A transgenic plant cell‐suspension system for expression of epitopes on chimeric Bamboo mosaic virus particles

We describe a novel strategy to produce vaccine antigens using a plant cell‐suspension culture system in lieu of the conventional bacterial or animal cell‐culture systems. We generated transgenic cell‐suspension cultures from Nicotiana benthamiana leaves carrying wild‐type or chimeric Bamboo mosaic virus (BaMV) expression constructs encoding the viral protein 1 (VP1) epitope of foot‐and‐mouth disease virus (FMDV). Antigens accumulated to high levels in BdT38 and BdT19 transgenic cell lines co‐expressing silencing suppressor protein P38 or P19. BaMV chimeric virus particles (CVPs) were subsequently purified from the respective cell lines (1.5 and 2.1 mg CVPs/20 g fresh weight of suspended biomass, respectively), and the resulting CVPs displayed VP1 epitope on the surfaces. Guinea pigs vaccinated with purified CVPs produced humoral antibodies. This study represents an important advance in the large‐scale production of immunopeptide vaccines in a cost‐effective manner using a plant cell‐suspension culture system.     

Chimeric plant virus particles as immunogens for inducing murine and human immune responses against human immunodeficiency virus type 1

The high-yield expression of a neutralizing epitope from human immunodeficiency virus type 1 (HIV-1) on the surface of a plant virus and its immunogenicity are presented. The highly conserved ELDKWA epitope from glycoprotein (gp) 41 was expressed as an N-terminal translational fusion with the potato virus X (PVX) coat protein. The resulting chimeric virus particles (CVPs), purified and used to immunize mice intraperitoneally or intranasally, were able to elicit high levels of HIV-1-specific immunoglobulin G (IgG) and IgA antibodies. Furthermore, the human immune response to CVPs was studied with severe combined immunodeficient mice reconstituted with human peripheral blood lymphocytes (hu-PBL-SCID). hu-PBL-SCID mice immunized with CVP-pulsed autologous dendritic cells were able to mount a specific human primary antibody response against the gp41-derived epitope. Notably, sera from both normal and hu-PBL-SCID mice showed an anti-HIV-1-neutralizing activity. Thus, PVX-based CVPs carrying neutralizing epitopes can offer novel perspectives for the development of effective vaccines against HIV and, more generally, for the design of new vaccination strategies in humans.     

Production of an engineered killer peptide in Nicotiana benthamiana by using a potato virus X expression system

The decapeptide killer peptide (KP) derived from the sequence of a single-chain, anti-idiotypic antibody acting as a functional internal image of a microbicidal, broad-spectrum yeast killer toxin (KT) was shown to exert a strong microbicidal activity against human pathogens. With the aim to exploit this peptide to confer resistance to plant pathogens, we assayed its antimicrobial activity against a broad spectrum of phytopathogenic bacteria and fungi. Synthetic KP exhibited antimicrobial activity in vitro towards Pseudomonas syringae, Erwinia carotovora, Botrytis cinerea, and Fusarium oxysporum. KP was also expressed in plants by using a Potato virus X (PVX)-derived vector as a fusion to the viral coat protein, yielding chimeric virus particles (CVPs) displaying the heterologous peptide. Purified CVPs showed enhanced antimicrobial activity against the above-mentioned plant pathogens and human pathogens such as Staphylococcus aureus and Candida albicans. Moreover, in vivo assays designed to challenge KP-expressing plants (as CVPs) with Pseudomonas syringae pv. tabaci showed enhanced resistance to bacterial attack. The results indicate that the PVX-based display system is a high-yield, rapid, and efficient method to produce and evaluate antimicrobial peptides in plants, representing a milestone for the large-scale production of high-added-value peptides through molecular farming. Moreover, KP is a promising molecule to be stably engineered in plants to confer broad-spectrum resistance to phytopathogens.     

