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Understanding cytotoxic T-lymphocyte escape during simian immunodeficiency virus infection
Abstract
Infection of rhesus macaques with simian immunodeficiency virus (SIV) is an excellent model system for studying viral adaptation to immune responses. In this review, we discuss how the SIV-infected macaque has provided unequivocal evidence for cytotoxic T-lymphocyte (CTL) selection of viral escape variants. This improved understanding of CTL escape may influence human immunodeficiency virus (HIV) vaccine design as well as our understanding of HIV pathogenesis.
... Immunodominance in CTL response is influenced by several factors [13], including MHC expression [14], T cell avidity [15, 16], proteosome processing [17], frequency of na¨ıvena¨ıve precursors specific for a viral epitope [18], and viral sequence variability [19]. It has been shown that in SIV infection CTL selective pressure is a cause of viral escape mutations20212223, and this phenomenon is one, but not the only, cause of vaccine failure [24, 25]. Escape variants have been identified and characterized in Mamu A * [26, 27] , B * [23], and B * 17-restricted [25] CTL epitopes, and it has been shown that these viral escape variants are poorly recognized by subsequent MHC-matched hosts but can revert to wild type if CTL selective pressure is removed by transmitting the mutated viruses to MHC mismatched animals282930; interestingly, SIV can also adapt to the fitness cost of certain escape mutations by generating compensatory mutations [29]. ...
... In addition, this increase in viral replication occurred despite late-emerging CTL responses of similar magnitude to the one seen for dominant epitopes. Previous studies using animals with Mamu A * 01 and/or Mamu A * 02 alleles have demonstrated that the evolution of CTL responses to subdominant epitopes can arise after loss of Mamu A * 01 immunodominant responses [36], but a loss of immunodominant responses is followed by increased viral replication, even if normal CTL responses to subdominant epitopes (Pol) are present [20, 37]; our data from the Mamu A * 01 + animal 16040 (Figure 2) agrees with this observation. In the same line of evidence, and outside the context of the Mamu A * 01 allele expression, the loss of the initial immunodominant Vif CTL epitope in animal 16044, resulted in increased viral replication and disease progression despite strong surge of subdominant CTLs (Figure 1). ...
The simian immunodeficiency virus- (SIV-) infected rhesus macaque is the preferred animal model for vaccine development, but the correlates of protection in this model are not completely understood. In this paper, we document the cytotoxic T lymphocyte (CTL) response to SIV and its effects on viral evolution in an effort to identify events associated with disease progression regardless of MHC allele expression. We observed the evolution of epitopes targeted by CTLs in a group of macaques that included long-term nonprogressing (LTNP), slowly progressing (SP), normally progressing (NP), and rapidly progressing (RP) animals. Collectively, our data (1) identify novel CTL epitopes from an SP animal that are not restricted by known protective alleles, (2) illustrate that, in this small study, RP and NP animals accrue more mutations in CTL epitopes than in SP or LTNP macaques, and (3) demonstrate that the loss of CTL responses to immunodominant epitopes is associated with viral replication increases, which are not controlled by secondary CTL responses. These findings provide further evidence for the critical role of the primary cell-mediated immune responses in the control of retroviral infections.
... Alternatively, SIV induced disease in rhesus monkeys represents an invaluable model system for studying the pathogenesis of human AIDS [18], because like humans, most rhesus monkeys develop AIDS after infection. These animals have been particularly useful in studying and understanding the initial phases of infection, and detailed studies of changes in viral sequences from infected animals over time as a function of CTL reactivity has enhanced our understanding of the means by which the virus escapes immune control [19]. Thus, comprehensive consideration of data generated from multiple experimental approaches is key to determining of the role of MHC class I in controlling HIV-1. ...
... SIV infection in non-human primates has served as an invaluable model for understanding AIDS pathogenesis (in rhesus monkeys) and viral resistance (in chimpanzee). The effect of rhesus MHC class I molecules on the evolution of SIV has been convincingly described [19], and a recent study in humans has suggested that selection pressure conferred by HLA molecules is responsible for specific genetic variation in HIV-1 [114]. HIV-1 may eventually have conspicuous evolutionary effects on HLA and other AIDS restriction genes, a prolonged process that could have occurred in chimpanzee [92].To prevent such an outcome, it will be necessary to approach the disease from many perspectives, and apply comprehensively the knowledge gained to the successful control of the virus. ...
Data indicate that resistance to HIV-1 disease involves an array of contrasting HLA genotypic effects that are subtle, but significant, particularly when these genetic effects are considered as a whole. Numerous reports attributing a role for HLA genotype in AIDS outcomes have been reported, and a few of these have been affirmed in multiple studies. Functional studies of immune cell recognition have provided clues to the underlying mechanisms behind some of the strongest HLA associations, suggesting the means by which relative resistance or susceptibility to the virus may occur. SIV infection in non-human primates has served as an invaluable model for understanding AIDS pathogenesis (in rhesus monkeys) and viral resistance (in chimpanzee). The effect of rhesus MHC class I molecules on the evolution of SIV has been convincingly described [19], and a recent study in humans has suggested that selection pressure conferred by HLA molecules is responsible for specific genetic variation in HIV-1 [114]. HIV-1 may eventually have conspicuous evolutionary effects on HLA and other AIDS restriction genes, a prolonged process that could have occurred in chimpanzee [92]. To prevent such an outcome, it will be necessary to approach the disease from many perspectives, andapply comprehensively the knowledge gained to the successful control of the virus.
... In contrast, escape variants of TL8 restricted by Mamu-A*01 are diversified and they appear very early postinfection, implying that this part of the Tat protein is nonessential and can tolerate mutations without compromising survival (26,27,30). Although the most dramatic loss in binding to Mamu-A*01 was caused by mutations in the P2 and P8 anchor positions, CTL escape can be achieved by mutations in any of the eight residues (26). ...
... Although the most dramatic loss in binding to Mamu-A*01 was caused by mutations in the P2 and P8 anchor positions, CTL escape can be achieved by mutations in any of the eight residues (26). In this study, we discuss the structural consequences of several predominant mutations, namely, T28P (P1 residue, also known as "S28P" in some studies), T29I (P2), S32L (P5), L35Q, L35P, and P35R (P8 residue) (26,30). The Tat T29I mutation is analogous to the Gag T182I mutation at the P2 location, resulting in a side chain that is unable to fit in the B pocket, as discussed above. ...
The infection of rhesus macaques (Macaca mulatta) by the SIV is the best animal model for studying HIV infection and for AIDS vaccine development. A prevalent MHC class I allele, Mamu-A*01, is known to correlate with containment of SIV, which has been extensively explored in studies of CTL-based vaccination concepts. We determined the crystal structures of Mamu-A*01 complexed with two immunodominant SIV epitopes: the nonamer CM9 of group-specific Ag (Gag, 181-189; CTPYDINQM) and the octamer TL8 of transcription activator (Tat, 28-35; TTPESANL). The overall structures of the two Mamu-A*01 complexes are similar to other MHC class I molecules. Both structures confirm the presence of an absolutely conserved proline anchor residue in the P3 position of the Ag, bound to a D pocket of the Mamu-A*01 H chain with optimal surface complementarity. Like other MHC/peptide complex structures, the P2 and C-terminal residues of the epitopes are also important for anchoring to the MHC molecule, whereas the middle residues form an arch and their side chains are directed into solvent. These two structures reveal details of how Mamu-A*01 interacts with two well-studied epitopes at the atomic level. We discuss the structural basis of CTL escape, based on molecular models made possible by these two structures. The results we present in this study are most relevant for the rational design of Mamu-A*01-restricted CTL epitopes with improved binding, as a step toward development of AIDS vaccines.
... In vivo, viral peptides are presented to CTLs bound on major histocompatibility class I (MHC-I) molecules, thereby dictating with their binding specificity the potential repertoire of CTL responses. Thus, it is important to understand the MHC-I restriction of CTL responses and its role in host immunity and viral diversification [11][12][13][14]. ...
An effective HIV vaccine will need to stimulate immune responses against the sequence diversity presented in circulating virus strains. In this study, we evaluate breadth and depth estimates of potential T-cell epitopes (PTEs) in transmitted founder virus sequence-derived cohort-specific peptide reagents against reagents representative of consensus and global sequences. CD8 T-cells from twenty-six HIV-1+ PBMC donor samples, obtained at 1-year post estimated date of infection, were evaluated. ELISpot assays compared responses to 15mer consensus (n = 121), multivalent-global (n = 320), and 10mer multivalent cohort-specific (n = 300) PTE peptides, all mapping to the Gag antigen. Responses to 38 consensus, 71 global, and 62 cohort-specific PTEs were confirmed, with sixty percent of common global and cohort-specific PTEs corresponding to consensus sequences. Both global and cohort-specific peptides exhibited broader epitope coverage compared to commonly used consensus reagents, with mean breadth estimates of 3.2 (global), 3.4 (cohort) and 2.2 (consensus) epitopes. Global or cohort peptides each identified unique epitope responses that would not be detected if these peptide pools were used alone. A peptide set designed around specific virologic and immunogenetic characteristics of a target cohort can expand the detection of CD8 T-cell responses to epitopes in circulating viruses, providing a novel way to better define the host response to HIV-1 with implications for vaccine development.
... There is a growing acceptance that CTL mediated viral clearance plays a critical role in controlling disease progression in pathogenic SIV infections (Kuroda et al 1999;Schmitz et a l.1999;Allen et al. 2001;Vogel et al. 2002). As discussed in Chapter 4, a broad sustained CTL response is often observed in the asymptomatic infections o f SIVsm, whereas the virulent infections o f macaque is characterised by the decline o f CTL activities. ...
I investigated variable selective pressures among amino acid sites in HIV-1 genes. Selective pressure at the amino acid level was measured using the nonsynonymous/synonymous substitution rate ratio. Likelihood ratio tests detected positive selection in every gene in the genome, with the majority located in gp160. Most HIV-1 genes were evolving at a subtype specific manner. As adaptive evolution is driven chiefly by immune detection, this change of selective constraint was indicative of variations in immune targeting. Differences in selective pressure contributed to the extensive genetic diversity observed across the genome and could be viewed as co-evolution of the subtypes. I measured the physiochemical properties of amino acids and found that those at positive selection sites were more diverse than those at variable sites. Furthermore, amino acid residues at exposed positive selection sites were more physiochemically diverse than at buried positive selection sites. I also examined the evolution of HIV-2 and SIVmac after the cross-species transmissions. My results indicated that HIV-2 did not appear to be evolving at a faster rate than the progenitor lineages. It is possible that fewer adaptive changes in the progenitor virus were required for successful infection. A notably different pattern was observed for SIVmac lineages. My findings showed that SIVmac lineages appeared to be evolving much faster than HIV-2. Also a fraction of sites in SIVmac lineages that were evolving by relaxed functional constraint became positively selected post zoonosis.
... This escapecornering strategy has long been proposed as a theoretical immune tactic against HIV (O'Connor et al., 2001;Altfeld and Allen, 2006; Allen and Altfeld, 2008) and has been mathematically modeled (Ferguson et al., 2013). That such a strategy can be effective is demonstrated for the first time here. ...
Recent studies have suggested greater HIV cure potential among infected children than adults. A major obstacle to HIV eradication in adults is that the viral reservoir is largely comprised of HIV-specific cytotoxic T lymphocyte (CTL) escape variants. We here evaluate the potential for CTL in HIV-infected slow-progressor children to play an effective role in “shock-and-kill” cure strategies. Two distinct subgroups of children were identified on the basis of viral load. Unexpectedly, in both groups, as in adults, HIV-specific CTL drove the selection of escape variants across a range of epitopes within the first weeks of infection. However, in HIV-infected children, but not adults, de novo autologous variant-specific CTL responses were generated, enabling the pediatric immune system to “corner” the virus. Thus, even when escape variants are selected in early infection, the capacity in children to generate variant-specific anti-HIV CTL responses maintains the potential for CTL to contribute to effective shock-and-kill cure strategies in pediatric HIV infection.
... They are highly virulent CCR5 tropic viruses that cause progression to AIDS in most infected animals within one year of infection. Similar to HIV-1, SIV infections cause a progressive loss of viral control due to the destruction of CD4+ T cells, CD8+ T cell exhaustion, and the selection of viral mutations that result in CD8+ T cell and neutralizing antibody escape (18)(19)(20)(21). The rapid onset of SIV-induced AIDS in rhesus macaques contrasts with most HIV-1 infections where AIDS does not develop until 8 to 10 years of infection. ...
... We suspect the adaptive responses generated to Gag and Env were unable to eliminate viral targets but succeeded in inducing an environment of chronic inflammation upon recognition, but not elimination, of their antigens. A primary mechanism by which HIV-1 evades immune recognition is by evolution within epitopes that is secondary to CTL selective pressures (9,10,35,43,58,(244)(245)(246)(247)271). Our observation that positive selection was not specific to CTL epitopes suggests an efficient epitope-specific response was not generated during natural HIV-1 infection. ...
Human immunodeficiency virus type-1 (HIV-1) affects more than 30 million people worldwide and has accounted for over 30 million deaths. The advent of combination antiretroviral therapy (cART) resulted in a drastic decrease in AIDS-associated morbidity and mortality. Despite this, cART fails to completely eradicate the virus from infected patients, as cessation of treatment results in a rebound in viremia and a resumption of disease progression. Partial immune reconstitution is achieved under suppressive therapy, thus causing research efforts to begin development of curative strategies for HIV-1-infected patients. We believe the best method of HIV-1 eradication will be through cytotoxic T lymphocyte (CTL) elimination of the latently-infected reservoir, which may be difficult given the propensity of the virus to undergo mutations that evade CTL recognition. Dendritic cells, the most potent antigen-presenting cells, can reveal broad and robust HIV-1-specific T cell responses in subjects on cART and can prime CD8+ T cells specific for various HIV-1 antigens. We therefore believe the best method of inducing a potent anti-HIV-1 CTL response will be through the use of a DC-based immunotherapy targeting the patient’s own, unique (autologous) virus. It is unclear, however, if the naïve T cell repertoire and function has been sufficiently restored to respond to autologous virus containing multiple mutational variants. In the present study, we longitudinally evaluated autologous HIV-1 evolution and changes in T cell responses throughout untreated and treated HIV-1 infection in subjects from the Multicenter AIDS Cohort Study (MACS). We show that dendritic cells reveal autologous antigen-specific T cell responses at all stages of disease progression and are potent inducers of polyfunctional T cell responses after long-term suppressive cART. We developed and utilized an in vitro model of DC immunotherapy targeting naïve and memory CD8+ T cell subsets to show for the first time that naïve CD8+ T cells from subjects on cART can be primed to target the latent HIV-1 reservoir. Taken together, these findings shed new light on T cell responses to autologous HIV-1 viral variants and support the use of a DC immunotherapy in subjects on cART.
