBV and HTNV.

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Interestingly, this very high level of cross-reactivity could not be found by Western Blot analysis of sera from HBcdDOB120 immunised mice; the sera reacted only weakly with rN proteins of PUUV-Sot and PUUV-Kaz and not at all with PUUV-Vra. This might mean that the cross-reactive antibody bind to conformational epitopes that were destroyed by the reducing SDS buffer used in the gels. On the other side this could reflect the lower sensitivity of Western Blots compared to ELISA. Thus, further investigation are needed to investigate if the conformational nature of the epitopes of cross-reactive antibodies induced by the immunisation with HBcdDOB120 is responsible for the lack of reactivity to denatured PUUV rN.

In this study N-specific IgG of all subclasses were detected in mice after immunisation with DOBV rN protein. In the same line, N-specific antibodies of the subclasses IgG1, IgG2a and IgG2b were found in BALB/c and C57BL/6 mice after two immunisations with PUUV rN protein, supplemented with the same adjuvants as used in this study [de Carvalho Nicacio, 01g]. However, in contrast to this study, no IgG3 was found. If this difference to the data outlined in this study is due to intrinsic properties of the rN proteins from different hantaviruses, due to the differences in expression systems, E. coli[de Carvalho Nicacio, 01h] and yeast (this study) or due to differences in the affinity of the detection antibody used remains to be investigated.

HBcdDOB120, as DOBV rN protein also induced N-specific IgG of all four subclasses. In line with that, immunisation of mice with chimeric HBcdHTN120 or HBcdPUUV120 VLPs or chimeric HaPyV VP1-derived VLPs harbouring 120 aa of PUUV-Vra N protein [Gedvilaite, 04] also resulted in the induction of N-specific antibodies of all four IgG subclasses. The presence of all all IgG subclasses, especially IgG1 and IgG2a, suggests the induction of a mixed Th1/Th2 response (see chapter 4.5).

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In respect to cellular immune response, HBcdDOB120 (this study) induced the same low levels of IL-2 and IL-4 secretion as the chimeric HaPyV VP1 VLPs harbouring 120 aa of PUU N protein [Gedvilaite, 04]. However, immunisation of bank voles with E. coli-derived rN proteins of DOBV, PUUV and ANDV [de Carvalho Nicacio, 02] resulted in the generation of PUUV N-specific lymphocytes. In contrast, this study showed that immunisation of mice with yeast-derived DOBV rN protein resulted in low levels of DOBV N-specific lymphocytes (further discussed in chapter 4.5). In the future it should be investigated what determines this difference in N-specific lymphocyte induction. Thus, proliferative response of DOBV N-specific lymphocytes of mice or bank voles immunised with E. coli-and yeast-derived DOBV rN protein, respectively, should be investigated in the same experiment.

Taken together, besides the induction of only low levels of N-specific lymphocytes after DOBV rN protein found in this study, HBcdDOB120 and DOBV rN proteins induce a similar immune response as other recombinant hantavirus constructs.

4.3 Freund's adjuvants enhances the immune response, but does not seem to modify the N-specific Th1/Th2 cell ratio

The antibody titres found after immunisation with HBcdDOB120 with adjuvant [data presented here and Geldmacher, 04e] were half to one order of magnitude higher compared to antibody titres in mice immunised with HBcdDOB120 without adjuvants (Geldmacher et al., unpubl. data). The immunisation scheme without adjuvant was in dosis and time schedule the same as the immunisation scheme described in Geldmacher et al., 2004b. Similarly, the N-specific antibody titre after immunisation of mice with HaPyV VP1-derived VLPs harbouring 120 aa of the PUU-Vra N protein with adjuvant was about one order of magnitude higher than that observed by immunisation with the same particles but without adjuvant [Gedvilaite, 04].

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The use of CFA and IFA as adjuvants during immunisations not only amplifies the immune response, but can lead to a more Th1 and more Th2 dominated immune response, respectively [Billiau, 01a]. Therefore the induction of a mixed Th1/Th2 response by HBcdDOB120 and DOBV rN protein as shown in this study might be due to the use of adjuvants. However, immunisation of mice without adjuvants with chimeric HBcdDOB120, HBcdHTN120 or HBcdPUUV120 particles (Geldmacher et al., unpubl. data, see paragraph above) or DOBV rN protein without adjuvant (Niedrig and Geldmacher, unpubl. data) also resulted in the induction of all IgG subclassed and thereby in a mixed Th1/Th2 response. The same was found after immunisation of mice with chimeric HaPyV VP1-derived VLPs harbouring 120 aa of PUUV-Vra N protein [Gedvilaite, 04]. This suggests that the chimeric particles and entire rN protein are able to mediate the induction of a mixed Th1/Th2 response and that the induction of subclasses are not biased by the presence of the added adjuvant.

