Carole Evelegh

Sequences in E1A proteins of human adenovirus 5 required for cell transformation, repression of a transcriptional enhancer, and induction of proliferating cell nuclear antigen

A range of deletion and other mutants in the coding region of the E1A gene of Ad5 has been assayed for transformation of baby rat kidney (BRK) cells in cooperation with ras, repression of the SV40 enhancer, and induction of proliferating cell nuclear antigen (PCNA). Transformation efficiency was drastically reduced by deletion of residues 4-25, 36-60, or 111-138 in exon 1 of the 289 residue (289R) and 243R E1A proteins. Deletion of other residues in exon 1 had little effect. With mutants in the region unique to the 289R protein, and in exon 2, the only effect on transformation seemed to be an increased tendency of mutant transformants, compared to wt, to migrate to form secondary foci. Repression assays, performed with E1A plasmids producing only the 243R protein, showed that deletion of residues 4-25 or 36-60 inhibited repression completely. Deletion of residues 128-138 reduced repression, but deletions elsewhere in exon 1 had little effect. Deletion of residues 188-204 in exon 2 reduced repression slightly, and deletion of all of exon 2 reduced it to about one-half. It is concluded that for transformation, there are two functional domains in E1A proteins, both in exon 1, both involved in binding different cellular proteins, and both probably concerned with different transforming functions. One of these domains, involving residues 4-25 and 36-60, also functions in repression, but the role of the second in repression is much less critical. All of the deletion mutants in exon 1 induced PCNA synthesis in BRK cells. This result, together with previously published work, suggests that the active site for PCNA induction either involves residues 61-69 or 82-85 in exon 1, which have not been deleted, or it does not depend on any single limited region of the E1A proteins.

Use of deletion and point mutants spanning the coding region of the adenovirus 5 E1A gene to define a domain that is essential for transcriptional activation

To help in identifying functional domains within Ad5 E1A proteins, we have constructed a series of mutants that create deletions throughout these products. We have also produced several mis-sense point mutations in the unique 13 S mRNA region. These mutated E1A regions have been tested in plasmid form for their ability to activate transcription of an E3-promoted CAT gene. From the results, a major domain for transactivation has been identified. This begins between residues 138 and 147, ends between residues 188 and 204, and encompasses the unique 13 S region. This domain is sensitive to mis-sense mutations. Transactivation was unaffected by small deletions in the N-terminal half of E1A proteins between residues 4 and 138, but was destroyed when this whole region was deleted. The C-terminal 71 residues may affect transactivation, but the results with the mutant in which this region was deleted were variable. The results obtained with these mutants are discussed in relation to the transactivation obtained by J. W. Lillie et al. [(1987). Cell 50, 1091-1100] with a synthetic peptide similar to the domain described here.

The Polyfunctionality of Human Memory CD8+ T Cells Elicited by Acute and Chronic Virus Infections Is Not Influenced by Age

As humans age, they experience a progressive loss of thymic function and a corresponding shift in the makeup of the circulating CD8+ T cell population from naïve to memory phenotype. These alterations are believed to result in impaired CD8+ T cell responses in older individuals; however, evidence that these global changes impact virus-specific CD8+ T cell immunity in the elderly is lacking. To gain further insight into the functionality of virus-specific CD8+ T cells in older individuals, we interrogated a cohort of individuals who were acutely infected with West Nile virus (WNV) and chronically infected with Epstein Barr virus (EBV) and Cytomegalovirus (CMV). The cohort was stratified into young (<40 yrs), middle-aged (41–59 yrs) and aged (>60 yrs) groups. In the aged cohort, the CD8+ T cell compartment displayed a marked reduction in the frequency of naïve CD8+ T cells and increased frequencies of CD8+ T cells that expressed CD57 and lacked CD28, as previously described. However, we did not observe an influence of age on either the frequency of virus-specific CD8+ T cells within the circulating pool nor their functionality (based on the production of IFNγ, TNFα, IL2, Granzyme B, Perforin and mobilization of CD107a). We did note that CD8+ T cells specific for WNV, CMV or EBV displayed distinct functional profiles, but these differences were unrelated to age. Collectively, these data fail to support the hypothesis that immunosenescence leads to defective CD8+ T cell immunity and suggest that it should be possible to develop CD8+ T cell vaccines to protect aged individuals from infections with novel emerging viruses.

