Non-viral TRAC-knocked-in CD19KICAR-T and gp350KICAR-T cells tested against Burkitt lymphomas with type 1 or 2 EBV infection: In vivo cellular dynamics and potency.

Introduction: The ubiquitous Epstein-Barr virus (EBV) is an oncogenic herpes virus associated with several human malignancies. EBV is an immune-evasive pathogen that promotes CD8+ T cell exhaustion and dysregulates CD4+ T cell functions. Burkitt lymphoma (BL) is frequently associated with EBV infections. Since BL relapses after conventional therapies are difficult to treat, we evaluated prospective off-the-shelf edited CAR-T cell therapies targeting CD19 or the EBV gp350 cell surface antigen. Methods: We used CRISPR/Cas9 gene editing methods to knock in (KI) the CD19CAR.CD28z or gp350CAR.CD28z into the T cell receptor (TCR) alpha chain (TRAC) locus. Results: Applying upscaled methods with the ExPERT ATx® MaxCyte system, KI efficacy was ~20% of the total ~2 × 108 TCR-knocked-out (KO) generated cells. KOTCRKICAR-T cells were co-cultured in vitro with the gp350+CD19+ BL cell lines Daudi (infected with type 1 EBV) or with Jiyoye (harboring a lytic type 2 EBV). Both types of CAR-T cells showed cytotoxic effects against the BL lines in vitro. CD8+ KICAR-T cells showed higher persistency than CD4+ KICAR-T cells after in vitro co-culture with BL and upregulation of the activation/exhaustion markers PD-1, LAG-3, and TIM-3. Two preclinical in vivo xenograft models were set up with Nod.Rag.Gamma mice injected intravenously (i.v.) with 2 × 105 Daudi/fLuc-GFP or with Jiyoye/fLuc-GFP cells. Compared with the non-treated controls, mice challenged with BL and treated with CD19KICAR-T cells showed delayed lymphoma dissemination with lower EBV DNA load. Notably, for the Jiyoye/fLuc-GFP model, almost exclusively CD4+ CD19KICAR-T cells were detectable at the endpoint analyses in the bone marrow, with increased frequencies of regulatory T cells (Tregs) and TIM-3+CD4+ T cells. Administration of gp350KICAR-T cells to mice after Jiyoye/GFP-fLuc challenge did not inhibit BL growth in vivo but reduced the EBV DNA load in the bone marrow and promoted gp350 antigen escape. CD8+PD-1+LAG-3+ gp350KICAR-T cells were predominant in the bone marrow. Discussion: The two types of KOTCRKICAR-T cells showed different therapeutic effects and in vivo dynamics. These findings reflect the complexities of the immune escape mechanisms of EBV, which may interfere with the CAR-T cell property and potency and should be taken into account for future clinical translation.

Dynamics of Torque Teno virus viremia could predict risk of complications after allogeneic hematopoietic stem cell transplantation.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an established treatment option for several hematological diseases. However, the first year post-transplantation is often complicated by infections and graft-versus-host disease (GVHD). Improvements in immunological monitoring could reduce such post-transplant complications. Torque Teno virus (TTV), a chronically persisting DNA virus, is reported to be a marker for immune function in immunocompromised patients. In the present study, the TTV kinetics were analyzed to investigate the potential role of TTV viremia as immune-competence read-out after allo-HSCT. Twenty-three monocentric allo-HSCT recipients were retrospectively tested for TTV-DNA in whole blood at given day post-transplant. Dynamics of TTV viremia was analyzed with respect to episodes of non-TTV viral reactivations (CMV, EBV, and BKPyV), acute GVHD, and recovery of immune cells. Recipients affected by persisting viral infections and/or GVHD during the first 100 days after allo-HSCT showed a significantly higher median TTV load at day +30 than patients with a less complicated clinical course (p = 0.005). This was also associated with a total lymphocyte count <5.5E+08 cells/L in this high-risk group (p = 0.039). These findings suggest that TTV could represent an additional parameter to identify patients at higher risk for complications in the first 100 days following allo-HSCT. Prospective studies, including the monitoring of lymphocyte subsets, are required to define the potential use of TTV in immunological monitoring after allo-HSCT.

