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Walter Wodal, Falko G Falkner,
Astrid Kerschbaum,
Claudia Gaiswinkler,
Richard Fritz,
Stefan Kiermayr,
Daniel Portsmouth,
Helga Savidis-Dacho,
Sogue Coulibaly,
Christina Piskernik,
Christine Hohenadl,
M Keith Howard,
Otfried Kistner,
P Noel Barrett,
Thomas R Kreil
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ABSTRACT: Influenza viruses of subtype A/H9N2 are enzootic in poultry across Asia and the Middle East and are considered to have pandemic potential. The development of new vaccine manufacturing technologies is a cornerstone of influenza pandemic preparedness.
A non-adjuvanted whole-virus H9N2 vaccine was developed using Vero cell culture manufacturing technology. The induction of hemagglutination inhibition (HI) and virus-neutralizing antibodies was assessed in CD1 mice and guinea pigs. A highly sensitive enzyme-linked lectin assay was used to investigate the induction of antibodies capable of inhibiting the enzymatic activity of the H9N2 neuraminidase. Protective efficacy against virus replication in the lung after challenge with the homologous virus was evaluated in BALB/c mice by a TCID(50) assay, and prevention of virus replication in the lung and associated pathology were evaluated by histology and immunohistochemistry. To investigate the ability of the vaccine to prevent severe disease, BALB/c mice were challenged with a highly virulent mouse-adapted H9N2 isolate which was generated by multiple lung-to-lung passage of wild-type virus.
The vaccine elicited high titers of functional H9N2-specific HA antibodies in both mice and guinea pigs, as determined by HI and virus neutralization assays. High titer H9N2-specific neuraminidase inhibiting (NAi) antibodies were also induced in both species. Vaccinated mice were protected from lung virus replication in a dose-dependent manner after challenge with the homologous H9N2 virus. Immunohistochemical analyses confirmed the lack of virus replication in the lung and an associated substantial reduction in lung pathology. Dose-dependent protection from severe weight loss was also provided after challenge with the highly virulent mouse-adapted H9N2 virus.
The induction of high titers of H9N2-specific HI, virus-neutralizing and NAi antibodies and dose-dependent protection from virus replication and severe disease in animal models suggest that the Vero cell culture-derived whole-virus vaccine will provide an effective intervention in the event of a H9N2 pandemic situation.
Vaccine 05/2012; 30(31):4625-31. · 3.77 Impact Factor
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ABSTRACT: Modified vaccinia virus Ankara (MVA) has become a promising vaccine vector due to its immunogenicity and its proven safety in humans. As a general approach for stringent and rapid selection of recombinant MVA, we assessed marker rescue of the essential viral D4R gene in an engineered deletion mutant that is fully replication defective in wild-type cells. Recombinant, replicating virus was obtained by re-introduction of the deleted viral gene as a dominant selection marker into the deletion mutant.
Virology Journal 12/2011; 8:529. · 2.34 Impact Factor
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Klaus K Orlinger,
Yvonne Hofmeister,
Richard Fritz,
Georg W Holzer, Falko G Falkner,
Bernd Unger,
Alexandra Loew-Baselli,
Eva-Maria Poellabauer,
Hartmut J Ehrlich,
P Noel Barrett,
Thomas R Kreil
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ABSTRACT: After vaccination of humans with tick-borne encephalitis virus (TBEV) vaccine, the extent of cross-neutralization between viruses of the European, Far Eastern, and Siberian subtypes of TBEV and Omsk hemorrhagic fever virus (OHFV) was analyzed. Hybrid viruses that encode the TBEV surface proteins for representative viruses within all subtypes, and OHFV, were constructed using the West Nile virus (WNV) backbone as vector. These viruses allow for unbiased head-to-head comparison in neutralization assays because they exhibit the antigenic characteristics of the TBEV strains from which the surface proteins were derived and showed equivalent biologic properties in cell culture. Human serum samples derived from a TBEV vaccine trial were analyzed and revealed comparable neutralizing antibody titers against European, Far Eastern, and Siberian subtype viruses, indicating equally potent cross-protection against these TBEV strains and a somewhat reduced but still protective neutralization capacity against more distantly related viruses, such as OHFV.
