Immunobiology of cytotoxic T-cell resistant virus variants: studies on lymphocytic choriomeningitis virus (LCMV)

Replication of the genetically variable lymphocytic choriomeningitis virus (LCMV) gives rise to a pool of variant viruses. Under the selection pressure exerted by a strong but narrow repertoire of antiviral cytotoxic T-cells (CTL) i.e. monoclonal or polyclonal monoepitope, variant viruses emerge that contain point mutations in the nucleotide sequence encoding antigenic CTL epitopes; these variants can be selected in both infected mice and cell cultures. These mutations permit infected cells to escape CTL recognition by altering the ability of the mutant peptides to bind MHC class-I-molecules or by interfering with the ability of T-cell receptors to interact with the mutant peptide/MHC complex. Because viral infections often trigger a polyclonal repertoire of antiviral CTL to multiple epitopes, the likelihood of selection of CTL resistant variants is probably low, but not impossible. Our empirical observations suggest that antigenic variations, even if they only occur in a part of the available CTL epitope, may exert significant effects on the subtle biological equilibrium established between virus and host immune system. This can reduce immunological control of the pathogen population, and so permit persistence of viral infection and promote disease progression.     

Molecular and functional dissection of the H-2Db-restricted subdominant cytotoxic T-cell response to lymphocytic choriomeningitis virus

Infection of H-2b mice with lymphocytic choriomeningitis virus (LCMV) generates an H-2Db-restricted cytotoxic T-lymphocyte (CTL) response whose subdominant component is directed against the GP92-101 (CSANNSHHYI) epitope. The aim of this study was to identify the functional parameters accounting for this subdominance. We found that the two naturally occurring (genetically encoded and posttranslationally modified) forms of LCMV GP92-101 were immunogenic, did not act as T-cell antagonists, and bound efficiently to but were unable to form stable complexes with H-2Db, a crucial factor for immunodominance. Thus, the H-2Db-restricted subdominant CTL response to LCMV resulted not from altered T-cell activation but from impaired major histocompatibility complex presentation properties.     

Restricted V-segment usage in T-cell receptors from cytotoxic T lymphocytes specific for a major epitope of lymphocytic choriomeningitis virus.

Cytotoxic T lymphocytes (CTL) play an important role in recovery from a number of viral infections. They are also implicated in virus-induced immunopathology as best demonstrated in lymphocytic choriomeningitis virus (LCMV) infection of adult immunocompetent mice. In the present study, the structure of the T-cell receptor (TCR) in LCMV-specific CTL in C57BL/6 (B6) mice was investigated. Spleen T cells obtained from LCMV-infected mice were cultured in vitro with virus-infected stimulator cells and then stained with anti-TCR V beta antibodies. A skewing of V beta usage was noticeable in T cells enriched for their reactivity to LCMV, suggesting that particular V segments are important for the recognition of LCMV T-cell epitopes in B6 mice. To gain more detailed information on the structure of the TCR specific for LCMV epitopes, we studied CTL clones. It has been shown that approximately 90% of LCMV-reactive CTL clones generated in H-2b mice are specific for a short peptide fragment of the LCMV glycoprotein, residues 278 to 286, recognized in the context of the class I major histocompatibility complex molecule, Db. Four CTL clones possessing the specificity were randomly selected from a collection of clones, and their TCR genes were isolated by cDNA cloning or by the anchored polymerase chain reaction. All four clones were found to use V alpha gene segments belonging to the V alpha 4 subfamily. By RNA blot analysis, two more clones with the same specificity were also shown to express the V alpha 4 mRNA. In contrast, three different V beta gene segments were used among the four clones examined. J beta 2.1 was used by three of the clones. Although amino acid sequences in the V(D)J junctional regions were dissimilar, aspartic acid was found in the V alpha J alpha and/or V beta D beta J beta junctions of all four of these clones, suggesting that this residue is involved in binding the LCMV fragment. Restricted usage of V alpha and possibly J beta segments in the CTL response to a major T-cell epitope of LCMV raises the possibility that immunopathology in LCMV infection can be treated with antibodies directed against such TCR segments. Thus, similar analysis of the TCR in other virus infections is warranted and may lead to therapeutic strategies for immunopathology due to virus infections.     

