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Original Article |
adrian.ciurea{at}dim.usz.ch
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Key Words: persistent infection • lymphocytic choriomeningitis virus • T helper cells • humoral responses • viral evasion
The following questions have been further addressed in this study. (a) Are the envelope GPs of nAb-escape LCMV variants less immunogenic? (b) How quickly and efficiently are nAbs generated against emerging escape variants? (c) Do variants evolve that are in general neutralization resistant? (d) Is the impairment of nAb responses due to deletion or anergy of virus-specific T helper cells?
We studied the nAb response during a long-term infection of CD8–/– mice with LCMV, strain WE 12. Due to the absence of CTLs in these mice, augmented virus production occurs, which is transiently controlled by polyclonal nAbs in the blood and only to a limited extent in solid organs 10. The results indicate that the broadening of nAb responses against emerging neutralization-resistant virus variants in this high viremia model infection was mainly limited by a decrease and eventual loss of virus-specific CD4+ T cell responses. Changes in replication properties or a decreased immunogenicity of some virus variants may additionally contribute to virus persistence.
LCMV strain WE originally was obtained from F. Lehmann-Grube (Heinrich Pette Institut, Hamburg, Germany) and propagated on L929 cells. Mice were infected with 2 x 106 PFU of LCMV-WE intravenously. LCMV titers in blood or virus titers of stock solutions were determined with an immunological focus assay 15.
LCMV nAb–escape variants were isolated from the blood of CD8–/– mice (120 and 240 d after infection), grown on MC57 cells for 48 h, and subsequently plaque purified two times in vitro as described 16. For de novo infections, mice were immunized with 2 x 104 PFU of selected virus variants intravenously.
Vesicular stomatitis virus (VSV) Indiana (VSV-IND; Mudd-Summers isolate) was originally obtained from Dr. D. Kolakovsky (University of Geneva, Geneva, Switzerland) and was grown on BHK21 cells. Mice were infected with 2 x 106 PFU of VSV-IND intravenously.
Neutralizing Activity.
Adoptive Transfers.
Intracellular IFN-
Molecular Analysis.
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Strong CD8+ CTL responses characterize the initial immunosurveillance of infections with poorly or noncytopathic viruses such as hepatitis B and C viruses, HIV, and the murine RNA virus lymphocytic choriomeningitis virus (LCMV 123). Nevertheless, this initial clearance of viremia by CD8+ CTLs does not always prevent the establishment of a chronic infection. Besides CTLs, neutralizing Abs (nAbs) in association with other noncytolytic factors (IFN-
, TNF-
, and chemokines) play a crucial role in controlling persisting virus infection 45678. However, virus escape from the nAb response during chronic infections does occur and may contribute to viral persistence 9. We have recently demonstrated in a model system that low CTL activity during LCMV infection facilitates the detection of neutralization-escape virus variants 10. Escape was due to single point mutations within the genes coding for the envelope glycoprotein (GP)-1. The ensuing amino acid changes affect the conformation of the neutralizing epitope 11.
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Mice and Viruses.
CD8–/– mice 13, LCMV-GP61-80–specific CD4+ TCR transgenic (tg) SMARTA mice 14, and control C57BL/6 (B6) mice were obtained from the Institut für Labortierkunde (University of Zürich, Zürich, Switzerland). In certain experiments, B6 mice were in vivo depleted of CD8+ T cells by treatment with a tested anti-CD8 monoclonal Ab on days 3 and 1 preceding the infection, as described 10. The degree of depletion was always >95% in blood and spleen. All animals were kept under specific pathogen-free conditions.
Neutralizing activity against LCMV was measured in a focus reduction assay 15. The neutralizing titer was defined as the dilution causing half-maximal reduction of plaques of LCMV when compared with the same amount of virus incubated with control sera from uninfected mice. VSV neutralization assay was performed as described 17. The highest dilution of serum that reduced the number of plaques by 50% was taken as the titer. To determine IgG Ab titers, undiluted serum was pretreated with 0.1 M β-mercaptoethanol.
Spleen cell suspensions were prepared from naive SMARTA tg mice previously in vivo depleted of CD8+ T cells by treatment with anti-CD8 monoclonal Abs. 5 x 105 SMARTA splenocytes were transferred intravenously into recipient B6 or CD8–/– mice, 1 d before infection with 2 x 106 PFU of LCMV-WE. Both SMARTA and CD8–/– mice have a pure B6 background. Clonal expansion and disappearance of transferred tg cells in the blood of recipients were monitored by FACS® analysis using TCR V2 and Vβ8.3 monoclonal Abs. Staining.
