Miles P. Davenport

Rapid Viral Escape at an Immunodominant Simian-Human Immunodeficiency Virus Cytotoxic T-Lymphocyte Epitope Exacts a Dramatic Fitness Cost

ABSTRACT Escape from specific T-cell responses contributes to the progression of human immunodeficiency virus type 1 (HIV-1) infection. T-cell escape viral variants are retained following HIV-1 transmission between major histocompatibility complex (MHC)-matched individuals. However, reversion to wild type can occur following transmission to MHC-mismatched hosts in the absence of cytotoxic T-lymphocyte (CTL) pressure, due to the reduced fitness of the escape mutant virus. We estimated both the strength of immune selection and the fitness cost of escape variants by studying the rates of T-cell escape and reversion in pigtail macaques. Near-complete replacement of wild-type with T-cell escape viral variants at an immunodominant simian immunodeficiency virus Gag epitope KP9 occurred rapidly (over 7 days) following infection of pigtail macaques with SHIVSF162P3. Another challenge virus, SHIVmn229, previously serially passaged through pigtail macaques, contained a KP9 escape mutation in 40/44 clones sequenced from the challenge stock. When six KP9-responding animals were infected with this virus, the escape mutation was maintained. By contrast, in animals not responding to KP9, rapid reversion of the K165R mutation occurred over 2 weeks after infection. The rapidity of reversion to the wild-type sequence suggests a significant fitness cost of the T-cell escape mutant. Quantifying both the selection pressure exerted by CTL and the fitness costs of escape mutation has important implications for the development of CTL-based vaccine strategies.

DNAzyme targeting c-jun suppresses skin cancer growth

Catalytic DNA molecules that target the transcription factor c-jun inhibit skin cancer growth in mice. Getting Under Cancer’s Skin Summer brings to mind barbecues, baseball, and trips to the local pool. Yet, outdoor fun can be hazardous to one’s health—too much sun exposure can increase the risk of developing skin cancer. Indeed, one in three cancers worldwide is skin-related, and currently available treatments may induce scarring or other toxicities. Cai et al. now report that the DNAzyme Dz13—which targets an mRNA that encodes a cancer-associated transcription factor, c-Jun—inhibits the growth of two common types of skin cancers: basal cell and squamous cell carcinomas. DNAzymes are single-stranded, all-DNA, catalytic molecules that specifically bind and cleave their target RNAs. The authors examined the effects of Dz13, which destroys c-jun mRNA, on animal models of skin cancer. Dz13 inhibited tumor growth, blocked neovascularization, and prevented metastasis in mouse models of skin cancer—effects that were mediated, in part, through the induction of antitumor immunity. Minimal toxicity was observed in Dz13-treated cynomolgus monkeys, minipigs, and rodents, and there were no off-target effects in more than 70 in vitro bioassays. Thus, Dz13 may prove to be a safe, effective therapy for skin cancer. Nonetheless, one is advised to pack the sun block in preparation for extra innings—or a fifth set. Worldwide, one in three cancers is skin-related, with increasing incidence in many populations. Here, we demonstrate the capacity of a DNAzyme-targeting c-jun mRNA, Dz13, to inhibit growth of two common skin cancer types—basal cell and squamous cell carcinomas—in a therapeutic setting with established tumors. Dz13 inhibited tumor growth in both immunodeficient and immunocompetent syngeneic mice and reduced lung nodule formation in a model of metastasis. In addition, Dz13 suppressed neovascularization in tumor-bearing mice and zebrafish and increased apoptosis of tumor cells. Dz13 inhibition of tumor growth, which required an intact catalytic domain, was due in part to the induction of tumor immunity. In a series of good laboratory practice–compliant toxicology studies in cynomolgus monkeys, minipigs, and rodents, the DNAzyme was found to be safe and well tolerated. It also did not interfere in more than 70 physiologically relevant in vitro bioassays, suggesting a reduced propensity for off-target effects. If these findings hold true in clinical trials, Dz13 may provide a safe, effective therapy for human skin cancer.

Low levels of SIV infection in sooty mangabey central memory CD4 + T cells are associated with limited CCR5 expression

Naturally simian immunodeficiency virus (SIV)-infected sooty mangabeys do not progress to AIDS despite high-level virus replication. We previously showed that the fraction of CD4+CCR5+ T cells is lower in sooty mangabeys compared to humans and macaques. Here we found that, after in vitro stimulation, sooty mangabey CD4+ T cells fail to upregulate CCR5 and that this phenomenon is more pronounced in CD4+ central memory T cells (TCM cells). CD4+ T cell activation was similarly uncoupled from CCR5 expression in sooty mangabeys in vivo during acute SIV infection and the homeostatic proliferation that follows antibody-mediated CD4+ T cell depletion. Sooty mangabey CD4+ TCM cells that express low amounts of CCR5 showed reduced susceptibility to SIV infection both in vivo and in vitro when compared to CD4+ TCM cells of rhesus macaques. These data suggest that low CCR5 expression on sooty mangabey CD4+ T cells favors the preservation of CD4+ T cell homeostasis and promotes an AIDS-free status by protecting CD4+ TCM cells from direct virus infection.

