MCMV-based vaccine vectors expressing full-length viral proteins provide long-term humoral immune protection upon a single-shot vaccination

Global pandemics caused by influenza or coronaviruses cause severe disruptions to public health and lead to high morbidity and mortality. There remains a medical need for vaccines against these pathogens. CMV (cytomegalovirus) is a β-herpesvirus that induces uniquely robust immune responses in which remarkably large populations of antigen-specific CD8+ T cells are maintained for a lifetime. Hence, CMV has been proposed and investigated as a novel vaccine vector for expressing antigenic peptides or proteins to elicit protective cellular immune responses against numerous pathogens. We generated two recombinant murine CMV (MCMV) vaccine vectors expressing hemagglutinin (HA) of influenza A virus (MCMVHA) or the spike protein of severe acute respiratory syndrome coronavirus 2 (MCMVS). A single injection of MCMVs expressing either viral protein induced potent neutralizing antibody responses, which strengthened over time. Importantly, MCMVHA-vaccinated mice were protected from illness following challenge with the influenza virus, and we excluded that this protection was due to the effects of memory T cells. Conclusively, we show here that MCMV vectors induce not only long-term cellular immunity but also humoral responses that provide long-term immune protection against clinically relevant respiratory pathogens.

Mechanistic diversity in MHC class I antigen recognition

Throughout its evolution, the human immune system has developed a plethora of strategies to diversify the antigenic peptide sequences that can be targeted by the CD8+ T cell response against pathogens and aberrations of self. Here we provide a general overview of the mechanisms that lead to the diversity of antigens presented by MHC class I complexes and their recognition by CD8+ T cells, together with a more detailed analysis of recent progress in two important areas that are highly controversial: the prevalence and immunological relevance of unconventional antigen peptides; and cross-recognition of antigenic peptides by the T cell receptors of CD8+ T cells.

What Is the Role of HLA-I on Cancer Derived Extracellular Vesicles? Defining the Challenges in Characterisation and Potential Uses of This Ligandome

The Human Leukocyte Antigen class I (HLA-I) system is an essential part of the immune system that is fundamental to the successful activation of cytotoxic lymphocytes, and an effective subsequent immune attack against both pathogen-infected and cancer cells. The importance of cytotoxic T cell activity and ability to detect foreign cancer-related antigenic peptides has recently been highlighted by the successful application of monoclonal antibody-based checkpoint inhibitors as novel immune therapies. Thus, there is an increased interest in fully characterising the repertoire of peptides that are being presented to cytotoxic CD8+ T cells by cancer cells. However, HLA-I is also known to be present on the surface of extracellular vesicles, which are released by most if not all cancer cells. Whilst the peptide ligandome presented by cell surface HLA class I molecules on cancer cells has been studied extensively, the ligandome of extracellular vesicles remains relatively poorly defined. Here, we will describe the current understanding of the HLA-I peptide ligandome and its role on cancer-derived extracellular vesicles, and evaluate the aspects of the system that have the potential to advance immune-based therapeutic approaches for the effective treatment of cancer.

Myelin Peptide–Mannan Conjugate Multiple Sclerosis Vaccines: Conjugation Efficacy and Stability of Vaccine Ingredient

Myelin peptide–mannan conjugates have been shown to be potential vaccines in the immunotherapy of multiple sclerosis. The conjugates are comprised from the epitope peptide and the polysaccharide mannan which transfers as a carrier the antigenic peptide to dendritic cells that process and present antigenic peptides at their surface in complex with MHC class I or class II resulting in T-cell stimulation. The conjugation of antigenic peptide with mannan occurs through the linker (Lys–Gly)5, which connects the peptide with the oxidized mannose units of mannan. This study describes novel methods for the quantification of the vaccine ingredient peptide within the conjugate, a prerequisite for approval of clinical trials in the pursuit of multiple sclerosis therapeutics. Myelin peptides, such as MOG35–55, MBP83–99, and PLP131–145 in linear or cyclic form, as altered peptide ligands or conjugated to appropriate carriers, possess immunomodulatory properties in experimental models and are potential candidates for clinical trials.