Persistence of Transgene Expression Influences CD8+ T-Cell Expansion and Maintenance following Immunization with Recombinant Adenovirus

ABSTRACT Previous studies determined that the CD8+ T-cell response elicited by recombinant adenovirus exhibited a protracted contraction phase that was associated with long-term presentation of antigen. To gain further insight into this process, a doxycycline-regulated adenovirus was constructed to enable controlled extinction of transgene expression in vivo. We investigated the impact of premature termination of transgene expression at various time points (day 3 to day 60) following immunization. When transgene expression was terminated before the maximum response had been attained, overall expansion was attenuated, yielding a small memory population. When transgene expression was terminated between day 13 and day 30, the memory population was not sustained, demonstrating that the early memory population was antigen dependent. Extinction of transgene expression at day 60 had no obvious impact on memory maintenance, indicating that maintenance of the memory population may ultimately become independent of transgene expression. Premature termination of antigen expression had significant but modest effects on the phenotype and cytokine profile of the memory population. These results offer new insights into the mechanisms of memory CD8+ T-cell maintenance following immunization with a recombinant adenovirus.

Cre Levels Limit Packaging Signal Excision Efficiency in the Cre/loxP Helper-Dependent Adenoviral Vector System

ABSTRACT Helper-dependent (HD) adenovirus vectors devoid of all viral coding sequences have a large cloning capacity and provide long-term transgene expression in vivo with negligible toxicity, making them attractive vectors for gene therapy. Currently, the most efficient means of producing HD vectors involves coinfecting 293 cells expressing Cre with the HD vector and a helper virus bearing a packaging signal flanked by loxP sites. Cre-mediated packaging signal excision renders the helper virus genome unpackageable but still able to replicate and provide helper functions for HD vector propagation. Typically, helper virus contamination is ≤1% pre- and ≤0.1% postpurification by CsCl banding. While these contamination levels are low, further reduction is desirable. However, this objective has not been realized since the Cre/loxP system was first developed. This lack of progress is due, at least in part, to our lack of understanding of the origins of the contaminating helper virus, thus rendering its reduction or elimination difficult to achieve. This study was designed to investigate the possible sources of contaminating helper virus persisting during HD vector amplification. The results revealed that Cre is limiting in helper virus-infected Cre-expressing 293 cells, thereby permitting helper viruses to escape packaging signal excision and propagate. The results of this study should provide a foundation for developing rational strategies to further reduce or possibly eliminate the contaminating helper virus.

CD8+ T-cell expansion and maintenance after recombinant adenovirus immunization rely upon cooperation between hematopoietic and nonhematopoietic antigen-presenting cells

We have recently reported that CD8(+) T-cell memory maintenance after immunization with recombinant human adenovirus type 5 (rHuAd5) is dependent upon persistent transgene expression beyond the peak of the response. In this report, we have further investigated the location and nature of the cell populations responsible for this sustained response. The draining lymph nodes were found to be important for primary expansion but not for memory maintenance, suggesting that antigen presentation through a nonlymphoid source was required. Using bone marrow chimeric mice, we determined that antigen presentation by nonhematopoietic antigen-presenting cells (APCs) was sufficient for maintenance of CD8(+) T-cell numbers. However, antigen presentation by this mechanism alone yielded a memory population that displayed alterations in phenotype, cytokine production and protective capacity, indicating that antigen presentation through both hematopoietic and nonhematopoietic APCs ultimately defines the memory CD8(+) T-cell response produced by rHuAd5. These results shed new light on the immunobiology of rHuAd5 vectors and provide evidence for a mechanism of CD8(+) T-cell expansion and memory maintenance that relies upon both hematopoietic and nonhematopoietic APCs.

