Pp65 Antigen Research Articles

CTIM-29. TUMOR RESPONSES IN A RANDOMIZED PHASE IIB TRIAL OF A CMV VACCINE IMMUNOTHERAPEUTIC CANDIDATE (VBI-1901) IN RECURRENT GLIOBLASTOMAS

Abstract Cytomegalovirus (CMV) antigens have been reported in over 90% of GBMs. CD4+ and CD8+ T cells are most frequently directed against the gB and pp65 antigens, respectively, which are immunogenic targets in a CMV-based GBM immunotherapeutic. First-recurrent GBM patients were enrolled, with Karnofsky Performance Status of at least 70, to receive VBI-1901 (a gB/pp65 enveloped virus-like particle [eVLP]) adjuvanted with GM-CSF and given intradermally (NCT03382977). Patients were vaccinated with VBI-1901 every 4 weeks, with surveillance brain MRI scans every 6 weeks. Among 16 subjects receiving the highest dose of VBI-1901 in the Phase I/IIa portion of the study, the disease control rate was 44%, including 2 durable partial responses, which translated into a mOS of 12.9 months. Based on these results, the study was amended into a randomized study. Approximately 60 adult subjects (18 years or older) with first recurrence of WHO 2016 grade IV Glioblastoma, IDH-wildtype will be randomized at a 1:1 ratio to two open-label cohorts to receive: VBI-1901 or standard of care (SOC) treatment with lomustine or carmustine. Only subjects with a CD4/CD8 ratio ≥1 or a CD4 count of ≥ 400/uL at screening are eligible, as people with a preserved immune system are more likely to respond to immunization with VBI-1901 based on the initial phases of the study. As of May 15, 2024, 23 patients have been randomized across 6 sites. Among evaluable patients (n=13), disease control rates of 43% and 0% have been observed in the VBI-1901 and SOC arms, respectively, including 1 partial response in the VBI-1901 arm. Full enrollment (n=60) is anticipated by the end of the year across 11 sites, and updated tumor response data and select biomarkers associated previously with tumor responses will be presented.

CTIM-01. OUTCOMES AND IMMUNE RESPONSES AFTER PEPTIDE VACCINATION TARGETING HUMAN CYTOMEGALOVIRUS ANTIGEN PP65 IN CHILDREN AND YOUNG ADULTS WITH RECURRENT HIGH-GRADE GLIOMA AND MEDULLOBLASTOMA: RESULTS OF A PHASE 1 TRIAL

Abstract INTRODUCTION The human cytomegalovirus (CMV) antigen pp65 is ubiquitously expressed in high-grade glioma (HGG) and medulloblastoma but not in adjacent brain. The primary objective of this phase I trial (NCT03299309) was to assess the safety and feasibility of a novel peptide vaccine targeting pp65 (PEP-CMV) in children/young adults with recurrent medulloblastoma and HGG. METHODS PEP-CMV is comprised of a synthetic long peptide of 26 amino acids and is administered as an emulsion in Montanide ISA 51. Patients receive a 5-day course of temozolomide to induce lymphopenia, tetanus/diphtheria toxoid site preconditioning to promote dendritic cell migration, then PEP-CMV intradermally in the groin every two weeks for 3 doses, then monthly. RESULTS 42 patients were enrolled. The mean age was 23.2 ± 9.3 years. 55% of patients were male. The median KPS was 80. The median number of prior chemotherapy regimens was 4 (range 1-15). Of the 38 patients who received PEP-CMV, the maximum grade adverse events (AE) possibly, probably, or definitely related to PEP-CMV were: 17 (45%) Grade 1 AEs, 16 (42%) Grade 2 AEs, 2 (5%) Grade 3 AEs (encephalopathy and pyramidal tract syndrome), and one (3%) Grade 4 AE (cerebral edema). One patient developed cytokine release syndrome (Grade 2). The median PFS was 2.5 months (95% CI:2.2, 3.2) and median OS was 6.4 months (95% CI:3.4, 7.9). The 12-month OS was 26.6% (95% CI:14, 41.1%). Of the 21 patients with evaluable immune monitoring data (received ≥3 vaccines/known CMV status), T cell reactivity on IFNγ pp65 ELISpot was increased after PEP-CMV delivery (median 2 vs 22, P=0.01). PFS and OS were significantly better in patients with low levels of CD56brightCD16dim phenotype NK cells: P=0.029 and P=0.047, respectively. Patients with naïve CD8+ counts over the median had significantly better PFS (P=0.006). CONCLUSIONS PEP-CMV is well-tolerated and elicits an antigen-specific immune response in heavily pretreated, multiply recurrent patients with a 12-month OS of 26.6%.

Outcomes and immune response after peptide vaccination targeting human cytomegalovirus antigen pp65 in children and young adults with recurrent high-grade glioma and medulloblastoma.