Vaccination of mice with DOBV rN and HBcdDOB120 protein both resulted in the induction of a long-lasting N-specific immunity. Even eight months after the last immunisation high N-specific antibody titres in both mice strain were found, which suggests a possible induction of memory B cells. Alternatively, long term storage of rN protein in the organism, as is believed to be caused by the adjuvants CFA and IFA [see Billiau, 01b], and thereby repeated activation of B cells could be responsible. The half life of IgG in the serum of mice is only several days [Vieira, 86; Vieira, 88] and thereby too short to explain the presence of such high antibody titre in the serum months after immunisation without the presence of N-specific B cells.

To study if memory B cells are induced, mice should be vaccinated with the two rN protein derivatives and the immune response should be characterised after several months, e.g. by means of a B cell ELISPOT. In the B cell ELISPOT, B cells get activated to secrete antigen-specific antibodies, including effector memory B cells as well as central memory B cells [Crotty, 04]. The same should be done for immunisation of mice without adjuvants.

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The data indicates that adding CFA and IFA as adjuvants in this study had increased the N-specific immunity but did not seem to bias the immune response towards Th1 or Th2. However, immunisations of DOBV rN protein without adjuvants was investigated in a very small number of mice (Niedrig and Geldmacher, unpubl. data). Thus, mice should be immunised with DOBV rN protein without adjuvants to test further if the same kind of immune response is induced as with adjuvants. Additionally, lymphocytes of these mice should be tested on their cytokine secretion pattern (e.g. IFN-γ versus IL5) by ELISPOT assay, intracellular cytokine staining or cytokine secretion assay to characterise the N-specific Th1/Th2 response more directly.

4.4 The antibody response induced by chimeric HBcd protein resembles the one induced by entire rN protein

In the bank vole challenge model [Lundkvist, 96], animals have been challenged two weeks after the third immunisation. The antibody titres detected in the present study two weeks after the third immunisation with complete rN protein (429 aa) are slightly higher than titres found after immunisation with HBcdDOB120 particles carrying 120 amino acids of the rN protein. The same slightly higher antibody titre is seen in BALB/c mice immunised with complete rN of PUUV-Vra compared to titres of mice immunised with VLPs carrying 120 aa of the PUUV rN protein [Gedvilaite, 04, Zvirbliene unpubl. data].

An immunisation with lower doses (10 µg, 20 µg) of HBcdDOB120 resulted in C57BL/6 mice in an about one fold lower reciprocal log antibody titre than BALB/c mice [Geldmacher, 04d, Geldmacher et al., unpubl. data and 4.3]. In contrast, the immunisation with a high dose (50 μg) of HBcdDOB120 induced the same N-specific antibody titre in BALB/c and C57BL/6 mice. The lower antibody response in C57BL/6 mice when immunised with a low dose of particles might be explained by the fact that C57BL/6 mice, compared to BALB/c mice, represent weaker responders to the HBc antigen [Milich, 87]. However this lower response in C57BL/6 mice could be overcome by immunising mice with a higher dose of chimeric particles as shown in this study.

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The titres of N-specific antibodies in mice immunised with HBcdDOB120 particles were only slightly lower than those observed in mice immunised with the entire yeast-expressed rN protein of DOBV-Slo. On the one hand this outcome reflects the fact that the amino-terminus of the N protein represents the immunodominant region of this protein [Jenison, 94b; Lundkvist, 96; Elgh, 96b; Gött, 97]. On the other hand these slightly lower titres might be explained by the presence of additional, more carboxy-terminally located epitopes in N protein [for references see Lundkvist, 02]. The strong cross-reactivity of sera from mice immunised with HBcdDOB120 or DOBV rN protein found in this study is also confirmed by binding of mAbs, directed against the amino-terminal region of N, to heterologous N proteins [Dzagurova, 95; Yoshimatsu, 96c; Razanskiene, 04].