... In primates, considerable research has been conducted on the Mhc of rhesus (Macaca mulatta) and cynomolgus macaques (Macaca fascicularis), since these species are widely used as models for human diseases and biology. Simian immunodeficiency virus infection of macaques, for instance, is an important model for the study of AIDS [4,7] The organization of the Mhc class I region of rhesus macaque -and probably most of the Old World Monkeys (OWM) -seems to be more complex than in humans and great apes. The Mhc-A and -B genes are shared between humans, great apes, and OWM, but OWM lack the Mhc-C gene, which arose by duplication in the Hominoid lineage [8]. ...
... These forces shape both the within-host dynamics of infections as well as the dynamics of spread on an epidemiological level. At the within-host level, the role of the opposing forces are manifest in chronic infections such as HIV, SIV, and HCV, where it has been shown that the virus population frequently escapes immune control by B-or T-cell responses12345678. Moreover, in HIV/SIV, slow disease progression is associated with efficient immune control via protective human leukocyte antigen (HLA) genes; see91011, and escape events can precipitate the loss of immune control [3,12,13]. ...
Human immunodeficiency virus (HIV-1) is, like most pathogens, under selective pressure to escape the immune system of its host. In particular, HIV-1 can avoid recognition by cytotoxic T lymphocytes (CTLs) by altering the binding affinity of viral peptides to human leukocyte antigen (HLA) molecules, the role of which is to present those peptides to the immune system. It is generally assumed that HLA escape mutations carry a replicative fitness cost, but these costs have not been quantified. In this study, we assess the replicative cost of mutations which are likely to escape presentation by HLA molecules in the region of HIV-1 protease and reverse transcriptase. Specifically, we combine computational approaches for prediction of in vitro replicative fitness and peptide binding affinity to HLA molecules. We find that mutations which impair binding to HLA-A molecules tend to have lower in vitro replicative fitness than mutations which do not impair binding to HLA-A molecules, suggesting that HLA-A escape mutations carry higher fitness costs than non-escape mutations. We argue that the association between fitness and HLA-A binding impairment is probably due to an intrinsic cost of escape from HLA-A molecules, and these costs are particularly strong for HLA-A alleles associated with efficient virus control. Counter-intuitively, we do not observe a significant effect in the case of HLA-B, but, as discussed, this does not argue against the relevance of HLA-B in virus control. Overall, this article points to the intriguing possibility that HLA-A molecules preferentially target more conserved regions of HIV-1, emphasizing the importance of HLA-A genes in the evolution of HIV-1 and RNA viruses in general.
... A related issue is epitope mutation. Numerous reports have demonstrated that HIV, SIV, and HCV CD8 T-cell epitopes can be mutated in response to selective pressure by CD8 T-cell responses (34,87,137). Even mutations that reduce the affinity of MHC binding, TCR interaction, or the efficiency of generation rather than abolish epitope production may considerably reduce T-cell stimulation. ...
Memory CD8 T cells are an important component of protective immunity against viral infections, and understanding their development will aid in the design of optimal vaccines. Recent work has shed light on the complex differentiation process that occurs during a CD8 T-cell response to viral infection. Dramatic cellular changes occur as T cells transition through the three characteristic phases of an antiviral response, initial activation and expansion, the death phase, and the formation of memory T cells. Each of these three phases of the T-cell response is accompanied by extensive transcriptional and functional changes that result in naive T cells expanding and gaining effector functions, survival of 5 to 10% of the effectors through the death phase, and the gradual acquisition of memory properties by the surviving virus-specific T cells. This review will discuss our current understanding of how functional and protective CD8 T-cell responses are generated and maintained following different types of infections.
Viral infections can be largely divided into two types: (i) acute infections, where virus is eliminated; and (ii) chronic infections, where virus persists. This second type of infection may be further classified into latent infections and those in which there is persistent viral replication. While acute infections usually result in effective antiviral immune responses, chronic infections can be associated with suboptimal T-cell function. We will first focus on acute infections and on recent work that has led to our current understanding of the CD8 T-cell differentiation program that occurs when antigen is eliminated following initial infection and then discuss how CD8 T-cell responses can be altered and impaired during chronic infections when virus persists.
... In primates , considerable research has been conducted on the Mhc of rhesus (Macaca mulatta) and cynomolgus macaques (Macaca fascicularis), since these species are widely used as models for human diseases and biology. Simian immunodeficiency virus infection of macaques, for instance, is an important model for the study of AIDS [4,7] The organization of the Mhc class I region of rhesus macaque – and probably most of the Old World Monkeys (OWM) – seems to be more complex than in humans and great apes. The Mhc-A and -B genes are shared between humans, great apes, and OWM, but OWM lack the Mhc-C gene, which arose by duplication in the Hominoid lineage [8]. ...
In sharp contrast to humans and great apes, the expanded Mhc-B region of rhesus and cynomolgus macaques is characterized by the presence of differential numbers and unique combinations of polymorphic class I B genes per haplotype. The MIB microsatellite is closely linked to the single class I B gene in human and in some great apes studied. The physical map of the Mhc of a heterozygous rhesus monkey provides unique material to analyze MIB and Mamu-B copy number variation and then allows one to decipher the compound evolutionary history of this region in primate species.
In silico research pinpointed 12 MIB copies (duplicons), most of which are associated with expressed B-genes that cluster in a separate clade in the phylogenetic tree. Generic primers tested on homozygous rhesus and pedigreed cynomolgus macaques allowed the identification of eight to eleven MIB copies per individual. The number of MIB copies present per haplotype varies from a minimum of three to six in cynomolgus macaques and from five to eight copies in rhesus macaques. Phylogenetic analyses highlight a strong transpecific sharing of MIB duplicons. Using the physical map, we observed that, similar to MIB duplicons, highly divergent Mamu-B genes can be present on the same haplotype. Haplotype variation as reflected by the copy number variation of class I B loci is best explained by recombination events, which are found to occur between MIBs and Mamu-B.
The data suggest the existence of highly divergent MIB and Mamu-B lineages on a given haplotype, as well as variable MIB and B copy numbers and configurations, at least in rhesus macaque. Recombination seems to occur between MIB and Mamu-B loci, and the resulting haplotypic plasticity at the individual level may be a strategy to better cope with pathogens. Therefore, evolutionary inferences based on the multiplicated MIB loci but also other markers close to B-genes appear to be promising for the study of B-region organization and evolution in primates.
... Model viral infections, such as lymphocytic choriomeningitis (LCMV) and simian immunodeficiency virus (SIV), have shown that the CD8 + T cell response is a crucial component in the control or elimination of viral infections [5,6,7]. Moreover, the power of CD8 + T cell responses have been elegantly shown to be a driving force in the selection of escape variants in SIV [8]. The immune response towards HIV-1 is complex; we set out to determine if T cell mediated responses in an HIV-1 infected individual can be stimulated through exposure to exogenous virus. ...
Unprotected sexual intercourse between individuals who are both infected with HIV-1 can lead to exposure to their partner's virus, and potentially to super-infection. However, the immunological consequences of continued exposure to HIV-1 by individuals already infected, has to our knowledge never been reported. We measured T cell responses in 49 HIV-1 infected individuals who were on antiretroviral therapy with suppressed viral loads. All the individuals were in a long-term sexual partnership with another HIV-1 infected individual, who was either also on HAART and suppressing their viral loads, or viremic (>9000 copies/ml). T cell responses to HIV-1 epitopes were measured directly ex-vivo by the IFN-gamma enzyme linked immuno-spot assay and by cytokine flow cytometry. Sexual exposure data was generated from questionnaires given to both individuals within each partnership. Individuals who continued to have regular sexual contact with a HIV-1 infected viremic partner had significantly higher frequencies of HIV-1-specific T cell responses, compared to individuals with aviremic partners. Strikingly, the magnitude of the HIV-1-specific T cell response correlated strongly with the level and route of exposure. Responses consisted of both CD4(+) and CD8(+) T cell subsets. Longitudinally, decreases in exposure were mirrored by a lower T cell response. However, no evidence for systemic super-infection was found in any of the individuals. Continued sexual exposure to exogenous HIV-1 was associated with increased HIV-1-specific T cell responses, in the absence of systemic super-infection, and correlated with the level and type of exposure.
... Utilizing the rhesus macaque/SIV infection model, a suppressive effect on virus replication was shown for CTLs [7]. However, the initial CTL responses are not able to contain the virus at a later stage, possibly due to the emergence of viral variants that evade the immune responses resulting in continued virus replica- tion [8,9] . Hence, an understanding of the CTL escape variants of HIV is important both in natural viral infections and also in the context of vaccine-induced immunity for developing effective CTL based polyvalent vaccines for containing diverse HIV-1 strains [10]. ...
The enormous genetic variability reported in HIV-1 has posed problems in the treatment of infected individuals. This is evident in the form of HIV-1 resistant to antiviral agents, neutralizing antibodies and cytotoxic T lymphocytes (CTLs) involving multiple viral gene products. Based on this, it has been suggested that a comprehensive analysis of the polymorphisms in HIV proteins is of value for understanding the virus transmission and pathogenesis as well as for the efforts towards developing anti-viral therapeutics and vaccines. This study, for the first time, describes an in-depth analysis of genetic variation in Vpr using information from global HIV-1 isolates involving a total of 976 Vpr sequences. The polymorphisms at the individual amino acid level were analyzed. The residues 9, 33, 39, and 47 showed a single variant amino acid compared to other residues. There are several amino acids which are highly polymorphic. The residues that show ten or more variant amino acids are 15, 16, 28, 36, 37, 48, 55, 58, 59, 77, 84, 86, 89, and 93. Further, the variant amino acids noted at residues 60, 61, 34, 71 and 72 are identical. Interestingly, the frequency of the variant amino acids was found to be low for most residues. Vpr is known to contain multiple CTL epitopes like protease, reverse transcriptase, Env, and Gag proteins of HIV-1. Based on this, we have also extended our analysis of the amino acid polymorphisms to the experimentally defined and predicted CTL epitopes. The results suggest that amino acid polymorphisms may contribute to the immune escape of the virus. The available data on naturally occurring polymorphisms will be useful to assess their potential effect on the structural and functional constraints of Vpr and also on the fitness of HIV-1 for replication.
... Finally, viral escape from CTL response has been considered as a major determinant of disease progression [103,117]. CTL pressure on HIV is believed to result in selection of viral mutants that have non-recognizable epitopes by CTL. Recent studies in hepatitis C virus (HCV) infection have indicated that incomplete CTL control of viral infection in the absence of adequate CD4 + T cell help results in the emergence of viral CTL escape mutants [59]. ...
An increasing body of evidence supports a key role for cytotoxic CD8+ T cells (CTL) in controlling HIV infection. Although a vigorous HIV-specific CD8+ T cell response is raised during the primary infection, these cells ultimately fail to control virus and prevent disease progression. The failure of CTL to control HIV infection has been attributed to a number of strategies HIV employs to evade the immune system. Recently, intrinsic defects in the CTL themselves have been proposed to contribute to the failure of CTL to control HIV. HIV-specific CD8+ T cells differ in their effector/memory phenotype from other virus-specific CD8+ T cells indicating that their differentiation status differs. This altered differentiation may affect effector functions as well as homing properties of these cells. Other studies have indicated that activation of HIV-specific CTL may be impaired and this contributes to their dysfunction. The effector function of these CTL may also be affected. There are conflicting reports about their ability to kill, whereas IFNgamma production does not appear to be impaired in these cells. In this review we focus on recent work indicating that apoptosis may be an important mechanism through which HIV evades the CTL response. In particular, HIV-specific CD8+ T cells are highly susceptible to CD95/Fas-induced apoptosis. This leads to the hypothesis that virus-specific cytotoxic T cells can be eliminated upon binding CD95L/FasL on HIV-infected cells. Understanding the intrinsic defects of CTL in HIV infection could lead to new therapeutic strategies and optimized vaccination protocols that enhance the HIV-specific cytotoxic response.
... D8 T lymphocytes exert important immune control over different viral infections (1,2). However, viruses can counteract CTL responses, for example by production of proteins that perturb the mechanisms of Ag processing and peptide transport or by selection of variants with mutated amino acids in the presented peptides (3). ...
Lymphocytic choriomeningitis virus infection of H-2(b) mice generates a strong CD8(+) CTL response mainly directed toward three immunodominant epitopes, one of which, gp33, is presented by both H-2D(b) and H-2K(b) MHC class I molecules. This CTL response acts as a selective agent for the emergence of viral escape variants. These variants generate altered peptide ligands (APLs) that, when presented by class I MHC molecules, antagonize CTL recognition and ultimately allow the virus to evade the cellular immune response. The emergence of APLs of the gp33 epitope is particularly advantageous for LCMV, as it allows viral escape in the context of both H-2D(b) and H-2K(b) MHC class I molecules. We have determined crystal structures of three different APLs of gp33 in complex with both H-2D(b) and H-2K(b). Comparison between these APL/MHC structures and those of the index gp33 peptide/MHC reveals the structural basis for three different strategies used by LCMV viral escape mutations: 1) conformational changes in peptide and MHC residues that are potential TCR contacts, 2) impairment of APL binding to the MHC peptide binding cleft, and 3) introduction of subtle changes at the TCR/pMHC interface, such as the removal of a single hydroxyl group.
... Functional studies of the rhesus immune response can benefit from these data immediately by incorporating the new knowledge of mamu class I and class II gene content into their experimental design. Many of these studies are focused on CTL responses and are looking in detail at the MHC-peptide complexes that are associated with viral escape and disease progression (O'Connor et al. 2001). The consideration of all of the antigen-presenting genes that are available for participation in a specific immune response could reveal in more detail how that response is initiated, how it progresses, and under what circumstances it is ultimately evaded or completed. ...
The major histocompatibility complex (MHC) is comprised of the class I, class II, and class III regions, including the MHC class I and class II genes that play a primary role in the immune response and serve as an important model in studies of primate evolution. Although nonhuman primates contribute significantly to comparative human studies, relatively little is known about the genetic diversity and genomics underlying nonhuman primate immunity. To address this issue, we sequenced a complete rhesus macaque MHC spanning over 5.3 Mb, and obtained an additional 2.3 Mb from a second haplotype, including class II and portions of class I and class III. A major expansion of from six class I genes in humans to as many as 22 active MHC class I genes in rhesus and levels of sequence divergence some 10-fold higher than a similar human comparison were found, averaging from 2% to 6% throughout extended portions of class I and class II. These data pose new interpretations of the evolutionary constraints operating between MHC diversity and T-cell selection by contrasting with models predicting an optimal number of antigen presenting genes. For the clinical model, these data and derivative genetic tools can be implemented in ongoing genetic and disease studies that involve the rhesus macaque.