In Silico Identification of Chikungunya Virus B- and T-Cell Epitopes with High Antigenic Potential for Vaccine Development

Reverse vaccinology is an outstanding strategy to identify antigens with high potential for vaccine development. Different parameters of five prediction programs were used to assess their sensitivity and specificity to identify B-cell epitopes of Chikungunya virus (CHIKV) strains reported in the IEDB database. The results, based on the use of 15 to 20 mer epitopes and the polyproteins to which they belong, were compared to establish the best parameters to optimize the prediction of antigenic peptides of the Mexican strain CHIKV AJV21562.1. LBtope showed the highest specificity when we used the reported epitopes and polyproteins but the worst sensitivity with polyproteins; ABCpred had similar specificity to LBtope only with the epitopes reported and showed moderate specificity when we used polyproteins for the predictions. Because LBtope was more reliable in predicting true epitopes, it was used as a reference program to predict and select six novel epitopes of the Mexican strain of CHIKV according to prediction frequency, viral genome localization, and non-homology with the human proteome. On the other hand, six bioinformatics programs were used with default parameters to predict T-cell epitopes in the CHIKV strains AJV21562.1 and AJV21561.1. The sequences of the polyproteins were analyzed to predict epitopes present in the more frequent HLA alleles of the Mexican population: DQA1*03011, DQA1*0401, DQA1*0501, DQB1*0201, DQB1*0301, DQB1*0302, and DQB1*0402. Fifteen predicted epitopes in the non-structural and 15 predicted epitopes in the structural polyprotein (9- to 16-mers) with the highest scores of each allele were compared to select epitopes with at least 80% identity. Next, the epitopes predicted with at least two programs were aligned to the human proteome, and 12 sequences without identity with the human proteome were identified as potential antigenic candidates. This strategy would be useful to evaluate vaccine candidates against other viral diseases affecting the countries of the Americas and to increase knowledge about these diseases.

Update on Intravenous Immunoglobulin in Neurology: Modulating Neuro-autoimmunity, Evolving Factors on Efficacy and Dosing and Challenges on Stopping Chronic IVIg Therapy

In the last 25 years, intravenous immunoglobulin (IVIg) has had a major impact in the successful treatment of previously untreatable or poorly controlled autoimmune neurological disorders. Derived from thousands of healthy donors, IVIg contains IgG1 isotypes of idiotypic antibodies that have the potential to bind pathogenic autoantibodies or cross-react with various antigenic peptides, including proteins conserved among the “common cold”-pre-pandemic coronaviruses; as a result, after IVIg infusions, some of the patients’ sera may transiently become positive for various neuronal antibodies, even for anti-SARS-CoV-2, necessitating caution in separating antibodies derived from the infused IVIg or acquired humoral immunity. IVIg exerts multiple effects on the immunoregulatory network by variably affecting autoantibodies, complement activation, FcRn saturation, FcγRIIb receptors, cytokines, and inflammatory mediators. Based on randomized controlled trials, IVIg is approved for the treatment of GBS, CIDP, MMN and dermatomyositis; has been effective in, myasthenia gravis exacerbations, and stiff-person syndrome; and exhibits convincing efficacy in autoimmune epilepsy, neuromyelitis, and autoimmune encephalitis. Recent evidence suggests that polymorphisms in the genes encoding FcRn and FcγRIIB may influence the catabolism of infused IgG or its anti-inflammatory effects, impacting on individualized dosing or efficacy. For chronic maintenance therapy, IVIg and subcutaneous IgG are effective in controlled studies only in CIDP and MMN preventing relapses and axonal loss up to 48 weeks; in practice, however, IVIg is continuously used for years in all the aforementioned neurological conditions, like is a “forever necessary therapy” for maintaining stability, generating challenges on when and how to stop it. Because about 35-40% of patients on chronic therapy do not exhibit objective neurological signs of worsening after stopping IVIg but express subjective symptoms of fatigue, pains, spasms, or a feeling of generalized weakness, a conditioning effect combined with fear that discontinuing chronic therapy may destabilize a multi-year stability status is likely. The dilemmas of continuing chronic therapy, the importance of adjusting dosing and scheduling or periodically stopping IVIg to objectively assess necessity, and concerns in accurately interpreting IVIg-dependency are discussed. Finally, the merit of subcutaneous IgG, the ineffectiveness of IVIg in IgG4-neurological autoimmunities, and genetic factors affecting IVIg dosing and efficacy are addressed.

