This study was undertaken to examine the genetic and viral target specificity of the cell-mediated immune response to Pichinde virus (PV) in mice, to ultimately facilitate the cloning of anti-PV cytotoxic T lymphocytes (CTL). Cloned populations provide a system for the study of the generation, function, and mechanism of action of CTL in viral infections. CTL are often genetically restricted to either the K or D end of H-2 Class I. Cytotoxicity assays performed using primary immunized murine splenocytes showed predominant restriction to the K locus in H-2ᵇ C57B1/6 mice. In limiting dilution assays, a significant population of anti-PV CTL (approximately one-half) were regulated by H-2Kᵇ. A panel of temperature sensitive (ts) mutants of wild type (wt) PV was used to study the viral target specificity of anti-PV CTL. These mutants were grouped according to surface expression and internal production of viral molecules as detected by indirect immunofluorescence and polyacrylamide gel electrophoresis respectively. Cytotoxicity assays with target cells infected with these mutants provided equivocal results regarding whether the major target antigen was an internally or externally expressed viral protein. The results suggest that surface expression of viral proteins may be related to target specificity in the anti-PV cytotoxic response. One temperature sensitive mutant, ts 488, provided a superior target for H-2ᵇ effectors than wt PV when used to infect 5R (H-2Kᵇ compatible) target cells. Limiting dilution studies indicated that anti-ts 488 CTL were predominantly regulated by H-2Kᵇ: approximately three-quarters of the CTL precursors were restricted to that end. Furthermore, limiting dilution assays indicated that ts 488 and wt PV were not fully cross reactive in generating lytic effectors. As a result, the mutant might not facilitate the generation of cloned lines of anti-wt PV CTL. The mutant and its augmented regulation by H-2Kᵇ may provide a novel system for the examination of the association and recognition of viral plus "self" antigens at the level of antigen presentation and target identification. / Thesis / Master of Science (MS)

Mouse monoclonal antibodies directed against Pichinde virus (PCV) were produced to evaluate their application as vehicles for the delivery of antiviral drugs or toxins to virus-infected cells. Four monoclonal antibodies, PC4.9A6, PC4.9D3, PC4.7C2, and PC4.8D3, were of the IgG2a subisotype and reacted with acetone-fixed and live PCV-infected Vero-76 cells. In vivo stained splenic macrophages derived from PCV-infected hamsters that had been injected with fluorescein-labeled PC4.9A6 (FITC9A6) demonstrated a 400% increase in total fluorescence over similarly treated, non-infected cells when analyzed by flow cytometry. This is an indication that FITC-9A6 does have some ability to specifically target PCV infected cells in vivo. Radioimmunoprecipitation of viral proteins showed that all the antibodies precipitated two different PCV proteins, one of 64,000 daltons and another of 38,000 daltons. These proteins are, respectively, PCV ix nucleoprotein (NP) and a breakdown product of NP that is present in PCV infected cells. An immunofluorescent assay (IFA) for PCV was developed. This IFA was used for antiviral drug assays against PCV. The assay was performed by adding fluorescein-labeled anti-PCV monoclonal antibody to fixed, virus infected cells at 24 h after infection and counting the fluorescent cells. The 50% effective dose (EDso) for ribavirin against PCV using this IFA was 6.0 IJ. g/ml. The EDso of ribavirin using inhibition of marginal PCV cytopathogenic effect after 12 days was 6.0 IJ. g/ml and using plaque reduction after 5 days is 2.5 IJ. g/ml, indicating that this IFA was of comparable sensitivity. An immunotoxin (IT) was produced by the conjugation of gelonin to PC4.9A6. This IT was tested in vivo in PCV-infected MHA hamsters. It was not active against the disease at the dosage tested and by the intraperitoneal (i.p.) treatment route employed in this study. The positive control, ribavirin, administered i.p. for 14 days at a dosage of 40 mg/ ml significantly increased the number of survivors. Three of 5 IT toxicity control animals developed some humoral response that inhibited PC4.9A6 binding to infected cells. They did not show any humoral response to the gelonin moiety of the IT.

