HLA B*5701-positive long-term nonprogressors/elite controllers are not distinguished from progressors by the clonal composition of HIV-specific CD8+ T cells

To better understand the qualitative features of effective human immunodeficiency virus (HIV)-specific immunity, we examined the TCR clonal composition of CD8(+) T cells recognizing conserved HIV p24-derived epitopes in HLA-B*5701-positive long-term nonprogressors/elite controllers (LTNP/EC) and HLA-matched progressors. Both groups displayed oligoclonal HLA-B5701-restricted p24-specific CD8(+) T-cell responses with similar levels of diversity and few public clonotypes. Thus, HIV-specific CD8(+) T-cell responses in LTNP/EC are not differentiated from those of progressors on the basis of clonal diversity or TCR sharing.     

Viral replication capacity as a correlate of HLA B57/B5801-associated nonprogressive HIV-1 infection

HLA B57 and the closely related HLA B5801 are over-represented among HIV-1 infected long-term nonprogressors (LTNPs). It has been suggested that this association between HLA B57/5801 and asymptomatic survival is a consequence of strong CTL responses against epitopes in the viral Gag protein. Moreover, CTL escape mutations in Gag would coincide with viral attenuation, resulting in low viral load despite evasion from immune control. In this study we compared HLA B57/5801 HIV-1 infected progressors and LTNPs for sequence variation in four dominant epitopes in Gag and their ability to generate CTL responses against these epitopes and the autologous escape variants. Prevalence and appearance of escape mutations in Gag epitopes and potential compensatory mutations were similar in HLA B57/5801 LTNPs and progressors. Both groups were also indistinguishable in the magnitude of CD8+ IFN-gamma responses directed against the wild-type or autologous escape mutant Gag epitopes in IFN-gamma ELISPOT analysis. Interestingly, HIV-1 variants from HLA B57/5801 LTNPs had much lower replication capacity than the viruses from HLA B57/5801 progressors, which did not correlate with specific mutations in Gag. In conclusion, the different clinical course of HLA B57/5801 LTNPs and progressors was not associated with differences in CTL escape mutations or CTL activity against epitopes in Gag but rather with differences in HIV-1 replication capacity.     

Decay kinetics of human immunodeficiency virus-specific CD8+ T cells in peripheral blood after initiation of highly active antiretroviral therapy.

We measured the longitudinal responses to 95 HLA class I-restricted human immunodeficiency virus (HIV) epitopes and an immunodominant HLA A2-restricted cytomegalovirus (CMV) epitope in eight treatment-naive HIV-infected individuals, using intracellular cytokine staining. Patients were treated with highly active antiretroviral therapy (HAART) for a median of 78 weeks (range, 34 to 121 weeks). Seven of eight patients maintained an undetectable viral load for the duration of therapy. A rapid decline in HIV-specific CD8(+) T-cell response was observed at initiation of therapy. After an undetectable viral load was achieved, a slower decrease in HIV-specific CD8(+) T-cell response was observed that was well described by first-order kinetics. The median half-life for the rate of decay was 38.8 (20.3 to 68.0) weeks when data were expressed as percentage of peripheral CD8(+) T cells. In most cases, data were similar when expressed as the number of responding CD8(+) T cells per microliter of blood. In subjects who responded to more than one HIV epitope, rates of decline in response to the different epitopes were similar and varied by a factor of 2.2 or less. Discontinuation of treatment resulted in a rapid increase in HIV-specific CD8(+) T cells. Responses to CMV increased 1.6- and 2.8-fold within 16 weeks of initiation of HAART in two of three patients with a measurable CMV response. These data suggest that HAART quickly starts to restore CD8(+) T-cell responses to other chronic viral infections and leads to a slow decrease in HIV-specific CD8(+) T-cell response in HIV-infected patients. The slow decrease in the rate of CD8(+) T-cell response and rapid increase in response to recurrent viral replication suggest that the decrease in CD8(+) T-cell response observed represents a normal memory response to withdrawal of antigen.     

Escape from CD8(+) T cell responses in Mamu-B*00801(+) macaques differentiates progressors from elite controllers

A small number of HIV-infected individuals known as elite controllers experience low levels of chronic phase viral replication and delayed progression to AIDS. Specific HLA class I alleles are associated with elite control, implicating CD8(+) T lymphocytes in the establishment of these low levels of viral replication. Most HIV-infected individuals that express protective HLA class I alleles, however, do not control viral replication. Approximately 50% of Mamu-B*00801(+) Indian rhesus macaques control SIVmac239 replication in the chronic phase in a manner that resembles elite control in humans. We followed both the immune response and viral evolution in SIV-infected Mamu-B*00801(+) animals to better understand the role of CD8(+) T lymphocytes during the acute phase of viral infection, when viral control status is determined. The virus escaped from immunodominant Vif and Nef Mamu-B*00801-restricted CD8(+) T lymphocyte responses during the critical early weeks of acute infection only in progressor animals that did not control viral replication. Thus, early CD8(+) T lymphocyte escape is a hallmark of Mamu-B*00801(+) macaques who do not control viral replication. By contrast, virus in elite controller macaques showed little evidence of variation in epitopes recognized by immunodominant CD8(+) T lymphocytes, implying that these cells play a role in viral control.     

