Overlapping Peptide Libraries Research Articles

Identification of broad-spectrum B-cell and T-cell epitopes of H9 subtype avian influenza virus HA protein using polypeptide scanning1

The H9N2 subtype avian influenza virus (AIV) hemagglutinin (HA) protein is a major immunogen in which HA1 is a genetic variant and HA2 is relatively conserved. Identifying broad-spectrum antigen epitopes targeting HA1 is crucial for vaccine design and detection. Based on the phylogenetic and serological analyses, we identified 2 antigenic groups and 3 representative viruses: A/chicken/Jiangsu/JY040218C/2019, A/pigeon/Jiangsu/JY020616/2019, and A/chicken/Jiangsu/WX090312/2018. An overlapping peptide library was synthesized using HA1 amino acid sequences of the viruses as templates. Through peptide scanning of the sera against different strains of H9N2 subtype AIV, we identified peptides from 4 regions (H9-2/3, H9-20/21, H9-26, and H9-29/30/31) that demonstrated broad-spectrum reactivity. Immunological assay results demonstrated that H9-21 (219RIFKPLIGPRPLVNGLMGRI239), H9-26 (269SGESHGRILKTDLKMGSCTV289), and H9-30 (309YAFGNCPKYI GVKSLKLAVG329) effectively induced antibody generation and conferred partial protective efficacy against the parent virus JY040218C. The results of lymphocyte proliferation and ELISpot assays indicated that peptides H9-15 (159MRWLTQKNNAYPTQDAQYTN179), H9-22 (229PLVNGLMGRINYYWSVLKP G249), and H9-23 (239NYYWSVLKPGQTLRIKSDGN259) could effectively stimulate the expression of interferon-gamma in peripheral blood lymphocytes of chickens immunized against different strains of H9N2 AIV. Collectively, 5 novel cell epitopes H9-15, H9-22, H9-23, H9-26, and H9-30, including the best B cell epitope H9-26 and the best T cells epitope H9-22, were identified that could be targeted for vaccine design or detection approaches against H9N2 AIVs.

A multifaceted approach for identification, validation, and immunogenicity of naturally processed and in silico-predicted highly conserved SARS-CoV-2 peptides

SARS-CoV-2 remains a major global public health concern. Antibody waning and immune escape variant emergence necessitate the development of next generation vaccines that induce cross-reactive durable immune responses. T cell responses to SARS-CoV-2 demonstrate higher conservation, antigenic breadth, and longevity than antibody responses. Therefore, we sought to identify pathogen-derived T cell epitopes for a potential peptide-based vaccine. We pursued an approach leveraging: 1) liquid chromatography and tandem mass spectrometry (LC-MS/MS)-based identification of peptides from ancestral SARS-CoV-2-infected cell lines, 2) epitope prediction algorithms, and 3) overlapping peptide libraries. From this strategy, we identified 380 unique SARS-CoV-2-derived peptide sequences, including 53 antigenic HLA class I and class II peptides from multiple structural and non-structural/accessory viral proteins. These peptide sequences were highly conserved across variants of concern/interest (VoC/VoIs), and are estimated to achieve coverage of >96% of the world population. Our findings validate this discovery pipeline for peptide identification and immunogenicity testing, and are a crucial step toward the development of a next-generation multi-epitope SARS-CoV-2 peptide vaccine, and a novel vaccine platform methodology.

Immune Profiling of Respiratory Syncytial Virus (RSV) for the Development of Targeted Immunotherapy

