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New perspectives for hepatitis B vaccines and immunization
Vaccine 27 (2009) 3271–3275
Contents lists available at ScienceDirect
Vaccine journal homepage: www.elsevier.com/locate/vaccine
New perspectives for hepatitis B vaccines and immunization Maria Rapicetta a,∗ , Emilio D’Ugo a , Claudio Argentini a , Stefania Catone a , Andrea Canitano a , Roberto Giuseppetti a , Reinhard Gluck b a b
Viral Hepatitis Unit, Department of Infectious, Parasitic and Immune-Mediated Disease, Istituto Superiore di Sanità, Rome, Italy Crucell-Berna Biotech Ltd., Rehhagstrasse 79, CH-3018 Bern, Switzerland
a r t i c l e
i n f o
Article history: Available online 5 February 2009 Keywords: HBV infection WHV Woodchuck HBV vaccine Immunotherapy
a b s t r a c t Present efforts of HBV vaccine research are aimed at defining targeted antigen compositions and adjuvancy systems for earlier and broader immune responses and optimization of immunotherapeutic approaches. We have demonstrated the applicability of the WHV/Marmota monax model for the evaluation of immunogenicity and protection of new formulations of HBV vaccines for human use. Protective activity was evaluated following the administrations of HBV CHO-PreS/S and adjuvanted S/Core vaccines. The administration of a complex constituted by HBV derived woodchuck PreS/S antibodies coupled with WHV particles was able to induce inhibition of viral replication. Future studies on treatment of HBV chronic infection should be addressed to the evaluation of therapies combined with antivirals, vaccines and immunomodulatory compounds. © 2009 Elsevier Ltd. All rights reserved.
1. Introduction Hepatitis B virus (HBV) infection is a major health problem worldwide. More than 370 million individuals are chronic carriers of the virus and need specific therapeutic interventions. Even if about 168 countries in the world have implemented National vaccination programs a lot of areas with elevated endemicity levels, particularly in developing regions, still remain to be covered. The currently applied recombinant HBV vaccines are safe and efficacious however they require a three-dose schedule administration that may influence patient compliance. Low or absence of response in older and obese individuals and in chronic disease and immunocompromised patients remain major concerns [1,2]. Furthermore the possible impact of circulation of viral variants cannot be neglected [3]. The major challenges in the research for improved HBV vaccines to be applied for prophylaxis and immunotherapy are represented by: formulations with epitope enhancement; inclusion of new adjuvants; application of cytokines and chemokines and of co-stimulatory molecules and implementation of DNA based preparations [4]. Of crucial importance is the availability of preclinical models of biological and pathogenetic significance. The woodchuck hepatitis B virus (WHV), the closest genetically related virus of HBV, and its natural host Marmota monax constitute a well-recognized
∗ Corresponding author. Tel.: +39 06 49903233; fax: +39 06 49902662. E-mail address: [email protected] (M. Rapicetta). 0264-410X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2009.01.063
animal model for pathogenetic and antiviral studies of HBV infection [5]. We have applied the WHV/woodchuck model for comparative evaluations of immunogenicity and protection levels of HBV vaccines with different antigen composition and adjuvant formulation. Immunotherapeutic approaches in natural and experimental WHV infections were also attempted. 2. Animals and methods 2.1. Woodchucks and sera The study was conducted on adult, wild-born woodchucks (M. monax) which were either WHV negative or natural chronic carriers of WHV (purchased from International Animal Exchange Inc., Ferndale, MI). They were maintained in isolation in an animal facility at the Istituto Superiore di Sanità (the Italian National Institute of Health) in Rome. The blood samples containing ethylenediaminetetraacetic acid (EDTA) at a final concentration of 10 IU/ml were centrifuged at 1500 rpm for 10 min. Plasma was recovered and stored at −80 ◦ C. 2.2. Vaccines, antivirals, immunocomplex CHO-HBsAg vaccine without adjuvants, which include both preS1 and preS2 full sequences and RC529 adjuvanted virosomal S/Core vaccine, were kindly provided by Berna Biotech of Bern, Switzerland. Lamivudine drug was provided by GlaxoWellcome Inc.
