Identification of dominant optimal HLA-B60- and HLA-B61-restricted cytotoxic T-lymphocyte (CTL) epitopes: rapid characterization of CTL responses by enzyme-linked immunospot assay

Human immunodeficiency virus type 1 (HIV-1)-specific cytotoxic T-lymphocyte (CTL) responses play a major role in the antiviral immune response, but the relative contribution of CTL responses restricted by different HLA class I molecules is less well defined. HLA-B60 or the related allele B61 is expressed in 10 to 20% of Caucasoid populations and is even more highly prevalent in Asian populations, but yet no CTL epitopes restricted by these alleles have been defined. Here we report the definition of five novel HLA-B60-restricted HIV-1-specific CTL epitopes, using peripheral blood mononuclear cells in enzyme-linked immunospot (Elispot) assays and using CTL clones and lines in cytolytic assays. The dominant HLA-B60-restricted epitope, Nef peptide KEKGGLEGL, was targeted by all eight subjects with B60 and also by both subjects with B61 studied. This study additionally establishes the utility of the Elispot assay as a more rapid and efficient method of defining novel CTL epitopes. This approach will help to define new CTL epitopes that may play an important role in the immune control of HIV-1.     

In vitro cytotoxicity against Marek's disease lymphoblastoid cell lines after enzymatic removal of Marek's disease tumor-associated surface antigen.

Marek's disease tumor-associated surface antigen (MATSA) has been claimed to be the target of cytotoxic lymphocytes in in vitro tests for Marek's disease immunity. Treatment with papain, but not with trypsin or mixed glycosidases, removed MATSA from certain Marek's disease lymphoblastoid cell lines. Tumor cells with and without MATSA were used as target cells for in vitro studies on cell-mediated immune responses with sensitized spleen cells in a chromium release assay. The removal of MATSA did not influence the results of the chromium release assay. Attempts to block the cell-mediated cytotoxicity in vitro by coating tumor cells with an anti-MATSA serum failed. It was concluded that cell-mediated immune responses against Marek's disease tumor cells are directed against an as yet undefined antigen(s).     

Injection of plasmid DNA into the gastric mucosa induces mucosal and systemic immunity

Nearly all mucosal surfaces participate in a common mucosal immune system, and application of an antigen to one mucosal surface elicits local as well as distant mucosal immune responses. However, whether the gastric mucosa is a part of this network has not been examined directly. We show here that the injection of plasmid DNA encoding beta-galactosidase into the gastric wall caused transfection of gastric mucosal epithelial cells, induced systemic and mucosal antibody responses at both local (digestive tract) and distant (genital and respiratory tracts) sites, and induced cytotoxic T lymphocyte responses in the spleen and the mesenteric and iliac lymph nodes.     

The cell-mediated immune response to ectromelia virus infection. I. Kinetics and characteristics of the primary effector T cell response in vivo.

The cell-mediated immune (CMI) response to ectromelia virus infection in mice was studied. Virus doses from 4 × 10² up to 5 × 10⁴ PFU of an attenuated strain inoculated intravenously (iv) all induced cytotoxic T cell responses in the spleen as measured in a ⁵¹Cr release assay using virus-infected target cells. Higher virus doses gave larger responses. There was little variation between individual animals, and mice ranging in age from 4–22 weeks gave similar responses. Following iv infection, virus grew logarithmically in spleen for 2 days, then titers declined to undetectable levels by day 5. The peak of the virus-specific cytotoxic T cell response occurred at 5–6 days post-infection, as determined by calculation of effector units based on a linear log-log relationship between killer cells added and targets lysed. T cells responsible for virus clearance in vivo gave similar kinetics, suggesting the possibility that both functions are mediated by the same T cell subset. Two other categories of cytotoxic activity were also generated at low levels in the spleen during ectromelia infection or during infection with a bacterium, Listeria monocytogenes. These activities were significantly sensitive to anti-δ and complement treatment, suggesting T cell dependence, but participation of other mechanisms has not been rigorously excluded. One category lysed allogenic target cells and reached a peak at 4 days post-infection. The other lysed H-2-compatible cells, syngeneic embryo cells, and some syngeneic tumor cells but not syngeneic macrophages, and was present at similar low levels through days 1–4. These different kinetics and evidence from “cold” target competition experiments suggested that the total cytotoxic activity of immune spleen cell populations was a composite of the activities of separate cellular subsets (probably mainly T cells), killing of any one target cell type being the responsibility of a subset with receptors at least partly specific for antigens on that target cell.     