Putative immunodominant human immunodeficiency virus-specific CD8(+) T-cell responses cannot be predicted by major histocompatibility complex class I haplotype

Recent studies of human immunodeficiency virus (HIV)-specific CD8(+) T cells have focused on responses to single, usually HLA-A2-restricted epitopes as surrogate measures of the overall response to HIV. However, the assumption that a response to one epitope is representative of the total response is unconfirmed. Here we assess epitope immunodominance and HIV-specific CD8(+) T-cell response complexity using cytokine flow cytometry to examine CD8(+) T-cell responses in 11 HLA-A2(+) HIV(+) individuals. Initial studies demonstrated that only 4 of 11 patients recognized the putative immunodominant HLA-A2-restricted p17 epitope SLYNTVATL, suggesting that the remaining subjects might lack significant HIV-specific CD8(+) T-cell responses. However, five of six SLYNTVATL nonresponders recognized other HIV epitopes, and two of four SLYNTVATL responders had greater responses to HIV peptides restricted by other class I alleles. In several individuals, no HLA-A2-restricted epitopes were recognized, but CD8(+) T-cell responses were detected to epitopes restricted by other HLA class I alleles. These data indicate that an individual's overall CD8(+) T-cell response to HIV is not adequately represented by the response to a single epitope and that individual major histocompatibility complex class I alleles do not predict an immunodominant response restricted by that allele. Accurate quantification of total HIV-specific CD8(+) T-cell responses will require assessment of the response to all possible epitopes.     

Comparisons of CD8+ T Cells Specific for Human Immunodeficiency Virus,Hepatitis C Virus,and Cytomegalovirus Reveal Differences in Frequency,Immunodominance, Phenotype,and Interleukin-2 Responsiveness

