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During chronic viral infections, CD8 T cells rapidly lose antiviral and immune-stimulatory functions in a sustained program termed exhaustion. In addition to this loss of function, CD8 T cells with the highest affinity for viral antigen can be physically deleted. Consequently, treatments designed to restore function to exhausted cells and control chronic viral replication are limited from the onset by the decreased breadth of the antiviral T cell response. Yet, it remains unclear why certain populations of CD8 T cells are deleted while others are preserved in an exhausted state. We report that CD8 T cell deletion during chronic viral infection can be prevented by therapeutically lowering viral replication early after infection. The initial resistance to deletion enabled long-term maintenance of antiviral cytolytic activity of the otherwise deleted high-affinity CD8 T cells. In combination with decreased virus titers, CD4 T cell help and prolonged interactions with costimulatory molecules B7-1/B7-2 were required to prevent CD8 T cell deletion. Thus, therapeutic strategies to decrease early virus replication could enhance virus-specific CD8 T cell diversity and function during chronic infection.

The ligand for CD40 (CD40L) is expressed on the surface of activated CD4+ T cells and its role in T-B cell collaborations and thymus-dependent humoral immunity is well established. Recently, by generating CD40L-knockout mice, we have confirmed its previously described role in humoral immunity and defined another important function of this molecule in the in vivo clonal expansion of antigen-specific CD4+ T cells. Here, we investigated the potential in vivo role of CD40L in antiviral immunity by examining the immune response mounted by CD40L-deficient mice following infection with lymphocytic choriomeningitis virus (LCMV), Pichinde virus, or vesicular stomatitis virus. Humoral immune responses of CD40L-deficient mice to these viruses were severely compromised, although moderate titres of antiviral IgM and some IgG2a were produced by virus-infected CD40L-deficient mice by a CD4+ T cell-independent mechanism. By contrast, CD40L-deficient mice made strong primary CTL responses to all three viruses. Interestingly however, although memory CTL activity was detectable in CD40L-deficient mice two months after infection with LCMV, the memory CTL response was much less efficient than in wild-type mice. Together, the results show that CD40-CD40L interactions are required for strong antiviral humoral immune responses, and reveal a novel role for CD40L in the establishment and/or maintenance of CD8+ CTL memory.

Measles virus (MV), one of the most infectious of human pathogens, still infects over 30 million humans and causes over 500,000 deaths each year [Griffin, D., 2001. Measles virus. In: Fields, B., Knipe, D., Howley, P. (Eds.), Fields Virology. Lippincott-Raven, Philadelphia, pp. 1401–1442; MMWR, 2005. Progress in reducing measles mortality–worldwide, 1999–2003. Morb. Mortal. Wkly Rep. 54, 200–203]. Death is primarily due to secondary microbial infections associated with the immunosuppression caused by MV. Studies of humans with genetic or acquired deficiencies of either the humoral or cellular arm of the immune system, and rodent models have implicated T cells in the control of the ongoing MV infection but the precise role and activities of the specific T cell subset or the molecules they produce is not clear. Using a transgenic mouse model in conjunction with depletion and reconstitution of individual B and T cell subsets alone or in combination, we show that neither CD4, CD8 nor B cells per se control acute MV infection. However, combinations of either CD4 T cells and B cells, or of CD4 and CD8 T cells are essential but CD8 T with B cells are ineffective. Interferon-γ and neutralizing antibodies, but neither perforin nor TNF-α alone are associated with clearance of MV infection. TNF-α combined with interferon-γ is more effective in protection than interferon alone. Further, the lack of an interferon-γ response leads to persistence of MV.

α-Dystroglycan (α-DG) was recently identified as a receptor for lymphocytic choriomeningitis virus (LCMV) and several other arenaviruses, including Lassa fever virus (W. Cao, M. D. Henry, P. Borrow, H. Yamada, J. H. Elder, E. V. Ravkov, S. T. Nichol, R. W. Compans, K. P. Campbell, and M. B. A. Oldstone, Science 282:2079–2081, 1998). Data presented in this paper indicate that the affinity of binding of LCMV to α-DG determines viral tropism and the outcome of infection in mice. To characterize this relationship, we evaluated the interaction between α-DG and several LCMV strains, variants, and reassortants. These viruses could be divided into two groups with respect to affinity of binding to α-DG, dependence on this protein for cell entry, viral tropism, and disease course. Viruses that exhibited high-affinity binding to α-DG displayed a marked dependence on α-DG for cell entry and were blocked from infecting mouse 3T6 fibroblasts by 1 to 4 nM soluble α-DG. In addition, high-affinity binding to α-DG correlated with an ability to infiltrate the white pulp (T-dependent) area of the spleen, cause ablation of the cytotoxic T-lymphocyte (CTL) response by day 7 postinfection, and establish a persistent infection. In contrast, viruses with a lower affinity of binding to α-DG were only partially inhibited from infecting α-DG−/−embryonic stem cells and required a concentration of soluble α-DG higher than 100 nM to prevent infection of mouse 3T6 fibroblasts. These viruses that bound at low affinity were mainly restricted to the splenic red pulp, and the host generated an effective CTL response that rapidly cleared the infection. Reassortants of viruses that bound to α-DG at high and low affinities were used to map genes responsible for the differences described to the S RNA, containing the virus attachment protein glycoprotein 1.

Adoptive transfer of virus-specific memory lymphocytes can be used to identify factors and mechanisms involved in the clearance of persistent virus infections. To analyze the role of B cells in clearing persistent infection with lymphocytic choriomeningitis virus (LCMV), we used B-cell-deficient μMT/μMT (B−/−) mice. B−/− mice controlled an acute LCMV infection with the same kinetics and efficiency as B-cell-competent (B+/+) mice via virus-specific, major histocompatibility complex (MHC) class I-restricted CD8+cytotoxic T lymphocytes (CTL). CTL from B−/− and B+/+ mice were equivalent in affinity to known LCMV CTL epitopes and had similar CTL precursor frequencies (pCTL). Adoptive transfer of memory cells from B+/+ mice led to virus clearance from persistently infected B+/+ recipients even after in vitro depletion of B cells, indicating that B cells or immunoglobulins are not required in the transfer population. In contrast, transfer of memory splenocytes from B−/− mice failed to clear virus. Control of virus was restored neither by transferring higher numbers of pCTL nor by supplementing B−/− memory splenocytes with LCMV-immune B cells or immune sera. Instead, B−/− mice were found to have a profound CD4 helper defect. Furthermore, compared to cultured splenocytes from B+/+ mice, those from B−/− mice secreted less gamma interferon (IFN-γ) and interleukin 2, with differences most pronounced for CD8 T cells. While emphasizing the importance of CD4 T-cell help and IFN-γ in the control of persistent infections, the CD4 T-helper and CD8 T-cell defects in B−/− mice suggest that B cells contribute to the induction of competent T effector cells.