Persistent CMV infection after allogeneic hematopoietic stem cell transplantation in a CMV-seronegative donor-to-positive recipient constellation: Development of multidrug resistance in the absence of anti-viral cellular immunity.

We describe a case of persistent cytomegalovirus (CMV) infection after allogeneic hematopoietic stem cell transplantation (allo-HSCT) with discordant and high-risk (D-/R+) constellation of CMV serostatus. Despite the use of different and innovative antiviral strategies, viral replication could not be suppressed successfully promoting a protracted CMV colitis associated with severe gastrointestinal graft-versus-host disease (GI GVHD). We illustrate that the development of multidrug viral resistance, the failure to mount a CMV-specific cellular immune response, as confirmed by QuantiFERON(®)-CMV (Qiagen) assay, and the refractory GVHD requiring prolonged immunosuppression were the main factors contributing to persistent viral replication and the fulminant unfavorable course.

Tumor prevention in HPV8 transgenic mice by HPV8-E6 DNA vaccination.

The genus beta human papillomavirus 8 (HPV8) is involved in the development of cutaneous squamous cell carcinomas (SCCs) in individuals with epidermodysplasia verruciformis. Immunosuppressed transplant recipients are prone to harbor particularly high betapapillomavirus DNA loads, which may contribute to their highly increased risk of SCC. Tumor induction in HPV8 transgenic mice correlates with increased expression of viral oncogenes E6 and E2. In an attempt to prevent skin tumor development, we evaluated an HPV8-E6-DNA vaccine, which was able to stimulate a detectable HPV8-E6-specific cell-mediated immune response in 8/15 immunized mice. When skin of HPV8 transgenic mice was grafted onto non-transgenic littermates, the grafted HPV8 transgenic tissue was not rejected and papillomas started to grow within 14 days all over the transplant of 9/9 non-vaccinated and 7/15 not successfully vaccinated mice. In contrast, no papillomas developed in 6/8 successfully vaccinated mice. In the other two of these eight mice, a large ulcerative lesion developed within the initial papilloma growth or papilloma development was highly delayed. As the vaccine completely or partially prevented papilloma development without rejecting the transplanted HPV8 positive skin, the immune system appears to attack only keratinocytes with increased levels of E6 protein, which would give rise to papillomas.

The CD3-zeta chimeric antigen receptor overcomes TCR Hypo-responsiveness of human terminal late-stage T cells.

Adoptive therapy of malignant diseases with tumor-specific cytotoxic T cells showed remarkable efficacy in recent trials. Repetitive T cell receptor (TCR) engagement of target antigen, however, inevitably ends up in hypo-responsive cells with terminally differentiated KLRG-1(+) CD57(+) CD7(-) phenotype limiting their therapeutic efficacy. We here revealed that hypo-responsiveness of CMV-specific late-stage CD8(+) T cells is due to reduced TCR synapse formation compared to younger cells. Membrane anchoring of TCR components contributes to T cell hypo-responsiveness since dislocation of galectin-3 from the synapse by swainsonine restored both TCR synapse formation and T cell response. Transgenic expression of a CD3-zeta signaling chimeric antigen receptor (CAR) recovered hypo-responsive T cells to full effector functions indicating that the defect is restricted to TCR membrane components while synapse formation of the transgenic CAR was not blocked. CAR engineered late-stage T cells released cytokines and mediated redirected cytotoxicity as efficiently as younger effector T cells. Our data provide a rationale for TCR independent, CAR mediated activation in the adoptive cell therapy to avoid hypo-responsiveness of late-stage T cells upon repetitive antigen encounter.

Evolution of protease inhibitor resistance in the gag and pol genes of HIV subtype G isolates.

