Structural relatedness of lysis proteins from colicinogenic plasmids and icosahedral coliphages

The host-lysis-inducing functions of phi X174 protein E and MS2 protein L were recently shown to reside on the N-terminal and C-terminal halves of the two respective lysis proteins. In the present study it is shown that the small lysis proteins encoded in various colicinogenic plasmids share local sequence similarities and certain structural characteristics with the essential peptides of their coliphage-coded counterparts. Despite their dissimilar sizes and origins, it is suggested that the colicinogenic lysis proteins are functionally analogous and evolutionarily related to those of icosahedral single- stranded DNA and RNA phages.      

Enzymic synthesis,chemical characterisation and Sda activity of GalNAcß1-4[NeuAcα2-3]Galß1-4GlcNAc and GalNAcß1-4[NeuAcα2-3]Galß1-4Glc

The tetrasaccharides GalNAcß1-4[NeuAc2-3]Galß1-4Glc and GalNAcß1-4[NeuAc2-3]Galß1-4GlcNAc were synthesised by enzymic transfer of GalNAc from UDP-GalNAc to 3-sialyllactose (NeuAc2-3Galß1-4Glc) and 3-sialyl-N-acetyllactosamine (NeuAc2-3Galß1-4GlcNAc). The structures of the products were established by methylation and¹H-500 MHz NMR spectroscopy. In Sd^a serological tests the product formed with 3-sialyl-N-acetyllactosamine was highly active whereas that formed with 3-sialyllactose had only weak activity.     

Applications of DNA shuffling to pharmaceuticals and vaccines

DNA shuffling is a practical process for directed molecular evolution which uses recombination to dramatically accelerate the rate at which one can evolve genes. Single and multigene traits that require many mutations for improved phenotypes can be evolved rapidly. DNA shuffling technology has been significantly enhanced in the past year, extending its range of applications to small molecule pharmaceuticals, pharmaceutical proteins, gene therapy vehicles and transgenes, vaccines and evolved viruses for vaccines, and laboratory animal models.     

Artificial induction of lactation in ewes: the use of prostaglandin

Injections of an anlogue of prostaglandin F2alpha (T.F.101) initiated secretion of copious amounts of fluid resembling normal ovine milk when given to non-pregnant ewes with developed mammary glands. Injections of T.F.101 elicited a substantial but transient increase in the levels of prolactin in plasma. Results for intact and ovariectomized ewes were similar.     

Measurement of the Infection and Dissemination of Bluetongue Virus in Culicoides Biting Midges Using a Semi-Quantitative RT-PCR Assay and Isolation of Infectious Virus

Background

Culicoides biting midges (Diptera: Ceratopogonidae) are the biological vectors of globally significant arboviruses of livestock including bluetongue virus (BTV), African horse sickness virus (AHSV) and the recently emerging Schmallenberg virus (SBV). From 2006–2009 outbreaks of BTV in northern Europe inflicted major disruption and economic losses to farmers and several attempts were made to implicate Palaearctic Culicoides species as vectors. Results from these studies were difficult to interpret as they used semi-quantitative RT-PCR (sqPCR) assays as the major diagnostic tool, a technique that had not been validated for use in this role. In this study we validate the use of these assays by carrying out time-series detection of BTV RNA in two colony species of Culicoides and compare the results with the more traditional isolation of infectious BTV on cell culture.

Methodology/Principal Findings

A BTV serotype 1 strain mixed with horse blood was fed to several hundred individuals of Culicoides sonorensis (Wirth & Jones) and C. nubeculosus (Mg.) using a membrane-based assay and replete individuals were then incubated at 25°C. At daily intervals 25 Culicoides of each species were removed from incubation, homogenised and BTV quantified in each individual using sqPCR (C_q values) and virus isolation on a KC-C. sonorensis embryonic cell line, followed by antigen enzyme-linked immunosorbent assay (ELISA). In addition, comparisons were also drawn between the results obtained with whole C. sonorensis and with individually dissected individuals to determine the level of BTV dissemination.

Conclusions/Significance

C_q values generated from time-series infection experiments in both C. sonorensis and C. nubeculosus confirmed previous studies that relied upon the isolation and detection of infectious BTV. Implications on the testing of field-collected Culicoides as potential virus vectors by PCR assays and the use of such assays as front-line tools for use in diagnostic laboratories in this role are discussed.     