Production of an Engineered Killer Peptide in Nicotiana benthamiana by Using a Potato virus X Expression System

The decapeptide killer peptide (KP) derived from the sequence of a single-chain, anti-idiotypic antibody acting as a functional internal image of a microbicidal, broad-spectrum yeast killer toxin (KT) was shown to exert a strong microbicidal activity against human pathogens. With the aim to exploit this peptide to confer resistance to plant pathogens, we assayed its antimicrobial activity against a broad spectrum of phytopathogenic bacteria and fungi. Synthetic KP exhibited antimicrobial activity in vitro towards Pseudomonas syringae, Erwinia carotovora, Botrytis cinerea, and Fusarium oxysporum. KP was also expressed in plants by using a Potato virus X (PVX)-derived vector as a fusion to the viral coat protein, yielding chimeric virus particles (CVPs) displaying the heterologous peptide. Purified CVPs showed enhanced antimicrobial activity against the above-mentioned plant pathogens and human pathogens such as Staphylococcus aureus and Candida albicans. Moreover, in vivo assays designed to challenge KP-expressing plants (as CVPs) with Pseudomonas syringae pv. tabaci showed enhanced resistance to bacterial attack. The results indicate that the PVX-based display system is a high-yield, rapid, and efficient method to produce and evaluate antimicrobial peptides in plants, representing a milestone for the large-scale production of high-added-value peptides through molecular farming. Moreover, KP is a promising molecule to be stably engineered in plants to confer broad-spectrum resistance to phytopathogens.     

Cowpea mosaic virus chimeric particles bearing the ectodomain of matrix protein 2 (M2E) of the influenza a virus: Production and characterization

The epitope presentation system for the ectodomain of the M2 protein (M2e) of the influenza A virus was constructed on the basis of the cowpea mosaic virus (CPMV) for expression in the plant Vigna unguiculata. CPMV is widely used as a vector to produce immunogenic chimeric virus particles (CVPs) bearing epitopes of various infectious human and animal pathogens. To produce chimeric CPMV particles in plants, two binary vectors were constructed to bear a modified gene coding for the CPMV S-coat protein with insertions of M2e epitopes of human influenza and bird influenza viruses. Antigenic and immunogenic properties of CVPs were investigated in mice immunization experiments. CVPs were shown to induce anti-M2e IgG production and to partly protect mice against a challenge with low doses of the influenza virus. However, low infectivity and immunogenicity of chimeric CPMV particles indicate that the plant virus-based systems for M2e epitope presentation requires further optimization in order to use plants as a possible source of flu vaccines.     

Use of Plant Viruses for Production of Plant-Derived Vaccines

Plants produce appropriately folded, post-translationally processed proteins that, as antigens, elicit efficacious immune responses in preclinical animal models and antigen-specific responses in humans. Plant-produced vaccine candidates have been produced using transgenic technologies and the utilization of plant viruses for the transient protein expression. The later approach has numerous advantages in recombinant protein production, including rapid protein expression and higher yields of antigenic proteins. In some cases, plant viruses are “decorated” with human or animal antigens from pathogens to form chimeric virus particles (CVPs). Immunization of animals with CVPs induces specific and often efficacious immune responses. While there are no plant-produced vaccines commercially available, the diversity and effectiveness of the products presently in development coupled with production advantages, including, reduced cost of production, the rapid scale-up capabilities, and the safety of the final product, should encourage continued investment and progress through clinical testing.     

FGF signaling regulates the number of posterior taste papillae by controlling progenitor field size

The sense of taste is fundamental to our ability to ingest nutritious substances and to detect and avoid potentially toxic ones. Sensory taste buds are housed in papillae that develop from epithelial placodes. Three distinct types of gustatory papillae reside on the rodent tongue: small fungiform papillae are found in the anterior tongue, whereas the posterior tongue contains the larger foliate papillae and a single midline circumvallate papilla (CVP). Despite the great variation in the number of CVPs in mammals, its importance in taste function, and its status as the largest of the taste papillae, very little is known about the development of this structure. Here, we report that a balance between Sprouty (Spry) genes and Fgf10, which respectively antagonize and activate receptor tyrosine kinase (RTK) signaling, regulates the number of CVPs. Deletion of Spry2 alone resulted in duplication of the CVP as a result of an increase in the size of the placode progenitor field, and Spry1(-/-);Spry2(-/-) embryos had multiple CVPs, demonstrating the redundancy of Sprouty genes in regulating the progenitor field size. By contrast, deletion of Fgf10 led to absence of the CVP, identifying FGF10 as the first inductive, mesenchyme-derived factor for taste papillae. Our results provide the first demonstration of the role of epithelial-mesenchymal FGF signaling in taste papilla development, indicate that regulation of the progenitor field size by FGF signaling is a critical determinant of papilla number, and suggest that the great variation in CVP number among mammalian species may be linked to levels of signaling by the FGF pathway.     

