Design and in vivo immunogenicity of a polyvalent vaccine based on SIVmac regulatory genes.

Most vaccine modalities for human immunodeficiency virus type 1 (HIV-1) tested for immunogenicity and efficacy in the SIVmac (simian immunodeficiency virus) macaque model do not include the viral regulatory proteins. Because viral regulatory proteins are expressed early during the virus life cycle and represent an additional source of antigens, their inclusion as a vaccine component may increase the overall virus-specific immune response in vaccinees. However, at least two of the early proteins, Tat and Nef, may be immunosuppressive, limiting their usefulness as components of an SIV vaccine. We have constructed a polyvalent chimeric protein in which the open reading frames for Tat and Nef have been reassorted and the nuclear localization sequence for Tat and Rev and the myristoylation site for Nef have been removed. The resulting DNA plasmid (pDNA-SIV-Retanef) (pDNA-SIV-RTN) encodes a protein of 55 kDa (Retanef) that localizes at the steady state in the cytoplasma of transfected cells. Both the DNA-SIV-RTN and the highly attenuated recombinant poxvirus vector NYVAC-SIV-RTN were demonstrated to be immunogenic in SIVmac251-infected macaques treated with ART as well as in naive macaques. An equivalent strategy may be used for the generation of polyvalent antigens encoding the regulatory proteins in a HIV-1 vaccine candidate.

Administration of recombinant rhesus interleukin-12 during acute simian immunodeficiency virus (SIV) infection leads to decreased viral loads associated with prolonged survival in SIVmac251-infected rhesus macaques.

The ability of recombinant rhesus interleukin-12 (rMamu-IL-12) administration during acute simian immunodeficiency virus SIVmac251 infection to influence the quality of the antiviral immune responses was assessed in rhesus macaques. Group I (n = 4) was the virus-only control group. Group II and III received a conditioning regimen of rMamu-IL-12 (10 and 20 microg/kg, respectively, subcutaneously [s.c.]) on days -2 and 0. Thereafter, group II received 2 microg of IL-12 per kg and group III received 10 microg/kg s.c. twice a week for 8 weeks. On day 0 all animals were infected with SIVmac251 intravenously. While all four group I animals and three of four group II animals died by 8 and 10 months post infection (p.i.), all four group III animals remained alive for >20 months p.i. The higher IL-12 dose led to lower plasma viral loads and markedly lower peripheral blood mononuclear cell and lymph node proviral DNA loads. During the acute viremia phase, the high-IL-12-dose monkeys showed an increase in CD3(-) CD8 alpha/alpha(+) and CD3(+) CD8 alpha/alpha(+) cells and, unlike the control and low-IL-12-dose animals, did not demonstrate an increase in CD4(+) CD45RA(+) CD62L(+) naive cells. The high-IL-12-dose animals also demonstrated that both CD8 alpha/alpha(+) and CD8 alpha/beta(+) cells produced antiviral factors early p.i., whereas only CD8 alpha/beta(+) cells retained this function late p.i. Long-term survival correlated with sustained high levels of SIV gag/pol and SIV env cytotoxic T lymphocytes and retention of high memory responses against nominal antigens. This is the first study to demonstrate the capacity of IL-12 to significantly protect macaques from SIV-induced disease, and it provides a useful model to more precisely identify correlates of virus-specific disease-protective responses.

Impairment of Gag-specific CD8(+) T-cell function in mucosal and systemic compartments of simian immunodeficiency virus mac251- and simian-human immunodeficiency virus KU2-infected macaques.