Previously, our lab demonstrated that heterologous immunity could result in either gain or loss of protective immunity and alteration in immune pathology following infection by a second un-related pathogen. One of the prototypical models to study T cell-mediated heterologous immunity involves two distantly related arenaviruses, namely lymphocytic choriomeningitis virus (LCMV) and Pichinde virus (PV). Each virus encodes a cross-reactive CD8 epitope that has six out of eight in amino acid (aa) similarity with respect to its counterpart at the position 205-212 of the nucleoprotein (NP205). Heterologous challenge between LCMV and PV results in 1) expansion of the cross-reactive NP205-specific CD8 T cell responses and alteration of the immunodominance hierarchy and 2) partial protective immunity (heterologous immunity). Our lab showed that cross-reactive NP205-specific CD8 T cell receptor (TCR) repertoires become extremely narrowed following a heterologous challenge between LCMV and PV. Therefore, I questioned if LCMV NP205 epitope escape variants could be isolated during a dominant but narrowed crossVI reactive NP205-specific CTL response. In the first part of my thesis, I describe the isolation of a LCMV NP-V207A CTL escape variant in vivo using PV-immune animals challenged with LCMV clone 13. The LCMV NP-V207A variant contains a point mutation, which results in the switching of valine to alanine at the third non-anchoring residue of the LCMV NP205 CD8 epitope. Immunization of mice with the LCMV NP-V207A variant results in a significantly diminished cross-reactive NP205-specific CD8 T cell response. This suggests that the point mutation is responsible for the loss in the immunogenicity of the LCMV NP205 CD8 epitope. In addition, an in vitrorescued(r) recombinant LCMV variant (r/V207A) that encodes the original mutation also induces a highly diminished cross-reactive NP205-specific CD8 T cell response in mice. In agreement with the result obtained from the intracellular cytokine assays (ICS), MHC-Ig dimers loaded with the LCMV NP205 (V-A) peptide could only detect a minute population of cross-reactive NP205-specific CD8 T cells in mice infected with r/V207A variant virus. All the data indicate that the point mutation results in a significant loss in immunogenicity of the LCMV NP205 CD8 epitope. So far, no direct link between the cross-reactive NP205-specific CD8 T cells and heterologous immunity had been established in this system. Therefore, we immunized mice with either LCMV WT or the LCMV NP-V207A variant virus and showed that a significant loss of heterologous immunity is associated with the group immunized with LCMV NP-V207A variant virus. Again, r/V207Aimmune animals also displayed a significant loss in heterologous immunity following PV challenge. This suggests that the cross-reactive NP205-specific CD8 T cells mediate the majority of heterologous immunity between LCMV and PV in vivo. In comparison to the PV-immune control group, PV clearance kinetics mediated by the cross-reactive NP205-specific CD8 T cells were significantly delayed. Finally, these data also suggest that bystander activation plays very little role in heterologous immunity between LCMV and PV. Many studies in murine systems and humans suggest that cross-reactive T cells are often associated with immune pathology. We showed that in mice that were sequentially immunized with PV and LCMV (PV+LCMV WT double immune mice), there was a development of a high incidence and high level of immune pathology known as acute fatty necrosis (AFN) following a final PV challenge. The data suggest that these cross-reactive NP205-specific CD8 T cells might play an important role in immune pathogenesis. Therefore, we asked if the cross-reactive NP205-specific CD8 T cells play a role in immune pathogenesis by comparing the incidence of AFN between the (PV+LCMV WT) and the (PV+LCMV NP-V207A) double immune mice following a final PV challenge. In agreement with our hypothesis, the result showed the (PV+LCMV NP-V207A) double immune mice developed a significantly lower incidence of AFN compared to the (PV+LCMV WT) double immune mice. However, linear correlation studies comparing the frequency of different antigen-specific CD8 T cell populations within the (PV+LCMV WT) double immune mice before challenge and the severity of AFN following the PV challenge suggest that two opposing antigen-specific CD8 T cell populations are involved in determining the final outcome of the immune pathology. The PV NP38-45-specific CD8 T cell response (PV NP38) appears to be more protective than the cross-reactive NP205-specific CD8 T cell response. In addition, a positive linear correlation between the ratio of cross-reactive NP205 to PV NP38 and the severity of AFN seem to suggest that these cross-reactive populations are important contributors to immune pathogenesis. Peptide titration studies examining the functional avidities to different antigenic specificities suggest that both populations consist of high avidity TCR and peptide MHC (TCR:pMHC) interactions. However, skewing within the cross-reactive NP205 specific CD8 T cell response towards the LCMV NP205 epitope response in one of the (PV+LCMV WT) double immune mice suggests that cross-reactive NP205 specific CD8 T cells could constitute a sub-optimal response to a PV challenge. In summary, I questioned what might be some of the immunological consequences of heterologous immunity in this model. First of all, we have established a direct link between the cross-reactive NP205-specific CD8 T cell response and heterologous immunity in LCMV and PV. Second of all, I demonstrated that a LCMV NP205 epitope escape variant could be selected in vivo under the conditions of heterologous immunity. In addition, I showed that PV clearance kinetic was significantly delayed in cross-reactive NP205-mediated heterologous immunity as compared to homologous challenge. Finally, we demonstrated that cross-reactive NP205-specific CD8 T cells could play an important role in immune pathogenesis in this model. However, correlation data indicate that two opposing antigen-specific CD8 T cell populations could ultimately decide the outcome and magnitude of immune pathology in each individual mouse. All the data presented above strongly suggest that the cross-reactive NP205 CD8 T cells play a crucial role in immune pathology in this model system by 1) interfering with the regular establishment of immunodominance hierarchy orders, or 2) exhibiting a sub-optimal protective immunity due to the nature of the cross-reactive epitope.