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Viral mutations, TCR antagonism and escape from the immune response
Viral mutations, TCR antagonism and escape from the immune response Alessandra Franco, Carlo Ferrari, Alessandro Sette and Francis V Chisari The Scripps Research Institute, La lolla, USA, Universita' degli Studi di Parma, Parma, Italy and Cytel Corporation, San Diego, USA Persistent viruses use several mechanisms to evade the immune response, including the generation of mutations that affect TCR recognition. It has recently been reported that spontaneous mutations at TCR contact sites within individual viral epitopes in certain persistent human viruses can abrogate or antagonize the recognition of the corresponding wild-type epitope, and it has been suggested that such mutations may contribute to viral persistence. Current Opinion in Immunology 1995, 7:524-531 Introduction Persistent viruses and their hosts share the common objective o f survival. Because of the high stakes involved, both sides use rather sophisticated strategies to achieve this goal. The host typically seeks to destroy the virus, while it is in the best interest of the virus to coexist with the host. From the host's point of view, viral clearance demands an effective and timely response as conditions favor the virus once the infection is well estabhshed. T lymphocytes play a critical role in this series of events, but they are not always successful. In fact, a strong T-cell response may even cause more harm than good if it occurs after most of the cells of a vital organ have already been infected. Conversely, a weak T-cell response may cause a chronic inflammatory disease that slowly destroys the infected organ while allowing the virus to persist. In the face o f these events, persistent viruses seek to avoid recognition, and the most successful persistent viruses have developed several clever strategies to confound, suppress or escape the host immune response. In this review, we will discuss one o f these strategies, specifically mutational escape from recognition by the TCR,; we will also consider the immunological parameters that must exist for the mutational inactivation of a single viral epitope to permit a virus to escape ehmination by the entire immune response.
Host strategies to clear viruses During the first few critical days after infection of an immunologically naive host, viral spread is controlled by a complex network of physiological events that
control viral entry, replication and dissemination, and also by the innate immunity o f the host, which is primarily mediated by the NK-cell system [1°°]. Soon thereafter, virus-specific T cells and antibodies play a crucial role in ehminating cells infected with virus, preventing cell-to-cell spread, and clearing extracellular viral particles [2°°]. The balance obtained between the capacity o f the virus to replicate and spread, and the ability o f the host to eliminate the virus during this early period probably determines the long-term outcome of the infection. During a viral infection, endogenously synthesized viral antigens are processed in a non-endosomal compartment and the derived peptides are transported to the lumen of the endoplasmic reticulum where they bind class I M H C molecules for presentation to CD8 + cytotoxic T lymphocytes (CTLs) [3,4]. In the case o f extracellular antigens, processing usually occurs in an endosomal compartment of professional antigen-presenting cells (APCs) (i.e. B cells, macrophages and dendritic cells) [3,5°°], and the derived peptides bind to class II M H C molecules for presentation to CD4 + T cells. T cells use an antigen-specific cell surface receptor (TCR) to recognize peptide ligands bound to MHC. This interaction initiates a signal that can induce T-cell activation and proliferation when it is coordinated with a second signal that is derived from the interaction of various adhesion molecules on the T cell and APC [6,7]. Importantly, when T C R recognition occurs in the absence of this costimulatory signal, the T cell may be anergized or undergo programmed cell death (apoptosis) [8]. As costimulatory molecules are preferentially expressed by professional APCs, infected somatic cells lacking costimulatory molecules may
Abbreviations APC--antigen-presenting cell; APL--altered peptide ligand; HBV--hepatitis B virus; HCV--hepatitisC virus; INF--interferon; IL--interleukin; TCR--T-cell receptor; Th--T helper. 524
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Viral mutations,TCR antagonismand escapefrom the immune responseFranco et paradoxically anergize virus-specific T cells thereby giving the virus an intrinsic advantage relative to the specific immune response. When conditions are optimal, however, T cells, especially CTLs, can clear viruses from infected cells by at least two specific mechanisms, the kinetics and relative strength of which influence the outcome of the encounter between virus and host: • Destruction of infected cells. CTLs lyse infected cells via perforin-granule exocytosis, as has been elegantly demonstrated in perforin knockout mice [9"'], or by inducing programmed cell death (apoptosis) through activation of the Fas, or other, intrinsic cell-death pathways [10,11,12]. Cell lysis is the classical mechanism generally considered responsible for control and/or clearance of most virus infections. In the setting of a massive viral infection, however, the number of infected cells and the virus production rate may be too high to be controlled by this process, which requires each infected cell to be reached and killed by individual CTLs [11,12]. Given the constraints of CTL movement in solid tissues [13], and the possibihty that CTLs may actually be inactivated by infected cells that do not provide the necessary costimulation required for continued CTL expansion, relatively few target cells may actually be killed by each CTL. This raises the possibility that certain infections may not be controllable by the direct CTL-mediated killing of infected cells. • Intracellular inactivation of virus in infected cells. CTLs can also inactivate the expression and replication of certain viruses non-cytolyticany by releasing antiviral cytokines in the infected tissue, possibly 'curing' infected cells that they can not physically reach in vivo. This mechanism has been shown to be responsible for the clearance of hepatitis B virus (HBV) from the hver in an HBV transgenic mouse model [14"] where it is 10- to 100-fold more effective at viral clearance than direct hepatocyte lysis. Similar non-cytolytic immunological mechanisms have been shown to downregulate HIV replication in vitro [15]. The balance between the destructive and curative functions of the immune response could influence the outcome of an infection. For example, if a given virus is not susceptible to control by T cell derived cytokines, or if the T-cell response to a given virus fails to produce the cytokines to which that virus is susceptible, viral clearance would depend entirely on the ability of a bruited number of CTLs to reach, recognize, and destroy all of the infected cells in the body. This may not be possible if the balance between the number of CTLs and the number of infected cells is unfavorable for the host. Under these conditions the infection could easily become persistent and the continuous, but subtotal, destruction of infected cells could actually cause a chronic inflammatory disease in the infected tissue. In addition to CD8 + CTLs, virus-specific CD4 + T lymphocytes also play an important role in viral clearance
al.
[16]. CD4 + T lymphocytes of the T-helper cell type 1 (Thl) phenotype produce the same lymphokines as CD8 + CTLs and can kill target cells in vitro, although their cytolytic activity in vivo has not been established [17]. In addition, Th cells may be required for CTL induction and expansion [18] and for establishment and maintenance of CTL memory [19]. Furthermore, Th cells provide help for antibody responses [20], which are important in limiting viral spread.