... It is thought that HIV replication in human and SIV in macaques is asynchronous and varies within and among compartments (Delassus et al., 1992; Martins et al., 1991; Sala et al., 1994). Possibly, this finding relates to the level of viral production and the dynamics of de novo infection of CD4+ T cells by cell-free virus or cell-associated virus and the strength of immunological pressure exerted by CTLs within a given locale (Allen et al., 2000; Borrow et al., 1997; Goulder et al., 1997; Jin et al., 1999; Johnson and Desrosiers, 2002; Kuroda et al., 1999a; O'Connor et al., 2001; Schmitz et al., 1999). Indeed, virus-specific CTL level in different compartments varies during both acute and chronic HIV/SIV infection in different tissues (Barouch et al., 2002; Hel et al., 2001a; Kuroda et al., 1999b; O'Connor et al., 2002; Schmitz et al., 2001; Stevceva et al., 2002a; Veazey et al., 2003 ). ...
Plasma virus in human immunodeficiency virus type 1/simian immunodeficiency virus (HIV-1/SIV) infection most likely results from the combination of viruses produced in different tissues. As immunological pressure may be higher in effector sites than secondary lymphoid tissues, we investigated quantitative and qualitative changes in viral RNA in blood and tissues of 10 Mamu-A*01-positive SIV-infected macaques in parallel with the frequency of CD8+ T cells recognizing the dominant Gag181-189 CM9 epitope. The plasma virus level in these macaques directly correlated with the viral RNA levels in lymph nodes, spleen, lungs, colon, and jejunum. In contrast, the frequency of the Gag181-189 CM9 tetramer did not correlate with SIV RNA levels in any compartment. We investigated the presence of viral immune escape in RNA from several tissues. The complete substitution of wild-type genotype with viral immune-escape variant within the Gag181-189 CM9 epitope was associated with low tetramer response in all tissues and blood of two macaques. In one macaque, the replacement of wild type with an immune-escape mutant was asynchronous. While the mutant virus was prevalent in blood and effector tissues (lungs, jejunum, and colon), secondary lymphoid organs such as spleen and lymph nodes still retained 80% and 40%, respectively, of the wild-type virus. These results may imply that there are differences in the immunological pressure exerted by cytotoxic T lymphocytes (CTLs) in tissue compartments of SIVmac251-infected macaques.
... HIV is associated with generation of strong CD8 T-cell responses which arise early and are maintained during viral persistence [6]. CD8 T cells play a key role in control of viral infection, as evidenced by substantial work in the SIV model [7], and the emergence of escape mutations in SIV and HIV [8,9]. In the case of HCV, strong immune responses in both CD4 and CD8 T-cell compartments may be observed in acute disease and in resolvers, where it appears that memory CD4 cells play an essential role in protection, although typically in chronic infection such responses are weak or undetectable [10,11]. ...
To investigate CD8 T-cell responses to HIV and hepatitis C virus (HCV) over time in a group of co-infected children with haemophilia to assess the influence of the virus infections on each other and on clinical outcome.
The HIV and HCV CD8 T-cell response of HLA-A2 co-infected individuals in the cohort were analysed at two time points, looking at the frequency and phenotype of HIV-specific T cells and assessing overall responses to the two viruses.
Peripheral blood mononuclear cells (PBMC) from 72 HLA-A2 co-infected individuals were analysed using an HIV HLA-A2 tetramer and by IFN-gamma ELISpot using a panel of HIV and HCV antigens. PBMC from a group of 26 HLA-A2 HIV mono-infected adults were also analysed as a comparison.
We identified two distinct patterns of response: some patients had a limited response to either virus whilst others made responses to a range of HIV epitopes. HCV responses were detected only in those who made multiple responses to HIV epitopes (P<0.0001). HCV infection had an influence on the phenotype of HIV-specific CD8 T cells, with a reduction in relative perforin and CD57 expression. Lack of functional or tetramer-positive HIV-specific T cells was associated with a decline in absolute CD4 T-cell counts between the time points (up to 7 years; P = 0.005).
HCV infection has an impact on the phenotype of HIV-specific CD8 T cells. In this well-defined cohort, failure to maintain effective CD8 T-cell responses against HIV may contribute to disease progression.
... Functional impairment of CD8 ϩ T cells has been described for patients infected with human immunodeficiency virus and in patients with hepatitis C virus (4,17,23,31,33,37,50). Both viruses are genetically highly variable and escape from CD8 ϩ T-cell responses by mutations in T-cell epitopes has been discussed as a major reason for the loss of CD8 ϩ T-cell activity in chronic infected carriers (35,36,40). However, other evidence suggests that several viral epitopes of human immunodeficiency virus and hepatitis C virus are recognized by CD8 ϩ T cells throughout the chronic phase of infection (16,27,53) and that viral loads in infected patients are more associated with functional properties of CD8 ϩ T cells than with viral mutations (16,23,31,33). ...
Virus-specific CD8+ T cells are critical for the control of acute Friend virus (FV) infections, but are rendered impotent by CD4+ regulatory T cells during the chronic phase of infection. The current study examines this CD8+ T-cell dysfunction by analyzing the production and release of cytolytic molecules by CD8+ T cells. CD8+ T cells with an activated phenotype (CD43+) from acutely infected mice produced all three key components of lytic granules: perforin, granzyme A, and granzyme B. Furthermore,
they displayed evidence of recent degranulation and in vivo cytotoxicity. In contrast, activated CD8+ T cells from chronically infected mice were deficient in cytolytic molecules and showed little evidence of recent degranulation
and poor in vivo cytotoxicity. Evidence from tetramer-positive CD8+ T cells with known virus specificity confirmed the findings from the activated subset of CD8+ T cells. Interestingly, perforin and granzyme A mRNA levels were not significantly reduced during chronic infection, indicating
control at a posttranscriptional level. Granzyme B deficiency was associated with a significant decrease in mRNA levels, but
posttranscriptional control also appeared to contribute to deficiency. These results demonstrate a broad impairment of cytotoxic
CD8+ T-cell effector function during chronic retroviral infection and explain the inability of virus-specific CD8+ T cells to eliminate persistent virus.
... Repeating this process for several MHC alleles representative of those found in captive macaque populations, we developed a consistent dataset of non-binding peptides, binding peptides and antigenic peptides for five common MHC molecules. Based on this knowledge, we were able to perform studies which have provided insight into kinetics of immune responses after infection, pathogenesis studies and viral escape mechanisms after natural infection and vaccination [12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28]. ...
Non-human primates, in general, and Indian rhesus macaques, specifically, play an important role in the development and testing of vaccines and diagnostics destined for human use. To date, several frequently expressed macaque MHC molecules have been identified and their binding specificities characterized in detail. Here, we report the development of computational algorithms to predict peptide binding and potential T cell epitopes for the common MHC class I alleles Mamu-A*01, -A*02, -A*11, -B*01 and -B*17, which cover approximately two thirds of the captive Indian rhesus macaque populations. We validated this method utilizing an SIV derived data set encompassing 59 antigenic peptides. Of all peptides contained in the SIV proteome, the 2.4% scoring highest in the prediction contained 80% of the antigenic peptides. The method was implemented in a freely accessible and user friendly website at . Thus, we anticipate that our approach can be utilized to rapidly and efficiently identify CD8+ T cell epitopes recognized by rhesus macaques and derived from any pathogen of interest.
... Consequently, survival did not correlate with the extent of viremia in this animal model. SIVsm/mac-infected animals that contain viremia to low levels generally remain clinically asymptomatic, with progression to disease occurring at a later time point and associated with rising plasma virus levels due to the development of immune escape variants (49). Indeed, ten Haaft et al. have postulated a rather arbitrary virus threshold of 10 4 copies/ml of plasma, above which pathogenic infection and progression to AIDS are observed (65). ...
Simian immunodeficiency virus (SIV) is known to result in an asymptomatic infection of its natural African monkey host. However, some SIV strains are capable of inducing AIDS-like symptoms and death upon experimental infection of Asian macaques. To further investigate the virulence of natural SIV isolates from African monkeys, pig-tailed (PT) macaques were inoculated intravenously with either of two recently discovered novel lentiviruses, SIVlhoest and SIVsun. Both viruses were apparently apathogenic in their natural hosts but caused immunodeficiency in PT macaques. Infection was characterized by a progressive loss of CD4(+) lymphocytes in the peripheral blood and lymph nodes, generalized lymphoid depletion, a wasting syndrome, and opportunistic infections, such as Mycobacterium avium or Pneumocystis carinii infections. However, unlike SIVsm/mac infection of macaques, SIVlhoest and SIVsun infections in PT macaques were not accompanied by high viral loads during the chronic disease stage. In addition, no significant correlation between the viral load at set point (12 weeks postinfection) and survival could be found. Five out of eight SIVlhoest-infected and three out of four SIVsun-infected macaques succumbed to AIDS during the first 5 years of infection. Thus, the survival of SIVsun- and SIVlhoest-infected animals was significantly longer than that of SIVagm- or SIVsm-infected macaques. All PT macaques maintained strong SIV antibody responses despite progression to SIV-induced AIDS. The development of immunodeficiency in the face of low viremia suggests that SIVlhoest and SIVsun infections of macaques may model unique aspects of the pathogenesis of human immunodeficiency virus infection in humans.
... The window of opportunity for an HIV vaccine is therefore narrowly limited to the very early stages of infection, before the virus can seed the lymphoid organs in mucosal tissues [63,64]. HIV also has developed multiple mechanisms to circumvent the host immune responses including its ability to downregulate the major histocompatibility complex (MHC) class I molecules and by doing so to minimize its recognition by CTL, as well as its high genetic evolution rates, which allows it to evade immune responses through the emergence of viral CTL [3,65666768 and neutralizing antibody [69,70] escape vari- ants. Another difficulty with the development of an effective HIV vaccine stems from the fact that the virus envelope glycoprotein conceals its conserved receptor-and coreceptorbinding sites in crypts that are masked by the hypervariable loops of the molecule and by glycan residues [45,70,71]. ...
Since the discovery of AIDS in 1981, the global spread of HIV has reached pandemic proportions, representing a global developmental and public health threat. The development of a safe, globally effective and affordable HIV vaccine offers the best hope for the future control of the pandemic. Significant progress has been made over the past years in the areas of basic virology, immunology, pathogenesis of HIV/AIDS and the development of antiretroviral drugs. However, the development of an HIV vaccine faces formidable scientific challenges related to the high genetic variability of the virus, the lack of immune correlates of protection, limitations with the existing animal models and logistical problems associated with the conduct of multiple clinical trials. More than 35 vaccine candidates have been tested in Phase I/II clinical trials, involving more than 10,000 volunteers, and two Phase III trials have been completed, themselves involving more than 7500 volunteers. Multiple vaccine concepts and vaccination strategies have been tested, including DNA vaccines, subunit vaccines, live vectored recombinant vaccines and various prime-boost vaccine combinations. This article reviews the state of the art in HIV vaccine development, summarizes the results obtained so far and discusses the challenges to be met in the development of the various vaccine candidates.
... For example, functional impairment of effector CD4 + T-cell and CD8 + T-cell responses has been described for patients infected with HIV (Trimble & Lieberman, 1998; Appay et al., 2000; Lieberman et al., 2001; Migueles et al., 2002; Hess et al., 2004). Since HIV is genetically highly variable, escape from T-cell responses by mutations in T-cell epitopes has been put forward as a major reason for the loss of T-cell activity in chronically infected carriers (O'Connor et al., 2001; Peyerl et al., 2004). However, evidence suggests that at least some viral epitopes of HIV are recognized by T cells throughout the chronic phase of infection (Draenert et al., 2004). ...
The ability to evade or suppress the host's immune response is a property of many viruses, indicating that this provides an advantage for the pathogen to spread efficiently or even to establish a persistent infection. The type and complexity of its genome and cell tropism but also its preferred type of host interaction are important parameters which define the strategy of a given virus to modulate the immune system in an optimal manner. Because they take a central position in any antiviral defence, the activation and function of T cells are the predominant target of many viral immunosuppressive regimens. In this review, two different strategies whereby this could be achieved are summarized. Retroviruses can infect professional antigen-presenting cells and impair their maturation and functional properties. This coincides with differentiation and expansion of silencing T cells referred to as regulatory T cells with suppressive activity, mainly to CD8+ effector T cells. The second concept, outlined for measles virus, is a direct, contact-mediated silencing of T cells which acquire a transient paralytic state.
... Consequently, the host-and virus-specific factors that contribute to the selection of CTL escape variant viruses have been inferred from the human or simian infections but not experimentally proven. For example, it is generally believed that mutations in CTL epitopes will occur if these changes do not significantly modify virus fitness and do not result in a novel T response to the mutated epitope (reviewed in reference 24). Similarly, an antiviral immune response that depends primarily (if not solely) on the CTL response would be prone to escape. ...
Mutation within virus-derived CD8 T-cell epitopes can effectively abrogate cytotoxic T-lymphocyte (CTL) recognition and impede
virus clearance in infected hosts. These so-called “CTL escape variant viruses” are commonly selected during persistent infections
and are associated with rapid disease progression and increased disease severity. Herein, we tested whether antiviral antibody-mediated
suppression of virus replication and subsequent virus clearance were necessary for preventing CTL escape in coronavirus-infected
mice. We found that compared to wild-type mice, B-cell-deficient mice did not efficiently clear infectious virus, uniformly
developed clinical disease, and harbored CTL escape variant viruses. These data directly demonstrate a critical role for antiviral
antibody in protecting from the selective outgrowth of CTL escape variant viruses.
... One of the major problems in human immunodeficiency virus vaccine development has been mutational escape from CD8 T-cell responses (4,5,11). Mutational escape is seen in both acute and chronic infections (1,8,12). During chronic infections, escape can also occur due to persistent stimulation by virus, leading to exhaustion of T cells (10,17). ...
In this study, we monitored the temporal breadths, frequencies, and functions of antiviral CD4 and CD8 T cells in 2 of 22
DNA/modified vaccinia virus Ankara-vaccinated macaques that lost control of a simian-human immunodeficiency virus 89.6P challenge
by 196 weeks postchallenge. Our results show that both mutation and exhaustion contributed to escape. With the reappearance
of viremia, responding CD8 and CD4 T cells underwent an initial increase and then loss of breadth and frequency. Antiviral
gamma interferon (IFN-γ)- and interleukin 2-coproducing cells were lost before IFN-γ-producing cells and CD4 cells before
CD8 cells. At euthanasia, all CD8, but no CD4, Gag epitopes detected during long-term control contained mutations.