Splenocytes (106 cells/well in 96-well plates) were stimulated in vitro with media or with the immunodominant LCMV-specific peptide GP61-80 (1 µg/ml). Brefeldin A (Sigma-Aldrich) was added after 1 h of culture. After a 5-h stimulation, the cells were harvested, washed once in PBS containing 2% FCS, and stained with PE-conjugated anti-CD4 monoclonal Abs (Caltag) for 30 min at 4°C. After washing of unbound Ab, the cells were fixed with PBS/4% paraformaldehyde for 10 min and then permeabilized with PBS/0.1% Saponin (Sigma-Aldrich). For intracellular staining, FITC-conjugated anti–IFN- monoclonal Abs were used and incubated with the cells for 30 min at 4°C. After washing twice with PBS/0.1% Saponin, the cells were resuspended in PBS containing 2% FCS and analyzed using a FACScanTM (Becton Dickinson). To control for nonspecific intracellular staining, parallel samples of stimulated and permeabilized CD4+ T cells were stained with FITC-conjugated isotype-matched monoclonal Abs of irrelevant specificity, which did not result in any staining signal.
Total RNA of MC57 cells infected either with LCMV-WE wild-type (wt) or with variant virus isolates for 48 h at an initial multiplicity of infection of 0.01 was extracted by using RNeasy kit (QIAGEN). Reverse transcription PCR was performed using the LCMV-GP1–specific primers R1 (5'-1037TCG TAG CAT GTC ACA GAA CTC TTC1014-3') for reverse transcription and the primer pairs 001/R1 and 001/RC1 (001, 5'-1CGC ACC GGG GAT CCT AGG CTT21-3'; RC1, 5'-965GAG CTC TGC AGC AAG GAT CAT CC942-3') for Hot Start PCR amplification. PCR products were sequenced by automated Taq cycle sequencing (Taq Dye Deoxy Terminator Cycle Sequencing kit; Applied Biosystems; Bio-Rad Laboratories) using the primers 001 and RC1.
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NAb Responses against Emerging nAb-escape Virus Mutants.
Virus titers as well as nAb titers were sequentially determined in the blood of five LCMV-WE–infected CD8–/– mice (animals M7–M11) for up to 240 d. As reported previously 10, nAb-mediated control of viremia, attained within 50–60 d after infection, was only transient and virus reappeared in the blood 2–4 wk after initial control (Fig. 1). This occurred despite the presence of relatively high titers of nAbs (Fig. 2, filled circles). Viremia was not controlled at later time points in CD8–/– mice (Fig. 1), suggesting that induction of new nAb responses against emerging neutralization-resistant virus variants had failed. To assess long-term virus-specific humoral and T helper responses in these mice, we next characterized the virus variants emerging in vivo. Virus was isolated from the blood of mice M7–M11 after the recrudescence of viremia (day 120). Sequence analysis of the gene encoding the envelope GP1 of LCMV isolates revealed amino acid alterations of the predominant viral clone (WE-M7 to WE-M11) within the bulk virus isolated from each animal (at least 5 out of 8–10 independent clones; Table ). One to three base pair exchanges per GP1 gene were identified, leading to amino acid substitutions within the three regions of GP1 that have been shown to correlate with virus escape from the nAb response 10. These mutations affected the efficiency of variant virus neutralization by polyclonal hyperimmune serum (pooled from B6 mice immunized with LCMV-WE) and by LCMV-WE-GP1–specific mAb (data not shown).
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Loss of CD4+ T Cell Responsiveness Precedes the Emergence of nAb-escape Virus Mutants.
Failure of the hosts to elicit new nAb responses against emerging nAb-escape virus variants could be due to insufficient CD4+ T helper responses at late time points. This could be the consequence of CD4+ T cell unresponsiveness induced during establishment of persistent infection 19, T helper epitope variation, or a decrease of infected CD4+ T cells.