Virus strategies to evade the host immune system Viruses have evolved several mechanisms to avoid recognition by host T cells and antibodies in order to establish persistent infection. These strategies include integration into the host genome [21]; silencing of viral gene expression (latency) [22]; inhibition of antigen processing and presentation [23-27]; synthesis of proteins homologous to known immune regulatory molecules [23,28]; infection of immunologically privileged sites that cannot be reached by virus-specific T cells, or that do not express class I or costimulatory molecules; mutations that either inhibit viral responsiveness to antiviral cytokines elaborated during the immune response (M Mattoubian M, Reddy K, Pedroza Martins L, Ahmed R, abstract, Proceedings IXth International Congress of Virology, Berlin, August 1993) preclude recognition by neutralizing antibodies [23], or modify residues that are critical for recognition by the M H C [29] or the T C R [30,31]. The latter can inactivate a T-cell epitope either by decreasing its binding at~inity for the M H C or T C R , or by actively anergizing or antagonizing the T-cell response to the wild-type epitope. Indeed, it has been recently reported that naturally occurring mutations within certain CTL epitopes in HBV [32"'] and HIV [33 "°] can code for viral peptides that function as T C R antagonists for wild-type epitope-specific CTLs. The mechanisms by which these altered peptide ligands (APLs) can interfere with T-cell recognition will now be discussed in some detail.
TCR antagonism and partial agonism Recently [34"',35"'], several groups have demonstrated that non-antigenic antigen analogs could partially or completely inhibit T-cell function. It is now known that changing the structure of the 'hgand with which the T C R interacts (i.e. converting an antigen-MHC complex to an antigen analog-MHC complex) can change the nature of the signal(s) transmitted to the T cell, with a corresponding change in the T-cell response. Two responses can occur when an antigen analog (i.e. an APL) engages the T C R : T C R antagonism and partial agonism.
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Immunity to infection T C R antagonism is a process whereby an APL carrying substitution(s) within a major T C R contact residue inhibits T-cell activation by the normal stimulatory antigen [36]. T C R antagonists effectively inhibit certain T-cell functions, such as T-cell proliferation or upregulation of IL-2 receptors in response to their natural ligands [34°°,37]. In addition, T C R antagonists can also inhibit early intracellular events associated with T-cell activation, such as Ca 2+ influx and phosphatidyl inositol turnover [37]. Partial agonism occurs when an APL can stimulate one T-cell function (agonism), while failing to stimulate or even inhibiting another (antagonism). In this case, the APL will elicit some, but not all, of the signals required for T-cell activation. For example, an HB-specific, I-Ek-restricted T-cell clone has been described that, when stimulated by APL, produces IL-4 but cannot proliferate [38]. Using the same antigenic system, Thl clones are able to display cytolytic activity, but they are unable to proliferate or produce lymphokines in the presence of APLs [39]. Particularly intriguing is the fact that some partial agonists are able to induce a profound and long-lasting state of T-cell anergy [40,41°°].
Molecular mechanisms How can T-cell activation be prevented or dampened when APLs interact with the TCR? The simple mechanism of receptor saturation by APL can be discarded for stoichiometric reasons as APL can antagonize the response to native antigen at very low molar ratios [34"°]. Instead, analysis of the relationship between structure and activity of T C R antagonists suggests that affinity plays an important role. According to this hypothesis, a certain a~nity threshold is required for T C R signaling - - engagement of the T C R below this threshold can result in T C R antagonism [42]. A model has been proposed [34 °'] suggesting that when antigen-MHC and A P L - M H C complexes are both present simultaneously on the cell surface, mixed oligomers will form with some T C R engaging antigen-MHC complexes and others engaging A P L - M H C complexes. According to this model, the mixed T C R - M H C - p e p t i d e clusters are incapable of proper signaling, probably because these mixed clusters are unstable, causing the T cell and APC to dissociate before the T C R can engage the critical number ofstimulatory antigen-MHC complexes required for T-cell activation, thus resulting in inhibition. To understand the events involved in T C R antagonism requires an appreciation of the molecular basis of T C R activation. The T C R is composed of an 0t-~ heterodimer and a multichain invariant CD3 complex. The intracellular tail of each subunit of the CD3 complex contains a tyrosine activation motif, which serves as a substrate for Src family protein tyrosine
kinases [43,44"°]. Tyrosine phosphorylation of the T C R chain and CD3 "?-8-e complex results in the activation of two independent signaling pathways [45,46,47]. APLs fail to trigger phosphorylation of the ~ chain (with physiological phosphorylation of the CD3 3~S-E complex). Two recent studies [48"',49 °'] have identified abnormalities in the signal transduction cascade that are induced when the T C R interacts with APL. In an experimental system where the APL is a previously described anergy-inducer [40], Allen and colleagues [48"'] demonstrated that presentation of APL to resting T-cell clones stimulated a unique pattern of T C R phospho-~ chain species and a consequent lack of association with Zap 70 kinase. Using a different experimental system, where the antagonist is a complex of native antigenic peptide bound to mutant M H C molecules, Germain and colleagues [49 °.] also demonstrated k-chain phosphorylation without Zap 70 activation. If these events can negate the signals that are normally induced by T C R contact with its nominal antigen-MHC complex, it might explain why very low molar ratios of APL to wild-type peptide can antagonize T-cell responses in some viral systems. Viral mutants that express T C R antagonist or partial agonist peptides should be able to evade specific T-cell responses in vivo. Indeed, the occurrence of T C R antagonist mutations during HBV and HIV infection has been recently demonstrated (see below).
