Andreas Heinold

Adaptation of the hepatitis B virus core protein to CD8(+) T-cell selection pressure.

Activation of hepatitis B virus (HBV)–specific CD8 T cells by therapeutic vaccination may promote sustained control of viral replication by clearance of covalently closed circular DNA from infected hepatocytes. However, little is known about the exact targets of the CD8 T‐cell response and whether HBV reproducibly evades CD8 T‐cell immune pressure by mutation. The aim of this study was to address if HBV reproducibly selects substitutions in CD8 T‐cell epitopes that functionally act as immune escape mutations. The HBV core gene was amplified and sequenced from 148 patients with chronic HBV infection, and the human leukocyte antigen (HLA) class I genotype (A and B loci) was determined. Residues under selection pressure in the presence of particular HLA class I alleles were identified by a statistical approach utilizing the novel analysis package SeqFeatR. With this approach we identified nine residues in HBV core under selection pressure in the presence of 10 different HLA class I alleles. Additional immunological experiments confirmed that seven of the residues were located inside epitopes targeted by patients with chronic HBV infection carrying the relevant HLA class I allele. Consistent with viral escape, the selected substitutions reproducibly impaired recognition by HBV‐specific CD8 T cells. Conclusion: Viral sequence analysis allows identification of HLA class I–restricted epitopes under reproducible selection pressure in HBV core; the possibility of viral escape from CD8 T‐cell immune pressure needs attention in the context of therapeutic vaccination against HBV. (Hepatology 2015;62:47‐56)

Functional distance between recipient and donor HLA-DPB1 determines nonpermissive mismatches in unrelated HCT

The role of HLA amino acid (AA) polymorphism for the outcome of hematopoietic cell transplantation (HCT) is controversial, in particular for HLA class II. Here, we investigated this question in nonpermissive HLA-DPB1 T-cell epitope (TCE) mismatches reflected by numerical functional distance (FD) scores, assignable to all HLA-DPB1 alleles based on the combined impact of 12 polymorphic AAs. We calculated the difference in FD scores (ΔFD) of mismatched HLA-DPB1 alleles in patients and their 10/10 HLA-matched unrelated donors of 379 HCTs performed at our center for acute leukemia or myelodysplastic syndrome. Receiver-operator curve-based stratification into 2 ΔFD subgroups showed a significantly higher percentage of nonpermissive TCE mismatches for ΔFD >2.665, compared with ΔFD ≤2.665 (88% vs 25%, P < .0001). In multivariate analysis, ΔFD >2.665 was significantly associated with overall survival (hazard ratio [HR], 1.40; 95% confidence interval [CI], 1.05-1.87; P < .021) and event-free survival (HR, 1.39; 95% CI, 1.05-1.82; P < .021), compared with ΔFD ≤2.665. These associations were stronger than those observed for TCE mismatches. There was a marked but not statistically significant increase in the hazards of relapse and nonrelapse mortality in the high ΔFD subgroup, whereas no differences were observed for acute and chronic graft-versus-host disease. Seven nonconservative AA substitutions in peptide-binding positions had a significantly stronger impact on ΔFD compared with 5 others (P = .0025), demonstrating qualitative differences in the relative impact of AA polymorphism in HLA-DPB1. The novel concept of ΔFD sheds new light onto nonpermissive HLA-DPB1 mismatches in unrelated HCT.

Impact of Sequence Variation in a Dominant HLA-A*02-Restricted Epitope in Hepatitis C Virus on Priming and Cross-Reactivity of CD8+ T Cells

ABSTRACT CD8+ T cells are an essential component of successful adaptive immune responses against hepatitis C virus (HCV). A major obstacle to vaccine design against HCV is its inherent viral sequence diversity. Here, we test the hypothesis that different sequence variants of an immunodominant CD8+ T cell epitope, all binding with high affinity to HLA class I, target different T cell receptor repertoires and thereby influence the quality of the CD8+ T cell response. The impacts of sequence differences in the HLA-A*02-restricted HCV NS31406–1415 epitope on in vitro priming of naive CD8+ T cells from seronegative donors and cross-reactivity of primed T cells with other epitope variants were characterized. Although the six epitope variants tested were all high-affinity binders to HLA-A*02:01, substantial differences in priming and cross-reactivity of CD8+ T cells were observed. The variant associated with the most reproducible priming and induction of T cells with broad cross-reactivity was a genotype 1b variant (KLSALGLNAV) that is more common in HCV isolates collected in Asia but is rare in sequences from Europe and North America. The superior immunogenicity and cross-reactivity of this relatively rare epitope variant were confirmed by using HCV-specific memory CD8+ T cells from people who inject drugs, who are frequently exposed to HCV. Collectively, the data suggest that sequence differences at the epitope level between HCV isolates substantially impact CD8+ T cell priming and the degree of cross-reactivity with other epitope variants. IMPORTANCE The results have important implications for vaccine design against highly variable pathogens and suggest that evidence-based selection of the vaccine antigen sequence may improve immunogenicity and T cell cross-reactivity. Cross-reactive CD8+ T cells are likely beneficial for immune control of transmitted viruses carrying epitope variants and for prevention of immune escape during acute infection. To this end, rare epitope variants and potentially even altered epitope sequences associated with priming of broadly cross-reactive T cell receptors should be considered for vaccine design and need further testing.