Introduction: RSV-associated respiratory viral infections are a major public health problem affecting the immunologically naïve/compromised population. For example, RSV-induced bronchiolitis and pneumonia are the leading cause of hospitalization in infants and young children worldwide, while in adult allogeneic hematopoietic transplant (HCT) recipients the incidence of progression from upper to lower respiratory tract infection (LRTI) is 40-60%, with mortality rates as high as 80%. At the national level, the Fall 2022-Spring 2023 RSV surge translated into a sharp rise in hospitalization rates surpassing last season's respective rate by almost 5-fold (https://www.cdc.gov/rsv/research/rsv-net). Our group has previously demonstrated the feasibility, safety and clinical efficacy of administering allogeneic ex vivo expanded multivirus-specific T cells (multi-VSTs) as a banked, off-the-shelf product for the treatment of EBV, CMV, BKV, HHV6, AdV and SARS-CoV-2 infections/disease in immunocompromised individuals. Given the RSV-associated morbidity and the paucity of therapeutic agents, we sought to characterize the cellular immune responses to RSV with a view to developing a targeted T-cell therapy product for those at highest risk of severe disease (i.e. HCT recipients, elderly/immunocompromised patients). Methods: To first identify immunogenic and protective RSV antigens we exposed PBMCs from healthy adult donors to pepmixes (overlapping peptide libraries) spanning the 11 RSV-encoded antigens and found that 7 induced highly reactive T cells. However, Nucleoprotein (N) and Fusion Protein (F) proved to be immunogenic in all donors and were thus advanced for VST manufacturing. We subsequently utilized our optimized VST manufacturing process and culture in a G-Rex device in medium supplemented with activating cytokines to generate RSV-specific VSTs (RSV-STs) with activity against this combination of immunodominant targets. Results: We achieved a mean 5.2±1-fold expansion (mean±SEM; n=13) of cells that were comprised almost exclusively of CD3+ T cells (94.08±1.15%), with a mixture of cytotoxic (CD8+; 28.91±5.16%) and helper (CD4+; 71.09±5.15%) T cells. These cells had a phenotype consistent with effector function and memory potential, as evidenced by upregulation of the activation markers CD25, CD69, and CD28 and expression of central (CD45RO+/CD62L+) and effector memory markers (CD45RO+/CD62L-) with minimal expression of exhaustion-related markers such as PD1, Tim3 and LAG3. The anti-viral activity of the expanded cells was confirmed in an IFNγ ELISpot using both stimulating antigens as an immunogen and all lines proved to be reactive against the target antigens (N: 3587±939 SFC/2x10 5; F: 3526±743). As demonstrated by surface and intracellular flow cytometry, the immune response was mediated by both CD4+ and CD8+ T cell subsets through IFNγ/TNFα production and upregulation of activation/costimulatory markers. Reactive cells were polyfunctional and primarily Th1-polarized as evidenced by IFNγ, Granzyme-B, IL-2 and GM-CSF production measured by FluoroSpot and Luminex array. Finally, the expanded cells were able to kill viral pepmix-loaded autologous PHA blasts with minimal/no activity against non-antigen-expressing autologous and allogeneic targets. Conclusion: Healthy donor-derived RSV-STs are Th1-polarized, polyfunctional and selectively able to kill viral antigen-expressing targets with no auto- or alloreactivity, indicative of their safety for clinical use. A clinical trial evaluating their safety and activity in HCT recipients as part of an off-the-shelf multi-respiratory virus-directed product is currently underway (NCT04933968, https://clinicaltrials.gov).

ASFV antigens selected from genotype I immunised pigs are immunogenic, but do not protect against genotype II challenge.

African swine fever virus (ASFV) has recent caused outbreaks of viral haemorrhagic fever in domestic pigs and wild boar in Africa, Asia, Europe, Oceania and North America. Control measures for African swine fever are limited in most countries to biosecurity at the farm gate followed by movement restrictions and selective or complete culling of pigs on affected premises. Modified live vaccines are being trialled in several countries, however development of safe and effective African swine fever subunit vaccines has been restricted by a poor understanding of the key antigens required for protection, particularly for the panzootic genotype II viruses. The cellular immune response is thought to be critical for protection against African swine fever and therefore to develop an effective subunit vaccine that stimulates an anti-ASFV T-cell response we screened lymphocytes from pigs which survived challenge with Georgia 2007/1. Using an overlapping peptide library corresponding to 168 annotated open reading frames and 24 potential minor open reading frames we identified seventeen proteins with strongly stimulated secretion of interferon gamma in an ELISpot assay. The phenotype of the T cells which were stimulated by these pools of peptides were then investigated by flow cytometry. Proteins stimulating predominantly CD8+ T cells were incorporated into bivalent replication deficient adenovirus vectors and tested as potential vaccine candidates in immunisation and challenge experiments in pigs.

Highly Active Cytotoxic CD4 + T Cells Targeting EBV Oncoproteins with Enhanced In Vivo Anti-Tumor and Self-Renewal Properties