Comprehensive screening for human immunodeficiency virus type 1 subtype-specific CD8 cytotoxic T lymphocytes and definition of degenerate epitopes restricted by HLA-A0207 and -C(W)0304 alleles

For this report, the rapid identification and characterization of human immunodeficiency virus type 1 (HIV-1)-derived broadly cross-subtype-reactive CD8 cytotoxic T lymphocyte (CTL) epitopes were performed. Using a gamma interferon (IFN-gamma) Elispot assay-based approach and a panel of recombinant vaccinia viruses expressing gag, env, pol, and nef genes representing the seven most predominant subtypes and one circulating recombinant form of HIV-1, the subtype specificity and cross-subtype reactivity of a CD8 response were directly measured from circulating peripheral blood mononuclear cells (PBMC). Enhanced sensitivity of detection of CD8 responses from cryopreserved PBMC was achieved using autologous vaccinia virus-infected B-lymphoblastoid cell lines as supplemental antigen-presenting cells. Of eleven subjects studied, six exhibited broadly cross-subtype-reactive CD8-mediated IFN-gamma production (at least seven of eight subtypes recognized) to at least one major gene product from HIV-1. Screening of subjects showing broadly cross-subtype-specific responses in the vaccinia virus-based enzyme-linked immunospot (Elispot) assay using a panel of overlapping peptides resulted in the identification of cross-subtype responses down to the 20-mer peptide level in less than 3 days. Three subjects showed broad cross-subtype reactivity in both the IFN-gamma Elispot assay and the standard chromium release cytotoxicity assay. Fine mapping and HLA restriction analysis of the response from three subjects demonstrated that this technique can be used to define epitopes restricted by HLA-A, -B, and -C alleles. In addition, the ability of all three epitopes to be processed from multiple subtypes of their parent proteins and presented in the context of HLA class I molecules following de novo synthesis is shown. While all three minimal epitopes mapped here had previously been defined as HIV-1 epitopes, two are shown to have novel HLA restriction alleles and therefore exhibit degenerate HLA binding capacity. These findings provide biological validation of HLA supertypes in HIV-1 CTL recognition and support earlier studies of cross-subtype CTL responses during HIV-1 infection.     

Study of the immune responses to nucleated cells. I. In vitro functional evaluation of the immune effectors responsible for complement-dependent cellular cytotoxicity

A modification of the ⁵¹Cr cytotoxic test has made it possible to assess under the same conditions not only cytotoxic serum antibodies, and cell-mediated immunity, but also cells releasing cytotoxic antibody. The measurement of these antibody-releasing cells was carried out with nucleated target cells, both normal and leukemic, across θ or H-2 antigenic differences. This test was found to be specific. The release of ⁵¹Cr from the labeled target cells was proportional to the ratio of immune cells to target cells, and for a given ratio to the incubation time, 60 min usually being the optimum time at ratios of 50–100 to 1. The test was not affected by treatment of the effector cells with an anti-θ serum; however, pretreatment of these cells with an anti-IgM serum, even without complement, inhibited the test with cells taken during primary responses. Both cytotoxic IgM and IgG antibodies were detected by the assay directly without the addition of enhancing serum; discrimination between these two γ-globulins can be made by suppressing the cytotoxicity due to either Ig class consequent to the addition of the appropriate specific anti-globulin serum during the incubation.     

Identification of human immunodeficiency virus type 1 subtype C Gag-, Tat-, Rev-, and Nef-specific elispot-based cytotoxic T-lymphocyte responses for AIDS vaccine design