The Journal of Infectious Diseases 06/2011; 203(11):1556-64. · 6.41 Impact Factor
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Georg W Holzer,
Sogue Coulibaly,
Gerald Aichinger,
Helga Savidis-Dacho,
Josef Mayrhofer,
Susanne Brunner,
Karl Schmid,
Otfried Kistner,
John G Aaskov, Falko G Falkner,
Hartmut Ehrlich,
P Noel Barrett,
Thomas R Kreil
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ABSTRACT: Ross River Virus has caused reported outbreaks of epidemic polyarthritis, a chronic debilitating disease associated with significant long-term morbidity in Australia and the Pacific region since the 1920s. To address this public health concern, a formalin- and UV-inactivated whole virus vaccine grown in animal protein-free cell culture was developed and tested in preclinical studies to evaluate immunogenicity and efficacy in animal models. After active immunizations, the vaccine dose-dependently induced antibodies and protected adult mice from viremia and interferon α/β receptor knock-out (IFN-α/βR(-/-)) mice from death and disease. In passive transfer studies, administration of human vaccinee sera followed by RRV challenge protected adult mice from viremia and young mice from development of arthritic signs similar to human RRV-induced disease. Based on the good correlation between antibody titers in human sera and protection of animals, a correlate of protection was defined. This is of particular importance for the evaluation of the vaccine because of the comparatively low annual incidence of RRV disease, which renders a classical efficacy trial impractical. Antibody-dependent enhancement of infection, did not occur in mice even at low to undetectable concentrations of vaccine-induced antibodies. Also, RRV vaccine-induced antibodies were partially cross-protective against infection with a related alphavirus, Chikungunya virus, and did not enhance infection. Based on these findings, the inactivated RRV vaccine is expected to be efficacious and protect humans from RRV disease.
Vaccine 05/2011; 29(24):4132-41. · 3.77 Impact Factor
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Annett Hessel,
Michael Schwendinger,
Georg W Holzer,
Klaus K Orlinger,
Sogue Coulibaly,
Helga Savidis-Dacho,
Marie-Luise Zips,
Brian A Crowe,
Thomas R Kreil,
Hartmut J Ehrlich,
P Noel Barrett, Falko G Falkner
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ABSTRACT: New highly pathogenic H5N1 influenza viruses are continuing to evolve with a potential threat for an influenza pandemic. So far, the H5N1 influenza viruses have not widely circulated in humans and therefore constitute a high risk for the non immune population. The aim of this study was to evaluate the cross-protective potential of the hemagglutinins of five H5N1 strains of divergent clades using a live attenuated modified vaccinia Ankara (MVA) vector vaccine.
The replication-deficient MVA virus was used to express influenza hemagglutinin (HA) proteins. Specifically, recombinant MVA viruses expressing the HA genes of the clade 1 virus A/Vietnam/1203/2004 (VN/1203), the clade 2.1.3 virus A/Indonesia/5/2005 (IN5/05), the clade 2.2 viruses A/turkey/Turkey/1/2005 (TT01/05) and A/chicken/Egypt/3/2006 (CE/06), and the clade 2.3.4 virus A/Anhui/1/2005 (AH1/05) were constructed. These experimental live vaccines were assessed in a lethal mouse model. Mice vaccinated with the VN/1203 hemagglutinin-expressing MVA induced excellent protection against all the above mentioned clades. Also mice vaccinated with the IN5/05 HA expressing MVA induced substantial protection against homologous and heterologous AH1/05 challenge. After vaccination with the CE/06 HA expressing MVA, mice were fully protected against clade 2.2 challenge and partially protected against challenge of other clades. Mice vaccinated with AH1/05 HA expressing MVA vectors were only partially protected against homologous and heterologous challenge. The live vaccines induced substantial amounts of neutralizing antibodies, mainly directed against the homologous challenge virus, and high levels of HA-specific IFN-γ secreting CD4 and CD8 T-cells against epitopes conserved among the H5 clades and subclades.
The highest level of cross-protection was induced by the HA derived from the VN/1203 strain, suggesting that pandemic H5 vaccines utilizing MVA vector technology, should be based on the VN/1203 hemagglutinin. Furthermore, the recombinant MVA-HA-VN, as characterized in the present study, would be a promising candidate for such a vaccine.