The identification of several simian immunodeficiency virus mac251 (SIV(mac251)) cytotoxic T-lymphocyte epitopes recognized by CD8(+) T cells of infected rhesus macaques carrying the Mamu-A*01 molecule and the use of peptide-major histocompatibility complex tetrameric complexes enable the study of the frequency, breadth, functionality, and distribution of virus-specific CD8(+) T cells in the body. To begin to address these issues, we have performed a pilot study to measure the virus-specific CD8(+) and CD4(+) T-cell response in the blood, lymph nodes, spleen, and gastrointestinal lymphoid tissues of eight Mamu-A*01-positive macaques, six of those infected with SIV(mac251) and two infected with the pathogenic simian-human immunodeficiency virus KU2. We focused on the analysis of the response to peptide p11C, C-M (Gag 181), since it was predominant in most tissues of all macaques. Five macaques restricted viral replication effectively, whereas the remaining three failed to control viremia and experienced a progressive loss of CD4(+) T cells. The frequency of the Gag 181 (p11C, C-->M) immunodominant response varied among different tissues of the same animal and in the same tissues from different animals. We found that the functionality of this virus-specific CD8(+) T-cell population could not be assumed based on the ability to specifically bind to the Gag 181 tetramer, particularly in the mucosal tissues of some of the macaques infected by SIV(mac251) that were progressing to disease. Overall, the functionality of CD8(+) tetramer-binding T cells in tissues assessed by either measurement of cytolytic activity or the ability of these cells to produce gamma interferon or tumor necrosis factor alpha was low and was even lower in the mucosal tissue than in blood or spleen of some SIV(mac251)-infected animals that failed to control viremia. The data obtained in this pilot study lead to the hypothesis that disease progression may be associated with loss of virus-specific CD8(+) T-cell function.

Role of CD28-B7 interactions in generation and maintenance of CD8 T cell memory.

Although the role of CD28-B7 interaction in the activation of naive T cells is well established, its importance in the generation and maintenance of T cell memory is not well understood. In this study, we examined the requirement for CD28-B7 interactions in primary T cell activation and immune memory. Ag-specific CD8 T cell responses were compared between wild-type (+/+) and CD28-deficient (CD28(-/-)) mice following an acute infection with lymphocytic choriomeningitis virus (LCMV). During the primary response, there was a substantial activation and expansion of LCMV-specific CD8 T cells in both +/+ and CD28(-/-) mice. However, the magnitude of the primary CD8 T cell response to both dominant and subdominant LCMV CTL epitopes was approximately 2- to 3-fold lower in CD28(-/-) mice compared with +/+ mice; the lack of CD28-mediated costimulation did not lead to preferential suppression of CD8 T cell responses to the weaker subdominant epitopes. As seen in CD28(-/-) mice, blockade of B7-mediated costimulation by CTLA4-Ig treatment of +/+ mice also resulted in a 2-fold reduction in the anti-LCMV CD8 T cell responses. Loss of CD28/B7 interactions did not significantly affect the generation and maintenance of CD8 T cell memory; the magnitude of CD8 T cell memory was approximately 2-fold lower in CD28(-/-) mice as compared with +/+ mice. Further, in CD28(-/-) mice, LCMV-specific memory CD8 T cells showed normal homeostatic proliferation in vivo and also conferred protective immunity. Therefore, CD28 signaling is not necessary for the proliferative renewal and maintenance of memory CD8 T cells.

Emergence and kinetics of simian immunodeficiency virus-specific CD8(+) T cells in the intestines of macaques during primary infection.

In this report, three Mamu-A*01(+) rhesus macaques were examined to compare the emergence of simian immunodeficiency virus (SIV)-specific CD8(+) T cells in the intestines and blood in early SIV infection using a major histocompatibility complex class I tetramer complexed with the Gag(181-189) peptide. Fourteen days after intravenous inoculation with SIVmac251, large numbers of SIV Gag(181-189)-specific CD8(+) T cells were detected in the intestinal mucosa (3.1 to 11.5% of CD3(+) CD8(+) lymphocytes) as well as in the blood (3.1 to 13.4%) of all three macaques. By 21 days postinoculation, levels of tetramer-binding cells had dropped in both the intestines and blood. At day 63, however, levels of SIV Gag(181-189)-specific CD8(+) T cells in the intestines had rebounded in all three macaques to levels that were higher (8.6 to 18.7%) than those at day 21. In contrast, percentages of tetramer-binding cells in the peripheral blood remained comparatively stable (2.5 to 4.5%) at this time point. In summary, SIV Gag(181-189)-specific CD8(+) T cells appeared in both the intestinal mucosa and peripheral blood at a comparable rate and magnitude in primary SIV infection. Given that the intestine is a major site of early viral replication as well as the site where most of the total body lymphocyte pool resides, these data indicate that it is also an early and important site of development of antiviral immune responses.