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M. Rapicetta et al. / Vaccine 27 (2009) 3271–3275
The Immunocomplex (IC) was constituted by pooled sera derived from two CHO-PreS/S vaccinated woodchucks (anti-HBs titre 2.5 mIU) mixed with WHV (107 WHV-DNA copies) and incubated 1 h at 37 ◦ C.
2.3. WHV serological assays Serum antibodies to WHV core antigen (anti-WHc), antibodies to WHV “e” antigen (anti-WHe) and WHV “e” antigen (WHeAg) were detected using electro-chemo-luminescence immuno-assays (Elecsys, Roche Molecular diagnostics). Specific cut-offs were calculated as described elsewhere [6]. Anti-HBs antibodies were detected using an immuno-assay ELISA AUSAB (Abbott Laboratories 100, Abbott Park Road, Abbott Park, Illinois).
2.4. WHV-DNA amplification, quantification and sequencing DNA was extracted from 200 l of plasma using QIAmp DNA Mini Kit (Qiagen), following the manufacturer’s instructions. For WHV-DNA detection WHV genome copies were determined using a TaqMan real-time PCR. Applied probes and primers and method were previously described [7]. The sensitivity of the realtime PCR was 50 copies per milliliter (copies/ml). Sequence analysis was carried out with ABI prism Dye terminator sequencing kit and ABI prism 7000 sequence detection system (Applied Biosystems).
3. Results 3.1. Antibody production and heterologous protection from PreS/S and S/Core HBV vaccines Fig. 1 reports the kinetics of antibody response following the administration, in 4–5 injections, of CHO derived PreS/S virosomal HBV vaccine and of RC529 adjuvanted S/Core vaccine and following the intravenous inoculation of challenge WHV (w197) in vaccinated woodchucks. From the comparison we could observe that sustained anti-HBs response (>10 mIU/ml) was present soon after the administration of the first dose for CHO-PreS/S vaccine and after 2–4 injections for S/Core vaccine. After WHV challenge, anti-HBs titres were higher in the group of PreS/S vaccinated woodchucks in comparison with S/Core vaccinated, which however maintained sustained anti-Core response. The viral and antibody markers, present in both groups, after the heterologous WHV challenge, are reported in Fig. 2. The antiHBe response was observed earlier in S/Core vaccinated animals and in one animal (w3996) preceded the viral challenge (data not shown) possibly due to the presence of small amount of truncated forms of Core antigen able to elicit anti-HBe response. The complete absence of the viral markers WHeAg and WHV-DNA (also by PCR) was observed in CHO-PreS/S vaccinated animals. A lower protection level was in general present in S/Core vaccinated woodchucks for which the transient presence of HBeAg was observed in one animal out of three. The still detected WHV-DNA was present however at titres up to 5 logs lower in respect to the control (see Fig. 3).
Fig. 1. Kinetics of antibody response in vaccinated woodchucks. Arrows indicate intramuscular vaccine administrations (respectively 50 and 20 g antigen content). Virus challenge (Ch) was performed with w197 inoculum containing 108 g eq/dose.
M. Rapicetta et al. / Vaccine 27 (2009) 3271–3275
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Fig. 2. Virus and antibody markers in vaccinated woodchucks after viral challenge.
Similar difference is also observed if we consider the WHV-DNA titres always observed following natural and experimental WHV infection [1011 log 10; personal communication]. 3.2. Kinetics of viral and antibody markers following the administration of lamivudine/PreS/S vaccine and PreS/S Ab/WHV immunocomplex (IC) in woodchucks In consideration of the capacity of inducing precocious and elevated levels of anti-HBs, the CHO-PreS/S vaccine was applied in
antiviral/vaccine therapeutic approach in woodchucks [7]. Two log decrease in titre of WHV-DNA was observed in the first 2 weeks. Soon after, the early appearance and selection of domain B FLLA motif A566T mutants was followed by the increase of viremia levels up to the initial titres (one animal follow-up is exemplified in Fig. 4). Thus, the still significant anti-HBs production was not able to interfere and to modulate the mutated, newly selected, viral population. We further performed several experiments based on woodchuck produced anti-PreS/S that were coupled with WHV to constitute
Fig. 3. Kinetics of viremia following viral challenge in S/Core vaccinated woodchucks.