A mechanism for TCR sharing between T cell subsets and individuals revealed by pyrosequencing.

The human naive T cell repertoire is the repository of a vast array of TCRs. However, the factors that shape their hierarchical distribution and relationship with the memory repertoire remain poorly understood. In this study, we used polychromatic flow cytometry to isolate highly pure memory and naive CD8+ T cells, stringently defined with multiple phenotypic markers, and used deep sequencing to characterize corresponding portions of their respective TCR repertoires from four individuals. The extent of interindividual TCR sharing and the overlap between the memory and naive compartments within individuals were determined by TCR clonotype frequencies, such that higher-frequency clonotypes were more commonly shared between compartments and individuals. TCR clonotype frequencies were, in turn, predicted by the efficiency of their production during V(D)J recombination. Thus, convergent recombination shapes the TCR repertoire of the memory and naive T cell pools, as well as their interrelationship within and between individuals.

Public clonotype usage identifies protective Gag-specific CD8+ T cell responses in SIV infection

Despite the pressing need for an AIDS vaccine, the determinants of protective immunity to HIV remain concealed within the complexity of adaptive immune responses. We dissected immunodominant virus-specific CD8+ T cell populations in Mamu-A*01+ rhesus macaques with primary SIV infection to elucidate the hallmarks of effective immunity at the level of individual constituent clonotypes, which were identified according to the expression of distinct T cell receptors (TCRs). The number of public clonotypes, defined as those that expressed identical TCR β-chain amino acid sequences and recurred in multiple individuals, contained within the acute phase CD8+ T cell population specific for the biologically constrained Gag CM9 (CTPYDINQM; residues 181–189) epitope correlated negatively with the virus load set point. This independent molecular signature of protection was confirmed in a prospective vaccine trial, in which clonotype engagement was governed by the nature of the antigen rather than the context of exposure and public clonotype usage was associated with enhanced recognition of epitope variants. Thus, the pattern of antigen-specific clonotype recruitment within a protective CD8+ T cell population is a prognostic indicator of vaccine efficacy and biological outcome in an AIDS virus infection.

Molecular analysis of HLA class II associations with hepatitis B virus clearance and vaccine nonresponsiveness

Clearance of acute hepatitis B virus (HBV) infection is associated with a vigorous CD4+ T‐cell response focusing on the core protein. HLA class II glycoproteins present viral peptides to CD4+ T cells and influence the immune responses. HLA‐DRB1*1301/2 have been associated with viral clearance, and HLA‐DRB1*0301 is associated with nonresponse to vaccination with envelope proteins. Binding affinities of overlapping peptides covering the core and envelope proteins of HBV were measured to HLA glycoproteins encoded by HLA‐DRA1*0101,‐DRB1*0101 (HLA‐DR1), HLA‐DRA1*0101,‐DRB1*0301 (HLA‐DR3), HLA‐DRA1*0101,‐DRB1*0701 (HLA‐DR7) and HLA‐DRA1*0101,‐DRB1*1301 (HLA‐DR13) molecules and compared with published peptide‐specific CD4+ T‐cell responses. There are more high‐affinity ligands (IC50 < 1 μmol/L) derived from the core protein than the surface antigen (P < .04 for HLA‐DR1/7/13), but there was no increase in the number or the affinity of ligands for HLA‐DR13. Clusters of particular core peptides bound to multiple HLA types, explaining the immunodominance of these regions for T‐cell responses. Within the envelope protein, the low‐affinity ligands (IC50 < 10 μmol/L) are found mainly in the surface antigen, with a marked paucity of ligands for HLA‐DR3 (HLA‐DR3 vs. non‐DR3; P < .05) consistent with the lower vaccination responses for this HLA type. Of all peptides tested, 8 to 10 bound mainly to one HLA type, allowing a substantially greater breadth of response in heterozygotes. In conclusion, these data offer a mechanistic explanation for the dominant response to the HBV core protein during infection and support the direct involvement of the HLA‐DRB1 gene in vaccine nonresponsiveness but not altered susceptibility to viral persistence. (HEPATOLOGY 2005;41:1383–1390.)

Biological determinants of immune reconstitution in HIV-infected patients receiving antiretroviral therapy: the role of interleukin 7 and interleukin 7 receptor α and microbial translocation.

