DPP4 Inhibitor Sitagliptin Enhances Lymphocyte Recruitment and Prolongs Survival in a Syngeneic Ovarian Cancer Mouse Model.

Immunity plays a key role in epithelial ovarian cancer (EOC) progression with a well-documented correlation between patient survival and high intratumoral CD8+ to T regulatory cell (Treg) ratios. We previously identified dysregulated DPP4 activity in EOCs as a potentially immune-disruptive influence contributing to a reduction in CXCR3-mediated T-cell infiltration in solid tumours. We therefore hypothesized that inhibition of DPP4 activity by sitagliptin, an FDA-approved inhibitor, would improve T-cell infiltration and function in a syngeneic ID8 mouse model of EOC. Daily oral sitagliptin at 50 mg/kg was provided to mice with established primary EOCs. Sitagliptin treatment decreased metastatic tumour burden and significantly increased overall survival and was associated with significant changes to the immune landscape. Sitagliptin increased overall CXCR3-mediated CD8+ T-cell trafficking to the tumour and enhanced the activation and proliferation of CD8+ T-cells in tumour tissue and the peritoneal cavity. Substantial reductions in suppressive cytokines, including CCL2, CCL17, CCL22 and IL-10, were also noted and were associated with reduced CD4+ CD25+ Foxp3+ Treg recruitment in the tumour. Combination therapy with paclitaxel, however, typical of standard-of-care for patients in palliative care, abolished CXCR3-specific T-cell recruitment stimulated by sitagliptin. Our data suggest that sitagliptin may be suitable as an adjunct therapy for patients between chemotherapy cycles as a novel approach to enhance immunity, optimise T-cell-mediated function and improve overall survival.

Predicted B Cell Epitopes Highlight the Potential for COVID-19 to Drive Self-Reactive Immunity.

COVID-19, caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), whilst commonly characterised as a respiratory disease, is reported to have extrapulmonary manifestations in multiple organs. Extrapulmonary involvement in COVID-19 includes autoimmune-like diseases such as Guillain-Barré syndrome and Kawasaki disease, as well as the presence of various autoantibodies including those associated with autoimmune diseases such a systemic lupus erythematosus (e.g. ANA, anti-La). Multiple strains of SARS-CoV-2 have emerged globally, some of which are found to be associated with increased transmissibility and severe disease. We performed an unbiased comprehensive mapping of the potential for cross-reactivity with self-antigens across multiple SARS-CoV-2 proteins and compared identified immunogenic regions across multiples strains. Using the Immune Epitope Database (IEDB) B cell epitope prediction tool, regions predicted as antibody epitopes with high prediction scores were selected. Epitope sequences were then blasted to eight other global strains to identify mutations within these regions. Of the 15 sequences compared, eight had a mutation in at least one other global strain. Predicted epitopes were then compared to human proteins using the NCBI blast tool. In contrast to studies focusing on short sequences of peptide identity, we have taken an immunological approach to selection criteria for further analysis and have identified 136 alignments of 6-23 amino acids (aa) in 129 human proteins that are immunologically likely to be cross-reactive with SARS-CoV-2. Additionally, to identify regions with significant potential to interfere with host cell function-or promote immunopathology, we identified epitope regions more likely to be accessible to pathogenic autoantibodies in the host, selected using a novel combination of sequence similarity, and modelling protein and alignment localization with a focus on extracellular regions. Our analysis identified 11 new predicted B-cell epitopes in host proteins, potentially capable of explaining key aspects of COVID-19 extrapulmonary pathology, and which were missed in other in silico studies which used direct identity rather than immunologically related functional criteria.

Pullulan-Coated Iron Oxide Nanoparticles for Blood-Stage Malaria Vaccine Delivery.