Biology of cloned cytotoxic T lymphocytes specific for lymphocytic choriomeningitis virus. I. Generation and recognition of virus strains and H-2b mutants

Cytotoxic T lymphocytes (CTL) were induced in C57BL/6 and (C57BL/6 X DBA/2)F1 mice after immunization with the Armstrong strain of lymphocytic choriomeningitis virus (LCMV-Arm) and were cloned by limiting dilution in vitro. The cytotoxic activity of these clones was LCMV specific and H-2 restricted. All clones induced in C57BL/6 (H-2b) mice with LCMV-Arm lysed target cells infected with each of five distinct strains of LCMV (Arm, Traub , WE, Pasteur, and UBC ), suggesting recognition of common regions of viral proteins in association with H-2b molecules. In contrast, one clone obtained from (B6 X D2)F1 mice and restricted to the H-2d haplotype only lysed cells infected with one of three strains of virus (Arm, Traub , WE) but not two others (Pasteur, UBC ), suggesting recognition of variable regions of viral proteins in the context of H-2d molecules. To assess the fine specificity for H-2 molecules, we tested H-2Kb-restricted CTL clones for their ability to kill LCMV-infected target cells bearing mutations in their H-2Kb, and we tested clones presumed to be restricted to the H-2Db region for their ability to all LCMV targets cells bearing a mutation in the H-2Db region. Several different patterns of killing of the mutant targets were observed, indicating that a number of different epitopes on the H-2b molecules were used as restricting determinants for LCMV antigen recognition by CTL. Thus, cross-reactive viral determinants were recognized in the context of several different restricting determinants. Mutations in the N or C1 domains of the H-2 molecule affected recognition by a single LCMV specific CTL clone. One implication of this result is that CTL recognize a conformational determinant on the H-2 molecule formed by the association of virus antigen(s) with H-2. An alternate explanation is that one site on the H-2 molecule is involved in the interaction of viral antigens with H-2, whereas another may serve as a binding site for the CTL receptor.     

Viral escape from the neutralizing antibody response: the lymphocytic choriomeningitis virus model

In addition to CD8+ cytotoxic T lymphocyte (CTL) responses, neutralizing antibodies contribute substantially to the long-term immune control of noncytopathic viruses, as demonstrated during infection with the lymphocytic choriomeningitis virus (LCMV). The high virus load during the initial phase of an infection and the ability of this RNA virus to spontaneously acquire mutations are important prerequisites for escaping an ongoing immune response. In this context, LCMV escape from the humoral response by single point mutations in neutralizing envelope protein determinants may occur, particularly during conditions of CTL deficiency, leading to virus persistence.     

Biology of cloned cytotoxic T lymphocytes specific for lymphocytic choriomeningitis virus: clearance of virus in vivo.

Our data show that 1 X 10(7) to 1.5 X 10(7) lymphocytic choriomeningitis virus-specific, H-2-restricted cloned cytotoxic T lymphocytes (CTL) administered intravenously into acutely infected mice totally cleared virus from the spleens (10(4) to 10(5) PFU per spleen reduced to less than 50 PFU per spleen) by 24 h. This activity was genetically restricted in that cloned CTL could reduce titers of infectious virus in syngeneic C57BL/6 mice but not allogeneic BALB/c mice. Dose-response analysis indicated that at least 3 X 10(6) to 5 X 10(6) cloned CTL injected intravenously were needed to reduce significant amounts of infectious virus in the spleens. No infectious virus could be recovered from the spleens for at least 4 days after injection of cloned CTL. Hence, CTL play a major role in elimination of infectious virus from spleens during lymphocytic choriomeningitis virus infection. Our results also indicate that cloned CTL propagated in vitro for long periods of time can mediate a biologically relevant effect in vivo. These cells should be of considerable value in defining the precise manner in which CTL bring about control of viral infection, analyzing lymphocyte trafficking, and the potential use of cloned CTL in immunotherapy against viral disease.     

Molecular definition of a major cytotoxic T-lymphocyte epitope in the glycoprotein of lymphocytic choriomeningitis virus.

