Maaike E. Ressing

Vaccination with HPV16 peptides of patients with advanced cervical carcinoma: clinical evaluation of a phase I-II trial

A phase I-II clinical trial was performed involving vaccination with HPV16 E7 peptides of patients suffering from HPV16 positive cervical carcinoma which was refractory to conventional treatment. Patients receiving the vaccine were HLA-A*0201 positive with HPV16 positive cervical carcinoma. The clinical trial was designed as a dose-escalation study, in which successive groups of patients received 100 micrograms, 300 micrograms or 1000 micrograms of each peptide, respectively. The vaccine consisted of two HPV16 E7 peptides and one helper peptide emulsified in Montanide ISA 51 adjuvant. 19 patients were included in the study, no adverse side-effects were observed. 2 patients showed stable disease for 1 year after vaccination; 15 patients showed progressive disease of whom 1 died during the vaccination treatment due to progressive disease; and 2 patients showed tumour-regression after chemotherapy following vaccination. A relative low count of lymphocytes before and after vaccination was present in 11/19 patients indicating that these patients were immunocompromised. This study shows that HPV16 E7 peptide vaccination is feasible, even in a group of patients with terminal disease. This paves the way for vaccinating patients with less advanced disease, whose immune system is less compromised by progressive disease.

Detection of t helper responses, but not of human papillomavirus-specific cytotoxic t lymphocyte responses, after peptide vaccination of patients with cervical carcinoma

Human papillomavirus type 16 (HPV16)-encoded E7 oncoprotein is constitutively expressed in cervical carcinoma cells and is required for cellular transformation to be maintained. The E7 protein, therefore, forms an attractive target for T-cell–mediated immune intervention to prevent or treat HPV16+ tumors. The authors performed a peptide-based phase I/II vaccination trial to induce anti-tumor immune responses in patients with recurrent or residual cervical carcinoma. Fifteen HLA-A*0201+ patients with HPV16+ cervical carcinoma received vaccinations with synthetic peptides representing 2 HPV16 E7-encoded, HLA-A*0201–restricted cytotoxic T lymphocyte epitopes and a pan-HLA-DR–binding T-helper epitope, PADRE, in adjuvant. No signs of toxicity were observed. Two patients had stable disease for more than 1 year after vaccination, 3 patients died of the disease during or shortly after the vaccination period, and 10 patients maintained progressive cervical carcinoma. Specific immune responses directed against the vaccine components were analyzed in peripheral blood samples. No cytotoxic T lymphocyte responses against the HPV16 E7 peptides were detectable. After vaccination, strong PADRE helper peptide-specific proliferation was detected in 4 of 12 patients. In conclusion, peptide vaccination with 2 HPV16 E7 cytotoxic T lymphocyte epitopes and a universal T helper epitope is well tolerated by patients with advanced cervical carcinoma. Despite a reduction of in vitro cytolytic or proliferative recall responses to some, but not all, conventional antigens in this patient group, peptide-specific proliferative responses were induced in 4 patients. Based on the current study, it is now feasible to perform peptide vaccination in earlier stages of HPV16-induced cervical disease.

Host shutoff during productive Epstein–Barr virus infection is mediated by BGLF5 and may contribute to immune evasion

Relatively little is known about immune evasion during the productive phase of infection by the γ1-herpesvirus Epstein–Barr virus (EBV). The use of a unique system to isolate cells in lytic cycle allowed us to identify a host shutoff function operating in productively EBV-infected B cells. This impairment of protein synthesis results from mRNA degradation induced upon expression of the early lytic-cycle gene product BGLF5. Recently, a γ2-herpesvirus, Kaposi sarcoma herpesvirus, has also been shown to encode a host shutoff function, indicating that host shutoff appears to be a general feature of γ-herpesviruses. One of the consequences of host shutoff is a block in the synthesis of HLA class I and II molecules, reflected by reduced levels of these antigen-presenting complexes at the surface of cells in EBV lytic cycle. This effect could lead to escape from T cell recognition and elimination of EBV-producing cells, thereby allowing generation of viral progeny in the face of memory T cell responses.

Natural T-helper immunity against human papillomavirus type 16 (hpv16) e7–derived peptide epitopes in patients with hpv16-positive cervical lesions: Identification of 3 human leukocyte antigen class ii–restricted epitopes

Tumor‐specific T‐helper (Th) immunity was found to play a pivotal role in the natural and vaccine‐induced immune defense against tumors. Since the majority of cervical cancers express human papillomavirus type 16 (HPV16) E7 oncoprotein, it is important to investigate the Th response against this target antigen in detail. By means of PBMC cultures from HLA‐typed healthy donors, we identified the central part of HPV16 E7 (E741–72) as the major immunogenic region within this antigen. Furthermore, we mapped 3 distinct Th epitopes within this region (DR15/E750–62, DR3/E743–77, DQ2/E735–50). In a parallel approach, employing IFN‐γ ELISPOT analysis, we detected Th immunity against HPV16 E7 in subjects with HPV16+ lesions. Several of these responses matched with the 3 Th epitopes defined in our study. A number of other HPV16+ subjects did not display any E7‐specific type 1 cytokine‐producing T‐cell immunity, indicating failure of the immune response. Our combined data argue for more extensive as well as longitudinal analysis of HPV16‐specific T‐cell immunity using the ELISPOT assay described, as well as for HPV‐specific vaccination of individuals with HPV+ lesions.