Measles virus (MV) still incites one of the most contagious infections of humankind. Despite the development and use of an excellent live attenuated virus vaccine, over one million infants and children continue to die each year from measles1–3. The main cause of morbidity and mortality is virus–induced immunosuppression of lymphocyte function, which allows secondary infections. Here we report an in vivo model for the study of MV–induced immunosuppression. Human peripheral blood leukocytes (PBLs) grafted onto mice with severe combined immunodeficiency disease (SCID mice) to create hu–PBLS–SCID mice produce human IgG that is suppressed by MV infection, immunosuppression is dependent on the involvement of live virus and is dramatically more severe for PBLs obtained from newborns than PBLs from adults. Suppression of IgG synthesis by PBLs from newborns occurs as early as ten days after administration of MV to hu–PBLS–SCID mice compared with 44 days required for PBLs from adults. Further, MV infection of SCID mice reconstituted with PBLs from newborns reduces IgG production 26 ± 5–fold (mean ± 1 s.e.m.) as compared with only a 6 ± 0.5–fold reduction in adults. MV RNA could be detected in live human PBLs recovered from SCID mice as long as 110 days after MV infection began. The profound immunosuppression we observe in PBLs from infants probably contributes to the morbidity and mortality observed in infants vaccinated with measles virus. Further, this model should be useful for accessing the potential immunosuppressive abilities of newly isolated field (wild–type) virus isolates and newly designed vaccines containing attenuated MV or subunit vaccines, as well as in dissecting the role played by maternal antibodies to MV on the ability of the virus to enhance or abort the virus–induced immunosuppression.

Infidelity of genome applications of RNA viruses leads to the generation of viral quasispecies both in vitro and in vivo. However, the biological significance of such generated variants in vivo is largely unknown and controversial. To study this issue, we continued our evaluation of the tropism of a lymphocytic choriomeningitis virus (LCMV) variant termed clone 13 with its parental virus clonal pool ARM 53b (wild-type parent) for neuronal cells in vivo. Earlier in vivo and in vitro studies noted that the wild-type virus contained a Phe at glycoprotein (GP) residue 260 which correlated with neuron tropism compared with LCMV variants containing a Leu at residue 260 which showed selected tropism for cells of the immune system (C.F. Evans, P. Borrow, J. C. de la Torre, and M. B. A. Oldstone J. Virol. 68:7367-7373, 1994; L. Villarete, T. Somasundaram, and R. Ahmed, J. Virol 68:7490-7496, 1994). Here we (i) evaluated the ability of the viral variants with either a Phe or Leu at GP residue 260 to replicate in vivo in the spleen, liver, or brain, (ii) analyzed the ability of these viruses to compete against each other for cell (neuron)-specific selection following a single viral inoculation of different ratios of both viruses, and (iii) utilized genetic reassortants of both viruses to test their ability to replicate in neurons in vivo. We found that viral variants containing either a Phe or Leu at GP residue 260 were equally capable of replicating in neurons, but when inoculated together, neurons selected for the viral population containing Phe at GP residue 260 over viruses containing a Leu at this position. This was in contrast to selection in the liver and spleen that favored viruses with Leu and not Phe at GP residue 260. Analysis of inoculations with viral reassortants indicated that genes encoded on the short RNA (the GP and nucleoprotein, not the L [polymerase] and Z proteins that are encoded by the large RNA) were associated with neurotropism. Since the nucleoprotein sequences of wild-type Armstrong and clone 13 are identical, it is likely that specific cytoplasmic factors of the neurons play a fundamental role in the selection of virus with Phe at GP residue 260.

The mechanism(s) by which infectious material is cleared by the host is an area of intensive study. This is especially so with the realization that persistent viral infection is a cause of chronic disease in humans and presents a major health problem. We have used the murine model of infection with lymphocytic choriomeningitis virus to evaluate immune clearance. Mice with a targeted disruption of the IFN-gamma gene mount effective cytotoxic T lymphocyte (CTL) responses after an acute viral challenge and clear virus. CD4+ T cells are not required but CD8+ T cells are mandatory. In contrast, CTL from mice with targeted disruption of the IFN-gamma gene are unable to clear virus from persistently infected mice. In addition to the requirement for IFN-gamma, CD4+ T cells are essential for maintaining a CD8(+)-mediated cure of persistent viral infection.

Lymphocytic choriomeningitis virus (LCMV) is a nonlytic murine virus that provides a valuable model system for studying the behavioral correlates of CNS viral infection. Newborn or immunosuppressed mice infected with LCMV develop a persistent tolerant infection characterized by continuous viral production. Virus can be found in various body organs including lung, liver, kidney, and brain. In brain, neurons are the predominant CNS cells infected and the greatest number of persistently infected neurons are found in the cerebral cortex, hippocampus, other limbic structures and parts of the hypothalamus. Despite continuous infection throughout the animal's life, neurons show no structural injury or dropout. Mice from the DBA/2J strain were infected with LCMV (1000 plaque-forming units) within 18 h of birth and tested for behavioral function as adults. Plaque assays indicated persistent infection in virus-injected mice. Mice were tested for their ability to learn a Y-maze spatial discrimination to avoid the onset of a mild footshock (0.43 mA). The number of correct avoidance responses made during training was taken as a measure of acquisition performance. The virus-infected mice showed a deficit in acquisition of the Y-maze discrimination compared to that seen in vehicle-injected and noninjected controls. Following additional training to reach control levels of performance, the infected mice and the controls were injected with the cholinergic antagonist scopolamine. Scopolamine (2.0 mg/kg) disrupted the performance of the infected mice significantly more than control performance, suggesting that a cholinergic dysfunction accounted for some of the learning deficit. A separate group of virus-infected mice exhibited hypoactivity during the first exposure to a locomotor testing apparatus.(ABSTRACT TRUNCATED AT 250 WORDS)

Lymphocytes and/or monocytes/macrophages carry viral genetic information in most, if not all, persistent and latent viral infections, and serve as potential reservoirs for maintaining or reintroducing the infection. Similarly, neurons can be persistently infected by several DNA and RNA viruses whose continued presence can alter the physiologic function of these cells, leading to disorders in neurotransmitters and disease. Here, we document that adoptive transfer of virus-specific cytotoxic T lymphocytes clears virus and viral nucleic acid sequences, in vivo, from individually infected lymphocytes, macrophages, and neurons. By plaquing, infectious center, Northern blot, and in situ hybridization at the single cell level, virus was efficiently removed from these cells.