Taken together, besides the slightly lower antibody titre and the lack of cross-reactivity to denatured PUU-Vra rN protein in the sera of HBcdDOB120 immunised sera, HBcdDOB120 and DOBV rN protein induce very similar antibody response.

4.5 Chimeric HBc particles as well as entire rN protein induce some N-specific lymphocytes

A switch in IgG subclasses indicates the presence of T helper cells (Th cells). The occurrence of the subclass IgG2a in mouse sera is strongly correlated to IFN-γ-secreting Th1 cells while the presence of IgG1 is strongly correlated to IL-4-secreting Th2 cells [Isakson, 82; Snapper, 87]. So far, a similarly strong correlation between the subclasses IgG2b and IgG3 to any cytokine has not been found [Snapper, 97]. Nevertheless, IgG subclass switching to IgG2b has been thought of being forced by TGF-β produced by several cell types [reviewed by Benoist, 98]. However, IgG2b generation is not driven by Th1 nor Th2 cells [Stevens, 88]. The production of IgG3 has been induced by IFN-γ in the presence of anti-IgD antibodies coupled to dextran and IL-5 (Snapper 1992), but inhibited by IFN-γ in the presence of LPS [Snapper, 87]. In addition Th1 as well as Th2 cells can induce an IgG subclass switch towards IgG3 [Stevens, 88]. The latter experiments indicated that an IgG subclass switch in B cells does not entirely depend on cytokine regimes.

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The majority of IFN-γ and IL-4 that induce IgG subclass switches is secreted by Th1 and Th2 cells, respectively. NK, NKT and mast cells also secrete IFN-γ and IL-4, respectively, but are present in only small amounts in the lymph nodes and spleen, where B cell isotype switching is thought to mainly take place.

The N-specific IgG subclass distribution after HBcdDOB120 vaccination might have been induced solely by the presentation of N protein on HBcd particles and not by the intrinsic antigenicity of the N insert. Truncated HBcd particles have been proven before to induce, compared to full length HBc, a IgG1 dominated IgG subclass response [Milich, 97a], due to the lack of the RNA binding domain in truncated HBcd [Riedl, 02]. However, as complete DOBV rN is also inducing a similar IgG subclass distribution, the IgG subclass distribution might be caused by an intrinsic property of the N protein and not by HBcd carrier characteristics.

HBcdDOB120 VLPs and DOBV rN protein both induced N-specific IgG antibodies of all subclasses. The IgG1 to IgG2a ratio is slightly smaller in the serum of animals immunised with HBcdDOB120 compared to the ratio in sera of animals immunised with DOBV rN protein. As outlined above, this indicates a stronger influence of IFN-γ in the mice immunised with HBcdDOB120 compared to the influence of IFN-γ in mice immunised with DOBV rN protein. Nevertheless the ratio of IgG1 to IgG2a at all time points is above 1 also in HBcdDOB120 immunised animals suggesting dominance of IL-4 and thus Th2 cells. This bias towards Th2 is expected when immunising with protein, as external proteins are known to induce a stronger antibody and Th2 cell than CTL and Th1 cell response. However, lymph node cells of animals immunised with HBcdDOB120 showed very little proliferation and cytokines after restimulation with DOBV rN.

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T cells, secreting IFN-γ and IL-4, leading to N-specific IgG2a and IgG1 switch, respectively (see above) have most probably been present during B cell activation after HBcdDOB120 and DOBV rN protein immunisation. It is possible that HBcd-specific T cells helped N-specific T cells in terms of proliferation by secreting IL-2. However, HBcd-specific T cell proliferation is low after immunisation with HBe [Milich, 97b], a protein very similar to the C-terminally truncated HBcd used in this study. However some HBc-specific T cell proliferation has been observed after immunising BALB/c mice with HBcd [Borisova, 93a]. Therefore, in this study the cytokines present at the IgG subclass switch of N-specific B cells probably could have come partly from N-specific and partly from HBcd-specific T cells. However, levels of N-specific T cells were low, as seen by the low proliferation and low secretion of IL-2, IL-4 and IFN-γ.