Functional avidity of T cells is a critical determinant for clearing viral infection and eliminating tumor. Understanding how functional avidity is maintained in T cells is imperative for immunotherapy. However, studies systematically characterize T cell with high functional avidity induced in vivo are still lacking. Previously, we and others found vaccinia vectored vaccine (VACV) induced antigen-specific CD8+ T cells with relatively high functional avidity to those from DNA vaccine. Herein, we used functional, immune phenotyping and transcriptomic studies to define the immune signature of these CD8+ T cells with high functional avidity. Antigen-specific CD8+ T cells induced by VACV executed superior in vivo killing activity and displayed a distinct transcriptional profile, whereas no significantly differences were found in composition of memory sub-populations and cytokine poly-functionality. Transcriptional analyses revealed unique features of VACV induced CD8+ T cells in several biological processes, including transport, cell cycle, cell communication and metabolic processes. In summary, we characterize CD8+ T cells of high functional avidity induced in vivo by VACV, which not only improves our understanding of adaptive T cell immunity in VACV vaccination, but also provides clues to modulate functional avidity of CD8+ T cells for T cell based immunotherapy.
This chapter reviews experimental animal studies of female genital tract infections by human immunodeficiency virus (HIV-1)-which leads to immunodeficiency and often death, human papillomavirus (HPV)-which causes genital warts and cervical cancer, and Chlamydia trachomatis-which causes infertility and ectopic pregnancy. The main animal model for human HIV-1 infection is rhesus monkeys infected with one of the many available strains of simian immunodeficiency viruses (SIV) or chimeric simian/human immunodeficiency viruses (SHIV). Experimental evidence indicates that the main immune effector process controlling virus replication in infected monkeys and humans is cell-mediated immunity. Antibody plays an important role in controlling most viral infections, but the envelope proteins of HIV-1, to which neutralizing antibody binds-are exceptionally variable. By the time an infected individual mounts a neutralizing antibody response, the virus envelope can change and no longer be recognized by that antibody. In comparison to antibody-mediated immunity against HIV-1, cell-mediated immunity has two advantages: it recognizes core proteins of the virus that are better conserved than the envelope proteins, and T cell antigen recognition is inherently more cross-reactive than antibody recognition.
The complex interplay between the host immune response and HIV has been the subject of intense research over the last 25 years. HIV and simian immunodeficiency virus (SIV) CD8 T cells have been of particular interest since they were demonstrated to be temporally associated with reduction in virus load shortly following transmission. Here, we briefly review the phenotypic and functional properties of HIV-specific and SIV-specific CD8 T-cell subsets during HIV infection and consider the influence of viral variation with specific responses that are associated with disease progression or control. The development of an effective HIV/AIDS vaccine combined with existing successful prevention and treatment strategies is essential for preventing new infections. In the context of previous clinical HIV/AIDS vaccine trials, we consider the challenges faced by therapeutic and vaccine strategies designed to elicit effective HIV-specific CD8 T cells.
Recognition of antigens by the adaptive immune system relies on a highly diverse T cell receptor repertoire. The mechanism that maintains this diversity is based on competition for survival stimuli; these stimuli depend upon weak recognition of self-antigens by the T cell antigen receptor. We study the dynamics of diversity maintenance as a stochastic competition process between a pair of T cell clonotypes that are similar in terms of the self-antigens they recognise. We formulate a bivariate continuous-time Markov process for the numbers of T cells belonging to the two clonotypes. We prove that the ultimate fate of both clonotypes is extinction and provide a bound on mean extinction times. We focus on the case where the two clonotypes exhibit negligible competition with other T cell clonotypes in the repertoire, since this case provides an upper bound on the mean extinction times. As the two clonotypes become more similar in terms of the self-antigens they recognise, one clonotype quickly becomes extinct in a process resembling classical competitive exclusion. We study the limiting probability distribution for the bivariate process, conditioned on non-extinction of both clonotypes. Finally, we derive deterministic equations for the number of cells belonging to each clonotype as well as a linear Fokker-Planck equation for the fluctuations about the deterministic stable steady state.
This thesis focuses on the impact of HLA class I restricted T cells on HIV-1 disease progression. It is generally accepted that cytotoxic T lymphocytes (CTL) play an important role in controlling HIV replication. In line with this, it has been well established that HLA class I alleles influence the rate of progression to AIDS. Both HLA heterozygosity as well as the presence of particular HLA alleles (e.g. HLA-B27 and B57) appear to be associated with relatively slow disease progression. The mechanism however via which these HLA alleles are associated with relatively slow disease progression remains largely unclear. In this thesis, several of the hypotheses were tested. We could show that CTL responses restricted via the protective HLA alleles HLA-B27 or B57 were lost at least as fast as CTL responses restricted via the non-protective HLA allele HLA-A2, indicating that maintenance of CTL responses per se is not the main determinant of protection against progression to AIDS. Furthermore, we showed that the low frequency of HLA-B27 and B57 in the human population is not the reason why these HLA alleles are associated with relatively slow HIV-1 disease progression. When comparing the number of CTL escape mutations that have been accumulating since the start of the epidemic for different HLA molecules, we did not find any evidence that adaptation of HIV to the human immune system has mainly occurred for CTL restricted by common HLA alleles. In contrast, we found that most CTL escape mutations have been accumulating for CTL restricted by the relatively rare HLA molecules HLA-B27 and B57. Our data suggest that instead of the frequency of HLA molecules in the human population it is the selection pressure exerted by the HLA-restricted CTL that determines the number of escape mutations that have accumulated throughout the epidemic. Moreover, we found that HLA-B27 and B57 have a strikingly different effect on the total HIV-specific T cell response in individuals expressing these HLA alleles. HLA-B57 clearly dominates the total CTL response, which might be mediated by the strong interaction between HLA-B57-peptide complexes and the T cell receptor. In sharp contrast, expression of HLA-B27 seems to preserve the total CTL response, leading to increased responses towards peptides restricted through not only HLA-B27 but also other HLA alleles expressed by the individual. These data suggest a different mechanism of protection for HLA-B27 and B57. Additionally, we investigated whether the presence of cytokine-producing HIV-specific CD8+ T cells early in infection was associated with AIDS-free survival time. We showed that regardless of subsequent clinical outcome, high frequencies of cytokine-producing CD4+ and CD8+ T cells can be found shortly after seroconversion. The fact that both progressors and long-term non-progressors have abundant T cell immunity of the specificity associated with low viral load shortly after seroconversion suggests that the more rapid loss of T cell immunity observed in progressors may be a consequence rather than a cause of disease progression.
The purpose of this review is to assess the influence of T-cell receptor clonotype diversity on the recognition and control of chronic viral infections, and specifically in the case of HIV infection.
The latest publications have examined the role of T-cell receptor repertoires specific for dominant epitopes in the ability to recognize variants and control viremia in chronic viral infections. In the hepatitis C virus and SIV models, diverse T-cell receptor repertoires appear to limit immune escape. In HIV infection, circulating clonotypes may have different functional abilities, showing another potential advantage of diverse clonotypic repertoires. A recent study suggests that at times narrow repertoires against a conserved epitope may be effective, perhaps through the ability to cross-recognize potential epitope variants.
The studies discussed in this review have identified T-cell receptor diversity as an important factor for understanding the immune recognition of highly variable viruses. Further studies are needed to determine whether T-cell receptor repertoire analysis of HIV epitope-specific immune responses will provide a more accurate correlate for the control of viremia than conventional immune function assays.
The high diversity of HIV variants driving the global AIDS epidemic has caused many to doubt whether an effective vaccine against the virus is possible. However, by identifying the selective forces that are driving the ongoing diversification of HIV and characterising their genetic consequences, it may be possible to design vaccines that pre-empt some of the virus' more common evasion tactics. One component of such vaccines might be the envelope protein, gp41. Besides being targeted by both the humoral and cellular arms of the immune system this protein mediates fusion between viral and target cell membranes and is likely to be a primary determinant of HIV transmissibility.
Using recombination aware analysis tools we compared site specific signals of selection in gp41 sequences from different HIV-1 M subtypes and circulating recombinant forms and identified twelve sites evolving under positive selection across multiple major HIV-1 lineages. To identify evidence of selection operating during transmission our analysis included two matched datasets sampled from patients with acute or chronic subtype C infections. We identified six gp41 sites apparently evolving under different selection pressures during acute and chronic HIV-1 infections. These sites mostly fell within functional gp41 domains, with one site located within the epitope recognised by the broadly neutralizing antibody, 4E10.
Whereas these six sites are potentially determinants of fitness and are therefore good candidate targets for subtype-C specific vaccines, the twelve sites evolving under diversifying selection across multiple subtypes might make good candidate targets for broadly protective vaccines.
Much recent work strongly supports the hypothesis that CD8(+) T lymphocytes (CTLs) exert important immune control over HIV and so are a major selective force in its evolution. We analyse this host-pathogen interplay and focus on new data that describe the overall 'effectiveness' of CTL responses (strength, spread, specificity and 'stamina') and the mechanisms by which HIV may evade this suppressive activity. CTLs directed against HIV recognise very large numbers of distinct epitopes across the genome, are largely functional, turn over rapidly, and possess a phenotype that is distinct from CD8(+) lymphocytes specific for other viruses. Mutation of HIV epitopes that alters or abolishes CTL recognition altogether appears to be the most important immune escape mechanism, as the variation that HIV generates defies the limits of the T cell repertoire. However, this immune evasion is still only well-studied in a few patients. The rules that govern immune escape, and the ultimate limits of CTL capacity to deal with the variant epitopes that currently circulate, are not understood. This information will determine the feasibility of current vaccine approaches that, so far, make no provision for the enormous antigenic plasticity of HIV.
Fifteen years after the first, definitive reports of HIV-1-specific, CD8+ T cells [147,148], there is ample evidence for the importance of these cells in control of HIV-1 infection. As much is known of their role in the natural history of HIV-1 infection and their cellular and molecular mechanisms of reactivity than of T-cell responses to any other human virus. Indeed, HIV-1-related research has led the scientific field in revealing many new, fundamental principles of cellular immunity in the last 15 years. From these data, there are multiple, posited mechanisms for loss of CD8+ T-cell control of HIV-1 infection. These include both intrinsic defects in T-cell function and loss of T-cell recognition of HIV-1 because of its extraordinary genetic diversity and disruption of antigen presentation. Efforts have begun on devising approaches to reverse these immune defects in infected individuals and develop vaccines that induce T-cell immunity for protection from infection. Combination antiretroviral drug regimens now provide exceptional, long-lasting control of HIV-1 infection, even though they do not restore anti-HIV-1 T-cell immunity fully in persons with chronic HIV-1 infection. Very encouraging results show that such treatment can maintain normal T-cell reactivity specific for this virus in some persons with early HIV-1 infection. Unfortunately, the antiviral treatment does not cure the host of this persistent, latent virus. This has led to new strategies for immunotherapeutic intervention to enhance the level and breadth of the T-cell repertoire specific for the host's residual virus in persons with chronic HIV-1 infection. Although the principles of immunotherapy stem from early in the last century, modern era approaches are integrating highly sophisticated, molecular and cell biology reagents and methods for control of HIV-1 infection. The most promising immunotherapies are autologous virus activated in vivo by STI or administered in autologous DC that have been engineered ex vivo. There are also compelling rationales supported by animal models and early clinical trials for use of cytokines and chemokines as recombinant proteins or DNA to augment anti-HIV-1 T-cell reactivity and trafficking of T cells and APC to tissue sites of infection. For prevention of HIV-1 infection, the discouragingly poor results of vaccine development in the late 1980s and early 1990s have led to very encouraging, recent studies in monkeys that show partially protective and possibly sterilizing immunity. Finally, clinical trials of new-generation DNA and live vector vaccines already have indications of improved induction of HIV-1-specific T-cell responses. Knowledge of HIV-1-specific T-cell immunity and its role in protection from HIV-1 infection and disease must continue to expand until the goal of complete control of HIV-1 infection is accomplished.
Hepatitis C virus (HCV) has infected over 170 million people world wide, and in the majority sets up a chronic infection associated with hepatic inflammation. How it evades host immunity, particularly CD8+ T cells (CTL) is unclear, but two major factors are likely to operate, viral escape mutation and T cell exhaustion. We have investigated the role of CTL in control of infection during acute disease using Class I peptide tetramers. Although the immune response is quite diverse and numerous epitopes can be targeted, we observe that, especially during acute disease, one epitope (NS3 1073-81) is commonly recognised in HLA-A2 positive individuals. However, the levels of response to this epitope (and others) are very much lower if persistence is established. We examined in detail whether the cause of this low level of reactivity is due to mutation within the epitope. We find that, in fact this epitope is highly conserved during chronic infection, at a clonal level, between individuals, and over time. Thus, although variation within the epitope does occur, lack of reactivity in peripheral blood against this epitope in chronic disease, and loss of control of virus cannot be explained entirely by viral escape. Escape through mutation probably does play an important role in persistence of HCV, but we also discuss other mechanisms which lead to attenuation of T cell responses which may be important in determining the outcome.
Twenty years after the discovery of HIV, there is still no vaccine. This year, an envelope vaccine aimed at stimulating neutralizing antibodies was unable to protect against infection in phase 3 trials. But more than 20 HIV vaccines designed to stimulate T-cell responses are being developed. Will any of them work?
Reconstitution of functional CD4(+) T cell responsiveness to in vitro stimuli is associated with continuous highly active antiretroviral therapy (HAART). Thirty-six antiretroviral naive patients received HAART over 16 weeks. Antigen-specific, mitogen and interleukin (IL)-2 induced lymphocyte proliferative responses and specific IL-2 and IL-4 production were assessed at each time-point, together with quantification of HIV-1 RNA load and lymphocyte populations. Reconstitution of recall responses was limited largely to persistent antigens such as Herpes simplex virus and Candida, rather than to HIV-1 or neo-antigens. Recall antigens, mitogens and IL-2-induced renewed responses were associated with in-vitro production of IL-2, but not IL-4. Differential responsiveness to low versus high concentration IL-2 stimulus increases in a stepwise manner, suggesting normalization of IL-2 receptor expression and improved functionality. These increases in in-vitro proliferative responses thus probably reflect short lived effector clones, driven by ongoing antigenic stimulus associated with persisting long-term organisms. In this context non-responsiveness to HIV-1 antigens suggests ongoing HIV-1 specific clonal T cell anergy.
Control of viremia in natural human immunodeficiency virus type 1 (HIV-1) infection in humans is associated with a virus-specific
T-cell response. However, still much is unknown with regard to the extent of CD8+ cytotoxic T-lymphocyte (CTL) responses required to successfully control HIV-1 infection and to what extent CTL epitope escape
can account for rises in viral load and ultimate progression to disease. In this study, we chose to monitor through full-length
genome sequence of replication-competent biological clones the modifications that occurred within predicted CTL epitopes and
to identify whether the alterations resulted in epitope escape from CTL recognition. From an extensive analysis of 59 biological
HIV-1 clones generated over a period of 4 years from a single individual in whom the viral load was observed to rise, we identified
the locations in the genome of five CD8+ CTL epitopes. Fixed mutations were identified within the p17, gp120, gp41, Nef, and reverse transcriptase genes. Using a
gamma interferon ELIspot assay, we identified for four of the five epitopes with fixed mutations a complete loss of T-cell
reactivity against the wild-type epitope and a partial loss of reactivity against the mutant epitope. These results demonstrate
the sequential accumulation of CTL escape in a patient during disease progression, indicating that multiple combinations of
T-cell epitopes are required to control viremia.