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M. Rapicetta et al. / Vaccine 27 (2009) 3271–3275
Fig. 4. Lamivudine PreS/S vaccine administration in woodchuck w2988. WHV-DNA kinetics and humoral response (anti-HBs measurements). Top figure: lamivudine was orally administered dissolved in orange juice, at daily dose of 100 mg/kg body weight, for 40 weeks. The presence was controlled in woodchuck sera by HPLC. FLLA motif mutant first detection. Bottom figure: arrows indicate vaccine administrations (50 g antigen content).
an immunocomplex (IC). The construct was applied to WHV negative animals, in comparison with experimentally WHV infected control animal, and to naturally infected WHV chronic carriers woodchucks. The preliminary results were encouraging, in that we have observed 4–5 log decrease of WHV-DNA titres both in previously WHV negative (in comparison to infection control; data not shown) and in WHV chronic carrier woodchucks. Fig. 5 reports the results relative to two WHV chronic carrier animals (w3953 and w840), that showed 4–5 logs decrease in titre of HBV-DNA, WHeAg negativization and anti-WHe seroconversion. 4. Discussion and conclusions The reported experimental data are based on the application of the hepadnavirus WHV/M. monax system, for comparative evaluations of different formulations of HBV vaccines. It was demonstrated
Domain B
that the model can be applied for studies on HBV vaccines for human use. In particular, following the standardization, for challenge experiments, of a biologically and molecularly characterized viral inoculum (w197), the protective efficacy of several HBV vaccine preparations was demonstrated. In this system, in spite of being heterologous, we were able to differentiate the various protection levels elicited by vaccines with different antigen compositions. The CHO-PreS/S vaccine was shown to be able to induce, in comparison with the S/Core vaccine, more precocious and higher levels of anti-HBs that were also applied for immunocomplex/WHV formulation. The research efforts for HBV vaccines are particularly dedicated to improvement of immunogenicity to ameliorate low response conditions and to avoid multiple injections, particularly relevant for immunization campaigns in low developed countries. Furthermore, of particular importance is the implementation of
Fig. 5. Immunogenic complex (IC) administration in WHV chronically infected woodchucks. IC was intravenously administered. Serum aliquot containing 2.5 mIU anti-HBs was mixed with equal aliquots of serum containing 107 WHV-DNA/50 l incubated 1 h at 37 ◦ C before injection.
M. Rapicetta et al. / Vaccine 27 (2009) 3271–3275
vaccines for immunotherapeutic application, for which a strong immune response in vivo is necessary and the yeast derived “S” vaccines have up to now failed. Immunomodulatory options and antivirals combination therapies might be more beneficial and effective than the application of the antiviral nucleoside analogs alone, that, up to now, have resulted ineffective in eradicating the infection, due to the development of drug resistant mutants. Our lamivudine PreS/S vaccine study was limited by the very early appearance and selection of the domain B FLLA motif resistant mutant not neutralized by the antibodies produced following vaccination, thus confirming that more potent antivirals and/or multiple targeted options with possible inclusion of immune complexes should be considered. Our preliminary studies on the application of the IC showed the significant decrease of 4–5 logs in viral load (manuscript in preparation). These data are in accordance with results of pilot studies in humans, in which similar complexes were applied in HBeAg positive patients leading to the decrease of HBVDNA and HBeAg titres and to anti-HBe seroconversion [8]. They are also in agreement with the results of other antiviral drug-immunomodulators combination studies performed in woodchucks [9]. In conclusion, it was demonstrated that WHV/woodchuck model can be used for HBV vaccine studies, resulting the PreS/S-CHO vaccine the first human vaccine able to elicit non-sterilizing protection in the woodchuck model. The level of the anti-HBs production seems to have a crucial role in neutralizing and protective immunity. The WHV/M. monax system could provide a well-characterized model for preclinical evaluation of combined therapeutic approaches for prevention and treatment of HBV chronic disease.
