Development of TMEV VP2_121-130 mutant viruses.

<p>(A) Real-time RT-PCR expression analysis of viral VP2 and plasmid neomycin phosphotransferase from BHK cells transfected with TMEV VP2_121-130 mutant cDNA. (B) Western blot analysis of whole cell lysates and viral supernatants for TMEV viral proteins from cells transfected with wild-type TMEV-DA cDNA for 3 and 7 days. (C) Western blot of whole cell lysates and supernatants from cells transfected with TMEV VP2_121-130 mutant plasmid cDNA (D) Western blot of supernatants derived from cells transfected with TMEV VP2₁₂₅ mutant plasmid cDNA.</p

Altered VP2_121-130 codon structure does not influence virus fidelity.

<p>(A) Thirteen silent nucleotide substitutions representing changes to 9 of the 10 codons of VP2_121-130 were introduced into the TMEV-DA plasmid cDNA by site-directed mutagenesis. (B) Sequence verification of codon alternate VP2_121-130 virus recovered from infected BHK cells. (C) Western blot analysis of virus supernatant recovered from cells infected with codon alternate TMEV-VP2_121-130.</p

CD8+ T-cell response and virus RNA levels in resistant and susceptible mice after intracranial infection with TMEV-wt, S125A and M130L viruses.

<p>(A) CD45+ cells isolated from the central nervous system of VP2 mutant infected C57BL/6 mice were analyzed for the presence of CD8+ cells. (^a significant compared to M130L by ANOVA, * significant compared to TMEV-wt by ANOVA). Figure is an experiment replicated 3 times with 3-5 animals per group. (B) CNS infiltrating lymphocytes were stained with H-2D^b/VP2₁₂₁ or H-2D^b/E7₄₉ tetramers and analyzed by flow cytometry to determine the percent of the CD8+ T-cell population that is positive for the immunodominant VP2₁₂₁ epitope (* significant compared to S125A and M130L by ANOVA). A representative example of 2 experiments using 3-5 animals per group. (C) Semi-quantitative RT-PCR analysis of RNA isolated by C57BL/6 mice infected with TMEV-wt, S125A or M130L mutants (n = 3/group). No significant differences in viral transcripts were detected between the groups. IgG specific responses to wild-type TMEV in C57BL/6 mice infected for 30 days. No significant differences were detected by ELISA between S125, M130L or pciDA (wild-type TMEV) (D) The same RT-PCR analysis in C on the susceptible strain FVB (* significant compared to TMEV-wild type by ANOVA, n = 5/group). TMEV-specific IgG response and antibody neutralization to wild-type TMEV in FVB mice infected for 30 days. No significant differences were detected by ELISA or neutralization assay between S125, M130L or pciDA (wild-type TMEV).</p

Peptide depletion reveals an overlap in the wild-type VP2_121-130 CD8+ T cell response after infection with S125A and M130L mutant viruses.

<p>(A) Mice were pre-depleted with control E7₄₉, wild-type VP2_121-130, VP2-S125A and VP2-M130L peptide prior to intracranial infection with wild-type virus. CNS infiltrating lymphocytes were analyzed by flow cytometry to determine the percentage and number of CD8+ T-cells specific for the immunodominant epitope after peptide depletion. Data are representative examples of three individual mice per group. (B) Percent and absolute number of H-2D^b/VP2₁₂₁+ CD8 T-cells derived from infected CNS tissue including a no peptide group (* significant by ANOVA).</p

VP2_121-130 mutants.

<p>VP2_121-130 mutants.</p

I-Mutant predicted ΔΔG values for TMEV VP2_121-130 amino acid substitutions.

<p>I-Mutant predicted ΔΔG values for TMEV VP2_121-130 amino acid substitutions.</p

Generation of FVB K^bα1α2D^b transgenic mice.

<p>(A) Segments of H-2K^b EcoRI and H-2D^b HindIII were used to generate a chimeric genomic construct with an H-2D^b Sal I/XbaI fragment on an H-2K^b backbone. (B) Expression of the construct yields a chimeric MHC class I molecule composed of the α1α2 domain from H-2D^b and the α3 domain from H-2K^b. (C) Verification of H-2D^b and K^bα1α2D^b transgene expression in 293T cells by flow cytometry. Data are mean fluorescence intensity of phycoerythrin labeled cells.</p

Surface expression of H-2 transgenes is regulated by genomic elements outside of sequences encoding peptide binding domains.

<p>(A) The mean fluorescence intensity (MFI) for peripheral blood lymphocytes stained with the H-2D^b specific antibody B22.249 from H-2^b haplotype mice (white) and transgenic mice (gray); (* Line 1 v. B10 or FVB D^b, p<0.05, ^a Line 2 v. B10 or FVB D^b, p<0.05). (B) Splenocytes from mice infected with TMEV for 21 days were isolated and tested by flow cytometry for H-2D^b expression levels using B22.249.R1 labeled with FITC. Data are expressed as the MFI of the FITC labeled cells (* p<0.05, Two-way ANOVA). (C) The same splenocytes in (B) were co-stained with AF6-88.5 labeled phycoerythrin to determined expression of the H-2K^b present on the chimeric K^bα1α2D^b molecule. Data are expressed as MFI of PE labeled cells (* p<0.001, Two-way ANOVA). (D) Brain resident cells from FVB, FVB D^b and FVB K^bα1α2D^b mice were isolated from naïve mice and analyzed by flow cytometry. Side-scatter and forward-scatter were analyzed for the presence of mononuclear resident cells using a lymphocyte gate. Resident antigen-presenting cells were further analyzed by a CD45 mid-level staining gate and assessed for expression of MHC class I. FVB (green), FVB D^b (black) and FVB K^bα1α2D^b (blue) brain cells were assessed for H-2D^b expression by FACS (* p<0.001, t-test). (E) Brain cells isolated from 6 day TMEV infected mice were gated as in (D) and assessed for changes in H-2D^b expression by FACS. Data are the MFI for H-2D^b-FITC.</p

Spinal cord demyelination and persistent virus infection in FVB K^bα1α2D^b transgenic mice.

<p>(A) Transgenic expression of H-2K^b fails to protect from TMEV induced demyelination, similar to non-transgenic control (B). (C) Expression of H-2D^b transgene protects FVB mice from TMEV induced demyelination present in littermate controls (D). (E) Expression of a chimeric K^bα1α2D^b molecule fails to protect from demyelination, similar to non-transgenic (F). (G) Relative TMEV RNA levels in spinal cords from transgenic mice infected for 21 days (* p<0.05 by ANOVA). (H) Relative TMEV RNA levels in the brains of transgenic mice after 24 days of infection.</p

Spinal cord pathology.

<p>Overall Chi Square analysis (P<0.001).</p>a<p>Fisher Exact Test NLM VS H-2K^b (P = 0.577).</p>b<p>Fisher Exact Test H-2D^b vs K^b/D^bα1α2 (P = 0.0198).</p>c<p>Fisher Exact Test NLM/H-2K^b vs K^b/D^bα1α2 (P = 0.0105).</p><p>30% TMEV resistance attributed to peptide presentation.</p><p>70% TMEV resistance attributed to differences between K and D loci.</p