Background: Epstein-Barr virus (EBV), a human gamma herpesvirus primarily of B-cell tropism that infects more than 90% of humans during childhood, establishing a lifelong latent persistence after the initial immune clearance. Alterations in the immune surveillance in subjects with impaired cellular immunity can lead to viral reactivation and uncontrolled B cell lymphoproliferation manifesting as post-transplant lymphoproliferative disease (PTLD) and malignant transformation into B-cell lymphomas and some epithelial tumors. Adoptive transfer of virus-specific cytotoxic T cells targeting viral oncoproteins of EBV (EBV-CTLs) may lead to rapid reconstitution of T cell immune surveillance and may efficiently control PTLD and some EBV-driven lymphomas. However, success of this approach has been hampered by suboptimal/variable potency of donor-derived EBV-CTLs and possibly their terminally differentiated/exhausted phenotype associated with limited in vivo persistence and activity, especially when targeting elusive Latency 1-associated diseases. We hypothesized that that EBV-specific CD4 + T cells differentiated using novel pro-inflammatory conditions might efficiently target viral oncoproteins concurrently retaining highly desirable stem-cell like properties required for successful eradication of tumor cells in vivo. Methods: In this study, we developed a method for generating novel and highly potent CD4 + cytotoxic T cells (CD4 +-CTLs) simultaneously targeting EBNA1, LMP1, and LMP2A. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors, and either total T cells or CD4 +T cells were purified. These cells were then stimulated with PBMCs loaded with overlapping peptide libraries spanning LMP1, LMP2A, and EBNA1 antigens, either under neutral conditions (IL2 and IL-7) or in the presence of pro-inflammatory (type 1/17) cytokines ex vivo for 12-14 days. The phenotypes, reactivity, and cytotoxic capability of these CD4 +-CTLs against autologous EBV-Lymphoblastoid cell lines (LCLs) were assessed in vitro and in vivo upon adoptive transfer into tumor-bearing NSG mice. Results: In comparison to EBV-CTLs generated from unselected PBMCs (standard conditions), purified CD4 + expanded in the presence of pro-inflammatory cytokines exhibited significantly higher frequency of antigen specific T cells with a marked skewing towards EBNA1 antigen, and displaying marked polyfunctionality, concurrently secreting tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and inducible granzyme B (GzmB), while maintaining interleukin-2 (IL-2) secretion capacity. Antigen-reactive CD4 +-CTLs induced in pro-inflammatory conditions retained a significant population with central memory (T CM) phenotype (CCR7 hi, CD27 hi), while those expanded in neutral conditions predominantly displayed an effector memory (T EM) phenotype. These CD4 +-CTLs demonstrated enhanced cytotoxicity against autologous EBV-LCLs and superior proliferative capacity in vitro upon secondary stimulation, whereas standard EBV-CTLs proliferated poorly. In the LCL-injected NSG mice, CD4 +-CTLs exhibited superior self-renewal and long-term persistence, resulting in a significant reduction/eradication of established LCL burden when compared to standard EBV-CTLs. Conclusion: Ex vivo priming with EBV antigens in the presence of pro-inflammatory cytokines reliably induces EBV-reactive CD4 +-CTLs with markedly enhanced antigen-specificity, polyfunctionality, cytotoxic potential, preserved stemness, proliferative capacity, and superior in vivo persistence and anti-tumor activity . These findings hold significant promise for developing improved adoptive immunotherapies to target EBV-associated diseases, especially of Latency-I origin and may be generalized as a strategy for targeting other viral oncoproteins expressed by human cancers. Figure 1: EBV-CD4 + CTLs generated under pro-inflammatory conditions display superior reactivity, persistence, and anti-tumor activity in vivo: (A.) The percentage of CD4 +Th cells primed with LMP1/2 and EBNA1 and expanded under indicated conditions producing TNF-α, IFN-γ, IL-2 and GZMB upon antigenic restimulation (n=29). (B.) Frequency of EBV-LCLs (CD23 +) and CD3 + cells detected in spleens on Day 26 post adoptive transfer of standard and pro-inflammatory EBV-CD4 + CTLs(n=4 per group).

Constitutive Interleukin-7 Cytokine Signaling Enhances the Persistence of Epstein-Barr Virus-Specific T-Cells.

The efficacy of therapeutic T-cells is limited by a lack of positive signals and excess inhibitory signaling in tumor microenvironments. We previously showed that a constitutively active IL7 receptor (C7R) enhanced the persistence, expansion, and anti-tumor activity of T-cells expressing chimeric antigen receptors (CARs), and C7R-modified GD2.CAR T-cells are currently undergoing clinical trials. To determine if the C7R could also enhance the activity of T-cells recognizing tumors via their native T-cell receptors (TCRs), we evaluated its effects in Epstein-Barr virus (EBV)-specific T-cells (EBVSTs) that have produced clinical benefits in patients with EBV-associated malignancies. EBVSTs were generated by stimulation of peripheral blood T-cells with overlapping peptide libraries spanning the EBV lymphoma antigens, LMP1, LMP2, and EBNA 1, followed by retroviral vector transduction to express the C7R. The C7R increased STAT5 signaling in EBVSTs and enhanced their expansion over 30 days of culture in the presence or absence of exogenous cytokines. C7R-EBVSTs maintained EBV antigen specificity but were dependent on TCR stimulation for continued expansion. C7R-EBVSTs produced more rapid lymphoma control in a murine xenograft model than unmodified EBVSTs and persisted for longer. The findings have led to a clinical trial, evaluating C7R-EBVSTs for the treatment of refractory or relapsed EBV-positive lymphoma (NCT04664179).

Identification of neutralizing epitopes on the D/A domain of the E2 glycoprotein of classical swine fever virus

Classical swine fever virus (CSFV) shares high antigenic homology with other members of the genus Pestivirus. Because several pestivirus species can also infect swine, eliciting cross-reactive antibodies, it is important to define CSFV-specific epitopes for the differential diagnosis of classical swine fever (CSF) by serology. For this purpose, epitope mapping of seven monoclonal antibodies (mAbs), recognizing sites on the D/A domain of glycoprotein E2, was performed using recombinant expressed antigenic domains and mutants of E2, as well as an overlapping peptide library. Three CSFV-specific epitopes, i.e., 780-IEEMGDDFGFGLCPF-794, 810-NGSAFYLVCPIGWTG-824, and 846-REKPF-850, were identified within the D/A domain of E2. Site-directed mutagenesis further confirmed that residues 783-MGD-785, 789-FGLCPF-794, 813-AFYLVCPIGWTG-824, and 846-REK-848 were critical residues in these regions. In addition, a F789S difference within the epitope 780-IEEMGDDFGFGLCPF-794 was responsible for the absence of binding of two mAbs to the E2 protein of the live attenuated CSFV vaccine strain Riems. Structural modeling revealed that, the three epitopes are located near each other, suggesting that they may form a more complex conformational epitope on the D/A domain in vivo. Six of the mAbs neutralized viruses of diverse genotypes, indicating that the target epitopes are involved in virus interaction with cells. The binding of CSFV to cells was significantly reduced after pre-incubation with either truncated E2 proteins comprising the D/A domain or with the CSFV-specific mAbs targeting the domain D/A. These epitopes identified on the D/A domain are important targets for virus neutralization that might be involved in the early steps of CSFV infection. These findings reveal potential candidates for improving the differential diagnosis of pestiviruses by serology.