Outcomes and immune response after peptide vaccination targeting human cytomegalovirus antigen pp65 in children and young adults with recurrent high-grade glioma and medulloblastoma.

Immunomonitoring via ELISPOT Assay Reveals Attenuated T-Cell Immunity to CMV in Immunocompromised Liver-Transplant Patients.

Assessing immune responses to cytomegalovirus (CMV) after liver transplant in patients on immunosuppressive therapy remains challenging. In this study, employing ELISPOT assays, 52 liver-transplant recipients were evaluated for antiviral T-cell activity in peripheral blood mononuclear cells (PBMCs), measuring interferon-γ (IFN-γ) secretion upon stimulation with CMV-specific peptides (CMV peptide pool, CMV IE-1, and pp65 antigens). Parameters such as stimulation index, mean spot size, and mean spot count were measured. The study found that heightened immunosuppression, especially with prednisolone in triple therapy, significantly dampened CMV-specific immune responses. This was demonstrated by decreased IFN-γ production by CMV-specific T-cells (CMV peptide pool: p = 0.036; OR = 0.065 [95% CI: 0.005-0.840], pp65 antigen: p = 0.026; OR = 0.048 [95% CI: 0.003-0.699]). Increased immunosuppression correlated with reduced IFN-γ secretion per cell, reflected in smaller mean spot sizes for the CMV peptide pool (p = 0.019). Notably, shorter post-transplant intervals correlated with diminished antiviral T-cell IFN-γ release at two years (CMV peptide pool: p = 0.019; IE antigen: p = 0.010) and five years (CMV peptide pool: p = 0.0001; IE antigen: p = 0.002; pp65 antigen: p = 0.047), as did advancing age (pp65 antigen: p = 0.016, OR = 0.932, 95% CI: 0.881-0.987). Patients with undetectable CMV antigens had a notably higher risk of CMV reactivation within six months from blood collection, closely linked with triple immunosuppression and prednisolone use. These findings highlight the intricate interplay between immunosuppression, immune response dynamics, and CMV reactivation risk, emphasizing the necessity for tailored immunosuppressive strategies to mitigate CMV reactivation in liver-transplant recipients. It can be concluded that, particularly in the early months post-transplantation, the use of prednisolone as a third immunosuppressant should be critically reconsidered. Additionally, the use of prophylactic antiviral therapy effective against CMV in this context holds significant importance.

Abstract LB117: Virus-inspired Particles (ViPs) harnessing cytomegalovirus immune memory for local and systemic cancer immunotherapy

Abstract VerImmune is pioneering a First-in-Class treatment known as “Anti-tumor Immune Redirection” (AIR). This approach repurposes a patient’s pre-existing anti-viral or childhood vaccine immunity towards tumor cells for targeted destruction. VerImmune’s lead product, VERI-101, leverages pre-existing CD8+ T-cell immune memory acquired from past human cytomegalovirus (HCMV) infections. VERI-101 consists of VerImmune’s proprietary platform technology known as ViPs (Virus-inspired Particles) that are conjugated on their surface with a CD8+ T cell viral peptide antigen. ViPs are based on a modified mouse papillomavirus capsid protein of which 60 copies self-assemble into a 20-30nm T=1 icosahedral structure. In the case of VERI-101, the ViP is conjugated with an HLA-A*0201 restricted peptide epitope (NLVPMVATV, [NLV]), derived from the CMV pp65 antigen with an upstream furin protease cleavage site. The mechanism of action of AIR-ViPs including VERI-101 has been previously presented and involves three steps. Briefly, (1) VERI-101 targets the surface of the tumor cells and (2) enables the presentation of the CMV viral epitopes on their surface MHC, together (3) stimulating a recall of the pre-existing CMV memory response to attack the cancer cells (Dorrier et al., 2023 AACR). Here, we sought to study the in vivo anti-tumor efficacy of AIR-ViPs via both intra-tumoral and systemic treatment routes in two murine tumor models with pre-existing murine CMV (MCMV) immunity: (1) MC38 subcutaneous tumors for local and (2) B16.F10 lung metastasis for systemic treatments. As human CMV is species-specific, instead of VERI-101, a surrogate product, VERI-003 which redirects MCMV immunity, was tested as a single agent or in combination with anti-PD-1 in these tumor models. Our intra-tumoral studies with VERI-003 alone showed a dose-dependent anti-tumor effect (from 5 to 100ug) compared to no treatment controls. Surprisingly, no dose dependence was observed across the same three dose levels when combined with anti-PD-1, although significantly better anti-tumor effects occurred leading to complete tumor clearances in 50% of mice. Statistically significant prolonged survival was observed in most treated groups compared to controls. Our systemic studies with VERI-003 were consistent with our intratumoral studies in showing statistically significant single agent anti-tumor efficacy against B16.F10 lung metastases compared to no treatment. Anti-tumor effects of anti-PD-1 and combination were also observed. Additional studies are on-going to assess dose dependence and analysis of PK/PD effects. Taken together, our initial in vivo results demonstrate the tumor antigen-agnostic therapeutic potential of AIR and our AIR-ViP technology as a novel class of immuno-therapeutic drugs that could be used either as a monotherapy or in combination with checkpoint inhibitors. Citation Format: Cayce Dorrier, Felagot Abebe, Olivia Pak, Emily De-Bodene, Joshua Wang. Virus-inspired Particles (ViPs) harnessing cytomegalovirus immune memory for local and systemic cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB117.

