Katrin Giesow

Glycoprotein M of bovine herpesvirus 1 (BHV-1) is nonessential for replication in cell culture and is involved in inhibition of bovine respiratory syncytial virus F protein induced syncytium formation in recombinant BHV-1 infected cells

Cell cultures infected with BHV-1/F(syn), a recombinant bovine herpesvirus 1 (BHV-1) which expresses a synthetic open reading frame encoding the fusion (F) protein of the bovine respiratory syncytial virus (BRSV), showed a cytopathic effect (CPE) indistinguishable from that induced by wildtype BHV-1 although transient transfection experiments demonstrated that expression of the F protein leads to formation of large syncytia. Since it has been shown that glycoprotein M (gM) of pseudorabies virus inhibits BRSV F-induced syncytium formation in transient plasmid transfection experiments [Pseudorbies virus glycoprotein M inhibits membrane fusion. J. Virol. 74 (2000) 6760], the gM ORF of wtBHV-1 and BHV-1/F(syn) was interrupted. Infection of cell cultures with the resulting gM(-) mutant of BHV-1/F(syn) led to formation of syncytia, whereas the CPE in gM(-)BHV-1 infected cells was comparable to the CPE in wtBHV-1 infected cultures. Our results demonstrate that gM is not essential for BHV-1 replication in cell culture and that gM is involved in inhibition of the cell fusion activity of the BHV-1 expressed BRSV F protein.

BacMam immunization partially protects pigs against sublethal challenge with African swine fever virus

Lack of vaccines and efficient control measures complicate the control and eradication of African swine fever (ASF). Limitations of conventional inactivated and attenuated virus-based vaccines against African swine fever virus (ASFV) highlight the need to use new technologies to develop efficient and safe vaccines against this virus. With this aim in mind, in this study we have constructed BacMam-sHAPQ, a baculovirus based vector for gene transfer into mammalian cells, expressing a fusion protein comprising three in tandem ASFV antigens: p54, p30 and the extracellular domain of the viral hemagglutinin (secretory hemagglutinin, sHA), under the control of the human cytomegalovirus immediate early promoter (CMVie). Confirming its correct in vitro expression, BacMam-sHAPQ induced specific T-cell responses directly after in vivo immunization. Conversely, no specific antibody responses were detectable prior to ASFV challenge. The protective potential of this recombinant vaccine candidate was tested by a homologous sublethal challenge with ASFV following immunization. Four out of six immunized pigs remained viremia-free after ASFV infection, while the other two pigs showed similar viremic titres to control animals. The protection afforded correlated with the presence of a large number of virus-specific IFNγ-secreting T-cells in blood at 17 days post-infection. In contrast, the specific antibody levels observed after ASFV challenge in sera from BacMam-sHAPQ immunized pigs were indistinguishable from those found in control pigs. These results highlight the importance of the cellular responses in protection against ASFV and point towards BacMam vectors as potential tools for future vaccine development.

Novel vectors for simultaneous high-level dual protein expression in vertebrate and insect cells by recombinant baculoviruses.

Gene transfer into cells of mammalian, avian or piscine origin by baculoviruses carrying expression cassettes active in vertebrate cells (BacMam method) is an attractive alternative to chemical or physical transfection methods or to the use of vectors originating from viruses of vertebrates. For simultaneous high-level expression of two proteins from recombinant baculoviruses we constructed novel dual expression vectors containing human and murine cytomegalovirus immediate-early enhancer/promoter elements in combination with the baculoviral polyhedrin and p10 promoters for simultaneous expression in vertebrate and insect cells. Transduction of ruminant cells with BacMam viruses containing the green fluorescent protein open reading frame downstream from the respective enhancer/promoter elements revealed that a dual expression cassette combining the murine cytomegalovirus immediate-early 1 sequence with the immediate early enhancer/promoter of human cytomegalovirus yields high levels of protein from both transcription units. Protein expression directed by several cytomegalovirus/baculovirus hybrid promoters proceeded efficiently in insect cells infected with the respective recombinants. However, for expression in vertebrate cells the murine ie1 enhancer/promoter upstream the baculoviral p10 promoter was most efficient.

Recombinant virus-expressed bovine cytokines do not improve efficacy of a bovine herpesvirus 1 marker vaccine strain

Cytokines play a key role as regulators of the immune response. To elucidate whether the efficacy of a live virus vaccine can be improved by co-expression of cytokines, expression cassettes for bovine interleukins (boIL)-2, -4, -6, and -12 and bovine interferon-gamma (boIFN-gamma) were integrated into the glycoprotein E (gE)-locus of the bovine herpesvirus 1 (BHV-1) vaccine virus strain GK/D. Cell culture analyses demonstrated that expression of the cytokines did not impair the replication of the recombinant viruses. To test safety and efficacy, groups of 4-6 months old BHV-1 seronegative calves were vaccinated intranasally with the parental virus strain GK/D or the recombinants, and challenged intranasally 3 weeks later with virulent BHV-1. The animals were monitored for clinical signs, virus excretion and antibody status after vaccination and challenge. All vaccines were well tolerated and protected the immunised calves from clinical disease following challenge, and reduced duration and titres of challenge virus shedding. Calves inoculated with the boIL-6, boIL-12 and boIFN-gamma expressing recombinants showed a significant reduction in vaccine virus shedding but secreted more challenge virus than the other vaccinees. These findings indicate that expression of these cytokines mediates a better control of the vaccine virus replication which, however, interferes with the immunogenicity of the vaccine. In summary, all recombinant viruses were safe and effective, but protection afforded by the recombinants was not improved as compared to vaccination with the parental virus strain GK/D.