Viral and bacterial diseases of Atlantic cod Gadus morhua, their prophylaxis and treatment: a review

This review summarises the state of knowledge of both viral and bacterial diseases of Atlantic cod Gadus morhua, and their diagnosis, prophylaxis and treatment. The most important losses have been at the larval and juvenile stages, and vibriosis has long been the most important bacterial disease in cod, with Listonella (Vibrio) anguillarum dominant among pathogenic isolates. Vaccination of cod against pathogens such as L. anguillarum and Aeromonas salmonicida clearly demonstrates that the cod immune system possesses an effective memory and appropriate mechanisms sufficient for protection, at least against some diseases. Well-known viruses such as the nodavirus that causes viral encephalopathy and retinopathy (VER), infectious pancreatic necrosis virus (IPNV) and viral haemorrhagic septicaemia virus (VHSV) have been isolated from Atlantic cod and can be a potential problem under intensive rearing conditions. No commercial vaccines against nodavirus are currently available, whereas vaccines against IPNV infections based upon inactivated virus as well as IPNV recombinant antigens are available. A number of investigations of the pharmacokinetic properties of antibacterial agents in cod and their efficacy in treating bacterial infections have been reviewed.     

How the mirror-image pattern specified by a janus mutation of Tetrahymena thermophila comes to expression

The initial changes of cell-surface organization that occurred as the recessive janA1 (janus) mutation of Tetrahymena thermophila first became expressed were elucidated in a special mating scheme in which old macronuclei homozygous for janA+ were synchronously replaced by new macronuclei homozygous for janA1. During this period of onset of expression, the number, regularity, and asymmetry of the ciliary rows remained unchanged. New normal (primary) oral apparatuses (OAs) continued to be formed posterior to old OAs, as in normal cells. At about four fissions after conjugation, abnormal (secondary) OAs with a partial reversal of asymmetry began to appear nearly opposite to the primary OAs, close to but not at the eventual circumferential position of janA1 secondary OAs. The array of contractile vacuole pores (CVPs), normally located adjacent to two ciliary rows centered near 22% of the cell circumference to the right of the primary oral meridian, underwent a two-step transformation: first, the number of adjacent ciliary rows bearing CVPs increased to 3, 4, and sometimes 5, then "skipped" rows appeared within this broadened CVP-arc to split the single set of CVPs into two separated subsets. The CVP transformations occurred gradually and progressively. They began prior to the expression of secondary OAs but accelerated as secondary OAs appeared. As the CVP arc became broader, its midpoint shifted somewhat to the right, away from the primary oral meridian, but ended up close to halfway between the primary and secondary oral meridians. The data provide a better fit to an intercalation model than to an alternative double-gradient model, suggesting that the janA1 mutation alters the large-scale organization of positional values by preventing the expression of a subset of these values and thus provoking reverse-intercalation of the remainder.     

Chimeric Plant Virus Particles Administered Nasally or Orally Induce Systemic and Mucosal Immune Responses in Mice