Clinical Significance of Cytomegalovirus Reactivation in Patients With Plasma Cell Dyscrasia Who Were Treated With Anti-CD38 Monoclonal Antibody: A Retrospective Analysis in a Single Institution

IntroductionAnti-CD38 monoclonal antibodies (mAbs) have improved the prognosis of patients with plasma cell dyscrasia (PCD), but are also associated with increased infectious adverse events. Cytomegalovirus (CMV) is a common latent pathogen that is reactivated in immunocompromised individuals. Although CMV reactivation has mostly been reported after high-dose chemotherapy followed by stem cell transplantation in patients with PCD, cases of reactivation during anti-CD38 mAb therapy have been reported recently. Due to limited studies, we aimed to determine the frequency and impact of CMV reactivation during anti-CD38 mAb therapy. Patients and MethodsThis retrospective analysis included 154 consecutive patients with PCD who were treated with anti-CD38 mAbs at a single institution. ResultsSeventy-six patients were evaluated for CMV reactivation by CMV pp65 antigen testing, and 29 (38%) patients, including nine with newly diagnosed PCD, showed positive results. Patients who tested positive for the CMV pp65 antigen had substantially lower serum albumin levels than those who tested negative. However, the two groups showed no marked difference in the concurrent anti-PCD medications or baseline absolute lymphocyte count. Although most patients showing positive results in the CMV pp65 antigen test had mild or no symptoms, with fever being the most common symptom, some patients developed CMV end-organ disease. In addition, CMV reactivation interfered with the course of anti-PCD treatment in most patients, necessitating dose reductions, delays, and discontinuation of chemotherapy. ConclusionThis study provides an overview of the clinical impact of CMV reactivation in patients with PCD treated with anti-CD38 mAb-containing regimens.

Circular mRNA-based TCR-T offers a safe and effective therapeutic strategy for treatment of cytomegalovirus infection

Circular mRNA (cmRNA) is particular useful due to its high resistance to degradation by exonucleases, resulting in greater stability and protein expression compared to linear mRNA. Tcell receptor (TCR)-engineered Tcells (TCR-T) represent a promising means of treating viral infections and cancer. This study aimed to evaluate the feasibility and efficacy of cmRNA in antigen-specific-TCR discovery and TCR-T therapy. Using human cytomegalovirus (CMV) pp65 antigen as a model, we found that the expansion of pp65-responsive Tcells was induced more effectively by monocyte-derived dendritic cells transfected with pp65-encoding cmRNA compared with linear mRNA. Subsequently, we developed cmRNA-transduced pp65-TCR-T (cm-pp65-TCR-T) that specifically targets the CMV-pp65 epitope. Our results showed that pp65-TCR could be expressed on primary Tcells for more than 7days. Moreover, both invitro killing and invivo CDX models demonstrated that cm-pp65-TCR-T cells specifically and persistently kill pp65-and HLA-expressing tumor cells, significantly prolonging the survival of mice. Collectively, our results demonstrated that cmRNA can be used as a more effective technical approach for antigen-specific TCR isolation and identification, and cm-pp65-TCR-T may provide a safe, non-viral, non-integrated therapeutic approach for controlling CMV infection, particularly in patients who have undergone allogeneic hematopoietic stem cell transplantation.

CTIM-27. PERIPHERAL BIOMARKER ANALYSIS OF T CELL-MEDIATED COLLAGEN REMODELING CORRELATES WITH TUMOR RESPONSES IN A PHASE IIA TRIAL OF VACCINE IMMUNOTHERAPEUTIC CANDIDATE (VBI-1901)