The role of p53 in regulating antiviral T cell responses.

It is now well established that viral infections can induce large expansions of Ag-specific CD8(+) T cells. These cells divide very rapidly with an estimated doubling time of approximately 6 h. When virus is cleared, the vast majority of these effector CD8 T cells undergo apoptosis. The remaining memory cells persist at constant levels and provide the basis for the accelerated recall response upon rechallenge. The molecular mechanisms that control the rapid proliferation and death of Ag-specific T cells are poorly understood. Because of its important role in controlling cell proliferation and death, we examined antiviral immune responses in p53(-/-) mice using lymphocytic choriomeningitis virus. We found that effector CD8 and CD4 responses were comparable but that memory levels were slightly higher in -/- mice compared with +/+ mice. The lack of a major difference in virus-specific T cell responses between +/+ and -/- mice suggests that p53 only plays a minor role in regulating the proliferation, apoptosis, and maintenance of Ag-specific T cells. Thus, it appears that the primary function of p53 is in controlling "illegitimate" proliferation and tumor development and not in regulating Ag-specific T cell responses.

Control of a mucosal challenge and prevention of AIDS by a multiprotein DNA/MVA vaccine.

Heterologous prime/boost regimens have the potential for raising high levels of immune responses. Here we report that DNA priming followed by a recombinant modified vaccinia Ankara (rMVA) booster controlled a highly pathogenic immunodeficiency virus challenge in a rhesus macaque model. Both the DNA and rMVA components of the vaccine expressed multiple immunodeficiency virus proteins. Two DNA inoculations at 0 and 8 weeks and a single rMVA booster at 24 weeks effectively controlled an intrarectal challenge administered 7 months after the booster. These findings provide hope that a relatively simple multiprotein DNA/MVA vaccine can help to control the acquired immune deficiency syndrome epidemic.

Evaluation of the percutaneous intramyocardial injection for local myocardial treatment.

Therapeutic angiogenesis requires the induction of new blood vessel formation for the treatment of peripheral vascular and coronary artery disease. Efficacious application of this new therapy requires optimizing multiple factors, including the therapeutic agent, dosing, frequency of administration, and delivery modality. In this study, a helical needle drug infusion catheter was applied for optimal application of percutaneous intramyocardial delivery (PIMD). (125)Iodine-labeled albumin was injected by PIMD into the left ventricle myocardium in eight swine. After 1 hr, PIMD resulted in a high concentration of radiolabel at the treatment site; 16.4% +/- 2.1% of delivered and 81.4% +/- 2.6% of the total cardiac activity was concentrated at the site of delivery. The depth of needle penetration correlated with the myocardial retention of delivered protein. The myocardial retention of radiolabel in animals with shallow injections was 10.1% +/- 0.8%, compared to 18.9% +/- 3.3% retention after deep injections. The specific activity at the treatment site (radioactive counts per gram of tissue) was 115 +/- 36, 226 +/- 55, and 47 +/- 10 times higher compared to liver, lung, and kidney, respectively. Continuous coronary sinus and aortic blood sampling indicates that within 15 min following intramyocardial injection, a significant amount of nonretained protein is found within the coronary sinus. This study defines some of the parameters that can affect optimal application of PIMD and demonstrates that PIMD is a safe and efficient method for local drug delivery.