PLoS ONE 01/2011; 6(1):e16247. · 4.09 Impact Factor
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ABSTRACT: Currently existing yellow fever (YF) vaccines are based on the live attenuated yellow fever virus 17D strain (YFV-17D). Although, a good safety profile was historically attributed to the 17D vaccine, serious adverse events have been reported, making the development of a safer, more modern vaccine desirable.
A gene encoding the precursor of the membrane and envelope (prME) protein of the YFV-17D strain was inserted into the non-replicating modified vaccinia virus Ankara and into the D4R-defective vaccinia virus. Candidate vaccines based on the recombinant vaccinia viruses were assessed for immunogenicity and protection in a mouse model and compared to the commercial YFV-17D vaccine. The recombinant live vaccines induced γ-interferon-secreting CD4- and functionally active CD8-T cells, and conferred full protection against lethal challenge already after a single low immunization dose of 10(5) TCID(50). Surprisingly, pre-existing immunity against wild-type vaccinia virus did not negatively influence protection. Unlike the classical 17D vaccine, the vaccinia virus-based vaccines did not cause mortality following intracerebral administration in mice, demonstrating better safety profiles.
The non-replicating recombinant YF candidate live vaccines induced a broad immune response after single dose administration, were effective even in the presence of a pre-existing immunity against vaccinia virus and demonstrated an excellent safety profile in mice.
PLoS ONE 01/2011; 6(9):e24505. · 4.09 Impact Factor
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ABSTRACT: A cDNA comprising the complete genome of West Nile Virus (WNV) was generated by chemical synthesis using published sequence data, independent of any preformed viral components. The synthetic WNV, produced by transfection of in vitro transcribed RNA into cell culture, exhibited undistinguishable biological properties compared to the corresponding animal-derived wild-type virus. No differences were found concerning viral growth in mammalian and insect cell lines and concerning expression of viral proteins in cells. There were also no significant differences in virulence in mice following intranasal challenge. After immunizations of mice with experimental vaccines derived from the synthetic and wild-type viruses, protection from lethal challenge was achieved with similar amounts of antigen. Both vaccine preparations also induced comparable levels of neutralizing antibodies in mice. In addition, the synthetic approach turned out to be very accurate, since the rescued WNV genome contained no undesired mutations. Thus, the first flavivirus based on chemical gene synthesis was indistinguishable from the parent virus. This demonstrates that virus isolates from animal sources are dispensable to derive seed viruses for vaccine production or research.
Vaccine 03/2010; 28(19):3318-24. · 3.77 Impact Factor
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Otfried Kistner,
Brian A Crowe,
Walter Wodal,
Astrid Kerschbaum,
Helga Savidis-Dacho,
Nicolas Sabarth, Falko G Falkner,
Ines Mayerhofer,
Wolfgang Mundt,
Manfred Reiter,
Leopold Grillberger,
Christa Tauer,
Michael Graninger,
Alois Sachslehner,
Michael Schwendinger,
Peter Brühl,
Thomas R Kreil,
Hartmut J Ehrlich,
P Noel Barrett
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ABSTRACT: The recent emergence and rapid spread of a novel swine-derived H1N1 influenza virus has resulted in the first influenza pandemic of this century. Monovalent vaccines have undergone preclinical and clinical development prior to initiation of mass immunization campaigns. We have carried out a series of immunogenicity and protection studies following active immunization of mice, which indicate that a whole virus, nonadjuvanted vaccine is immunogenic at low doses and protects against live virus challenge. The immunogenicity in this model was comparable to that of a whole virus H5N1 vaccine, which had previously been demonstrated to induce high levels of seroprotection in clinical studies. The efficacy of the H1N1 pandemic vaccine in protecting against live virus challenge was also seen to be equivalent to that of the H5N1 vaccine. The protective efficacy of the H1N1 vaccine was also confirmed using a severe combined immunodeficient (SCID) mouse model. It was demonstrated that mouse and guinea pig immune sera elicited following active H1N1 vaccination resulted in 100% protection of SCID mice following passive transfer of immune sera and lethal challenge. The immune responses to a whole virus pandemic H1N1 and a split seasonal H1N1 vaccine were also compared in this study. It was demonstrated that the whole virus vaccine induced a balanced Th-1 and Th-2 response in mice, whereas the split vaccine induced mainly a Th-2 response and only minimal levels of Th-1 responses. These data supported the initiation of clinical studies with the same low doses of whole virus vaccine that had previously been demonstrated to be immunogenic in clinical studies with a whole virus H5N1 vaccine.
