"Immunogenicity of hantavirus Dobrava
nucleocapsid protein derivatives in mice"

Dissertation

zur Erlangung des akademischen Grades

doctor rerum naturalium(Dr. rer. nat.)


im Fach Biologie

eingereicht an der

Mathematisch-Naturwissenschaftlichen Fakultät I

der Humboldt-Universität zu Berlin

von

Diplombiologin Astrid Geldmacher
geboren am 12. Mai 1971 in Erlangen

Präsident der Humboldt-Universität zu Berlin

Prof. Dr. Jürgen Mlynek

Dean: Dekan der Mathematisch-Naturwissenschaftliche Fakultät I
Prof. Thomas Buckhout, PhD

Approvals:
1. Prof. Dr. Richard Lucius
2. Prof. Dr. Detlev H. Krüger
3. Prof. Dr. Paul Pumpens

Datum der Promotion: 02. Mai 2005

Zusammenfassung

Das in Europa vorkommende Dobravavirus (DOBV) wird durch zwei unterschiedliche Nagetierwirte, die Gelbhalsmaus Apodemus flavicollis und die Brandmaus A. agrarius, übertragen. DOBV kann bei humanen Infektionen zum Auslösen eines "Hämorrhagischen Fiebers mit renalem Syndrom" (HFRS) unterschiedlicher Schweregrade führen. Wie alle Hantaviren ist das DOBV ein umhülltes Virus, das in seiner Hülle die Glykoproteine G1 und G2 trägt. Im Inneren der Viruspartikel befinden sich die drei mit Nukleokapsid (N) Protein assoziierten Negativstrang-RNA Genomsegmente, sowie die RNA-abängige RNA-Polymerase.

Das N Protein von Hantaviren ist stark immunogen, sowohl in natürlich vorkommenden Infektionen von Menschen als auch in natürlichen und experimentellen Infektionen von Nagetieren. Des weiteren rufen Impfungen von Nagetieren mit N Protein eine starke N-spezifische Immunantwort hervor. Eine Impfung mit rekombinanten N Protein Derivativen schützt in Nagetiermodellen vor einer Hantavirusinfektion. Dies konnte unter anderem für chimaere Hepatitis B Virus (HBV) Corepartikel und das komplette rekombinante N (rN) Protein gezeigt werden.

In der vorliegenden Arbeit wurde die Immunogenität von zwei auf dem DOBV N Protein basierende Protein Derivativen in Mäusen getestet. Zum einen wurden in E. coli exprimierte chimaere HBV Corepartikel verwendet, die von verkürztem Core-(HBcd)-Protein gebildet wurden, das die 120 amino-terminalen Aminosäuren (AS) des DOBV N Proteins trugen (HBcdDOB120). Das zweite Protein, komplettes DOBV rN Protein (429 AS), wurde in Hefen exprimiert. Anschließend wurden BALB/c (H2-d) und C57BL/6 (H2-b) Mäuse dreimal subkutan mit 50 μg HBcdDOB120 oder DOBV rN Protein in komplettem Freund's, inkomplettem Freund's und anschliessend ohne Adjuvants immunisiert. Für die Immunisierungen wurde ein Schema verwendet, mit dem bereits das Potential verschiedener Hantavirus Impfstoffkandidaten im Nagetiermodell getestet wurde. Vor jeder Impfung, sowie zwei Wochen und 29 Wochen nach der dritten Impfung wurde der N-spezifische Antikörpertiter im Serum bestimmt.

Sowohl BALB/c, als auch C57BL/6 Mäuse entwickelten eine starke N-spezifische Antikörperantwort nach Impfung mit sowohl HBcdDOB120, als auch nach Impfung mit DOBV rN-Protein, mit maximalen Titern von über 1:1.000.000. Die Antikörperantwort war langanhaltend und N-spezifische Titer waren 29 nach der dritten Impfung mit HBcdDOB120 und DOBV rN Protein immer noch höher als 1:35.000 in allen Mäusen. Beide Proteine induzierten Antikörper, die eine starke Kreuzreaktivität gegenüber den rN Proteinen der Hantaviren Puumala, Hantaan, Andes und Sin Nombre aufwiesen.

HBcdDOB120 und DOBV rN-Protein induzierten in BALB/c und C57BL/6 Mäusen N-spezifische Antikörper aller Subklassen (IgG1, IgG2a, IgG2b und IgG3), was auf eine gemischte Th1/Th2 Antwort schließen lies. Ebenfalls auf eine gemischte Th1/Th2 Immunantwort deuteten die N-spezifischen IFN-γ und IL-4 sekretierenden Lymphozyten von HBcdDOB120 oder DOBV rN Protein immunisierten Tieren nach in vivo Restimulierung. Die Frequenz der durch die Immunisierungen induzierte N-spezifischen Lymphozyten war allerdings gering.

