Presentation of endogenous viral peptide epitopes by anti-CD40 stimulated human B cells following recombinant vaccinia infection

Human cytomegalovirus (HCMV) can establish both nonproductive (latent) and productive (lytic) infections. Many of the proteins expressed during these phases of infection could be expected to be targets of the immune response; however, much of our understanding of the CD8؉ -T-cell response to HCMV is mainly based on the pp65 antigen. Very little is known about T-cell control over other antigens expressed during the different stages of virus infection; this imbalance in our understanding undermines the importance of these antigens in several aspects of HCMV disease pathogenesis. In the present study, an efficient and rapid strategy based on predictive bioinformatics and ex vivo functional T-cell assays was adopted to profile CD8؉ -T-cell responses to a large panel of HCMV antigens expressed during different phases of replication. These studies revealed that CD8؉ -T-cell responses to HCMV often contained multiple antigen-specific reactivities, which were not just constrained to the previously identified pp65 or IE-1 antigens. Unexpectedly, a number of viral proteins including structural, early/late antigens and HCMV-encoded immunomodulators (pp28, pp50, gH, gB, US2, US3, US6, and UL18) were also identified as potential targets for HCMV-specific CD8؉ -T-cell immunity. Based on this extensive analysis, numerous novel HCMV peptide epitopes and their HLA-restricting determinants recognized by these T cells have been defined. These observations contrast with previous findings that viral interference with the antigen-processing pathway during lytic infection would render immediate-early and early/late proteins less immunogenic. This work strongly suggests that successful HCMV-specific immune control in healthy virus carriers is dependent on a strong T-cell response towards a broad repertoire of antigens.

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“…ϩ -T-cell epitopes, 23 have not previously been identified. These responses were not constrained to a single HCMV antigen.…”

Section: Resultsmentioning

confidence: 95%

“…LCLs were infected with recombinant vaccinia virus at an MOI of 10:1 for 1 h at 37°C, as described earlier (21,22). After overnight infection, cells were washed with growth medium and labeled with 51 Cr for use as targets in cytotoxicity assays (23). Cytotoxicity assay.…”

Section: Methodsmentioning

confidence: 99%

Ex Vivo Profiling of CD8⁺-T-Cell Responses to Human Cytomegalovirus Reveals Broad and Multispecific Reactivities in Healthy Virus Carriers

Elkington

Walker

Crough

et al. 2003

J Virol

290

293

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“…The target cells used in Fig. 2 for the A-type-specific CTL were therefore autologous lymphoblastoid cell lines (LCLs) transformed with B-type virus (B-type LCLs), and the target cells for CS31 and YW22 were autologous EBV-negative B-cell blasts, established by using anti-CD40 antibody and recombinant interleukin 4 (25).…”

Section: Methodsmentioning

confidence: 99%

“…2 were infected with recombinant VV at a multiplicity of infection of 5:1 followed by [14][15][16] hr of incubation at 37°C prior to use in the standard 5-hr 51Cr-release assay with the CTL clones as effectors (25). W. the polyepitope W at a multiplicity of infection of 0.01 for 2 hr followed by two washes.…”

Section: Methodsmentioning

confidence: 99%

Minimal epitopes expressed in a recombinant polyepitope protein are processed and presented to CD8+ cytotoxic T cells: implications for vaccine design.

Thomson

Khanna

Gardner

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

130

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The epitopes recognized by CD8+ cytotoxic T lymphocytes (CTL) are generated from cytosolic proteins by proteolytic processing. CD8+ a3 cytotoxic T lymphocytes (CTL) recognize short peptides (epitopes) associated with specific alleles of the class I major histocompatibility complex (1). The peptide epitopes are mainly generated from cytosolic proteins by proteolysis, a process believed to involve the multicatalytic proteosome complex (2-8). The influence of sequences flanking CTL epitopes on the proteolytic processing and presentation of these epitopes remains controversial (9-14). Moving CTL epitopes into new positions within a protein in some cases prevents (9) and in other cases has no effect on (10, 11) presentation of the epitope. A profound effect on presentation was observed when an epitope was expressed with different truncations of the natural flanking sequences (12). However, only one of a series of substitutions made to the amino acid residues flanking a CTL epitope was shown to effect presentation (13,14). The subdominance of a CTL epitope has been attributed to the inability of proteosomes to generate the peptide from the parent protein (15), although recently the subdominance of this epitope has been attributed to low affinity of the peptide for its restriction element (16). Although some of these data may illustrate that certain flanking sequences prevent proteolytic generation of an epitope, it is not clear whether the presence of any specific natural flanking sequences is needed to direct the activity of proteolytic processing enzymes.To determine whether any natural sequences outside CTL epitopes are a requirement for class I processing, a recombinant vaccinia virus (VV) was constructed that coded for a single artificial polyepitope protein containing nine class I-restricted CD8+ CTL epitopes in sequence (see Fig.

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“…Melanoma cells were infected with recombinant vaccinia virus at a multiplicity of infection of 10:1 for 1 hr at 37°C and then incubated in growth medium as described earlier (Khanna et al, 1991). After 16 to 20 hr, cells were washed with growth medium and processed for CTL assays or for immunoblotting to assess the expression of recombinant protein (Khanna et al, 1991(Khanna et al, , 1993b.…”

Section: Vaccinia Virus Recombinantsmentioning

confidence: 99%

Constitutive transduction of peptide transporter and HLA genes restores antigen processing function and cytotoxic T cell-mediated immune recognition of human melanoma cells

et al. 1998

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Presentation of endogenous viral peptide epitopes by anti-CD40 stimulated human B cells following recombinant vaccinia infection

Cited by 18 publications

References 18 publications

Ex Vivo Profiling of CD8⁺-T-Cell Responses to Human Cytomegalovirus Reveals Broad and Multispecific Reactivities in Healthy Virus Carriers

Ex Vivo Profiling of CD8⁺-T-Cell Responses to Human Cytomegalovirus Reveals Broad and Multispecific Reactivities in Healthy Virus Carriers

Minimal epitopes expressed in a recombinant polyepitope protein are processed and presented to CD8+ cytotoxic T cells: implications for vaccine design.

Constitutive transduction of peptide transporter and HLA genes restores antigen processing function and cytotoxic T cell-mediated immune recognition of human melanoma cells

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Presentation of endogenous viral peptide epitopes by anti-CD40 stimulated human B cells following recombinant vaccinia infection

Cited by 18 publications

References 18 publications

Ex Vivo Profiling of CD8+-T-Cell Responses to Human Cytomegalovirus Reveals Broad and Multispecific Reactivities in Healthy Virus Carriers

Ex Vivo Profiling of CD8+-T-Cell Responses to Human Cytomegalovirus Reveals Broad and Multispecific Reactivities in Healthy Virus Carriers

Minimal epitopes expressed in a recombinant polyepitope protein are processed and presented to CD8+ cytotoxic T cells: implications for vaccine design.

Constitutive transduction of peptide transporter and HLA genes restores antigen processing function and cytotoxic T cell-mediated immune recognition of human melanoma cells

Contact Info

Product

Resources

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Ex Vivo Profiling of CD8⁺-T-Cell Responses to Human Cytomegalovirus Reveals Broad and Multispecific Reactivities in Healthy Virus Carriers

Ex Vivo Profiling of CD8⁺-T-Cell Responses to Human Cytomegalovirus Reveals Broad and Multispecific Reactivities in Healthy Virus Carriers