H-2 k (C3H/Hej) cytotoxic T lymphocytes (CTL) specific for lymphocytic choriomeningitis virus (LCMV) were cloned. Three clones recognizing internal viral antigens were studied. One such CTL clone recognized neither the glycoprotein nor nucleoprotein encoded by the viral short RNA segment, but reacted with a protein encoded by the long RNA segment, either the viral polymerase, or the Z protein. This one clone, in addition to primary CTL harvested from immunized C3H mice, failed to lyse target cells expressing the Z protein, suggesting recognition was to the viral polymerase. Two other clones recognized the viral nucleoprotein, amino acids 93–100, as determined by protein deletion and peptide mapping studies. When introduced directly into the central nervous systems of LCMV-infected histocompatible mice, all clones were active in vivo and capable of causing immunopathologically mediated death.

Virus-antibody immune complexes form during infection with most RNA and DNA viruses, including those with human immunodeficiency virus (HIV). Yet a subset of individuals so infected apparently does not mount such responses. To understand the principles involved, we studied the formation and deposition of virus-antibody immune complexes in the circulation in a model system utilizing mice persistently infected with lymphocytic choriomeningitis virus (LCMV). Although mice of several genetic haplotypes could be persistently infected with LCMV, mount anti-LCMV antibody responses, and form immune complexes levels varied among murine strains. Earlier, genetic analysis of high and low immune complex formers, their F1 crosses, and appropriately selected recombinant inbred strains located the ability to mount heightened immune responses in genes within the MHC. Further, variations among LCMV strains in the capacity to incite high levels of immune complex formation were found. Persistent infection with LCMV Armstrong (ARM) strain was associated with high levels of complexes in the circulation and marked deposits in the glomeruli of high-responder SWR/J mice. In contrast, persistent infection of SWR/J mice with LCMV Traub strain led to very low levels of circulating complexes and minimal immune complex deposition in tissues. The amount of virus carried during both infections was roughly equivalent indicating that the genetics of both the host and the virus play essential roles in whether or not immune complexes develop. Antibody responses in SWR/J mice persistently infected with LCMV ARM were 5- to 10-fold higher than responses of age- and sex-matched mice infected with LCMV Traub.(ABSTRACT TRUNCATED AT 250 WORDS)

Subacute sclerosing panencephalitis (SSPE) is a progressive fatal neurodegenerative disease associated with persistent infection of the central nervous system (CNS) by measles virus (MV), biased hypermutations of the viral genome affecting primarily the matrix (M) gene with the conversion of U to C and A to G bases, high titers of antibodies to MV, and infiltration of B cells and T cells into the CNS. Neither the precipitating event nor biology underlying the MV infection is understood, nor is their any satisfactory treatment. We report the creation of a transgenic mouse model that mimics the cardinal features of SSPE. This was achieved by initially infecting mice expressing the MV receptor with lymphocytic choriomeningitis virus Cl 13, a virus that transiently suppressed their immune system. Infection by MV 10 days later resulted in persistent MV infection of neurons. Analysis of brains from infected mice showed the biased U to C hypermutations in the MV M gene and T and B lymphocyte infiltration. These sera contained high titers of antibodies to MV. Thus, a small animal model is now available to both molecularly probe the pathogenesis of SSPE and to test a variety of therapies to treat the disease.

Among infectious agents, measles virus (MV) remains a scourge responsible for 1 million deaths per year and is a leading cause of childhood deaths in developing countries. Although MV infection itself is not commonly lethal, MV-induced suppression of the immune system results in a greatly increased susceptibility to opportunistic bacterial infections that are largely responsible for the morbidity and mortality associated with this disease. Despite its clinical importance, the underlying mechanisms of MV-induced immunosuppression remain unresolved. To begin to understand the basis of increased susceptibility to bacterial infections during MV infection, we inoculated transgenic mice expressing the MV receptor, CD46, with MV and Listeria monocytogenes. We found that MV-infected mice were more susceptible to infection with Listeria and that this corresponded with significantly decreased numbers of macrophages and neutrophils in the spleen and substantial defects in IFN-gamma production by CD4(+) T cells. The reduction in CD11b(+) macrophages and IFN-gamma-producing T cells was due to reduced proliferative expansion and not to enhanced apoptosis or to altered distribution of these cells between spleen, blood, and the lymphatic system. These results document that MV infection can suppress both innate and adaptive immune responses and lead to increased susceptibility to bacterial infection.

CD4 and CD8 T lymphocytes infiltrate the parenchyma of mouse brains several weeks after intracerebral, intraperitoneal, or oral inoculation with the Chandler strain of mouse scrapie, a pattern not seen with inoculation of prion protein knockout (PrP−/−) mice. Associated with this cellular infiltration are expression of MHC class I and II molecules and elevation in levels of the T-cell chemokines, especially macrophage inflammatory protein 1β, IFN-γ-inducible protein 10, and RANTES. T cells were also found in the central nervous system (CNS) in five of six patients with Creutzfeldt-Jakob disease. T cells harvested from brains and spleens of scrapie-infected mice were analyzed using a newly identified mouse PrP (mPrP) peptide bearing the canonical binding motifs to major histocompatibility complex (MHC) class IH-2borH-2dmolecules, appropriate MHC class I tetramers made to include these peptides, and CD4 and CD8 T cells stimulated with 15-mer overlapping peptides covering the whole mPrP. Minimal to modest Kbtetramer binding of mPrP amino acids (aa) 2 to 9, aa 152 to 160, and aa 232 to 241 was observed, but such tetramer-binding lymphocytes as well as CD4 and CD8 lymphocytes incubated with the full repertoire of mPrP peptides failed to synthesize intracellular gamma interferon (IFN-γ) or tumor necrosis factor alpha (TNF-α) cytokines and were unable to lyse PrP−/−embryo fibroblasts or macrophages coated with51Cr-labeled mPrP peptide. These results suggest that the expression of PrPscin the CNS is associated with release of chemokines and, as shown previously, cytokines that attract and retain PrP-activated T cells and, quite likely, bystander activated T cells that have migrated from the periphery into the CNS. However, these CD4 and CD8 T cells are defective in such an effector function(s) as IFN-γ and TNF-α expression or release or lytic activity.