To better understand the components of an effective immune response to human immunodeficiency virus (HIV), the CD8⁺ T-cell responses to HIV, hepatitis C virus (HCV), and cytomegalovirus (CMV) were compared with regard to frequency, immunodominance, phenotype, and interleukin-2 (IL-2) responsiveness. Responses were examined in rare patients exhibiting durable immune-mediated control over HIV, termed long-term nonprogressors (LTNP) or elite controllers, and patients with progressive HIV infection (progressors). The magnitude of the virus-specific CD8⁺ T-cell response targeting HIV, CMV, and HCV was not significantly different between LTNP and progressors, even though their capacity to proliferate to HIV antigens was preserved only in LTNP. In contrast to HIV-specific CD8⁺ T-cell responses of LTNP, HLA B5701-restricted responses within CMV pp65 were rare and did not dominate the total CMV-specific response. Virus-specific CD8⁺ T cells were predominantly CD27⁺45RO⁺ for HIV and CD27⁻45RA⁺ for CMV; however, these phenotypes were highly variable and heavily influenced by the degree of viremia. Although IL-2 induced significant expansions of CMV-specific CD8⁺ T cells in LTNP and progressors by increasing both the numbers of cells entering the proliferating pool and the number of divisions, the proliferative capacity of a significant proportion of HIV-specific CD8⁺ T cells was not restored with exogenous IL-2. These results suggest that immunodominance by HLA B5701-restricted cells is specific to HIV infection in LTNP and is not a feature of responses to other chronic viral infections. They also suggest that poor responsiveness to IL-2 is a property of HIV-specific CD8⁺ T cells of progressors that is not shared with responses to other viruses over which immunologic control is maintained.Gaining a better understanding of the immunologic control of human immunodeficiency virus type 1 (HIV-1) is among the most critical goals for the rational design of HIV vaccines and immunotherapies. Although most HIV-infected patients develop high-level viremia, CD4⁺ T-cell depletion, and progressive disease, a rare subgroup of patients variably termed long-term nonprogressors (LTNP) or elite controllers restrict HIV replication to below 50 copies of HIV RNA/ml plasma and remain disease free for up to 25 years without antiretroviral therapy (ART). Measurements of HIV-specific immune responses in these patients, in comparison with progressors, are providing insights into mechanisms that mediate immunologic control or loss of control in humans. Although the mechanisms of restriction of HIV replication remain incompletely understood, a number of lines of evidence suggest that it is mediated by HIV-specific CD8⁺ T cells (reviewed in reference 51). High frequencies of HIV-specific CD8⁺ T cells specific for the autologous virus are observed in both LTNP and untreated progressors, suggesting that differences in immunologic control are mediated not by quantitative but more likely by qualitative features of the immune response.A number of qualitative features of the HIV-specific CD8⁺ T-cell response of LTNP or progressors have recently been proposed as the cause of immunologic control or loss of control, respectively. HLA B*5701 is highly overrepresented in LTNP, and the HIV-specific CD8⁺ T-cell response is highly focused on B5701-restricted peptides in B*5701⁺ LTNP but not in B*5701⁺ progressors (19, 50). In addition, there is a difference in surface markers between HIV- and cytomegalovirus (CMV)-specific CD8⁺ T cells thought to represent differences in maturation of the T-cell response (8). The CD8⁺ T cells of progressors are diminished in proliferative capacity and perforin upregulation in response to autologous HIV-infected CD4⁺ T cells (49). Recently, it has been proposed that this diminished proliferative capacity is due to a lack of paracrine or autocrine interleukin-2 (IL-2) production by HIV-specific CD4⁺ T cells or CD8⁺ T cells (41, 42, 75). Interpretation of proliferation studies is complicated by the fact that the effects of IL-2 were measured on the basis of 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) dye dilution of major histocompatibility complex (MHC) tetramer-positive cells. Because cell division over 6 days is an exponential function, IL-2 may induce small increases in the percentage of cells dividing or in the number of cell divisions that can result in large changes in the percent CFSE^lo cells, and yet the majority of antigen-specific cells may not proceed through the cell cycle. In addition, there are very limited data regarding whether the features of immunodominance, surface phenotype, and IL-2 responsiveness of HIV-specific CD8⁺ T cells extend to other chronic virus infections.In the present study, we examined these qualitative features within the response to HIV, CMV, or hepatitis C virus (HCV) across patient groups. We observed that the high degree of focus upon B5701-restricted peptides found in LTNP does not extend to the HCV- or CMV-specific responses. The phenotype of HIV- or CMV-specific CD8⁺ T cells was highly variable and heavily influenced by the degree of viremia. In addition, when both the number of divisions and the percentage of cells dividing were analyzed, proliferation of HIV-specific CD8⁺ T cells was refractory to IL-2 stimulation, unlike that of CMV-specific cells. These results offer important insights into qualitative features of the HIV-specific CD8⁺ T-cell response, whether they extend to responses to other viruses, and whether they are associated with the presence or absence of immunologic control.     

Hepatitis B virus splice-generated protein induces T-cell responses in HLA-transgenic mice and hepatitis B virus-infected patients

Hepatitis B virus splice-generated protein (HBSP), encoded by a spliced hepatitis B virus RNA, was recently identified in liver biopsy specimens from patients with chronic active hepatitis B. We investigated the possible generation of immunogenic peptides by the processing of this protein in vivo. We identified a panel of potential epitopes in HBSP by using predictive computational algorithms for peptide binding to HLA molecules. We used transgenic mice devoid of murine major histocompatibility complex (MHC) class I molecules and positive for human MHC class I molecules to characterize immune responses specific for HBSP. Two HLA-A2-restricted peptides and one immunodominant HLA-B7-restricted epitope were identified following the immunization of mice with DNA vectors encoding HBSP. Most importantly, a set of overlapping peptides covering the HBSP sequence induced significant HBSP-specific T-cell responses in peripheral blood mononuclear cells from patients with chronic hepatitis B. The response was multispecific, as several epitopes were recognized by CD8(+) and CD4(+) human T cells. This study provides the first evidence that this protein generated in vivo from an alternative reading frame of the hepatitis B virus genome activates T-cell responses in hepatitis B virus-infected patients. Given that hepatitis B is an immune response-mediated disease, the detection of T-cell responses directed against HBSP in patients with chronic hepatitis B suggests a potential role for this protein in liver disease progression.     

