Cell division modulates prion accumulation in cultured cells.

The phenotypic effect of prions on host cells is influenced by the physical properties of the prion strain and its level of accumulation. In mammalian cell cultures, prion accumulation is determined by the interplay between de novo prion formation, catabolism, cell division, and horizontal cell-to-cell transmission. Understanding this dynamic enables the analytical modeling of protein-based heritability and infectivity. Here, we quantitatively measured these competing effects in a subline of neuroblastoma (N2a) cells and propose a concordant reaction mechanism to explain the kinetics of prion propagation. Our results show that cell division leads to a predictable reduction in steady-state prion levels but not to complete clearance. Scrapie-infected N2a cells were capable of accumulating different steady-state levels of prions, dictated partly by the rate of cell division. We also show that prions in this subline of N2a cells are transmitted primarily from mother to daughter cells, rather than horizontal cell-to-cell transmission. We quantitatively modeled our kinetic results based on a mechanism that assumes a subpopulation of prions is capable of self-catalysis, and the levels of this subpopulation reach saturation in fully infected cells. Our results suggest that the apparent effectiveness of antiprion compounds in culture may be strongly influenced by the growth phase of the target cells.

High degree of inter-clade cross-reactivity of HIV-1-specific T cell responses at the single peptide level.

OBJECTIVES: To determine HIV-1-specific T cell responses in clade B infected individuals recognizing the clade A, B and C consensus sequences in order to assess the degree of inter-clade cross-reactivity of these immune responses at the single epitope level. METHODS: HIV-1-specific T cell responses were assessed cross-sectionally in 27 chronically HIV-1-infected individuals from a population infected mainly with clade B viral strains, using an interferon-gamma Elispot assay with a total of 1230 overlapping peptides spanning the entire amino acid sequence of the clade A, B and C 2001 consensus sequences. RESULTS: No significant difference was observed between the total magnitude or breadth of T cell responses recognizing either the clade A, B or C consensus sequences. However, at the single peptide level, 194 T cell responses were identified that recognized only one of the three different clade-specific peptide variants (A: B: C, 34: 105: 55), 125 T cell responses recognized two of the three peptide variants (AB: AC: BC, 71: 15: 39) and 166 T cell responses (34%) were cross-reactive with all three different peptide variants. Peptides recognized in all three consensus sequence variants had a significantly lower entropy (P < 0.0001) and a significantly higher inter-clade homology (P < 0.0001). CONCLUSIONS: Viral epitopes within regions of low HIV-1 clade B diversity and high inter-clade homology can be recognized in the clade A, B and C variants and indicate a wide degree of cross-isolate and cross-clade recognition by HIV-1-specific T cells. These regions may therefore be of particular relevance for the design of HIV-1 vaccines.

HIV-1 Nef is preferentially recognized by CD8 T cells in primary HIV-1 infection despite a relatively high degree of genetic diversity.

OBJECTIVE: To compare the magnitude, breadth and protein specificity of HIV-1-specific CD8 T-cell responses against the clade B consensus sequence during primary and chronic HIV-1 infection and to analyze the impact of viral diversity on the localization of detected responses. METHODS: HIV-1-specific CD8 T-cell responses against the clade B consensus sequence in individuals with acute (n = 10), early (n = 19) and chronic (n = 10) infection were longitudinally assessed using an interferon-gamma EliSpot assay. RESULTS: CD8 T-cell responses against clade B consensus sequences were preferentially directed against central regions of Nef during primary HIV-1 infection, despite a relatively higher degree of genetic diversity compared with other subsequently targeted regions. In subjects with acute and early infection, Nef-specific CD8 T-cell responses against the consensus Nef sequence represented 94 and 46% of the total magnitude of HIV-1-specific CD8 T-cell responses, respectively. Subjects with untreated chronic infection exhibited broadly diversified CD8 T-cell responses against more conserved viral regions, with only 17% of virus-specific T-cell responses targeting Nef. The initial immunodominance of Nef persisted in individuals with treated acute infection, but shifted rapidly to Gag, Env and Pol in subjects with continuous antigen exposure. CONCLUSION: These data show that despite relatively high sequence variability, viral regions within the clade B consensus sequence of Nef are preferentially recognized during primary HIV-1 infection. Later diversification of responses to other proteins during prolonged antigen exposure provides evidence of the initial preferential immunogenicity of Nef epitopes compared to similarly conserved regions within other viral proteins.

CD8+ T lymphocyte responses target functionally important regions of Protease and Integrase in HIV-1 infected subjects.