The most severe human immunodeficiency virus type 1 (HIV-1) epidemic is occurring in southern Africa. It is caused by HIV-1 subtype C (HIV-1C). In this study we present the identification and analysis of cumulative cytotoxic T-lymphocyte (CTL) responses in the southern African country of Botswana. CTLs were shown to be an important component of the immune response to control HIV-1 infection. The definition of optimal and dominant epitopes across the HIV-1C genome that are targeted by CTL is critical for vaccine design. The characteristics of the predominant virus that causes the HIV-1 epidemic in a certain geographic area and also the genetic background of the population, through the distribution of common HLA class I alleles, might impact dominant CTL responses in the vaccinee and in the general population. The enzyme-linked immunospot (Elispot) gamma interferon assay has recently been shown to be a reliable tool to map optimal CTL epitopes, correlating well with other methods, such as intracellular staining, tetramer staining, and the classical chromium release assay. Using Elispot with overlapping synthetic peptides across Gag, Tat, Rev, and Nef, we analyzed HIV-1C-specific CTL responses of HIV-1-infected blood donors. Profiles of cumulative Elispot-based CTL responses combined with diversity and sequence consensus data provide an additional characterization of immunodominant regions across the HIV-1C genome. Results of the study suggest that the construction of a poly-epitope subtype-specific HIV-1 vaccine that includes multiple copies of immunodominant CTL epitopes across the viral genome, derived from predominant HIV-1 viruses, might be a logical approach to the design of a vaccine against AIDS.     

Comprehensive epitope analysis of human immunodeficiency virus type 1 (HIV-1)-specific T-cell responses directed against the entire expressed HIV-1 genome demonstrate broadly directed responses,but no correlation to viral load

Cellular immune responses play a critical role in the control of human immunodeficiency virus type 1 (HIV-1); however, the breadth of these responses at the single-epitope level has not been comprehensively assessed. We therefore screened peripheral blood mononuclear cells (PBMC) from 57 individuals at different stages of HIV-1 infection for virus-specific T-cell responses using a matrix of 504 overlapping peptides spanning all expressed HIV-1 proteins in a gamma interferon-enzyme-linked immunospot (Elispot) assay. HIV-1-specific T-cell responses were detectable in all study subjects, with a median of 14 individual epitopic regions targeted per person (range, 2 to 42), and all 14 HIV-1 protein subunits were recognized. HIV-1 p24-Gag and Nef contained the highest epitope density and were also the most frequently recognized HIV-1 proteins. The total magnitude of the HIV-1-specific response ranged from 280 to 25,860 spot-forming cells (SFC)/10(6) PBMC (median, 4,245) among all study participants. However, the number of epitopic regions targeted, the protein subunits recognized, and the total magnitude of HIV-1-specific responses varied significantly among the tested individuals, with the strongest and broadest responses detectable in individuals with untreated chronic HIV-1 infection. Neither the breadth nor the magnitude of the total HIV-1-specific CD8+-T-cell responses correlated with plasma viral load. We conclude that a peptide matrix-based Elispot assay allows for rapid, sensitive, specific, and efficient assessment of cellular immune responses directed against the entire expressed HIV-1 genome. These data also suggest that the impact of T-cell responses on control of viral replication cannot be explained by the mere quantification of the magnitude and breadth of the CD8+-T-cell response, even if a comprehensive pan-genome screening approach is applied.     

Bacterial magnetic particles (BMPs)-PEI as a novel and efficient non-viral gene delivery system

BACKGROUND: Non-viral methods of gene delivery, especially using polyethylenimine (PEI), have been widely used in gene therapy or DNA vaccination. However, the PEI system has its own drawbacks, which limits its applications. METHODS: We have developed a novel non-viral delivery system based on PEI coated on the surface of bacterial magnetic nanoparticles (BMPs). The ability of BMPs-PEI complexes to bind DNA was determined by retardation of plasmid DNA in agarose gel electrophoresis. The transfection efficiency of BMPs-PEI/DNA complexes into eukaryotic cells was determined by flow cytometric analysis. The MTT assay was invited to investigate the cytotoxicity of BMPs-PEI/DNA complexes. The expression efficiency in vivo of BMPs-PEI bound to the plasmid pCMVbeta encoding beta-galactosidase was evaluated intramuscularly inoculated into mice. The immune responses of in vivo delivery of BMPs-PEI bound plasmid pcD-VP1 were determined by MTT assay for T cell proliferation and ELISA for detecting total IgG antibodies. RESULTS: BMPs-PEI complexes could bind DNA and provide protection from DNase degradation. The transfection efficiency of BMPs-PEI/DNA complexes was higher than that in PEI/DNA complexes. Interestingly, in contrast to PEI, the BMPs-PEI complex was less cytotoxic to cells in vitro. We further demonstrated that the BMPs-PEI system can deliver an exogenous gene to animals and allow it to be expressed in vivo. Such expression resulted in higher levels of humoral and cellular immune responses against the target antigen compared to controls. CONCLUSIONS: We have developed a novel BMPs-PEI gene delivery system with a high transfection efficiency and low toxicity, which presents an attractive strategy for gene therapy and DNA vaccination.     