It has been found that part of the HBV surface antigen (HBs) inserted into a C-terminally truncated HBcd induced more insert-specific T cell proliferation [Borisova, 93b] compared to this study. In the same line, it has been shown that a papillomavirus E17 protein epitope inserted into particles composed of full length HBc protein can induce E17-specific T cell proliferation as well as IL-2 and IL-4 secretion [Tindle, 94]. In both cases, the epitope was inserted at the same region of HBc as the 120 aa of the DOBV N protein into the C-terminally truncated HBcd used in this study. As has been shown previously, HBc/epitope fusion proteins elicited low or no epitope-specific CTL responses in mice [Street, 99; Storni, 02] indicating limited induction of a Th1/CTL response. The two protein derivatives used in this study also did not generate any cytolytic T cells in five experiments (Geldmacher unpubl. data). However, those experiments should be repeated with a positive control group of mice immunised with an N protein construct that induces N-specific CTLs. This would make it possible to investigate whether the target cells that expressed N protein could be lysed by N-specific CTLs.

In contrast to this study, a stronger induction of N-specific T cells was found in bank voles after immunisation with E. coli-derived DOBV rN protein [de Carvalho Nicacio, 02, see chapter 4.2]. One explanation for this difference in T cell induction could be the difference in time from immunisation to the sacrificing of the animals. When a moderately high proliferation of N-specific spleenocytes were found after immunisation with E. coli-derived rN proteins days after immunisation [de Carvalho Nicacio, 02], while a longer time schedule was used in this study (see Fig. 2B). However, two other experiments in which BALB/c mice were sacrificed a few days after HBcdDOB120 and DOBV rN protein immunisation (Geldmacher, unpubl. data) the same proliferation as shown in Fig. 10 was found: no or hardly any N-specific proliferation after HBcdDOB120 and an SI of up to 5 in lymph node cells from DOBV rN protein immunised mice. This indicates that the difference in N-specific proliferative response when comparing the data described by De Carvalho Nicacio [de Carvalho Nicacio, 02] and the data described in this study is not due to differences in immunisation schedule.

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Lymphocytes from mice immunised with DOBV rN proliferated after restimulation with DOBV rN, but proliferated in some antigen concentrations as strongly after restimulation with a yeast-expressed negative control protein, rG2. However, it has recently been found out that there is a problem with measuring the concentration of rG2, as it is not well coloured by Bradford reagent nor by Coomassie and thereby concentration is often underjudged (Razanskiene, personal communication). Therefore, if amounts of restimulating G2 was underestimated by two fold, then some N-specific T cells were induced after DOBV rN protein immunisation. Another proof for the induction of N-specific T cells after immunisation with DOBV rN protein is the high N-specific IL-2 secretion by lymphocytes. However, further investigations studying the induction of N-specific T cells are needed, e.g. by measuring proliferation using a different protein as negative control.

4.6 Compared to DOBV rN protein HBcdDOB120 seems to need less T cell help to induce an N-specific immune response.

After the first immunisation the antibody titre in BALB/c and C57BL/6 mice immunised with HBcdDOB120 is slightly higher than in animals immunised with complete rN protein. The sligthly higher titre of animals immunised with HBcdDOB120 after the first immunisation could be due to the T cell independent B cell activation by the repetitive nature of the HBc antigen as described for nude B10.BR mice [Milich, 86a]. In another study, chimeric HBc particles have been shown to induce T cell independent insert-specific antibodies in mice [Fehr, 98]. When antigens, so called T cell independent antigens, do not require T cells for activating B cells, they are thought to induce a more rapid antibody response compared to T cell dependent antigens [DeFranco, 98].

Besides the particulate nature, LPS contamination of HBcdDOB120 [Geldmacher, 04c] is another possible cause for the more rapid antibody response induced by HBcdDOB120 compared to the antibody response induced by DOBV rN protein. LPS can also act as a T cell independent antigen and polyclonally activate B cells [reviewed by DeFranco, 98]. When immunising mice with HBcdDOB120 particles, the contaminating LPS might still be in the proximity of the particles after immunisation. The LPS could then activate DOBV N-specific B cells that would otherwise be activated much later, after N-specific T cell help became available.

