Wild type measles virus attenuation independent of type I IFN

<p>Abstract</p> <p>Background</p> <p>Measles virus attenuation has been historically performed by adaptation to cell culture. The current dogma is that attenuated virus strains induce more type I IFN and are more resistant to IFN-induced protection than wild type (wt).</p> <p>Results</p> <p>The adaptation of a measles virus isolate (G954-PBL) by 13 passages in Vero cells induced a strong attenuation of this strain in vivo. The adapted virus (G954-V13) differs from its parental strain by only 5 amino acids (4 in P/V/C and 1 in the M gene). While a vaccine strain, Edmonston Zagreb, could replicate equally well in various primate cells, both G954 strains exhibited restriction to the specific cell type used initially for their propagation. Surprisingly, we observed that both G954 strains induced type I IFN, the wt strain inducing even more than the attenuated ones, particularly in human plasmacytoid Dendritic Cells. Type I IFN-induced protection from the infection of both G954 strains depended on the cell type analyzed, being less efficient in the cells used to grow the viral strain.</p> <p>Conclusion</p> <p>Thus, mutations in M and P/V/C proteins can critically affect MV pathogenicity, cellular tropism and lead to virus attenuation without interfering with the α/β IFN system.</p

Interplay between virus-specific effector response and Foxp3 regulatory T cells in measles virus immunopathogenesis.

Measles is a highly contagious childhood disease associated with an immunological paradox: although a strong virus-specific immune response results in virus clearance and the establishment of a life-long immunity, measles infection is followed by an acute and profound immunosuppression leading to an increased susceptibility to secondary infections and high infant mortality. In certain cases, measles is followed by fatal neurological complications. To elucidate measles immunopathology, we have analyzed the immune response to measles virus in mice transgenic for the measles virus receptor, human CD150. These animals are highly susceptible to intranasal infection with wild-type measles strains. Similarly to what has been observed in children with measles, infection of suckling transgenic mice leads to a robust activation of both T and B lymphocytes, generation of virus-specific cytotoxic T cells and antibody responses. Interestingly, Foxp3(+)CD25(+)CD4(+) regulatory T cells are highly enriched following infection, both in the periphery and in the brain, where the virus intensively replicates. Although specific anti-viral responses develop in spite of increased frequency of regulatory T cells, the capability of T lymphocytes to respond to virus-unrelated antigens was strongly suppressed. Infected adult CD150 transgenic mice crossed in an interferon receptor type I-deficient background develop generalized immunosuppression with an increased frequency of CD4(+)CD25(+)Foxp3(+) T cells and strong reduction of the hypersensitivity response. These results show that measles virus affects regulatory T-cell homeostasis and suggest that an interplay between virus-specific effector responses and regulatory T cells plays an important role in measles immunopathogenesis. A better understanding of the balance between measles-induced effector and regulatory T cells, both in the periphery and in the brain, may be of critical importance in the design of novel approaches for the prevention and treatment of measles pathology

High Pathogenicity of Wild-Type Measles Virus Infection in CD150 (SLAM) Transgenic Mice

Measles virus (MV) infection causes an acute childhood disease, associated in certain cases with infection of the central nervous system and development of a severe neurological disease. We have generated transgenic mice ubiquitously expressing the human protein SLAM (signaling lymphocytic activation molecule), or CD150, recently identified as an MV receptor. In contrast to all other MV receptor transgenic models described so far, in these mice infection with wild-type MV strains is highly pathogenic. Intranasal infection of SLAM transgenic suckling mice leads to MV spread to different organs and the development of an acute neurological syndrome, characterized by lethargy, seizures, ataxia, weight loss, and death within 3 weeks. In addition, in this model, vaccine and wild-type MV strains can be distinguished by virulence. Furthermore, intracranial MV infection of adult transgenic mice generates a subclinical infection associated with a high titer of MV-specific antibodies in the serum. Finally, to analyze new antimeasles therapeutic approaches, we created a recombinant soluble form of SLAM and demonstrated its important antiviral activity both in vitro and in vivo. Taken together, our results show the high susceptibility of SLAM transgenic mice to MV-induced neurological disease and open new perspectives for the analysis of the implication of SLAM in the neuropathogenicity of other morbilliviruses, which also use this molecule as a receptor. Moreover, this transgenic model, in allowing a simple readout of the efficacy of an antiviral treatment, provides unique experimental means to test novel anti-MV preventive and therapeutic strategies

MV infection induces a specific antibody and cytotoxic response in CD150 transgenic mice.

<p>(A, B) Production of anti-MV nucleoprotein (N) antibodies (IgG) was measured in serum of individual mice by ELISA and the number of tested animals is indicated in parenthesis. Titers are expressed as relative units. (A) Mice were immunized with low titre of MV (200 PFU) and serum was collected on days 13 and 28, or (B) mice were immunized with higher dose of MV (10³ PFU) and serum was collected on day 13 post infection. (C) To analyze cellular anti-viral response, splenocytes were harvested and restimulated with target cells expressing MV N gene (P815-N) for one week (3 to 8 pooled mice per group). Cytotoxic activity was measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004948#s4" target="_blank">Methods</a>; results are expressed as the mean percentage of N-specific cytotoxic activity from duplicate cultures (+/−SD) and the data are from one representative experiment out of three. Cytotoxic activity of lymphocytes obtained from MV-infected CD150 mice was significantly higher compared to the other groups, (p<0.05, Mann-Whitney U test).</p

Characterization of Treg function following MV infection.