Measles virus (MV) infects 40 million persons and kills one million per year primarily by suppressing the immune system and afflicting the central nervous system (CNS). The lack of a suitable small animal model has impeded progress of understanding how MV causes disease and the development of novel therapies and improved vaccines. We tested a transgenic mouse line in which expression of the MV receptor CD46 closely mimicked the location and amount of CD46 found in humans. Virus replicated in and was recovered from these animals' immune systems and was associated with suppression of humoral and cellular immune responses. Infectious virus was recovered from the CNS, replicated primarily in neurons, and spread to distal sites presumably by fast axonal transport. Thus, a small animal model is available for analysis of MV pathogenesis.

Cytotoxic T lymphocytes (CTL) play a pivotal role in preventing persistent viral infections and aborting acute infections. H-2Db-restricted CTL optimally recognize a specific peptide of 9 to 11 amino acids (aa) derived from a viral protein and held in place (restricted) by a MHC class I glycoprotein on the surfaces of infected cells. Only three peptide sequences with the appropriate Db motif from lymphocytic choriomeningitis virus Armstrong strain (LCMV) are known to be presented to CTL by H-2Db molecules; they are from the glycoproteins (GP), residues 33-41 KAVYNFATC (GP1) and 276-286 SGVENPGGYCL (GP2), and the nucleoprotein (NP), 396-404 FQPQNGQFI. Incubation of virally infected H-2b cells with CTL clones that recognize only GP1, GP2, or NP leads to the selection of viral variants which upon infecting cells bearing H-2b molecules, escape recognition by CTL of the appropriate specificity. Nucleic acid sequencing showed a single mutation in GP1 (aa 38 F--L), GP2 (aa 282 G--D), or NP (aa 403 F--L) in the variant viruses. When wild-type (wt) LCMV peptides and the three variant peptides (GP1, GP2, NP) were synthesized and subjected to a competitive inhibition binding assay, no differences in binding affinity for H-2Db were found between the wt and variant peptides. Uninfected cells coated with the wt peptide were recognized and lysed by the appropriate CTL clone or by in vivo-primed bulk CTL, but similar targets coated with the GP1, GP2, or NP variant peptides were not. This result, coupled with computer graphic analysis of these variant peptides with the recently solved three-dimensional structure for the Db MHC class I molecule, placed the side chain of the mutated residues on the outer surface of the MHC-peptide complex and accessible to the T cell receptor. Ala substitution at GP residue 38 or 282 or at NP 403 also abrogated CTL recognition and lysis. Inoculation of any one of the mutated viral variants into mice produced an effective CTL response to the other two nonmutated GP or NP peptides, suggesting that production of biologically relevant CTL escape virus variants in vivo requires selection of mutations in more than one and likely all the CTL epitopes, a low probability event.

Viruses that persist must develop strategies to escape immunologic surveillance in order to survive. Investigation of lymphocytic choriomeningitis virus (LCMV)-induced persistence has indicated that this virus avoids immune clearance mainly by aborting the viral specific cytotoxic T lymphocyte (CTL) response, a response that is necessary for terminating viral infection. This study demonstrates that persistence established in immunologically immature newborns selectively depletes the LCMV-specific CTL response but does not hinder CTL responses to the RNA and DNA viruses influenza, vaccinia, or herpes simplex. In contrast, persistence established in immunologically mature adults leads not to selective but rather to generalized immunosuppression during which CTL responses to LCMV, influenza, vaccinia, and herpes simplex viruses are all ablated or down-regulated. These results indicate that the state of maturity of the immune system at the time of virus-induced immunosuppression can result in two distinct phenotypes. These observations may account for the differing patterns of infection caused by hepatitis B virus or human immunodeficiency virus initiated in the neonatal period compared to that initiated in adulthood.

Of nine established murine haplotypes, mice of three types (H-2d, H-2u, and H-2q) possess major histocompatibility complex class I glycoproteins able to present an identical viral peptide for recognition and lysis by virus-specific cytotoxic T lymphocytes. Incorporation of this viral epitope into a recombinant vaccinia vaccine and administration of a single dose protects mice with these three haplotypes from an ordinarily lethal challenge of virus. Hence, a common epitope can exist. The sharing of the ability to bind such epitopes among different MHC haplotypes underscores the feasibility of developing an effective cytotoxic T-lymphocyte vaccine for outbred populations like humans.

Cytotoxic T lymphocytes (CTL) recognize virus peptide fragments complexed with class I major histocompatibility complex (MHC) molecules on the surface of virus-infected cells. Recognition is mediated by a membrane-bound T-cell receptor (TCR) composed of alpha and beta chains. Studies of the CTL response to lymphocytic choriomeningitis virus (LCMV) in H-2b mice have revealed that three distinct viral epitopes are recognized by CTL of the H-2b haplotype and that all of the three epitopes are restricted by the Db MHC molecule. The immunodominant Db-restricted CTL epitope, located at LCMV glycoprotein amino acids 278 to 286, was earlier noted to be recognized by TCRs that consistently contained V alpha 4 segments but had heterogeneous V beta segments. Here we show that CTL clones recognizing the other two H-2Db-restricted epitopes, LCMV glycoprotein amino acids 34 to 40 and nucleoprotein amino acids 397 to 407 (defined in this study), utilize TCR alpha chains which do not belong to the V alpha 4 subfamily. Hence, usage of V alpha and V beta in the TCRs recognizing peptide fragments from one virus restricted by a single MHC molecule is not sufficiently homogeneous to allow manipulation of the anti-viral CTL response at the level of TCRs. The diversity of anti-viral CTL likely provides the host with a wider option for attacking virus-infected cells and prevents the emergence of virus escape mutants that might arise if TCRs specific for the virus were homogeneous.