3275
Acknowledgements This work was supported by Ministry of Health “Viral Vaccines Project” (No. 99 I) and by MIUR Project 6254 on “HBV Therapeutic Vaccination: Application of Marmota monax Model”. We also wish to acknowledge Romina Tomasetto for secretarial and editing support; the study’s veterinarian (Gianluca Panzini) and the staff of the animal housing (Franco Varano, Nicola Bellizzi and Maurizio Bonanno). References [1] Keeffe EB. Acute hepatitis A and B in patients with chronic liver disease: prevention through vaccination. Am J Med 2005;118(10):21S–7S. [2] Chow KM, Lau MC, Leung CB, Szeto CC, Li PKT. Antibody response to hepatitis B vaccine in end-stage renal disease patients. Nephron Clin Pract 2006;103:c89–93. [3] Hunt CM, McGill JM, Allen MI, Condreay LD. Clinical relevance of hepatitis B viral mutations. Hepatology 2000;31(5):1037–44. [4] Berzofsky JA, Ahlers JD, Belyakov IM. Strategies for designing and optimizing new generation vaccines. Nat Rev 2001;1:209–19. [5] Menne S, Cote PJ. The woodchuck as an animal model for pathogenesis and therapy of chronic hepatitis B virus infection. World J Gastroenterol 2007;13(1):104–24. [6] La Sorsa V, Argentini C, Bruni R, Villano U, Giuseppetti R, Rapicetta M. In vivo transmission and dynamics of deleted genomes after experimental infection of woodchuck hepatitis B virus in adult animals. Virus Genes 2002;25:147–57. [7] D’Ugo E, Kondili LA, Canitano A, Catone S, Giuseppetti R, Gallinella B, et al. Rapid emergence of a viral resistant mutant in WHV chronically infected woodchucks treated with lamivudine and a pre-S/S CHO-derived hepatitis B virus vaccine. Vaccine 2007;25:4895–902. [8] Yao X, Zheng B, Zhou J, Dao-Zhen X, Zhao K, Shu-Hui S, et al. Therapeutic effect of hepatitis B surface antigen-antibody complex is associated with cytolytic and non-cytolytic immune responses in hepatitis B patients. Vaccine 2007;25(10):1771–9. [9] Roggendorf M, Schulte I, Xu Y, Lu M. Therapeutic vaccination in chronic hepatitis B: preclinical studies in the woodchuck model. J Viral Hep 2007;14(Suppl. 1):51–7.
Contents lists available at ScienceDirect
Vaccine journal homepage: www.elsevier.com/locate/vaccine
New perspectives for hepatitis B vaccines and immunization Maria Rapicetta a,∗ , Emilio D’Ugo a , Claudio Argentini a , Stefania Catone a , Andrea Canitano a , Roberto Giuseppetti a , Reinhard Gluck b a b
Viral Hepatitis Unit, Department of Infectious, Parasitic and Immune-Mediated Disease, Istituto Superiore di Sanità, Rome, Italy Crucell-Berna Biotech Ltd., Rehhagstrasse 79, CH-3018 Bern, Switzerland
a r t i c l e
i n f o
Article history: Available online 5 February 2009 Keywords: HBV infection WHV Woodchuck HBV vaccine Immunotherapy
a b s t r a c t Present efforts of HBV vaccine research are aimed at defining targeted antigen compositions and adjuvancy systems for earlier and broader immune responses and optimization of immunotherapeutic approaches. We have demonstrated the applicability of the WHV/Marmota monax model for the evaluation of immunogenicity and protection of new formulations of HBV vaccines for human use. Protective activity was evaluated following the administrations of HBV CHO-PreS/S and adjuvanted S/Core vaccines. The administration of a complex constituted by HBV derived woodchuck PreS/S antibodies coupled with WHV particles was able to induce inhibition of viral replication. Future studies on treatment of HBV chronic infection should be addressed to the evaluation of therapies combined with antivirals, vaccines and immunomodulatory compounds. © 2009 Elsevier Ltd. All rights reserved.
