Organ damage in Atlantic herring larvae as a result of ocean acidification

The dissolution of anthropogenically emitted excess carbon dioxide lowers the pH of the world's ocean water. The larvae of mass spawning marine fishes may be particularly vulnerable to such ocean acidification (OA), yet the generality of earlier results is unclear. Here we show the detrimental effects of OA on the development of a commercially important fish species, the Atlantic herring (Clupea harengus). Larvae were reared at three levels of CO2: today (0.0385 kPa), end of next century (0.183 kPa), and a coastal upwelling scenario (0.426 kPa), under near-natural conditions in large outdoor tanks. Exposure to elevated CO2 levels resulted in stunted growth and development, decreased condition, and severe tissue damage in many organs, with the degree of damage increasing with CO2 concentration. This complements earlier studies of OA on Atlantic cod larvae that revealed similar organ damage but at increased growth rates and no effect on condition.

ROLE OF MULTIPLE TOLL-LIKE RECEPTOR ACTIVATION IN REGULATING CYTOTOXIC T CELL PRIMING TO LYMPOCYTIC CHORIOMENINGITIS VIRUS INFECTIONS

Foreign pathogens are recognized by toll-like receptors (TLR), present on various immune cells such as professional antigen-presenting cells (pAPCs). On recognition of its ligand, these receptors activate pAPCs, which may in turn influence naïve CD8+ T cell activation and affect their abilities to clear viral infection. However, how TLR ligands (TLR-L) can regulate CD8+ T cell responses have not been fully elucidated. This thesis will focus on examining how the presence of components from foreign pathogens, e.g. viral or bacterial infection, can contribute to shaping host immunity during concurrent viral infections. Since nitric oxide (NO), an innate effector immune molecule, was recently suggested to regulate proteasome activity; we sought to examine if NO can influence MHC-I antigen presentation during viral infections. The data in this section of the thesis provides evidence that combined TLR engagement can alter the presentation of certain CD8+ epitopes due to NO-induced inhibition in proteasome activity. Taken together, the data demonstrate that TLR ligation can influence the adaptive immune response due to induction of specific innate effector molecules such as NO. Next, the influence of combined TLR engagement on CD8+ T cell immunodominance hierarchies during viral infections was examined. In this section, we established that dual TLR2 and TLR3 stimulation alters immunodominance hierarchies of LCMV epitopes as a result of reduced uptake of cell-associated antigens and reduced cross-presentation of NP396 consequently suppressing NP396-specific CD8+ T cell responses. These findings are significant as they highlight a new role for TLR ligands in regulating anti-viral CD8+ T cell responses through impairing cross-presentation of cell-associated antigens depending on the type of TLR present in the environment during infections. Finally, we addressed TLR ligand induced type I interferon production and the signalling pathways that regulate them in two different mouse macrophage populations – those derived from the spleen or bone marrow. In this study, we observed that concomitant TLR2 stimulation blocked the induction of type I IFN induced by TLR4 in bone marrow-derived macrophages, but not spleen-derived macrophages in SOCS3-dependent manner. Taken together, the data presented in this thesis have defined new facets of how anti-viral responses are regulated by TLR activation, especially if multiple receptors are engaged simultaneously.

The Influence of 1,25-Dihydroxyvitamin D3 on the Cross-Priming of Lymphocytic Choriomeningitis Virus Nucleoprotein

