PLASMD BEARING A CDNA COPY OF THE GENOME OF BOVINE VIRAL DIARRHEA VIRUS, CHIMERIC DERIVATIVES THEREOF, AND METHOD OF PRODUCING AN INFECTIOUS BOVINE WRAL DARRHEAVIRUS USING SAD PLASMID

A plasmid bearing a cDNA copy of the genome of bovine viral diarrhea virus (BVDV), chimeric derivatives of the plasmid and a method of producing an infectious bovine viral diarrhea virus using the plasmid are disclosed. The invention relates to a plasmid DNA molecule that replicates easily in E. coli and contains a sufficient portion of the genome of BVDV, cloned as cDNA, to be a suitable template to produce RNA in vitro which, upon transfection into bovine cells, gives rise to infectious BVDV. The BVDV created by the process of the invention can be engineered for use as a vector in many advantageous applications

Antigenic diversity is generated by distinct evolutionary mechanisms in African trypanosome species

Antigenic variation enables pathogens to avoid the host immune response by continual switching of surface proteins. The protozoan blood parasite Trypanosoma brucei causes human African trypanosomiasis ("sleeping sickness") across sub-Saharan Africa and is a model system for antigenic variation, surviving by periodically replacing a monolayer of variant surface glycoproteins (VSG) that covers its cell surface. We compared the genome of Trypanosoma brucei with two closely related parasites Trypanosoma congolense and Trypanosoma vivax, to reveal how the variant antigen repertoire has evolved and how it might affect contemporary antigenic diversity. We reconstruct VSG diversification showing that Trypanosoma congolense uses variant antigens derived from multiple ancestral VSG lineages, whereas in Trypanosoma brucei VSG have recent origins, and ancestral gene lineages have been repeatedly co-opted to novel functions. These historical differences are reflected in fundamental differences between species in the scale and mechanism of recombination. Using phylogenetic incompatibility as a metric for genetic exchange, we show that the frequency of recombination is comparable between Trypanosoma congolense and Trypanosoma brucei but is much lower in Trypanosoma vivax. Furthermore, in showing that the C-terminal domain of Trypanosoma brucei VSG plays a crucial role in facilitating exchange, we reveal substantial species differences in the mechanism of VSG diversification. Our results demonstrate how past VSG evolution indirectly determines the ability of contemporary parasites to generate novel variant antigens through recombination and suggest that the current model for antigenic variation in Trypanosoma brucei is only one means by which these parasites maintain chronic infections

Potent single-domain antibodies that arrest respiratory syncytial virus fusion protein in its prefusion state

Human respiratory syncytial virus (RSV) is the main cause of lower respiratory tract infections in young children. The RSV fusion protein (F) is highly conserved and is the only viral membrane protein that is essential for infection. The prefusion conformation of RSV F is considered the most relevant target for antiviral strategies because it is the fusion-competent form of the protein and the primary target of neutralizing activity present in human serum. Here, we describe two llama-derived single-domain antibodies (VHHs) that have potent RSV-neutralizing activity and bind selectively to prefusion RSV F with picomolar affinity. Crystal structures of these VHHs in complex with prefusion F show that they recognize a conserved cavity formed by two F protomers. In addition, the VHHs prevent RSV replication and lung infiltration of inflammatory monocytes and T cells in RSV-challenged mice. These prefusion F-specific VHHs represent promising antiviral agents against RSV

Contact transmission of influenza virus between ferrets imposes a looser bottleneck than respiratory droplet transmission allowing propagation of antiviral resistance

Influenza viruses cause annual seasonal epidemics and occasional pandemics. It is important to elucidate the stringency of bottlenecks during transmission to shed light on mechanisms that underlie the evolution and propagation of antigenic drift, host range switching or drug resistance. The virus spreads between people by different routes, including through the air in droplets and aerosols, and by direct contact. By housing ferrets under different conditions, it is possible to mimic various routes of transmission. Here, we inoculated donor animals with a mixture of two viruses whose genomes differed by one or two reverse engineered synonymous mutations, and measured the transmission of the mixture to exposed sentinel animals. Transmission through the air imposed a tight bottleneck since most recipient animals became infected by only one virus. In contrast, a direct contact transmission chain propagated a mixture of viruses suggesting the dose transferred by this route was higher. From animals with a mixed infection of viruses that were resistant and sensitive to the antiviral drug oseltamivir, resistance was propagated through contact transmission but not by air. These data imply that transmission events with a looser bottleneck can propagate minority variants and may be an important route for influenza evolution

Antibody Responses to Recombinant Oligopeptides of <i>Pf</i>EMP1 Isolated From Children Infected With <i>Plasmodium falciparum</i>