Humoral and cellular responses to a single dose of Fendrix in renal transplant recipients with non-response to previous hepatitis B vaccination

Approximately 70% of kidney transplant recipients are non‐responders to conventional hepatitis B virus (HBV) vaccines. We examined whether Fendrix™, an HBV vaccine containing 3‐O‐desacyl‐4′‐monophosphoryl lipid A (MPL) as adjuvant, could induce HBV immunity in these patients and compared their vaccination efficacy with healthy controls tested previously by the same assays. We selected 35 kidney transplant recipients who had been vaccinated at least thrice against HBV but had never displayed anti‐HBs antibodies. We re‐assessed their anti‐HBs antibody titres and further determined cellular HBV immunity by proliferation assay and interferon (IFN)‐γ ELISpot. Seventeen recipients did neither display humoral nor cellular immunity and could be tested prior to and at month 1 after vaccination. Of note, HLA antigens associated with non‐response to HBV vaccination (HLA‐DRB1*03 and HLA‐DQB1*02) were over‐represented in these 17 recipients. At month 1 after a single vaccination with Fendrix™, we observed a significant increase in anti‐HBs antibodies (P = 0.02). In seven of 17 recipients, we detected anti‐HBs antibodies ≥10 IU/l (10–264), in four HBV‐specific lymphocyte proliferation (stimulation index of 2.6–8.7) and in one specific IFN‐γ responses (12 spots increment). The vaccination response to Fendrix™ was significantly higher (P = 0.035) than the response to HBVaxPro™ in young healthy controls. In summary, the results show that a single vaccination with Fendrix™ could already induce HBV‐specific humoral and/or cellular responses in ten of 17 kidney transplant patients. Thus, Fendrix™ appears as an efficient vaccine in this patient cohort.

HLA-Bw4 80(T) and multiple HLA-Bw4 copies combined with KIR3DL1 associate with spontaneous clearance of HCV infection in people who inject drugs

BACKGROUND & AIMS Natural killer (NK) cell function is regulated by inhibitory and activating receptors including killer cell immunoglobulin-like receptors (KIRs). Here, we analyzed the impact of different KIR/KIR-ligand genotypes on the outcome of hepatitis C virus (HCV) infection in people who inject drugs (PWID). METHODS KIR/KIR-ligand genotypes associated with spontaneous clearance of HCV infection were identified in a cohort of PWID from Germany (n=266) and further validated in a second anti-HCV positive cohort of PWID recruited in North America (n=342). NK cells of PWID and healthy donors were functionally characterized according to their KIR/KIR-ligand genotype by flow cytometry. RESULTS Multivariate logistic regression analysis revealed that KIR3DL1/HLA-Bw4 80(T) was associated with spontaneous clearance of HCV infection in PWID, which was confirmed in the PWID cohort from North America. Compared with PWID with detectable HCV RNA, the frequency of individuals with multiple HLA-Bw4 alleles was significantly higher in anti-HCV positive PWID with resolved HCV infection (29.7% vs. 15.2%; p=0.0229) and in anti-HCV seronegative PWID (39.2%; p=0.0006). KIR3DL1+ NK cells from HLA-Bw4 80(T)-positive PWID showed superior functionality compared to HLA-Bw4 80(I)-positive PWID. This differential impact was not observed in healthy donors; however, the HLA-Bw4 copy number strongly correlated with the functionality of KIR3DL1+ NK cells. CONCLUSIONS HLA-Bw4-80(T) and multiple HLA-Bw4 copies in combination with KIR3DL1 are associated with protection against chronic hepatitis C in PWID by distinct mechanisms. Better licensing of KIR3DL1+ NK cells in the presence of multiple HLA-Bw4 copies is beneficial prior to seroconversion whereas HLA-Bw4 80(T) may be beneficial during acute hepatitis C. Lay summary: Natural killer (NK) cells are part of the innate immune system and are regulated by a complex network of activating and inhibiting receptors. The regulating receptor-ligand pairs of an individual are genetically determined. Here, we identified a particular set of ligand and receptor genes that are associated with better functionality of NK cells and better outcome upon exposure to HCV in a high-risk group.

Clinical Utility of Quantitative PCR for Chimerism and Engraftment Monitoring after Allogeneic Stem Cell Transplantation for Hematologic Malignancies