Development of a size-restricted pIX-deleted helper virus for amplification of helper-dependent adenovirus vectors

Helper-dependent adenovirus vectors (hdAd), which are deleted of all viral protein-coding sequences, can mediate long-term expression of a therapeutic transgene and lead to life-long, phenotypic correction in animal models of genetic disease. Here, we describe a new system for the generation of hdAd, which utilizes the DNA size restrictions imposed on an Ad virion deleted of protein IX (pIX): such virions are reported to package up to only ∼35 kb of viral DNA. A pIX− helper virus (∼37.3 kb) was easily grown on complementing 293pIX cells. Upon infection of noncomplementing cells, this virus was not capable of forming infectious virions, but provided replicative and packaging functions for propagation of a 30-kb hdAd. The pIX− helper virus was effective in amplifying an hdAd and, in combination with Cre-mediated excision in the viral-packaging signal, resulted in a 1000-fold reduction in helper virus contamination in hdAd stocks compared to Cre/lox alone, as determined by plaque assay. However, through slot blot analysis of DNA isolated from virions, we determined that the ratio of hdAd to helper DNA was 500:1, similar to the ratio observed when using Cre/lox alone. Surprisingly, a large amount of the 37.3-kb helper DNA was being packaged into the pIX-deleted virions, but these virions were incapable of establishing productive infections in plaque assays, for reasons which are still unclear. Nevertheless, the pIX− hdAd generated in this system infected cells and expressed a transgene at levels similar to those obtained with a pIX+ hdAd. These data suggest that, although further studies are necessary to characterize the nature of the defective helper virions formed in this system, deletion of pIX from the helper virus genome does provide an effective method to prevent recovery of functional helper virus during hdAd amplification.

The Memory T Cell Response to West Nile Virus in Symptomatic Humans following Natural Infection Is Not Influenced by Age and Is Dominated by a Restricted Set of CD8+ T Cell Epitopes

We examined the West Nile virus (WNV)-specific T cell response in a cohort of 52 patients with symptomatic WNV infections, including neuroinvasive and non-invasive disease. Although all virus proteins were shown to contain T cell epitopes, certain proteins, such as E, were more commonly targeted by the T cell response. Most patients exhibited reactivity toward 3–4 individual WNV peptides; however, several patients exhibited reactivity toward >10 individual peptides. The relative hierarchy of T cell reactivities in all patients showed a fixed pattern that was sustained throughout the 12-mo period of the current study. Surprisingly, we did not observe any relationship between age and either the breadth or magnitude of the T cell response following infection. We also did not observe a relationship between disease severity and either the breadth or magnitude of the T cell response. The T cell epitopes were distributed in a non-random fashion across the viral polyprotein and a limited number of epitopes appeared to dominate the CD8+ T cell response within our cohort. These data provide important new insight into the T cell response against WNV in humans.

Use of a murine secreted alkaline phosphatase as a non-immunogenic reporter gene in mice

The development of any vector system as a gene delivery system requires its optimization in vitro and in vivo. Preliminary studies frequently involve the use of a reporter gene, which allows for the rapid and simple assay of vector function through monitoring expression levels of the reporter gene. However, evaluation of vector efficacy can be compromised by immune responses directed against immunogenic reporter proteins.

The magnitude of the CD8+ T cell response produced by recombinant virus vectors is a function of both the antigen and the vector

Virus-based recombinant vaccines have proven highly effective at generating protective CD8+ T cell responses. Multiple vector platforms are available, however, little is known about the relative influence of the different vectors on the transgene-specific CD8+ T cell population. To address this question, we compared several characteristics of the CD8+ T cell response elicited by recombinant adenovirus (rAd) and vaccinia virus (rVV). We found that following rAd immunization the transgene-specific CD8+ T cell response peaked around day 12 and was larger and more sustained than the response produced by rVV. In addition, the CD8+ T cell response generated by rAd was directed primarily against the transgene, whereas the CD8+ T cell response produced by rVV principally targeted the vector backbone. In addition, we also observed that transgene selection also impacted on the magnitude of the CD8+ T cell response elicited by both vectors. Despite differences in the magnitude of the anti-transgene CD8+ T cell response, both vectors elicited CD8+ T cell populations with similar cytokine production, functional avidity and cytolytic activity. In addition, plasmid priming prior to immunization with either rAd or rVV only impacted the magnitude of the transgene gene specific CD8+ T cell response. Our study demonstrates that both vector and transgene selection can influence the magnitude of the CD8+ T cell response, but they do not influence functionality.