For viruses to establish persistent infections in their hosts, they must possess some mechanism for evading clearance by the immune system. When inoculated into adult immunocompetent mice, wild-type lymphocytic choriomeningitis virus (LCMV ARM) induces a CD8(+)-mediated cytotoxic T lymphocyte (CTL) response that clears the infection within 7-14 d (CTL+ [P-]). By contrast, variant viruses isolated from lymphoid tissues of persistently infected mice fail to induce a CTL response and are thus able to establish a persistent infection in adult mice (CTL- [P+]). This report compares the interaction of CTL+ (P-) and CTL- (P+) viruses with cells of the immune system. Both types of virus initially bind to 2-4% of CD4+ and CD8+ T lymphocytes and replicate within cells of both subsets. The replication of CTL- (P+) and CTL+ (P-) viruses in lymphocytes in vivo is similar for the first 5 d after initiating infection. Thereafter, in mice infected with CTL- (P+) variants, lymphocytes retain viral genetic information, and infectious virus can be recovered throughout the animals' lives. In contrast, when adult mice are infected with wild-type CTL+ (P-) LCMV ARM, virus is not recovered from lymphocytes for greater than 7 d after infection. A CD8(+)-mediated anti-LCMV CTL response is induced in such mice. Clearance of infected lymphocytes is produced by these LCMV-specific CTLs, as shown by their ability to lyse lymphocytes expressing LCMV determinants in vitro and the fact that depletion of CD8+ lymphocytes before infection with CTL+ (P-) viruses results in levels of infected lymphocytes similar to those found in undepleted CTL- (P+)-infected mice. Hence, CTL-mediated lysis of T lymphocytes carrying infectious virus is a critical factor determining whether virus persists or the infection is terminated.

Retarded growth and disordered glucose metabolism secondary to growth hormone (GH) deficiency are associated with persistent lymphocytic choriomeningitis virus (LCMV) infection of GH-producing cells in the anterior lobe of the pituitary gland. Infected C3H/ST mice, which are H-2k haplotype, become GH deficient, and LCMV replicates in most (more than 90%) of their GH-producing cells. In contrast, BALB/WEHI and SWR/J mice, which are H-2d and H-2q, respectively, do not develop this GH deficiency, and less than 20% of their GH-producing cells are infected by virus. Yet all three strains infected at birth with LCMV strain Armstrong (ARM) carry equivalent amounts of virus in their blood, brain, heart, kidney, liver, spleen, and thymus throughout life. Of five additional H-2k murine strains tested, C3H/HEJ and CBA/N mice develop this GH-like disorder, whereas neither AKR/J, B10/BR, nor BALB/KAE mice do, indicating that the H-2K haplotype does not control the GH susceptibility. Furthermore C3H/SW mice, which have the H-2b haplotype on the C3H background, develop the disease, again negating any correlation with H-2k but inferring that the C3H background is responsible. One half of the hybrid offspring produced by crossing the C3H/ST GH-deficient strain with BALB/WEHI-resistant mice develop the disease, but the trait is not sex linked. F1 hybrid backcrosses with the susceptible C3H/ST parental strain or resistant BALB/WEHI strain indicate the involvement of more than two genes. Hence the development of a GH deficiency by LCMV-infected C3H/ST mice is not linked to the MHC haplotype, is not sex linked, and is not due to a dominant gene. Multiple genes are involved and these are related to C3H background.

Lymphocytic choriomeningitis virus (LCMV) infection prevents the usual insulin-dependent diabetes mellitus of aged BB rats (Dyrberg, Schwimmbeck & Oldstone, 1988; Schwimmbeck, Dyrberg & Oldstone, 1988). In this study earlier observations are extended by noting that LCMV infection substantially alters the immune responsesof BB diabetic-prone (dp) rats. The control, uninfected rats make vigorous primary and secondary antibody responses when challenged with keyhole limpet haemocyanin (KLH), human immunoglobulin (HuIg) or sheep red blood cells (SRBC). Such rats fail to mount a primary response to bovine serum albumin (BSA) but do produce a moderate secondary response. They mount good antibody responses to LCMV but fail to generate either primary or secondary LCMV-specific cytotoxic T-lymphocyte (CTL) responses or CTL responses to Pichinde virus. In contrast, BB dp rats acutely infected with LCMV generate no primary immune responses to SRBC, KLH or BSA and only meager responses when challenged with HuIg. They mount secondary responses to KLH, HuIg and BSA that approximate those of their uninfected litter mates, but have a comparatively lower response to SRBC. LCMV binds to and infects lymphocytes of the BB dp rat. Binding is enhanced over that observed with lymphocytes from BB diabetic-resistant (dr) rats, which are able to generate CTL immune responses to LCMV and Pichinde viruses. Hence, lymphocytes from BB dp rats are uniquely susceptible to binding and replication of LCMV. During the acute stage of LCMV infection, a general primary T-cell immunosuppression occurs with respect to a variety of viral and non-viral antigens. Over time, responsiveness to T-cell dependent antigens returns except for the ability to generate CTL responses to LCMV or the autoimmune response(s) required to cause insulin-dependent diabetes mellitus.

BioBreeding Worcester diabetes-prone (BBdp) rats develop insulin-dependent autoimmune-driven diabetes mellitus spontaneously and intravenous administration of 1 x 10(7) p.f.u. of lymphocytic choriomeningitis virus (LCMV) to young adult mice prevents disease. The virus is lymphotropic, binding to and replicating in such cells. BBdp rats fail to generate virus-specific major histocompatibility complex-restricted cytotoxic T lymphocyte (CTL) responses when challenged with this dose or other doses of LCMV, Pichinde virus or vaccinia virus. Yet such rats clear virus effectively and show no evidence of persistent infection. Associated with this clearance of virus and establishment of immunity is the production of neutralizing antibodies. In contrast, diabetes-resistant (BBdr) rats generate virus-specific CTL responses. Furthermore LCMV binds to fewer lymphoid cells of BBdr rats (in comparison to those of BBdp rats) and replicates in fewer lymphocytes (by one order of magnitude) from these rats. Thus, unlike mice in which CTLs play a dominant role in the control of LCMV infection, BBdp rats do not overcome this infection via CTLs. In addition, both the BBdp rats and their BBdr counterpart may provide useful models for determining whether or how individual lymphocyte subsets function in the induction of CTL responses, for the analysis of virus-induced immunosuppression and for the use of viruses or their products as therapeutic modalities.

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.