Effect of viral mutations on T-cell recognition Mutations occur randomly in all viruses at frequencies that are determined by the enzymes they use for replication. They are particularly common in R N A viruses (e.g. hepatitis C virus [HCV] and HIV) and in certain DNA viruses (e.g. HBV) that use R N A polymerase or reverse transcriptase, respectively, to replicate, because these enzymes lack proof-reading activity. Mutations that increase the intrinsic growth rate of a virus could be positively selected, and those that decrease host recognition should be negatively selected by the immune response. The latter can theoretically include mutations that interfere with the ability of a virus to respond to antiviral cytokines, as well as mutations that inhibit the binding of antigenic peptides to the M H C or to the T C R , and these can either fail to activate, partially activate or antagonize the responsiveness of T cells that recognize the original wild-type antigenic peptide. The binding of antigenic peptides to M H C molecules and recognition of the resulting peptide-MHC complex by the T C R involves interaction of specific amino acid residues of the peptide with corresponding determinants in both the M H C and the T C R [3]. Amino acid changes in antigenic viral peptides that affect M H C anchor residues can abolish or reduce their binding to
Viral mutations, TCR antagonism and escape from the immune response Franco et al. M H C class I molecules and result in their selection due to decreased immune recognition. This mechanism has been invoked to explain the prevalence o f a particular Epstein-Barr virus (EBV) isolate in a population that displays an uncommonly high frequency of the HLAA l l allele. The variant virus contains a single mutation within a CTL epitope that profoundly reduces its binding to HLA-A11 class I molecules, thus abrogating T-cell recognition o f this epitope by CTLs that are restricted to HLA-A11 [29]. These results suggest that variant EBV strains can be selected by CTLs leading to their prevalence at the population level, perhaps over an extended period o f time. Whether this process can also represent a primary cause o f viral persistence in individual patients remains uncertain in view of the enormous coding capacity of this virus, the exceptional strength o f the EBV-specific T-cell response, and the polymorphism of the human MHC. Mutations that affect TC1L binding residues within a viral epitope can also contribute to viral escape by creating peptides that affect T-cell recognition. In most cases, the mutant peptide will still function as a T C R agonist. In other cases, however, the mutation can abolish T C R recognition or actively inhibit the T-cell response by creating peptides that function as T C R antagonists or partial agonists. Viral escape due to mutational loss of a T C R contact site within a CTL epitope has been demonstrated in a murine model in which mice bearing a transgenic T C R specific for an immunodominant lymphocytic choriomeningitis virus epitope (glycoprotein residue 32-42) were unable to clear the virus after infection [50]. In this system, a spontaneous mutation occurred in the epitope that abolished its antigenicity for the transgenic T C R . As the immune response in these animals was focused exclusively on that epitope, the variant virus was selected. This model represents a good example of the profound effect that viral mutations can have when the T-cell response is monoclonal and selectively focused on the epitope where the mutation arises. This kind of immune response does not usually occur, however, during most viral infections in man. Nonetheless, loss o f C T L recognition has been described in a longitudinal study of HIV infected patients where CTL responses to several HLA-B8 restricted CTL epitopes in the HIV gag protein waned as a result o f the mutational loss o f T C R contact sites in the wild-type peptides [30]. At the same time, however, other mutations emerged in an HLA-B27 restricted CTL epitope in the same HIV gag protein that created peptide agonists that were more immunogenic than the wild-type sequence. Thus, although the principle of immune selection o f viral escape mutants due to loss o f antigen recognition is well established, its role in viral persistence during most natural viral infections still remains to be fully clarified. Mutational inactivation of a single viral epitope should lead to persistence only if it confers a survival advantage to the mutant that cannot be counteracted by
the residual immune response to all o f the remaining epitopes. On the one hand, this is unlikely when the CTL response is vigorous and multispecific as it is during the acute stage of various viral infections [51,52,53]. Similarly, immune selection o f mutant viruses is not likely to occur when the immune response is weak or undetectable as it is during many persistent viral infections [51,52]. O n the other hand, oligoclonal expansions o f T cells carrying the same TC1L V~ chains have been described in some patients acutely infected by HIV [54], suggesting either that some epitopes are strongly immunodominant, or that these patients may express such a limited repertoire o f antiviral effector cells allowing viral escape to occur. Similar findings of monoclonal or oligoclonal TCIL expansions have also been reported during other persistent viral infections [55,56,57]. Viral mutations that affect recognition of an epitope by some CTL clones do not automatically affect all CTL clones specific for the same epitope. In fact, different T-cell clones specific for a given epitope can bind different T C R contact residues [58"°,59]. Thus, the situation is very complex, and longitudinal studies correlating the C T L response against multiple viral epitopes, with the sequence o f those epitopes during the evolution of an acute to a persistent infection are needed to clarify the actual pathogenetic role o f mutations in viral persistence.
Role of TCR antagonism in viral infections In addition to its involvement in thymic education, peripheral tolerance, autoreactive T-cell responses, and T-cell memory [34"°,35°°], T C R antagonism may also play a role in viral escape from immune recognition. For example, in two recent reports, naturally occurring variants of the HBV [32 °°] and the human immunodeficiency virus 1 (HIV-1) [33 °°] that contained mutations at T C R contact sites in immunodominant epitopes were shown to antagonize CTL recognition of the corresponding wild-type epitopes in selected chronically infected patients. In both instances, antagonism occurred at very low concentrations of the variant peptide, much lower than the concentration required to induce activation by the native antigen. This was surprising considering that in other experimental systems an equimolar dose or an excess of antagonist peptide is required to inhibit the T-cell response [36,37,58°°]. The recent demonstration of negative signals delivered to the TClq. by A P L - M H C complexes due to improper phosphorylation o f the CD3 ~ chain, [48",49 °°] may help to explain these results. The importance o f this phenomenon in both the HBV and HIV model is illustrated by the fact that recognition of the corresponding naturally processed wild-type epitopes was also efficiently antagonized by the variant peptides. In general, the antagonist viral peptides in these reports could not sensitize target cells for lysis by CTL clones
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Immunityto infection specific for the wild-type peptide, except at high concentrations or after prolonged incubation, and this is compatible with the notion that antagonism results from a weak interaction between the antagonist peptide and the T C R [42]. Interestingly, some APLs were antigenic at high doses and antagonistic at low doses, suggesting that the immunological consequences of this kind of viral mutation could be determined by the amount of antigen present and, therefore, by viral load. The antagonist effect in the HBV system was not limited to cytotoxicity, as IFN-3' production was also inhibited by mutant epitopes in some of the T-cell clones studied. Most of the antagonist peptides were poorly immunogenic compared to the wild-type peptide, suggesting they were also unable to elicit an efficient alternative T-cell response to themselves [57"]. Collectively, these results suggest that naturally occurring viral variants may be able to specifically inhibit the destruction of cells that simultaneously express both wild-type and variant viruses at their surface, and thereby protect the cells containing mutant virus from immune elimination by CTL specific for the wild-type epitope (negative selection). Furthermore, in one instance it was shown that CTL recognition of a wild-type HIV peptide on one target cell could be antagonized by recognition of a variant peptide present on a different cell [33"]. Although this 'trans-antagonism' phenomenon was not observed in the HBV system, and is otherwise difficult to reconcile with our current understanding of the molecular basis for antagonism, these results raise the possibility that the presence of antagonistic epitopes on cells containing a mutant virus might be able to protect neighboring cells containing wild-type virus.