Evidence for human immunodeficiency virus type 1 (HIV-1) superinfection was sought among 37 HIV-1-positive street-recruited
active injection drug users (IDUs) from the San Francisco Bay area. HIV-1 sequences from pairs of samples collected 1 to 12
years apart, spanning a total of 215 years of exposure, were generated at p17 gag, the V3-V5 region of env, and/or the first exon of tat and phylogenetically analyzed. No evidence of HIV-1 superinfection was detected in which a highly divergent HIV-1 variant
emerged at a frequency >20% of the serum viral quasispecies. Based on the reported risk behavior of the IDUs and the HIV-1
incidence in uninfected subjects in the same cohort, a total of 3.4 new infections would have been expected if existing infection
conferred no protection from superinfection. Adjusted for risk behaviors, the estimated relative risk of superinfection compared
with initial infection was therefore 0.0 (95% confidence interval, 0.00, 0.79; P = 0.02), indicating that existing infection conferred a statistically significant level of protection against superinfection
with an HIV-1 strain of the same subtype, which was between 21 and 100%.
With the continued spread of the HIV/AIDS epidemic at alarming proportions there is a sense of urgency for an effective prophylactic HIV vaccine. However, in addition to the social, geopolitical and public health problems, the scientific challenges often seem insurmountable. Empirical approaches to develop an HIV/AIDS vaccine have been unsuccessful and this, coupled with the recent failure of the first Phase III clinical trials, calls for a strong rational approach based on a deeper scientific understanding of the correlates of immunity observed in both preclinical and clinical settings. While the field has been polarized between those who have been proponents of vaccines that induce strong cytotoxic T-cell responses, and those who advocate inducing neutralizing antibody responses, we have maintained middle ground. Based on our early preclinical observations in rigorous nonhuman primate vaccine efficacy studies, we have focused on vaccine strategies that induce potent T-helper immune responses capable of driving both cytotoxic, as well as broad highly effective neutralizing antibodies. The critical issue remains in the selection of the specific vaccine antigens. To date, our approach has been to utilize multiple structural as well as regulatory HIV antigens containing highly conserved epitopes. The current challenge faced is to design novel antigens based on mimicking envelope structures capable of inducing broad neutralizing antibodies. Our aim is to combine these with immunization strategies capable of eliciting potent cellular as well as humoral immune responses with the ultimate goal of providing mucosal barriers to HIV entry.
Preclinical studies in nonhuman primates (NHP) play key roles in AIDS vaccine development efforts. In addition to their traditional utilization to gauge vaccine safety and immunogenicity, NHP models are currently employed to an unprecedented extent and in unprecedented ways in contemporary basic and applied vaccine development efforts. Current studies employ NHP models to probe fundamental mechanisms of primate immune system regulation, to investigate pathogenic mechanisms of AIDS, and to optimize immunization strategies involving novel vaccine vectors. The use of experimental challenges of immunized NHPs with either simian immunodeficiency virus or chimeric simian/human immunodeficiency virus to generate preclinical vaccine efficacy data has emerged as an important criterion for facilitating entry of a given vaccine candidate into early phase clinical evaluation in humans. However, for studies of the biology of AIDS virus transmission, AIDS virus disease pathogenesis and AIDS virus vaccine efficacy that are predicated on experimental viral challenge to be most valuable, additional efforts need to be devoted to generating challenge models that more closely recapitulate HIV-1 infection in humans. Towards this end, improved communication between clinical and preclinical investigators, to promote a bidirectional flow of information focusing on individual research needs and shared goals should enable the NHP models to most effectively expedite progress toward the development of a safe and effective AIDS vaccine.
The classical paradigm for T cell dynamics suggests that the resolution of a primary acute virus infection is followed by the generation of a long-lived pool of memory T cells that is thought to be highly stable. Very limited alteration in this repertoire is expected until the immune system is re-challenged by reactivation of latent viruses or by cross-reactive pathogens. Contradicting this view, we show here that the T cell repertoire specific for two different latent herpes viruses in the peripheral blood displayed significant contemporaneous co-fluctuations of virus-specific CD8(+) T cells. The coordinated responses to two different viruses suggest that the fluctuations within the T cell repertoire may be driven by sub-clinical viral reactivation or a more generalized 'bystander' effect. The later contention was supported by the observation that, while absolute number of CD3(+) T cells and their subsets and also the cell surface phenotype of antigen-specific T cells remained relatively constant, a loss of CD62L expression in the total CD8(+) T cell population was coincident with the expansion of tetramer-positive virus-specific T cells. This study demonstrates that the dynamic process of T cell expansion and contractions in persistent viral infections is not limited to the acute phase of infection, but also continues during the latent phase of infection.
The failure of HIV-specific CD8+ T cells to control HIV has suggested a functional defect for these cells in HIV infection. Recently, HIV-specific CD8+ T cells from HIV-infected patients were shown to exhibit reduced levels of Bcl-2 and Bcl-xL and to be highly prone to CD95/Fas-induced apoptosis. We hypothesize that this apoptosis of HIV-specific CD8+ T cells may affect their maturation and/or impair effector function. In this review, we discuss the apoptotic defect, the intracellular mediators of this proapoptotic state and the potential mechanisms that result in the priming of HIV-specific CD8+ T cells to apoptosis. Thus, HIV may employ a counterattack mechanism in which HIV-specific CD8+ T cells undergo apoptosis upon engagement of CD95L/FasL on apoptosis-resistant HIV-infected cells. Understanding the mechanisms governing the survival of HIV-specific CD8+ T cells is a prerequisite both for therapeutic interventions and vaccines targeting an effective long-lasting immunoprotective cytotoxic T lymphocyte (CTL) response.
Human HIV infection is characterised by great variability in outcome. Much of this variability is due either to viral variation or host genetic factors, particularly major histocompatibility complex differences within genetically diverse populations. The study of non-human primates infected with well characterised simian immunodeficiency virus strains has recently allowed further dissection of the critical role of genetic influences on both susceptibility to infection and progression to AIDS. This review summarises the important role of many host genetic factors on HIV infection and highlights important variables that will need to be taken into account in evaluating effective HIV vaccines.
Hepatitis C virus (HCV) is a variable RNA virus that can readily establish persistent infection. Cellular immune responses are important in the early control of the virus. Evidence from animal models suggests that mutation in epitopes recognized by CD8+ T lymphocytes may play an important role in the establishment of persistence but in human persistent infection, equivalent evidence is lacking. We investigated this by analysing a unique resource: viruses from a set of chronically HCV-infected individuals in whom the CD8+ T-cell responses in liver had previously been accurately mapped. Virus was sequenced in seven individuals at 10 epitopes restricted by 10 human leucocyte antigen (HLA) molecules. Two main patterns emerged: in the majority of epitopes sequenced, no variation was seen. In three epitopes, mutations were identified which were compatible with immune escape as assessed using phylogenetic and/or functional studies. These data suggest that - even where specific intrahepatic T cells are detectable - many epitopes do not undergo mutation in chronic human infection. On the contrary, virus may escape from intrahepatic CD8+ T-cell responses in a 'patchy' manner in certain specific epitopes. Furthermore, longitudinal studies to identify the differences between 'selecting' and 'nonselecting' intrahepatic CD8+ T-cell responses are needed in HCV infection.
Because of the importance of developing HIV vaccine strategies that generate cytotoxic T lymphocyte (CTL) responses with a maximal breadth of epitope recognition, we have explored a variety of novel strategies designed to overcome the usual propensity of CTLs to focus recognition on a limited number of dominant epitopes. In studies of rhesus monkeys expressing the Mamu-A*01 MHC class I allele, we show that variously configured multiepitope plasmid DNA vaccine constructs elicit CTL populations that do not evidence skewing of recognition to dominant epitopes. Nevertheless, repeated boosting of these vaccinated monkeys with different live recombinant vaccine vectors uncovers and amplifies the usual CTL epitope dominance hierarchy. Importantly, in vitro peptide stimulation of peripheral blood mononuclear cells from monkeys that have received only a multiepitope plasmid DNA priming immunization uncovers this dominance hierarchy. Therefore, the dominance hierarchy of the vaccine-elicited epitope-specific CTL populations is inherent in the T lymphocytes of the monkeys after initial exposure to epitope peptides, and the ultimate breadth of epitope recognition cannot be modified thereafter. This finding underscores the enormous challenge associated with increasing the breadth of CTL recognition through vaccination.
Lentiviral envelope antigenic variation and associated immune evasion are believed to present major obstacles to effective vaccine development. Although this perception is widely assumed by the scientific community, there is, to date, no rigorous experimental data assessing the effect of increasing levels of lentiviral Env variation on vaccine efficacy. It is our working hypothesis that Env is, in fact, a primary determinant of vaccine effectiveness. We previously reported that a successful experimental attenuated equine infectious anemia virus vaccine, derived by mutation of the viral S2 accessory gene, provided 100% protection from disease after virulent virus challenge. Here, we sought to comprehensively test our hypothesis by challenging vaccinated animals with proviral strains of defined, increasing Env variation, using variant envelope SU genes that arose naturally during experimental infection of ponies with equine infectious anemia virus. The reference attenuated vaccine combined with these variant Env challenge strains facilitated evaluation of the protection conferred by ancestral immunogens, because the Env of the attenuated vaccine is a direct ancestor to the variant proviral strain Envs. The results demonstrated that ancestral Env proteins did not impart broad levels of protection against challenge. Furthermore, the results displayed a significant inverse linear correlation of Env divergence and protection from disease. This study demonstrates potential obstacles to the use of single isolate ancestral Env immunogens. Finally, these findings reveal that relatively minor Env variation can pose a substantial challenge to lentiviral vaccine immunity, even when attenuated vaccines are used that, to date, achieve the highest levels of vaccine protection.
• ancestral immunogen
• equine infectious anemia virus
• lentivirus
Cytotoxic T lymphocyte escape occurs in many human infections, as well as mice infected with the JHM strain of mouse hepatitis virus, which exhibit CTL escape variants with mutations in a single epitope from the spike glycoprotein (S510). In all CTL epitopes prone to escape, only a subset of all potential variants is generally detected, even though many of the changes that are not selected would result in evasion of the T cell response. It is postulated that these unselected mutations significantly impair virus fitness. To define more precisely the basis for this preferential selection, we combine x-ray crystallographic studies of the MHC class I (D(b))/S510 complexes with viral reverse genetics to identify a prominent TCR contact residue (tryptophan at position 4) prone to escape mutations. The data show that a mutation that is commonly detected in chronically infected mice (tryptophan to arginine) potently disrupts the topology of the complex, explaining its selection. However, other mutations at this residue, which also abrogate the CTL response, are never selected in vivo even though they do not compromise virus fitness in acutely infected animals or induce a significant de novo CTL response. Thus, while structural analyses of the S510/D(b) complex provide a strong basis for why some CTL escape variants are selected, our results also show that factors other than effects on virus fitness limit the diversification of CD8 T cell epitopes.
A unique cohort of HIV-1-infected long term nonprogressors (LTNP) with normal CD4+ T cell counts and <50 copies/ml of plasma were prospectively recruited for study. HLA typing revealed a dramatic association between the HLA B*5701 class I allele and nonprogressive infection [85% (11 of 13) vs. 9.5% (19 of 200) in progressors; P < 0.001]. Antigen-specific CD8+ T cells were enumerated by flow cytometric detection of intracellular IFN-γ in response to HIV antigens and HLA B*57-gag tetramer staining. No quantitative differences in the total HIV-specific CD8+ T cell responses were observed between B*57+ LTNP and five B*57+ progressors (P = 0.4). Although similar frequencies of peptide specific CD8+ T cells were also found, the gag-specific CD8+ T cell response in the LTNP group was highly focused on peptides previously shown to be B*57-restricted. These findings indicate that, within this phenotypically and genotypically distinct cohort, a host immune factor is highly associated with restriction of virus replication and nonprogressive disease. They also strongly suggest a mechanism of virus specific immunity that directly operates through the B*5701 molecule. Further characterization of qualitative differences in the virus-specific responses that distinguish HLA B*57+ LTNP from progressors may ultimately define mechanisms of effective immune mediated restriction of virus replication.
Although the immunologic basis of protective immunity in human immunodeficiency virus type 1 (HIV-1) infection has not yet been defined, virus-specific cytotoxic T lymphocytes (CTL) are likely to be an important host defense and may be a critical feature of an effective vaccine. These observations, along with the inclusion of the HIV-1 envelope in the majority of vaccine candidates presently in clinical trials, underscore the importance of the precise characterization of the cellular immune responses to this protein. Although humoral immune responses to the envelope protein have been extensively characterized, relatively little information is available regarding the envelope epitopes recognized by virus-specific CTL and the effects of sequence variation within these epitopes. Here we report the identification of two overlapping CTL epitopes in a highly conserved region of the HIV-1 transmembrane envelope protein, gp41, using CTL clones derived from two seropositive subjects. An eight-amino acid peptide was defined as the minimum epitope recognized by HLA-B8-restricted CTL derived from one subject, and in a second subject, an overlapping nine-amino acid peptide was identified as the minimal epitope for HLA-B14-restricted CTL clones. Selected single amino acid substitutions representing those found in naturally occurring HIV-1 isolates resulted in partial to complete loss of recognition of these epitopes. These data indicate the presence of a highly conserved region in the HIV-1 envelope glycoprotein that is immunogenic for CTL responses. In addition, they suggest that natural sequence variation may lead to escape from immune detection by HIV-1-specific CTL. Since the region containing these epitopes has been previously shown to contain an immunodominant B cell epitope and also overlaps with a major histocompatibility complex class II T cell epitope recognized by CD4+ CTL from HIV-1 rgp160 vaccine recipients, it may be particularly important for HIV-1 vaccine development. Finally, the identification of minimal CTL epitopes presented by class I HLA molecules should facilitate the definition of allele-specific motifs.