Abstract Cytomegalovirus (CMV) antigens have been reported in over 90% of GBMs. CD4+ and CD8+ T cells are most frequently directed against the gB and pp65 antigens, respectively, which are immunogenic targets in a CMV-based GBM immunotherapeutic. First-recurrent GBM patients with KPS³70 received VBI-1901 (a gB/pp65 enveloped virus-like particle [eVLP]) adjuvanted with GM-CSF intradermally (NCT03382977). Patients received VBI-1901 q4w, with serologic immune-monitoring q2w after each vaccination and surveillance brain MRI scans q6w. Among 16 patients treated with the highest (10µg dose) of VBI-1901, the mOS was 12.9 months with a 12-month OS rate of 62.5%. T cell-mediated collagen remodeling is necessary for T cell migration and activity. The peripheral C4G biomarker targets granzyme-B-cleaved type IV collagen, which is associated with T cell infiltration and has previously been shown to potentially identify recurrent GBM patients responding to nivolumab and bevacizumab. Here, C4G was measured in available plasma samples from patients with progressive disease (PD) (n = 9), stable disease (SD) (n = 5) or partial tumor responses (PR) (n = 2) prior to (baseline) and after (cycle 1-4) treatment with VBI-1901. Although numerically lower in patients with PD, there were no differences in baseline levels of C4G among the groups. However, there were statistically significant increases in C4G levels in the 2 patients with PR both after 1st/2nd doses (p = 0.0324) and after 3rd/4th doses (p = 0.0027) of treatment (1st/2nd doses; PD: 7.6ng/mL, SD: 10.0ng/mL, PR: 21.0ng/mL. 3rd/4th doses; PD: 6.1ng/mL, SD: 8.5ng/mL, PR: 16.8ng/mL). On-going analyses are evaluating additional peripheral markers of tumor microenvironment (TME) remodeling. Evaluating the balance of immune infiltration vs. tumor cell growth in patients with brain tumors is challenging and these data suggest a means of assessing this dynamic using a peripheral biomarker. These data may be particularly useful in discerning pseudoprogression associated with T cell infiltration from tumor progression.

Human T cells loaded with superparamagnetic iron oxide nanoparticles retain antigen-specific TCR functionality.

Immunotherapy of cancer is an emerging field with the potential to improve long-term survival. Thus far, adoptive transfer of tumor-specific T cells represents an effective treatment option for tumors of the hematological system such as lymphoma, leukemia or myeloma. However, in solid tumors, treatment efficacy is low owing to the immunosuppressive microenvironment, on-target/off-tumor toxicity, limited extravasation out of the blood vessel, or ineffective trafficking of T cells into the tumor region. Superparamagnetic iron oxide nanoparticles (SPIONs) can make cells magnetically controllable for the site-specific enrichment. In this study, we investigated the influence of SPION-loading on primary human T cells for the magnetically targeted adoptive T cell therapy. For this, we analyzed cellular mechanics and the T cell response after stimulation via an exogenous T cell receptor (TCR) specific for the melanoma antigen MelanA or the endogenous TCR specific for the cytomegalovirus antigen pp65 and compared them to T cells that had not received SPIONs. SPION-loading of human T cells showed no influence on cellular mechanics, therefore retaining their ability to deform to external pressure. Additionally, SPION-loading did not impair the T cell proliferation, expression of activation markers, cytokine secretion, and tumor cell killing after antigen-specific activation mediated by the TCR. In summary, we demonstrated that SPION-loading of T cells did not affect cellular mechanics or the functionality of the endogenous or an exogenous TCR, which allows future approaches using SPIONs for the magnetically enrichment of T cells in solid tumors.

Impact of Baseline and Week 2 and Week 4 Posttransplant CMV Cell-Mediated Immunity on Risk of CMV Infections and Mortality in Recipients of Allogeneic Hematopoietic Cell Transplant.

Cytomegalovirus (CMV) infection is a common opportunistic infection after allogeneic hematopoietic cell transplant (alloHCT). We explored whether a change in CMV cell-mediated immunity during the first month after transplant predicts the risk of development of CMV infection and all-cause mortality. This follow-up analysis is based on data from the REACT study, a multicenter prospective observational study of recipients of alloHCT who were CMV-seropositive. Production of interferon γ following ex vivo stimulation with CMV antigens IE1 (immediate early 1) and pp65 (phosphoprotein 65) was assessed by CMV ELISPOT assay at baseline and 2 and 4 weeks after transplant. Clinically significant CMV infection (CS-CMVi) was defined as CMV viremia and/or disease necessitating antiviral therapy. We evaluated the impact of CMV CMI changes on the risk of CS-CMVi and post transplant mortality. The analysis included 226 recipients of alloHCT with CMV cell-mediated immunity data at baseline and 2 and/or 4 weeks after transplant. CS-CMVi occurred in 64 patients (28%). On Cox regression analyses, independent predictors of CS-CMVi included a negative Δ change from baseline to week 2 of pp65 spot counts (hazard ratio, 3.65 [95% CI, 1.65-8.04]; P = .001) to week 4 of IE1 spot counts (hazard ratio, 2.79 [95% CI, 1.46-5.35]; P = .002), anti-thymocyte globulin conditioning regimen, type of transplant, female sex, and corticosteroid use. Kaplan-Meir analysis showed a significant association of a negative IE1 change from baseline to week 4 and increased all-cause mortality after transplant (log rank test = 0.041). A decrease in CMV-specific T-cell responses during the first month after transplant may predict CS-CMVi and is associated with all-cause mortality in recipients of alloHCT.