Engineering Glycoprotein B of Bovine Herpesvirus 1 To Function as Transporter for Secreted Proteins: a New Protein Expression Approach

ABSTRACT Glycoprotein B (gB) of bovine herpesvirus 1 (BHV-1) is essential for BHV-1 replication and is required for membrane fusion processes leading to virus penetration into the target cell and direct spreading of BHV-1 from infected to adjacent noninfected cells. Like many of the herpesvirus gB homologs, BHV-1 gB is proteolytically processed by furin, an endoproteinase localized in the trans-Golgi network. Cleavage by furin is a common mechanism for the activation of a number of viral fusion (F) proteins. Among these, the F proteins of both human and bovine respiratory syncytial virus (RSV) have the so-far unique feature that cleavage of the respective F protein precursors occurs at two furin recognition sites, resulting in the release of a 27-amino-acid intervening peptide which is secreted into the extracellular space. We showed recently that the intervening peptide of bovine RSV can be replaced by bovine interleukins which are secreted into the medium of cells infected with the respective bovine RSV recombinants (P. König, K. Giesow, K. Schuldt, U. J. Buchholz, and G. M. Keil, J. Gen. Virol. 85:1815-1824, 2004). To elucidate whether the approach to transport heterologous proteins as furin-excisable polypeptides functions in principle also in glycoproteins which are cleaved by furin only once, we inserted a second furin cleavage site into BHV-1 gB and integrated a 16-amino-acid peptide sequence, the 246-amino-acid green fluorescent protein (GFP), or the 167 amino acids for mature bovine alpha interferon (boIFN-α) as an intervening polypeptide. The resulting gB variants rescued gB-negative BHV-1 mutants, the resulting BHV-1 recombinants were fully infectious, and infected cells secreted biologically active GFP and boIFN-α, respectively. In contrast to the gB2Fu and gB2FuGFP precursor molecules, which were efficiently cleaved at both furin sites, the majority of pgB2FuIFN-α was not cleaved at the site between the amino-terminal (NH2) subunit and boIFN-α, whereas cleavage at the newly introduced site was normal. This resulted in virus particles that also contain the NH2-subunit/boIFN-α fusion protein within their envelopes. Our results demonstrate that BHV-1 gB can be used as a transporter for peptides and proteins which could be important for development of novel vaccines. In addition, the general principle might be useful for other applications, e.g., in gene therapy and also in nonviral systems.

A novel bromodeoxyuridine-resistant wild boar lung cell line facilitates generation of African swine fever virus recombinants

Manipulation of African swine fever virus (ASFV) genomes, in particular those from field strains, is still a challenge. We have shown recently that generation of a green-fluorescent-protein-expressing, thymidine-kinase-negative (TK−) mutant of the low-pathogenic African swine fever virus field strain NHV was supported by a TK− Vero cell line. Since NHV, like other ASFV field strains, does not replicate well in Vero cells, a bromodeoxyuridine (BrdU)- resistant cell line derived from wild boar lung (WSL) cells, named WSL-Bu, was selected. WSL cells were used because they are suitable for productive replication of NHV and other ASFV field strains. Here, we show that WSL-Bu cells enable positive selection of both TK− and TK+ ASFV recombinants, which allows for novel strategies for construction of ASFV mutants. We further demonstrate for a low-pathogenic ASFV strain that TK expression is required for infectious replication in macrophages infected at low multiplicity and that vaccinia TK fully complements ASFV TK in this respect.

High-Level Protein Expression Following Single and Dual Gene Cloning of Infectious Bronchitis Virus N and S Genes Using Baculovirus Systems

Baculovirus is an efficient system for the gene expression that can be used for gene transfer to both insect and different vertebrate hosts. The nucleocapsid gene (N) of the infectious bronchitis virus was cloned in a baculovirus expression system for insect cell expression. Dual expression vectors containing IBV N and spike (S) proteins of the avian infectious bronchitis virus were engineered under the control of human and murine cytomegalovirus immediate-early enhancer/promoter elements in combination with the baculoviral polyhedrin and p10 promoters for simultaneous expression in both vertebrate and insect cells. Transduction of the N gene in the insect Sf9 cells revealed a high level of protein expression. The expressed protein, used in ELISA, effectively detected chicken anti-IBV antibodies with high specificity. Transduction of mammalian and avian cells with BacMam viruses revealed that dual expression cassettes yielded high levels of protein from both transcription units.