Biologically active 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) binds the vitamin D receptor (VDR) to exert its effect on target cells. VDR expression is found in a number of immune cells including professional antigen-presenting cells such as dendritic cells. It has been found that the actions of 1,25-(OH)2D3 on the immune system are mainly immunosuppressive. The cross-presentation pathway allows for exogenously derived antigens to be presented by pAPCs on MHC-I molecules to CD8+ T cells. CD8+ T cell activation results in the expansion of epitope-specific T cell populations that confer host protection. These epitopes can be organized into an immunodominance hierarchy. Previous work demonstrated that introducing LCMV-NP via the cross-priming pathway significantly alters the immunodominance hierarchy of a subsequent LCMV infection. Building upon these observations, our study assessed the effects of LCMV-NP cross priming in the presence of a single dose of 1,25-(OH)2D3. Treatment with 1,25-(OH)2D3 was found to have biological effects in our model system. In vitro pAPCs were demonstrated to up-regulate IL-10 and CYP24A1 mRNA, in addition to the transactivation of cellular VDR, as demonstrated by a relocalization to the nuclear region. Mice treated with 1,25-(OH)2D3 were found to produce up-regulated IL-10 and CYP24A1 transcripts. Expression of VDR was increased at both the transcript and protein level. Our results demonstrate that a single dose of 1,25-(OH)2D3 does not affect the cross-priming pathway in this system. Treatment with 1,25-(OH)2D3 did not influence the ability of differentiated pAPCs to phagocytose or cross-present exogenous antigen to epitope-specific CD8+ T cells. Furthermore, 1,25-(OH)2D3 did not alter cross-priming or the establishment of the LCMV immunodominance hierarchy in vivo. By confirming that 1,25-(OH)2D3 does not suppress cross-priming in our model, our study helps to expand the understanding of the immunomodulatory role of exogenous 1,25-(OH)2D3 on the outcome of virus infection. Collectively, our data supports the observation that the role of 1,25-(OH)2D3 in the immune system is not always associated with suppressive effects.

Measles virus superinfection immunity and receptor redistribution in persistently infected NT2 cells

A recombinant measles virus (MV) expressing red fluorescent protein (MVDsRed1) was used to produce a persistently infected cell line (piNT2-MVDsRed1) from human neural precursor (NT2) cells. A similar cell line (piNT2-MVeGFP) was generated using a virus that expresses enhanced green fluorescent protein. Intracytoplasmic inclusions containing the viral nucleocapsid protein were evident in all cells and viral glycoproteins were present at the cell surface. Nevertheless, the cells did not release infectious virus nor did they fuse to generate syncytia. Uninfected NT2 cells express the MV receptor CD46 uniformly over their surface, whereas CD46 was present in cell surface aggregates in the piNT2 cells. There was no decrease in the overall amount of CD46 in piNT2 compared to NT2 cells. Cell-to-cell fusion was observed when piNT2 cells were overlaid onto confluent monolayers of MV receptor-positive cells, indicating that the viral glycoproteins were correctly folded and processed. Infectious virus was released from the underlying cells, indicating that persistence was not due to gross mutations in the virus genome. Persistently infected cells were superinfected with MV or canine distemper virus and cytopathic effects were not observed. However, mumps virus could readily infect the cells, indicating that superinfection immunity is not caused by general soluble antiviral factors. As MVeGFP and MVDsRed1 are antigenically indistinguishable but phenotypically distinct it was possible to use them to measure the degree of superinfection immunity in the absence of any cytopathic effect. Only small numbers of non-fusing green fluorescent piNT2-MVDsRed1 cells (1 : 300 000) were identified in which superinfecting MVeGFP entered, replicated and expressed its genes.

Dynamics of Viral RNA Synthesis during Measles Virus Infection

We propose a reference model of the kinetics of a viral RNA-dependent RNA polymerase (vRdRp) activities and its regulation during infection of eucaryotic cells. After measles virus infects a cell, mRNAs from all genes immediately start to accumulate linearly over the first 5 to 6 h and then exponentially until approximately 24 h. The change from a linear to an exponential accumulation correlates with de novo synthesis of vRdRp from the incoming template. Expression of the virus nucleoprotein (N) prior to infection shifts the balance in favor of replication. Conversely, inhibition of protein synthesis by cycloheximide favors the latter. The in vivo elongation speed of the viral polymerase is approximately 3 nucleotides/s. A similar profile with fivefold-slower kinetics can be obtained using a recombinant virus expressing a structurally altered polymerase. Finally, virions contain only encapsidated genomic, antigenomic, and 5'-end abortive replication fragment RNAs.