Abstract 6662: Studying immune responses against human endogenous retroviruses in immune checkpoint blockade-treated lung adenocarcinoma

Abstract Background: Clinical response to immune checkpoint blockade (ICB) is predicated upon the existence of tumor-reactive T cells, but their precise targets remain poorly defined. Some patients with low tumor mutational burden (TMB) lung adenocarcinoma (LUAD) derive clinical benefit from ICB, suggesting that non-mutated antigens may serve as T cell targets. Human endogenous retroviruses (hERVs) can be differentially expressed in malignant cells due to epigenetic dysregulation and can be targeted by T cells. We hypothesize that the detection of T cell responses against hERVs correlates with a favorable clinical response to ICB in LUAD. Methods: Tumor tissues from patients with Stage I-III LUAD who underwent upfront surgical resection were enzymatically digested. Viable CD45-EPCAM+ cells were sorted. Total RNA was extracted and RNA sequencing was performed. The hervQUANT workflow was used to bioinformatically identify differentially-expressed, open reading frame (ORF)-encoding hERV transcripts. Publicly available RNAseq datasets derived from human medullary thymic epithelial cells (mTECs) and normal tissues were queried to exclude hERV transcripts expressed in these tissues. Overlapping peptide libraries (OPLs) spanning the shared hERV ORFs were synthesized and used in Functional Expansion of Specific T Cells (FEST) assays to detect hERV-specific T cell clones in the peripheral blood of a cohort of patients who met these criteria: (1) had advanced/metastatic LUAD, (2) were treated with ICB, (3) had peripheral blood mononuclear cells isolated at 4-8 weeks post-initiation of ICB and (4) had formal radiographic assessment of response to ICB. Results: We identified 41 differentially-expressed, ORF-encoding hERV transcripts that were shared by ≥4 (out of 11) CD45-EPCAM+ LUAD samples. OPLs covering hERV ORFs with mean expression of ≥ 3 CPM in CD45-EPCAM+ LUAD cells and <3 CPM in mTECs were synthesized. Low expression in normal tissues was also confirmed (<3 CPM). A total of 318 patients were screened and 30 patients (22 radiographic responders and 8 non-responders) met the inclusion criteria. The median age was 66.5 years for responders and 58 years for non-responders. The median PD-L1 TPS was 72.5% and 70%, respectively. The median TMB was 8 and 2.55, respectively. FEST assays utilizing hERV OPLs revealed 4 potential hERV-reactive T cell clonotypes in the peripheral blood of 2 patients with LUAD treated with ICB. Studies are ongoing to validate and characterize these clonotypes. Testing of the remainder of the selected patient cohort is also ongoing. Conclusions: Potential hERV-reactive T cell clonotypes are detectable in ICB-treated patients with LUAD. Correlation with clinical response to ICB will be evaluated upon validation of these clonotypes, but these results are promising findings in the ongoing efforts to identify immunogenic sources of non-mutated antigens in cancer. Citation Format: Khaled Sanber, Sydney Connor, Steven Vensko, Christopher Cherry, Zhen Zeng, Drew Pardoll, Patrick Forde, Benjamin Vincent, Kellie Smith. Studying immune responses against human endogenous retroviruses in immune checkpoint blockade-treated lung adenocarcinoma [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 6662.

Identification of Major B-Cell Linear Epitopes of Peach Allergen Pru p 3 Using Immune Slot-Blot Microarray Assay.

Pru p 3, one of the most representative proteins of the lipid transfer proteins (LTPs), is responsible for clinical allergic reactions to food of peach origin. The identification of Pru p 3 epitopes is not comprehensive due to different methods and principles of epitope screening. In addition, evaluation of the stability of the epitopes and the validation of the immunological key amino acids still need further research. Therefore, in the present study, an immune slot-blot microarray assay was performed to screen the epitopes from Pru p 3 overlapping peptide library, and a new epitope (P-1, AA1-16, ITCGQVSSALAPCIPY) was identified and two identified epitopes were deeply investigated (P-2, AA12-27, PCIPYVRGGGAVPPAC; P-3, AA23-38, VPPACCNGIRNVNNLA). The stability of these epitopes was then verified by thermal processing treatment and digestion experiments. Moreover, the key amino acids of the three identified epitopes were obtained by epitope amino acid mutation combined with slot-blot experiments. These findings may contribute to the further understanding of Pru p 3 and the prevention of peach allergy.

Broadly recognized, cross-reactive SARS-CoV-2 CD4 T cell epitopes are highly conserved across human coronaviruses and presented by common HLA alleles.