Disordered growth and glucose metabolism secondary to growth hormone deficiency is associated with persistent lymphocytic choriomeningitis virus (LCMV) infection. C3H/St, BALB/WEHI, and SWR/J mice infected at birth with LCMV:ARM carried virus in their blood and organs throughout life but only C3H/St mice developed growth hormone insufficiency. BALB/WEHI and SWR/J infected mice contained normal amounts of growth hormone in their pituitaries and a relatively small proportion of the cells containing growth hormone replicated the virus. In susceptible C3H/St mice, the disease-causing viral strains (LCMV:ARM, E-350, and Pasteur) replicated to higher titers and infected the vast majority of cells producing growth hormone in the anterior lobe of the pituitary. In contrast, LCMV strains Traub and WE replicated in far fewer growth hormone-producing cells and failed to disorder growth hormone synthesis. In another paper (Y. Riviere, R. Ahmed, P. Southern, and M. B. A. Oldstone (1985), Virology 142, 175-182) these findings are used to make reassortants between LCMV:ARM (disease positive) and LCMV:WE (disease nil) and the pathogenic effect is mapped to the small RNA segment of LCMV:ARM. Peptides cleaved by trypsin and chymotrypsin from growth hormone molecules isolated from infected cells or control cells were equivalent when examined by two-dimensional electrophoresis. Further, transfer of antibody to interferon failed to alter the growth hormone insufficiency in these mice, although it corrected LCMV-induced liver disease of BALB mice, suggesting that interferon did not play a dominant role in this disease. The selective tropism of LCMV:ARM for cells containing growth hormone over cells that contain prolactin was observed in both infected animals and in cultured GH-3 cells.

We define a nine-amino acid (aa) sequence of VAL-GLU-ASN-PRO-GLY-GLY-TYR-CYS-LEU as a major epitope for immunologic recognition of lymphocytic choriomeningitis virus (LCMV) by H-2b-restricted CTL. The epitope was characterized using molecular genetics to bracket broadly and chemistry to precisely identify aa residues 278-286 of the viral glycoprotein. The epitope's composition is characteristic of a reverse (beta turn) but not an amphipathic alpha helix. A series of peptides with a single aa substitution in position 278 of VAL with other nonpolar (hydrophobic) amino acids (LEU, ILE, ALA, or GLY) coat targets that are recognized and lysed by CTL clones recognizing this epitope. In contrast, substitution of VAL with either large aromatic amino acids (that add bulk: PHE, TYR) or polar side chains (SER, THR) segregates CTL clones normally recognizing aa 278-286 into two groups, one that remains lytic (permissive) despite these changes and another that fails to lyse, indicating CTL can discriminate at a single aa. A change in charge at this position (VAL----ASP or GLU), in general, reduces CTL lysis while a change of VAL to LYS or ASN has minimal affect for four of the five CTL clones analyzed. CTL reactivity with the viral epitope is restricted by the Db but not the Kb of the murine MHC haplotype. A 16-aa peptide of Db that spans alpha 1 residues 37-52 blocks CTL lysis, whereas the corresponding Kb peptide that differs from Db in a single aa in position 50 does not.

Viruses that cause in vivo persistent infections avoid the host's immunologic surveillance machinery. A major component of that armamentarium is virus-specific MHC-restricted cytotoxic T lymphocyte (CTL) response of the host. Studies with lymphocytic choriomeningitis virus (LCMV) have uncovered a parental virus (CTL+) that in immunocomponent adults induces CTL and terminates acute infection and a variant (CTL− that fails to elicit CTL responses and establishes a persistent state ( R. Ahmed et al (1984) , J. Exp. Med. 160, 521–540). The biologic properties, similarities, and differences between CTL+ and CTL− viruses as regards their interactions with lymphocytes of newborn and adult mice is recorded here. CTL+ and CTL− viruses persist in lymphocytes of newborn inoculated mice, primarily within the T helper subset. Approximately 2% of lymphocytes express viral nucleic acid sequences while only 0.04% score as infectious centers suggesting incomplete viral replication. These levels were maintained over the course of infection. In contrast, CTL− virus but not CTL+ persists in lymphocytes of mice inoculated when adults. Lymphocytes easily scored as infecting centers but rarely displayed nucleic acid sequences suggesting a different balance of incomplete to complete virion replication. Further, infectious centers decreased by 10-fold from the 3rd to 68th day of infection and the total numbers of T lymphocytes in the circulation decreased suggesting CTL− may replicate in and destroy lymphocytes of adult mice. In the following paper the primary nucleotide structure of the LCMV small RNA segment, the segment responsible for generation of CTL and encoding the proteins recognized by CTL, for CTL+ and CTL− viruses is reported.

Persistent infections caused by such agents as the human immunodeficiency virus, hepatitis B virus, Epstein–Barr virus, etc., present formidable medical problems. A defining characteristic of these infections is that anti-viral cytotoxic T lymphocytes (CTL) may be lost or, if present, fail to clear the infection. Here we report a vaccination strategy which was successful in generating lytic CTL in persistently infected mice. Vaccination with an immunodominant CTL epitope derived from the nucleoprotein of lymphocytic choriomeningitis virus (LCMV) delivered in the form of a lipopeptide incorporating a universal CD4 helper epitope successfully induced lytic MHC-restricted CTL in mice persistently infected with LCMV since birth. However, induction of such CTL did not eliminate the virus, most likely because the CTL were generated at low frequencies and had 2 to 3 logs lower affinity than CTL generated in uninfected mice inoculated with the vaccine. Both CTL populations from either uninfected or persistently infected mice produced significant and similar amounts of interferon-γ and IL-6. Vaccine-induced low-affinity CTL were still inadequate at complete removal of the virus when combined with LCMV-specific CD4 helper T lymphocytes. Thus, our results establish that CTL can be generated in persistently infected mice and that a crucial factor for clearing viral infection is the affinity of the CTL.

The noncytopathic lymphocytic choriomeningitis virus displays a tropism for the anterior lobe of the murine pituitary gland. Virus replicates in cells that make growth hormone. This results in a diminished synthesis of growth hormone with a concomitant clinical picture of retarded growth and hypoglycemia. However, there is no morphologic evidence of either cell necrosis or inflammation in the anterior lobe of the pituitary. Hence, during infection in vivo, a noncytopathic virus may turn off the "differentiation" or "luxury" function of a cell while not killing that cell (loss of vital function). This is turn can disrupt homeostasis and cause disease. This model illustrates a novel way whereby viruses may cause disease.