A CD8+ T cell immune evasion protein specific to Epstein-Barr virus and its close relatives in Old World primates

γ1-Herpesviruses such as Epstein-Barr virus (EBV) have a unique ability to amplify virus loads in vivo through latent growth-transforming infection. Whether they, like α- and β-herpesviruses, have been driven to actively evade immune detection of replicative (lytic) infection remains a moot point. We were prompted to readdress this question by recent work (Pudney, V.A., A.M. Leese, A.B. Rickinson, and A.D. Hislop. 2005. J. Exp. Med. 201:349–360; Ressing, M.E., S.E. Keating, D. van Leeuwen, D. Koppers-Lalic, I.Y. Pappworth, E.J.H.J. Wiertz, and M. Rowe. 2005. J. Immunol. 174:6829–6838) showing that, as EBV-infected cells move through the lytic cycle, their susceptibility to EBV-specific CD8+ T cell recognition falls dramatically, concomitant with a reductions in transporter associated with antigen processing (TAP) function and surface human histocompatibility leukocyte antigen (HLA) class I expression. Screening of genes that are unique to EBV and closely related γ1-herpesviruses of Old World primates identified an early EBV lytic cycle gene, BNLF2a, which efficiently blocks antigen-specific CD8+ T cell recognition through HLA-A–, HLA-B–, and HLA-C–restricting alleles when expressed in target cells in vitro. The small (60–amino acid) BNLF2a protein mediated its effects through interacting with the TAP complex and inhibiting both its peptide- and ATP-binding functions. Furthermore, this targeting of the major histocompatibility complex class I pathway appears to be conserved among the BNLF2a homologues of Old World primate γ1-herpesviruses. Thus, even the acquisition of latent cycle genes endowing unique growth-transforming ability has not liberated these agents from evolutionary pressure to evade CD8+ T cell control over virus replicative foci.

The Epstein-Barr Virus G-Protein-Coupled Receptor Contributes to Immune Evasion by Targeting MHC Class I Molecules for Degradation

Epstein-Barr virus (EBV) is a human herpesvirus that persists as a largely subclinical infection in the vast majority of adults worldwide. Recent evidence indicates that an important component of the persistence strategy involves active interference with the MHC class I antigen processing pathway during the lytic replication cycle. We have now identified a novel role for the lytic cycle gene, BILF1, which encodes a glycoprotein with the properties of a constitutive signaling G-protein-coupled receptor (GPCR). BILF1 reduced the levels of MHC class I at the cell surface and inhibited CD8+ T cell recognition of endogenous target antigens. The underlying mechanism involves physical association of BILF1 with MHC class I molecules, an increased turnover from the cell surface, and enhanced degradation via lysosomal proteases. The BILF1 protein of the closely related CeHV15 γ1-herpesvirus of the Rhesus Old World primate (80% amino acid sequence identity) downregulated surface MHC class I similarly to EBV BILF1. Amongst the human herpesviruses, the GPCR encoded by the ORF74 of the KSHV γ2-herpesvirus is most closely related to EBV BILF1 (15% amino acid sequence identity) but did not affect levels of surface MHC class I. An engineered mutant of BILF1 that was unable to activate G protein signaling pathways retained the ability to downregulate MHC class I, indicating that the immune-modulating and GPCR-signaling properties are two distinct functions of BILF1. These findings extend our understanding of the normal biology of an important human pathogen. The discovery of a third EBV lytic cycle gene that cooperates to interfere with MHC class I antigen processing underscores the importance of the need for EBV to be able to evade CD8+ T cell responses during the lytic replication cycle, at a time when such a large number of potential viral targets are expressed.

Epstein-Barr virus evasion of CD8(+) and CD4(+) T cell immunity via concerted actions of multiple gene products.

Upon primary infection, EBV establishes a latent infection in B cells, characterized by maintenance of the viral genome in the absence of viral replication. The Epstein-Barr Nuclear Antigen 1 (EBNA1) plays a crucial role in maintenance of the viral DNA episome and is consistently expressed in all EBV-associated malignancies. Compared to other EBV latent gene products, EBNA1 is poorly recognized by CD8(+) T lymphocytes. Recent studies are discussed that shed new light on the mechanisms that underlie this unusual lack of CD8(+) T cell activation. Whereas the latent phase is characterized by the expression of a limited subset of viral gene products, the full repertoire of over 80 EBV lytic gene products is expressed during the replicative phase. Despite this abundance of potential T cell antigens, which indeed give rise to a strong response of CD4(+) and CD8(+) T lymphocytes, the virus can replicate successfully. Evidence is accumulating that this paradoxical situation is the result of actions of multiple viral gene products, inhibiting discrete stages of the MHC class I and class II antigen presentation pathways. Immediately after initiation of the lytic cycle, BNLF2a prevents peptide-loading of MHC class I molecules through inhibition of the Transporter associated with Antigen Processing, TAP. This will reduce presentation of viral antigens by the large ER-resident pool of MHC class I molecules. Synthesis of new MHC class I molecules is blocked by BGLF5. Viral-IL10 causes a reduction in mRNA levels of TAP1 and bli/LMP2, a subunit of the immunoproteasome. MHC class I molecules present at the cell surface are downregulated by BILF1. Also the antigen presenting capacity of MHC class II molecules is severely compromised by multiple EBV lytic gene products, including gp42/gH/gL, BGLF5, and vIL-10. In this review, we discuss how concerted actions of these EBV lytic proteins result in highly effective interference with CD8(+) and CD4(+) T cell surveillance, thereby providing the virus with a window for undisturbed generation of viral progeny.