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In nude mice, which lack most T cells, HBc particles are able to induce IgG antibodies, most of which were of the IgG2b subclass [Milich, 87]. Other studies showed a predominance of IgG3 in the sera of fully immunocompetent animals immunised with T cell independent antigens [Snapper, 87; Snapper, 92; Snapper, 97]. When comparing the ratio of the DOBV rN-induced IgG2b and IgG3 antibody titre to the total IgG antibody titre a slightly higher ratio in sera of animals immunised with HBcdDOB120 compared to the ratio in sera of animals immunised with DOBV rN protein were observed. In addition to the rapid antibody response after immunisation with HBcdDOB120 described above, these high IgG2b and IgG3 titres might be further evidence for the partially T cell independent nature of HBcdDOB120 particles. Additionally, the high IgG3 titre indicated that not LPS contamination is causing the T cell independent nature of the HBcdDOB120 particles as the presence of IFN-γ seems to inhibit IgG3 in LPS-activated B cells [Snapper, 87]. However, only the ratios of IgG2b, but not IgG3 to total IgG were significantely higher in HBcdDOB120 immunised BALB/c (but not C57BL/6) mice compared to animals immunised with DOBV rN, a protein that is very probably not a T cell independent B cell activator.

Additional studies are needed to tackle the question of the T cell independent nature of HBcdDOB120. Thus immunisations without adjuvants might elucidate if antibody titres in sera of HBcdDOB120 primed mice are higher than in DOBV rN protein primed mice. Furthermore immunisation of mostly T cell deficient nude mice with HBcdDOB120 particles are needed to investigate if this more rapid response is due to the T cell independent nature of the particles. Additionally, HBcdDOB120 without LPS contamination [as described in Geldmacher, 04b] and non-particulate HBcdDOB120 could be generated to see if the immune response is induced by the particulate nature of the protein or due to the T cell independent nature of the LPS contaminating the particles.

4.7 Protection against hantaviruses can be confered by N-specific T cells as well as N-specific antibodies

In 1985 it was suggested that T cell mediated immunity plays a crucial role in fighting hantavirus infection in laboratory rodents that - in contrast to the natural rodent reservoir - cannot be persistently infected [Nakamura, 85a]. The possibility to readily infect T cell deficient nude mice but only transiently infect immunocompetent BALB/c mice with HTNV [Asada, 87a] also indicated that T cells are important in protection against hantavirus infection. However, T cells are needed for an efficient B cell response. Consequently the lack of a T cell help to B cells might be in part responsible for the persisting infection of nude mice and for the failure to persistently infect immunocompetent mice with hantaviruses.

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In the same line it has been shown in adoptive transfer experiments that CD4+ and CD8+ cells are of importance in the protection against a HTNV challenge in mice [Asada, 87e]. The biggest drop in protection was observed when Lyt1+ (CD5+) cells were lysed before transferring spleen cells from HTNV immunised mice to naive mice. As CD5 is expressed on T cells and a subset of B cells this underlines again the necessity for these cell types in protection against hantavirus infection. However, the specificity the transferred cells had not been studied.

Splenocytes of bank voles immunised with rN proteins of different hantaviruses proliferated to a similar extent after restimulation with PUUV rN protein [de Carvalho Nicacio, 02]. However, N-specific stimulation indices were slightly higher after immunisation with rN protein of PUUV and Topographov (TOPV). As the rN protein of these two hantaviruses protected all 16 bank voles against PUUV infection, this suggests a role for N-specific T cells in protection [de Carvalho Nicacio, 02]. In contrast to N-specific splenocyte proliferation, N-specific antibody response was not directly associated to protection. PUUV N-specific antibody response was highest in PUUV rN protein, but second highest in the ANDV rN protein immunised group, in which only three of eight bank voles were protected against a PUUV challenge [de Carvalho Nicacio, 02]. This might suggest a stronger importance of N-specific T cells than of N-specific antibodies in protection against the virus. However, the level of N-specific lymphocyte proliferation as well as N-specific antibody titre both did not correlate significantly with protection. Additionally, splenocytes of DOBV rN protein immunised bank voles proliferated to a similar level as cells from ANDV rN protein immunised animals, even though DOBV rN protein protected twice as many animals (7 of 10) as ANDV rN protein (3 of 8) [de Carvalho Nicacio, 02]. As the titre of PUUV N-reactive antibodies was higher in ANDV rN protein immunised bank voles, antibody level could not explain this difference in protection. One explanation for the higher protection of DOBV rN protein immunised animals could be that DOBV rN protein harbours more conserved CTL epitopes that are identical in the PUUV N protein. However, as protein immunisation does normally not induce a lot of CTLs (discussed in chapter 4.5), this explanations seems unlikely.