LCMV-specific CD4+ T cell responses were compared between LCMV-infected CD8–/– mice, which show a high and sustained viremia, particularly in solid organs, and control B6 mice, which rapidly control the virus (10; Fig. 1). We monitored the number of splenic CD4+ T cells specific for the LCMV-immunodominant epitope GP61–80 and expressing intracellular IFN- after antigenic stimulation at different time points after infection (Fig. 3 A). In agreement with previous studies 202122, this response peaked at day 9 in control B6 mice (
3% of CD4+ T cells) and decreased to 0.4% by day 50. This percentage was stable in the memory phase (up to 240 d). By contrast, only 0.6% of CD4+ T cells stained positive for intracellular IFN- at the peak of the response in CD8–/– mice. This percentage rapidly dropped to background levels by day 20. In CD8–/– test animals M7 to M11, only background levels of virus-specific CD4+ T helper activity were detected on day 240 (Fig. 3 A), irrespective of the ability of the mice to produce low or high nAb titers against the immunizing LCMV-WE-wt virus (Fig. 2). We were not able to monitor an additional T helper response against the nucleoprotein NP309–328 epitope because on day 8 after infection, frequencies of specific CD4+ T cells were below background levels in LCMV-WE–infected CD8–/– mice. To ascertain that the responses measured in the in vitro assays were not affected by the absence of CD8+ T cells, we performed the following control experiment. LCMV-WE–immune B6 mice (70 d after infection) were either depleted of CD8+ T cells or left untreated. We then evaluated the number of splenic LCMV-specific, IFN-–expressing CD4+ T cells. Similar percentages of GP61-80–specific CD4+ T cells were measured in the spleens of mice of the two groups (data not shown). As indicated by sequence analysis, failure to detect GP61-80–specific CD4+ T cells in CD8–/– mice was not due to epitope variation (Table , and data not shown).
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and Vβ chains (Fig. 3 B). Compared with B6 mice, clonal expansion of the transferred tg CD4+ T cells was compromised in LCMV-infected CD8–/– mice. Moreover, we found that these cells disappeared more rapidly from the blood of recipient LCMV-infected CD8–/– mice. The tg CD4+ T cells were still present in high numbers on day 16 in the blood of LCMV-infected B6 mice and were still above background levels by about day 40. No obvious differences were seen in uninfected B6 and CD8–/– mice, suggesting that the effects seen were a consequence of high level viral replication. To investigate whether the reduction of LCMV-specific T helper cells was due to an overall decline of the CD4+ T cell population in the high viremia model, we assessed the absolute number of splenic CD4+ T cells by flow cytometry before and after LCMV infection. No depletion of the general CD4+ T cell pool was detected after infection of CD8–/– mice with LCMV-WE (Fig. 4).
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As documented here, several factors may allow the persistence of emerging neutralization-resistant variants. A weaker immunogenicity of the escape variants seems not to be a general phenomenon, as most of the variants were able to induce autologous nAb responses after de novo infections similar to wt LCMV-WE. However, we observed an asymmetric pattern of cross-reactivity between neutralizing responses induced by wt and escape virus isolates. Whereas the original wt strain induced a response against itself but not against the emerging escape variants, the mutant viruses were able to induce nAbs that inhibited both wt and escape viruses. Thus, the variant viruses reveal via nAbs a recapitulation of their evolution and exhibit a new sort of coevolutionarily directed connectivity that one might call "archetypical". In some aspects, this recall of genetic history is reminiscent of findings during influenza virus infections in the context of preexisting immune memory: after infection with an escape mutant after antigenic drift, influenza-immune individuals will generate higher Ab titers against the influenza surface hemagglutinin experienced during the original infection than against the mutated hemagglutinin of the drifted variant. This has been called original antigenic sin 3536.
Other factors may contribute to the long-term persistence of nAb-resistant viruses, depending on the variant analyzed. Some variants may have better intrinsic replication capacities in vivo. Alternatively, changes in cell or tissue tropism, as well as possible differences in resistance to interferons, may account for the observed enhanced persistence. All these alterations may be the consequence of some of the documented mutations in the GP1 gene, as demonstrated at several occasions for different LCMV strains 373839. In addition, mutations in other genes cannot be excluded, particularly on the L strand which encodes the viral polymerase and which was not sequenced in this study.
However, the most important mechanism leading to the persistence of emerging nAb-escape variants is the induction of specific CD4+ T cell unresponsiveness, as demonstrated here. The absence of an adequate T helper response at the moment of viral escape impaired the induction of new nAb responses against the virus mutants and allowed them to persist. Interestingly, the primary nAb response in CD8–/– mice after infection with LCMV-WE was very potent, despite the fact that the CD4+ T cell expansion was much lower than in normal mice and the helper response was rapidly lost. Several factors may account for this finding. First, virus-specific B cells, infected through the LCMV-GP1–recognizing receptor, are not killed in the absence of CTL responses 40. Second, the antigen load is much higher in CD8–/– mice. On the other hand, memory B cell responses may be less dependent on functional T help 41, explaining how high nAb titers can be maintained in CD8–/– mice against the immunizing LCMV-WE-wt despite vanishing T helper responses.