Effect of foot-and-mouth disease virus capsid precursor protein and 3C protease expression on bovine herpesvirus 1 replication

Several reports have previously shown that expression of the foot-and-mouth disease virus (FMDV) capsid precursor protein encoding region P1-2A together with the 3C protease (P1-2A/3C) results in correct processing of the capsid precursor into VP0, VP1 and VP3 and formation of FMDV capsid structures that are able to induce a protective immune response against FMDV challenge after immunization using naked DNA constructs or recombinant viruses. To elucidate whether bovine herpesvirus 1 (BHV-1) might also be suitable as a viral vector for empty capsid generation, we aimed to integrate a P1-2A/3C expression cassette into the BHV-1 genome, which, however, failed repeatedly. In contrast, BHV-1 recombinants that expressed an inactive 3C protease or the P1-2A polyprotein alone could be easily generated, although the recombinant that expressed P1-2A exhibited a defect in direct cell–cell spread and release of infectious particles. These results suggested that expression of the original, active FMDV 3C protease is not compatible with BHV-1 replication. This conclusion is supported by the isolation of recombinant BHV-1/3C*, which contained mutations within the 3C ORF (3C* ORF)—probably introduced spontaneously during generation of BHV-1/3C*—instead of the authentic 3C ORF contained in the transfer plasmids. Within the 3C* ORF, the codons for glycine 38 and phenylalanine 48 were both substituted by codons for serine. The resulting 3C* protease exhibits a highly reduced activity for proteolytic processing of the P1-2A polyprotein and thus might be a good candidate for the generation of live attenuated FMDV variants.

Protein display by bovine herpesvirus type 1 glycoprotein B.

Glycoprotein B (gB) of bovine herpesvirus 1 (BHV-1), a major component of the viral envelope, is essential for membrane fusion during entry and cell-to-cell spread. It is cleaved in the trans-Golgi network by the proprotein convertase furin. Integration of the open reading frame (ORF) encoding a mutated gB with a second furin cleavage site and mature boIFN-alpha as intervening peptide between the amino-terminal (NH(2)) and carboxy-terminal (COOH) gB subunits yielded recombinant BHV-1/gB2FuIFN-alpha which, unexpectedly, express gB with an enlarged NH(2)-subunit of 90kDa. Here we show that boIFN-alpha-specific antibodies bind to the 90kDa gB subunit and efficiently neutralize BHV-1/gB2FuIN-alpha infectivity. We also show that inactivated BHV-1/gB2FuIN-alpha virions induce an antiviral state in cells incubated with UV-inactivated particles. These results demonstrate that the 90kDa protein is a NH(2)-subunit/boIFN-alpha fusion protein whose boIFN-alpha domain is biologically active. To verify that BHV-1 gB is suitable for the display of (glyco)proteins on the surface of virions we constructed BHV-1 recombinants expressing within gB the first 273 amino acids of the NH(2)-subunit (HA1) of avian influenza haemagglutinin, either flanked by two furin cleavage sites or with only one cleavage site between a gB/NH(2)_HA1 fusion protein and the COOH subunit. The resulting recombinant BHV-1/gB2FuHA1 expressed gB from which 55kDa HA1 was excised and secreted. In contrast, gB from BHV-1/gB_NH(2)HA1 infected cells retained HA1 as fusion protein with the NH(2)-subunit. Immunoblotting and neutralization analyses revealed that HA1 is incorporated into the envelope BHV-1/gB/NH(2)_HA1 particles and exposed to the exterior of virions. Thus, this novel approach enables display of polypeptides and (glyco)proteins of at least 273 amino acids on viral particles which is of particular interest for development of novel diagnostics and vaccines as well as for, e.g. gene therapy applications especially when biologically active ligands need to be presented.

Genetic fusion of peste des petits ruminants virus haemagglutinin and fusion protein domains to the amino terminal subunit of glycoprotein B of bovine herpesvirus 1 interferes with transport and function of gB for BHV-1 infectious replication

Peste des petits ruminants is an emerging, often fatal viral disease of domestic and wild small ruminants caused by peste des petits ruminants virus. The haemagglutinin and the fusion protein are viral envelope glycoproteins and essential for the infection process and both induce a protective immune response in infected or vaccinated animals. Attempts to generate pseudotyped bovine herpesvirus 1 recombinants firstly by integration of expression cassettes for PPRV-H and PPRV-F into the herpesviral genome or secondly to generate pseudotyped BHV-1 by genetically fusing relevant parts of both PPRV glycoproteins to the amino-terminal subunit of glycoprotein B, approaches that had been successful for heterologous viral membrane glycoproteins in the past, failed repeatedly. We therefore analyzed at which intracellular stage generation of viable BHV-1 hybrid-gB recombinants might be inhibited. Results obtained from transient protein expression experiments revealed that, dependent on the fusion protein, transport of the hybrid glycoproteins beyond the endoplasmic reticulum is impeded. Thus, expression of heterologous glycoproteins using BHV-1 interferes more than expected from published experience with BHV-1 gB transport and consequently with virus replication.