Analyses with segmental reassortants of lymphocytic choriomeningitis virus (LCMV) RNA have shown that cytotoxic T lymphocytes (CTL) are induced by and recognize proteins encoded by the viral short segment, which specifies two virus structural proteins, glycoprotein (GP) and nucleoprotein (NP). Expression of cDNA copies of these genes in vaccinia virus vectors demonstrates that C57BL/6 (H2bb) mice mount significant CTL responses to both GP and NP. We have used LCMV-specific H2bb-restricted CTL clones and a family of serial C-terminal truncations of the LCMV GP expressed in vaccinia virus to map the precise specificities of the anti-GP clones. Of the 18 CTL clones studied, 1 recognizes NP and the other 17 recognize GP. The reactivities of 14 of the 17 anti-GP CTL clones against the deleted GP molecules have been fully characterized, and two clear patterns of anti-GP activity have emerged, defining at least two CTL epitopes. The first epitope, recognized by only two of the clones, lies within GP residues 1 to 218. The second is recognized by all 12 of the remaining clones and was mapped, by using the GP deletions, to a 22-amino-acid region comprising GP residues 272 to 293. A synthetic peptide representing this area sensitized uninfected syngeneic target cells to lysis both by bulk CTL obtained from the spleen after a primary immunization and by appropriate CTL clones. Two sets of criteria are available which are said to identify potential T-cell epitopes, one based on primary amino acid sequence and the second based on protein secondary structure. Neither of these predictive schemes would have identified region 272 to 293 as a CTL recognition motif, indicating that such programs are of limited usefulness as presently conceived. Analysis of the CTL clones shows clearly that all three families (anti-NP and anti-GP 1 to 218 and 272 to 293) direct efficient cross-reactive killing against a variety of serologically distinct strains of LCMV.     

Cellular entry of lymphocytic choriomeningitis virus

In contrast to most enveloped viruses that enter the host cell via clathrin-dependent endocytosis, the Old World arenavirus lymphocytic choriomeningitis virus (LCMV) enters cells via noncoated vesicles that deliver the virus to endosomes, where pH-dependent membrane fusion occurs. Here, we investigated the initial steps of LCMV infection. We found that the attachment of LCMV to its cellular receptor α-dystroglycan occurs rapidly and is not dependent on membrane cholesterol. However, subsequent virus internalization is sensitive to cholesterol depletion, indicating the involvement of a cholesterol-dependent pathway. We provide evidence that LCMV entry involves an endocytotic pathway that is independent of clathrin and caveolin and that does not require the GTPase dynamin. In addition, neither the structural integrity nor the dynamics of the actin cytoskeleton are required for infection. These findings indicate that the prototypic Old World arenavirus LCMV uses a mechanism of entry that is different from clathrin-mediated endocytosis, which is used by the New World arenavirus Junin virus, and pathways used by other enveloped viruses.     

Comparison of cytotoxic T lymphocyte efficacy in acute and persistent lymphocytic choriomeningitis virus infection

Immune responses mediated by cytotoxic T lymphocytes (CTLs) have often been found to be functionally impaired in persistent infections. It is assumed that this impairment contributes to persistence of the infection. In this study, we compare the killing efficacy of CD8(+) T-cell responses in mice acutely and persistently infected with the lymphocytic choriomeningitis virus, using an in vivo CTL killing assay. To infer the killing efficacy of CTLs, we developed a new mathematical model describing the disappearance of peptide-pulsed cells from the blood of the mice over time. We estimate a lower half-life for peptide-pulsed cells in acute infection than in persistent infection, which indicates a higher killing efficacy of the CD8(+) T-cell response in acute infection. However, by controlling for the different levels of CTLs in acutely and persistently infected mice, we find that CTLs in persistent infection are only two times less efficacious than CTLs in acute infections. These results strongly suggest that the in vivo cytotoxicity of CD8(+) T-cell responses in persistent infection is modulated via the number of CTLs rather than their individual functionality.     

Determination of structural principles underlying three different modes of lymphocytic choriomeningitis virus escape from CTL recognition

Lymphocytic choriomeningitis virus infection of H-2(b) mice generates a strong CD8(+) CTL response mainly directed toward three immunodominant epitopes, one of which, gp33, is presented by both H-2D(b) and H-2K(b) MHC class I molecules. This CTL response acts as a selective agent for the emergence of viral escape variants. These variants generate altered peptide ligands (APLs) that, when presented by class I MHC molecules, antagonize CTL recognition and ultimately allow the virus to evade the cellular immune response. The emergence of APLs of the gp33 epitope is particularly advantageous for LCMV, as it allows viral escape in the context of both H-2D(b) and H-2K(b) MHC class I molecules. We have determined crystal structures of three different APLs of gp33 in complex with both H-2D(b) and H-2K(b). Comparison between these APL/MHC structures and those of the index gp33 peptide/MHC reveals the structural basis for three different strategies used by LCMV viral escape mutations: 1) conformational changes in peptide and MHC residues that are potential TCR contacts, 2) impairment of APL binding to the MHC peptide binding cleft, and 3) introduction of subtle changes at the TCR/pMHC interface, such as the removal of a single hydroxyl group.     