Virus-antibody immune complex formation, deposition, and disease is a common manifestation in most mice persistently infected with lymphocytic choriomeningitis virus (LCMV). Although mice of several strains persistently infected with LCMV mount continuous anti-LCMV immune responses to the three virion structural polypeptides, the amount of antibody(s) made varies among strains. The formation of antibody to LCMV correlates with the detection of C1q binding materials (immune complexes) in the circulation; however, there is no correlation between the total amount of IgG and the C1q binding material. SWR/J mice (H2qq) are high responders (high levels of anti-LCMV antibodies, high C1q binding responses), whereas BALB/W mice (H-2dd) are low responders (low levels of anti-LCMV antibodies, low C1q binding responses). Sera from 37 SWR/J mice, 12 wk of age, bound 55.1% of offered 125I C1q compared to 8.4% for 37 age and sex-matched BALB/W mice. SWR/J mice made approximately 60-fold more antibody to LCMV than did BALB/W mice. To define the genetic factors involved, we used the C1q binding assay, high responder and low responder mice, their hybrid offspring, backcrosses of the hybrids to both high and low responder parents, and selected recombinant inbred mouse strains. From studies with BALB/W mice persistently infected with LCMV, we defined low C1q responder mice as those having binding activity of 18.4% or less (BALB/W mean C1q binding value + 2 SD). By using this criteria for 12-wk-old mice, 55/57 (96%) of SWR/J, 27/37 (73%) of F1(SxB or BxS), 13/21 (62%) of qq or 11/20 (55%) qd from F1 x SWR/J, and 6/13 (45%) qd from F1 x BALB/W were high C1q responders. In contrast, none of the 17 dd mice from the F1 x BALB/W cross were high responders. Thus, F1 hybrid mice are high responders (dominant) and data from backcrossing F1 hybrids to either high or low responder parents suggested the complexes associated with C1q binding was controlled by gene(s) in the H-2 complex. Support for the role of Ir gene(s) was provided by experiments showing LCMV persistently infected BALB/kae (H-2 KkIkDk) and recombinant inbred strains B10.A (KkIkDd) and A.TL (KsIkDd) were high C1q responders, whereas B10.A(5R) (KbIbDd) and BALB/cby (KdIdDd) were not. The wide scatter in C1q binding levels observed among C1q high responder mice and the production of C1q binding levels in F1 mice that are intermediate between high and low responder strains suggested that, in addition to H-2-linked genes, other non-H-2-linked genes also play a role in this response. The amount of C1q binding complexes in LCMV persistently infected mouse strains and their crosses was unrelated to the amounts of infectious virus carried in their sera. Despite the low C1q binding by sera of H-2dd mice that originated from mating F1 hybrid (qd) x BALB/WEHI (dd), these mice carried equivalent amounts of infectious virus as their qd littermates or qd and qq mice originated from F1 x SWR/J mice.

The efficiency of cloned class I MHC restricted CTL specific for the nucleoprotein or glycoprotein of lymphocytic choriomeningitis virus in either mediating virus clearance or immunopathologic disease in mice during acute infection was quantitated. Cloned CTL specific for either an internal (nucleoprotein) or surface (glycoprotein) protein of lymphocytic choriomeningitis virus, when administered intracerebrally 5 days after the initiation of infection induced fatal immunopathology, indicating that both internal and surface viral Ag play a role in immune mediated disease in vivo. Dose-response analysis indicated that only 10(2) to 10(3) cloned CTL injected intracerebrally were required to induce mortality in 50% of inoculated syngeneic mice. Thus relatively few virus-specific CTL are required to induce an acute immunopathologic disease in the central nervous system. In contrast, if cloned CTL are adoptively transferred at the time of initiation of viral infection they provide protection as demonstrated by their ability to eliminate virus from the brain and thus terminate the acute infection.

The cytotoxic T-lymphocyte (CTL) response to lymphocytic choriomeningitis virus infection determines the outcome of infection. Here we show that this response in BALB/c mice (H-2d), when analyzed both at the primary CTL level and using CTL clones, is predominantly monospecific. The vast majority of CTL have a common specificity for a single epitope in the virus nucleoprotein, which can be minimally identified by amino acids GVYMG. This epitope is presented by the Ld class I glycoprotein. We used these data to design a subunit CTL vaccine, whose effectiveness is demonstrated in the accompanying report (L. S. Klavinskis, J. L. Whitton, and M. B. A. Oldstone, J. Virol. 63:4311-4316, 1989). Further analysis indicates that, while CTL clones share a common minimal epitope, they differ in their ability to recognize cells infected with a related but distinct strain of lymphocytic choriomeningitis virus. Studies on the molecular nature of CTL cross-reactivity indicate that CTL induced by similar sequences may cross-react in a unidirectional manner. These novel observations suggest that CTL vaccines, to achieve optimal effectiveness, should not simply include virus sequences which will yield a CTL response; the immunizing sequences should also be selected to ensure that the fine specificities of the induced CTL are such that they maximize the chance of recognizing serotypically diverse strains.

Murine F9 and PCC4 teratoma cells do not express H-2 major transplantation antigens according to virus-specific T-lymphocyte cytotoxic or serological assays. However, such cells can be infected with and readily replicate many types of viruses (coxsackie B 3, mouse hepatitis, Sindbis, Semliki Forest [SFV], lymphocytic choriomeningitis, Pichinde, vesicular stomatitis, herpes simplex type 1) to the same extent as do murine F12 teratoma cells and mouse embryo fibroblasts, all of which express the H-2 determinants. In contrast, F9 and PCC4 cells are not productively infected with murine cytomegalovirus, whereas F12 and mouse embryo fibroblast cells are. In addition to replicating in H-2-negative murine teratoma cells, SFV replicates in H-2-negative murine lymphoblastoid cells. The ability of SFV to infect cells without H-2 antigens and then to effect viral antigenic expression in the cells' cytoplasm and on their surface with similar kinetics and in equivalent amounts as cells with H-2 antigens indicates that the H-2 receptor is not needed for SFV infection. Daudi cells, which lack HLA antigens, block the replication of SFV. This occurs at some point after receptor binding, as demonstrated by diminished viral mRNA. In addition, a possible membrane defect precludes viral exit in Daudi cells transfected with SFV infectious RNA. These results indicate that a cell's possession of H-2 antigens is not a requirement for SFV infection and that major histocompatibility complex antigens are not specific receptors for this virus.