<p>(A) Analysis of suppressor activity of Tregs isolated from CD150 mice, inoculated with MV (open symbol) or with medium (full symbol), in cocultures with CD4⁺CD25⁻ effector T cells from either uninfected CD150 mice (left panel), or infected CD150 mice (right panel) in the presence of irradiated CD4⁺ T cell–depleted splenic APCs and Con A (3 to 5 pooled mice per group). Proliferation of Tregs from either control or CD150 infected mice in response to Con A was ∼300–600 cpm. The results are shown as the mean percentage of proliferation inhibition in triplicate cultures±SD. Results are representative of three different experiments. (B) Splenocytes isolated from either uninfected (left panel) or MV-infected (right panel) CD150 mice and their nontransgenic littermates (wild type) were stimulated with either irradiated Balb/c or C57Bl/6 splenocytes in MLR in triplicate cultures, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004948#s4" target="_blank">Methods</a>. Proliferation is expressed as mean proliferation index±SD and is representative from two independent experiments (* P<0,01, Student t-test).</p

MV infection induces a strong activation of the immune response in CD150 transgenic mice.

<p>CD150 transgenic mice and nontransgenic littermates (control) were inoculated i.n. with MV or medium (uninfected). Spleens were harvested 13 dpi and cells were analyzed by flow cytometry. (A) Expression of MV hemagglutinin (H) antigen on the surface of spleen cells from uninfected (grey line) and MV-infected (black line) CD150 transgenic mice, gated on CD19⁺ (B cells) or CD3⁺ (T cells) cells. Numbers are percentages of cells expressing MV H antigen in infected conditions. (B) Staining for CD4⁺ and CD8⁺ T cells and (C) CD19⁺ B cells, among analyzed splenocytes. Results are representative of 8 different experiments, each involving 3–6 mice. Differences between CD150 transgenic infected mice and the other groups were statistically significant (p<0.05, Student t-test).</p

T lymphocyte infiltration at the sites of MV-brain infection.

<p>Brain sections from suckling CD150-transgenic (A–E) and nontransgenic littermate mice (F) infected with MV were analyzed by immunohistofluorescence for MV nucleoprotein (N) localization (A–F, in red) and the presence of CD4⁺ (A, in green), CD8⁺ (B, in green) and Foxp3⁺ T cells (C–F, in green). Cell nuclei were counterstained with DAPI (in blue). Infiltrating T CD4⁺ and CD8⁺ lymphocytes were detected in brains from CD150 transgenic mice (A and B, respectively) at the sites of MV infection identified by a N-specific labelling (red dots) but not in their nontransgenic littermates (not shown). Images are shown at 40× original magnification and are representative of three to five mice per group.</p

MV-induced immunosuppression in adult transgenic mice.

<p>(A) Transgenic CD150×IFNα/βR KO mice were infected i.n. with MV at different ages and monitored for survival by Kaplan-Meier analysis. (B, C) Groups of 5 mice, 6 to 7 week-old, were infected i.n. with MV or left untreated. Splenocytes were harvested at 11 dpi and stained for CD4 and CD25 followed by anti-Foxp3 intracellular staining and analyzed by flow cytometry. Results are presented: (B) as the percentage of CD25⁺Foxp3⁺ cells within CD4⁺ compartment, for each analyzed animal and (C) as a percentage of CD25⁺ cells within the CD4⁺Foxp3⁺ population. Horizontal bars correspond to mean values. (D) Groups of 10 mice, 6 to 7 weeks old, were infected i.n. with MV or left untreated. Seven days later, mice were sensitized with DNFB 0.5% on the ventral skin or left unsensibilized (unsens). All mice were challenged 5 days later with DNFB 0.1% on the left ear. Results are presented as the individual ear swelling of two independent experiments and horizontal bars correspond to mean values. (** p<0.01, * p<0.05, Mann-Whitney test).</p

MV infection increases the proportion of CD4⁺CD25⁺Foxp3⁺ Tregs.

<p>(A) Splenocytes from CD150 or nontransgenic mice (control), inoculated i.n. with either MV or medium (uninfected), were harvested 13 dpi and stained for CD4 and CD25 followed by anti-Foxp3 intracellular staining and analyzed by flow cytometry. (B, C) CD150×Foxp3-GFP transgenic mice and Foxp3-GFP littermates (control) were inoculated i.n. with MV. Brains were harvested 8 dpi and analyzed by flow cytometry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004948#s4" target="_blank">Methods</a>. (B) Proportion of infiltrating CD4⁺ and CD8⁺ T lymphocytes in the brain (two left panels); expression of the CD44 activation marker on CD4⁺ T lymphocytes (right panel, CD150 transgenic in red and nontransgenic control in blue). (C) Tregs detected by the co-expression of Foxp3 and CD25 or ICOS. Results are representative of 4 independent experiments, each involving 4–7 mice per group. Differences between infected and noninfecetd mice were statistically significant (p<0.05, Student t-test).</p