Different viruses, different scenarios Non-cytopathic persistent viruses must be able to evade immune surveillance either by not inducing an effective antiviral immune response or by evolving strategies to escape an otherwise efficient response. The importance of the frst scenario is illustrated by the well-documented fact that the T-cell response to HBV appears to be weak and inefficient in patients with chronic HBV infection, whereas it is vigorous and multispecific in patients with acute HBV infection who successfully clear the virus [2"']. The recent report [32"] that naturally occurring viral mutants could act as TCI< antagonists in two patients with chronic HBV infection, suggests that selection of TClk antagonist peptides may also contribute to the persistence of this virus in some patients. It should be noted, however, that mutations within known CTL epitopes do not appear to be commonplace in chronic hepatitis (B R.ehermann and FV Chisari, unpublished data). Furthermore, the characteristically weak immune response to HBV in most chronically
infected patients is probably not strong enough to select for the outgrowth of mutant viruses. These observations suggest that CTL escape mutation may not be primarily responsible for HBV persistence in most patients who are chronically infected by this virus, even though mutations could contribute to HBV persistence when the CTL response is narrowly focused. On the basis of available data, however, this type of immune response, and the corresponding selection pressure, does not appear to be a common event during HBV infection. The situation may be somewhat different in early chronic HIV infection where the CTL response is characteristically very strong yet, like the response to HBV, it is unable to clear the virus [54,55]. The incredibly high rate of HIV production and the exceptionally high mutation rate of this virus may cause so many different viruses to be generated each day that they exceed the capacity of the immune system to respond to them effectively simply on a numerical basis [60]. In this regard, the ostensibly vigorous immune response to the virus appears to be unable to compete with its capacity to replicate and its ability to generate mutants. Mutational inactivation of CTL epitopes might thus play an earher and more important role in the estabhshment of viral persistence for this virus than for HBV. It is important to emphasize, however, that the overwhelming rates of viral replication and spread relative to the ability of the immune system to produce enough CTLs to reach and destroy all of the infected cells, plus the immunosuppressive effects of the virus itself, may be more important than mutations for the development of persistent infection. The situation may be different again during chronic HCV infection where an extensive quasi-species of viral variants can coexist with a multispecific CTL response [53,61] that is intermediate in strength between the response of patients chronically infected by HBV and HIV. Unlike HBV and HIV where the viral titer is high, the viral titer is very low during chronic HCV infection [62], so viral persistence cannot be ascribed to an overwhelming viral load in this instance. In this setting, therefore, escape mutants may play a greater role in the primary establishment of HCV persistence than is likely for these other viruses.
Conclusions We suggest that the balance between the kinetics and magnitude of viral spread and the immune response during the early days of an infection probably determines the ultimate outcome of infection by viruses that have the capacity to become persistent. An ineffective immune response could be due to many factors, including neonatal tolerance, immunological exhaustion by high viral loads, infection ofimmunologically privileged sites, modulation of recognition molecules on the surface of
Viral mutations, TCR antagonism and escape from the immune response Franco et al. infected cells, immune suppression and altered cytokine production. One o f the viral factors that could influence this balance is the development o f mutations that nullify, deviate, or antagonize the antiviral T-cell response. This mechanism should cause viral persistence, however, only if the residual immune response to the remaining non-mutated viral epitopes is unable to clear the virus. In view o f the multispecificity of the CTL response to most viruses that ultimately persist in their host, current data favor the notion that negative selection o f CTL escape mutants is most likely to occur only after a persistent infection is already established. In this setting, occurrence o f these viral mutations could solidify the chronicity o f the infection and perhaps even make it irreversible. Whether such mutations can also serve as the primary cause o f viral persistence in the context o f a hierarchical multispecific T-cell response remains to be proven.
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41. •o
Sloan-Lancaster J, Evavold BD, Allen PM: Th2 cell clonal anergy as a consequence of partial activation. J Exp Med 1994, 180:1195-1205. Describes that T helper cell type 2 (Th2) clones are susceptible to anergy after stimulation with an altered peptide ligand and live antigen-presenting cells. 42.
Alexander J, Snoke K, Ruppert J, Sidney J, Wall M, Southwood S, Arrhenius T, Gaeta FCA, Colon SM, Grey HM, Sette A: Functional consequences of engagement of the T cell receptor by low affinity ligands. J Immuno/ 1993, 150:1-7.
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Weiss A: T cell antigen receptor signal transduction: a tale of tails and cytoplasmic protein-tyrosine kinases. Ceil 1993, 73:209-212.
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Chan AC, Desai DM, Weiss A: The role of protein tyrosine kinases and protein tyrosine phosphatases in T cell antigen receptor signal transduction. Annu Rev Immunol 1994, 12:555-592. A complete description of the current understanding of the intracellular events that follow T-cell recognition leading to activation. 45.
Iwashima M, Irving BA, Van Oers NS, Chan AC, Weiss A: Sequential interaction of the TCR with two distinct cytoplasmic tyrosine kinases. Science 1994, 263:1136-1139.
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Letourneur F, Klausner RD: Activation of T cells by a tyrosine kinase activation domain in the cytoplasmic tail of the CD3 epsilon. Science 1992, 255:79-82.
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Wegener AMK, Letourner F, Hoevler A, Broker T, Malissen B: The T cell receptor/CD3 complex is composed of at least two autonomous transduction modules. Ceil 1992, 68:93-95.
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Sloan-Lancaster J, Shaw AS, Rothbard JB, Allen PM: Partial T cell signaling: altered phospho-zeta and lack of Zap 70 recruitment in APL-induced T cell anergy. Ceil 1994, 79:913-922. See annotation [49""]. 49. ""
Madrenas J, Wange RL, Wang JL, Isakov N, Samelson LE, Germain RN: Zeta phosphorylation without zap 70 activation induced by TCR antagonists or partial agonists. Science 1995, 267:515-518. Using different antigenic systems, this study and [48eel demonstrate that TCR antagonist and partial agonist ligands stimulate a distinct pattern of k-chain phosphorylation and fail to activate Zap 70 kinase. These results indicate that an early step in the tyrosine-phosphorylation cascade is affected by TCR engagement with altered peptide ligand-MHC complexes. 50.
Pircher H, Moskophidis D, Rohrer U, Burki K, Hengattner H, Zinkemagel RM: Viral escape by selection of cytotoxic T cellresistant virus variants in vivo. Nature 1990, 346:629-633.
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Nayersina R, Fowler P, Guilhot S, Missale G, Cerny A, Schlicht HJ, Vitiello A, Chesnut R, Person JL, Redeker AG, Chisari FV: HLA A2 restricted cytotoxic T lymphocyte response to multiple hepatitis B surface antigen epitopes during hepatitis B virus infection. J Immunol 1993, 150:4659-4671.
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Rehermann 8, Fowler P, Sidney J, Person J, Redeker A, Brown M, Moss B, Sette A, Chisari FV: The cytotoxic T lymphocyte response to multiple hepatitis B virus polymerase epitopes during and after acute viral hepatitis. J Exp Med 1995, 181:1047-1058.
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Cerny A, McHutchison JG, Pasquinelli C, Brown ME, Brothers MA, Grabscheid B, Fowler P, Houghton M, Chisari FV: Cytotoxic T lymphocyte response to hepatitis C virus-derived peptides containing the HLA A2.1 binding motif. J C/in Invest 1995, 95:521-530.