PLoS ONE 01/2010; 5(2):e9349. · 4.09 Impact Factor
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Annett Hessel,
Michael Schwendinger,
Daniela Fritz,
Sogue Coulibaly,
Georg W Holzer,
Nicolas Sabarth,
Otfried Kistner,
Walter Wodal,
Astrid Kerschbaum,
Helga Savidis-Dacho,
Brian A Crowe,
Thomas R Kreil,
P Noel Barrett, Falko G Falkner
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ABSTRACT: The development of novel influenza vaccines inducing a broad immune response is an important objective. The aim of this study was to evaluate live vaccines which induce both strong humoral and cell-mediated immune responses against the novel human pandemic H1N1 influenza virus, and to show protection in a lethal animal challenge model.
For this purpose, the hemagglutinin (HA) and neuraminidase (NA) genes of the influenza A/California/07/2009 (H1N1) strain (CA/07) were inserted into the replication-deficient modified vaccinia Ankara (MVA) virus--a safe poxviral live vector--resulting in MVA-H1-Ca and MVA-N1-Ca vectors. These live vaccines, together with an inactivated whole virus vaccine, were assessed in a lung infection model using immune competent Balb/c mice, and in a lethal challenge model using severe combined immunodeficient (SCID) mice after passive serum transfer from immunized mice. Balb/c mice vaccinated with the MVA-H1-Ca virus or the inactivated vaccine were fully protected from lung infection after challenge with the influenza H1N1 wild-type strain, while the neuraminidase virus MVA-N1-Ca induced only partial protection. The live vaccines were already protective after a single dose and induced substantial amounts of neutralizing antibodies and of interferon-gamma-secreting (IFN-gamma) CD4- and CD8 T-cells in lungs and spleens. In the lungs, a rapid increase of HA-specific CD4- and CD8 T cells was observed in vaccinated mice shortly after challenge with influenza swine flu virus, which probably contributes to the strong inhibition of pulmonary viral replication observed. In addition, passive transfer of antisera raised in MVA-H1-Ca vaccinated immune-competent mice protected SCID mice from lethal challenge with the CA/07 wild-type virus.
The non-replicating MVA-based H1N1 live vaccines induce a broad protective immune response and are promising vaccine candidates for pandemic influenza.
PLoS ONE 01/2010; 5(8):e12217. · 4.09 Impact Factor
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Josef Mayrhofer,
Sogue Coulibaly,
Annett Hessel,
Georg W Holzer,
Michael Schwendinger,
Peter Brühl,
Marijan Gerencer,
Brian A Crowe,
Shen Shuo,
Wanjing Hong,
Yee Joo Tan,
Barbara Dietrich,
Nicolas Sabarth,
Helga Savidis-Dacho,
Otfried Kistner,
P Noel Barrett, Falko G Falkner
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ABSTRACT: The timely development of safe and effective vaccines against avian influenza virus of the H5N1 subtype will be of the utmost importance in the event of a pandemic. Our aim was first to develop a safe live vaccine which induces both humoral and cell-mediated immune responses against human H5N1 influenza viruses and second, since the supply of embryonated eggs for traditional influenza vaccine production may be endangered in a pandemic, an egg-independent production procedure based on a permanent cell line. In the present article, the generation of a complementing Vero cell line suitable for the production of safe poxviral vaccines is described. This cell line was used to produce a replication-deficient vaccinia virus vector H5N1 live vaccine, dVV-HA5, expressing the hemagglutinin of a virulent clade 1 H5N1 strain. This experimental vaccine was compared with a formalin-inactivated whole-virus vaccine based on the same clade and with different replicating poxvirus-vectored vaccines. Mice were immunized to assess protective immunity after high-dose challenge with the highly virulent A/Vietnam/1203/2004(H5N1) strain. A single dose of the defective live vaccine induced complete protection from lethal homologous virus challenge and also full cross-protection against clade 0 and 2 challenge viruses. Neutralizing antibody levels were comparable to those induced by the inactivated vaccine. Unlike the whole-virus vaccine, the dVV-HA5 vaccine induced substantial amounts of gamma interferon-secreting CD8 T cells. Thus, the nonreplicating recombinant vaccinia virus vectors are promising vaccine candidates that induce a broad immune response and can be produced in an egg-independent and adjuvant-independent manner in a proven vector system.
