Ex Vivo Stimulation and Expansion of both CD4⁺and CD8⁺T Cells from Peripheral Blood Mononuclear Cells of Human Cytomegalovirus-Seropositive Blood Donors by Using a Soluble Recombinant Chimeric Protein, IE1-pp65

Abstract

Human cytomegalovirus (HCMV) infection is common and usually well controlled. Immunocompromised patients such as those undergoing bone marrow transplantation and infected newborns are especially vulnerable to HCMV disease (5,20,23).The immune control of HCMV replication appears to be mainly mediated by cellular immune responses. CD4 ϩ and CD8 ϩ T lymphocytes have been proposed to play a major role in the control of viral replication and in protection from disease. The contribution of anti-IE1-and anti-pp65-specific Tcell precursors to the total anti-HCMV immunity is now well established (7,8,15,19,34). IE1 is the major protein produced in the immediate-early phase of the HCMV replication cycle, and the matrix protein pp65 has been shown to be internalized immediately after the viral input without de novo synthesis and then to be available for presentation to specific CD8 ϩ cytotoxic T lymphocytes (CTL) (2, 19). The establishment of a rapid T-cell response before the synthesis of new infectious virions could provide an efficient means to avoid spreading of the virus. This strongly supports the idea that both CD4 ϩ and CD8 ϩ T cells directed against IE1 and pp65 could be critical for the generation of effective vaccines against HCMV and in anti-HCMV cell therapy.The transfer of anti-HCMV effector T cells to allogeneic bone marrow recipients results in protection from HCMV diseases associated with transplantation. The procedure is based on the use of HCMV-infected autologous fibroblasts to stimulate anti-HCMV-specific T cells in vitro (33). The authors showed that persistence of cytotoxic CD8 ϩ T cells in recipients was facilitated by a simultaneous recovery of CD4 ϩ helper T cells after bone marrow transplantation (33). More recently, the use of Epstein-Barr virus (EBV)-transformed B cells transduced with a recombinant retrovirus expressing pp65 has been suggested to allow the concomitant expansion of both anti-EBV-and anti-HCMV-specific T cells (31). A similar strategy used autologous B lymphoblastoid cells stably transfected with cDNA coding for either pp65 or IE1 for the generation of specific CD8 ϩ T-cell clones (26). Our approach to circumvent the use of infectious virus in ex vivo expansion protocols for cellular immunotherapy is based on a procedure allowing the simultaneous triggering of the anti-IE1 and -pp65 responses by means of a recombinant chimeric protein, IE1-pp65.In this paper, we report the construction and purification of a recombinant IE1-pp65 protein from insect cells. We demonstrate that an important proportion of anti-HCMV CD4 ϩ T cells was directed against IE1-pp65 in HCMV-seropositive donors and that the protein induced activation of HLA-DR3-restricted anti-IE1 CD4 ϩ T-cell clones, as assessed through gamma interferon (IFN-␥) secretion and cytotoxicity. Moreover, soluble IE1-pp65 was able to stimulate and to expand anti-pp65 CD8 ϩ T cells from peripheral blood mononuclear

Exogenous introduction of an immunodominant peptide from the non-structural IE1 protein of human cytomegalovirus into the MHC class I presentation pathway by recombinant dense bodies

“…Consequently, fusion of IE1 to pp65 would potentially generate an excellent recDB-based vaccine candidate. Such a fusion protein, expressed by insect cells, was effective at stimulating the expansion of anti-HCMV CD4 and CD8 T-cell clones from HCMVseropositive donors in vitro (Vaz-Santiago et al, 2001). However, including the complete IE1 polypeptide for the design of a vaccine may be subject to safety concerns, as IE1 is known to activate both cellular and viral genes (Mocarski et al, 2007).…”

Infection of APC by Human Cytomegalovirus Controlled Through Recognition of Endogenous Nuclear Immediate Early Protein 1 by Specific CD4+ T Lymphocytes

“…Additionally, a high frequency of CD4 ϩ T cells specific for CMV Ags has been widely reported (12)(13)(14)(15). Ag specificity has been shown against several proteins such as immediate early (IE) 3 protein 1, IE2, glycoprotein B (gB), and phosphoprotein (pp) 65, either at the population (16 -18) or the clonal (19,20) level.…”

Induction of HLA-G-restricted human cytomegalovirus pp65 (UL83)-specific cytotoxic T lymphocytes in HLA-G transgenic mice

“…U-373MG(U373) human astrocytoma cells (ATCC), U373/G [U373 cells stably transfected with pcDNA-G1 (Blaschitz et al, 1997)] and U373/G-IE1-pp65 [U373/G cells stably transfected with IE1-pp65 cDNA (Vaz-Santiago et al, 2001)] were used as HCMV-infected target cells.…”

“…To study whether the in vivo processing of pp65 was sufficient to induce a specific antiviral response, we also injected transgenic mice with DCs loaded with the recombinant fusion protein IE1-pp65, which has been produced and purified previously (Vaz-Santiago et al, 2001). Splenocytes were recovered after immunization and stimulated in vitro with peptide-loaded DCs.…”

“…3(A), significant lysis of EL4/G-vac-pp65 target cells (25 % specific lysis) was observed as compared to EL4/G target cells (6 % specific lysis) and to EL4/Gvacwt (15 % specific lysis), suggesting that these peptides were processed naturally (10 % anti-pp65 specific lysis). In addition, the absence of killing of the HLA-G-negative EL4 target cells loaded with peptides as well as the decrease of cytotoxicity observed when the specific anti-HLA-G mAb 87G was added to EL4/G-vac-pp65 cells demonstrated that the cytotoxic activity was HLA-G restricted.To study whether the in vivo processing of pp65 was sufficient to induce a specific antiviral response, we also injected transgenic mice with DCs loaded with the recombinant fusion protein IE1-pp65, which has been produced and purified previously (Vaz-Santiago et al, 2001). Splenocytes were recovered after immunization and stimulated in vitro with peptide-loaded DCs.…”