Polyploid measles virus with hexameric genome length

Particles of most virus species accurately package a single genome, but there are indications that the pleomorphic particles of parainfluenza viruses incorporate multiple genomes. We characterized a stable measles virus mutant that efficiently packages at least two genomes. The first genome is recombinant and codes for a defective attachment protein with an appended domain interfering with fusion-support function. The second has one adenosine insertion in a purine run that interrupts translation of the appended domain and restores function. In that genome, a one base deletion in a different purine run abolishes polymerase synthesis, but restores hexameric genome length, thus ensuring accurate RNA encapsidation, which is necessary for efficient replication. Thus, the two genomes are complementary. The infection kinetics of this mutant indicate that packaging of multiple genomes does not negatively affect growth. We also show that polyploid particles are produced in standard infections at no expense to infectivity. Our results illustrate how the particles of parainfluenza viruses efficiently accommodate cargoes of different volume, and suggest a mechanism by which segmented genomes may have evolved.

Identification of mouse langerin/CD207 in Langerhans cells and some dendritic cells of lymphoid tissues

Human (h)Langerin/CD207 is a C-type lectin of Langerhans cells (LC) that induces the formation of Birbeck granules (BG). In this study, we have cloned a cDNA-encoding mouse (m)Langerin. The predicted protein is 66% homologous to hLangerin with conservation of its particular features. The organization of human and mouse Langerin genes are similar, consisting of six exons, three of which encode the carbohydrate recognition domain. The mLangerin gene maps to chromosome 6D, syntenic to the human gene on chromosome 2p13. mLangerin protein, detected by a mAb as a 48-kDa species, is abundant in epidermal LC in situ and is down-regulated upon culture. A subset of cells also expresses mLangerin in bone marrow cultures supplemented with TGF-beta. Notably, dendritic cells in thymic medulla are mLangerin-positive. By contrast, only scattered cells express mLangerin in lymph nodes and spleen. mLangerin mRNA is also detected in some nonlymphoid tissues (e.g., lung, liver, and heart). Similarly to hLangerin, a network of BG form upon transfection of mLangerin cDNA into fibroblasts. Interestingly, substitution of a conserved residue (Phe(244) to Leu) within the carbohydrate recognition domain transforms the BG in transfectant cells into structures resembling cored tubules, previously described in mouse LC. Our findings should facilitate further characterization of mouse LC, and provide insight into a plasticity of dendritic cell organelles which may have important functional consequences.

Safety and Immunogenicity of a Novel Recombinant Subunit Respiratory Syncytial Virus Vaccine (BBG2Na) in Healthy Young Adults

A novel recombinant respiratory syncytial virus (RSV) subunit vaccine, designated BBG2Na, was administered to 108 healthy adults randomly assigned to receive 10, 100, or 300 μg of BBG2Na in aluminum phosphate or saline placebo. Each subject received 1, 2, or 3 intramuscular injections of the assigned dose at monthly intervals. Local and systemic reactions were mild, and no evidence of harmful properties of BBG2Na was reported. The highest ELISA and virus-neutralizing (VN) antibody responses were evident in the 100- and 300-μg groups; second or third injections provided no significant boosts against RSV-derived antigens. BBG2Na induced ⩾2-fold and ⩾4-fold increases in G2Na-specific ELISA units in up to 100% and 57% of subjects, respectively; corresponding RSV-A–specific responses were 89% and 67%. Furthermore, up to 71% of subjects had ⩾2-fold VN titer increases. Antibody responses to 2 murine lung protective epitopes were also highly boosted after vaccination. Therefore, BBG2Na is safe, well tolerated, and highly immunogenic in RSV-seropositive adults

Analysis of receptor (CD46, CD150) usage by measles virus

This is a collaborative paper between Juergen Schneider-Schaulies's group and ours. It shows the differential use of the two measles receptors in various strains.