Macaque long-term nonprogressors resist superinfection with multiple CD8+ T cell escape variants of simian immunodeficiency virus

Human immunodeficiency virus (HIV)-positive individuals can be superinfected with different virus strains. Individuals who control an initial HIV infection are therefore still at risk for subsequent infection with divergent viruses, but the barriers to such superinfection remain unclear. Here we tested long-term nonprogressors' (LTNPs') susceptibility to superinfection using Indian rhesus macaques that express the major histocompatibility complex class I (MHC-I) allele Mamu-B 17, which is associated with control of the pathogenic AIDS virus SIVmac239. The Mamu-B 17-restricted CD8(+) T cell repertoire is focused almost entirely on 5 epitopes. We engineered a series of SIVmac239 variants bearing mutations in 3, 4, or all 5 of these epitopes and used them to serially challenge 2 Mamu-B 17-positive LTNPs. None of the escape variants caused breakthrough replication in LTNPs, although they readily infected Mamu-B 17-negative naive macaques. In vitro competing coculture assays and examination of viral evolution in hosts lacking Mamu-B 17 suggested that the mutant viruses had negligible defects in replicative fitness. Both LTNPs maintained robust immune responses, including simian immunodeficiency virus (SIV)-specific CD8(+) and CD4(+) T cells and neutralizing antibodies. Our results suggest that escape mutations in epitopes bound by "protective" MHC-I molecules may not be sufficient to establish superinfection in LTNPs.     

Patterns of CD8+ immunodominance may influence the ability of Mamu-B*08-positive macaques to naturally control simian immunodeficiency virus SIVmac239 replication

Certain major histocompatibility complex (MHC) class I alleles are strongly associated with control of human immunodeficiency virus and simian immunodeficiency virus (SIV). CD8(+) T cells specific for epitopes restricted by these molecules may be particularly effective. Understanding how CD8(+) T cells contribute to control of viral replication should yield important insights for vaccine design. We have recently identified an Indian rhesus macaque MHC class I allele, Mamu-B*08, associated with elite control and low plasma viremia after infection with the pathogenic isolate SIVmac239. Here, we infected four Mamu-B*08-positive macaques with SIVmac239 to investigate why some of these macaques control viral replication. Three of the four macaques controlled SIVmac239 replication with plasma virus concentrations below 20,000 viral RNA copies/ml at 20 weeks postinfection; two of four macaques were elite controllers (ECs). Interestingly, two of the four macaques preserved their CD4(+) memory T lymphocytes during peak viremia, and all four recovered their CD4(+) memory T lymphocytes in the chronic phase of infection. Mamu-B*08-restricted CD8(+) T-cell responses dominated the acute phase and accounted for 23.3% to 59.6% of the total SIV-specific immune responses. Additionally, the ECs mounted strong and broad CD8(+) T-cell responses against several epitopes in Vif and Nef. Mamu-B*08-specific CD8(+) T cells accounted for the majority of mutations in the virus at 18 weeks postinfection. Interestingly, patterns of viral variation in Nef differed between the ECs and the other two macaques. Natural containment of AIDS virus replication in Mamu-B*08-positive macaques may, therefore, be related to a combination of immunodominance and viral escape from CD8(+) T-cell responses.     

Selection, transmission, and reversion of an antigen-processing cytotoxic T-lymphocyte escape mutation in human immunodeficiency virus type 1 infection

Numerous studies now support that human immunodeficiency virus type 1 (HIV-1) evolution is influenced by immune selection pressure, with population studies showing an association between specific HLA alleles and mutations within defined cytotoxic T-lymphocyte epitopes. Here we combine sequence data and functional studies of CD8 T-cell responses to demonstrate that allele-specific immune pressures also select for mutations flanking CD8 epitopes that impair antigen processing. In persons expressing HLA-A3, we demonstrate consistent selection for a mutation in a C-terminal flanking residue of the normally immunodominant Gag KK9 epitope that prevents its processing and presentation, resulting in a rapid decline in the CD8 T-cell response. This single amino acid substitution also lies within a second HLA-A3-restricted epitope, with the mutation directly impairing recognition by CD8 T cells. Transmission of the mutation to subjects expressing HLA-A3 was shown to prevent the induction of normally immunodominant acute-phase responses to both epitopes. However, subsequent in vivo reversion of the mutation was coincident with delayed induction of new CD8 T-cell responses to both epitopes. These data demonstrate that mutations within the flanking region of an HIV-1 epitope can impair recognition by an established CD8 T-cell response and that transmission of these mutations alters the acute-phase CD8(+) T-cell response. Moreover, reversion of these mutations in the absence of the original immune pressure reveals the potential plasticity of immunologically selected evolutionary changes.     

Circulating anti-Tax cytotoxic T lymphocytes from human T-cell leukemia virus type I-infected people, with and without tropical spastic paraparesis, recognize multiple epitopes simultaneously.