The influence of HLA A, B, C, DR alleles and HLA haplotypes on cytomegalovirus-specific cell mediated immunity in seropositive Korean kidney transplant candidates.

We evaluated the effect of specific HLA alleles and haplotypes on cytomegalovirus (CMV)-specific cell mediated immunity (CMI) in kidney transplant (KT) candidates. CMV-specific ELISPOT against pp65 and IE-1 antigens (hereafter referred to as pp65 and IE-1, respectively) was performed in 229 seropositive KT candidates. We analyzed the results related to 44 selected HLA alleles (9 HLA-A, 15 HLA-B, 9 HLA-C, and 11 HLA-DR) and 13 HLA haplotypes commonly found in study participants. The pp65 and IE-1 results in 229 seropositive candidates were 227.5 (114.5-471.5) and 41.0 (8.8-185.8) (median [interquartile range]) spots/2 × 105 PBMCs, respectively. The pp65 and IE-1 results showed significant differences between candidates with different HLA alleles (A*02 vs. A*26 [p = 0.016], A*24 vs. A*30 [p = 0.031], B*07 vs. B*46 [p = 0.005], B*54 vs. B*35 [p = 0.041], B*54 vs. B*44 [p = 0.018], B*54 vs. B*51 [p = 0.025], and C*06 vs. C*14 [p = 0.034]). HLA-A*02 and B*54 were associated with increased pp65 and IE-1 results, respectively (p = 0.005 and p < 0.001, respectively). In contrast, the HLA-A*26 and B*46 alleles were associated with a decreased pp65 response, whereas the A*30 allele was associated with a decreased IE-1 response (p < 0.05). The pp65 results correlated with the HLA-A allele frequencies (R = 0.7546, p = 0.019) and the IE-1 results correlated with the HLA-C allele frequencies of the study participants (R = 0.7882, p = 0.012). Among 13 haplotypes, HLA-A*30 ~ B*13 ~ C*06 ~ DRB1*07 showed decreased CMV-CMIs compared to the other HLA haplotypes, probably due to a combination of HLA alleles associated with lower CMV-CMIs. Our results demonstrated that CMV-specific CMIs may be influenced by the HLA allele as well as the HLA haplotype. To better predict CMV reactivation, it is important to estimate risk in the context of HLA allele and haplotype information.

Abstract 690: Modification of mRNA-encoded HPV16 antigens to include endolysosomal trafficking domains drives cross-presentation and results in superior in vivo and ex vivo antigen-specific responses

Abstract Many cancer immunotherapies require the strong activation of the T-cell mediated adaptive immune response to elicit their full anti-tumor potential. Antigen processing and presentation is a complex, multistep process that can be significantly enhanced via antigen modification, leading to better antigen-specific immune responses. Using mRNA, we can express exogenous antigens inside the cell and facilitate the subcellular localization of these antigens to enhance antigen processing and presentation. In this work, we introduced endolysosomal trafficking domains from CD1d molecules to drive subcellular localization of antigenic proteins to the late endosomes (LE) to enhance class I and class II antigen loading. CD1d are molecules that naturally traffic between the membrane and the late endosomes, where antigens are processed and loaded to both class II and class I molecules in a TAP-independent manner. We compared the immunogenicity of a novel endolysosomal trafficking domain, human CD1d, coupled to a chimeric HPV16 E6 and E7 (HPV16E6E7-hCD1d) antigen to antigen designs intended to be either a) secreted (Sec-HPV16E6E7), b) retained in the endoplasmic reticulum (ER) by an ER retention signal KDEL (HPV16E6E7-KDEL) or c) unmodified antigen sequence (HPV16E6E7). To ensure intracellular translation and expression, all antigens were encoded and delivered as mRNAs. The immunogenicity of each antigen design was assessed in HPV16 positive cervical intraepithelial neoplasia (CIN) donor derived PBMCs and healthy controls. Using confocal microscopy, we observed that the hCD1d trafficking domain directed the translated E6E7 protein into late endosomes, which was not observed for any of the other antigen designs. Importantly, antigen localization to late endosomes resulted in significantly improved immunogenicity in vitro as well as in vivo. We observed a consistent increase in numbers of activated antigen-specific T cells across multiple CIN donor PBMCs treated with HPV16E6E7-hCD1d compared to the other 3 designs, as measured by Interferon gamma secretion, surface marker expression, and T-cell proliferation. To assess the generalizability of this approach, we also compared immunogenicity in human donor PBMCs using the human cytomegalovirus antigen pp65, and observed similar results. Lastly, the immunogenicity of each antigen design was tested in vivo by intramuscular injection of nanoparticle formulated mRNAs in C56/Bl6 mice, and inclusion of the murine analogue (mCD1d) resulted in significantly increased generation of HPV16-specific T cell responses. Based on these findings, we are developing a novel mRNA-based cancer therapeutic that includes the addition of hCD1d trafficking domain in the HPV16-specific antigen component. Citation Format: Weiqun Liu, Daniel Fernandez, Colin J. McKinlay, Daniel O. Frimannsson, Meredith L. Leong, W. Martin Kast, Samuel Deutsch, Ole Audun W. Haabeth. Modification of mRNA-encoded HPV16 antigens to include endolysosomal trafficking domains drives cross-presentation and results in superior in vivo and ex vivo antigen-specific responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 690.