Persistent virus infection despite chronic cytotoxic T-lymphocyte activation in gamma interferon-deficient mice infected with lymphocytic choriomeningitis virus

The role of gamma interferon (IFN-gamma) in the permanent control of infection with a noncytopathic virus was studied by comparing immune responses in wild-type and IFN-gamma-deficient (IFN-gamma -/-) mice infected with a slowly invasive strain of lymphocytic choriomeningitis virus (LCMV Armstrong). While wild-type mice rapidly cleared the infection, IFN-gamma -/- mice became chronically infected. Virus persistence in the latter mice did not reflect failure to generate cytotoxic T-lymphocyte (CTL) effectors, as an unimpaired primary CTL response was observed. Furthermore, while ex vivo CTL activity gradually declined in wild-type mice, long-standing cytolytic activity was demonstrated in IFN-gamma -/- mice. The prolonged effector phase in infected IFN-gamma -/- mice was associated with elevated numbers of CD8(+) T cells. Moreover, a higher proportion of these cells retained an activated phenotype and was actively cycling. However, despite the increased CD8(+) T-cell turnover, which might have resulted in depletion of the memory CTL precursor pool, no evidence for exhaustion was observed. In fact, at 3 months postinfection we detected higher numbers of LCMV-specific CTL precursors in IFN-gamma -/- mice than in wild-type mice. These findings indicate that in the absence of IFN-gamma, CTLs cannot clear the infection and are kept permanently activated by the continuous presence of live virus, resulting in a delicate new balance between viral load and immunity. This interpretation of our findings is supported by mathematical modeling describing the effect of eliminating IFN-gamma-mediated antiviral activity on the dynamics between virus replication and CTL activity.     

Virus specificity of cytotoxic T lymphocytes generated during acute lymphocytic choriomeningitis virus infection: role of the H-2 region in determining cross-reactivity for different lymphocytic choriomeningitis virus strains

We have compared the relatedness of five different strains of lymphocytic choriomeningitis virus (LCMV) as assessed by LCMV-specific cytotoxic T lymphocytes (CTL). Several different mouse strains were injected with each of the five LCMV strains, and the cross-reactivity of virus-specific CTL generated during the acute infection was tested by killing on a panel of target cells infected with the various LCMV strains. We found that the cross-reactivity pattern of LCMV-specific CTL generated in mice of H-2d haplotype (BALB/c WEHI and DBA/2) was strikingly different from that in mice of H-2b haplotype (C57BL/6 and C3H.Sw/Sn), suggesting that the fine specificity of LCMV-specific CTL is a function of the H-2 region. The characteristic cross-reactivity patterns were also observed in (C57BL/6 X DBA/2)F1 mice, demonstrating that the repertoire of the H-2b- and H-2d-restricted LCMV-specific CTL is not changed as a result of complementation by gene products of the other major histocompatibility haplotype. Studies with congenic BALB.B10 and (BALB.B10 X BALB/c)F1 mice firmly established that the characteristic cross-reactivity patterns of LCMV-specific CTL map to the H-2 region and are not influenced by background genes outside the major histocompatibility locus. These results suggest that LCMV determinants seen in the context of H-2d-restricting elements are different from those seen in the context of H-2b-restricting elements. Moreover, our studies show that CTL can be used as probes for dissecting differences among various LCMV strains, but the degree of relatedness between the different LCMV strains is not absolute when measured by CTL recognition. Since the H-2 region regulates the fine specificity of CTL generated during LCMV infection in its natural host, the degree of cross-protective immunity developed during a viral infection apparently depends on the major histocompatibility haplotype. The importance of these findings lies in understanding susceptibility or resistance of various host populations to viral infections and in designing vaccination programs to provide immunity.     

Murine infection with lymphocytic choriomeningitis virus following gastric inoculation.