Although quantitative PCR (qPCR) has been explored for chimerism monitoring after allogeneic stem cell transplantation (SCT), evidence regarding its clinical utility compared with standard short tandem repeat (STR) is still limited. We retrospectively studied commercial qPCR and STR chimerism with respective positivity thresholds of .1% and 1% in 359 peripheral blood (PB) and 95 bone marrow (BM) samples from 30 adult patients after first HLA-matched SCT for myeloid malignancies or acute lymphatic leukemia. Concordance between the 2 methods was 79.5%, with all discordant samples positive in qPCR but negative in STR. Of the latter, sporadic qPCR positivity without clinical correlates was seen mostly in BM samples early post-transplant. In 7 of 21 patients with available follow-up samples in the first months after transplantation, qPCR but not STR revealed low levels (<1%) of sustained host chimerism in PB, reflecting delayed engraftment or persistent mixed chimerism (PMC). These conditions were associated with donor-recipient cytomegalovirus (CMV) serostatus and early CMV reactivation but not with immunosuppressive regimens or clinical outcome. qPCR predicted all 8/8 relapses with samples in the 6 months before onset by sustained positivity in both PB and BM compared with 1/8 relapses predicted by STR mainly in BM. The response kinetics to donor lymphocyte infusions for the treatment of PMC or relapse was shown by qPCR but not STR to be protracted over several months in 3 patients. Our results demonstrate the superior clinical utility of qPCR compared with STR for monitoring subtle changes of host chimerism associated with different clinical conditions, making a case for its use in the clinical follow-up of transplant patients.

Immunosuppression with mTOR inhibitors prevents the development of donor-specific antibodies after liver transplant

Donor‐specific antibodies (DSAs) are an important cause of complications after solid organ transplant. Risk factors and, thus, strategies for preventing DSA development are not well defined.

A single nucleotide polymorphism upstream of the HLA-C locus is associated with an anti-HCV seronegative state in a high-risk exposed cohort

In this study, we examined the impact of the rs9264942 single-nucleotide polymorphism, previously shown to be associated with human immunodeficiency virus infection status and HLA-C expression, on the hepatitis C virus status in 359 people who inject drugs (PWID). The linkage of rs9264942 alleles to HLA-C antigens assigned to different expression levels was confirmed. Multivariate analysis revealed the age (P = .003) and the rs9264942 genotype (P = .006) to be independent factors for the classification to the PWID groups. Our study showed that the presence of the rs9264942 C/C genotype was associated with persistent seronegativity.

Author Correction: Glomerulocapillary miRNA response to HLA-class I antibody in vitro and in vivo

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Adoptive transfer of cellular immunity against cytomegalovirus by virus-specific lymphocytes from a third-party family donor

Infection remains a major challenge after hematopoietic stem cell transplantation [1]. In transplant recipients, reactivation of herpes viruses, especially of the human cytomegalovirus (CMV), is associated with severe and fatal complications. CMV is a life-long persisting virus with a seroprevalence between 45 and 100% in the general adult population [2]. Whereas symptomatic CMV infection of healthy individuals is rare, in immunodeficient patients, such as transplant recipients, CMV infection or reactivation commonly manifests as a life-threatening disease affecting different organ systems. Transplant recipients with CMV viremia are usually treated with antiviral drugs [3]. However, myelotoxicity as well as mutations of the viral DNA sequence may limit the use of these drugs [3–5]. Thus, in selected cases alternative therapeutic options are mandatory. We here present the follow-up of a 21-year-old female with acute myeloid leukemia, who was treated with CMVspecific T cells (virus-specific T cells, VSTs). Written informed patient consent has been obtained. Viral load and cellular CMV immunity were closely monitored for almost 3 years. The patient was transplanted in cytomorphologic complete remission but with cytogenetically minimal residual disease. She received allogeneic peripheral blood stem cells from a 9/10 HLA-matched, unrelated male donor in May 2015. Myeloablative conditioning consisted of fludarabin, carmustine, antithymocyte globulin, and melphalan, and the immunosuppressive regimen included cyclosporine A and budesonide. Anti-infective prophylaxis/ therapy included amoxicillin, metronidazole, itraconazole, valganciclovir, cotrimoxazol, calciumfolinate, and pentamidine. She received 9.4 × 10 CD34+ cells/kg body weight. Three weeks after peripheral blood stem cell transplantation (PBSCT) the patient suffered from acute graft vs. host disease (GvHD) of the skin (stage 1, grade 1), which did not require systemic treatment. The unrelated donor was CMV IgMand IgG-negative, and the patient was CMV IgM-negative but IgG-positive. Thus, the recipient was at high risk of CMV reactivation. In July 2015 anti-CMV IgM and IgG became positive in the recipient and the viral load reached up to 1,022,908 copies (1,595,800 IU)/ml (day 35) as shown in Fig. 1. Despite antiviral therapy (sequentially with valganciclovir, ganciclovir, and foscarnet) and CMV-specific immunoglobulins no adequate decrease of the viral load was observed. At the peak of CMV reactivation, the patient suffered from a generalized seizure, which was considered cyclosporinerelated. Treatment with cyclosporine and budesonide was switched to mycophenolate mofetil and prednisone. The patient received levetirazepam and no further seizure occurred. It was then decided to treat the patient with CMVspecific lymphocytes of her HLA haploidentical sister who was CMV IgG-positive. Manufacturing of CMV-specific T cells was carried out at Hannover Medical School with the CliniMACS Plus Instrument and GMP PepTivator HCMVpp65 for antigenic restimulation, as described previously [6, 7]. Enrichment of IFN-γ-secreting CMV-specific T cells was performed by immunomagnetic separation using the CliniMACS Cytokine Capture System (Miltenyi Biotec, * Monika Lindemann monika.lindemann@uk-essen.de