The CD4+ T cell dysfunction in CD8–/– mice does not affect the overall CD4+ T cell population and is only confined to the LCMV-specific compartment. Furthermore, as exemplified by the adoptive transfer of tg SMARTA splenocytes into LCMV-infected CD8–/– mice, LCMV-specific CD4+ T cell numbers remain equal to levels in uninfected control animals for at least 3 wk, indicating that the cells might undergo a phase of functional unresponsiveness before being physically deleted.
It has to be emphasized that CD8–/– mice are able to mount vigorous CD4+ T cell responses when infected with a low dose of a slowly replicating LCMV strain (LCMV-Armstrong; up to 14% at day 9; reference 21). An influence of elevated virus titers on the fate of T helper responses is indicated by the fact that a similar low percentage of LCMV-specific CD4+ T cells was reached during infection with the clone 13 strain of LCMV 21, which is able to establish persistent infections in immunocompetent mice 37. The high viral replication level achieved in CD8–/– mice (but not B6 mice) infected with LCMV-WE is comparable to the overwhelming infection of immunocompetent hosts by the rapidly replicating Docile strain of LCMV, where virus-specific T helper cell loss has also been shown 19. As CD8+ T cells may also contribute positively to T helper cell responsiveness via bystander factors 42, their absence in CD8–/– mice might also impact on the loss of specific CD4+ T cells.
Decrease of virus-specific T helper cells seems to be a critical evolutionary phenomenon which allows the survival of the host by preventing lethal immunopathology during persisting noncytopathic virus infection 19. The molecular mechanisms involved in the loss of T cell function remain unclear. As hypothesized for the described exhaustion of CTLs 2443, high antigenic load after wide viral spread may induce a general activation of all available virus-specific CD4+ T cells. These cells then would die of activation-induced apoptosis preceded by a period of anergy. Replenishment of virus-specific T helper cells would not be possible as a consequence of negative T cell selection in the infected thymus 44. Other contributing factors like interleukin starvation have been postulated, but their involvement has not been conclusively demonstrated.
The relevance of this in vivo model of inactivation of specific CD4+ T cells is based on increasing evidence for a role of T helper cells in the immune control of noncytopathic viral infections, by supporting not only nAb responses, but also CTLs 45. Studies of LCMV-infected mice deficient in CD4+ T cells either by in vivo treatment with anti-CD4 monoclonal Ab or by gene targeting have demonstrated a loss of long-term functional CTL responses and the enhancement of viral persistence in the absence of T helper cells 452546474849. Low CD4+ T cell responses have also been shown to correlate with high viral load 5051 and with low CTL responses 52 during HIV infection. Similarly, hepatitis C virus–specific CD4+ T cells seem to be essential not only for initial viral clearance, but also for long-term viral control 53. With regard to the impact of early viral load on subsequent impairment of T helper responses, it has been recently shown that early suppression of HIV-1 by highly active antiretroviral therapy may preserve CD4+ and CD8+ T cell function and may be associated with immune control 5455. The preserved T helper cell function may allow multiple rounds of nAb-escape and may therefore lead to the observed broadly cross-reactive nAb responses in HIV-infected long-term nonprogressors 56.
Together with earlier observations demonstrating an influence of nAb-producing B cells on LCMV long-term control 451057, this study provides further evidence for the interactions between cellular and humoral immune responses for efficient virus control. However, an adequate balance between the two arms of the acquired immune system is needed in order to avoid virus escape due to virus variant selection through a unilateral immune response.
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This work was supported by Swiss National Foundation Grants 31.50900.97 and 31.50884.97, and the Kanton of Zurich.
Submitted: 25 July 2000
Revised: 14 December 2000
Accepted: 25 December 2000
P. Klenerman's present address is Nuffield Department of Medicine, John Radcliffe Hospital, Oxford OX3 9DU, UK.
Abbreviations used in this paper: GP, glycoprotein; LCMV, lymphocytic choriomeningitis virus; nAb, neutralizing Ab; tg, transgenic; VSV, vesicular stomatitis virus; VSV-IND, VSV Indiana; wt, wild-type.
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) were infected with 2 x 106 PFU of LCMV-WE-wt intravenously, and sequential blood samples were analyzed for virus titers. Data shown are the mean for five mice ± SEM.