The humoral immune responses to the D2 peptide of fibronectin-binding protein B (FnBP) of Staphylococcus aureus, expressed on the plant virus cowpea mosaic virus (CPMV), were evaluated after mucosal delivery to mice. Intranasal immunization of these chimeric virus particles (CVPs), either alone or in the presence of ISCOM matrix, primed CPMV-specific T cells and generated high titers of CPMV- and FnBP-specific immunoglobulin G (IgG) in sera. Furthermore, CPMV- and FnBP-specific IgA and IgG could also be detected in the bronchial, intestinal, and vaginal lavage fluids, highlighting the ability of CVPs to generate antibody at distant mucosal sites. IgG2a and IgG2b were the dominant IgG subclasses in sera to both CPMV and FnBP, demonstrating a bias in the response toward the T helper 1 type. The sera completely inhibited the binding of human fibronectin to the S. aureus FnBP. Oral immunization of the CVPs also generated CPMV- and FnBP-specific serum IgG; however, these titers were significantly lower and more variable than those generated by the intranasal route, and FnBP-specific intestinal IgA was undetectable. Neither the ISCOM matrix nor cholera toxin enhanced these responses. These studies demonstrate for the first time that recombinant plant viruses have potential as mucosal vaccines without the requirement for adjuvant and that the nasal route is most effective for the delivery of these nonreplicating particles.Replicating vaccines such as live-attenuated bacterial (13) and virus (36, 40, 45) vaccines, as well as naked DNA vaccines (31), induce stronger and longer-lasting immune responses than conventional killed-subunit vaccines and also elicit protective cell-mediated immunity, often without the need for adjuvant. There are however, safety concerns over the use of these vaccines (24, 49), where persistence or reversion to virulence of the live vaccine strains and integration of the naked DNA vaccine into the host chromosome are of major concern. Recent technological advances, such as the use of more-effective adjuvants for both mucosal and systemic delivery (12, 16), liposome and ISCOM encapsulation of proteins and peptides (3, 19, 27), multiple antigenic peptides (35), and virus-like particles (VLPs) (1), have led to the development of more-effective subunit vaccines. To circumvent the safety concerns of replicating vaccines and to avoid the need for peptide synthesis and chemical coupling to a carrier such as keyhole limpet hemocyanin, we have been examining the utility of the plant virus cowpea mosaic virus (CPMV) as a carrier of peptides for immune recognition. CPMV is composed of 2 subunits, the small (S) and large (L) coat proteins, of which there are 60 copies of each per virus particle (46). Foreign peptides up to 37 amino acids in length can be expressed on either the L or S proteins; hence, 60 to 120 copies of a peptide can be displayed on a single virus particle (4b, 34). A peptide from the human immunodeficiency virus (HIV) gp41 glycoprotein is highly immunogenic when displayed on CPMV, eliciting high titers of HIV neutralizing antibodies (28, 29). Furthermore, a peptide derived from the VP2 protein of canine parvovirus (CPV) expressed on CPMV is immunogenic when administered to mink and subsequently protected the mink from a lethal challenge with the CPV-related mink enteritis virus (10).Most infectious viral and bacterial diseases involve colonization or invasion through mucosal surfaces by the pathogen, and hence it is important to develop vaccines that induce strong protective mucosal immune responses as a first line of defense. Where the organism, such as Vibrio cholerae and enterotoxigenic Escherichia coli, is restricted to the mucosa, strong mucosal immunity is often sufficient. However, when the organism disseminates from the mucosa into the bloodstream, a strong systemic response is also required to engender sterile immunity. Hence, the ideal mucosal vaccine should generate local immune responses at mucosal surfaces but also elicit generalized vaccine-specific immunity in the systemic lymphoid organs. The potential of CPMV-based vaccines for mucosal vaccination has not previously been determined.Oral immunization with particulate antigens, especially when presented as viable organisms, which can colonize the mucosa better than killed organisms, is effective at inducing local and generalized secretory and systemic immune responses (5, 43). However, the acidic pH and the presence of degradative enzymes in the gastrointestinal tract mean that when nonreplicating antigens are used, high concentrations are often required to elicit high levels of immunity (6). Another way to elicit mucosal immunity but circumvent the problems of oral immunization is to vaccinate via the intranasal route (2). Intranasal immunization requires up to 10-fold less immunogen for effective immunization and avoids the problems of low pH. Live vaccines (15, 37), virus-like particles (4, 25, 32), and synthetic peptides (17, 33, 44) in the absence of adjuvant have been shown to stimulate strong immunity when administered by this route. Furthermore, stimulation of the nasal mucosa, like stimulation of the intestinal mucosa, has been shown to be effective at generating protective immunity at distant mucosal sites (reviewed in reference 2).To assess the potential of CVPs as mucosal vaccines, mice were immunized intranasally or orally with CPMV expressing a peptide derived from the fibronectin-binding protein B (FnBP) D2 motif of Staphylococcus aureus (14, 42). The three fibronectin-binding domains, termed D1, D2, and D3, of FnBP have been shown to be immunogenic in mice and rats (7, 41). The CVPs were shown to be more immunogenic when administered (without adjuvant) via the intranasal route than when administered by the oral route, generating high titers of D2-specific antibody in serum and mucosa, and the serum antibody inhibited fibronectin binding to FnBP.     