TEAM: A MULTIPLE TESTING ALGORITHM ON THE AGGREGATION TREE FOR FLOW CYTOMETRY ANALYSIS.

In immunology studies, flow cytometry is a commonly used multivariate single-cell assay. One key goal in flow cytometry analysis is to detect the immune cells responsive to certain stimuli. Statistically, this problem can be translated into comparing two protein expression probability density functions (pdfs) before and after the stimulus; the goal is to pinpoint the regions where these two pdfs differ. Further screening of these differential regions can be performed to identify enriched sets of responsive cells. In this paper, we model identifying differential density regions as a multiple testing problem. First, we partition the sample space into small bins. In each bin, we form a hypothesis to test the existence of differential pdfs. Second, we develop a novel multiple testing method, called TEAM (Testing on the Aggregation tree Method), to identify those bins that harbor differential pdfs while controlling the false discovery rate (FDR) under the desired level. TEAM embeds the testing procedure into an aggregation tree to test from fine- to coarse-resolution. The procedure achieves the statistical goal of pinpointing density differences to the smallest possible regions. TEAM is computationally efficient, capable of analyzing large flow cytometry data sets in much shorter time compared with competing methods. We applied TEAM and competing methods on a flow cytometry data set to identify T cells responsive to the cytomegalovirus (CMV)-pp65 antigen stimulation. With additional downstream screening, TEAM successfully identified enriched sets containing monofunctional, bifunctional, and polyfunctional T cells. Competing methods either did not finish in a reasonable time frame or provided less interpretable results. Numerical simulations and theoretical justifications demonstrate that TEAM has asymptotically valid, powerful, and robust performance. Overall, TEAM is a computationally efficient and statistically powerful algorithm that can yield meaningful biological insights in flow cytometry studies.

Different recovery patterns of CMV-specific and WT1-specific T cells in patients with acute myeloid leukemia undergoing allogeneic hematopoietic cell transplantation: Impact of CMV infection and leukemia relapse.

In allogeneic hematopoietic cell transplantation (allo-HSCT), both virus-specific T cells and leukemia-specific T cells need to be reconstituted to protect patients from virus infections and primary disease relapse. Cytomegalovirus (CMV) infection remains an important cause of morbidity and mortality after allo-HSCT. Emerging data indicate that CMV reactivation is associated with reduced risk of leukemia relapse in patients with acute myeloid leukemia (AML) undergoing allo-HSCT. In a cohort of 24 WT1+ AML patients during the first year following HSCT, CMV specific CD8+ T cells (CMV-CTL) reconstituted much faster than WT1-specific CD8+ T cell (WT1-CTL) after allo-SCT. Moreover, CMV-CTL expressed lower levels of exhaustion markers and were more functional as identified by production of IFN-γ/TNF-α and expression of Eomes/T-bet. Interestingly, our patients with CMV reactivation presented higher frequency of CMV-CTL, lower levels of Eomes+T-bet- and higher levels of Eomes+T-bet+ expression in response to WT1 and CMV pp65 antigen during the first year after transplantation as compared to patients without CMV reactivation. Kinetics of CMV-CTL and WT1-CTL after transplantation might be associated with measurable residual disease and later leukemia relapse. Our results support that CMV reactivation, aside from the CMV-CTL reconstitution, could influence WT1-CTL reconstitution after allo-HSCT, thus potentially contributing to the remission/relapse of AML.

Immune checkpoint analysis of T-cell responses to pp65 and IE-1 antigens in end-stage lung diseases.

Lung transplant (LTX) patients are at high risk of cytomegalovirus (CMV) infection, which is often associated with high mortality and morbidity. Reactivation of CMV causes cell injury due to the cytopathic effect of viral replication and triggering of T cell immunity. The aim of this study was to compare expression of immune checkpoints (ICs) (PD-1, CTLA-4, LAG-3 and TIGIT) in CD4, CD8 and CD56 and activation markers CD137, CD154 and CD69 of end-stage patients awaiting lung transplant. Eighteen pre-LTX positive for anti-CMV IgG titres and 18 healthy subjects were enrolled. IC and activation markers have been evaluated through flow cytometric analysis in HC and pre-LTX patients. Reactive (QF+) and unreactive (QF-) patients were stratified according to QuantiFERON-CMV assays. ICs' and activation markers' expression were determined before and after in vitro stimulation with pp-65 and IE-1 antigens. Lower expression of PD-1 was observed in CD4 and CD8 cells of pre-LTX patients than controls, whereas CTLA4 appeared upregulated in CD56 and CD8 cells. TIGIT is increased on the surface of CD4, CD8 and NK cells after peptide stimulation in QF-negative patients and PD-1 is only downregulated after stimulation in the QF-positive patients. This study provides new evidence of immune dysregulation in patients with end-stage lung disorders, particularly in relation to immune checkpoint cell biology. The change in QF+ mostly happens on cytotoxic cells NK and CD8, while the changes in QF- were observed in adaptive immune cells, including CD4 and CD8.