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Pantaleo G, Demarest JF, Soudeyns H, Graziosi C, Denis F, Adelsberger JW, Borrow P, Saag MS, Shaw GM, Sekaly RP, Fauci AS: Major expansion of CD8 ÷ cells with a predominant V-beta usage during the primary immune response to HIV. Nature 1994, 370:463~,67.
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Kalams SA, Johnson RP, Trocha AK, Dynan MJ, Ngo S, D'Aquila RT, Kurnick JT, Walker BD: Longitudinal analysis of T cell receptor (TCR) gene usage by human immunodeficiency virus 1 envelope specific T lymphocyte clones reveals a limited TCR repertoire. J Exp Med 1994, 179:1261-1271.
35. JamesonSC, Bevan MJ: T cell receptor antagonists and partial "" agonist. Immunity 1995, 2:1-11. This review, along with [34e'], is a complete review on TCR antagonism and partial agonism. 36.
De Magistris MT, Alexander J, Coggeshall M, Altman A, Gaeta FCA, Grey HM, Sette A: Analog antigen/MHC complexes act as antagonist of the T cell receptor. Cell 1992, 68: 625-634.
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Ruppert J, Alexander J, Snoke K, Coggeshall M, Herbert E, McKenzie D, Grey HM, Sette A: Effect of T cell receptor antagonism on interaction between T cells and antigen-presenting cells and on T signaling events. Proc Nat/ Acad Sci USA 1993, 90:2671-75.
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Evavold BD, Allen PM: Separation of IL4 production from Th cell proliferation by an altered T cell receptor ligand. Science ]991, 252:1308-1310. Evavold BD, Sloan-Lancaster J, Hsu BL, Allen PM: Separation of T helper 1 clone cytolysis from proliferation and lymphokine production using analog peplides. J Immunol 1993,150:3131-3140. Sloan-Lancaster J, Evavold BD, Allen PM: Induction of T cell anergy by altered T cell receptor ligand on live antigen-presenting cells. Nature 1993, 363:156-159.
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Argaet VPC, Schmidt CW, Burrows SR, Silings SL, Kurilla MG, Doolan DL, Suhrbier A, Moss DJ, Kieff E, Sculley TB, Misko IS: Dominant selection of an invariant T cell antigen receptor in response to persistent infection by Epstein-Barr virus. ] Exp Med 1994, 180:2335-2340. Bertoletti A, Costanzo C, Chisari FV, Levrero M, Artini M, Sette A, Penna A, Giuberti 1", Fiaccadori F, Ferrari C: Cytotoxic T lymphocyte response to a wild type hepatitis B virus epitope in patients chronically infected by variant viruses carrying substitutions within the epitope. J Exp Med 1994, 180:933-943.
Franco A, Southwood S, Arrhenius 1-, Kuchroo VK, Grey HM, Sette A, Ishioka G: T cell receptor antagonist peptides are highly effective inhibitors of experimental allergic encephalomyelitis. Eur J Immunol 1994, 24:940-946. In vivo validation of the TCR antagonism concept. TCR antagonism peptides are able to inhibit all experimental autoimmnue disease, such as experimental autoimmune encephalomyelitis, mediated by a polyclonal T-cell response. TCR antagonism is shown to be much more efficient than MHC blockade.
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Markowitz M: Rapid turnover of plasma virions and CD4 lymphocytes in HIV-t infection. Nature 1995, 373:123-126. 61.
Koziel MJ, Dudley D, Afdhal N, Choo W-L, Houghton M, Ralston R, Walker BD: Hepatitis C virus specifici cytotoxic T lymphocytes recognize epitopes in the core and envelope proteins of HCV. J Virol 1993, 67:7522-7532.
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Simmonds P: Variabilityof hepatitis C virus. Hepatology 1995, 21:570--583.
58. °"
59.
Kuchroo VK, Greer JM, Kaul D, Ishioka G, Franco A, Sette A, Sobel RA, Less MB: A single TCR antagonist peptide inhibits experimental allergic encephalomyelitis by a diverse T cell repertoire. J Immunol 1994, 153:3326-3336.
A Franco and FV Chisari, The Scripps Research Institute, Department of Molecular and Experimental Medicine, 10666 North Torrey Pines Road, La Jolla, California 92037, USA. E-mail:[email protected] C Ferrari, Universita degli Studi di Parma, Cattedra di Malattie lnfettive, Via A. Gramsci 14, 43100 Parma, Italy. A Sette, Cytel Corporation, 3525 John Hopkins Court, San Diego, California 92121, USA. Author for correspondence: FV Chrisari.
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Host strategies to clear viruses During the first few critical days after infection of an immunologically naive host, viral spread is controlled by a complex network of physiological events that
control viral entry, replication and dissemination, and also by the innate immunity o f the host, which is primarily mediated by the NK-cell system [1°°]. Soon thereafter, virus-specific T cells and antibodies play a crucial role in ehminating cells infected with virus, preventing cell-to-cell spread, and clearing extracellular viral particles [2°°]. The balance obtained between the capacity o f the virus to replicate and spread, and the ability o f the host to eliminate the virus during this early period probably determines the long-term outcome of the infection. During a viral infection, endogenously synthesized viral antigens are processed in a non-endosomal compartment and the derived peptides are transported to the lumen of the endoplasmic reticulum where they bind class I M H C molecules for presentation to CD8 + cytotoxic T lymphocytes (CTLs) [3,4]. In the case o f extracellular antigens, processing usually occurs in an endosomal compartment of professional antigen-presenting cells (APCs) (i.e. B cells, macrophages and dendritic cells) [3,5°°], and the derived peptides bind to class II M H C molecules for presentation to CD4 + T cells. T cells use an antigen-specific cell surface receptor (TCR) to recognize peptide ligands bound to MHC. This interaction initiates a signal that can induce T-cell activation and proliferation when it is coordinated with a second signal that is derived from the interaction of various adhesion molecules on the T cell and APC [6,7]. Importantly, when T C R recognition occurs in the absence of this costimulatory signal, the T cell may be anergized or undergo programmed cell death (apoptosis) [8]. As costimulatory molecules are preferentially expressed by professional APCs, infected somatic cells lacking costimulatory molecules may
Abbreviations APC--antigen-presenting cell; APL--altered peptide ligand; HBV--hepatitis B virus; HCV--hepatitisC virus; INF--interferon; IL--interleukin; TCR--T-cell receptor; Th--T helper. 524
© Current Biology Ltd ISSN 0952-7915
