Bert Jan Haijema

Acquisition of macrophage tropism during the pathogenesis of feline infectious peritonitis is determined by mutations in the feline coronavirus spike protein.

ABSTRACT In feline coronavirus (FCoV) pathogenesis, the ability to infect macrophages is an essential virulence factor. Whereas the low-virulence feline enteric coronavirus (FECV) isolates primarily replicate in the epithelial cells of the enteric tract, highly virulent feline infectious peritonitis virus (FIPV) isolates have acquired the ability to replicate efficiently in macrophages, which allows rapid dissemination of the virulent virus throughout the body. FIPV 79-1146 and FECV 79-1683 are two genetically closely related representatives of the two pathotypes. Whereas FECV 79-1683 causes at the most a mild enteritis in young kittens, FIPV 79-1146 almost invariably induces a lethal peritonitis. The virulence phenotypes correlate with the abilities of these viruses to infect and replicate in macrophages, a feature of FIPV 79-1146 but not of FECV 79-1683. To identify the genetic determinants of the FIPV 79-1146 macrophage tropism, we exchanged regions of its genome with the corresponding parts of FECV 79-1683, after which the ability of the FIPV/FECV hybrid viruses to infect macrophages was tested. Thus, we established that the FIPV spike protein is the determinant for efficient macrophage infection. Interestingly, this property mapped to the C-terminal domain of the protein, implying that the difference in infection efficiency between the two viruses is not determined at the level of receptor usage, which we confirmed by showing that infection by both viruses was equally blocked by antibodies directed against the feline aminopeptidase N receptor. The implications of these findings are discussed.

Switching Species Tropism: an Effective Way To Manipulate the Feline Coronavirus Genome

ABSTRACT Feline infectious peritonitis virus (FIPV), a coronavirus, is the causative agent of an invariably lethal infection in cats. Like other coronaviruses, FIPV contains an extremely large positive-strand RNA genome of ca. 30 kb. We describe here the development and use of a reverse genetics strategy for FIPV based on targeted RNA recombination that is analogous to what has been described for the mouse hepatitis virus (MHV) (L. Kuo et al., J. Virol. 74:1393-1406, 2000). In this two-step process, we first constructed by targeted recombination a mutant of FIPV, designated mFIPV, in which the ectodomain of the spike glycoprotein was replaced by that of MHV. This switch allowed for the selection of the recombinant virus in murine cells: mFIPV grows to high titers in these cells but has lost the ability to grow in feline cells. In a second, reverse process, mFIPV was used as the recipient, and the reintroduction of the FIPV spike now allowed for selection of candidate recombinants by their regained ability to grow in feline cells. In this fashion, we reconstructed a wild-type recombinant virus (r-wtFIPV) and generated a directed mutant FIPV in which the initiation codon of the nonstructural gene 7b had been disrupted (FIPVΔ7b). The r-wtFIPV was indistinguishable from its parental virus FIPV 79-1146 not only for its growth characteristics in tissue culture but also in cats, exhibiting a highly lethal phenotype. FIPVΔ7b had lost the expression of its 7b gene but grew unimpaired in cell culture, confirming that the 7b glycoprotein is not required in vitro. We establish the second targeted RNA recombination system for coronaviruses and provide a powerful tool for the genetic engineering of the FIPV genome.

Murine Coronavirus with an Extended Host Range Uses Heparan Sulfate as an Entry Receptor

ABSTRACT Only a relatively few mutations in its spike protein allow the murine coronavirus to switch from a murine-restricted tropism to an extended host range by being passaged in vitro. One such virus that we studied had acquired two putative heparan sulfate-binding sites while preserving another site in the furin-cleavage motif. The adaptation of the virus through the use of heparan sulfate as an attachment/entry receptor was demonstrated by increased heparin binding as well as by inhibition of infection through treatment of cells and the virus with heparinase and heparin, respectively.

A Protective and Safe Intranasal RSV Vaccine Based on a Recombinant Prefusion-Like Form of the F Protein Bound to Bacterium-Like Particles

