MHC Class II–Alpha Chain Knockout Mice Support Increased Viral Replication That Is Independent of Their Lack of MHC Class II Cell Surface Expression and Associated Immune Function Deficiencies

MHCII molecules are heterodimeric cell surface proteins composed of an α and β chain. These molecules are almost exclusively expressed on thymic epithelium and antigen presenting cells (APCs) and play a central role in the development and function of CD4 T cells. Various MHC-II knockout mice have been generated including MHC-IIAα(-/-) (I-Aα(-/-)), MHC-IIAβ(-/-) (I-β(-/-)) and the double knockout (I-Aαxβ(-/-)). Here we report a very striking observation, namely that alphaviruses including the avirulent strain of Semliki Forest virus (aSFV), which causes asymptomatic infection in wild-type C57BL6/J (B6) mice, causes a very acute and lethal infection in I-Aα(-/-), but not in I-β(-/-) or I-Aαxβ(-/-), mice. This susceptibility to aSFV is associated with high virus titres in muscle, spleen, liver, and brain compared to B6 mice. In addition, I-Aα(-/-) mice show intact IFN-I responses in terms of IFN-I serum levels and IFN-I receptor expression and function. Radiation bone marrow chimeras of B6 mice reconstituted with I-Aα(-/-) bone marrow expressed B6 phenotype, whereas radiation chimeras of I-Aα(-/-) mice reconstituted with B6 bone marrow expressed the phenotype of high viral susceptibility. Virus replication experiments both in vivo and in vitro showed enhanced virus growth in tissues and cell cultures derived form I-Aα(-/-) compared to B6 mice. This enhanced virus replication is evident for other alpha-, flavi- and poxviruses and may be of great benefit to producers of viral vaccines. In conclusion, I-Aα(-/-) mice exhibit a striking susceptibility to virus infections independent of their defective MHC-II expression. Detailed genetic analysis will be carried out to characterise the underlining genetic defects responsible for the observed phenomenon.This work was supported by institutional research support to Prof Mullbacher laboratory at the John Curtin School of Medical Research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Intranasal Flu Vaccine Protective against Seasonal and H5N1 Avian Influenza Infections

Background Influenza A (flu) virus causes significant morbidity and mortality worldwide, and current vaccines require annual updating to protect against the rapidly arising antigenic variations due to antigenic shift and drift. In fact, current subunit or split flu vaccines rely exclusively on antibody responses for protection and do not induce cytotoxic T (Tc) cell responses, which are broadly cross-reactive between virus strains. We have previously reported that γ-ray inactivated flu virus can induce cross-reactive Tc cell responses. Methodology/Principal Finding Here, we report that intranasal administration of purified γ-ray inactivated human influenza A virus preparations (γ-Flu) effectively induces heterotypic and cross-protective immunity. A single intranasal administration of γ-A/PR8[H1N1] protects mice against lethal H5N1 and other heterotypic infections. Conclusions/Significance Intranasal γ-Flu represents a unique approach for a cross-protective vaccine against both seasonal as well as possible future pandemic influenza A virus infections.Mohammed Alsharifi, Yoichi Furuya, Timothy R. Bowden, Mario Lobigs, Aulikki Koskinen, Matthias Regner, Lee Trinidad, David B. Boyle and Arno Müllbache

Gamma-Irradiated Influenza Virus Uniquely Induces IFN-I Mediated Lymphocyte Activation Independent of the TLR7/MyD88 Pathway

Background: We have shown previously in mice, that infection with live viruses, including influenza/A and Semliki Forest virus (SFV), induces systemic partial activation of lymphocytes, characterized by cell surface expression of CD69 and CD86, but not CD25. This partial lymphocytes activation is mediated by type-I interferons (IFN-I). Importantly, we have shown that c-irradiated SFV does not induce IFN-I and the associated lymphocyte activation. Principal Findings: Here we report that, in contrast to SFV, c-irradiated influenza A virus elicits partial lymphocyte activation in vivo. Furthermore, we show that when using influenza viruses inactivated by a variety of methods (UV, ionising radiation and formalin treatment), as well as commercially available influenza vaccines, only c-irradiated influenza virus is able to trigger IFN-I-dependent partial lymphocyte activation in the absence of the TLR7/MyD88 signalling pathways. Conclusions: Our data suggest an important mechanism for the recognition of c-irradiated influenza vaccine by cytosolic receptors, which correspond with the ability of c-irradiated influenza virus to induce cross-reactive and cross-protective cytotoxic T cell responses.Yoichi Furuya, Jennifer Chan, En-Chi Wan, Aulikki Koskinen, Kerrilyn R. Diener, John D. Hayball, Matthias Regner, Arno Müllbacher, Mohammed Alsharif