Sequence homology between SARS-CoV-2 and common-cold human coronaviruses (HCoVs) raises the possibility that memory responses to prior HCoV infection can affect T cell response in COVID-19. We studied T cell responses to SARS-CoV-2 and HCoVs in convalescent COVID-19 donors and identified a highly conserved SARS-CoV-2 sequence, S811-831, with overlapping epitopes presented by common MHC class II proteins HLA-DQ5 and HLA-DP4. These epitopes are recognized by low-abundance CD4 T cells from convalescent COVID-19 donors, mRNA vaccine recipients, and uninfected donors. TCR sequencing revealed a diverse repertoire with public TCRs. T cell cross-reactivity is driven by the high conservation across human and animal coronaviruses of T cell contact residues in both HLA-DQ5 and HLA-DP4 binding frames, with distinct patterns of HCoV cross-reactivity explained by MHC class II binding preferences and substitutions at secondary TCR contact sites. These data highlight S811-831 as a highly conserved CD4 T cell epitope broadly recognized across human populations.

Parkinson's Disease-Specific Autoantibodies against the Neuroprotective Co-Chaperone STIP1.

Parkinson’s disease (PD) is a debilitating movement disorder characterised by the loss of dopaminergic neurons in the substantia nigra. As neuroprotective agents mitigating the rate of neurodegeneration are unavailable, the current therapies largely focus only on symptomatic relief. Here, we identified stress-inducible phosphoprotein 1 (STIP1) as a putative neuroprotective factor targeted by PD-specific autoantibodies. STIP1 is a co-chaperone with reported neuroprotective capacities in mouse Alzheimer’s disease and stroke models. With human dopaminergic neurons derived from induced pluripotent stem cells, STIP1 was found to alleviate staurosporine-induced neurotoxicity. A case-control study involving 50 PD patients (average age = 62.94 ± 8.48, Hoehn and Yahr >2 = 55%) and 50 age-matched healthy controls (HCs) (average age = 63.1 ± 8) further revealed high levels of STIP1 autoantibodies in 20% of PD patients compared to 10% of HCs. Using an overlapping peptide library covering the STIP1 protein, we identified four PD-specific B cell epitopes that were not recognised in HCs. All of these epitopes were located within regions crucial for STIP1’s chaperone function or prion protein association. Our clinical and neuro-immunological studies highlight the potential of the STIP1 co-chaperone as an endogenous neuroprotective agent in PD and suggest the possible involvement of autoimmune mechanisms via the production of autoantibodies in a subset of individuals.

Identification of a dominant murine T-cell epitope in recombinant protein P29 from Echinococcus granulosus

Echinococcus granulosus causes echinococcosis, an important zoonotic disease worldwide and a major public health issue. Vaccination is an economical and practical approach for controlling E. granulosus. We have previously revealed that a recombinant protein P29 (rEg.P29) is a good vaccine candidate against E. granulosus. However, T cell immunogenic epitopes have not been identified. In the present study, we use rEg.P29-immunized mice as models to screen immunogenic epitopes for the construction of a novel multi-epitope vaccine. We search for immunodominant epitopes from an overlapping peptide library to screen the peptides of rEg.P29. Our results confirm that rEg.P29 immunization in mice elicits the activation of T cells and induces cellular immune responses. Further analyses show that a T cell epitope within amino acids 86–100 of rEg.P29 elicits significant antigen-specific IFN-γ production in CD4+ and CD8+ T cells and promotes specific T-cell activation and proliferation. Collectively, these results provide a reference for the construction of a novel vaccine against broad E. granulosus genotypes based on epitopes of rEg.P29.

T Cell Response following Anti-COVID-19 BNT162b2 Vaccination Is Maintained against the SARS-CoV-2 Omicron B.1.1.529 Variant of Concern

The progression of the COVID-19 pandemic has led to the emergence of variants of concern (VOC), which may compromise the efficacy of the currently administered vaccines. Antigenic drift can potentially bring about reduced protective T cell immunity and, consequently, more severe disease manifestations. To assess this possibility, the T cell responses to the wild-type Wuhan-1 SARS-CoV-2 ancestral spike protein and the Omicron B.1.1.529 spike protein were compared. Accordingly, peripheral blood mononuclear cells (PBMC) were collected from eight healthy volunteers 4–5 months following a third vaccination with BNT162b2, and stimulated with overlapping peptide libraries representing the spike of either the ancestral or the Omicron SARS-CoV-2 virus variants. Quantification of the specific T cells was carried out by a fluorescent ELISPOT assay, monitoring cells secreting interferon-gamma (IFNg), interleukin-10 (IL-10) and interleukin-4 (IL-4). For all the examined individuals, comparable levels of reactivity to both forms of spike protein were determined. In addition, a dominant Th1 response was observed, manifested mainly by IFNg-secreting cells and only limited numbers of IL-10- and IL-4-secreting cells. The data demonstrate stable T cell activity in response to the emerging Omicron variant in the tested individuals; therefore, the protective immunity to the variant following BNT162b2 vaccination is not significantly affected.

