Picarda, G. | Search bioRxiv

bioRxiv2021

Profiling Human CMV-specific T cell responses reveals novel immunogenic ORFs

Despite the prevalence and medical significance of human cytomegalovirus (HCMV) infections, a systematic analysis of the targets of T cell recognition in humans that spans the entire genome and includes recently described potential novel ORFs is not available. Here, we screened a library of epitopes predicted to bind HLA class II that spans over 350 different HCMV ORFs and includes [~]150 previously described and [~]200 recently described potential novel ORFs using an ex vivo IFN{gamma} fluorospot assay. We identified 235 unique HCMV specific epitopes derived from 100 ORFs, some previously described as immunodominant and others that were not previously described to be immunogenic. Of those, 41 belong to the set of recently reported novel ORFs, thus providing evidence that at least some of these are actually expressed in vivo in humans. These data reveal that the breadth of the human T cell response to HCMV is much greater than previously thought. The ORFs and epitopes identified will help elucidate how T cell immunity relates to HCMV pathogenesis and instruct ongoing HCMV vaccine research. ImportanceTo understand the crucial role of adaptive immunity in controlling cytomegalovirus infection and disease, we systematically analyzed the CMV ORFeome to identify new CMV epitopes targeted primarily by CD4 T cells in humans. Our study identified >200 new T cell epitopes derived from both canonical and novel ORFs, highlighting the substantial breadth of anti-CMV T cell response and providing new targets for vaccine design.

immunology↗

bioRxiv2019

Structure of human cytomegalovirus UL144, an HVEM orthologue, bound to the B and T cell Lymphocyte Attenuator

Human cytomegalovirus (HCMV) is a {beta}-herpesvirus that has co-evolved with the host immune system to establish lifelong persistence. HCMV encodes many immune-modulatory molecules, including the glycoprotein UL144. UL144 is a structural mimic of the TNFRSF member HVEM, which binds to various ligands LIGHT, LT, BTLA, CD160 and gD. However, in contrast to HVEM, UL144 selectively binds to only BTLA, inhibiting T cell activation. Here, we report the crystal structure of the UL144/BTLA complex, providing key insights into the molecular mechanisms underlying this virus-host protein interaction. Our structure reveals that UL144 utilizes residues from its N-terminal CRD1 to interact with BTLA in an orientation similar, but not exactly, to that of HVEM. The structural modifications at the CRD1 region of UL144 compared to HVEM have a significant impact on the fine-tuning of BTLA-binding. In addition, the N-terminal CRD2 loop of UL144 is shorter compared to the corresponding region of HVEM, altering the relative orientation of CRD2 with respect to CRD1. Employing structure-guided mutagenesis we have identified a mutant of BTLA (L123A) that interferes with binding to HVEM while preserving interaction towards UL144. Furthermore, our results illuminate structural differences between UL144 and HVEM that explain the inability of UL144 to bind to either LIGHT or CD160. In summary, the specific molecular differences that UL144 has evolved to exclusively target BTLA highlight it as a suitable scaffold for designing superior BTLA agonists that have high potential for potently inhibiting immune responses.\n\nImportanceThe co-evolution of HCMV with its host over millions of years has allowed the virus to develop an efficient and specific immune modulatory protein, UL144, that binds exclusively to an immune inhibitory receptor BTLA. The crystal structure of the UL144/BTLA complex presented in this manuscript provides key insights into the molecular mechanisms underlying the virus-host protein interaction. The structure guided mutagenesis revealed select structural hot spots of the UL144/BTLA interaction. The structural details of this viral protein that has evolved to target only BTLA helps in successful design of BTLA agonists to target various T and B cell mediated autoimmune diseases.

immunology↗