Cytokine gene expression patterns and immune responses to systemic Candida albicans infection in inbred mice.

Aims of the research:To characterise the tissue histology and tissue distribution patterns of C. albicans during systemic murine candidiasis.To develop a reliable, reproducible and sensitive SQ-RT-PCR for the quantitation of in vivo cytokine gene transcription.To use this technique to determine the in vivo pattern of tissue specific cytokine gene expression during systemic candidiasis.To determine if cytokine gene expression patterns vary between resistant BALB/c and sensitive CBA/CaH mice during primary systemic candidiasis.To determine if differences in tissue distribution of C. albicans in infected mice is matched by differences in tissue responses to infection.To determine if cytokine mRNA expression patterns during secondary systemic candidiasis, are different to those during primary systemic candidiasis.To determine if cytokine gene expression patterns vary between resistant BALB/c and sensitive CBA/CaH mice during secondary systemic candidiasis

Murine cytomegalovirus strains co-replicate at multiple tissue sites and establish co-persistence in salivary glands in the absence of Ly49H-mediated competition

Infection with multiple genetically distinct strains of pathogen is common and can lead to positive (complementation) or negative (competitive) within-host interactions. These interactions can alter aspects of the disease process and help shape pathogen evolution. Infection of the host with multiple strains of cytomegalovirus (CMV) occurs frequently in humans and mice. Profound, NK-cell-mediated (apparent) competition has been identified in C57BL/6 mice, and prevented the replication and shedding of certain co-infecting CMV strains. However, the frequency of such strong competition has not been established. Other within-host interactions such as complementation or alternative forms of competition remain possible. Moreover, high rates of recombination in both human CMV and murine CMV (MCMV) suggest prolonged periods of viral co-replication, rather than strong competitive suppression. An established model was employed to investigate the different possible outcomes of multi-strain infection in other mouse strains. In this study, co-replication of up to four strains of MCMV in the spleen, liver and salivary glands was observed in both MCMV-susceptible and MCMV-resistant mice. In the absence of apparent competition, no other forms of competition were unmasked. In addition, no evidence of complementation between viral strains was observed. Importantly, co-replication of MCMV strains was apparent for up to 90 days in the salivary glands. These data indicated that competition was not the default outcome of multi-strain CMV infection. Prolonged, essentially neutral, co-replication may be the norm, allowing for multi-strain transmission and prolonged opportunities for recombination

Membrane association of a model CD4 + T‐cell vaccine antigen confers enhanced yet incomplete protection against murid herpesvirus‐4 infection

Vaccination against γ-herpesviruses has proved difficult. CD4 T cells are essential to contain infection, but how best to prime them and whether this can reduce viral loads remain unclear. To address these questions, we used ovalbumin (OVA) as a model antigen, delivering it with murine cytomegalovirus (MCMV) to protect mice against OVA-expressing murine herpesvirus-4 (MuHV-4). Membrane-associated OVA (mOVA) was more effective than soluble OVA, both to prime CD4 T cells and as an effector target. It was also a better target than an OVA epitope limited to infected cells, suggesting that protective CD4 T cells recognize infected cell debris rather than infected cells themselves. While MCMV-mOVA protected acutely against MuHV-4-mOVA, long-term protection was incomplete, even when OVA-specific CD8 T cells and B cells were also primed. Thus, even optimized single-target vaccines may poorly reduce long-term γ-herpesvirus infections

Murine cytomegalovirus disseminates independently of CX3CR1, CCL2 or its m131/m129 chemokine homologue

Cytomegaloviruses (CMVs) use myeloid cells to move within their hosts. Murine CMV (MCMV) colonizes the salivary glands for long-term shedding, and reaches them via CD11c infected cells. A need to recruit patrolling monocytes for systemic spread has been proposed, based on poor salivary gland infection in fractalkine receptor (CX3CR1)-deficient mice. We found no significant CX3CR1 dependence of salivary gland infection. CCL2 and the viral m131/m129 chemokine homologue were also redundant for acute MCMV spread, arguing against a need for inflammation or infection to recruit additional monocytes to the entry site. M131/m129 promoted salivary gland infection, but only after the initial seeding of infected cells to this site. Our data support the idea that MCMV disseminates by infecting and mobilizing tissue-resident dendritic cells