Journal of Virology 04/2009; 83(10):5192-203. · 5.40 Impact Factor
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Otfried Kistner,
M Keith Howard,
Martin Spruth,
Walter Wodal,
Peter Brühl,
Marijan Gerencer,
Brian A Crowe,
Helga Savidis-Dacho,
Ian Livey,
Manfred Reiter,
Ines Mayerhofer,
Christa Tauer,
Leopold Grillberger,
Wolfgang Mundt, Falko G Falkner,
P Noel Barrett
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ABSTRACT: The rapid spread and the transmission to humans of avian influenza virus (H5N1) have induced world-wide fears of a new pandemic and raised concerns over the ability of standard influenza vaccine production methods to rapidly supply sufficient amounts of an effective vaccine. We report here on a robust and flexible strategy which uses wild-type virus grown in a continuous cell culture (Vero) system to produce an inactivated whole virus vaccine. Candidate vaccines based on clade 1 and clade 2 influenza H5N1 strains were developed and demonstrated to be highly immunogenic in animal models. The vaccines induce cross-neutralising antibodies, highly cross-reactive T-cell responses and are protective in a mouse challenge model not only against the homologous virus but also against other H5N1 strains, including those from another clade. These data indicate that cell culture-grown whole virus vaccines, based on the wild-type virus, allow the rapid high yield production of a candidate pandemic vaccine.
Vaccine 09/2007; 25(32):6028-36. · 3.77 Impact Factor
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Kuo-Ming Lip,
Shuo Shen,
Xiaoming Yang,
Choong-Tat Keng,
Aihua Zhang,
Hsueh-Ling Janice Oh,
Zhi-Hong Li,
Le-Ann Hwang,
Chih-Fong Chou,
Burtram C Fielding,
Timothy H P Tan,
Josef Mayrhofer, Falko G Falkner,
Jianlin Fu,
Seng Gee Lim,
Wanjin Hong,
Yee-Joo Tan
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ABSTRACT: We have previously shown that an Escherichia coli-expressed, denatured spike (S) protein fragment of the severe acute respiratory coronavirus, containing residues 1029 to 1192 which include the heptad repeat 2 (HR2) domain, was able to induce neutralizing polyclonal antibodies (C. T. Keng, A. Zhang, S. Shen, K. M. Lip, B. C. Fielding, T. H. Tan, C. F. Chou, C. B. Loh, S. Wang, J. Fu, X. Yang, S. G. Lim, W. Hong, and Y. J. Tan, J. Virol. 79:3289-3296, 2005). In this study, monoclonal antibodies (MAbs) were raised against this fragment to identify the linear neutralizing epitopes in the functional domain and to investigate the mechanisms involved in neutralization. Eighteen hybridomas secreting the S protein-specific MAbs were obtained. Binding sites of these MAbs were mapped to four linear epitopes. Two of them were located within the HR2 region and two immediately upstream of the HR2 domain. MAbs targeting these epitopes showed in vitro neutralizing activities and were able to inhibit cell-cell membrane fusion. These results provide evidence of novel neutralizing epitopes that are located in the HR2 domain and the spacer region immediately upstream of the HR2 of the S protein.