An HLA-directed molecular and bioinformatics approach identifies new HLA-A11 HIV-1 subtype E cytotoxic T lymphocyte epitopes in HIV-1-infected Thais.

Only limited cytotoxic T lymphocyte (CTL) epitope mapping has been done in nonsubtype B HIV-infected persons. We used molecular immunogenetic tools to determine HIV-specific CTL responses in HIV-1 Env subtype E-infected female sex workers (FSWs) from northern Thailand, where more than 50% of the population is HLA-A11 positive. EpiMatrix, a computer-based T cell epitope prediction algorithm, and a manual editing approach were used to predict 77 possible HLA-A11 CTL epitopes in HIV-1, some of which were conserved between subtypes B and E. MHC binding of these peptides was determined in an HLA-A11 stabilization assay, and binding peptides were tested for CTL recognition in eight HLA-A11-positive FSWs. Subtype E versions of known HLA-A2 subtype B HIV epitopes were also tested in four HLA-A2 positive FSWs. CTL responses were detected in all HLA-A11-positive and in three of four HLA-A2-positive persons. Among the 12 FSWs responses to peptides were found to Pol in 9 (75%), Env in 7 (58%), Nef in 5 (42%), and Gag in 5 (42%), and to conserved epitopes in 8 (67%). To identify HLA-A11 CTL epitopes in the absence of prediction tools, it would have been necessary to test almost 3000 10-mer peptides. EpiMatrix and manual predictions reduced this number to 77, of which 26 were MHC binding and 12 were CTL epitopes. Six of these HLA-A11 CTL epitopes have not been previously reported and are located in RT, gp120, and gp41. This report of CTL responses in subtype E-infected individuals defines epitopes that may be useful in HIV pathogenesis or vaccine studies.

In vivo selection of a lymphocytic choriomeningitis virus variant that affects recognition of the GP33-43 epitope by H-2Db but not H-2Kb.

CD8 T cells drive the protective immune response to lymphocytic choriomeningitis virus (LCMV) infection and are thus a determining force in the selection of viral variants. To examine how escape mutations affect the presentation and recognition of overlapping T-cell epitopes, we isolated an LCMV variant that is not recognized by T-cell receptor (TCR)-transgenic H-2Db-restricted LCMV GP33-41-specific cytotoxic T lymphocytes (CTL). The variant virus carried a single-amino-acid substitution (valine to alanine) at position 35 of the viral glycoprotein. This region of the LCMV glycoprotein encodes both the Db-restricted GP33-43 epitope and a second epitope (GP34-42) presented by the Kb molecule. We determined that the V-to-A CTL escape mutant failed to induce a Db GP33-43-specific CTL response and that Db-restricted GP33-43-specific CTL induced by the wild-type LCMV strain were unable to kill target cells infected with the variant LCMV strain. In contrast, the Kb-restricted response was much less affected. We found that the V-to-A substitution severely impaired peptide binding to Db but not to Kb molecules. Strikingly, the V-to-A mutation did not change any of the anchor residues, and the dramatic effect on binding was therefore unexpected. The strong decrease in Db binding explains why the variant virus escapes the Db GP33-43-specific response but still elicits the Kb-restricted response. These findings also illustrate that mutations within regions encoding overlapping T-cell epitopes can differentially affect the presentation and recognition of individual epitopes.

Antiviral CD8+ T cell responses in neonatal mice: susceptibility to polyoma virus-induced tumors is associated with lack of cytotoxic function by viral antigen-specific T cells.