Auch in Mäusen, die hohe HBc-spezifische Antikörpertiter aufwiesen konnte eine starke N-spezifischen Immunantwort mittels Impfung mit HBcdDOB120 induziert werden. Das heisst, auf chimären Core Partikel basierende Impfstoffe sollten selbst in anti-HBc-positiven Individuen nach einer HBV Infektion wirksam sein.

HBcdDOB120 und Hefe-exprimiertes DOBV rN Protein stellen vielversprechende Vakzinekandidaten dar, die auf ihre Protektivität hin getestet werden sollten, sobald ein DOBV Infektionsmodell verfügbar ist. Da HBcdDOB120 sowie DOBV rN Protein eine starke Antikörperantwort und nur eine schwache T-Zellantwort induzieren sollte zusätzlich die Rolle von N-spezifischen Antikörpern im Schutz gegen die Virusinfektion weiter charakterisiert werden.

Eigene Schlagworte: Hantaviren, Virus-ähnliche Partikel, Dobrava Virus, rekombinantes Nukleokapsidprotein, Antikörperantwort, Präexistierende Immunantwort, Impfstoff

Summary

In Europe, the human pathogenic Dobrava virus (DOBV) is carried by the yellow-necked mouse Apodemus flavicollis and the stiped field mouse A. agrarius and causes "haemorrhagic fever with renal syndrome" of different severity in humans. Like other hantaviruses, DOBV is an enveloped virus with the glycoproteins G1 and G2 embedded in the envelope. Inside the virions are the RNA-dependent RNA-polymerase and the three negative-strand RNA segments which are associated with the nucleocapsid (N) protein.

The N protein is very immunogenic in natural infections of humans and in natural as well as experimental infections of rodents. Even immunisations of rodents with N protein induces a strong N-secific immune response. Moreover, immunisation with N protein derivatives could protect rodents from a hantavirus infection. This was shown for several derivatives, including chimeric hepatitis B virus core (HBc) particles and entire recombinant N (rN) protein.

In this study, the immunogenicity of the two following derivatives based on the DOBV N protein was tested in mice. Chimeric HBV core particles, consisting of truncated HBc (HBcd) particles carrying the amino-terminal 120 amino acids (aa) of the DOBV N protein (HBcdDOB120) were expressed in E. coli. The second derivative, the entire DOBV rN protein (429 aa) was expressed in the yeastSaccharomyces cerevisiae. Hence BALB/c (H2-d) and C57BL/6 (H2-b) mice were immunised subcoutanously three times with 50 μg HBcdDOB120 or DOBV rN protein in complete Freund's, incomplete Freund's and without adjuvant, respectively. The immunisations were thereby identical to the immunisation sheme used previously in a hantavirus challenge model. Before each immunisation as well as two and 29 weeks after the last immunisation N-specific antibody titers in the serum were determined.

Mice of both strains elicited strong N-specific antibody responses after HBcdDOB120 as well as after DOBV rN protein immunisation, with endpoint titers as high as 1:1,000,000. The antibody response was long-lived and N-specific titers were above 1:35,000 in all mice 29 weeks after the third immunisation with either derivative. Both derivatives induced antibodies that were highly cross-reactiveto the rN proteins of the hantaviruses Puumala, Hantaan, Andes and Sin Nombre.

HBcdDOB120 and DOBV rN protein induced in BALB/c and C57BL/6 mice N-specific antibodies of all IgG subclasses (IgG1, IgG2a, IgG2b and IgG3) suggesting a mixed Th1/Th2 immune response. In the same line, IFN-γ and IL-4 was secreted by N-specific lymphocytes from mice immunised with HBcdDOB120 or DOBV rN protein after in vitro restimulation which also indicated a mixed Th1/Th2 response. However, the frequency of N-specific lymphocytes that were induced by HBcdDOB120 and DOBV rN protein seemed to be low.

In mice that exhibited a high HBc-specific antibody titer HBcdDOB120 induced a strong N-specific immune response. Therefore, vaccines based on chimeric HBcd particles will probably be effective even in anti-HBc positive individuals after HBV infection.

HBcdDOB120 and yeast-expressed DOBV rN protein represent a promising vaccine candidate that should be tested for their protective potential in an DOBV challenge model as soon as one gets available. Additionally, as protection might be partially based on N-specific antibodies, their role in protecting against a hantavirus infection should be characterised further.