CD8+ T cells were freshly isolated from a human T-cell leukemia virus type I (HTLV-I)-infected patient with tropical spastic paraparesis. These cells, which were specific for HTLV-I Tax, simultaneously recognized a minimum of five, and possibly as many as seven, distinct peptide epitopes within the protein. A further Tax epitope was recognized after a short period of culture without exogenous peptide stimulation. All but one of these epitopes were clustered in the N-terminal third of Tax, and one of the epitopes was clearly immunodominant on two separate occasions of testing. Recognition of the immunodominant epitope was restricted by human leukocyte antigen (HLA) B15, and recognition of all the others was by HLA A2. Similar patterns of cytotoxic T lymphocyte recognition of the HLA A2-restricted Tax peptides in two healthy HTLV-I-seropositive individuals, each of whom carried the HLA A2 allele, were observed.     

Dominance of CD8 responses specific for epitopes bound by a single major histocompatibility complex class I molecule during the acute phase of viral infection.

Cytotoxic T-lymphocyte (CTL) responses are thought to control human immunodeficiency virus replication during the acute phase of infection. Understanding the CD8(+) T-cell immune responses early after infection may, therefore, be important to vaccine design. Analyzing these responses in humans is difficult since few patients are diagnosed during early infection. Additionally, patients are infected by a variety of viral subtypes, making it hard to design reagents to measure their acute-phase immune responses. Given the complexities in evaluating acute-phase CD8(+) responses in humans, we analyzed these important immune responses in rhesus macaques expressing a common rhesus macaque major histocompatibility complex class I molecule (Mamu-A*01) for which we had developed a variety of immunological assays. We infected eight Mamu-A*01-positive macaques and five Mamu-A*01-negative macaques with the molecularly cloned virus SIV(mac)239 and determined all of the simian immunodeficiency virus-specific CD8(+) T-cell responses against overlapping peptides spanning the entire virus. We also monitored the evolution of particular CD8(+) T-cell responses by tetramer staining of peripheral lymphocytes as well as lymph node cells in situ. In this first analysis of the entire CD8(+) immune response to autologous virus we show that between 2 and 12 responses are detected during the acute phase in each animal. CTL against the early proteins (Tat, Rev, and Nef) and against regulatory proteins Vif and Vpr dominated the acute phase. Interestingly, CD8(+) responses against Mamu-A*01-restricted epitopes Tat(28-35)SL8 and Gag(181-189)CM9 were immunodominant in the acute phase. After the acute phase, however, this pattern of reactivity changed, and the Mamu-A*01-restricted response against the Gag(181-189)CM9 epitope became dominant. In most of the Mamu-A*01-positive macaques tested, CTL responses against epitopes bound by Mamu-A*01 dominated the CD8(+) cellular immune response.     

HIV-1 Replication Fitness of HLA-B*57/58:01 CTL Escape Variants Is Restored by the Accumulation of Compensatory Mutations in Gag

Expression of HLA-B*57 and the closely related HLA-B*58:01 are associated with prolonged survival after HIV-1 infection. However, large differences in disease course are observed among HLA-B*57/58:01 patients. Escape mutations in CTL epitopes restricted by these HLA alleles come at a fitness cost and particularly the T242N mutation in the TW10 CTL epitope in Gag has been demonstrated to decrease the viral replication capacity. Additional mutations within or flanking this CTL epitope can partially restore replication fitness of CTL escape variants. Five HLA-B*57/58:01 progressors and 5 HLA-B*57/58:01 long-term nonprogressors (LTNPs) were followed longitudinally and we studied which compensatory mutations were involved in the restoration of the viral fitness of variants that escaped from HLA-B*57/58:01-restricted CTL pressure. The Sequence Harmony algorithm was used to detect homology in amino acid composition by comparing longitudinal Gag sequences obtained from HIV-1 patients positive and negative for HLA-B*57/58:01 and from HLA-B*57/58:01 progressors and LTNPs. Although virus isolates from HLA-B*57/58:01 individuals contained multiple CTL escape mutations, these escape mutations were not associated with disease progression. In sequences from HLA-B*57/58:01 progressors, 5 additional mutations in Gag were observed: S126N, L215T, H219Q, M228I and N252H. The combination of these mutations restored the replication fitness of CTL escape HIV-1 variants. Furthermore, we observed a positive correlation between the number of escape and compensatory mutations in Gag and the replication fitness of biological HIV-1 variants isolated from HLA-B*57/58:01 patients, suggesting that the replication fitness of HLA-B*57/58:01 escape variants is restored by accumulation of compensatory mutations.     