For some enveloped viruses there is evidence that nucleocapsid specific antibodies can protect against a virus infection: p17-specific antibodies protected cells against HIV [Papsidero, 89], nucleocapsid specific antibodies against rabies virus [Lafon, 87] and against the Toscana virus, a representative of the genus Phlebovirus, family of Bunyaviridae [Cusi, 01]. For hantaviruses, it was shown in vitro (VeroE6 cells) and in vivo (suckling mice model) that protection against infection can be mediated by N-specific mAbs from infection. Yoshimatsu did not measure the exact amount of antibodies used in passive transfer in the suckling mouse model [Yoshimatsu, 93] but did show some delay in death of mice challenged with HTNV that got passively transferred N-specific serum [Yoshimatsu, 96b]. In the same line, N-specific antibodies have been demonstrated to protect adult bank voles from a hantavirus infection (Lundkvist, personal communication). But, large amounts of mabs (0.5 mg) were needed for getting 50 % of animals protected and most of the N-specific mabs did not protect even when given in such a high amount (Lundkvist personal communication). The same level of protection was caused by passive transfer of spleen cells from mice immunised with HTN rN protein, which protected 43 % of suckling mice from an HTNV infection [Yoshimatsu, 93]. Therefore, protection against a hantavirus challenge induced by immunisation with N-constructs can partly be due to N-specific antibodies.

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The mechanism by which antibodies specific for the internal N protein protect against the enveloped virus is not known. In the in vitro protection experiments mentioned above, the antibodies were scrape loaded onto the cells. Thereby, the N-specific antibodies can go into the cell and it was postulated that they inhibit uncoating or translation of the RNA [Yoshimatsu, 96a]. In vivo however, this mechanisms can not be imagined on big scale. ADCC was suggested as another potential protective mechanism for N-specific antibodies [de Carvalho Nicacio, 01i], i.e. by binding of antibodies to cell surface located N protein and thereby marking these cells for destruction by NK cells. However, in contrast to the glycoproteins [Ogino, 04], N protein could not be found on the surface of HTNV infected cells [Yoshimatsu, 93]. As Old World hantaviruses are thought to bud at the Golgi compartment (see chapter 1.1) it is unlikely that N protein is located on the surface of PUUV infected cells.

A possibly more likely explanation for the protection of N-specific antibodies against a hantavirus infection is the following. Antibodies bind to uncomplete virions, e.g. nucleocapsids, that are released by hantavirus infected cells and that these antibody/nucleocapsid complexes are more rapidly taken up by Fc receptor bearing APCs. These so called immune complexes might have been presented on MHC class I and II better than protein alone [Rafiq, 02b]. Thereby nucleocapsid would be presented more rapidly and in bigger quantities on MHC class I and II to T cells. As the incubation time from hantavirus infection to outbreak of disease is relatively long (> 10 days), the rapid Fc dependent uptake of nucleocapsid protein leads to a more rapid and more vigorous T cell response that then can protect against the virus.

Taken together, the immune response induced by HBcdDOB120 is similar to the immune response induced by DOBV rN protein as shown in this study. Thus, the immunological mechanisms induced by HBcdDOB120 and DOBV rN protein leading to protection against a hantavirus infection may be similar.The same derivatives based on PUUV rN protein induced protection against a hantavirus challenge. Immunisations with HBcdPUU120 protected 88 % (Lundkvist unpub. data) and PUUV rN protein protected 100% of bank voles agaisnt a PUUV challenge [Dargeviciute, 02]. The proteins used in this study should be tested in an DOBV challenge model. Suckling mice can be infected with DOBV [Klingström, 03]. But, the immune system of suckling mice is not fully developed.. Recently it was shown that C57BL/6 mice might be used as a DOBV challenge model [Klingström, 04], even though the infection seems to be only transient. Thus both models are not ideal as a DOBV challenge model for the evaluation of vaccine candidates

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In conclusion it is not clear what kind of immune response is needed to fight hantaviruses, but it can be deduced from the studies mentioned above and the results from this investigation, that N-specific antibodies as well as T cells can play a role in protecting against a hantavirus infection. Further investigations are needed to address the question of which arm of the immune response is most important in protection against hantaviruses.

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