Journal of Virology 02/2006; 80(2):941-50. · 5.40 Impact Factor
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ABSTRACT: The Escherichia coli thymidine kinase/thymidylate kinase (tk/tmk) fusion gene encodes an enzyme that efficiently converts the prodrug 3'-azido-2',3'-dideoxythymidine (AZT) into its toxic triphosphate derivative, a substance which stops DNA chain elongation. Integration of this marker gene into vaccinia virus that normally is not inhibited by AZT allowed the establishment of a powerful selection procedure for recombinant viruses. In contrast to the conventional vaccinia thymidine kinase (tk) selection that is performed in tk-negative cell lines, AZT selection can be performed in normal (tk-positive) cell lines. The technique is especially useful for the generation of replication-deficient vaccinia viruses and may also be used for gene knock-out studies of essential vaccinia genes.
Virology 08/2005; 337(2):235-41. · 3.35 Impact Factor
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ABSTRACT: Recombinant vaccinia viruses that express defective retroviral vectors upon a single infection event in normal host cells were constructed. The gag-pol and envelope genes and a retroviral vector unit were inserted as vaccinia virus promoter-controlled transcription units at three separate loci. The triple recombinant virus was used to infect such diverse cell types as monkey and rabbit kidney, human lung, and primary chicken cells, resulting in the production of transduction-competent defective retroviral vectors. Infection of Chinese hamster ovary cells, which are nonpermissive for vaccinia virus replication, also resulted in production of retroviral vectors and secondary permanent transduction of the host cells. Since vaccinia virus supports the expression of cytotoxic proteins, the vesicular stomatitis virus G glycoprotein could be chosen as the envelope allowing a broad host range of transduction. Functionality of particles was monitored by expression of the green fluorescent protein in transduced 3T3 cell clones. This is the first description of a single chimeric virus encoding and releasing functional retroviral vectors, providing proof of principle of the new concept. No replication-competent retrovirus was detectable by sensitive reverse transcriptase assays. Since vaccinia virus has a broad host range, is extremely robust, and can be obtained at high titers and safe nonreplicating vaccinia virus strains are available, the hybrid system may open new perspectives for gene delivery.
Journal of Virology 07/2003; 77(12):7017-25. · 5.40 Impact Factor
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ABSTRACT: Current hepatitis B virus (HBV) vaccines consist of preparations of recombinant HBV major surface antigen (sAg) and are protective in about 90-95% of vaccinated subjects. In improved vaccines, the frequency of nonresponders to the classical vaccine could be reduced by including additional epitopes from the preS-domains of the middle and large surface antigens. In this report, the development and characterization of a CHO cell line for HBsAg, expressing major, middle, and large antigens are described. Despite the previously reported retention of secreted proteins by the preS1 domain, cell lines could be amplified that secreted large amounts of the complete set of antigens. A producer line was established that expressed 1mg HBsAg per 100ml suspension culture per week during exponential growth. The productivity per cell increased further by at least threefold when the culture reached the stationary phase at high cell densities. In the production cell line, several hundred copies of the HBV vector were integrated at two adjacent sites into chromosome 2. The cell line was adapted to growth in a defined protein-free medium minimizing the risk of adventitious agents introduced by animal derived supplements. The cell line stably produced antigen over several months. In the candidate vaccine, both preS2 and preS1 domains were present at ratios similar to HBsAg from human sera. In summary, a production cell line for an improved HBV vaccine is presented with properties such as high productivity, long term stability of expression, and growth in protein-free media.
Protein Expression and Purification 06/2003; 29(1):58-69. · 1.59 Impact Factor
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Methods in molecular medicine 02/2003; 76:565-78.
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ABSTRACT: We report the rescue of a defective vaccinia virus, forming the basis for a stringent selection protocol to generate replicating recombinant virus without the need for marker cassettes and selection agents. Plaques of recombinant virus could be isolated solely by their ability to grow in wild-type cells normally supporting the growth of vaccinia virus. All growth-competent clones analyzed contained the gene of interest in the intended genomic locus and displayed foreign gene expression to the same levels as was seen with classical recombinants obtained by insertion into the vaccinia virus thymidine kinase locus. The system is based on a defective vaccinia virus, expressing exclusively early genes, termed eVAC-1, and an insertion plasmid vector providing the essential function, the uracil DNA glycosylase gene. In addition, the defective virus is free of selection and color marker genes, thus also representing a basic vector for the generation of defective recombinants.
Virology 10/1998; · 3.35 Impact Factor