1. Introduction Hepatitis B virus (HBV) infection is a major health problem worldwide. More than 370 million individuals are chronic carriers of the virus and need specific therapeutic interventions. Even if about 168 countries in the world have implemented National vaccination programs a lot of areas with elevated endemicity levels, particularly in developing regions, still remain to be covered. The currently applied recombinant HBV vaccines are safe and efficacious however they require a three-dose schedule administration that may influence patient compliance. Low or absence of response in older and obese individuals and in chronic disease and immunocompromised patients remain major concerns [1,2]. Furthermore the possible impact of circulation of viral variants cannot be neglected [3]. The major challenges in the research for improved HBV vaccines to be applied for prophylaxis and immunotherapy are represented by: formulations with epitope enhancement; inclusion of new adjuvants; application of cytokines and chemokines and of co-stimulatory molecules and implementation of DNA based preparations [4]. Of crucial importance is the availability of preclinical models of biological and pathogenetic significance. The woodchuck hepatitis B virus (WHV), the closest genetically related virus of HBV, and its natural host Marmota monax constitute a well-recognized
∗ Corresponding author. Tel.: +39 06 49903233; fax: +39 06 49902662. E-mail address: [email protected] (M. Rapicetta). 0264-410X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2009.01.063
animal model for pathogenetic and antiviral studies of HBV infection [5]. We have applied the WHV/woodchuck model for comparative evaluations of immunogenicity and protection levels of HBV vaccines with different antigen composition and adjuvant formulation. Immunotherapeutic approaches in natural and experimental WHV infections were also attempted. 2. Animals and methods 2.1. Woodchucks and sera The study was conducted on adult, wild-born woodchucks (M. monax) which were either WHV negative or natural chronic carriers of WHV (purchased from International Animal Exchange Inc., Ferndale, MI). They were maintained in isolation in an animal facility at the Istituto Superiore di Sanità (the Italian National Institute of Health) in Rome. The blood samples containing ethylenediaminetetraacetic acid (EDTA) at a final concentration of 10 IU/ml were centrifuged at 1500 rpm for 10 min. Plasma was recovered and stored at −80 ◦ C. 2.2. Vaccines, antivirals, immunocomplex CHO-HBsAg vaccine without adjuvants, which include both preS1 and preS2 full sequences and RC529 adjuvanted virosomal S/Core vaccine, were kindly provided by Berna Biotech of Bern, Switzerland. Lamivudine drug was provided by GlaxoWellcome Inc.
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M. Rapicetta et al. / Vaccine 27 (2009) 3271–3275
The Immunocomplex (IC) was constituted by pooled sera derived from two CHO-PreS/S vaccinated woodchucks (anti-HBs titre 2.5 mIU) mixed with WHV (107 WHV-DNA copies) and incubated 1 h at 37 ◦ C.
2.3. WHV serological assays Serum antibodies to WHV core antigen (anti-WHc), antibodies to WHV “e” antigen (anti-WHe) and WHV “e” antigen (WHeAg) were detected using electro-chemo-luminescence immuno-assays (Elecsys, Roche Molecular diagnostics). Specific cut-offs were calculated as described elsewhere [6]. Anti-HBs antibodies were detected using an immuno-assay ELISA AUSAB (Abbott Laboratories 100, Abbott Park Road, Abbott Park, Illinois).
2.4. WHV-DNA amplification, quantification and sequencing DNA was extracted from 200 l of plasma using QIAmp DNA Mini Kit (Qiagen), following the manufacturer’s instructions. For WHV-DNA detection WHV genome copies were determined using a TaqMan real-time PCR. Applied probes and primers and method were previously described [7]. The sensitivity of the realtime PCR was 50 copies per milliliter (copies/ml). Sequence analysis was carried out with ABI prism Dye terminator sequencing kit and ABI prism 7000 sequence detection system (Applied Biosystems).
3. Results 3.1. Antibody production and heterologous protection from PreS/S and S/Core HBV vaccines Fig. 1 reports the kinetics of antibody response following the administration, in 4–5 injections, of CHO derived PreS/S virosomal HBV vaccine and of RC529 adjuvanted S/Core vaccine and following the intravenous inoculation of challenge WHV (w197) in vaccinated woodchucks. From the comparison we could observe that sustained anti-HBs response (>10 mIU/ml) was present soon after the administration of the first dose for CHO-PreS/S vaccine and after 2–4 injections for S/Core vaccine. After WHV challenge, anti-HBs titres were higher in the group of PreS/S vaccinated woodchucks in comparison with S/Core vaccinated, which however maintained sustained anti-Core response. The viral and antibody markers, present in both groups, after the heterologous WHV challenge, are reported in Fig. 2. The antiHBe response was observed earlier in S/Core vaccinated animals and in one animal (w3996) preceded the viral challenge (data not shown) possibly due to the presence of small amount of truncated forms of Core antigen able to elicit anti-HBe response. The complete absence of the viral markers WHeAg and WHV-DNA (also by PCR) was observed in CHO-PreS/S vaccinated animals. A lower protection level was in general present in S/Core vaccinated woodchucks for which the transient presence of HBeAg was observed in one animal out of three. The still detected WHV-DNA was present however at titres up to 5 logs lower in respect to the control (see Fig. 3).