Visualization and quantification of T cell-mediated cytotoxicity using cell-permeable fluorogenic caspase substrates

We have developed a non-radioactive flow-cytometry assay to monitor and quantify the target-cell killing activities mediated by cytotoxic T lymphocytes (CTLs). This flow-cytometry CTL (FCC) assay is predicated on measurement of CTL-induced caspase activation in target cells through detection of the specific cleavage of fluorogenic caspase substrates. Here we show that this assay reliably detects antigen-specific CTL killing of target cells, and demonstrate that it provides a more sensitive, more informative and safer alternative to the standard 51Cr-release assay most often used to quantify CTL responses. The FCC assay can be used to study CTL-mediated killing of primary host target cells of different cell lineages, and enables the study of antigen-specific cellular immune responses in real time at the single-cell level. As such, the FCC assay can provide a valuable tool for studies of infectious disease pathogenesis and development of new vaccines and immunotherapies.     

Generation of cytotoxic T lymphocytes during coxsackievirus B-3 infection. I. Model and viral specificity1.

The production of cytotoxic cells in the spleen of adult male BALB/c mice infected with Coxsackievirus B-3 has been examined.An in vitro 51Cr release assay was used to measure cytotoxic activity against virus-infected and uninfected neonatal sygeneic fibroblasts. Cytotoxicity of immune spleen cells against virus-infected targets was detected on the 3rd day after infection, reached a peak on day 7, and then declined to low levels by days 12 and 14. Spleen cells obtained 3 and 5 days after infection also exerted cytotoxicity against uninfected fibroblasts, but by the 7th day there was little or no reactivity against uninfected target cells, although activity against infected fibroblasts was maximal at this time. Reciprocal assays performed by using Coxsackie and vaccinia viruses provided evidence of virus specificity of the cytotoxic reaction. When spleen cells were obtained 7 days after infection, the Coxsackievirus-immune population was not cytotoxic for vaccinia-infected fibroblasts, and the vaccinia-immune population was not cytotoxic for Coxsackievirus-infected targets, although each immune cell preparation caused significant lysis of fibroblasts infected with the homologous virus. Additional studies showed that primary mouse or hyperimmune rabbit anti-Coxsackieviral serum could not block immune spleen cell cytotoxicity or induce complement-mediated lysis of infected targets. The findings indicate that Coxsackievirus infection results in surface membrane alterations, but no evidence was obtained that antiviral antibody could react with the infected cells.     

Cytotoxic T-lymphocyte (CTL) responses directed against regulatory and accessory proteins in HIV-1 infection

The HIV-1 regulatory proteins Tat and Rev and the accessory proteins Vpr, Vpu, and Vif are essential for viral replication, and their cytoplasmic production suggests that they should be processed for recognition by cytotoxic T lymphocytes. However, only limited data is available evaluating to which extent these proteins are targeted in natural infection and optimal cytotoxic T lymphocyte (CTL) epitopes within these proteins have not been defined. In this study, CTL responses against HIV-1 Tat, Rev, Vpr, Vpu, and Vif were analyzed in 70 HIV-1 infected individuals and 10 HIV-1 negative controls using overlapping peptides spanning the entire proteins. Peptide-specific interferon-gamma (IFN-gamma) production was measured by Elispot assay and flow-based intracellular cytokine quantification. HLA class I restriction and cytotoxic activity were confirmed after isolation of peptide-specific CD8+ T-cell lines. All regulatory and accessory proteins served as targets for HIV-1- specific CTL and multiple CTL epitopes were identified in functionally important regions of these proteins. In certain individuals HIV-1-specific CD8+ T-cell responses to these accessory and regulatory proteins contributed up to a third to the magnitude of the total HIV-1-specific CTL response. These data indicate that despite the small size of these proteins regulatory and accessory proteins are targeted by CTL in natural HIV-1 infection, and contribute importantly to the total HIV-1-specific CD8+ T-cell responses. These findings are relevant for the evaluation of the specificity and breadth of immune responses during acute and chronic#10; infection, and will be useful for the design and testing of candidate human immunodeficiency virus (HIV) vaccines.     