Molecular evolution of P transposable elements in the genus Drosophila. III. The melanogaster species group

Phylogenetic relationships were determined for 76 partial P-element sequences from 14 species of the melanogaster species group within the Drosophila subgenus Sophophora. These results are examined in the context of the phylogeny of the species from which the sequences were isolated. Sequences from the P-element family fall into distinct subfamilies, or clades, which are often characteristic for particular species subgroups. When examined locally among closely related species, the evolution of P elements is characterized by vertical transmission, whereby the P-element phylogeny traces the species phylogeny. On a broader scale, however, the P-element phylogeny is not congruent with the species phylogeny. One feature of P-element evolution in the melanogaster group is the presence of more than one P-element subfamily, differing by as much as 36%, in the genomes of some species. Thus, P elements from several individual species are not monophyletic, and a likely explanation for the incongruence between P-element and species phylogenies is provided by the comparison of paralogous sequences. In certain instances, horizontal transfer seems to be a valid alternative explanation for lack of congruence between species and P-element phylogenies. The canonical P-element subfamily, which represents the active, autonomous transposable element, is restricted to D. melanogaster. Thus, its origin clearly lies outside of the melanogaster species group, consistent with the earlier conclusion of recent horizontal transfer.      

Optimizing Human Synovial Fluid Preparation for Two-Dimensional Gel Electrophoresis

Background

Prenatal screening for Down Syndrome (DS) would benefit from an increased number of biomarkers to improve sensitivity and specificity. Improving sensitivity and specificity would decrease the need for potentially risky invasive diagnostic procedures.

Results

We have performed an in depth two-dimensional difference gel electrophoresis (2D DIGE) study to identify potential biomarkers. We have used maternal plasma samples obtained from first and second trimesters from mothers carrying DS affected fetuses compared with mothers carrying normal fetuses. Plasma samples were albumin/IgG depleted and expanded pH ranges of pH 4.5 - 5.5, pH 5.3 - 6.5 and pH 6 - 9 were used for two-dimensional gel electrophoresis (2DE). We found no differentially expressed proteins in the first trimester between the two groups. Significant up-regulation of ceruloplasmin, inter-alpha-trypsin inhibitor heavy chain H4, complement proteins C1s subcomponent, C4-A, C5, and C9 and kininogen 1 were detected in the second trimester in maternal plasma samples where a DS affected fetus was being carried. However, ceruloplasmin could not be confirmed as being consistently up-regulated in DS affected pregnancies by Western blotting.

Conclusions

Despite the in depth 2DE approach used in this study the results underline the deficiencies of gel-based proteomics for detection of plasma biomarkers. Gel-free approaches may be more productive to increase the number of plasma biomarkers for DS for non-invasive prenatal screening and diagnosis.     

Novel Recombinant Mycobacterium bovis BCG,Ovine Atadenovirus,and Modified Vaccinia Virus Ankara Vaccines Combine To Induce Robust Human Immunodeficiency Virus-Specific CD4 and CD8 T-Cell Responses in Rhesus Macaques