Polyoma virus is a potent oncogenic pathogen when inoculated into newborn mice of particular H-2(k) strains. Using D(k) tetramers containing the dominant antipolyoma CD8(+) T cell epitope, middle T protein (MT)389-397, and intracellular interferon gamma staining, we enumerated MT389-specific CD8(+) T cells in infected neonates having opposite susceptibilities to polyoma virus-induced tumors. In resistant mice, MT389-specific CD8(+) T cells dramatically expanded during acute infection in neonates to a frequency rivaling that in adults; furthermore, in both neonatal and adult mice, this antipolyoma CD8(+) T cell response exhibited nearly identical T cell receptor (TCR) functional avidities and TCR functional fingerprints. Susceptible mice mounted an MT389-specific CD8(+) T cell response of only fourfold lower magnitude than resistant mice; but, in clear contrast to resistant mice, these CD8(+) T cells lacked ex vivo MT389-specific cytotoxic activity. However, MT389-specific CD8(+) T cells in resistant and susceptible mice expressed similar TCR avidities, perforin levels, and surface type O-glycan levels indicative of mature CD8(+) T cell effectors. Upon in vitro restimulation with infected antigen-presenting cells, CD8(+) T cells from acutely infected susceptible neonates acquired strong MT389-specific cytotoxicity. These findings indicate that polyoma-specific CD8(+) T cells are armed with, but restrained from deploying, their cytotoxic effector function in mice susceptible to polyoma virus tumorigenesis.

Functional impairment of simian immunodeficiency virus-specific CD8+ T cells during the chronic phase of infection.

In an attempt to determine why high frequencies of circulating virus-specific CD8+ T cells are unable to control human immunodeficiency virus and simian immunodeficiency virus (SIV) replication, we assessed the functional nature of SIV-specific CD8+ lymphocytes. After vaccination and early after infection, nearly all tetramer-staining CD8+ cells produced gamma interferon in response to their specific stimulus. However, by 4 months postinfection with pathogenic SIVmac239, signs of functional impairment in the CD8+ T-cell compartment were detected which might prevent these T cells from efficiently controlling the infection during the chronic phase.

Simian immunodeficiency virus (SIV) infection of a rhesus macaque induces SIV-specific CD8(+) T cells with a defect in effector function that is reversible on extended interleukin-2 incubation.

A vigorous expansion of antigen-specific CD8(+) T cells lacking apparent effector function was observed in a rhesus macaque acutely infected with the simian immunodeficiency virus (SIV) strain SIVmac239. Antigen-specific CD8(+) T cells were identified using antigenic-peptide class I major histocompatibility complex tetramers. As many as 8.3% of CD8(+) cells recognized the Mamu-A*01-associated SIV epitope Gag(181-189) (CTPYDINQM); however, these cells demonstrated no effector function when presented with peptide-incubated targets, as measured by intracellular cytokine staining for gamma interferon (IFN-gamma), interleukin-2 (IL-2) production, or direct cellular lysis. Similar results were observed with three other SIV peptide antigens. Nonresponsiveness did not correlate with apoptosis of the CD8(+) cells, nor were cells from this macaque impaired in their ability to present peptide antigens. Associated with the nonresponsive state was a lack of IL-2 production and decreased IL-2 receptor expression. Exogenous IL-2 treatment for 1 week in the absence of antigenic stimulation restored antigen-specific responses and the quantitative correlation between tetramer recognition and antigen-responsive IFN-gamma secretion. This case report suggests a regulatory mechanism that may impede the effector function of antigen-specific T cells during acute infection with SIV or human immunodeficiency virus in some cases. This mechanism may participate in the failure of the immune system to limit infection.

CD8(+) lymphocytes from simian immunodeficiency virus-infected rhesus macaques recognize 14 different epitopes bound by the major histocompatibility complex class I molecule mamu-A*01: implications for vaccine design and testing.