Fig. 1. Kinetics of antibody response in vaccinated woodchucks. Arrows indicate intramuscular vaccine administrations (respectively 50 and 20 g antigen content). Virus challenge (Ch) was performed with w197 inoculum containing 108 g eq/dose.
M. Rapicetta et al. / Vaccine 27 (2009) 3271–3275
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Fig. 2. Virus and antibody markers in vaccinated woodchucks after viral challenge.
Similar difference is also observed if we consider the WHV-DNA titres always observed following natural and experimental WHV infection [1011 log 10; personal communication]. 3.2. Kinetics of viral and antibody markers following the administration of lamivudine/PreS/S vaccine and PreS/S Ab/WHV immunocomplex (IC) in woodchucks In consideration of the capacity of inducing precocious and elevated levels of anti-HBs, the CHO-PreS/S vaccine was applied in
antiviral/vaccine therapeutic approach in woodchucks [7]. Two log decrease in titre of WHV-DNA was observed in the first 2 weeks. Soon after, the early appearance and selection of domain B FLLA motif A566T mutants was followed by the increase of viremia levels up to the initial titres (one animal follow-up is exemplified in Fig. 4). Thus, the still significant anti-HBs production was not able to interfere and to modulate the mutated, newly selected, viral population. We further performed several experiments based on woodchuck produced anti-PreS/S that were coupled with WHV to constitute
Fig. 3. Kinetics of viremia following viral challenge in S/Core vaccinated woodchucks.
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M. Rapicetta et al. / Vaccine 27 (2009) 3271–3275
Fig. 4. Lamivudine PreS/S vaccine administration in woodchuck w2988. WHV-DNA kinetics and humoral response (anti-HBs measurements). Top figure: lamivudine was orally administered dissolved in orange juice, at daily dose of 100 mg/kg body weight, for 40 weeks. The presence was controlled in woodchuck sera by HPLC. FLLA motif mutant first detection. Bottom figure: arrows indicate vaccine administrations (50 g antigen content).
an immunocomplex (IC). The construct was applied to WHV negative animals, in comparison with experimentally WHV infected control animal, and to naturally infected WHV chronic carriers woodchucks. The preliminary results were encouraging, in that we have observed 4–5 log decrease of WHV-DNA titres both in previously WHV negative (in comparison to infection control; data not shown) and in WHV chronic carrier woodchucks. Fig. 5 reports the results relative to two WHV chronic carrier animals (w3953 and w840), that showed 4–5 logs decrease in titre of HBV-DNA, WHeAg negativization and anti-WHe seroconversion. 4. Discussion and conclusions The reported experimental data are based on the application of the hepadnavirus WHV/M. monax system, for comparative evaluations of different formulations of HBV vaccines. It was demonstrated
Domain B
that the model can be applied for studies on HBV vaccines for human use. In particular, following the standardization, for challenge experiments, of a biologically and molecularly characterized viral inoculum (w197), the protective efficacy of several HBV vaccine preparations was demonstrated. In this system, in spite of being heterologous, we were able to differentiate the various protection levels elicited by vaccines with different antigen compositions. The CHO-PreS/S vaccine was shown to be able to induce, in comparison with the S/Core vaccine, more precocious and higher levels of anti-HBs that were also applied for immunocomplex/WHV formulation. The research efforts for HBV vaccines are particularly dedicated to improvement of immunogenicity to ameliorate low response conditions and to avoid multiple injections, particularly relevant for immunization campaigns in low developed countries. Furthermore, of particular importance is the implementation of
Fig. 5. Immunogenic complex (IC) administration in WHV chronically infected woodchucks. IC was intravenously administered. Serum aliquot containing 2.5 mIU anti-HBs was mixed with equal aliquots of serum containing 107 WHV-DNA/50 l incubated 1 h at 37 ◦ C before injection.