OBJECTIVES: To analyse HIV Gag cleavage site (CS) and non-CS mutations in HIV non-B isolates from patients failing antiretroviral therapy. PATIENTS AND METHODS: Twenty-one HIV isolates were obtained from patients infected with HIV subtype G during an outbreak in Russia 20 years ago. Most patients were failing antiretroviral therapy when genotyping was performed. RESULTS: HIV Gag CS mutations accumulated in protease inhibitor (PI)-resistant HIV isolates and were correlated with the presence of three or more PI resistance mutations. Only 1 of 11 HIV isolates carrying major protease mutations did not harbour treatment-associated CS mutations. Natural polymorphism 453T, often found in HIV non-B subtypes, seems to favour the selection of CS mutation 453I rather than treatment-associated CS mutation 453L. Resistance-associated non-CS mutations (123E and 200I) were also observed in PI-resistant clinical isolates. Non-CS mutations in the frameshift-regulating site, which controls the synthesis of Gag-Pol, did not affect frameshift efficiency in dual luciferase assays. Of note, one of four HIV isolates from patients failing PI therapies without protease mutations harboured Gag mutations associated with PI resistance (123E and 436R) and reverse transcriptase inhibitor mutations conferring resistance to the backbone drug. CONCLUSIONS: HIV Gag CS mutations commonly occurred in HIV isolates from patients failing PI therapies and natural polymorphisms at the same position influence their emergence. Non-CS mutations previously associated with PI resistance were also observed in clinical isolates. Gag mutations might indicate the evolution of PI resistance even in the absence of protease mutations.

Short communication: selection of thymidine analogue resistance mutational patterns in children infected from a common HIV type 1 subtype G source.

In HIV-1, thymidine analogue mutations (TAMs) cluster in one of two groups (215Y, 41L, 210W, or 215F, 219E/Q), representing two independent mutational patterns (T215Y and T215F cluster, respectively). The mechanisms by which these pathways are selected are not fully understood. To investigate possible factors driving the selection of the TAMs, we analyzed the TAM patterns with regard to the respective treatment, viral load, and HLA in 18 children all infected from a common source of HIV-1 clade G virus and initially treated with zidovudine. The HIV reverse transcriptase sequences of 14/18 children carried at least one TAM. At first sampling date, the T215Y-linked pattern was observed in five cases and the T215F cluster was seen in nine. During the follow-up period, three patients changed their patterns. Children treated with identical NRTI combinations at the first sampling date developed different pathways. Under AZT/d4T therapies, an association was found between the HLA B*13 (in combination with HLA DRB1*0701) and the mutation T215Y. The mutation T215Y reverted in three out of four patients who discontinued AZT/d4T treatment. We speculate that in the context of these subtype G viruses, the development of the T215Y mutation may be strongly disfavored whereas the presence of HLA B*13 may counteract this effect and permit its development.

Reactivation of an occult hepatitis B virus escape mutant in an anti-HBs positive, anti-HBc negative lymphoma patient.

BACKGROUND: Hepatitis B virus (HBV) often persists after resolution, but its replication is suppressed by antiviral T cells. Immunosuppressive treatment may lead to viral reactivation and severe hepatitis. Early antiviral therapy prevents reactivation but some occult HBV infections are not easily detectable. RESULTS: Here we describe a patient with a progressive non-Hodgkin lymphoma who had probably not been vaccinated against HBV and, before immunosuppression, showed antibodies (anti-HBs) against the viral surface antigen (HBsAg) as the only possible marker of occult HBV infection. Under immunosuppression he developed viremia (>10(8)copies/mL). The virus exhibited three S gene mutations (L109R, C137W, G145R) which led to false negative HBsAg results and diminished binding of vaccine-induced anti-HBs. CONCLUSIONS: Reliable screening and monitoring of severely immunosuppressed patients for HBV should include, in addition to anti-HBc and HBsAg, anti-HBs and sensitive HBV DNA assays. Furthermore, active vaccination or hepatitis B immune globulin may not protect against such mutants.