Quantitative analysis of the antiviral activity of CD8(+) T cells from human immunodeficiency virus-positive asymptomatic patients with different rates of CD4(+) T-cell decrease

We have measured in 22 asymptomatic human immunodeficiency virus type 1-infected patients (10 rapid progressors and 12 slow progressors) the proviral load of CD4(+) T cells homogeneously superinfected by the same dose of a non-syncytium-inducing virus in the presence or in the absence of autologous CD8(+) T cells. We demonstrated that the antiviral activity of CD8(+) T cells was highly predictive of the rate of peripheral CD4(+) T-cell decline.     

Maternal Transmission of Human Immunodeficiency Virus Escape Mutations Subverts HLA-B57 Immunodominance but Facilitates Viral Control in the Haploidentical Infant

Expression of HLA-B57 is associated with restricted replication of human immunodeficiency virus (HIV), but the mechanism for its protective effect remains unknown. If this advantage depends upon CD8 T-cell recognition of B57-restricted epitopes, mother-to-child transmission of escape mutations within these epitopes could nullify its protective effect. However, if the B57 advantage is largely mediated by selection for fitness-attenuating viral mutations within B57-restricted epitopes, such as T242N in TW10-Gag, then the transmission of such mutations could facilitate viral control in the haploidentical infant. We assessed the consequences of B57-associated mutations on replication capacity, viral control, and clinical outcome after vertical transmission in 13 mother-child pairs. We found that expression of HLA-B57 was associated with exceptional control of HIV during infancy, even when mutations within TW10 and most other B57-restricted epitopes were transmitted, subverting the natural immunodominance of HLA-B57. In contrast, most B57-negative infants born to B57-positive mothers progressed rapidly to AIDS. The presence of T242N led to a reproducible reduction in viral fitness, as demonstrated by in vitro assays using NL4-3 constructs encoding p24 sequences from individual mothers and infants. Associated compensatory mutations within p24-Gag were observed to reverse this impairment and to influence the propensity of T242N to revert after transmission to B57-negative hosts. Moreover, primary failure to control viremia was observed in one infant to whom multiple compensatory mutations were transmitted along with T242N. These parallel in vivo and in vitro data suggest that HLA-B57 confers its advantage primarily by driving and maintaining a fitness-attenuating mutation in p24-Gag.Expression of HLA-B57 is associated with low levels of viremia and prolonged survival after human immunodeficiency virus (HIV) infection, but the mechanism underlying this association is not well understood. Because the principal role of class I HLA molecules is to bind viral peptides for presentation to cytotoxic CD8⁺ T cells, numerous studies have sought to identify quantitative or qualitative features of the B57-restricted CD8 T-cell response to HIV that correlate with effective viral containment. However, despite exhaustive studies of the HIV-specific CD8 response in B57-positive individuals, few differences in the frequency, function, or epitope specificity of CD8 cells have been identified that reliably distinguish B57-positive controllers from the many B57-positive individuals with progressive HIV infection (1, 28-30). Early in the course of HIV infection, CD8 cells consistently select for a T242N substitution in the B57-restricted epitope TW10, which lies in a highly conserved region of p24-Gag capsid protein. Although this mutation permits the virus to escape from CD8 T-cell surveillance, it incurs a cost to viral replicative capacity (5, 24, 26). Thus, it has been hypothesized that the B57 advantage may be an indirect effect of CD8 response, whereby viral control does not rely on active surveillance by B57-restricted CD8 T cells but instead upon an “imprint” of the B57-restricted CD8 response that forces the viral capsid protein into a less fit state. Recent studies have suggested that such escape mutations may even result in lower HIV RNA levels in the next host to whom the virus is transmitted (9, 18). Selection for T242N is frequently followed by the emergence of upstream mutations at residues H219, I223, and M228 in the cyclophilin A (CypA) binding loop that have been shown to partially compensate for its fitness defect (5, 24, 26, 32), which may explain why many B57-positive individuals ultimately develop high viral loads despite the presence of T242N. The timing with which these compensatory mutations emerge and accumulate varies among B57-positive individuals, possibly due to constraints imposed by interacting Gag residues (7), and this variability may contribute to the observed heterogeneity in clinical outcomes.Mother-to-child transmission of HIV affords a unique model for differentiating between the impact of CD8 T cells and the viral escape mutations that they induce. Because transmission occurs between a haploidentical mother-child pair, it is possible to assess the impact of CD8 T-cell responses to, and mutations within, epitopes restricted by shared and unshared HLA molecules. Each infant expresses at least three HLA molecules shared with his or her mother, and vertical transmission of maternally selected escape mutations within epitopes restricted by these shared HLA molecules may effectively eliminate these potential cytotoxic-T-lymphocyte (CTL) targets from the infecting viral inoculum. However, infants also express paternally inherited HLA molecules for which no viral “imprint” exists. If the HLA-B57 advantage depends upon active surveillance by CD8 cells, mother-to-child transmission of B57-associated escape mutations may nullify this benefit among infants who share the B57 allele with their mothers, as has been demonstrated in the case of another protective HLA class I allele B*27, where transmission of a mutation within the highly dominant KK10-Gag epitope leads to failure of viral control in B27-positive infants (20). On the other hand, if viral attenuation is a major contributor to the B57 advantage, then the advantage associated with HLA-B57 expression would likely be passed along to the child, who would benefit from the reduced replication capacity of the transmitted virus.We assessed HIV sequence evolution and clinical outcomes in 13 HIV-infected mother-child pairs, one or both of whom expressed HLA-B*B57, in order to determine the impact of viral sequence changes that occur when B57-mediated immune pressure is introduced, or removed, after transmission.     