Residual active granzyme B in cathepsin C–null lymphocytes is sufficient for perforin-dependent target cell apoptosis

Cathepsin C activates serine proteases expressed in hematopoietic cells by cleaving an N-terminal dipeptide from the proenzyme upon granule packaging. The lymphocytes of cathepsin C–null mice are therefore proposed to totally lack granzyme B activity and perforin-dependent cytotoxicity. Surprisingly, we show, using live cell microscopy and other methodologies, that cells targeted by allogenic CD8+ cytotoxic T lymphocyte (CTL) raised in cathepsin C–null mice die through perforin-dependent apoptosis indistinguishable from that induced by wild-type CTL. The cathepsin C–null CTL expressed reduced but still appreciable granzyme B activity, but minimal granzyme A activity. Also, in contrast to mice with inactivation of both their granzyme A/B genes, cathepsin C deficiency did not confer susceptibility to ectromelia virus infection in vivo. Overall, our results indicate that although cathepsin C clearly generates the majority of granzyme B activity, some is still generated in its absence, pointing to alternative mechanisms for granzyme B processing and activation. Cathepsin C deficiency also results in considerably milder immune deficiency than perforin or granzyme A/B deficiency

Intrinsic Defect in T Cell Production of Interleukin (IL)-13 in the Absence of Both IL-5 and Eotaxin Precludes the Development of Eosinophilia and Airways Hyperreactivity in Experimental Asthma

Interleukin (IL)-5 and IL-13 are thought to play key roles in the pathogenesis of asthma. Although both cytokines use eotaxin to regulate eosinophilia, IL-13 is thought to operate a separate pathway to IL-5 to induce airways hyperreactivity (AHR) in the allergic lung. However, identification of the key pathway(s) used by IL-5 and IL-13 in the disease process is confounded by the failure of anti–IL-5 or anti–IL-13 treatments to completely inhibit the accumulation of eosinophils in lung tissue. By using mice deficient in both IL-5 and eotaxin (IL-5/eotaxin−/−) we have abolished tissue eosinophilia and the induction of AHR in the allergic lung. Notably, in mice deficient in IL-5/eotaxin the ability of CD4+ T helper cell (Th)2 lymphocytes to produce IL-13, a critical regulator of airways smooth muscle constriction and obstruction, was significantly impaired. Moreover, the transfer of eosinophils to IL-5/eotaxin−/− mice overcame the intrinsic defect in T cell IL-13 production. Thus, factors produced by eosinophils may either directly or indirectly modulate the production of IL-13 during Th2 cell development. Our data show that IL-5 and eotaxin intrinsically modulate IL-13 production from Th2 cells and that these signaling systems are not necessarily independent effector pathways and may also be integrated to regulate aspects of allergic disease

Caspase-Dependent Inhibition of Mousepox Replication by gzmB

BACKGROUND: Ectromelia virus is a natural mouse pathogen, causing mousepox. The cytotoxic T (Tc) cell granule serine-protease, granzyme B, is important for its control, but the underlying mechanism is unknown. Using ex vivo virus immune Tc cells, we have previously shown that granzyme B is able to activate several independent pro-apoptotic pathways, including those mediated by Bid/Bak/Bax and caspases-3/-7, in target cells pulsed with Tc cell determinants. METHODS AND FINDINGS: Here we analysed the physiological relevance of those pro-apoptotic pathways in ectromelia infection, by incubating ectromelia-immune ex vivo Tc cells from granzyme A deficient (GzmB(+) Tc cells) or granzyme A and granzyme B deficient (GzmAxB(-/-) Tc cell) mice with ectromelia-infected target cells. We found that gzmB-induced apoptosis was totally blocked in ectromelia infected or peptide pulsed cells lacking caspases-3/-7. However ectromelia inhibited only partially apoptosis in cells deficient for Bid/Bak/Bax and not at all when both pathways were operative suggesting that the virus is able to interfere with apoptosis induced by gzmB in case not all pathways are activated. Importantly, inhibition of viral replication in vitro, as seen with wild type cells, was not affected by the lack of Bid/Bak/Bax but was significantly reduced in caspase-3/-7-deficient cells. Both caspase dependent processes were strictly dependent on gzmB, since Tc cells, lacking both gzms, neither induced apoptosis nor reduced viral titers. SIGNIFICANCE: Out findings present the first evidence on the biological importance of the independent gzmB-inducible pro-apoptotic pathways in a physiological relevant virus infection model