793 Targeting engineered interleukin-2 (IL-2) to antigen specific T cells via novel biologic platforms

BackgroundA key challenge with IL-2 immunotherapy for cancers is lack of selectivity for anti-tumor immune cells and safety liabilities related to indiscriminate activation of immune cells. The CUE-100 series of Immuno-STATs (ISTs) are designed to selectively activate tumor-specific T cells while avoiding IL-2 toxicities due to systemic activation. CUE-100 series ISTs are rationally engineered Fc fusion proteins comprised of bivalent tumor-peptide-HLA (pHLA) complexes and four affinity-attenuated IL-2 molecules to preferentially engage and activate tumor-specific T cells directly in the patient. Emerging clinical data from our lead candidate CUE-101, which targets HPV-specific T cells in 2L+ R/M HNSCCC, provides PoC for the approach and builds confidence for broad applications in numerous cancers. Building on the CUE-100 series framework, our Neo-STAT (NST) platform contains HLA molecules manufactured with an “empty” peptide-binding pocket, into which diverse tumor-peptides can be chemically conjugated, hence addressing tumor heterogeneity in a cost- and time-efficient manner. Our RDI-STAT (Re-Directed Immuno-STAT) platform further expands the CUE-100 series by redirecting the pre-existing protective viral-specific T cell repertoire to target tumor cells via scFv moieties. RDI-STATs are designed to circumvent potential tumor escape mechanisms linked to HLA loss or defects in antigen-presenting pathways. We present here preclinical data supporting the mechanism of action of these platforms to enhance anti-tumor immune responses.MethodsNSTs were engineered with “empty” HLA-A*0201, into which relevant antigenic peptides were conjugated, and assessed for capacity to expand T cells. RDI-STATs were engineered with TAA-specific scFv and viral-specific pHLA complexes, and assessed for their capacity to induce redirected killing of tumor cells while avoiding systemic activation of all T cells.ResultsThe NST platform demonstrated that different T cell epitopes can be efficiently conjugated into the HLA-binding pocket, and that these molecules activate and expand antigen specific T cells in vitro. RDI-STATs were able to expand anti-viral T cell repertoires and drive anti-viral T cell redirected killing of TAA-expressing cells. In contrast to pan anti-CD3 bispecific molecules, RDI-STATs demonstrated significantly lower induction of pro-inflammatory cytokines.ConclusionsThe IST, NST, and RDI-STAT platforms provide novel opportunities for selective targeting of IL-2 to tumor-relevant T cells while avoiding global immune activation and cytokine release. The scalability and versatility of NSTs highlight the potential to target multiple TAA T cell responses, while RDI-STATs highlight a novel means to harness antiviral immunity against cancer, especially in cases where the tumor may escape immune detection due to loss of HLA.

211 SQZ™ eAPCs generated from PBMCs by delivery of multiple mRNAs encoding for antigens, costimulatory proteins, and engineered cytokines