Keywords: Hantavirus, virus-like particle, Dobrava virus, recombinant nucleocapsid protein, antibodies, preexisting immunity, vaccine

Table of contents

• 1 Introduction
  • 1.1  Structure of hantaviruses
  • 1.2 Geographic distribution and natural hosts of hantaviruses
  • 1.3 Diseases caused by hantaviruses
  • 1.4 Treatment of hantavirus infections
  • 1.5 Vaccine development
    • 1.5.1  Whole virus vaccines
    • 1.5.2 Recombinant proteins as potential hantavirus vaccines
    • 1.5.3 Recombinant virus-like particles
    • 1.5.4 The need of adjuvants in subunit vaccines
    • 1.5.5 Hantavirus proteins suitable as a subunit vaccine
  • 1.6 Animal models for hantavirus research
  • 1.7 Nucleocapsid protein specific immune response
    • 1.7.1  Antibody response
    • 1.7.2 Cellular immune response
  • 1.8 Objectives of the study
• 2 Material and methods
  • 2.1  Protein derivatives for immunisation
    • 2.1.1  Expression and purification of VLPs
    • 2.1.2 Expression and purification of full-length rN protein
  • 2.2 Characterisation of the recombinant protein derivatives
    • 2.2.1  SDS-PAGE and Western Blot
    • 2.2.2 Determination of protein concentration
  • 2.3 Immunisation of mice
    • 2.3.1  Mice strains
    • 2.3.2 Immunisation of mice to investigate the influence of HBc-specific preexisting immunity
    • 2.3.3 Immunisation of mice with HBcdDOB120 and DOBV rN protein
    • 2.3.4 Bleeding and storage of blood
  • 2.4 Characterisation of the immune response of mice
    • 2.4.1  ELISA
    • 2.4.2 Immunofluorecence assay (IFA)
    • 2.4.3 Preparation of single cell suspensions from lymph nodes
    • 2.4.4 Proliferation and cytokine assays for the determination of N-specific lymphocytes
  • 2.5 Data analysis
• 3 Results
  • 3.1  HBcdDOB120 and DOBV rN were expressed in E. coli and S. cerevisiae, respectively
  • 3.2 Preexisiting antibodies to HBc did not abrogate the antibody response to DOBV rN protein after immunisation with HBcdDOB120
  • 3.3 HBcdDOB120 and DOBV rN induced antibodies that reacted to virus infected cells
  • 3.4 HBcdDOB120 and DOBV rN induced a strong and long lasting antibody response
  • 3.5 HBcdDOB120 and DOBV rN protein induced antibodies are highly cross-reactive to the rN proteins of other hantaviruses
  • 3.6 HBcdDOB120 and DOBV rN induced N-specific antibodies of all IgG subclasses
  • 3.7 Proliferation of N-specific lymphocytes was low after immunisation with HBcdDOB120 or DOBV rN protein
  • 3.8 Higher cytokine levels were secreted after immunisation with DOBV rN protein than after immunisation with HBcdDOB120
• 4 Discussion
  • 4.1  A preexisiting immunity to the carrier protein rather boosts the immunity to the antigenic insert
  • 4.2 DOBV N proteins induce a similar immune response as other hantavirus N proteins
  • 4.3 Freund's adjuvants enhances the immune response, but does not seem to modify the N-specific Th1/Th2 cell ratio
  • 4.4 The antibody response induced by chimeric HBcd protein resembles the one induced by entire rN protein
  • 4.5 Chimeric HBc particles as well as entire rN protein induce some N-specific lymphocytes
  • 4.6 Compared to DOBV rN protein HBcdDOB120 seems to need less T cell help to induce an N-specific immune response.
  • 4.7 Protection against hantaviruses can be confered by N-specific T cells as well as N-specific antibodies
• Literature
• Abbreviations
• Acknowledgment
• Publications
• Eidesstattliche Erklärung

Tables

• TABLE 1: Natural reservoir and geographical distribution of selected hantaviruses and their associated diseases. [Krüger, 01; For a more complete summary of hantaviruses see Hooper, 01c; Khaiboullina, 02]
• TABLE 2: Amino-acid identities (in %) in the glycoprotein precursor protein and the RNA-dependent RNA polymerase of different hantaviruses after alignment by Clustal Method.
• TABLE 3: Amino acid (aa) identities (in %) of the entire nucleocapsid proteins and their amino terminal 120 aa of different hantavirus strains used in this study. The aa identities were determined by alignment using the Clustal method.
• 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.

Images

• FIGURE 1: Schematic drawing of a hantavirus particle. Hantaviruses are enveloped negative-strand RNA viruses. The virus particle consists of an RNA-dependent RNA-polymerase (RdRp), two glycoproteins (G1 and G2) and the nucleocapsid (N) protein encoded by the three RNA segment, the large (L), the medium (M) and the small (S) segment, respectively. The RNA segments are associated with the N protein.
• 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) .
• 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.
• 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.
• 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).
• 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.
• 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).
• 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).
• 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).
• 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.
• 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).
• 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).
• 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).