Presence of Epstein-Barr virus in esophageal cancer is restricted to tumor infiltrating lymphocytes.

As representatives of low and high incidence countries respectively, 72 esophageal squamous cell carcinomas and 40 adenocarcinomas from Germany, and 43 esophageal squamous cell carcinomas from Russia were tested for the presence of Epstein-Barr virus (EBV) DNA by PCR and in situ hybridization. Thirty-four percent of the squamous cell carcinomas (SCC) and 26% of the adenocarcinomas (AC) contained EBV DNA as detected by nested PCR. Quantitative analysis using real time PCR revealed one copy of the EBV genome per every 27-200,000 cells. EBER RNA in situ hybridization showed no EBV-specific transcripts in the nuclei of the tumor cells. However, EBER transcripts were expressed in the nuclei of tumor infiltrating lymphocytes in 7 SCC and 1 AC of 24 EBV DNA positive cases. The present data provide no evidence for the persistence of EBV in the tumor cells of esophageal cancer. In contrast to a previous report from Taiwan, EBV is unlikely to play a role in esophageal carcinogenesis.

Viral escape and T-cell immunity during ganciclovir treatment of cytomegalovirus infection: case report of a pancreatico-renal transplant recipient.

BACKGROUND: Pancreas-kidney transplant recipients are at high risk for cytomegalovirus (CMV) disease despite prophylactic ganciclovir therapy. Because the impact of antiviral therapy on anti-CMV immune reactions is unknown, CMV-specific T-cell subsets in primary and recurrent CMV infection were analyzed in a pancreas-kidney transplant case study. METHODS: Major histocompatibility complex class I tetramers were used to detect peripheral CMV pp65-specific CD8 T cells. Intracellular cytokine staining was used to determine the frequency of CMV-specific CD4 T cells. Conventional virologic parameters and routine laboratory parameters were monitored. For ganciclovir resistance testing, CMV-UL97 genotyping was performed. RESULTS: Despite prophylactic ganciclovir therapy, primary CMV infection induced in vivo expansion of activated CMV-specific CD8 T cells. Interestingly, viral dissemination during recurrent CMV disease was a result of partially ganciclovir-resistant CMV. Recovery after discontinued ganciclovir treatment was associated with the expansion of CMV-specific CD4 T cells. CONCLUSION: Immunologic monitoring may contribute to clinical management of recurrent CMV disease.

Antibodies to human papillomavirus 16 L1 virus-like particles as an independent prognostic marker in cervical cancer.

OBJECTIVE: Infection with high-risk human papillomavirus (HPV) types such as HPV-16 is a major risk factor for the development of cervical cancer. HPV-16 capsid antibodies are detectable in approximately 50% of patients with HPV-16 DNA-positive cervical cancer. We investigated the prognostic significance of HPV capsid antibodies for survival in patients with cervical cancer in comparison with conventional clinicopathologic features such as staging, histologic grading, histology, age, and treatment modality. STUDY DESIGN: Serum samples from 68 patients with cervical cancer and 65 healthy female control subjects were analyzed by enzyme-linked immunosorbent assay for HPV-specific immunoglobulin G (IgG) antibodies to baculovirus expressed HPV-6, HPV-11, HPV-16, and HPV-18 L1 virus-like particles (VLPs). RESULTS: HPV-16 L1 IgG antibodies were detectable in 6 of 65 (9%) of the control subjects and in 19 of 68 (28%) of the patients with cervical cancer (P =.007). In the subgroup of patients with HPV-16 DNA-positive cervical cancer (comprising 50% of the investigated samples), HPV-16 L1 antibodies were detected in 40%. HPV-16 L1 seropositivity was in univariate and multivariate analysis in addition to International Federation of Gynecology and Obstetrics stage, the only independent positive prognostic factor for overall survival (P =.01). CONCLUSION: Antibodies to HPV-16 L1 were found to be an independent prognostic factor for overall survival in patients with cervical cancer. Thus, HPV-16 infection may be involved not only in oncogenesis but also in tumor development and behavior.