Cytotoxic T lymphocyte (CTL)-mediated cytolysis is induced via the interaction of the specific T-cell antigen receptor and the peptidic viral antigen associated with the major histocompatibility complex class I antigen. Here we demonstrate in vitro that lymphocytic choriomeningitis virus (LCMV) can escape the cytotoxic activity of LCMV-specific cloned CTLs by single amino acid changes within the recognized T-cell epitope defined by residues 275-289 of the LCMV glycoprotein [LCMV-GP-(275-289)]. LCMV-infected fibroblasts at a multiplicity of infection of 10(-3) exposed to virus-specific CTL at an effector-to-target cell ratio of 4:1 4 hr after infection was optimal for virus mutant selection. The selections were carried out with three LCMV-GP-(275-289)-specific CTL clones expressing T-cell antigen receptors containing the identical variable gene segments V alpha 4 and V beta 10 but different junctional regions; selection was also possible with LCMV-GP-(275-289)-specific cytotoxic polyclonal T cells. The most common escape mutation was an amino acid change of asparagine (AAT) to aspartic acid (GAT) at position 280; an additional mutation was glycine (GGT) to aspartic acid (GAT) at position 282. The results presented show that relevant point mutations within the T-cell epitope of LCMV-GP-(275-289) occur frequently and that they are selectable in vitro by CTLs.
Better understanding of the pathogenesis of acquired immunodeficiency syndrome (AIDS) would be greatly facilitated by a relevant animal model that uses molecularly cloned virus of defined sequence to induce the disease. Such a system would also be of great value for AIDS vaccine research. An infectious molecular clone of simian immunodeficiency virus (SIV) was identified that induces AIDS in common rhesus monkeys in a time frame suitable for laboratory investigation. These results provide another strong link in the chain of evidence for the viral etiology of AIDS. More importantly, they define a system for molecular dissection of the determinants of AIDS pathogenesis.
A study was conducted to determine the fidelity of DNA synthesis catalyzed in vitro by the reverse transcriptase from a human immunodeficiency virus type 1 (HIV-1). Like other retroviral reverse transcriptases, the HIV-1 enzyme does not correct errors by exonucleolytic proofreading. Measurements with M13mp2-based fidelity assays indicated that the HIV-1 enzyme, isolated either from virus particles or from Escherichia coli cells infected with a plasmid expressing the cloned gene, was exceptionally inaccurate, having an average error rate per detectable nucleotide incorporated of 1/1700. It was, in fact, the least accurate reverse transcriptase described to date, one-tenth as accurate as the polymerases isolated from avian myeloblastosis or murine leukemia viruses, which have average error rates of approximately 1/17,000 and approximately 1/30,000, respectively. DNA sequence analyses of mutations generated by HIV-1 polymerase showed that base substitution, addition, and deletion errors were all produced. Certain template positions were mutational hotspots where the error rate could be as high as 1 per 70 polymerized nucleotides. The data are consistent with the notion that the exceptional diversity of the HIV-1 genome results from error-prone reverse transcription.
Immune evasion by the human immunodeficiency virus (HIV) is unexplained but may involve the mutation of viral antigens. When
cytotoxic T lymphocytes engaged CD4-positive cells that were acutely infected with HIV bearing natural variant epitopes in
reverse transcriptase, substantial inhibition of specific antiviral lysis was observed. Mutant viruses capable of these transactive
effects could facilitate the persistence of a broad range of HIV variants in the face of an active and specific immune response.
The proviral genome of the 32H reisolate of simian immunodeficiency of macaques (SIVmac32H) has been cloned and sequenced. Including both long terminal repeats, it is 10277 base pairs in length and contains open reading frames for all known SIV genes (gag, pol, vif, vpx, vpr, tat, rev, env and nef). This is the first report of an infectious SIVmac molecular clone which contains no premature termination codons. Three molecular clones of SIVmac32H have been constructed differing in sequence only within their last 1.2 kb. Two of the molecular clones, SIVmac32H(pJ5) and SIVmac32H (pC8), differ in the nef coding region by an in-frame deletion of four amino acids in pC8 and two conservative amino acid changes; other nucleotide changes in the 3' LTR were not associated with known functionally critical motifs. The third clone, SIVmac32H(pB1), contains the last 1.2 kb of the SIVmac251 clone pBK28. The biological properties of virus produced after electroporation of these clones into C8166 cells has been assessed by infection of rhesus and cynomolgus macaques, time to seroconversion and by induction of cytopathic effects upon co-cultivation of infected rhesus peripheral blood lymphocytes with C8166 cells. The viruses obtained from these clones have identical growth kinetics in vitro but differ in their ability to persist in macaques. Macaques infected with pJ5 derived virus remain viraemic longer than macaques infected with pC8-derived virus. PCR analysis of circulating provirus indicates that the nef gene evolved over time in pJ5 virus-infected macaques, whereas late in infection in pC8 virus-infected macaques the nef gene remained invariant in sequence. These results support the observation that a nef deletion mutant of SIVmac239 lost its pathogenic potential and resulted in low-level viraemia when rhesus macaques were infected. Virus challenge pools for vaccine studies have been prepared for pJ5 using both human and monkey cell substrates and these stocks have been titrated both in vitro and in vivo. Virus has also been prepared from pC8 and titrated in vitro. This virus pool is being assessed as an attenuated live-virus vaccine in macaques. Since only virus originating from the SIVmac239 molecular clone is known to cause AIDS-like symptoms in rhesus macaques consistently, the SIVmac32H molecular clones should tell us more about which viral sequence features are important for the pathogenesis of AIDS.
Transmission of human immunodeficiency virus 1 (HIV-1) from an infected women to her offspring during gestation and delivery was found to be influenced by the infant's major histocompatibility complex class II DRB1 alleles. Forty-six HIV-infected infants and 63 seroreverting infants, born with passively acquired anti-HIV antibodies but not becoming detectably infected, were typed by an automated nucleotide-sequence-based technique that uses low-resolution PCR to select either the simpler Taq or the more demanding T7 sequencing chemistry. One or more DR13 alleles, including DRB1*1301, 1302, and 1303, were found in 31.7% of seroreverting infants and 15.2% of those becoming HIV-infected [OR (odds ratio) = 2.6 (95% confidence interval 1.0-6.8); P = 0.048]. This association was influenced by ethnicity, being seen more strongly among the 80 Black and Hispanic children [OR = 4.3 (1.2-16.4); P = 0.023], with the most pronounced effect among Black infants where 7 of 24 seroreverters inherited these alleles with none among 12 HIV-infected infants (Haldane OR = 12.3; P = 0.037). The previously recognized association of DR13 alleles with some situations of long-term nonprogression of HIV suggests that similar mechanisms may regulate both the occurrence of infection and disease progression after infection. Upon examining for residual associations, only only the DR2 allele DRB1*1501 was associated with seroreversion in Caucasoid infants (OR = 24; P = 0.004). Among Caucasoids the DRB1*03011 allele was positively associated with the occurrence of HIV infection (P = 0.03).
The precise role played by HIV-specific cytotoxic T lymphocytes (CTL) in HIV infection remains controversial. Despite strong CTL responses being generated during the asymptomatic phase, the virus persists and AIDS ultimately develops. It has been argued that the virus is so variable, and the virus turnover so great that escape from CTL recognition would occur continually, but so far there is limited evidence for CTL escape. The opposing argument is that evidence for CTL escape is present but hard to find because multiple anti-HIV immune responses are acting simultaneously during the asymptomatic phase of infection. We describe six donors who make a strong CTL response to an immunodominant HLA-B27-restricted epitope. In the two donors who progressed to AIDS, CTL escape to fixation by the same mutation was observed, but only after 9-12 years of epitope stability. CTL escape may play an important role in the pathogenesis of HIV infection.
The recent development of potent antiviral drugs not only has raised hopes for effective treatment of infections with HIV
or the hepatitis B virus, but also has led to important quantitative insights into viral dynamics in vivo. Interpretation of the experimental data depends upon mathematical models that describe the nonlinear interaction between
virus and host cell populations. Here we discuss the emerging understanding of virus population dynamics, the role of the
immune system in limiting virus abundance, the dynamics of viral drug resistance, and the question of whether virus infection
can be eliminated from individual patients by drug treatment.
Cytotoxic T lymphocytes (CTLs) lyse virally infected cells that display viral peptide epitopes in association with major histocompatibility complex (MHC) class I molecules on the cell surface. However, despite a strong CTL response directed against viral epitopes, untreated people infected with the human immunodeficiency virus (HIV-1) develop AIDS. To resolve this enigma, we have examined the ability of CTLs to recognize and kill infected primary T lymphocytes. We found that CTLs inefficiently lysed primary cells infected with HIV-1 if the viral nef gene product was expressed. Resistance of infected cells to CTL killing correlated with nef-mediated downregulation of MHC class I and could be overcome by adding an excess of the relevant HIV-1 epitope as soluble peptide. Thus, Nef protected infected cells by reducing the epitope density on their surface. This effect of nef may allow evasion of CTL lysis by HIV-1-infected cells.
Despite detailed analysis of the HIV-1-specific cytotoxic T lymphocyte response by various groups, its relation to viral load and viral sequence variation remains controversial. We analyzed HLA-A*0201 restricted cytotoxic T lymphocyte responses in 17 HIV-1-infected individuals with viral loads ranging from < 400 to 221,000 HIV RNA molecules per milliliter of plasma. In 13 out of 17 infected subjects, CTL responses against the SLYNTVATL epitope (p17 Gag; aa 77-85) were detectable, whereas two other HLA-A*0201 restricted epitopes (ILKEPVHGV, IV9; and VIYQYMDDL, VL9) were only recognized by six and five individuals out of 17 individuals tested, respectively. Naturally occurring variants of the SL9 epitope were tested for binding to HLA-A*0201 and for recognition by specific T cell clones generated from five individuals. Although these variants were widely recognized, they differed by up to 10,000-fold in terms of variant peptide concentrations required for lysis of target cells. A comparison of viral sequences derived from 10 HLA-A*0201-positive individuals to sequences obtained from 11 HLA-A*0201-negative individuals demonstrated only weak evidence for immune selective pressure and thus question the in vivo efficacy of immunodominant CTL responses present during chronic HIV-1 infection.
Genetic evolution of the simian immunodeficiency virus (SIV) envelope glycoprotein was evaluated in a group of six macaques (Macaca nemestrina) infected with the molecularly cloned, moderately pathogenic SIVsm62d. The extent of envelope evolution was subsequently evaluated within the context of the individual pattern of viremia and disease outcome. Two macaques in this cohort developed AIDS by 1.5 years postinoculation (progressors), whereas the remaining four macaques remained asymptomatic (nonprogressors). Compared with the nonprogressor macaques, the two progressor macaques exhibited higher persistent plasma viremia, higher homologous neutralizing antibody titers, and more extensive mutation and evolution in the V1 region of envelope. Although clearly distinct in each of these parameters from the progressors, the four nonprogressors exhibited more individual variability with respect to the extent of persistent viremia and genetic evolution of the V1 region of envelope. The extent of V1 envelope varied from no apparent V1 evolution in a macaque with good viral containment to extensive evolution in one macaque with persistent viremia. This study underscores the critical role of persistent replication in the genetic evolution of SIV.
Combination treatments with agents that inhibit protease and reverse transcriptase of human immunodeficiency virus type 1 (HIV-1) decrease mortality and slow disease progression.1 The development of resistance to these drugs, however, limits the benefit of such treatments.2,3 There have been reports of the transmission of HIV-1 variants that are resistant to nucleoside and non-nucleoside inhibitors of reverse transcriptase.4–9 The transmission of HIV-1 variants that are resistant to protease inhibitors could represent an important emerging clinical and public health problem. We report a case of transmission of an HIV-1 variant with multiple mutations that conferred resistance to both protease . . .
We examined the regulation of virus-specific CD8 T cell responses during chronic lymphocytic choriomeningitis virus (LCMV) infection of mice. Our study shows that within the same persistently infected host, different mechanisms can operate to silence antiviral T cell responses; CD8 T cells specific to one dominant viral epitope were deleted, whereas CD8 T cells responding to another dominant epitope persisted indefinitely. These virus-specific CD8 T cells expressed activation markers (CD69(hi), CD44(hi), CD62Llo) and proliferated in vivo but were unable to elaborate any antiviral effector functions. This unresponsive phenotype was more pronounced under conditions of CD4 T cell deficiency, highlighting the importance of CD8- CD4 T cell collaboration in controlling persistent infections. Importantly, in the presence of CD4 T cell help, adequate CD8 effector activity was maintained and the chronic viral infection eventually resolved. The persistence of activated virus-specific CD8 T cells without effector function reveals a novel mechanism for silencing antiviral immune responses and also offers new possibilities for enhancing CD8 T cell immunity in chronically infected hosts.
Simian immunodeficiency virus (SIV) infection of the rhesus macaque is currently the best animal model for AIDS vaccine development. One limitation of this model, however, has been the small number of cytotoxic T-lymphocyte (CTL) epitopes and restricting major histocompatibility complex (MHC) class I molecules available for investigating virus-specific CTL responses. To identify new MHC class I-restricted CTL epitopes, we infected five members of a family of MHC-defined rhesus macaques intravenously with SIV. Five new CTL epitopes bound by four different MHC class I molecules were defined. These included two Env epitopes bound by Mamu-A*11 and -B*03 and three Nef epitopes bound by Mamu-B*03, -B*04, and -B*17. All four restricting MHC class I molecules were encoded on only two haplotypes (b or c). Interestingly, resistance to disease progression within this family appeared to be associated with the inheritance of one or both of these MHC class I haplotypes. Two individuals that inherited haplotypes b and c separately survived for 299 and 511 days, respectively, while another individual that inherited both haplotypes survived for 889 days. In contrast, two MHC class I-identical individuals that did not inherit either haplotype rapidly progressed to disease (survived <80 days). Since all five offspring were identical at their Mamu-DRB loci, MHC class II differences are unlikely to account for their patterns of disease progression. These results double the number of SIV CTL epitopes defined in rhesus macaques and provide evidence that allelic differences at the MHC class I loci may influence rates of disease progression among AIDS virus-infected individuals.
Sequence analysis of human immunodeficiency virus type 1 (HIV-1) from 74 persons with acute infections identified eight strains with mutations in the reverse transcriptase (RT) gene at positions 41, 67, 68, 70, 215, and 219 associated with resistance to the nucleoside analogue zidovudine (AZT). Follow-up of the fate of these resistant HIV-1 strains in four newly infected individuals revealed that they were readily replaced by sensitive strains. The RT of the resistant viruses changed at amino acid 215 from tyrosine (Y) to aspartic acid (D) or serine (S), with asparagine (N) as a transient intermediate, indicating the establishment of new wild types. When we introduced these mutations and the original threonine (T)-containing wild type into infectious molecular clones and assessed their competitive advantage in vitro, the order of fitness was in accord with the in vivo observations: 215Y < 215D = 215S = 215T. As detected by real-time nucleic acid sequence-based amplification with two molecular beacons, the addition of AZT or stavudine (d4T) to the viral cultures favored the 215Y mutant in a dose-dependent manner. Our results illustrate that infection with nucleoside analogue-resistant HIV leads in newly infected individuals to mutants that are sensitive to nucleoside analogues, but only a single mutation removed from drug-resistant HIV. Such mutants were shown to be transmissible, stable, and prone to rapid selection for resistance to AZT or d4T as soon as antiretroviral therapy was administered. Monitoring of patients for the presence of new HIV-1 wild types with D, S, or N residues at position 215 may be warranted in order to estimate the threat to long-term efficacy of regimens including nucleoside analogues.