37. Allogeneic, Off-the-Shelf, SARS-CoV-2-specific T Cells Demonstrate Reactivity Against Emerging Variant Strains

BackgroundThe impact of COVID-19 has been profound with >170,000,000 confirmed cases worldwide and emerging variants being a cause of global concern. Defects in T-cell function and trafficking have been described among those with severe illness, and immunodeficiency is a risk factor for persistent viral shedding and prolonged symptoms. Because of our prior clinical data demonstrating that allogeneic, off-the-shelf virus-specific T cells (VSTs) can safely and effectively treat viral infections, we investigated the feasibility of targeting COVID-19 using banked, SARS-CoV-2-specific VSTs.MethodsWe first screened PBMCs from convalescent individuals against 18 structural and non-structural/accessory (NSPs/APs) SARS-CoV-2 proteins and identified 5 [Spike (S), Membrane (M), Nucleoprotein (N), NSP4, and AP7a] as immunodominant which were then advanced to our VST production process.ResultsUsing overlapping peptide libraries spanning these antigens as a stimulus, we achieved a mean 7.6±0.9 fold expansion (n=13) of VSTs (96±0.5%), with a mixture of cytotoxic (CD8+) and helper (CD4+) T cells that expressed activation and central/effector memory markers. These VSTs were potent, Th1-polarized and polyfunctional, producing IFNγ, TNFα, GM-CSF and Granzyme B. Moreover, the VSTs were able to kill pepmix-loaded autologous targets with no evidence of auto- or alloreactivity, attesting to their virus selectivity and safety for clinical use (Figure 1). Finally, though initially generated against the reference strain NC_045512.2 (Wuhan), these VSTs were able to recognize other clinically important variants including B1.1.7 (UK), B1.351 (South Africa) and P1 (Brazil). This demonstrates the cross-reactive potential of these polyclonal and diverse VSTs, which were developed to provide potent antiviral effects and minimize the risk of immune escape due to sequence variation. Figure 1: SARS-CoV-2 Specific T cells Have Demonstrated Selective Cytolytic Activity against SARS-CoV-2 While Leaving Non-Virus Infected Targets Intact. ConclusionIn conclusion, it is feasible to generate polyclonal SARS-CoV-2 VSTs that provide coverage against variant strains using GMP-compliant manufacturing methodologies. We have advanced this product to the bedside for administration in a Phase I, randomized clinical trial [VSTs+ standard of care (SOC) vs SOC] in high-risk patients hospitalized with COVID-19 (NCT04401410).Disclosures Spyridoula Vasileiou, PhD, AlloVir (Consultant) Manik Kuvalekar, MSc, AlloVir (Consultant) Aster Workineh, MSc, AlloVir (Employee) Ayumi Watanabe, BSc, AlloVir (Consultant) Yovana Velazquez, BSc, AlloVir (Consultant) Helen Elisabeth Heslop, MD, AlloVir (Shareholder)Cell Medica (Grant/Research Support)Gilead Sciences (Consultant)Kiadis Pharma (Consultant)Marker Therapeutics (Consultant, Shareholder)Mesoblast (Consultant)Novartis (Consultant)PACT Pharma (Consultant)Tessa Therapeutics (Consultant, Research Grant or Support) Premal Lulla, MD, Johnson & Johnson (Shareholder) Ann Marie Leen, PhD, AlloVIr (Consultant, Shareholder)Marker Therapeutics (Consultant, Shareholder)

Early disappearance of tumor antigen-reactive T cells from peripheral blood correlates with superior clinical outcomes in melanoma under anti-PD-1 therapy

BackgroundAnti-programmed cell death protein 1 (PD-1) antibodies are now routinely administered for metastatic melanoma and for increasing numbers of other cancers, but still only a fraction of patients respond. Better...

Long Term Immune Response Produced by the SputnikV Vaccine.

SputnikV is a vaccine against SARS-CoV-2 developed by the Gamaleya National Research Centre for Epidemiology and Microbiology. The vaccine has been shown to induce both humoral and cellular immune responses, yet the mechanisms remain largely unknown. Forty SputnikV vaccinated individuals were included in this study which aimed to demonstrate the location of immunogenic domains of the SARS-CoV-2 S protein using an overlapping peptide library. Additionally, cytokines in the serum of vaccinated and convalescent COVID-19 patients were analyzed. We have found antibodies from both vaccinated and convalescent sera bind to immunogenic regions located in multiple domains of SARS-CoV-2 S protein, including Receptor Binding Domain (RBD), N-terminal Domain (NTD), Fusion Protein (FP) and Heptad Repeats (HRs). Interestingly, many peptides were recognized by immunized and convalescent serum antibodies and correspond to conserved regions in circulating variants of SARS-CoV-2. This breadth of reactivity was still evident 90 days after the first dose of the vaccine, showing that the vaccine has induced a prolonged response. As evidenced by the activation of T cells, cellular immunity strongly suggests the high potency of the SputnikV vaccine against SARS-CoV-2 infection.