Mycobacterium bovis bacillus Calmette-Guérin (BCG), which elicits a degree of protective immunity against tuberculosis, is the most widely used vaccine in the world. Due to its persistence and immunogenicity, BCG has been proposed as a vector for vaccines against other infections, including HIV-1. BCG has a very good safety record, although it can cause disseminated disease in immunocompromised individuals. Here, we constructed a recombinant BCG vector expressing HIV-1 clade A-derived immunogen HIVA using the recently described safer and more immunogenic BCG strain AERAS-401 as the parental mycobacterium. Using routine ex vivo T-cell assays, BCG.HIVA⁴⁰¹ as a stand-alone vaccine induced undetectable and weak CD8 T-cell responses in BALB/c mice and rhesus macaques, respectively. However, when BCG.HIVA⁴⁰¹ was used as a priming component in heterologous vaccination regimens together with recombinant modified vaccinia virus Ankara-vectored MVA.HIVA and ovine atadenovirus-vectored OAdV.HIVA vaccines, robust HIV-1-specific T-cell responses were elicited. These high-frequency T-cell responses were broadly directed and capable of proliferation in response to recall antigen. Furthermore, multiple antigen-specific T-cell clonotypes were efficiently recruited into the memory pool. These desirable features are thought to be associated with good control of HIV-1 infection. In addition, strong and persistent T-cell responses specific for the BCG-derived purified protein derivative (PPD) antigen were induced. This work is the first demonstration of immunogenicity for two novel vaccine vectors and the corresponding candidate HIV-1 vaccines BCG.HIVA⁴⁰¹ and OAdV.HIVA in nonhuman primates. These results strongly support their further exploration.Vaccine strategies must balance safety with immunogenicity. Recombinant attenuated subunit vaccines are generally regarded as safe, but not sufficiently immunogenic as stand-alone vaccines (17). Heterologous prime-boost regimens employing diverse attenuated viruses or bacteria as vectors delivering a common, often T cell-based, immunogen have been shown to induce stronger responses than multiple repeated dosings of the same vaccine modalities (19, 22, 39, 54). This is because heterologous regimens allow boosting of pathogen insert-specific responses while avoiding the accumulation of antivector immunity, which can significantly decrease vaccine “take” (1, 41). Results of the STEP study, which used a candidate single-vector human immunodeficiency virus type 1 (HIV-1) vaccine (6, 17, 41), have highlighted the need for novel alternative vaccine vectors and strategies. Such alternatives could complement the limited mainstream vectors and provide additional safety and immunogenicity through increased flexibility, for example, through the availability of personalized vaccination regimens based on preexisting immune status and/or responsiveness to vaccination.Mycobacterium bovis bacillus Calmette-Guérin (BCG) remains the world''s most widely used vaccine, with over three billion doses administered since its deployment in 1920s. It is the only licensed vaccine against tuberculosis and is administered at birth as part of the WHO Expanded Programme on Immunization (EPI). Due to its many attractive features, BCG or related mycobacterial vectors have also been explored in the context of vaccines against a number of infectious agents such as Leishmania, Borrelia burgdorferi, Streptococcus pneumoniae, Bordetella pertussis, malaria, cottontail rabbit papillomavirus, measles virus, and indeed human and simian immunodeficiency viruses (34). Many of these vaccines showed immunogenicity and protection in murine models, and some were also immunogenic in nonhuman primates (8, 56, 67, 68). In human adults, recombinant BCG (rBCG) vaccines alone failed to provide consistent protection against Lyme disease (13). In addition to adult applications, we have suggested the use of rBCG expressing an HIV-1-derived immunogen as the priming component of a vaccine platform against mother-to-child transmission of HIV-1 through infected breast milk (32), where it would be critical to elicit a protective HIV-1-specific response as soon as possible after birth.To compare vectors and heterologous prime-boost regimens directly, we have advocated and pioneered the development of a panel of vaccine modalities delivering the same shared immunogen (18). Our first such model immunogen is called HIVA (21). This is a T-cell immunogen comprising HIV-1 consensus clade A Gag and a string of partially overlapping immunodominant CD8 T-cell epitopes originating from Gag, Pol, Nef, and Env, which has already been tested extensively in human volunteers (20). To facilitate iterative preclinical improvements of the HIVA vaccines, epitopes recognized by murine (58) and rhesus macaque (44) CD8 T cells were also incorporated. Furthermore, we have formulated HIVA into various vaccine modalities, including plasmid DNA (21), modified vaccinia virus Ankara (MVA) (21), human adenovirus serotype 5 (HAdV-5) (5), Semliki Forest virus replicons (18, 49), recombinant lysine auxotroph BCG strain Pasteur (32), and baculovirus-expressed and purified, bluetongue virus-derived chimeric NS1 tubules (37); the immunogenicity of these vectors has been compared directly and in heterologous combinations. More recently, we reported on the immunogenicity of a novel and promising vaccine vector derived from ovine atadenovirus type 7 (OAdV) (5); OAdV is the prototype member of the genus Atadenovirus, which is structurally and biologically distinct from Mastadenovirus (e.g., HAdV-5) (2, 50). Importantly, no immunity to OAdV has so far been detected in human sera (26). In mice, OAdV.HIVA induced strong polyfunctional HIVA-specific T cell responses with distinct kinetics from those induced by HAdV5.HIVA and displayed demonstrable single-dose efficacy against a surrogate virus challenge (5). OAdV is approved for use in a phase I human clinical trial (http://clinicaltrials.gov identifier no. {"type":"clinical-trial","attrs":{"text":"NCT00625430","term_id":"NCT00625430"}}NCT00625430). All of the vectors/modalities we explore are perceived to be safe and acceptable for use in humans.Here, as a step toward translating our results into human volunteers, we constructed a novel vaccine designated BCG.HIVA⁴⁰¹ vectored by AERAS-401, a Danish 1331 strain of BCG with improved immunogenicity and safety (57), and demonstrated priming of T cells to the HIVA transgene product in rhesus macaques. These BCG.HIVA⁴⁰¹-primed HIV-1-specific CD4 and CD8 T-cell responses were readily boosted with MVA.HIVA and OAdV.HIVA vaccines to elicit broad and robust HIV-1-specific T cell responses.