Respiratory syncytial virus (RSV) is an important cause of respiratory tract disease in infants and the elderly. Currently, no licensed vaccine against RSV is available. Here we describe the development of a safe and effective intranasal subunit vaccine that is based on recombinant fusion (F) protein bound to the surface of immunostimulatory bacterium-like particles (BLPs) derived from the food-grade bacterium Lactococcus lactis. Different variants of F were analyzed with respect to their conformation and reactivity with neutralizing antibodies, assuming that F proteins mimicking the metastable prefusion form of RSV F expose a more extensive and relevant epitope repertoire than F proteins corresponding to the postfusion structure. Our results indicate that the recombinant soluble ectodomain of RSV F readily adopts a postfusion conformation, generation of which cannot be prevented by C-terminal addition of a trimerization motif, but whose formation is prevented by mutation of the two furin cleavage sites in F. While the putative postfusion form of F is recognized well by the monoclonal antibody Palivizumab, this is much less so for the more potently neutralizing, prefusion-specific antibodies D25 and AM22. Both addition of the trimerization motif and mutation of the furin cleavage sites increased the reactivity of F with D25 and AM22, with the highest reactivity being observed for F proteins in which both these features were combined. Intranasal vaccination of mice or cotton rats with BLPs loaded with this latter prefusion-like F protein (BLP-F), resulted in the potent induction of F-specific immunoglobulins and in significantly decreased virus titers in the lungs upon RSV challenge. Moreover, and in contrast to animals vaccinated with formalin-inactivated RSV, animals that received BLP-F exhibited high levels of F-specific secretory IgA in the nose and RSV-neutralizing antibodies in sera, but did not show symptoms of enhanced disease after challenge with RSV.

Cleavage of Group 1 Coronavirus Spike Proteins: How Furin Cleavage Is Traded Off against Heparan Sulfate Binding upon Cell Culture Adaptation

ABSTRACT A longstanding enigmatic feature of the group 1 coronaviruses is the uncleaved phenotype of their spike protein, an exceptional property among class I fusion proteins. Here, however, we show that some group 1 coronavirus spike proteins carry a furin enzyme recognition motif and can actually be cleaved, as demonstrated for a feline coronavirus. Interestingly, this feature can be lost during cell culture adaptation by a single mutation in the cleavage motif; this, however, preserves a heparan sulfate binding motif and renders infection by the virus heparan sulfate dependent. We identified a similar cell culture adaptation for the human coronavirus OC43.

Coronaviruses as Vectors: Stability of Foreign Gene Expression

ABSTRACT Coronaviruses are enveloped, positive-stranded RNA viruses considered to be promising vectors for vaccine development, as (i) genes can be deleted, resulting in attenuated viruses; (ii) their tropism can be modified by manipulation of their spike protein; and (iii) heterologous genes can be expressed by simply inserting them with appropriate coronaviral transcription signals into the genome. For any live vector, genetic stability is an essential requirement. However, little is known about the genetic stability of recombinant coronaviruses expressing foreign genes. In this study, the Renilla and the firefly luciferase genes were systematically analyzed for their stability after insertion at various genomic positions in the group 1 coronavirus feline infectious peritonitis virus and in the group 2 coronavirus mouse hepatitis virus. It appeared that the two genes exhibit intrinsic differences, the Renilla gene consistently being maintained more stably than the firefly gene. This difference was not caused by genome size restrictions, by different effects of the encoded proteins, or by different consequences of the synthesis of the additional subgenomic mRNAs. The loss of expression of the firefly luciferase was found to result from various, often large deletions of the gene, probably due to RNA recombination. The extent of this process appeared to depend strongly on the coronaviral genomic background, the luciferase gene being much more stable in the feline than in the mouse coronavirus genome. It also depended significantly on the particular genomic location at which the gene was inserted. The data indicate that foreign sequences are more stably maintained when replacing nonessential coronaviral genes.

Antiviral activity of carbohydrate-binding agents against Nidovirales in cell culture.

Abstract Coronaviruses are important human and animal pathogens, the relevance of which increased due to the emergence of new human coronaviruses like SARS-CoV, HKU1 and NL63. Together with toroviruses, arteriviruses, and roniviruses the coronaviruses belong to the order Nidovirales. So far antivirals are hardly available to combat infections with viruses of this order. Therefore, various antiviral strategies to counter nidoviral infections are under evaluation. Lectins, which bind to N-linked oligosaccharide elements of enveloped viruses, can be considered as a conceptionally new class of virus inhibitors. These agents were recently evaluated for their antiviral activity towards a variety of enveloped viruses and were shown in most cases to inhibit virus infection at low concentrations. However, limited knowledge is available for their efficacy towards nidoviruses. In this article the application of the plant lectins Hippeastrum hybrid agglutinin (HHA), Galanthus nivalis agglutinin (GNA), Cymbidium sp. agglutinin (CA) and Urtica dioica agglutinin (UDA) as well as non-plant derived pradimicin-A (PRM-A) and cyanovirin-N (CV-N) as potential antiviral agents was evaluated. Three antiviral tests were compared based on different evaluation principles: cell viability (MTT-based colorimetric assay), number of infected cells (immunoperoxidase assay) and amount of viral protein expression (luciferase-based assay). The presence of carbohydrate-binding agents strongly inhibited coronaviruses (transmissible gastroenteritis virus, infectious bronchitis virus, feline coronaviruses serotypes I and II, mouse hepatitis virus), arteriviruses (equine arteritis virus and porcine respiratory and reproductive syndrome virus) and torovirus (equine Berne virus). Remarkably, serotype II feline coronaviruses and arteriviruses were not inhibited by PRM-A, in contrast to the other viruses tested.