BACKGROUND: CD8+ T cell responses are known to be important to the control of HIV-1 infection. While responses to reverse transcriptase and most structural and accessory proteins have been extensively studied, CD8 T cell responses specifically directed to the HIV-1 enzymes Protease and Integrase have not been well characterized, and few epitopes have been described in detail. METHODS: We assessed comprehensively the CD8 T cell responses to synthetic peptides spanning Protease and Integrase in 56 HIV-1 infected subjects with acute, chronic, or controlled infection using IFN-gamma-Elispot assays and intracellular cytokine staining. Fine-characterization of novel CTL epitopes was performed on peptide-specific CTL lines in Elispot and 51Chromium-release assays. RESULTS: Thirteen (23%) and 38 (68%) of the 56 subjects had detectable responses to Protease and Integrase, respectively, and together these targeted most regions within both proteins. Sequence variability analysis confirmed that responses cluster largely around conserved regions of Integrase, but responses against a large, highly conserved region of the N-terminal DNA-binding domain of Integrase were not readily detected. CD8 T cell responses targeted regions of Protease that contain known Protease inhibitor mutation residues, but strong Protease-specific CD8 T cell responses were rare. Fine-mapping of targeted epitopes allowed the identification of three novel, HLA class I-restricted, frequently-targeted optimal epitopes. There were no significant correlations between CD8 T cell responses to Protease and Integrase and clinical disease category in the study subjects, nor was there a correlation with viral load. CONCLUSIONS: These findings confirm that CD8 T cell responses directed against HIV-1 include potentially important functional regions of Protease and Integrase, and that pharmacologic targeting of these enzymes will place them under both drug and immune selection pressure.

Differences in the expressed HLA class I alleles effect the differential clustering of HIV type 1-specific T cell responses in infected Chinese and caucasians.

China is a region of the world with a rapidly spreading HIV-1 epidemic. Studies providing insights into HIV-1 pathogenesis in infected Chinese are urgently needed to support the design and testing of an effective HIV-1 vaccine for this population. HIV-1-specific T cell responses were characterized in 32 HIV-1-infected individuals of Chinese origin and compared to 34 infected caucasians using 410 overlapping peptides spanning the entire HIV-1 clade B consensus sequence in an IFN-gamma ELISpot assay. All HIV-1 proteins were targeted with similar frequency in both populations and all study subjects recognized at least one overlapping peptide. HIV-1-specific T cell responses clustered in seven different regions of the HIV-1 genome in the Chinese cohort and in nine different regions in the caucasian cohort. The dominant HLA class I alleles expressed in the two populations differed significantly, and differences in epitope clustering pattern were shown to be influenced by differences in class I alleles that restrict immunodominant epitopes. These studies demonstrate that the clustering of HIV-1-specific T cell responses is influenced by the genetic HLA class I background in the study populations. The design and testing of candidate vaccines to fight the rapidly growing HIV-1 epidemic must therefore take the HLA genetics of the population into account as specific regions of the virus can be expected to be differentially targeted in ethnically diverse populations.

The majority of currently circulating human immunodeficiency virus type 1 clade B viruses fail to prime cytotoxic T-lymphocyte responses against an otherwise immunodominant HLA-A2-restricted epitope: implications for vaccine design.

Human immunodeficiency virus type 1 (HIV-1) mutates to escape immune selection pressure, but there is little evidence of selection mediated through HLA-A2, the dominant class I allele in persons infected with clade B virus. Moreover, HLA-A2-restricted responses are largely absent in the acute phase of infection as the viral load is being reduced, suggesting that circulating viruses may lack immunodominant epitopes targeted through HLA-A2. Here we demonstrate an A2-restricted epitope within Vpr (Vpr59-67) that is targeted by acute-phase HIV-1-specific CD8+ T cells, but only in a subset of persons expressing HLA-A2. Individuals in the acute stage of infection with viruses containing the most common current sequence within this epitope (consensus sequence) were unable to mount epitope-specific T-cell responses, whereas subjects infected with the less frequent I60L variant all developed these responses. The I60L variant epitope was a stronger binder to HLA-A2 and was recognized by epitope-specific T cells at lower peptide concentrations than the consensus sequence epitope. These data demonstrate that HLA-A2 is capable of contributing to the acute-phase cytotoxic T-lymphocyte response in infected subjects, but that most currently circulating viruses lack a dominant immunogenic epitope presented by this allele, and suggest that immunodominant epitopes restricted by common HLA alleles may be lost as the epidemic matures.