Viral mutations,TCR antagonismand escapefrom the immune responseFranco et paradoxically anergize virus-specific T cells thereby giving the virus an intrinsic advantage relative to the specific immune response. When conditions are optimal, however, T cells, especially CTLs, can clear viruses from infected cells by at least two specific mechanisms, the kinetics and relative strength of which influence the outcome of the encounter between virus and host: • Destruction of infected cells. CTLs lyse infected cells via perforin-granule exocytosis, as has been elegantly demonstrated in perforin knockout mice [9"'], or by inducing programmed cell death (apoptosis) through activation of the Fas, or other, intrinsic cell-death pathways [10,11,12]. Cell lysis is the classical mechanism generally considered responsible for control and/or clearance of most virus infections. In the setting of a massive viral infection, however, the number of infected cells and the virus production rate may be too high to be controlled by this process, which requires each infected cell to be reached and killed by individual CTLs [11,12]. Given the constraints of CTL movement in solid tissues [13], and the possibihty that CTLs may actually be inactivated by infected cells that do not provide the necessary costimulation required for continued CTL expansion, relatively few target cells may actually be killed by each CTL. This raises the possibility that certain infections may not be controllable by the direct CTL-mediated killing of infected cells. • Intracellular inactivation of virus in infected cells. CTLs can also inactivate the expression and replication of certain viruses non-cytolyticany by releasing antiviral cytokines in the infected tissue, possibly 'curing' infected cells that they can not physically reach in vivo. This mechanism has been shown to be responsible for the clearance of hepatitis B virus (HBV) from the hver in an HBV transgenic mouse model [14"] where it is 10- to 100-fold more effective at viral clearance than direct hepatocyte lysis. Similar non-cytolytic immunological mechanisms have been shown to downregulate HIV replication in vitro [15]. The balance between the destructive and curative functions of the immune response could influence the outcome of an infection. For example, if a given virus is not susceptible to control by T cell derived cytokines, or if the T-cell response to a given virus fails to produce the cytokines to which that virus is susceptible, viral clearance would depend entirely on the ability of a bruited number of CTLs to reach, recognize, and destroy all of the infected cells in the body. This may not be possible if the balance between the number of CTLs and the number of infected cells is unfavorable for the host. Under these conditions the infection could easily become persistent and the continuous, but subtotal, destruction of infected cells could actually cause a chronic inflammatory disease in the infected tissue. In addition to CD8 + CTLs, virus-specific CD4 + T lymphocytes also play an important role in viral clearance
al.
[16]. CD4 + T lymphocytes of the T-helper cell type 1 (Thl) phenotype produce the same lymphokines as CD8 + CTLs and can kill target cells in vitro, although their cytolytic activity in vivo has not been established [17]. In addition, Th cells may be required for CTL induction and expansion [18] and for establishment and maintenance of CTL memory [19]. Furthermore, Th cells provide help for antibody responses [20], which are important in limiting viral spread.
Virus strategies to evade the host immune system Viruses have evolved several mechanisms to avoid recognition by host T cells and antibodies in order to establish persistent infection. These strategies include integration into the host genome [21]; silencing of viral gene expression (latency) [22]; inhibition of antigen processing and presentation [23-27]; synthesis of proteins homologous to known immune regulatory molecules [23,28]; infection of immunologically privileged sites that cannot be reached by virus-specific T cells, or that do not express class I or costimulatory molecules; mutations that either inhibit viral responsiveness to antiviral cytokines elaborated during the immune response (M Mattoubian M, Reddy K, Pedroza Martins L, Ahmed R, abstract, Proceedings IXth International Congress of Virology, Berlin, August 1993) preclude recognition by neutralizing antibodies [23], or modify residues that are critical for recognition by the M H C [29] or the T C R [30,31]. The latter can inactivate a T-cell epitope either by decreasing its binding at~inity for the M H C or T C R , or by actively anergizing or antagonizing the T-cell response to the wild-type epitope. Indeed, it has been recently reported that naturally occurring mutations within certain CTL epitopes in HBV [32"'] and HIV [33 "°] can code for viral peptides that function as T C R antagonists for wild-type epitope-specific CTLs. The mechanisms by which these altered peptide ligands (APLs) can interfere with T-cell recognition will now be discussed in some detail.
TCR antagonism and partial agonism Recently [34"',35"'], several groups have demonstrated that non-antigenic antigen analogs could partially or completely inhibit T-cell function. It is now known that changing the structure of the 'hgand with which the T C R interacts (i.e. converting an antigen-MHC complex to an antigen analog-MHC complex) can change the nature of the signal(s) transmitted to the T cell, with a corresponding change in the T-cell response. Two responses can occur when an antigen analog (i.e. an APL) engages the T C R : T C R antagonism and partial agonism.
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Immunity to infection T C R antagonism is a process whereby an APL carrying substitution(s) within a major T C R contact residue inhibits T-cell activation by the normal stimulatory antigen [36]. T C R antagonists effectively inhibit certain T-cell functions, such as T-cell proliferation or upregulation of IL-2 receptors in response to their natural ligands [34°°,37]. In addition, T C R antagonists can also inhibit early intracellular events associated with T-cell activation, such as Ca 2+ influx and phosphatidyl inositol turnover [37]. Partial agonism occurs when an APL can stimulate one T-cell function (agonism), while failing to stimulate or even inhibiting another (antagonism). In this case, the APL will elicit some, but not all, of the signals required for T-cell activation. For example, an HB-specific, I-Ek-restricted T-cell clone has been described that, when stimulated by APL, produces IL-4 but cannot proliferate [38]. Using the same antigenic system, Thl clones are able to display cytolytic activity, but they are unable to proliferate or produce lymphokines in the presence of APLs [39]. Particularly intriguing is the fact that some partial agonists are able to induce a profound and long-lasting state of T-cell anergy [40,41°°].