Risk factors associated with cytomegalovirus reactivation in patients receiving immunosuppressive therapy for rheumatic diseases: a retrospective study

Immunosuppressive treatment is a common cause of cytomegalovirus (CMV) reactivation. However, there is no consensus regarding the risk factors for CMV reactivation in rheumatic diseases. Therefore, this study aimed to elucidate the risk factors associated with CMV reactivation. We retrospectively collected the data of 472 patients with rheumatic diseases whose CMV pp65 antigen (C7-HRP) titer was measured. We divided the patients into those with and those without C7-HRP. We retrospectively collected data on age, sex, primary condition and organ involvement, and blood test results. We also investigated the use of immunosuppressants and the maximum and cumulative doses of prednisolone (PSL). We performed univariate and multivariate analyses to identify risk factors for CMV reactivation. Multivariate analysis showed that higher age (71.2 vs. 64.4 years, p = 0.0022), hypoalbuminemia (2.9 vs. 3.4 g/dL, p = 0.0104), higher creatinine level (1.2 vs. 0.9 mg/dL, p = 0.0026), cyclosporine use (8.2 vs. 3.6%, p = 0.0101), and higher maximum (552.4 vs. 243.3 mg, p < 0.0001) and cumulative (2785.9 vs. 1330.5 mg, p < 0.0001) doses of PSL were associated with CMV reactivation. Older age, hypoalbuminemia, higher creatinine level, cyclosporine use, and higher maximum and cumulative doses of PSL were significant risk factors for CMV reactivation in rheumatic diseases.

CTIM-14. COMPREHENSIVE BIOMARKER ANALYSIS OF RESPONDERS AND NON-RESPONDERS IN A PHASE IIA TRIAL OF A CMV VACCINE IMMUNOTHERAPEUTIC CANDIDATE (VBI-1901)

Abstract Cytomegalovirus (CMV) antigens have been reported in over 90% of GBMs. CD4+ and CD8+ T cells are most frequently directed against the gB and pp65 antigens, respectively, which are immunogenic targets in a CMV-based GBM immunotherapeutic. First-recurrent GBM patients were enrolled, with Karnofsky Performance Status of at least 70, to receive VBI-1901 (a gB/pp65 enveloped virus-like particle [eVLP]) adjuvanted with GM-CSF and given intradermally (NCT03382977). Patients were vaccinated with VBI-1901 every 4 weeks, with serologic immune-monitoring 2 weeks after each vaccination and surveillance brain MRI scans every 6 weeks. Sixteen patients (8 women, 8 men) with a median age of 55 (33-67 yrs) were enrolled into the GM-CSF arm. Seven tumor responses, including two durable partial responses, were observed which led to an 18-month OS rate of 25% and mOS of 56 weeks. Detailed immunological testing was performed to identify potential correlations at baseline and after treatment between biomarkers and tumor responses. Analyses included class I and II HLA typing, modulation of plasma cytokine and chemokine responses, boosting of CMV-specific antibody and IFN-g ELISPOT responses, and modulation of CMV-specific CD4 Tem cells, of which only differential trafficking of the CD4 Tem cells appears to distinguish patients with tumor responses from those with tumor progression. Single cell, RNAseq is underway in an attempt to understand mechanistic differences in these immune cell populations in Tumor Responders vs. Non-Responders. The U.S. FDA granted Fast Track Designation to VBI-1901 adjuvanted with GM-CSF in first-recurrent GBM patients, and an expansion of the ongoing trial with this formulation in this patient population, with the addition of randomization with a contemporaneous control arm, is anticipated to begin in Q3 2022.

Limited T-Cell-Stimulating Effect of Cytochalasin-B-Induced Membrane Vesicles Isolated from Artificial Antigen-Presenting Cells.