The DNase of Gammaherpesviruses Impairs Recognition by Virus-Specific CD8+ T Cells through an Additional Host Shutoff Function

ABSTRACT The DNase/alkaline exonuclease (AE) genes are well conserved in all herpesvirus families, but recent studies have shown that the AE proteins of gammaherpesviruses such as Epstein-Barr virus (EBV) and Kaposis sarcoma-associated herpesvirus (KSHV) exhibit an additional function which shuts down host protein synthesis. One correlate of this additional shutoff function is that levels of cell surface HLA molecules are downregulated, raising the possibility that shutoff/AE genes of gammaherpesviruses might contribute to viral immune evasion. In this study, we show that both BGLF5 (EBV) and SOX (KSHV) shutoff/AE proteins do indeed impair the ability of virus-specific CD8+ T-cell clones to recognize endogenous antigen via HLA class I. Random mutagenesis of the BGLF5 gene enabled us to genetically separate the shutoff and AE functions and to demonstrate that the shutoff function was the critical factor determining whether BGLF5 mutants can impair T-cell recognition. These data provide further evidence that EBV has multiple mechanisms to modulate HLA class I-restricted T-cell responses, thus enabling the virus to replicate and persist in the immune-competent host.

The UL41-encoded virion host shutoff (vhs) protein and vhs-independent mechanisms are responsible for down-regulation of MHC class I molecules by bovine herpesvirus 1

The virion host shutoff (vhs) protein of alphaherpesviruses causes a rapid shutoff of host cell protein synthesis. We constructed a bovine herpesvirus 1 (BHV1) deletion mutant in which the putative vhs gene, UL41, has been disrupted. Whereas protein synthesis is inhibited within 3 h after infection with wild-type BHV1, no inhibition was observed after infection with the BHV1(vhs-) deletion mutant. These results indicate that the BHV1 UL41 gene product is both necessary and sufficient for shutoff of host cell protein synthesis at early times post-infection. Using the vhs deletion mutant, we investigated the mechanism of BHV1-induced down-regulation of MHC class I cell surface expression. In contrast to BHV1 wild-type infection, the BHV1(vhs-) mutant allows detection of MHC class I molecules at much later time-points after infection. This illustrates the role the vhs protein plays in MHC class I down-regulation. However, even after infection with BHV1(vhs-), MHC class I cell surface expression is impaired. In BHV1(vhs-)-infected cells, MHC class I molecules are retained within the endoplasmic reticulum (ER). Moreover, the transporter associated with antigen presentation (TAP) is still blocked. Temporal control of viral protein expression using chemical inhibitors shows that viral protein(s) expressed within the early phase of BHV1 infection are responsible for ER retention of MHC class I molecules. These results indicate that multiple mechanisms are responsible for down-regulation of MHC class I molecules in BHV1-infected cells.

Human Cytomegalovirus-Encoded US2 Differentially Affects Surface Expression of MHC Class I Locus Products and Targets Membrane-Bound, but Not Soluble HLA-G1 for Degradation

Human CMV (HCMV) can elude CTL as well as NK cells by modulating surface expression of MHC class I molecules. This strategy would be most efficient if the virus would selectively down-regulate viral Ag-presenting alleles, while at the same time preserving other alleles to act as inhibitors of NK cell activation. We focused on the HCMV unique short (US) region encoded protein US2, which binds to newly synthesized MHC class I H chains and supports their dislocation to the cytosol for subsequent degradation by proteasomes. We studied the effect of US2 on surface expression of individual class I locus products using flow cytometry. Our results were combined with crystal structure data of complexed US2/HLA-A2/β2-microglobulin and alignments of 948 HLA class I database sequences of the endoplasmic reticulum lumenal region inplicated in US2 binding. This study suggests that surface expression of all HLA-A and -G and most HLA-B alleles will be affected by US2. Several HLA-B alleles and all HLA-C and -E alleles are likely to be insensitive to US2-mediated degradation. We also found that the MHC class I endoplasmic reticulum-lumenal domain alone is not sufficient for degradation by US2, as illustrated by the stability of soluble HLA-G1 in the presence of US2. Furthermore, we showed that the membrane-bound HLA-G1 isoform, but also tailless HLA-A2, are targeted for degradation. This indicates that the cytoplasmic tail of the MHC class I H chain is not required for its dislocation to the cytosol by US2.