Bacterium-Like Particles for Efficient Immune Stimulation of Existing Vaccines and New Subunit Vaccines in Mucosal Applications

The successful development of a mucosal vaccine depends critically on the use of a safe and effective immunostimulant and/or carrier system. This review describes the effectiveness and mode of action of an immunostimulating particle, derived from bacteria, used in mucosal subunit vaccines. The non-living particles, designated bacterium-like particles are based on the food-grade bacterium Lactococcus lactis. The focus of the overview is on the development of intranasal BLP-based vaccines to prevent diseases caused by influenza and respiratory syncytial virus, and includes a selection of Phase I clinical data for the intranasal FluGEM vaccine.

The carbohydrate-binding plant lectins and the non-peptidic antibiotic pradimicin A target the glycans of the coronavirus envelope glycoproteins.

Abstract Objectives Many enveloped viruses carry carbohydrate-containing proteins on their surface. These glycoproteins are key to the infection process as they are mediators of the receptor binding and membrane fusion of the virion with the host cell. Therefore, they are attractive therapeutic targets for the development of novel antiviral therapies. Recently, carbohydrate-binding agents (CBA) were shown to possess antiviral activity towards coronaviruses. The current study further elucidates the inhibitory mode of action of CBA. Methods Different strains of two coronaviruses, mouse hepatitis virus and feline infectious peritonitis virus, were exposed to CBA: the plant lectins Galanthus nivalis agglutinin, Hippeastrum hybrid agglutinin and Urtica dioica agglutinin (UDA) and the non-peptidic mannose-binding antibiotic pradimicin A. Results and conclusions Our results indicate that CBA target the two glycosylated envelope glycoproteins, the spike (S) and membrane (M) protein, of mouse hepatitis virus and feline infectious peritonitis virus. Furthermore, CBA did not inhibit virus–cell attachment, but rather affected virus entry at a post-binding stage. The sensitivity of coronaviruses towards CBA was shown to be dependent on the processing of the N-linked carbohydrates. Inhibition of mannosidases in host cells rendered the progeny viruses more sensitive to the mannose-binding agents and even to the N-acetylglucosamine-binding UDA. In addition, inhibition of coronaviruses was shown to be dependent on the cell-type used to grow the virus stocks. All together, these results show that CBA exhibit promising capabilities to inhibit coronavirus infections.

Inactivated influenza vaccine adjuvanted with Bacterium-like particles induce systemic and mucosal influenza A virus specific T-cell and B-cell responses after nasal administration in a TLR2 dependent fashion

BACKGROUND Nasal vaccination is considered to be a promising alternative for parenteral vaccination against influenza virus as it is non-invasive and offers the opportunity to elicit strong antigen-specific responses both systemic and locally at the port of entry of the pathogen. Previous studies showed that non-living bacterium-like particles (BLPs) from the food-grade bacterium Lactococcus lactis are effective stimulators of local and systemic immune responses when administered intranasally. Moreover, in vitro, BLPs specifically interact with human Toll-like receptor 2 (TLR2), suggestive of a role for TLR2 dependent immune activation by BLPs. METHODS In the present study, we examined the role of TLR2 in vivo in immune activation after nasal administration of BLP mixed with split influenza vaccine (BLP-SV) of influenza A virus (IAV) using TLR2 knockout mice. RESULTS The systemic Th1 cell and subsequent B-cell responses induced after intranasal BLP-SV vaccination depended on the interaction of BLPs with TLR2. Notably, the BLP-SV-induced class switch to IgG2c depended on the interaction of BLP with TLR2. Local induced IAV-specific Th1 cell responses and the mucosal B-cell responses also depended on interaction of BLP with TLR2. Strongly reduced SIgA levels were observed in TLR2 knockout mice both in the nasal and vaginal lavages. In addition, detailed analysis of the T-cell response revealed that nasal BLP-SV vaccination promoted Th1/Th17 immune responses that coincided with increased IAV-specific IgG2c antibody production. DISCUSSION Altogether these results indicate that nasal BLP-SV vaccination induces IAV-specific T-cell and B-cell responses, both systemically and at the site of virus entry in a TLR2-dependent manner.