Although the immunologic basis of protective immunity in human immunodeficiency virus type 1 (HIV-1) infection has not yet been defined, virus-specific cytotoxic T lymphocytes (CTL) are likely to be an important host defense and may be a critical feature of an effective vaccine. These observations, along with the inclusion of the HIV-1 envelope in the majority of vaccine candidates presently in clinical trials, underscore the importance of the precise characterization of the cellular immune responses to this protein. Although humoral immune responses to the envelope protein have been extensively characterized, relatively little information is available regarding the envelope epitopes recognized by virus-specific CTL and the effects of sequence variation within these epitopes. Here we report the identification of two overlapping CTL epitopes in a highly conserved region of the HIV-1 transmembrane envelope protein, gp41, using CTL clones derived from two seropositive subjects. An eight-amino acid peptide was defined as the minimum epitope recognized by HLA-B8-restricted CTL derived from one subject, and in a second subject, an overlapping nine-amino acid peptide was identified as the minimal epitope for HLA-B14-restricted CTL clones. Selected single amino acid substitutions representing those found in naturally occurring HIV-1 isolates resulted in partial to complete loss of recognition of these epitopes. These data indicate the presence of a highly conserved region in the HIV-1 envelope glycoprotein that is immunogenic for CTL responses. In addition, they suggest that natural sequence variation may lead to escape from immune detection by HIV-1-specific CTL. Since the region containing these epitopes has been previously shown to contain an immunodominant B cell epitope and also overlaps with a major histocompatibility complex class II T cell epitope recognized by CD4+ CTL from HIV-1 rgp160 vaccine recipients, it may be particularly important for HIV-1 vaccine development. Finally, the identification of minimal CTL epitopes presented by class I HLA molecules should facilitate the definition of allele-specific motifs.
A selective advantage against infectious disease associated with increased heterozygosity at the human major histocompatibility
complex [human leukocyte antigen (HLA) class I and class II] is believed to play a major role in maintaining the extraordinary allelic diversity of these genes.
Maximum HLA heterozygosity of class I loci (A, B, and C) delayed acquired immunodeficiency syndrome (AIDS) onset among patients infected with human immunodeficiency virus–type 1
(HIV-1), whereas individuals who were homozygous for one or more loci progressed rapidly to AIDS and death. The HLA class I alleles B*35 andCw*04 were consistently associated with rapid development of AIDS-defining conditions in Caucasians. The extended survival of 28
to 40 percent of HIV-1–infected Caucasian patients who avoided AIDS for ten or more years can be attributed to their being
fully heterozygous at HLA class I loci, to their lacking the AIDS-associated alleles B*35 and Cw*04, or to both.
Effective preventive and therapeutic intervention in individuals exposed to or infected with human immunodeficiency virus (HIV) depends, in part, on a clear understanding of the interactions between the virus and those elements of the host immune response which control viral replication. Recent advances have provided compelling evidence that cytotoxic T lymphocytes (CTLs) constitute an essential component of protective antiretroviral immunity. Here, we review briefly the significance of this work in the context of previous studies, and outline the mechanisms through which HIV evades CTL activity.
The relatively low fidelity of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) was implicated as a major factor that contributes to the genetic variability of the virus. Extension of mismatched 3′ termini of the primer DNA was shown to be a major determinant of the infidelity of HIV-1 RT. Human immunodeficiency virus type 2 (HIV-2) also shows extensive genetic variations. Therefore, we have analyzed the fidelity of the DNA-dependent DNA polymerase activity of HIV-2 RT and compared it with those of RTs of HIV-1 and murine leukemia virus (MLV). Like other retroviral RTs, the HIV-2 RT was shown to lack a 3′→5′ exonuclease activity. The ability of HIV-2 RT to extend preformed 3′-terminal A:A, A:C and A:G mispairs was examined by quantitating the amount and length of extended primers. The results demonstrate a relatively efficient mispair extension by HIV-2 RT with a specificity of A:C⪢A:A>A:G. The mispair extension appears to be affected mainly by the increase of apparent Km values rather than by the change in Vmax values. The relative extension frequencies from all mispairs with HIV-1 and HIV-2 RTs was 6- to 9-fold greater than that of MLV RT, suggesting that the HIV enzymes are substantially more error-prone than MLV RT.
THE nucleotide sequence of the DNA of phiX174 phage established by Sanger's group1,2 has revealed that this phage has two overlapping genes in which the same stretch of DNA can code for two proteins which are translated in different reading frames. That two overlapping genes occur in the same DNA genome suggests that this overlapping expression is not unique to phiX174 but of more general significance, at least in organisms with small DNA molecules. Once the overlapping expression is established, all mutations in one gene would also alter the other gene within the region of overlap. Some of these mutations, however, may affect one protein but not the other because of codon degeneracy. In general, the evolutionary constraints on a sequence representing both proteins must be very severe. In this report we estimate the extent to which the evolution of a gene is reduced by the acquisition of overlapping expression.
Human immunodeficiency viruses type 1 and 2 (HIV-1 and HIV-2, respectively) exhibit extensive genetic variations. It was postulated that much of this genetic variability stems from the low fidelity of the reverse transcription step. Both HIV reverse transcriptases (RTs) were shown to be particularly error-prone during the in vitro DNA-dependent DNA synthesis relative to other retroviral RTs. Extension of mismatched 3'-termini of the primer DNA was shown to be a major determinant in the infidelity of HIV RTs. However, reverse transcriptases generally exhibit dual template specificities. Therefore, we determined in the current study the fidelity of RNA-dependent DNA synthesis catalyzed in vitro by the RTs of HIV-1 and HIV-2 in comparison with that of murine leukemia virus (MLV) RT. Consequently, we examined the ability of these enzymes to extend preformed 3'-terminal A.A, A.C, and A.G mispairs by quantitating the amount and length of extended primers in a primer extension assay using ribosomal RNA as a template. The results demonstrate that the three RTs studied exhibited efficient extensions from 3'-terminal mispairs with a specificity of A.C greater than A.A greater than A.G. Nevertheless, the HIV RTs are qualitatively as well as quantitatively more error-prone than MLV RT. The mispair extension efficiency appears to be affected mainly by the increase of apparent Km values, rather than by the change in Vmax values.(ABSTRACT TRUNCATED AT 250 WORDS)
Studies to date assessing HIV escape from CTL in vivo have yielded conflicting results. Previous studies have demonstrated that simian immunodeficiency virus of macaques (SIVmac)-infected rhesus monkeys expressing the MHC class I allele Mamu-A*01 reproducibly develop a gag-specific CTL response limited to a 9-amino acid epitope of the SIVmac gag protein (residues 182-190 within peptide 11C). To determine whether CTL have a role in selecting for AIDS virus mutants, we examined mutations in SIVmac proviral DNA encoding this gag CTL epitope in PBL of infected rhesus monkeys. Three Mamu-A*01+ rhesus monkeys were infected with SIVmac and assessed for gag- and peptide 11C-specific CTL responses. This specific CTL response was maintained in two monkeys, but lost in the third animal 2 yr after infection. The generation of proviral gag mutations was then determined by sequencing 500-bp proviral fragments amplified from fresh PBL obtained from the monkeys more than 2.5 yr after infection. Although numerous point mutations were characterized in 131 polymerase chain reaction-generated clones of SIVmac gag, only four mutations within the gag CTL epitope-coding region of the genome were identified. Comparison of synonymous and nonsynonymous nucleotide substitutions in the regions encoding peptide 11C (p11C) and the flanking gag protein indicated a lack of selective pressure for viral mutations in the CTL epitope coding region. Interestingly, a predominant gag mutant encoding a single amino acid change in p11C was found in a monkey which lost its CTL activity. However, even in this setting there was no evidence for selection of mutations in the CTL epitope coding region when compared with the flanking region. Furthermore, synthetic peptides corresponding to all naturally occurring variants in the gag epitope-coding region were recognized by cloned and bulk cultured effector cells of the infected monkeys with persistent CTL. These results indicate that SIVmac gag- and p11C-specific CTL do not select for mutations in the immunodominant epitope-coding region and that the naturally occurring mutants do not appear to escape CTL recognition.
Viruses persist in an immune population, as in the case of influenza, or in an individual, as postulated for human immunodeficiency virus, when they are able to escape existent neutralizing antibody responses by changing their antigens. It is now shown that viruses can in principle escape the immunosurveillance of virus-specific cytotoxic T cells by mutations that alter the relevant T-cell epitope.
A large number of human immunodeficiency virus type 1 (HIV-1) specific HLA-restricted cytotoxic T cell (CTL) epitopes have been mapped, including an HLA-B27-restricted immunodominant epitope within p25gag. Accordingly, this segment of the HIV-1 provirus was amplified by the polymerase chain reaction from DNA derived from fresh uncultured peripheral blood mononuclear cells (PBMC) of four HLA-B27 HIV-1-infected individuals. In all cases the majority of infected PBMC bore sequences encoding the HLA-B27-restricted peptide. CTL escape mutants had not accumulated in vivo 8 and 14 months later despite demonstrable CTL activity in vitro. These data emphasize the importance of silently infected lymphocytes in evading immune surveillance.
In a longitudinal study of HIV seropositive patients, there were fluctuations in the specificity of cytotoxic T cells for the virus. This was matched by variability in proviral gag DNA epitope sequences in the lymphocytes of these patients. Some of these viral variants are not recognized by autologous T cells. Accumulation of such mutations in T-cell antigenic targets would provide a mechanism for immune escape.
In an attempt to generate a suitable animal model to study the infectivity and possible pathogenicity of human immunodeficiency viruses, we intravenously inoculated juvenile rhesus macaques and African green monkeys with a molecularly cloned virus, human immunodeficiency virus type 2 HIV-2sbl/isy, as well as with the uncloned HIV-2nih-z virus. Infection was monitored by virus recovery from the peripheral blood cells and by seroconversion against HIV-2 antigens measured by Western immunoblot, radioimmunoprecipitation, and enzyme-linked immunosorbent assay. We successfully infected two out of two macaques with the molecularly cloned virus and one macaque out of two with the HIV-2nih-z. No evidence of infection was seen in the African green monkeys with either virus. We followed the infected animals for 2 years. The animals remained healthy, although we observed intermittent lymphadenopathy and a transient decrease in the absolute number of circulating CD4+ T lymphocytes in both animals infected with the molecularly cloned virus. Virus isolation from the peripheral blood cells of the infected animals was successful only within the first few months after inoculation. Evidence of persistent infection was provided by the detection of proviral DNA by polymerase chain reaction analysis of the blood cells of the inoculated animals and by the stability of antiviral antibody titers. To evaluate the genetic drift of the proviral DNA, we molecularly cloned viruses which were reisolated 1 and 5 months postinoculation from one of these animals. Comparison of the DNA sequences of the envelope genes of both these isolates indicated that a low degree of variation (0.2%) in the envelope protein had occurred in vivo during the 5-month period. These data suggest that the use of HIV-2sbl/isy in rhesus macaques may represent a good animal model system to study prevention of viral infection. In particular, molecularly cloned virus can be manipulated for functional studies of viral genes in the pathogenesis of acquired immune deficiency syndrome and provides a reproducible source of virus for vaccine studies.
The SIV macaque model is an excellent surrogate for SIV infection of humans. Genital mucosal transmission of SIV presents the opportunity for testing the effectiveness of spermicides, pharmacologic agents and vaccines in preventing the heterosexual transmission of HIV. Because the incubation period is usually shorter and the disease tempo more rapid than seen with HIV infection, the endpoint for therapeutic, prophylaxis and vaccine trials can be reached sooner in the monkey model. Initial vaccine experiments using inactivated whole SIV mac did not protect rhesus macaques against IV or genital mucosal challenge with a moderately high dose of homologous live virus but did appear to delay disease in the IV challenge group. Similarly, a modified live SIVmac immunogen also failed to protect rhesus monkeys against IV challenge with live virus but did delay disease. It appears, therefore, that a strong immediate immune response to SIVmac, whether naturally or artificially induced can reduce the level of viremia and delay the onset of clinical SAIDS. We believe that these inactivated whole virus and modified live virus approaches are worth pursuing further and they may guide us towards an eventual effective vaccine for AIDS.
Information on the extent of genetic variability among non-human primate lentiviruses related to human immunodeficiency virus (HIV) is sorely lacking. Here we describe the isolation of two molecular clones from the simian immunodeficiency virus (SIV) and their use to derive restriction endonuclease maps of five SIV isolates from rhesus macaques and one from a cynomolgus macaque. Although similar, all six viral isolates are readily distinguishable; the single isolate from a cynomolgus macaque is the most different. The restriction endonuclease map of one macaque isolate (SIVMAC-251) is identical to that published by others for STLV-IIIAGM of African green monkeys and for HTLV-IV of humans. Nucleotide sequences from the envelope region of cloned SIVMAC-251 have more than 99% identify to previously published sequences for STLV-IIIAGM (refs 2, 4) and HTLV-IV (ref. 4). These results and other observations provide strong evidence that isolates previously referred to as STLV-IIIAGM and HTLV-IV by others are not authentic, but were derived from cell cultures infected with SIVMAC-251.
Most asymptomatic individuals infected with HIV-1 have a cytotoxic T lymphocyte (CTL) response to the virus Gag proteins which can be demonstrated in vitro. Epitopes have been mapped in p17 Gag and p24 Gag restricted by HLA-B8 (p17-3 and p24-13) and -B27 (p24-14). Viruses isolated from patients who make CTL responses to these peptides vary within the genetic sequences encoding these epitopes and some mutations lead to reduction in killing activity in vitro. This was attributed to either failure of the variant epitope to bind major histocompatibility complex class I or failure of T-cell receptors to bind the presented peptide. But peptide variants of class I-restricted epitopes cause 'antagonism', that is, the presence of a variant epitope (in the form of peptide) inhibits normal lysis of targets presenting the original epitope. This mirrors similar findings in class II-restricted systems. Here we report that naturally occurring variant forms of p17-3, p24-13 and p24-14 may cause antagonism of CTL lines derived from the same individuals. The effect is present if the epitopes are derived from synthetic peptides and when they are processed from full-length proteins expressed by either recombinant vaccinia constructs or replicating HIV.
