Productive replication of peste des petits ruminants virus Nigeria 75/1 vaccine strain in vero cells correlates with inefficiency of maturation of the viral fusion protein.

Peste des petits ruminants virus (PPRV), a member of the genus Morbillivirus, in the family Paramyxoviridae expresses two membrane glycoproteins, the fusion (F) and haemagglutinin (H) glycoproteins which mediate virus-to-cell fusion and cell-to-cell fusion leading to the induction of syncytia in PPRV infected cells. In the context of the characterization of the virulent lineage IV strain PPRV Kurdistan 2011, isolated from wild goats from the Kurdistan region in Iraq, we observed that both PPRV Kurdistan 2011 and the PPRV Nigeria 75/1 vaccine strain led to induction of large syncytia in Vero-dogSLAM cells within 48 h whereas both failed to induce detectable cell-cell fusion events in two Vero cell lines of differing passage histories. We were unable to detect syncytium formation in transiently transfected cells expressing PPRV F or H alone whereas co-expression of F and H induced large syncytia - in Vero-dogSLAM cells only. In VeroMontpellier cells expressing PPRV F and H, fused cells were rarely detectable indicating that PPRV mediated cell fusion activity is impaired in this cell line. Surprisingly, on Vero-dogSLAM cells the vaccine strain grew to titers of 105.25 TCID50/ml, whereas infectious virus yield was about 200-fold higher on VeroMontpellier and Vero-76 cells. In contrast, the virulent Kurdistan 2011 strain grew to a maximum titer of 107.0 TCID50/ml on Vero-dogSLAM cells and only 104.5 TCID50/ml on normal Vero cells. This was as expected since Vero cells lacking the SLAM receptor for PPRV are regarded as not so permissive for infection. To elucidate the divergent productive replication behaviour of PPRV Nigeria 75/1 vaccine strain on Vero vs Vero-dogSLAM cells, we examined whether intracellular transport and/or maturation of the viral envelope glycoproteins F and H might be implicated with this phenomenon. The results indicate that F in contrast to the H glycoprotein matures inefficiently during intracellular transport in VeroMontpellier cells, thus leading to an absence of detectable syncytia formation. However, in the case of the PPRV Nigeria 75/1 vaccine strain this did not impair efficient virus assembly and release.

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.

BacMam Platform for Vaccine Antigen Delivery.

Recombinant baculo viruses based on Autographa californica multiple nuclear polyhedrosis virus carrying vertebrate cell active expression cassettes, so-called BacMam viruses, are increasingly used as gene delivery vectors for vaccination of animals against pathogens. Different approaches for generation of BacMams exist and a variety of transfer vectors to improve target protein expression in vivo have been constructed. Here we describe a use of transfer vector which contains an insect cell-restricted expression cassette for the green fluorescent protein and thus enables easy monitoring of BacMam virus rescue, fast plaque purification of recombinants and their convenient titer determination and which has been proven to be efficacious for gene delivery in vaccination/challenge experiments.

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.

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.

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.

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.

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.

High-level expression of biologically active bovine alpha interferon by Bovine herpesvirus 1 interferes only marginally with recombinant virus replication in vitro.

An artificial open reading frame (ORF) for bovine alpha interferon (boIFN-alpha) with the codon preference of Bovine herpesvirus 1 (BHV-1) glycoprotein B was constructed to assess the effect of expression of boIFN-alpha by BHV-1 from an expression cassette. Transient expression of the ORF revealed that transfected cells secreted substantial amounts of biologically active boIFN-alpha, which moderately inhibited replication of BHV-1 after stimulation of bovine cells with 10(4) U ml(-1). The boIFN-alpha-encoding expression cassette was recombined into the glycoprotein E locus of the glycoprotein E-negative BHV-1 vaccine strain GKD. Cells infected with the resulting recombinant BHV-1/boIFN-alpha secreted up to 10(7) U boIFN-alpha per ml cell culture supernatant, which is about 40- to more than 100-fold the activity reached with other virus expression systems. Bioassays demonstrated that the BHV-1-expressed interferon induced a rapid and sustained antiviral state in stimulated bovine cells. Analysis of the in vitro growth properties of the recombinant revealed, depending on the cell line used, no or only slight inhibition in direct spreading from cell to cell and a modest delay in virus egress from infected cells. Final titres, however, were comparable to those reached by the parent strain. Penetration into cells was not affected. The results from this study demonstrate that BHV-1/boIFN-alpha expresses high levels of boIFN-alpha, grows to high titres in cell culture and thus represents a potential alternative means to deliver endogenously produced boIFN-alpha in situ for a period of time.

Engineering glycoprotein B of bovine herpesvirus 1 to function as transporter for secreted proteins: a new protein expression approach.

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. Konig, 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-alpha) 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-alpha, respectively. In contrast to the gB2Fu and gB2FuGFP precursor molecules, which were efficiently cleaved at both furin sites, the majority of pgB2FuIFN-alpha was not cleaved at the site between the amino-terminal (NH2) subunit and boIFN-alpha, whereas cleavage at the newly introduced site was normal. This resulted in virus particles that also contain the NH2-subunit/boIFN-alpha 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 protein expression strategy using recombinant bovine respiratory syncytial virus (BRSV): modifications of the peptide sequence between the two furin cleavage sites of the BRSV fusion protein yield secreted proteins, but affect processing and function of the BRSV fusion protein.

The bovine respiratory syncytial virus (BRSV) fusion (F) protein is cleaved at two furin cleavage sites, which results in generation of the disulfide-linked F(1) and F(2) subunits and release of an intervening peptide of 27 aa (pep27). A series of mutated open reading frames encoding F proteins that lacked the entire pep27, that contained an arbitrarily chosen 23 aa sequence instead of pep27 or in which pep27 was replaced by the amino acid sequences for the bovine cytokines interleukin 2 (boIL2), interleukin 4 (boIL4) or gamma interferon (boIFN-gamma) was constructed. Transient expression experiments revealed that the sequence of the intervening peptide influenced intracellular transport, maturation of the F protein and F-mediated syncytium formation. Expression of boIL2, boIL4 or boIFN-gamma in place of pep27 resulted in secretion of the cytokines into the culture medium. All mutated F proteins except the boIFN-gamma-containing variant could be expressed by and were functional for recombinant BRSV. Characterization of the cell culture properties of the recombinants demonstrated that the amino acid sequence between the two furin cleavage sites affected entry into target cells, direct spreading of virions from cell to cell and virus growth. Secretion of boIL2 and boIL4 into the medium of cells infected with the respective recombinants demonstrated that the F protein can be used to express secreted heterologous bioactive peptides or (glyco)proteins, which might be of interest for the development of novel RSV vaccines.

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.

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.