Artificial antigen-presenting cells (aAPCs) that stably express particular HLA and co-stimulatory molecules by gene transfer have been developed to effectively stimulate T cells. To investigate whether cytochalsin-B-induced membrane vesicles derived from aAPCs (AP-CIMVs) have similar antigen-presenting functions as a cell-free system, T cell responses to different types of antigen presentation were measured using Jurkat reporter cells. First, the aggregation of AP-CIMV, which affects the measurement of function, was inhibited by nuclease treatment to produce uniform AP-CIMVs. The Green fluorescent protein (GFP) expression in Jurkat reporter cells was induced in a dose-dependent manner in groups stimulated with anti-CD3 antibody-coated AP-CIMVs and aAPCs, and anti-CD3/CD28 Dynabead. When Jurkat reporter cells expressing specific T cell receptors were stimulated by AP-CIMVs and aAPCs loaded with CMV pp65 peptide, AP-CIMVs showed similar stimulatory effects to that by aAPC. However, when these Jurkat reporter cells were stimulated by aAPCs endogenously expressing CMV pp65 antigen and their AP-CIMVs, the GFP expression rate by AP-CIMVs was 8.4%, which was significantly lower than 53.2% by aAPCs. Although this study showed a limited T-cell-stimulating effect of AP-CIMVs on endogenously processed antigen presentation, these results provide useful information for the development of improved cell-free systems for T cell stimulation in the future.

Cytomegalovirus infection of brain tumors and CMV immunotherapy

Objective. The article presents the literature of the last ten years and the results of our own research on the importance of cytomegalovirus (CMV) in the development of brain tumors, especially glioblastoma and medulloblastoma. Two alternative views are discussed - the pros and cons of the role of the virus in the induction and stimulation of tumor growth.Materials and methods. 256 samples of biotic material of tissues of various brain tumors were studied. Among them are histologically diagnosed: in 123 cases glial tumors of various grade of malignancy, in 51 cases meningiomas, in 25 cases medulloblastomas, in 16 cases oligodendroastrocytomas of the second grade of malignancy, in 14 cases metastatic tumors. Tumor fragments were obtained from biopsy material 1.5-2.0 hours after surgical removal. To detect the presence of CMV in the tumor tissue real-time polymerase chain reaction (PCR) using "DNA sorb A and B" kits was performed, the company "Amplisens" (Russia), according to the manufacturer’s instructions and BioRal device (USA) with standard DNA detection kits of CMV "DNA Technology" (Russia). Cytological imprints on slides were also made from tumor tissue fragments, which were examined by indirect immunofluorescence method with monoclonal antibodies to CMV pP-65 protein using the "MonoScan CMV" kit.Results. The frequency of detection of CMV antigen or its DNA in brain tissue depends on the research method - the immunofluorescence method detects pP-65 antigen by monoclonal antibodies 2-2.5 times more often than the PCR method of CMV in tumor tissue. In the tissue of different histogenesis of brain tumors both the pP-65 antigen and CMV DNA are detected with different frequencies. CMV was most often detected in tumors of glial origin and medulloblastomas. No CMV DNA was detected in the peripheral blood of patients with brain tumors at the time of admission for examination and surgical treatment, indicating an earlier contamination of the tumor focus with this virus. Data on the mechanisms of CMV induction and stimulation of tumor growth by activating cell proliferation, including nerve stem cells, are presented. Works using specific antiviral therapy and CMV specific cell immunotherapy in the treatment of gliomas have been analyzed in detail.Conclusions. The paper concludes on the important clinical and prognostic value of determining CMV infection in brain tumors and indicates the need for CMV viral and cellular immunotherapy in the combined treatment of malignant brain tumors.

Characterization of NKG2-A/-C, Kir and CD57 on NK Cells Stimulated with pp65 and IE-1 Antigens in Patients Awaiting Lung Transplant

Introduction: Cytomegalovirus (CMV) is the leading opportunistic infection in lung transplant (LTx) recipients. CMV is associated with graft failure and decreased survival. Recently, new antiviral therapies have been proposed. The present study aimed to investigate NK and T cell subsets of patients awaiting LTx. We analyzed the cellular populations between reactive and non-reactive QuantiFERON (QF) CMV patients for the prediction of immunological response to infection. Methods: Seventeen pre-LTx patients and 15 healthy controls (HC) have been enrolled. QF and IFN-γ ELISA assay detections were applied. NK cell subsets and T cell and proliferation assay were detected before and after stimulation with pp-65 and IE-1 CMV antigens after stratification as QF+ and QF−. Furthermore, we quantified the serum concentrations of NK− and T-related cytokines by bead-based multiplex analysis. Results: CD56brCD16lowNKG2A+KIR+ resulted in the best discriminatory cellular subsets between pre-LTx and HC. Discrepancies emerged between serology and QF assay. Better proliferative capability emerged from patients who were QF+, in particular in CD8 and CD25-activated cells. CD56brCD16low, adaptive/memory-like NK and CD8Teff were highly increased only in QF+ patients. Conclusions: QF more than serology is useful in the detection of patients able to respond to viral infection. This study provides new insights in terms of immunological responses to CMV in pre-LTX patients, particularly in NK and T cells biology.