Identification of major epitopes of Mycobacterium tuberculosis AG85B that are recognized by HLA-A*0201-restricted CD8+ T cells in HLA-transgenic mice and humans

CD8(+) T cells are thought to play an important role in protective immunity to tuberculosis. Although several nonprotein ligands have been identified for CD1-restricted CD8(+) CTLs, epitopes for classical MHC class I-restricted CD8(+) T cells, which most likely represent a majority among CD8(+) T cells, have remained ill defined. HLA-A*0201 is one of the most prevalent class I alleles, with a frequency of over 30% in most populations. HLA-A2/K(b) transgenic mice were shown to provide a powerful model for studying induction of HLA-A*0201-restricted immune responses in vivo. The Ag85 complex, a major component of secreted Mycobacterium tuberculosis proteins, induces strong CD4(+) T cell responses in M. tuberculosis-infected individuals, and protection against tuberculosis in Ag85-DNA-immunized animals. In this study, we demonstrate the presence of HLA class I-restricted, CD8(+) T cells against Ag85B of M. tuberculosis in HLA-A2/K(b) transgenic mice and HLA-A*0201(+) humans. Moreover, two immunodominant Ag85 peptide epitopes for HLA-A*0201-restricted, M. tuberculosis-reactive CD8(+) CTLs were identified. These CD8(+) T cells produced IFN-gamma and TNF-alpha and recognized Ag-pulsed or bacillus Calmette-Guérin-infected, HLA-A*0201-positive, but not HLA-A*0201-negative or uninfected human macrophages. This CTL-mediated killing was blocked by anti-CD8 or anti-HLA class I mAb. Using fluorescent peptide/HLA-A*0201 tetramers, Ag85-specific CD8(+) T cells could be visualized in bacillus Calmette-Guérin-responsive, HLA-A*0201(+) individuals. Collectively, our results demonstrate the presence of HLA class I-restricted CD8(+) CTL against a major Ag of M. tuberculosis and identify Ag85B epitopes that are strongly recognized by HLA-A*0201-restricted CD8(+) T cells in humans and mice. These epitopes thus represent potential subunit components for the design of vaccines against tuberculosis.     

Combination of Immune and Viral Factors Distinguishes Low-Risk versus High-Risk HIV-1 Disease Progression in HLA-B*5701 Subjects

HLA-B*5701 is the host factor most strongly associated with slow HIV-1 disease progression, although rates can vary within this group. Underlying mechanisms are not fully understood but likely involve both immunological and virological dynamics. The present study investigated HIV-1 in vivo evolution and epitope-specific CD8(+) T cell responses in six HLA-B*5701 patients who had not received antiretroviral treatment, monitored from early infection for up to 7 years. The subjects were classified as high-risk progressors (HRPs) or low-risk progressors (LRPs) based on baseline CD4(+) T cell counts. Dynamics of HIV-1 Gag p24 evolution and multifunctional CD8(+) T cell responses were evaluated by high-resolution phylogenetic analysis and polychromatic flow cytometry, respectively. In all subjects, substitutions occurred more frequently in flanking regions than in HLA-B*5701-restricted epitopes. In LRPs, p24 sequence diversity was significantly lower; sequences exhibited a higher degree of homoplasy and more constrained mutational patterns than HRPs. The HIV-1 intrahost evolutionary rate was also lower in LRPs and followed a strict molecular clock, suggesting neutral genetic drift rather than positive selection. Additionally, polyfunctional CD8(+) T cell responses, particularly to TW10 and QW9 epitopes, were more robust in LRPs, who also showed significantly higher interleukin-2 (IL-2) production in early infection. Overall, the findings indicate that HLA-B*5701 patients with higher CD4 counts at baseline have a lower risk of HIV-1 disease progression because of the interplay between specific HLA-linked immune responses and the rate and mode of viral evolution. The study highlights the power of a multidisciplinary approach, integrating high-resolution evolutionary and immunological data, to understand mechanisms underlying HIV-1 pathogenesis.     