Cytotoxic T lymphocytes (CTL) directed against human immunodeficiency virus (HIV)-1 are detectable in the majority of infected individuals, and their early appearance as the initial viremia is suppressed is thought to represent a potent antiviral response. Variation which arises in CTL epitopes can affect recognition by CTL, and we have observed previously that variant epitopes in HIV-1 gag which arise in HIV-1-seropositive donors may act as T cell receptor (TCR) antagonists of their own CTL (Klenerman et al., Nature 1994. 369: 403). The most important question arising from these observations is the extent of these immune escape mechanisms in vivo.
Here we show that fresh, uncultured lymphocytes taken directly from HIV-1-infected patients are susceptible to TCR antagonism by variants present within their own virus. In contrast to HLA Class II-restricted T cell responses, where anergy may be induced, we find that in vitro, natural variants may stimulate and sustain growth of CTL. These CTL lines retain lytic specificity exclusively for the original peptide. If this represents events in vivo, natural HIV altered peptide ligands (APL) have the capacity to inhibit the range of CTL directed against an epitope, not simply those clones selected in vitro. Partial activation of CTL by APL could also act to drive an ineffectual CTL response incapable of lysing infected cells bearing these natural antigenic variants. Distortion of lymphocyte populations and function by APL might represent a further mechanism of immune evasion by HIV.
A model is presented for sequence evolution on the basis of which one can analyze combinations of noncoding, singly coding, and multiply coding regions of aligned homologous DNA sequences. It is a generalization of Kimura's (J. Mol. Evol. 16:111–120, 1980) and Li et al.'s (J. Mol. Evol. 36:96–99, 1985) transition-transversion models with selection on replacement substitutions.
Based on a hierarchy of hypotheses, one will be able to estimate selection factors and transition and transversion distances for different combinations of regions ranging from many regions, each with their private set of parameters, to one set of parameters for all regions.
The method is demonstrated on two aligned HIV I retroviruses.
Several recent reports indicate that the long, clinically latent phase that characterizes human immunodeficiency virus (HIV) infection of humans is not a period of viral inactivity, but an active process in which cells are being infected and dying at a high rate and in large numbers. These results lead to a simple steady-state model in which infection, cell death, and cell replacement are in balance, and imply that the unique feature of HIV is the extraordinarily large number of replication cycles that occur during infection of a single individual. This turnover drives both the pathogenic process and (even more than mutation rate) the development of genetic variation. This variation includes the inevitable and, in principle, predictable accumulation of mutations such as those conferring resistance to antiviral drugs whose presence before therapy must be considered in the design of therapeutic strategies.
A comprehensive system for genetic typing of the HLA class I A locus is described, based on PCR amplification and typing with nonradioactively labeled SSO probes. Exons 1-3 of the A locus are amplified and typing is performed with a set of 30 nonradioactively labeled oligonucleotide probes. This system resolves 34 of 39 known alleles and 561 (94%) of 595 possible genotypes. Among a sample of 354 individuals from Sweden and China, 97.5% of the genotypes were resolved. Probes were directed preferentially at replacement substitutions in foreign antigen-binding sites, in order to detect not only the known alleles but also new combinations of polymorphic motifs, indicative of previously unrecognized alleles. Three individuals were found with a new combination of polymorphic motifs, suggesting the presence of at least one previously undescribed allele in the populations sampled. This typing system is useful for disease association studies, tissue typing, and in forensic medicine.
Like other pathogenic viruses, HIV-1 down-modulates surface expression of major histocompatibility complex class I (MHC-I) molecules in infected cells, thus impairing lysis by cytotoxic T lymphocytes. We have observed that this phenomenon depends on the expression of Nef. nef is an early gene of primate lentiviruses, which is necessary for maintaining high virus loads and inducing AIDS. Nef is not necessary for viral replication in vitro and stimulates the endocytosis of CD4. We show that the expression of MHC-I at the surface of lymphoid, monocytic and epithelial cells was reduced in the presence of Nef protein from various HIV-1 strains. Whereas MHC-I protein synthesis and transport through the endoplasmic reticulum and cis Golgi apparatus occurred normally in Nef(+) cells, surface MHC-I molecules were rapidly internalized, accumulated in endosomal vesicles and were degraded. The stimulation of MHC-I endocytosis by Nef represents a previously undocumented viral mechanism for evading the immune response.
The retroviral DNA polymerase, or reverse transcriptase, lacks a 3' exonuclease proofreading activity. This causes a high mutation rate with the result that genetic diversity and drug resistance are increased. Only combination chemotherapy has the potential to out-manoeuvre these viruses. However, there is no evidence that ongoing variation allows escape from immune surveillance, so contributing to pathogenesis.
Human immunodeficiency virus type 1 (HIV-1) Gag-specific cytotoxic T lymphocyte (CTL) responses were studied in seven seropositive long-term asymptomatic individuals (CDC A1) with stable CD4 counts for more than 8 years. Using a set of partially overlapping peptides covering the whole Gag, five 15-20-mer peptides were found to contain CTL epitopes. Further characterization of these epitopes revealed a new HLA-A25-restricted CTL epitope in p24, p24(203-212) ETINEEAAEW. This region of Gag is highly conserved in clades B and D of HIV-1. Naturally occurring amino acid sequences, containing p24(203)D (consensus HIV-1 clades A, C, F, G and H) or p24(204)I (HIV-2ROD) were not recognized by CTL recognizing the index peptide. No virus variants with mutations in this sequence were found in peripheral blood mononuclear cells from the HIV-1-infected individual concerned during the 8 year observation period, indicating that the virus had not escaped from the observed CTL response.
The HIV-1-specific cytotoxic T lymphocyte (CTL) response is temporally associated with the decline in viremia during primary HIV-1 infection, but definitive evidence that it is of importance in virus containment has been lacking. Here we show that in a patient whose early CTL response was focused on a highly immunodominant epitope in gp 160, there was rapid elimination of the transmitted virus strain and selection for a virus population bearing amino acid changes at a single residue within this epitope, which conferred escape from recognition by epitope-specific CTL. The magnitude (> 100-fold), kinetics (30-72 days from onset of symptoms) and genetic pathways of virus escape from CTL pressure were comparable to virus escape from antiretroviral therapy, indicating the biological significance of the CTL response in vivo. One aim of HIV-1 vaccines should thus be to elicit strong CTL responses against multiple codominant viral epitopes.
Cytotoxic T lymphocytes (CTLs) are thought to play a crucial role in the termination of the acute primary HIV-1 syndrome, but clear evidence for this presumption has been lacking. Here we demonstrate positive selection of HIV-1 proviral sequences encoding variants within a CTL epitope in Nef, a gene product critical for viral pathogenicity, during and after seroconversion. These positively selected HIV-1 variants carried epitope sequence changes that either diminished or escaped CTL recognition. Other proviruses had mutations that abolished the Nef epitope altogether. These results provide clear evidence that CTLs exert selection pressure on the viral population in acute HIV-1 infection.
With the aim of elucidating the evolution of a hepadnavirus family, we constructed molecular phylogenetic trees for 27 strains of hepatitis B virus (HBV) using both the unweighted pair-grouping and neighbor-joining methods. All five gene regions, P, C, S, X, and preS, were used to construct the phylogenetic trees. Using the phylogenetic trees obtained, we classified these strains into five major groups in which the strains were closely related to each other. Our classification reinforced our previous view that genetic classification is not always compatible with conventional classification determined by serological subtypes. Moreover, constraints on the evolutionary process of HBV were analyzed for amino-acid-altering (nonsynonymous) and silent (synonymous) substitutions, because two-thirds of the open reading frame (ORF), P, contains alternating overlapping ORFs. In our unique analysis of this interesting gene structure of HBV, the most frequent synonymous substitutions were observed in the nonoverlapped parts of the P and C genes. On the other hand, the number of synonymous substitutions per nucleotide site for the S gene was quite low and appeared a strongly constrained evolution. Because the P gene overlaps the S gene in a different frame, the low rate of synonymous substitution for the S gene can be explained by the evolutionary constraints which are imposed on the overlapping gene region. In other words, synonymous substitutions in the S gene can cause amino acid changes in its overlapping region in a different frame. Thus, the evolution of HBV is constrained evolutionarily by the overlapping genes. We propose calling this mode of viral evolution "constrained evolution." The evolution of HBV represents a typical constrained evolution.
In genetic language a peculiar arrangement of biological information is provided by overlapping genes in which the same region of DNA can code for functionally unrelated messages. In this work, the informational content of overlapping genes belonging to prokaryotic and eukaryotic viruses was analyzed. Using information theory indices, we identified in the regions of overlap a first pattern, exhibiting a more uniform base composition and more severe constraints in base ordering with respect to the nonoverlapping regions. This pattern was found to be peculiar to coliphage, avian hepatitis B virus, human lentivirus, and plant luteovirus families. A second pattern, characterized by the occurrence of similar compositional constraints in both types of coding regions, was found to be limited to plant tymoviruses. At the level of codon usage, a low degree of correlation between overlapping and nonoverlapping coding regions characterized the first pattern, whereas a close link was found in tymoviruses, indicating a fine adaptation of the overlapping frame to the original codon choice of the virus. As a result of codon usage correlation analysis, deductions concerning the origin and evolution of several overlapping frames were also proposed. Comparison of amino acid composition revealed an increased frequency of amino acid residues with a high level of degeneracy (arginine, leucine, and serine) in the proteins encoded by overlapping genes; this peculiar feature of overlapping genes can be viewed as a way with which they may expand their coding ability and gain new, specialized functions.
Adaptive immunity is unique to the vertebrates, and the molecules involved (including immunoglobulins, T cell receptors and the major histocompatibility complex molecules) seem to have diversified very rapidly early in vertebrate history. Reconstruction of gene phylogenies has yielded insights into the evolutionary origin of a number of molecular systems, including the complement system and the major histocompatibility complex (MHC). These analyses have indicated that the C5 component of complement arose by gene duplication prior to the divergence of C3 and C4, which suggests that the alternative complement pathway was the first to evolve. In the case of the MHC, phylogenetic analysis supports the hypothesis that MHC class II molecules evolved before class I molecules. The fact that the MHC-linked proteasome components that specifically produce peptides for presentation by class I MHC appear to have originated before the separation of jawed and jawless vertebrates suggests that the MHC itself may have been present at this time. Immune system gene families have evolved by gene duplication, interlocus recombination and (in some cases) positive Darwinian selection favoring diversity at the amino acid level.
RNA virus quasispecies are subjected to processes of positive Darwinian selection, to a very active and continuous negative selection and to random genetic drift. The course of RNA virus evolution is often unpredictable, and recent results suggest that even highly conserved motifs, once regarded as essential for infectivity, may be rendered dispensable by singular evolutionary events. An immediate consequence of the quasispecies genetic organization of RNA viruses is a surprising ability to gain fitness once a minimal replication ability is established in a biological environment. The unique features of RNA genetics should not be underestimated since they are at the basis of the emergence of new viral diseases and of the current difficulties to control many diseases associated variable viruses.
We compared nef gene sequences isolated by polymerase chain reaction from peripheral blood lymphocyte DNA of macaques that had been inoculated with either biologically (E11S) or molecularly (clone 8) cloned SIV/Mne. Two samples from each animal obtained either early (weeks 2-8) or late (weeks 21-137) after infection were analyzed. Three substitutions in the predicted Nef amino acid sequence were seen in all animals at the late time point, and two other substitutions were seen in all except one. Two of the common exchanges are located approximately 40 residues apart in the Nef core sequence but are juxtaposed on the tertiary structure as judged by computer modeling using the structure of the HIV Nef core protein as a guide. Most recurrent in vivo changes replaced a residue found in the cloned Nef sequence with one present in a consensus derived by aligning the Nef sequences of the SIV/Sm clade. Recombinant virus containing a macaque-adapted (MA nef) nef on the clone 8 backbone was 3-fold more infectious on SMAGI cells than the original virus. A lymphocyte line infected with SIV-clone 8-MAnef contained a large proportion of cells carrying provirus with defective nef genes. These findings suggest that the nef gene of the cloned SIV/Mne had undergone attenuating mutations during propagation in tissue culture that were "corrected" in vivo.
Infection with human immunodeficiency virus type 1 (HIV-1) and progression to acquired immune deficiency syndrome (AIDS) are controlled by both host genetic factors and viral factors. The HLA (human leukocyte antigen) region in humans controls immune response functions and tissue rejection and influences susceptibility to neoplasia, autoimmune diseases, and infectious diseases including HIV. Twenty-eight African American and 12 Caucasian patients participated in the study. HLA-DQB1 and HLA-DRB1 genotyping was performed using PCR and sequence-specific oligonucleotide probe reverse hybridization and analyzed with the LiPA Key Typing System and LiPA software. DQB1*0603 was found to be positively associated with HIV-1 infection and with HIV-1 infection in Caucasians but not African Americans. DQB1*03032 frequencies indicate a positive association with protection from HIV-1 infection. It was further found to be protective against HIV-1 infection in Caucasians but not in African Amens. DQB1*0201 was observed more frequently in HIV(+) African Americans than HIV(-) African Americans, suggesting a positive association with HIV-1 infection in this ethnic group. HLA-DRB1*04 exhibited a positive association with HIV-1 infection in Caucasians. These data show that there are HLA class II alleles associated with susceptibility to and protection from HIV-1 infection and that these differ between ethnic groups.
Although animal models have been useful in guiding vaccine development, HIV/AIDS models have not yielded a clear correlate of immunity nor given consistent results on the potential efficacy of various vaccine approaches. Further development and improved uniformity in the use of animal models would maximize their potential to meet the urgent worldwide need for a safe and effective HIV vaccine.
To address the subtle interactions between antiviral cytotoxic T-cell (CTL) immune responses and the evolution of viral quasispecies variants in vivo, we performed a longitudinal study in a simian immunodeficiency virus (SIV)-infected rhesus macaque that had a long experimental SIV infection before developing simian AIDS. Before being infected with SIV, this animal was immunized with a mixture of seven lipopeptides derived from SIV Nef and Gag proteins and showed a bispecific antiviral CTL response directed toward Nef 169-178 and 211-225 peptides. After SIV infection, CTL activity against the Nef 169-178 epitope was no longer detectable, as assessed from peripheral blood mononuclear cells stimulated by autologous SIV. CTL activity against the 211-225 epitope was lost after 3 months, and an additional CTL response to the amino acids 112-119 Nef epitope emerged. Analysis of the Nef proviral sequence revealed the presence of immune escape variants first in the 211-225 epitope and much later in the 112-119 epitope. In contrast, epitope 169-178 showed only two mutations among all viral sequencing performed. We conclude that in this macaque, bispecific CTL exerted a strong selective pressure and escape virus mutants finally emerged. We identified CTL recognizing a conserved Nef epitope 112-119 (SYKLAIDM), essential for viral replication, which could be associated with a prolonged AIDS-free period. These results stress the importance of the induction of broader multispecific CTLs directed against highly conserved and functional T-cell epitopes by vaccination, with the aim of keeping HIV infection in check.