Correlation of human CD56+ cell cytotoxicity and IFN-gamma production

The use of an IFN-gamma ELISPOT assay to evaluate cellular immune responses has gained increasing popularity, especially as a surrogate measure for cytotoxic T lymphocyte (CTL) responses. We have compared the IFN-gamma ELISPOT assay and the traditional(51)Cr release assay for detection of human natural killer (NK) cell activity. The cell populations used for evaluation of these assays included freshly isolated and IL-2-activated peripheral blood mononuclear cells (PBMC). CD56-positive cells were demonstrated to be the primary source of the IFN-gamma signal when PBMC were evaluated with NK-sensitive targets in the IFN-gamma ELISPOT assay. IFN-gamma ELISPOT and(51)Cr release assays showed excellent correlation suggesting that NK activity can be reliably evaluated with methods other than the traditional(51)Cr release assays.     

Cell-mediated immunity to Friend virus-induced leukemia. III. Characteristics of secondary cell-mediated cytotoxic response.

By employing the 125IUdR release cytotoxicity assay, we have been able to measure the primary and secondary cell-mediated cytotoxic response of C57BL/6 mice to FBL-3 cells, a syngeneic Friend virus-induced leukemia. It was found that the secondary cell-mediated cytotoxic response occurred more rapidly after challenge (within 3 days) than the primary response, and the levels of reactivity were considerably higher. As in the primary response, the secondary cytotoxic reactivity of spleen cells was T cell dependent, being eliminated by pretreatment with anti-theta antibody plus complement. However, the secondary reactivity of pertioneal exudate (PE) cells was not entirely T-cell dependent. The specificity of the secondary cytotoxic response was analyzed by primary or secondary immunization with various tumor cells and by testing of cytotoxic lymphocytes against a variety of target cells. When spleen cells were used for testing, only tumor cells induced by Friend, Moloney, or Rauscher (FMR) leukemia viruses could produce secondary cell-mediated cytotoxic responses against FBL-3 cells. This correlated well with the specificity observed in the in vivo tumor transplantation protection studies. Similarly, spleen cells immune to FBL-3 had appreciable cytotoxicity against tumor cells induced by FMR viruses. The FBL-3 immune mice also gave significant protection against the challenge of FMR leukemias. When PE cells were used for testing, they gave higher levels of cytotoxicity against tumor cells induced by FMR viruses, but also gave less, but appreciable, cytotoxicity against non-FMR tumors. The latter reactivity might be related to the antigens induced by the murine endogenous type C viruses.     

The effects of vasoactive intestinal peptide on human natural killer cell function

Vasoactive intestinal peptide (VIP) can be found at nerve endings in various tissues and has recently been shown to interact with human lymphocytes through an adenylate cyclase-linked receptor. Because various neuroendocrine factors are thought to influence immune responsiveness, we studied the effect of VIP on natural killer (NK) effector function. Human lymphocytes were incubated with 51Cr-labeled K562 target cells in a 4-hr cytotoxicity assay in the absence or presence of increasing concentrations of VIP. As expected from its activation of adenylate cyclase, VIP was inhibitory at 10(-6) to 10(-10) M. Interestingly, however, when lymphocytes were preincubated with VIP for 30 or 60 min, then washed and added to target cells, a significant augmentation of NK activity ensued. Binding studies revealed that preincubation with VIP resulted in increased numbers of effector-target conjugates, whereas cytotoxic activity in agarose was not affected at the single cell level. Studies with synthetic analogs of VIP revealed that the integrity of the 14-28 C-terminal amino acid sequence was essential for its activity in cytotoxicity. These data strongly suggest a functional role for VIP in modulating immune responses during neuroendocrine interactions with the immune system.     

Results and harmonization guidelines from two large-scale international Elispot proficiency panels conducted by the Cancer Vaccine Consortium (CVC/SVI)