Molecular mechanisms How can T-cell activation be prevented or dampened when APLs interact with the TCR? The simple mechanism of receptor saturation by APL can be discarded for stoichiometric reasons as APL can antagonize the response to native antigen at very low molar ratios [34"°]. Instead, analysis of the relationship between structure and activity of T C R antagonists suggests that affinity plays an important role. According to this hypothesis, a certain a~nity threshold is required for T C R signaling - - engagement of the T C R below this threshold can result in T C R antagonism [42]. A model has been proposed [34 °'] suggesting that when antigen-MHC and A P L - M H C complexes are both present simultaneously on the cell surface, mixed oligomers will form with some T C R engaging antigen-MHC complexes and others engaging A P L - M H C complexes. According to this model, the mixed T C R - M H C - p e p t i d e clusters are incapable of proper signaling, probably because these mixed clusters are unstable, causing the T cell and APC to dissociate before the T C R can engage the critical number ofstimulatory antigen-MHC complexes required for T-cell activation, thus resulting in inhibition. To understand the events involved in T C R antagonism requires an appreciation of the molecular basis of T C R activation. The T C R is composed of an 0t-~ heterodimer and a multichain invariant CD3 complex. The intracellular tail of each subunit of the CD3 complex contains a tyrosine activation motif, which serves as a substrate for Src family protein tyrosine
kinases [43,44"°]. Tyrosine phosphorylation of the T C R chain and CD3 "?-8-e complex results in the activation of two independent signaling pathways [45,46,47]. APLs fail to trigger phosphorylation of the ~ chain (with physiological phosphorylation of the CD3 3~S-E complex). Two recent studies [48"',49 °'] have identified abnormalities in the signal transduction cascade that are induced when the T C R interacts with APL. In an experimental system where the APL is a previously described anergy-inducer [40], Allen and colleagues [48"'] demonstrated that presentation of APL to resting T-cell clones stimulated a unique pattern of T C R phospho-~ chain species and a consequent lack of association with Zap 70 kinase. Using a different experimental system, where the antagonist is a complex of native antigenic peptide bound to mutant M H C molecules, Germain and colleagues [49 °.] also demonstrated k-chain phosphorylation without Zap 70 activation. If these events can negate the signals that are normally induced by T C R contact with its nominal antigen-MHC complex, it might explain why very low molar ratios of APL to wild-type peptide can antagonize T-cell responses in some viral systems. Viral mutants that express T C R antagonist or partial agonist peptides should be able to evade specific T-cell responses in vivo. Indeed, the occurrence of T C R antagonist mutations during HBV and HIV infection has been recently demonstrated (see below).
Effect of viral mutations on T-cell recognition Mutations occur randomly in all viruses at frequencies that are determined by the enzymes they use for replication. They are particularly common in R N A viruses (e.g. hepatitis C virus [HCV] and HIV) and in certain DNA viruses (e.g. HBV) that use R N A polymerase or reverse transcriptase, respectively, to replicate, because these enzymes lack proof-reading activity. Mutations that increase the intrinsic growth rate of a virus could be positively selected, and those that decrease host recognition should be negatively selected by the immune response. The latter can theoretically include mutations that interfere with the ability of a virus to respond to antiviral cytokines, as well as mutations that inhibit the binding of antigenic peptides to the M H C or to the T C R , and these can either fail to activate, partially activate or antagonize the responsiveness of T cells that recognize the original wild-type antigenic peptide. The binding of antigenic peptides to M H C molecules and recognition of the resulting peptide-MHC complex by the T C R involves interaction of specific amino acid residues of the peptide with corresponding determinants in both the M H C and the T C R [3]. Amino acid changes in antigenic viral peptides that affect M H C anchor residues can abolish or reduce their binding to
Viral mutations, TCR antagonism and escape from the immune response Franco et al. M H C class I molecules and result in their selection due to decreased immune recognition. This mechanism has been invoked to explain the prevalence o f a particular Epstein-Barr virus (EBV) isolate in a population that displays an uncommonly high frequency of the HLAA l l allele. The variant virus contains a single mutation within a CTL epitope that profoundly reduces its binding to HLA-A11 class I molecules, thus abrogating T-cell recognition o f this epitope by CTLs that are restricted to HLA-A11 [29]. These results suggest that variant EBV strains can be selected by CTLs leading to their prevalence at the population level, perhaps over an extended period o f time. Whether this process can also represent a primary cause o f viral persistence in individual patients remains uncertain in view of the enormous coding capacity of this virus, the exceptional strength o f the EBV-specific T-cell response, and the polymorphism of the human MHC. Mutations that affect TC1L binding residues within a viral epitope can also contribute to viral escape by creating peptides that affect T-cell recognition. In most cases, the mutant peptide will still function as a T C R agonist. In other cases, however, the mutation can abolish T C R recognition or actively inhibit the T-cell response by creating peptides that function as T C R antagonists or partial agonists. Viral escape due to mutational loss of a T C R contact site within a CTL epitope has been demonstrated in a murine model in which mice bearing a transgenic T C R specific for an immunodominant lymphocytic choriomeningitis virus epitope (glycoprotein residue 32-42) were unable to clear the virus after infection [50]. In this system, a spontaneous mutation occurred in the epitope that abolished its antigenicity for the transgenic T C R . As the immune response in these animals was focused exclusively on that epitope, the variant virus was selected. This model represents a good example of the profound effect that viral mutations can have when the T-cell response is monoclonal and selectively focused on the epitope where the mutation arises. This kind of immune response does not usually occur, however, during most viral infections in man. Nonetheless, loss o f C T L recognition has been described in a longitudinal study of HIV infected patients where CTL responses to several HLA-B8 restricted CTL epitopes in the HIV gag protein waned as a result o f the mutational loss o f T C R contact sites in the wild-type peptides [30]. At the same time, however, other mutations emerged in an HLA-B27 restricted CTL epitope in the same HIV gag protein that created peptide agonists that were more immunogenic than the wild-type sequence. Thus, although the principle of immune selection o f viral escape mutants due to loss o f antigen recognition is well established, its role in viral persistence during most natural viral infections still remains to be fully clarified. Mutational inactivation of a single viral epitope should lead to persistence only if it confers a survival advantage to the mutant that cannot be counteracted by
the residual immune response to all o f the remaining epitopes. On the one hand, this is unlikely when the CTL response is vigorous and multispecific as it is during the acute stage of various viral infections [51,52,53]. Similarly, immune selection o f mutant viruses is not likely to occur when the immune response is weak or undetectable as it is during many persistent viral infections [51,52]. O n the other hand, oligoclonal expansions o f T cells carrying the same TC1L V~ chains have been described in some patients acutely infected by HIV [54], suggesting either that some epitopes are strongly immunodominant, or that these patients may express such a limited repertoire o f antiviral effector cells allowing viral escape to occur. Similar findings of monoclonal or oligoclonal TCIL expansions have also been reported during other persistent viral infections [55,56,57]. Viral mutations that affect recognition of an epitope by some CTL clones do not automatically affect all CTL clones specific for the same epitope. In fact, different T-cell clones specific for a given epitope can bind different T C R contact residues [58"°,59]. Thus, the situation is very complex, and longitudinal studies correlating the C T L response against multiple viral epitopes, with the sequence o f those epitopes during the evolution of an acute to a persistent infection are needed to clarify the actual pathogenetic role o f mutations in viral persistence.