Viruses that cause in vivo persistent infections need to selectively compromise the host's immunologic surveillance machinery in order to survive. To understand the molecular basis of how this is accomplished we have analyzed persistent virus infection by lymphocytic choriomeningitis in its normal host, the mouse. Earlier we noted by infectious center analysis that five in 10(4) lymphocytes carried by persistently infected mice contained infectious materials throughout the course of infection. A previous publication extended these results, in BALB mice by showing that the L3T4+ lymphocyte subset in lymph nodes and spleens was predominantly involved. Using cDNA labeled probes to the viral genome and in situ hybridization we report that 1 to 2% of circulating lymphocytes from several mouse strains contain viral RNA sequences for the three viral structural genes. By FACS analysis, the Thy-1.2+, L3T4+ subset primarily harbors virus while viral sequences are usually not detected in the Lyt-2+ subset as early as 6 days after initiating infection in newborns and throughout the course of the persistence. These findings suggest that incomplete, presumably defective, virus is generated in a subset of Th lymphocytes during persistent infection and that during this time infection of cytotoxic T cell subsets is minimal.

Mice of several strains persistently infected with lymphocytic choriomeningitis virus (LCMV) mount continuous anti-LCMV immune responses leading to the formation and tissue deposition of immune complexes. Such mice carry infectious virus-immunoglobulin (presumably anti-LCMV antibody) complexes in the circulation. We have now determined that anti-LCMV antibody both complexed and free is found in the circulation of mice persistently infected with LCMV. This antibody reacts specifically against the three main LCMV structural polypeptides: nucleoprotein, 63,000 m.w. and two glycopeptides, GP-1 and GP-2 with m.w. of 45,000 and 35,000, respectively. A C1q binding assay was developed and found to be effective in measuring C1Q binding substances (presumably virus-anti-viral Ig complexes) in the circulations of several strains of mice persistently infected with LCMV. With different strains of mice, the levels, time of formation, and fate of C1q binding materials varied markedly. Formation of antibodies to LCMV was correlated with the detection of C1q binding materials. Mice (SWR/J) persistently infected with lactic dehydrogenase virus also form infectious virus-Ig in their sera but deposit minimal amounts of complexes in their tissues. In such mice, C1q binding substances did not form in the circulation.

Abnormal glucose metabolism (hyperglycemia and/or aberrant glucose tolerance test) occurred over the life-times of mice persistently infected with lymphocytic choriomeningitis virus (LCMV). Persistent infection could be initiated in both newborn and adult mice. For newborns, inoculation with any of several strains of LCMV (Armstrong, E350, Pasteur, Traub or WE) caused continuous infection, but such infection of adults required a selected lymphotropic variant of LCMV Armstrong (Clone 13). Throughout these animals' lives, viral materials (nucleic acid sequences and proteins) accumulated in multiple tissues, including the beta-cells of the islets of Langerhans; infectious virus was present in blood and tissues, and the LCMV-specific H-2 restricted CTL response was poor. Adult mice that had been inoculated with virus as newborns displayed neither histopathologic injury nor infiltrates of mononuclear cells in the islets of Langerhans despite moderate viral replication in beta-cells. In contrast, mice inoculated as adults with Clone 13 LCMV consistently developed inflammatory infiltrates in perivascular spaces of the islets of Langerhans, and their beta-cells expressed LCMV antigens. Addition of a subdiabetogenic dose of streptozotocin, a specific beta-cell toxin, magnified the virus-induced abnormality in glucose metabolism. This indicated a potentiating role between persistent virus infection initiated at birth or in adulthood and an environmental factor in causing abnormalities in glucose metabolism.

Thirty-six monoclonal antibodies from mice and three from rats were raised against a peptide corresponding to the immunodominant domain of the transmembrane gp41 protein of human immunodeficiency virus (HIV) type 1 (LGLWGCSGKLIC; amino acid residues 598 to 609). Of these, three monoclonal antibodies from the mice and one from a rat also reacted with the corresponding peptide derived from the HIV type 2 transmembrane gp41 protein (amino acid residues 593 to 603; NSWGCAFRQVC). Immunochemical studies using a variety of synthetic peptides indicated that the cross-reactivity was due to antibody binding to CSGKLIC of HIV type 1 or CAFRQVC of HIV type 2. Single amino acid substitutions for a cysteine at either the amino or the carboxy end of the peptide interrupted antibody binding, indicating that the site recognized was the Cys-XXXXX-Cys loop. Similar results were obtained when the 11-mer HIV type 2 gp41 peptide (amino acids 593 to 603) was inoculated into mice to raise monoclonal antibodies. In this instance, of 30 monoclonal antibodies developed, 4 reacted with both HIV type 1 and HIV type 2 peptides. The conformation of a seven-residue peptide, CSGKLIC, corresponding to residues 603 to 609 of the gp41 immunodominant epitope of HIV-1 was investigated by proton nuclear magnetic resonance spectroscopy. The immunologically active form of CSGKLIC contains an intramolecular disulfide bond and maintains a preference for a folded conformation, apparently including a type I reverse turn about the residues SGKL. No such preference is observed for the reduced form of the peptide, which contains two thiol groups. The presence of the disulfide bond is thus integral to the formation of the structure of the loop in solution. In agreement with this finding, elimination of the possibility of loop formation by substitution of S for C at the amino or carboxy termini of the 7-mer is accompanied by the failure of antibody binding to this peptide.

CD8 + cytotoxic T lymphocytes (CTL) play a key role in the control of many virus infections, and the need for vaccines to elicit strong CD8 + T-cell responses in order to provide optimal protection in such infections is increasingly apparent. However, the mechanisms involved in the induction and maintenance of CD8 + CTL memory are currently poorly understood. In this study, we investigated the involvement of CD40 ligand (CD40L)-mediated interactions in these processes by analyzing the memory CTL response of CD40L-deficient mice following infection with lymphocytic choriomeningitis virus (LCMV). The maintenance of memory CD8 + CTL precursors (CTLp) at stable frequencies over time was not impaired in CD40L-deficient mice. By contrast, the initial generation of memory CTLp was affected. CD40L-deficient mice produced lower levels of CD8 + CTLp during the primary immune response to LCMV than did wild-type controls, despite the fact that the LCMV-specific effector CTL response of CD40L-deficient mice was indistinguishable from that of control animals. The differentiation of naı̈ve CD8 + T cells into effector and memory CTL thus involves pathways that can be discriminated from each other by their requirement for CD40L-mediated interactions. Expression of CD40L by CTLp themselves was not an essential step during their expansion and differentiation from naı̈ve CD8 + cells into memory CTLp; instead, the reduction in memory CTLp generation in CD40L-deficient mice was likely a consequence of defects in the CD4 + T-cell response mounted by these animals. These results thus suggest a previously unappreciated role for CD40L in the generation of CD8 + memory CTLp, the probable nature of which is discussed.