Identification of a specific surface epitope of OmpC for Escherichia coli O157:H7 with protein topology facilitated affinity mass spectrometry

The goal of this work was to identify the target protein and epitope of a previously reported Escherichia coli O157:H7 (ECO157)–specific monoclonal antibody (mAb) 2G12. mAb 2G12 has shown high specificity for the recovery and detection of ECO157. To achieve this goal, the target protein was first separated by two-dimensional gel electrophoresis (2-DE) and located by Western blot (WB). The protein spots were identified to be the outer membrane protein (Omp) C by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF–MS). After that, the target protein was purified by immunoaffinity chromatography (IAC) and subjected to in situ enzymatic cleavage of the vulnerable peptides. Eight eluted peptides of OmpC identified by liquid chromatography–tandem mass spectrometry (LC–MS/MS) were further mapped onto the homologous protein structure of E. coli OmpC (2IXX). The topology of OmpC showed that three peptides had extracellular loops. Epitope mapping with overlapping peptide library and sequence homology analysis revealed that the epitope consisted of a specific peptide, “LGVING,” and an adjacent conservative peptide, “TQTYNATRVGSLG.” Both peptides loop around the overall structure of the epitope. To test the availability of the epitope when ECO157 was grown under different osmolarity, pH, and nutrition levels, the binding efficacy of mAb 2G12 with ECO157 grown in these conditions was evaluated. Results further demonstrated the good stability of this epitope under potential stressful environmental conditions. In summary, this study revealed that mAb 2G12 targeted one specific and one conservative extracellular loop (peptide) of the OmpC present on ECO157, and the epitope was stable and accessible on ECO157 cells grown in different environment.Key points• OmpC is the target of a recently identified ECO157-specific mAb 2G12.• Eight peptides were identified from the OmpC by using LC–MS/MS.• The specificity of mAb 2G12 is mainly determined by the “LGVING” peptide.

BK virus-specific T cells for immunotherapy of progressive multifocal leukoencephalopathy: an open-label, single-cohort pilot study

Progressive multifocal leukoencephalopathy, a rare disease of the CNS caused by JC virus and occurring in immunosuppressed people, is typically fatal unless adaptive immunity is restored. JC virus is a member of the human polyomavirus family and is closely related to the BK virus. We hypothesised that use of partly HLA-matched donor-derived BK virus-specific T cells for immunotherapy in progressive multifocal leukoencephalopathy would be feasible and safe. We did an open-label, single-cohort pilot study in patients (aged 18 years or older) with clinically definite progressive multifocal leukoencephalopathy and disease progression in the previous month at the National Institutes of Health (NIH) Clinical Center (Bethesda, MD, USA). Overlapping peptide libraries derived from large T antigen and major capsid protein VP1 of BK virus with high sequence homology to JC virus counterparts were used to generate polyomavirus-specific T cells cross-recognising JC virus antigens. Polyomavirus-specific T cells were manufactured from peripheral blood mononuclear cells of first-degree relative donors aged 18 years or older. These cells were administered to patients by intravenous infusion at 1 × 106 polyomavirus-specific T cells per kg, followed by up to two additional infusions at 2 × 106 polyomavirus-specific T cells per kg. The primary endpoints were feasibility (no manufacturing failure based on meeting release criteria, achieving adequate numbers of cell product for clinical use, and showing measurable antiviral activity) and safety in all patients. The safety monitoring period was 28 days after each infusion. Patients were followed up with serial MRI for up to 12 months after the final infusion. This trial is registered at ClinicalTrials.gov, NCT02694783. Between April 7, 2016, and Oct 19, 2018, 26 patients were screened, of whom 12 were confirmed eligible and received treatment derived from 14 matched donors. All administered polyomavirus-specific T cells met the release criteria and recognised cognate antigens in vitro. 12 patients received at least one infusion, ten received at least two, and seven received a total of three infusions. The median on-study follow-up was 109·5 days (range 23-699). All infusions were tolerated well, and no serious treatment-related adverse events were observed. Seven patients survived progressive multifocal leukoencephalopathy for longer than 1 year after the first infusion, whereas five died of progressive multifocal leukoencephalopathy within 3 months. We showed that generation of polyomavirus-specific T cells from healthy related donors is feasible, and these cells can be safely used as an infusion for adoptive immunotherapy of progressive multifocal leukoencephalopathy. Although not powered to assess efficacy, our data provide additional support for this strategy as a potential life-saving therapy for some patients. Intramural Research Program of the National Institute of Neurological Disorders and Stroke of the NIH.