Role of TCR antagonism in viral infections In addition to its involvement in thymic education, peripheral tolerance, autoreactive T-cell responses, and T-cell memory [34"°,35°°], T C R antagonism may also play a role in viral escape from immune recognition. For example, in two recent reports, naturally occurring variants of the HBV [32 °°] and the human immunodeficiency virus 1 (HIV-1) [33 °°] that contained mutations at T C R contact sites in immunodominant epitopes were shown to antagonize CTL recognition of the corresponding wild-type epitopes in selected chronically infected patients. In both instances, antagonism occurred at very low concentrations of the variant peptide, much lower than the concentration required to induce activation by the native antigen. This was surprising considering that in other experimental systems an equimolar dose or an excess of antagonist peptide is required to inhibit the T-cell response [36,37,58°°]. The recent demonstration of negative signals delivered to the TClq. by A P L - M H C complexes due to improper phosphorylation o f the CD3 ~ chain, [48",49 °°] may help to explain these results. The importance o f this phenomenon in both the HBV and HIV model is illustrated by the fact that recognition of the corresponding naturally processed wild-type epitopes was also efficiently antagonized by the variant peptides. In general, the antagonist viral peptides in these reports could not sensitize target cells for lysis by CTL clones
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Immunityto infection specific for the wild-type peptide, except at high concentrations or after prolonged incubation, and this is compatible with the notion that antagonism results from a weak interaction between the antagonist peptide and the T C R [42]. Interestingly, some APLs were antigenic at high doses and antagonistic at low doses, suggesting that the immunological consequences of this kind of viral mutation could be determined by the amount of antigen present and, therefore, by viral load. The antagonist effect in the HBV system was not limited to cytotoxicity, as IFN-3' production was also inhibited by mutant epitopes in some of the T-cell clones studied. Most of the antagonist peptides were poorly immunogenic compared to the wild-type peptide, suggesting they were also unable to elicit an efficient alternative T-cell response to themselves [57"]. Collectively, these results suggest that naturally occurring viral variants may be able to specifically inhibit the destruction of cells that simultaneously express both wild-type and variant viruses at their surface, and thereby protect the cells containing mutant virus from immune elimination by CTL specific for the wild-type epitope (negative selection). Furthermore, in one instance it was shown that CTL recognition of a wild-type HIV peptide on one target cell could be antagonized by recognition of a variant peptide present on a different cell [33"]. Although this 'trans-antagonism' phenomenon was not observed in the HBV system, and is otherwise difficult to reconcile with our current understanding of the molecular basis for antagonism, these results raise the possibility that the presence of antagonistic epitopes on cells containing a mutant virus might be able to protect neighboring cells containing wild-type virus.
Different viruses, different scenarios Non-cytopathic persistent viruses must be able to evade immune surveillance either by not inducing an effective antiviral immune response or by evolving strategies to escape an otherwise efficient response. The importance of the frst scenario is illustrated by the well-documented fact that the T-cell response to HBV appears to be weak and inefficient in patients with chronic HBV infection, whereas it is vigorous and multispecific in patients with acute HBV infection who successfully clear the virus [2"']. The recent report [32"] that naturally occurring viral mutants could act as TCI< antagonists in two patients with chronic HBV infection, suggests that selection of TClk antagonist peptides may also contribute to the persistence of this virus in some patients. It should be noted, however, that mutations within known CTL epitopes do not appear to be commonplace in chronic hepatitis (B R.ehermann and FV Chisari, unpublished data). Furthermore, the characteristically weak immune response to HBV in most chronically
infected patients is probably not strong enough to select for the outgrowth of mutant viruses. These observations suggest that CTL escape mutation may not be primarily responsible for HBV persistence in most patients who are chronically infected by this virus, even though mutations could contribute to HBV persistence when the CTL response is narrowly focused. On the basis of available data, however, this type of immune response, and the corresponding selection pressure, does not appear to be a common event during HBV infection. The situation may be somewhat different in early chronic HIV infection where the CTL response is characteristically very strong yet, like the response to HBV, it is unable to clear the virus [54,55]. The incredibly high rate of HIV production and the exceptionally high mutation rate of this virus may cause so many different viruses to be generated each day that they exceed the capacity of the immune system to respond to them effectively simply on a numerical basis [60]. In this regard, the ostensibly vigorous immune response to the virus appears to be unable to compete with its capacity to replicate and its ability to generate mutants. Mutational inactivation of CTL epitopes might thus play an earher and more important role in the estabhshment of viral persistence for this virus than for HBV. It is important to emphasize, however, that the overwhelming rates of viral replication and spread relative to the ability of the immune system to produce enough CTLs to reach and destroy all of the infected cells, plus the immunosuppressive effects of the virus itself, may be more important than mutations for the development of persistent infection. The situation may be different again during chronic HCV infection where an extensive quasi-species of viral variants can coexist with a multispecific CTL response [53,61] that is intermediate in strength between the response of patients chronically infected by HBV and HIV. Unlike HBV and HIV where the viral titer is high, the viral titer is very low during chronic HCV infection [62], so viral persistence cannot be ascribed to an overwhelming viral load in this instance. In this setting, therefore, escape mutants may play a greater role in the primary establishment of HCV persistence than is likely for these other viruses.
Conclusions We suggest that the balance between the kinetics and magnitude of viral spread and the immune response during the early days of an infection probably determines the ultimate outcome of infection by viruses that have the capacity to become persistent. An ineffective immune response could be due to many factors, including neonatal tolerance, immunological exhaustion by high viral loads, infection ofimmunologically privileged sites, modulation of recognition molecules on the surface of
Viral mutations, TCR antagonism and escape from the immune response Franco et al. infected cells, immune suppression and altered cytokine production. One o f the viral factors that could influence this balance is the development o f mutations that nullify, deviate, or antagonize the antiviral T-cell response. This mechanism should cause viral persistence, however, only if the residual immune response to the remaining non-mutated viral epitopes is unable to clear the virus. In view o f the multispecificity of the CTL response to most viruses that ultimately persist in their host, current data favor the notion that negative selection o f CTL escape mutants is most likely to occur only after a persistent infection is already established. In this setting, occurrence o f these viral mutations could solidify the chronicity o f the infection and perhaps even make it irreversible. Whether such mutations can also serve as the primary cause o f viral persistence in the context o f a hierarchical multispecific T-cell response remains to be proven.
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Acknowledgments The authors thank Janette Sanders for preparation of the manuscript. These studies were supported by grants R O I A120001, CA40489, AI26626 and RR00833 from the National Institutes of Health. This is manuscript number 9298-MEM from The Scripps Research Institute.
14. ""
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A Franco and FV Chisari, The Scripps Research Institute, Department of Molecular and Experimental Medicine, 10666 North Torrey Pines Road, La Jolla, California 92037, USA. E-mail:[email protected] C Ferrari, Universita degli Studi di Parma, Cattedra di Malattie lnfettive, Via A. Gramsci 14, 43100 Parma, Italy. A Sette, Cytel Corporation, 3525 John Hopkins Court, San Diego, California 92121, USA. Author for correspondence: FV Chrisari.
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