Respiratory viral infection predisposing for bacterial disease: a concise review

Although bacterial superinfection in viral respiratory disease is a clinically well documented phenomenon, the pathogenic mechanisms are still poorly understood. Recent studies have revealed some of the mechanisms involved. Physical damage to respiratory cells as a result of viral infection may lead to opportunistic adherence of bacteria. Enhanced bacterial adherence by specific mechanisms has been documented for respiratory cells infected with influenza A virus, respiratory syncytial virus and adenovirus in both in vitro and in vivo models. To date, results of various experimental studies indicate that different mechanisms for increased bacterial adherence induced by viruses are operating for specific viral-bacterial combinations. In the present review, a number of key findings obtained during the past two decades is presented and discussed.     

How the mirror-image pattern specified by a janus mutation of Tetrahymena thermophila comes to expression

The initial changes of cell-surface organization that occurred as the recessive janAl (janus) mutation of Tetrahymena thermophila first became expressed were elucidated in a special mating scheme in which old macronuclei homozygous for janA⁺ were synchronously replaced by new macronuclei homozygous for janAl. During this period of onset of expression, the number, regularity, and asymmetry of the ciliary rows remained unchanged. New normal (primary) oral apparatuses (OAs) continued to be formed posterior to old OAs, as in normal cells. At about four fissions after conjugation, abnormal (secondary) OAs with a partial reversal of asymmetry began to appear nearly opposite to the primary OAs, close to but not at the eventual circumferential position of janAl secondary OAs. The array of contractile vacuole pores (CVPs), normally located adjacent to two ciliary rows centered near 22% of the cell circumference to the righ of the primary oral meridian, underwent a two-step transformation: first, the number of adjacent ciliary rows bearing CVPs increased to 3, 4, and sometimes 5, then “skipped” rows appeared within this broadened CVP-arc to split the single set of CVPs into two separated subsets. The CVP transformations occurred gradually and progressively. They began prior to the expression of secondary OAs but accelerated as secondary OAs appeared. As the CVP are became broader, its midpoint shifted somewhat to the right, away from the primary oral meridian, but ended up close to halfway between the primary and secondary oral meridians. The data provide a better fit to an intercalation model than to an alternative double gradient model, suggesting that the janAl mutation alters the large-scale organization of positional values by preventing the expression of a subset of these values and thus provoking reverse-intercalation of the remainder.     

Gene transactivation mediated by the TAT gene of human immunodeficiency virus in transgenic mice.

Transgenic mice were generated carrying either the long terminal repeat of Human Immunodeficiency Virus fused to the bacterial chloramphenicol acetyl transferase reporter gene or a control element of the murine alpha A crystallin gene fused to the tat gene of human immunodeficiency virus. By crossing these two strains, progeny were obtained which carried both transgenes. The bacterial reporter gene was specifically transactivated in the eyes of these animals.     

Bacterial Neuraminidase Rescues Influenza Virus Replication from Inhibition by a Neuraminidase Inhibitor

Influenza virus neuraminidase (NA) cleaves terminal sialic acid residues on oligosaccharide chains that are receptors for virus binding, thus playing an important role in the release of virions from infected cells to promote the spread of cell-to-cell infection. In addition, NA plays a role at the initial stage of viral infection in the respiratory tract by degrading hemagglutination inhibitors in body fluid which competitively inhibit receptor binding of the virus. Current first line anti-influenza drugs are viral NA-specific inhibitors, which do not inhibit bacterial neuraminidases. Since neuraminidase producing bacteria have been isolated from oral and upper respiratory commensal bacterial flora, we posited that bacterial neuraminidases could decrease the antiviral effectiveness of NA inhibitor drugs in respiratory organs when viral NA is inhibited. Using in vitro models of infection, we aimed to clarify the effects of bacterial neuraminidases on influenza virus infection in the presence of the NA inhibitor drug zanamivir. We found that zanamivir reduced progeny virus yield to less than 2% of that in its absence, however the yield was restored almost entirely by the exogenous addition of bacterial neuraminidase from Streptococcus pneumoniae. Furthermore, cell-to-cell infection was severely inhibited by zanamivir but restored by the addition of bacterial neuraminidase. Next we examined the effects of bacterial neuraminidase on hemagglutination inhibition and infectivity neutralization activities of human saliva in the presence of zanamivir. We found that the drug enhanced both inhibitory activities of saliva, while the addition of bacterial neuraminidase diminished this enhancement. Altogether, our results showed that bacterial neuraminidases functioned as the predominant NA when viral NA was inhibited to promote the spread of infection and to inactivate the neutralization activity of saliva. We propose that neuraminidase from bacterial flora in patients may reduce the efficacy of NA inhibitor drugs during influenza virus infection. (295 words).     