The Cancer Vaccine Consortium of the Sabin Vaccine Institute (CVC/SVI) is conducting an ongoing large-scale immune monitoring harmonization program through its members and affiliated associations. This effort was brought to life as an external validation program by conducting an international Elispot proficiency panel with 36 laboratories in 2005, and was followed by a second panel with 29 participating laboratories in 2006 allowing for application of learnings from the first panel. Critical protocol choices, as well as standardization and validation practices among laboratories were assessed through detailed surveys. Although panel participants had to follow general guidelines in order to allow comparison of results, each laboratory was able to use its own protocols, materials and reagents. The second panel recorded an overall significantly improved performance, as measured by the ability to detect all predefined responses correctly. Protocol choices and laboratory practices, which can have a dramatic effect on the overall assay outcome, were identified and lead to the following recommendations: (A) Establish a laboratory SOP for Elispot testing procedures including (A1) a counting method for apoptotic cells for determining adequate cell dilution for plating, and (A2) overnight rest of cells prior to plating and incubation, (B) Use only pre-tested serum optimized for low background: high signal ratio, (C) Establish a laboratory SOP for plate reading including (C1) human auditing during the reading process and (C2) adequate adjustments for technical artifacts, and (D) Only allow trained personnel, which is certified per laboratory SOPs to conduct assays. Recommendations described under (A) were found to make a statistically significant difference in assay performance, while the remaining recommendations are based on practical experiences confirmed by the panel results, which could not be statistically tested. These results provide initial harmonization guidelines to optimize Elispot assay performance to the immunotherapy community. Further optimization is in process with ongoing panels.     

Cancer immunotherapy: the past,the present and the future

One of the most controversial issues in immunology for over a century has been whether an effective immune response can be elicited against malignant tumours. Whether the immunology community has believed cancer immunotherapy is feasible or impossible has been largely determined by the prevailing immunological paradigms at that time. In fact, during the last 110 years it is possible to trace at least five dramatic fluctuations in attitude towards cancer immunotherapy. It now appears, however, that overwhelming evidence is available to support the view that both the innate and adaptive immune responses can recognize and eliminate tumours. On the other hand, it remains to be seen if these immune responses can be harnessed to control cancer as, at the time of diagnosis, many tumours have already been immunoselected to be highly resistant to immune elimination. Based on these observations it is argued that immunotherapy approaches, other than the generation of tumour-specific cytotoxic T lymphocytes, must be explored. Alternative strategies include recruiting tumouricidal myeloid cells into tumours, generating antiangiogenic immune responses and directing innate immunity to hypoxia-induced ligands on tumour cells.     

Microfluorometer assay to measure the expression of beta-galactosidase and green fluorescent protein reporter genes in single Drosophila flies

beta-galactosidase and green fluorescent protein (GFP) are among the most commonly used reporter genes to monitor gene expression in various organisms including Drosophila melanogaster. Their expression is usually detected in a qualitative way by direct microscopic observations of cells, tissues, or whole animals. To measure in vivo the inducibility of two antimicrobial peptide genes expressed during the Drosophila innate immune response, we have adapted two reporter gene systems based on the beta-galactosidase enzymatic activity and GFP. We have designed a 96-well microplate fluorometric assay sensitive enough to quantify the expression of both reporter genes in single flies. The assay has enabled us to process efficiently and rapidly a large number of individual mutant flies generated during an ethylmethane sulfonate saturation mutagenesis of the Drosophila genome. This method may be used in any screen that requires the quantification of reporter gene activity in individual insects.     

Limited sequence evolution within persistently targeted CD8 epitopes in chronic human immunodeficiency virus type 1 infection

Studies in acute human immunodeficiency virus type 1 (HIV-1) infection indicate viral evolution under CD8 T-cell immune selection pressure, but the effects of ongoing immune pressure on epitope evolution during chronic infection are not well described. In this study, we performed a detailed longitudinal analysis of viral sequence variation within persistently targeted cytotoxic T-lymphocyte (CTL) epitopes in two HIV-1-infected persons during 6 years of persistent viremia. Responses were quantitated using freshly isolated peripheral blood lymphocytes in direct lytic assays as well as by gamma interferon (IFN-gamma) Elispot assays on cryopreserved cells. Seven targeted epitopes were identified in each person. In the majority of cases, the dominant epitope sequence did not change over time, even in the presence of responses of sufficient magnitude that they were detectable using fresh peripheral blood mononuclear cells in direct lytic assays. Only 4 of the 14 autologous epitopes tested represented potential CTL escape variants; however, in most cases strong responses to these epitopes persisted for the 6 years of study. Although persistent IFN-gamma responses were detected to all epitopes, direct lytic assays demonstrated declining responses to some epitopes despite the persistence of the targeted sequence in vivo. These data indicate limited viral evolution within persistently targeted CD8 T-cell epitopes during the chronic phase of infection and suggest that these regions of the virus are either refractory to sequence change or that persistently activated CD8 T-cell responses in chronic infection exert little functional selection pressure.