BACKGROUND Multiple host factors may influence CD4(+) T cell reconstitution in human immunodeficiency virus (HIV)-infected patients after suppressive antiretroviral therapy (ART). We hypothesized that residual immune activation and polymorphisms in the interleukin 7 (IL-7) receptor α (IL-7Rα) gene were important for immune recovery. METHODS We examined HIV-infected patients receiving suppressive ART (n = 96) for their IL-7Rα haplotypes and measured levels of lipopolysaccharide (LPS), soluble CD14, and IL-7 in plasma samples collected before and after ART initiation. Levels of soluble IL-7Rα were measured in HIV-infected patients with IL-7Rα haplotype 2 (n = 11) and those without IL-7Rα haplotype 2 (n = 22). Multivariate analysis was used to identify variables associated with faster recovery to CD4(+) T cell counts of >500 and >200 cells/μL. RESULTS Both LPS and soluble CD14 levels were significantly decreased with ART (P < .001, respectively) but remained elevated compared with uninfected controls. In a multivariate analysis, faster recovery to a CD4(+) T cell count of >500 cells/μL was significantly associated with higher baseline CD4(+) T cell count, younger age, lower pre-ART LPS level, higher pre-ART soluble CD14 level, lower pre-ART IL-7 level, and IL-7Rα haplotype 2 (hazard ratio, 1.50; 95% confidence interval, 1.03-2.19; P = .034). HIV-infected patients with haplotype 2 had significantly lower soluble IL-7Rα levels compared with those of patients without haplotype 2 (P < .001). CONCLUSION Both the extent of immune depletion prior to ART and IL-7Rα haplotype 2 are important determinants of time to CD4(+) T cell recovery to counts of >500 cells/μL.

Sharing of T cell receptors in antigen-specific responses is driven by convergent recombination

Public responses where identical T cell receptors (TCRs) are clonally dominant and shared between different individuals are a common characteristic of CD8+ T cell-mediated immunity. Focusing on TCR sharing, we analyzed ≈3,400 TCR β chains (TCRβs) from mouse CD8+ T cells responding to the influenza A virus DbNP366 and DbPA224 epitopes. Both the “public” DbNP366-specific and “private” DbPA224-specific TCR repertoires contain a high proportion (≈36%) of shared TCRβs, although the numbers of mice sharing TCRβs in each repertoire varies greatly. Sharing of both the TCRβ amino acid and TCRβ nucleotide sequence was negatively correlated with the prevalence of random nucleotide additions in the sequence. However, the extent of TCRβ amino acid sequence sharing among mice was strongly correlated with the level of diversity in the encoding nucleotide sequences, suggesting that a key feature of public TCRs is that they can be made in a variety of ways. Using a computer simulation of random V(D)J recombination, we estimated the relative production frequencies and variety of production mechanisms for TCRβ sequences and found strong correlations with the sharing of both TCRβ amino acid sequences and TCRβ nucleotide sequences. The overall conclusion is that “convergent recombination,” rather than a bias in recombination or subsequent selection, provides the mechanistic basis for TCR sharing between individuals responding to identical peptide plus MHC class I glycoprotein complexes.

Clonal selection, clonal senescence, and clonal succession: the evolution of the T cell response to infection with a persistent virus.

We have analyzed the CD8+ T cell response to EBV and find that a larger primary burst size is associated with proportionally greater decay during the development of memory. Consequently, immunodominance and clonal dominance are less marked in memory than primary responses. An intuitive interpretation of this finding is that there is a limit to the number of cell divisions a T cell clone can undergo, and that the progeny of clones that have expanded massively during a primary immune response are more prone to die as a result of senescence. To test this hypothesis, we have derived a mathematical model of the response of different T cell clones of varying avidity for Ag in the primary and persistent phases of viral infection. When cellular survival and replication are linked to T cell avidity for Ag and Ag dose, then high-avidity T cells dominate both the primary and secondary responses. We then incorporated a limit in the number of cell divisions of individual T cell clones to test whether such a constraint could reproduce the observed association between cell division number and alterations in the contribution of clones to the response to persistent infection. Comparison of the model output with the experimental results obtained from primary and persistent EBV infection suggests that there is indeed a role for cellular senescence in shaping the immune response to persistent infection.

Kinetics of Virus-Specific CD8+ T Cells and the Control of Human Immunodeficiency Virus Infection

ABSTRACT Several primate models indicate that cytotoxic T lymphocyte-inducing vaccines may be unable to prevent human immunodeficiency virus infection but may have a long-term benefit in controlling viral replication and delaying disease progression. Here we show that analysis of the kinetics of antigen-specific CD8+ T-cell expansion suggests a delay in activation following infection that allows unimpeded early viral replication. Viral kinetics do not differ between controls and vaccinees during this delay phase. An increase in virus-specific CD8+ T-cell numbers around day 10 postinfection coincides with a slowing in viral replication in vaccinees and reduces peak viral loads by around 1 log. However, this response is too little too late to prevent establishment of persistent infection.