M. Rapicetta et al. / Vaccine 27 (2009) 3271–3275
vaccines for immunotherapeutic application, for which a strong immune response in vivo is necessary and the yeast derived “S” vaccines have up to now failed. Immunomodulatory options and antivirals combination therapies might be more beneficial and effective than the application of the antiviral nucleoside analogs alone, that, up to now, have resulted ineffective in eradicating the infection, due to the development of drug resistant mutants. Our lamivudine PreS/S vaccine study was limited by the very early appearance and selection of the domain B FLLA motif resistant mutant not neutralized by the antibodies produced following vaccination, thus confirming that more potent antivirals and/or multiple targeted options with possible inclusion of immune complexes should be considered. Our preliminary studies on the application of the IC showed the significant decrease of 4–5 logs in viral load (manuscript in preparation). These data are in accordance with results of pilot studies in humans, in which similar complexes were applied in HBeAg positive patients leading to the decrease of HBVDNA and HBeAg titres and to anti-HBe seroconversion [8]. They are also in agreement with the results of other antiviral drug-immunomodulators combination studies performed in woodchucks [9]. In conclusion, it was demonstrated that WHV/woodchuck model can be used for HBV vaccine studies, resulting the PreS/S-CHO vaccine the first human vaccine able to elicit non-sterilizing protection in the woodchuck model. The level of the anti-HBs production seems to have a crucial role in neutralizing and protective immunity. The WHV/M. monax system could provide a well-characterized model for preclinical evaluation of combined therapeutic approaches for prevention and treatment of HBV chronic disease.
3275
Acknowledgements This work was supported by Ministry of Health “Viral Vaccines Project” (No. 99 I) and by MIUR Project 6254 on “HBV Therapeutic Vaccination: Application of Marmota monax Model”. We also wish to acknowledge Romina Tomasetto for secretarial and editing support; the study’s veterinarian (Gianluca Panzini) and the staff of the animal housing (Franco Varano, Nicola Bellizzi and Maurizio Bonanno). References [1] Keeffe EB. Acute hepatitis A and B in patients with chronic liver disease: prevention through vaccination. Am J Med 2005;118(10):21S–7S. [2] Chow KM, Lau MC, Leung CB, Szeto CC, Li PKT. Antibody response to hepatitis B vaccine in end-stage renal disease patients. Nephron Clin Pract 2006;103:c89–93. [3] Hunt CM, McGill JM, Allen MI, Condreay LD. Clinical relevance of hepatitis B viral mutations. Hepatology 2000;31(5):1037–44. [4] Berzofsky JA, Ahlers JD, Belyakov IM. Strategies for designing and optimizing new generation vaccines. Nat Rev 2001;1:209–19. [5] Menne S, Cote PJ. The woodchuck as an animal model for pathogenesis and therapy of chronic hepatitis B virus infection. World J Gastroenterol 2007;13(1):104–24. [6] La Sorsa V, Argentini C, Bruni R, Villano U, Giuseppetti R, Rapicetta M. In vivo transmission and dynamics of deleted genomes after experimental infection of woodchuck hepatitis B virus in adult animals. Virus Genes 2002;25:147–57. [7] D’Ugo E, Kondili LA, Canitano A, Catone S, Giuseppetti R, Gallinella B, et al. Rapid emergence of a viral resistant mutant in WHV chronically infected woodchucks treated with lamivudine and a pre-S/S CHO-derived hepatitis B virus vaccine. Vaccine 2007;25:4895–902. [8] Yao X, Zheng B, Zhou J, Dao-Zhen X, Zhao K, Shu-Hui S, et al. Therapeutic effect of hepatitis B surface antigen-antibody complex is associated with cytolytic and non-cytolytic immune responses in hepatitis B patients. Vaccine 2007;25(10):1771–9. [9] Roggendorf M, Schulte I, Xu Y, Lu M. Therapeutic vaccination in chronic hepatitis B: preclinical studies in the woodchuck model. J Viral Hep 2007;14(Suppl. 1):51–7.