Individuals living in malaria endemic areas acquire immunity against clinical disease through repeated encounters with the Plasmodium falciparum parasite during their childhood. As a result, the majority of severe cases of malaria are restricted to younger children. Naturally acquired immunity is thought to be partly mediated by antibodies directed at parasite derived antigens on the surface of red blood cells infected with mature forms of P. falciparum called variant surface antigens (VSA). Of these, P. falciparum erythrocyte membrane protein 1 is well characterized and associated with pathology and immune evasion. Sera obtained from young children living in endemic areas show limited recognition of PfEMP1 unlike immune adult sera that exhibit recognition of a wide range of PfEMP1. It is not yet clear whether this wide recognition of PfEMP1 by adult immune sera is exclusively a product of variant specific response accumulated over many exposures or whether it includes a cross-reactive response that recognizes a diverse set of antigens. Here, I have used the recombinant DBLα-tag region of PfEMP1 generated from parasites isolated from children infected with malaria to show that individuals develop a strong variant-specific response to their infecting isolate, accompanied by partly cross-reactive response. Based on antigenic and genetic similarities between different DBLα-tag variants, I identified potential epitope regions in this study that may account for the cross reactive responses. However, these appear to be conformational and predominantly lie towards the N-terminal end. Once verified, such regions may form good candidates for inclusion in a multi-epitope vaccine construct that affords broad protection against malaria

Cloning of hemagglutinin (HA) protein of influenza A virus - Potential for sialic acid linkage discrimination

The initial step in infection of a cell by influenza A virus is the attachment of a virus particle to the target cell. This is accomplished by interaction of a glycoprotein, hemagglutinin (HA), found on the surface of the viral lipid membrane with cell-surface oligosaccharides containing sialic acids. All influenza virus attachment requires terminal sialic acid residues and two major linkages between sialic acid (Neu5Ac) and the penultimate galactose (Gal) residues of carbohydrate side chains are found in nature, Neu5Ac(α2,3)-Gal and Neu5Ac(α2,6)-Gal. The HA’s of different subtypes of influenza A virus exhibit different recognition specificities for these linkages and these linkage specificities have been correlated with host range specificity. The ability of the HA protein to differentiate sialic acid linkages makes it an interesting candidate for use in the characterization of glycoprotein's potentially facilitating the discrimination of alternate glycoforms of biopharmaceutical therapeutics and their subsequent purification

Affinity and dose of TCR engagement yield proportional enhancer and gene activity in CD4+ T cells.

Affinity and dose of T cell receptor (TCR) interaction with antigens govern the magnitude of CD4+ T cell responses, but questions remain regarding the quantitative translation of TCR engagement into downstream signals. We find that while the response of mouse CD4+ T cells to antigenic stimulation is bimodal, activated cells exhibit analog responses proportional to signal strength. Gene expression output reflects TCR signal strength, providing a signature of T cell activation. Expression changes rely on a pre-established enhancer landscape and quantitative acetylation at AP-1 binding sites. Finally, we show that graded expression of activation genes depends on ERK pathway activation, suggesting that an ERK-AP-1 axis plays an important role in translating TCR signal strength into proportional activation of enhancers and genes essential for T cell function

Plasmodium falciparum antigenic variation. Mapping mosaic var gene sequences onto a network of shared, highly polymorphic sequence blocks

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a potentially important family of immune targets, encoded by an extremely diverse gene family called var. Understanding of the genetic organization of var genes is hampered by sequence mosaicism that results from a long history of non-homologous recombination. Here we have used software designed to analyse social networks to visualize the relationships between large collections of short var sequences tags sampled from clinical parasite isolates. In this approach, two sequences are connected if they share one or more highly polymorphic sequence blocks. The results show that the majority of analysed sequences including several var-like sequences from the chimpanzee parasite Plasmodium reichenowi can be either directly or indirectly linked together in a single unbroken network. However, the network is highly structured and contains putative subgroups of recombining sequences. The major subgroup contains the previously described group A var genes, previously proposed to be genetically distinct. Another subgroup contains sequences found to be associated with rosetting, a parasite virulence phenotype. The mosaic structure of the sequences and their division into subgroups may reflect the conflicting problems of maximizing antigenic diversity and minimizing epitope sharing between variants while maintaining their host cell binding functions

Immunological assessment of plant-derived avian flu H5/HA1 variants.

Polypeptide variants of the HA1 antigenic domain of the H5N1 avian influenza virus hemagglutinin (HA) molecule were produced in plants using transient and stable expression systems and fused with His/c-myc tags or with mouse or human Fc antibody fragments. The resulting peptides were purified and used for intramuscular immunization of mice. While the recombinant HA1 variants induced a significant serum humoral immune response in the mice, none of the HA1 preparations induced virus-neutralizing antibodies. Fusion with the Fc fragment improved overall yield of the constructs and allowed purification requiring only a single step, but led to no detectable fusion-related enhancement of immunogenicity or quality of immune response