The m15 Locus of Murine Cytomegalovirus Modulates Natural Killer Cell Responses to Promote Dissemination to the Salivary Glands and Viral Shedding

As the largest herpesviruses, the 230 kb genomes of cytomegaloviruses (CMVs) have increased our understanding of host immunity and viral escape mechanisms, although many of the annotated genes remain as yet uncharacterised. Here we identify the m15 locus of murine CMV (MCMV) as a viral modulator of natural killer (NK) cell immunity. We show that, rather than discrete transcripts from the m14, m15 and m16 genes as annotated, there are five 3′-coterminal transcripts expressed over this region, all utilising a consensus polyA tail at the end of the m16 gene. Functional inactivation of any one of these genes had no measurable impact on viral replication. However, disruption of all five transcripts led to significantly attenuated dissemination to, and replication in, the salivary glands of multiple strains of mice, but normal growth during acute infection. Disruption of the m15 locus was associated with heightened NK cell responses, including enhanced proliferation and IFNγ production. Depletion of NK cells, but not T cells, rescued salivary gland replication and viral shedding. These data demonstrate the identification of multiple transcripts expressed by a single locus which modulate, perhaps in a concerted fashion, the function of anti-viral NK cells

Competition is evident early, before the maturation of acquired immunity.

<p>B6 mice were inoculated i.p. with 1×10⁴ pfu of either C4C, K181 or K181^Δm157 (closed symbols) or a total of 1×10⁴ pfu of a mixed inoculum of equal proportions of with C4C and K181 or C4C and K181^Δm157 (open symbols). <b>A</b>. Viral DNA levels in the spleens of B6 mice three days after single strain infection. <b>B</b>. Viral DNA levels in the spleens of mice co-infected with C4C and K181. <b>C</b>. Viral DNA levels in the spleens of mice co-infected with C4C and K181^Δm157<b>D</b>. Viral DNA levels in the livers of B6 mice three days after single strain infection. <b>E</b>. Viral DNA levels in the livers of mice co-infected with C4C and K181. <b>F</b>. Viral DNA levels in the spleen of mice co-infected with C4C and K181^Δm157. Note competition was evident in the spleen (B) but not in the liver (E). Data are mean ± SEM for 5 animals per group. Dotted line indicates limit of detection. DNA levels in mice following single strain infection were compared by one-way Anova, asterisks indicate level of significance after Tukey's post hoc analysis (*** P<0.001, ** P<0.01, * P<0.05).</p

Blocking the NK cell receptor Ly49H eliminates viral competition.

<p>B6 mice were treated with 200 µg of the monoclonal antibody 3D10 (anti-Ly49H) or 200 µg of isotype control antibody 9E10 (anti-cMyc) on day −2, 0, 4, 8, 12 and 16 of infection. On day 0 mice were inoculated i.p. with 1×10⁴ pfu of either a single MCMV strain (closed symbols) or a total of 1×10⁴ pfu of a mixed inoculum of C4A and C4C (half filled diamonds - plaque assay or open symbols – strain specific PCR). <b>A</b>. Titers of infectious virus were determined by plaque assay in salivary glands of B6 mice infected with either C4A or C4C (closed symbols), or in anti-Ly49H or isotype control treated mice that were also co-infected with C4A and C4C (half filled diamonds). <b>B</b>. Viral DNA levels in B6 mice treated with anti-Ly49H and co-infected with C4A and C4C. Note both C4A and C4C DNA were detectable following anti-Ly49H treatment. <b>C</b>. Viral DNA levels in B6 mice treated with isotype control mAb and co-infected with C4A and C4C. Note competition was retained in these mice with C4A undetectable in the salivary glands. The x-axis represents the limit of detection for plaque assays (100 pfu/g of tissue) and the multiplex qPCR (1×10⁴ copy number/g of tissue), n = 5 co-infected mice/group, 3 single strain infected mice/group.</p