It is becoming increasingly clear that any human immunodeficiency virus (HIV) vaccine should induce a strong CD8(+) response. Additional desirable elements are multispecificity and a focus on conserved epitopes. The use of multiple conserved epitopes arranged in an artificial gene (or EpiGene) is a potential means to achieve these goals. To test this concept in a relevant disease model we sought to identify multiple simian immunodeficiency virus (SIV)-derived CD8(+) epitopes bound by a single nonhuman primate major histocompatibility complex (MHC) class I molecule. We had previously identified the peptide binding motif of Mamu-A*01(2), a common rhesus macaque MHC class I molecule that presents the immunodominant SIV gag-derived cytotoxic T lymphocyte (CTL) epitope Gag_CM9 (CTPYDINQM). Herein, we scanned SIV proteins for the presence of Mamu-A*01 motifs. The binding capacity of 221 motif-positive peptides was determined using purified Mamu-A*01 molecules. Thirty-seven peptides bound with apparent K(d) values of 500 nM or lower, with 21 peptides binding better than the Gag_CM9 peptide. Peripheral blood mononuclear cells from SIV-infected Mamu-A*01(+) macaques recognized 14 of these peptides in ELISPOT, CTL, or tetramer analyses. This study reveals an unprecedented complexity and diversity of anti-SIV CTL responses. Furthermore, it represents an important step toward the design of a multiepitope vaccine for SIV and HIV.

Gene expression in antigen-specific CD8+ T cells during viral infection.

Following infection with intracellular pathogens, Ag-specific CD8(+) T cells become activated and begin to proliferate. As these cells become activated, they elaborate effector functions including cytokine production and cytolysis. After the infection has been cleared, the immune system returns to homeostasis through apoptosis of the majority of the Ag-specific effector cells. The surviving memory cells can persist for extended periods and provide protection against reinfection. Little is known about the changes in gene expression as Ag-specific cells progress through these stages of development, i.e., naive to effector to memory. Using recombinant MHC class I tetramers, we isolated Ag-specific CD8(+) T cells from mice infected with lymphocytic choriomeningitis virus at various time points and performed semiquantitative RT-PCR. We examined expression of: 1) genes involved in cell cycle control, 2) effector and regulatory functions, and 3) susceptibility to apoptosis. We found that Ag-specific CD8(+) memory T cells contain high steady-state levels of Bcl-2, BAX:, IFN-gamma, and lung Kruppel-like factor (LKLF), and decreased levels of p21 and p27 mRNA. Moreover, the pattern of gene expression between naive and memory cells is distinct and suggests that these two cell types control susceptibility to apoptosis through different mechanisms.

Increased CD95/Fas-induced apoptosis of HIV-specific CD8(+) T cells.

Why HIV-specific CD8(+) T cells ultimately fail to clear or control HIV infection is not known. We show here that HIV-specific CD8(+) T cells exhibit increased sensitivity to CD95/Fas-induced apoptosis. This apoptosis is 3-fold higher compared to CMV-specific CD8(+) T cells from the same patients. HIV-specific CD8(+) T cells express the CD45RA(-)CD62L(-) but lack the CD45RA(+)CD62L(-) T cell effector memory (T(EM)) phenotype. This skewing is not found in CMV- and EBV-specific CD8(+) T cells in HIV-infected individuals. CD95/Fas-induced apoptosis is much higher in the CD45RA(-)CD62L(-) T(EM) cells. However, cytotoxicity and IFNgamma production by HIV-specific CD8(+) T cells is not impaired. Our data suggest that the survival and differentiation of HIV-specific CD8(+) T cells may be compromised by CD95/Fas apoptosis induced by FasL-expressing HIV-infected cells.

CD8 binding to MHC class I molecules is influenced by T cell maturation and glycosylation.

CD8 serves both as an adhesion molecule for class I MHC molecules and as a coreceptor with the TCR for T cell activation. Here we study the developmental regulation of CD8-mediated binding to noncognate peptide/MHC ligands (i.e., those not bound by the TCR). We show that CD8's ability to bind soluble class I MHC tetramers and to mediate T cell adhesion under shear flow conditions diminishes as double-positive thymocytes mature into CD8(+) T cells. Furthermore, we provide evidence that this decreased CD8 binding results from increased T cell sialylation upon T cell maturation. These data suggest that CD8's ability to interact with class I MHC is not fixed and is developmentally regulated through the T cell's glycosylation state.