A newly identified linear epitope on non-RBD region of SARS-CoV-2 spike protein improves the serological detection rate of COVID-19 patients

BackgroundSerological test is helpful in confirming and tracking infectious diseases in large population with the advantage of fast and convenience. Using the specific epitope peptides identified from the whole antigen as the detection antigen is sensitive and relatively economical. The development of epitope peptide-based detection kits for COVID-19 patients requires comprehensive information about epitope peptides. But the data on B cell epitope of SARS-CoV-2 spike protein is still limited. More importantly, there is a lack of serological data on the peptides in the population. In this study, we aimed to identify the B cell epitope peptides of spike protein and detect the reactivity in serum samples, for further providing data support for their subsequent serological applications.ResultsTwo B cell linear epitopes, P104 and P82, located in non-RBD region of SARS-CoV-2 S protein were identified by indirect ELISA screening of an overlapping peptide library of the S protein with COVID-19 patients’ convalescent serum. And the peptides were verified by testing with 165 serum samples. P104 has not been reported previously; P82 is contained in peptide S21P2 reported before. The positive reaction rates of epitope peptides S14P5 and S21P2, the two non-RBD region epitopes identified by Poh et al., and P82 and P104 were 77.0%, 73.9%, 61.2% and 30.3%, respectively, for 165 convalescent sera, including 30 asymptomatic patients. Although P104 had the lowest positive rate for total patients (30.3%), it exhibited slight advantage for detection of asymptomatic infections (36.7%). Combination of epitopes significantly improved the positive reaction rate. Among all combination patterns, (S14P5 + S21P2 + P104) pattern exhibited the highest positive reaction rate for all patients (92.7%), as well as for asymptomatic infections (86.7%), confirming the feasibility of P104 as supplementary antigen for serological detection. In addition, we analyzed the correlation between epitopes with neutralizing antibody, but only S14P5 had a medium positive correlation with neutralizing antibody titre (rs = 0.510, P < 0.01).ConclusionOur research proved that epitopes on non-RBD region are of value in serological detection especially when combination more than one epitope, thus providing serological reaction information about the four epitopes, which has valuable references for their usage.

Evaluating Cyclin A1 As an Immunotherapeutic Target in Acute Myeloid Leukemia (AML)

T cell based immunotherapy targeting leukemia associated antigens (LAAs) such as Wilm's tumor 1 antigen (WT1) has proven both safe and clinically effective, with the induction of durable responses in patients with relapsed AML. However, because of heterogeneous LAA expression among leukemic blasts, targeting a single LAA has led to tumor immune escape and relapse, highlighting the need to identify and simultaneously target an array of tumor-expressed LAAs. To expand the target antigen spectrum we sought to immunologically characterize cyclin A1 (CCNA1), a meiosis-specific cell cycle antigen that is selectively expressed in AML blasts with limited normal tissue expression and which has been implicated as playing a role in leukemogenesis based on preclinical animal models.To assess the suitability of CCNA1 as an immunotherapeutic target we first sought to expand reactive populations ex vivo using an overlapping peptide library (15mers overlapping by 11 amino acids) spanning the entire antigen as a source of stimulation. In 12 donors tested we achieved a mean 5.2±0.65 fold expansion of T cells, of which 11 proved to recognize CCN1 as measured by an IFNg ELISpot [mean 377.3 spot forming cells (SFCs)/1x105 T cells (range 38 to 1188 SFCs) ]. The antigen specific lines were polyclonal (23±4% CD4+ and 64±5% CD8+ T cells) that expressed markers for both central and effector memory (29±7% CD45RO+CD62L+ and 28±6% CD45RO+CD62L-), required for long term in vivo persistence. Importantly both helper (CD4+) and cytotoxic (CD8+) subsets contained CCNA1-directed T cell populations, which secreted multiple cytokines (1.47±0.07% CD4+IFNg+TNFa+ and 9.52±0.25% CD8+IFNg+TNFa+) as measured by intracellular cytokine staining, confirming their polyfunctionality. Moreover, CCNA1-specific T cells were able to selectively kill pepmix-loaded autologous target cells and a partially HLA-matched cell line that endogenously-expresses cyclin A1 (44±7% and 55±1%, respectively; E:T 20:1). without non-specific activity against control (unloaded) targets (9±2%; E:T 20:1). This killing was confirmed to be HLA-restricted since the addition of an MHC class I blocking antibody diminished specific lysis (decrease from 44% to 26%). Finally, using individual peptides organized into minipools, we were able to identify 4 novel HLA class I-restricted epitope peptides.To assess the clinical relevance of CCNA1-directed T cells we assessed whether patients with AML had detectable circulating T cells and in 5 of 9 patients who had undergone an allogeneic hematopoietic stem cell transplant as disease treatment we could detect CCNA1-specific T cells (mean 117±52.8 SFC/2x105 expanded PBMCs), and detection of reactive cells correlated with a lower risk for relapse.In summary, we have identified CCNA1 as a suitable candidate antigen to target using immunotherapeutic approaches for future clinical trials of adoptively transferred T cells. DisclosuresLulla:Leukemia Texas: Membership on an entity's Board of Directors or advisory committees; ASH Scholar Award: Research Funding; Leukemia Texas Research grant: Research Funding; Junior Faculty sees funding-Baylor College of Medicine: Research Funding; Lymphoma SPORE: Research Funding; ASBMT Young Investigator Award: Research Funding. Tzannou:ViraCyte LLC: Consultancy. Heslop:Celgene: Research Funding; Cell Medica: Patents & Royalties: EBV specific T cells, Research Funding; Viracyte: Equity Ownership; Marker Therapeutics: Equity Ownership. Vera:Wilson Wolf Manufacturing: Consultancy; ViraCyte LLC: Equity Ownership; Marker Therapeutics: Equity Ownership. Leen:ViraCyte LLC: Equity Ownership.