BackgroundAntigen-specific CD8+ T cells are critical components of mounting an effective immune response against tumors. Generation of antigen-specific T cells require interactions with multiple signals produced by antigen presenting cells (APCs). These signals are comprised of three components: (signal 1) the peptide-MHC complex binding to the T cell receptor, (signal 2) costimulatory molecules on the surface of APCs, and (signal 3) inflammatory cytokines binding to cognate receptors on T cells.MethodsTo engineer all major cell subsets of human peripheral blood mononuclear cells (PBMCs) to become enhanced APCs (eAPCs), we used Cell Squeeze® technology to deliver multiple mRNA encoding for non-self-antigens (signal 1), CD86 (signal 2), and/or membrane-bound cytokines (signal 3). The signal 3 molecules, membrane-bound IL-12 (mbIL-12) and membrane-bound IL-2 (mbIL-2), are chimeric proteins designed to increase the localized concentration of the cytokines and limit off-target effects. Flow cytometry and western blots were used to confirm the translation of each of the delivered mRNA. The increased capabilities of these enhanced APCs were assessed in vitro by culturing the APCs with antigen-specific T cells for multiple days before measuring the functionality of antigen-specific T cells via intracellular cytokine staining or ELISA.ResultsWe demonstrate that Cell Squeeze® processing of PBMCs with mRNA encoding for signals 1, 2, and 3 results in highly effective enhanced APCs in vitro. In a single squeeze process, efficient delivery and translation of up to five mRNA is observed in all major PBMC cell subsets including T cells, B cells, NK cells, and monocytes. Once translated, the chimeric mbIL-2 and mbIL-12 can bind to their cognate receptors and exhibit minimal shedding from the surface. We show that enhanced APCs can present antigenic peptides derived from mRNA encoding for a foreign antigen on MHC complexes in an HLA agnostic manner, which drives antigen-specific T cell responses. The addition of CD86, mbIL-2, and mbIL-12 further enhance the activation and potency of antigen-specific T cells, as measured by an increase in the secretion of inflammatory cytokines upon restimulation (i.e. IFNγ).ConclusionsCell squeezing of human PBMCs with mRNA encoding for signals 1, 2, and 3 has the potential to generate enhanced APCs that drive robust CD8+ T cell response against multiple targets across several disease areas. The versatility of the Cell Squeeze® technology potentially enables rapid exchange of mRNA to other antigens or T cell activation signals.

Development of surface engineered antigenic exosomes as vaccines for respiratory syncytial virus

Respiratory syncytial virus (RSV) is one of the main pathogens associated with lower respiratory tract infections in infants and young children worldwide. Exosomes secreted by antigen presenting cells (APCs) can elicit immune responses by carrying major histocompatibility complex (MHC) class I molecules complexed with antigenic peptides and other co-stimulating factors. Therefore, we developed novel immunomagnetic nanographene particles to sequentially isolate, surface engineer, and release intact dendritic cell (DC) exosomes for use as a potential vaccine platform against RSV. The H-2Db-restricted, immunodominant peptides from RSV (M187–195 and NS161–75) were introduced to MHC-I on DC-derived exosomes to express peptide/MHC-I (pMHC-I) complexes. A mouse model of RSV infection was used to define the immunogenicity of surface engineered exosomes for activating virus-specific immune responses. Ex vivo assays demonstrated that engineered exosomes carrying RSV-specific peptides can elicit interferon-gamma (IFN-γ) production by virus-specific CD8+ T cells isolated from RSV-infected C57BL/6 mice. In vivo assays demonstrated that subcutaneous administration of both M187–195 and NS161–75 engineered exosomes to mice, with or without additional adjuvant, appeared safe and well tolerated, however, did not prime antigen-specific CD8+ T cell responses. Surface engineered exosomes are immunogenic and promising for further development as a vaccine platform.

Characterization of Two Immunodominant Antigenic Peptides in NSP2 of PRRSV-2 and Generation of a Marker PRRSV Strain Based on the Peptides

Porcine reproductive and respiratory syndrome (PRRS) is a widespread disease with great economic importance in the pig industry. Although vaccines against the PRRS virus (PRRSV) have been employed for more than 20 years, differentiating infected from vaccinated animal remains challenging. In this study, all 907 non-structural protein 2 (NSP2) full-length sequences of PRRSV-2 available from GenBank were aligned. Two peptides, at positions 562–627 (m1B) and 749–813 (m2B) of NSP2, were selected, and their potential for use in differential diagnosis was assessed. Both m1B and m2B were recognized by PRRSV-positive pig serum in peptide-coated enzyme-linked immunosorbent assays. Further epitope identification yielded five overlapping short peptides for the immunodominant regions of m1B and m2B. Using the infectious clone of PRRSV HuN4-F112 as a template, the deletion mutants rHuN4-F112-m1B, rHuN4-F112-m2B, and rHuN4-F112-C5-m1B-m2B were generated and successfully rescued in Marc-145 cells. Growth kinetics revealed that deletion of m1B and m2B did not significantly affect virus replication. Hence, m1B and m2B show potential as molecular markers for developing a PRRSV vaccine.