Adsorption of LLCMK2 cell-grown Sendai virus onto human red blood cells and its release from the virus adsorbed cells

An early stage of virus adsorption was studied in a system of Sendai virus metabolically labeled with [3H]leucine in LLCMK2 cells and of human red blood cells (RBCs). The efficiency of viral release from the virus-bound RBCs by incubation at 37 C depended on the number of virus particles which had been used for adsorption onto the RBC at 4 C. When 7.8 x 10(2) virus particles were previously adsorbed onto the RBC at 4 C, most of the viruses were dissociated from the RBC at 37 C. In the case of adsorption of 3 to 12 virus particles per RBC, however, most of the viruses were not dissociated from the RBC by incubation at 37 C. Such RBC-bound viruses were released by incubation with various bacterial neuraminidases (Clostridium perfringens, etc.) or with a large number of LLCMK2 cell-grown Sendai virus (LLCMK2-Sendai) particles, but not released by treatment with hemagglutinin-neuraminidase protein (Sendai-gp) isolated from egg-grown Sendai virus.     

Systematic Excision of Vector Sequences from the BAC-Cloned Herpesvirus Genome during Virus Reconstitution

Recently the mouse cytomegalovirus (MCMV) genome was cloned as an infectious bacterial artificial chromosome (BAC) (M. Messerle, I. Crnkovic, W. Hammerschmidt, H. Ziegler, and U. H. Koszinowski, Proc. Natl. Acad. Sci. USA 94:14759-14763, 1997). The virus obtained from this construct is attenuated in vivo due to deletion of viral sequences and insertion of the BAC vector. We reconstituted the full-length MCMV genome and flanked the BAC vector with identical viral sequences. This new construct represents a versatile basis for construction of MCMV mutants since virus generated from the construct loses the bacterial sequences and acquires wild-type properties.     

Dynamics of local defensive reactions in the lungs in acute experimental inflammatory process induced by respiratory syncytial virus

Study has been made of broncho-associated lymphoid tissue (BALT), the number and functional activity of lung phagocytes, with 42 mice BALB/c, induced by respiratory-syncytial virus. With virus inflammation a decreased number of lung macrophages, their stronger attraction and increased lung chemotaxis have been observed. The addition of bacterial infection was accompanied by an increase in lung neutrophil, lymphocyte and macrophage, a decrease in lung neutrophil chemotaxis, an increase in macrophage phagocytosis and BALT hyperplasia.     

Cloning and mutagenesis of the murine gammaherpesvirus 68 genome as an infectious bacterial artificial chromosome

Gammaherpesviruses cause important infections of humans, in particular in immunocompromised patients. Recently, murine gammaherpesvirus 68 (MHV-68) infection of mice has been developed as a small animal model of gammaherpesvirus pathogenesis. Efficient generation of mutants of MHV-68 would significantly contribute to the understanding of viral gene functions in virus-host interaction, thereby further enhancing the potential of this model. To this end, we cloned the MHV-68 genome as a bacterial artificial chromosome (BAC) in Escherichia coli. During propagation in E. coli, spontaneous recombination events within the internal and terminal repeats of the cloned MHV-68 genome, affecting the copy number of the repeats, were occasionally observed. The gene for the green fluorescent protein was incorporated into the cloned BAC for identification of infected cells. BAC vector sequences were flanked by loxP sites to allow the excision of these sequences using recombinase Cre and to allow the generation of recombinant viruses with wild-type genome properties. Infectious virus was reconstituted from the BAC-cloned MHV-68. Growth of the BAC-derived virus in cell culture was indistinguishable from that of wild-type MHV-68. To assess the feasibility of mutagenesis of the cloned MHV-68 genome, a mutant virus with a deletion of open reading frame 4 was generated. Genetically modified MHV-68 can now be analyzed in functionally modified mouse strains to assess the role of gammaherpesvirus genes in virus-host interaction and pathogenesis.