Laboratory studies of arenaviruses have been limited to parenteral routes of infection; however, recent epidemiological studies implicate virus ingestion as a natural route of infection. Accordingly, we developed a model for oral and gastric infection with lymphocytic choriomeningitis virus to enable studies of mucosal transmission and vaccination by this additional route.     

Characterization of ribonucleoproteins and ribosomes isolated from lymphocytic choriomeningitis virus.

Disruption of purified lymphocytic choriomeningitis (LCM) virus with Nonidet P-40 in 0.5 M KCl followed by sucrose gradient centrifugation in 0.3 M KCl led to the isolation of two viral nucleoproteins (RNPs) as well as 40S and 60S ribosomal subunits. The largest viral RNP sedimented heterogenously at 123S to 148S and was associated with 23S and 31S viral RNA. The other viral RNP sedimented at 83S and was associated with 23S viral RNA. The buoyant density in CsCl was determined to be 1.32 g/cm3 for the viral RNP. Densities of 1.52 and 1.60 g/cm3 were determined for the 40S and 60S subunits, similar to those of the BHK-21 cells subunits dissociated by 0.5 M KCl. The viral RNPs were partly sensitive to RNase.     

Anti-asialo GM1 eliminates both inflammatory process and cytotoxic T-cell function in the lymphocytic choriomeningitis adoptive transfer model

The induction of severe inflammatory process and fatal neurological disease by transfer of lymphocytic choriomeningitis virus (LCMV)-immune T cells into cyclophosphamide (Cy)-suppressed LCMV-infected mice is greatly inhibited by treatment of these recipients with antibody to the asialo GM1 ganglioside (anti-ASGM1). Examination of cytotoxic activity in lymphoid tissue of the Cy-suppressed recipients at 72 hr after cell transfer revealed that anti-ASGM1 treatment prevented the development of the cytotoxic T lymphocyte (CTL) response, even though the dose of antibody used did not significantly decrease CTL generation in unsuppressed mice. Abrogation of CTL activity was also observed following antibody treatment of NK-deficient (bg/bg) Cy-suppressed recipients, indicating that the anti-ASGM1 was unlikely to be operating via removal of NK cells that are in some way involved in the development of the CTL response. The possibility that anti-ASGM1 may act directly on T cells should be considered in all protocols involving the use of this reagent in immunosuppressed mice.     

Clearance of lymphocytic choriomeningitis virus in antibody- and B-cell-deprived mice.

The role of antibody in immune recovery from infection with lymphocytic choriomeningitis virus (LCMV) strain WE was evaluated in B-cell-depleted mice. Mice were treated from birth with either affinity-purified rabbit anti-mouse immunoglobulin M (IgM), normal rabbit immunoglobulin, or, alternatively, an affinity-purified monoclonal rat anti-mouse IgM antibody (LO-MM-9); untreated mice served as controls. B-cell depletion was considered complete in specifically treated mice according to the following criteria: absence of a significant response to the B-cell mitogen lipopolysaccharide, absence of B cells expressing immunoglobulin on their surfaces, absence of detectable IgM or IgG in serum, and presence in the serum of free anti-IgM antibodies. In organs of mu-suppressed BALB/c mice, LCMV-WE replicated, dependent upon organ, at the same rate or more rapidly and, in general, to higher titers than in normal rabbit immunoglobulin-treated mice; untreated mice eliminated the virus most rapidly and showed lower virus titers. In addition, LCMV-primed control mice cleared a second LCMV challenge very rapidly and contained no virus by day 3, whereas mu-suppressed mice had virus in their blood and organs (except the spleen) up to days 3 to 6. The observed effects of anti-mu treatment may reflect the action of neutralizing antibodies (which so far have been difficult to demonstrate in vivo) or other antibody-dependent antiviral mechanisms which, together with T cells, efficiently control LCMV clearance.     

Dianhydrodulcitol treatment of lymphocytic choriomeningitis virus infection in suckling mice.

Mice 2--4 days of age were pretreated with a single 5 mg/kg dose of dianhydrodulcitol (DAD) and later infected intracerebrally with lymphocytic choriomeningitis (LCM) virus. These animals had a lower mortality rate and died later than the untreated control animals. Thus DAD pretreatment prevented in part of the animals the development of lethal meningitis, the consequence of LCM virus infection, reducing the cellular immune response. This effect of DAD could equally be observed in animals infected at the age of 16--18 days and of 4 weeks.