Quantitation of CD8(+) T-lymphocyte responses to multiple epitopes from simian virus 40 (SV40) large T antigen in C57BL/6 mice immunized with SV40, SV40 T-antigen-transformed cells, or vaccinia virus recombinants expressing full-length T antigen or epitope minigenes

The cytotoxic T-lymphocyte response to wild-type simian virus 40 large tumor antigen (Tag) in C57BL/6 (H2(b)) mice is directed against three H2-D(b)-restricted epitopes, I, II/III, and V, and one H2-K(b)-restricted epitope, IV. Epitopes I, II/III, and IV are immunodominant, while epitope V is immunorecessive. We investigated whether this hierarchical response was established in vivo or was due to differential expansion in vitro by using direct enumeration of CD8(+) T lymphocytes with Tag epitope/major histocompatibility complex class I tetramers and intracellular gamma interferon staining. The results demonstrate that epitope IV-specific CD8(+) T cells dominated the Tag-specific response in vivo following immunization with full-length Tag while CD8(+) T cells specific for epitopes I and II/III were detected at less than one-third of this level. The immunorecessive nature of epitope V was apparent in vivo, since epitope V-specific CD8(+) T cells were undetectable following immunization with full-length Tag. In contrast, high levels of epitope V-specific CD8(+) T lymphocytes were recruited in vivo following immunization and boosting with a Tag variant in which epitopes I, II/III, and IV had been inactivated. In addition, analysis of the T-cell receptor beta (TCRbeta) repertoire of Tag epitope-specific CD8(+) cells revealed that multiple TCRbeta variable regions were utilized for each epitope except Tag epitope II/III, which was limited to TCRbeta10 usage. These results indicate that the hierarchy of Tag epitope-specific CD8(+) T-cell responses is established in vivo.     

Identification of novel HLA-DR1-restricted epitopes from the hepatitis B virus envelope protein in mice expressing HLA-DR1 and vaccinated human subjects

Helper T lymphocytes that control CD8(+) T-cell and antibody responses are key elements for the resolution of infection by the hepatitis B virus and for the development of effective immunological memory after hepatitis B vaccination. We have used H-2 class II-deficient mice that express the human MHC class II molecule, HLA-DR1, to identify novel hepatitis B virus envelope-derived T helper epitopes. We confirmed the immunogenicity of a previously described HLA-DR1-restricted epitope, and identified three novel epitopes. CD4(+) T-cell immune responses against these epitopes were detected in peripheral blood mononuclear cells from HLA-DR1(+) individuals vaccinated against hepatitis B. We showed that subjects receiving the currently available hepatitis B vaccines do not develop cross-reactive T helper responses against one of the novel epitopes which are structurally variable between different hepatitis B virus subtypes. These findings highlight the need for developing vaccines against a wider range of viral subtypes, and establish humanized mice as a convenient tool for identifying new immunogenic epitopes from pathogens.     

Consequences of suboptimal priming are apparent for low-avidity T-cell responses

The emergence of the novel reassortant A(H1N1)-2009 influenza virus highlighted the threat to the global population posed by an influenza pandemic. Pre-existing CD8(+) T-cell immunity targeting conserved epitopes provides immune protection against newly emerging strains of influenza virus, when minimal antibody immunity exists. However, the occurrence of mutations within T-cell antigenic peptides that enable the virus to evade T-cell recognition constitutes a substantial issue for virus control and vaccine design. Recent evidence suggests that it might be feasible to elicit CD8(+) T-cell memory pools to common virus mutants by pre-emptive vaccination. However, there is a need for a greater understanding of CD8(+) T-cell immunity towards commonly emerging mutants. The present analysis focuses on novel and immunodominant, although of low pMHC-I avidity, CD8(+) T-cell responses directed at the mutant influenza D(b)NP(366) epitope, D(b)NPM6A, following different routes of infection. We used a C57BL/6J model of influenza to dissect the effectiveness of the natural intranasal (i.n.) versus intraperitoneal (i.p.) priming for generating functional CD8(+) T cells towards the D(b)NPM6A epitope. In contrast to comparable CD8(+) T-cell responses directed at the wild-type epitopes, D(b)NP(366) and D(b)PA(224), we found that the priming route greatly affected the numbers, cytokine profiles and TCR repertoire of the responding CD8(+) T cells directed at the D(b)NPM6A viral mutant. As the magnitude, polyfunctionality, and T-cell repertoire diversity are potential determinants of the protective efficacy of CD8(+) T-cell responses, our data have implications for the development of vaccines to combat virus mutants.