Evolution of the T cell repertoire during primary, memory, and recall responses to viral infection.

Many viral infections induce a broad repertoire of CD8(+) T cell responses that initiate recognition and elimination of infected cells by interaction of TCRs with viral peptides presented on infected cells by MHC class I proteins. Following clearance of the infection, >90% of activated CD8(+) T cells die, leaving behind a stable pool of memory CD8(+) T cells capable of responding to subsequent infections with enhanced kinetics. To probe the mechanisms involved in the generation of T cell memory, we compared primary, memory, and secondary challenge virus-specific T cell repertoires using a combination of costaining with MHC class I tetramers and a panel of anti-Vss Abs, as well as complementarity-determining region 3 length distribution analysis of TCR Vss transcripts from cells sorted according to tetramer binding. Following individual mice over time, we found identity between primary effector and memory TCR repertoires for each of three immunodominant epitopes from lymphocytic choriomeningitis virus. During secondary responses, we found quantitative changes in epitope-specific T cell hierarchies but little evidence for changes in Vss usage or complementarity-determining region 3 length distributions within epitope-specific populations. We conclude that 1) selection of memory T cell populations is stochastic and not determined by a distinct step of clonal selection necessary for survival from the acute responding population, and 2) maturation of the T cell repertoire during secondary lymphocytic choriomeningitis virus infection alters the relative magnitudes of epitope-specific responses but does not significantly modify the repertoire of T cells responding to a given epitope.

Peptide-MHC class I tetrameric complexes display exquisite ligand specificity.

The production of synthetic MHC-peptide tetramers has revolutionized cellular immunology by revealing enormous CD8(+) T cell expansions specific for peptides from various pathogens. A feature of these reagents, essential for their staining function, is that they bind T cells with relatively high avidity. This could, theoretically, promote cross-reactivity with irrelevant T cells leading to overestimates of epitope-specific T cell numbers. Therefore, we have investigated the fine specificity of CTL staining with these reagents for comparison with functional data. Using a panel of CTL clones with distinct fine specificity patterns for analogs of an HLA-B8-binding EBV epitope, together with B8 tetramers incorporating these peptides, we show a very good correlation between tetramer staining and peptide activity in cytotoxicity assays. Significant staining only occurred with tetramers that incorporate strong stimulatory agonist peptides and not weak agonists that are unlikely to induce full T cell activation at physiological levels of presentation. In almost every case where a peptide analog had >10-fold less activity than the optimal EBV peptide in cytotoxicity assays, the corresponding tetramer stained with >10-fold less intensity than the EBV epitope tetramer. Furthermore, by examining an EBV-specific clonotypic T cell expansion in EBV-exposed individuals, we show similar fine specificity in tetramer staining of fresh peripheral T cells. Collectively, our data demonstrate the exquisite specificity of class I MHC-peptide tetramers, underlining their accuracy in quantifying only those T cells capable of recognizing the low levels of cell surface peptide presented after endogenous Ag processing.

Tat-specific cytotoxic T lymphocytes select for SIV escape variants during resolution of primary viraemia.

Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infections are characterized by early peaks of viraemia that decline as strong cellular immune responses develop. Although it has been shown that virus-specific CD8-positive cytotoxic T lymphocytes (CTLs) exert selective pressure during HIV and SIV infection, the data have been controversial. Here we show that Tat-specific CD8-positive T-lymphocyte responses select for new viral escape variants during the acute phase of infection. We sequenced the entire virus immediately after the acute phase, and found that amino-acid replacements accumulated primarily in Tat CTL epitopes. This implies that Tat-specific CTLs may be significantly involved in controlling wild-type virus replication, and suggests that responses against viral proteins that are expressed early during the viral life cycle might be attractive targets for HIV vaccine development.