MHC class II-alpha chain knockout mice support increased viral replication that is independent of their lack of MHC class II cell surface expression and associated immune function deficiencies

MHCII molecules are heterodimeric cell surface proteins composed of an α and β chain. These molecules are almost exclusively expressed on thymic epithelium and antigen presenting cells (APCs) and play a central role in the development and function of CD4 T cells. Various MHC-II knockout mice have been generated including MHC-IIAα-/- (I-Aα-/-), MHC-IIAβ-/- (I-β-/-) and the double knockout (I-Aαxβ-/-). Here we report a very striking observation, namely that alphaviruses including the avirulent strain of Semliki Forest virus (aSFV), which causes asymptomatic infection in wild-type C57BL6/J (B6) mice, causes a very acute and lethal infection in I-Aα-/-, but not in I-β-/- or I-Aαxβ-/-, mice. This susceptibility to aSFV is associated with high virus titres in muscle, spleen, liver, and brain compared to B6 mice. In addition, I-Aα-/- mice show intact IFN-I responses in terms of IFN-I serum levels and IFN-I receptor expression and function. Radiation bone marrow chimeras of B6 mice reconstituted with I-Aα-/- bone marrow expressed B6 phenotype, whereas radiation chimeras of I-Aα-/- mice reconstituted with B6 bone marrow expressed the phenotype of high viral susceptibility. Virus replication experiments both in vivo and in vitro showed enhanced virus growth in tissues and cell cultures derived form I-Aα-/- compared to B6 mice. This enhanced virus replication is evident for other alpha-, flavi- and poxviruses and may be of great benefit to producers of viral vaccines. In conclusion, I-Aα-/- mice exhibit a striking susceptibility to virus infections independent of their defective MHC-II expression. Detailed genetic analysis will be carried out to characterise the underlining genetic defects responsible for the observed phenomenon.Mohammed Alsharifi, Aulikki Koskinen, Danushka K. Wijesundara, Jayaram Bettadapura, Arno Müllbache

CD8+ T cell-mediated immune responses in West Nile virus (Sarafend strain) encephalitis are independent of gamma interferon

The flavivirus West Nile virus (WNV) can cause fatal encephalitis in humans and mice. It has recently been demonstrated, in an experimental model using WNV strain Sarafend and C57BL/6 mice, that both virus- and immune-mediated pathology is involved in WNV encephalitis, with CD8+ T cells being the dominant subpopulation of lymphocyte infiltrates in the brain. Here, the role of activated WNV-immune CD8+ T cells in mouse WNV encephalitis was investigated further. Passive transfer of WNV-immune CD8+ T cells reduced mortality significantly and prolonged survival times of mice infected with WNV. Early infiltration of WNV-immune CD8+ T cells into infected brains is shown, suggesting a beneficial contribution of these lymphocytes to recovery from encephalitis. This antiviral function was not markedly mediated by gamma interferon (IFN-γ), as a deficiency in IFN-γ did not affect mortality to two strains of WNV (Sarafend and Kunjin) or brain virus titres significantly. The cytolytic potential, as well as precursor frequency, of WNV-immune CD8+ T cells were not altered by the absence of IFN-γ. This was reflected in transfer experiments of WNV-immune CD8+ T cells from IFN-γ-/- mice into WNV-infected wild-type mice, which showed that IFN-γ-deficient T cells were as effective as those from WNV-immune wild-type mice in ameliorating disease outcome. It is speculated here that one of the pleiotropic functions of IFN-γ is mimicked by WNV-Sarafend-mediated upregulation of cell-surface expression of major histocompatibility complex antigens, which may explain the lack of phenotype of IFN-γ-/- mice in response to WNV

Restricted Semliki Forest virus replication inperforin and Fas-ligand double-deficient mice

Previously, we have shown that mice defective in granule exocytosis and/ or Fas.L/Fas-mediated cytolytic pathways are significantly more resistant to alphavirus, Semliki Forest virus (SFV), infection compared with wild-type mice. Here, we evaluated SFV replication in different tissues of mice defective in both cytolytic pathways (perf-/-xg/d) relative to that in wild-type counterparts and found that viral replication in perf-/-xg/d mice is remarkably restricted. Although the mechanism responsible for this observation is yet to be established, the lower virus titres found in these mice indicate that the role of cytolytic effector molecules in antiviral immunity needs to be re-evaluated