Vaccines against blood-stage malaria often aim to induce antibodies to neutralize parasite entry into red blood cells, interferon gamma (IFNγ) produced by T helper 1 (Th1) CD4+ T cells or interleukin 4 (IL-4) produced by T helper 2 (Th2) cells to provide B cell help. One vaccine delivery method for suitable putative malaria protein antigens is the use of nanoparticles as vaccine carriers. It has been previously shown that antigen conjugated to inorganic nanoparticles in the viral-particle size range (~40-60 nm) can induce protective antibodies and T cells against malaria antigens in a rodent malaria challenge model. Herein, it is shown that biodegradable pullulan-coated iron oxide nanoparticles (pIONPs) can be synthesized in this same size range. The pIONPs are non-toxic and do not induce conventional pro-inflammatory cytokines in vitro and in vivo. We show that murine blood-stage antigen MSP4/5 from Plasmodium yoelii could be chemically conjugated to pIONPs and the use of these conjugates as immunogens led to the induction of both specific antibodies and IFNγ CD4+ T cells reactive to MSP4/5 in mice, comparable to responses to MSP4/5 mixed with classical adjuvants (e.g., CpG or Alum) that preferentially induce Th1 or Th2 cells individually. These results suggest that biodegradable pIONPs warrant further exploration as carriers for developing blood-stage malaria vaccines.

Functional Recognition by CD8+ T Cells of Epitopes with Amino Acid Variations Outside Known MHC Anchor or T Cell Receptor Recognition Residues.

Peptide-based vaccines can be safer and more cost effective than whole organism vaccines. Previous studies have shown that inorganic polystyrene nanoparticles (PSNPs) covalently conjugated to the minimal immunodominant peptide epitope from murine liver stage malaria (SYIPSAEKI) induced potent CD8+ T cell responses. Many pathogens, including malaria, have polymorphic T cell epitope regions. Amino acid changes in positions that are contact residues for the T cell receptor (TCR) often alter the specific cross-reactivity induced by the peptide antigen, and it is largely assumed that changes outside of these residues have little impact. Herein, each amino acid residue (except major histocompatibility complex (MHC) anchors) was systematically changed to an alanine. Peptide epitopes with altered amino acids outside T cell contact residues were still recognized by T cells induced by PSNPs-SYIPSAEKI (KI) vaccines, albeit at lower levels, except for the variant SYIPSAAKI (A7). PSNPs-SYIPSAAKI vaccines further elicited high responses to the index KI peptide. None of the epitopes displayed altered peptide ligand (APL) antagonism in vitro, and re-stimulating SYIPSAEKI and SYIPSAAKI together synergistically enhanced IFN-γ production by the T cells. These results show epitope variation in non-TCR recognition residues can have effects on T cell reactivity, suggesting that such natural variation may also be driven by immune pressure. Additionally, when re-modelling peptides to enhance the cross-reactivity of vaccines, both TCR recognition and non-recognition residues should be considered.

Antioxidant-Based Medicinal Properties of Stingless Bee Products: Recent Progress and Future Directions.

Stingless bees are a type of honey producers that commonly live in tropical countries. Their use for honey is being abandoned due to its limited production. However, the recent improvements in stingless bee honey production, particularly in South East Asia, have brought stingless bee products back into the picture. Although there are many stingless bee species that produce a wide spread of products, known since old eras in traditional medicine, the modern medical community is still missing more investigational studies on stingless bee products. Whereas comprehensive studies in the current era attest to the biological and medicinal properties of honeybee (Apis mellifera) products, the properties of stingless bee products are less known. This review highlights for the first time the medicinal benefits of stingless bee products (honey, propolis, pollen and cerumen), recent investigations and promising future directions. This review emphasizes the potential antioxidant properties of these products that in turn play a vital role in preventing and treating diseases associated with oxidative stress, microbial infections and inflammatory disorders. Summarizing all these data and insights in one manuscript may increase the commercial value of stingless bee products as a food ingredient. This review will also highlight the utility of stingless bee products in the context of medicinal and therapeutic properties, some of which are yet to be discovered.

Biodegradable PLGA-b-PEG Nanoparticles Induce T Helper 2 (Th2) Immune Responses and Sustained Antibody Titers via TLR9 Stimulation.

Sustained immune responses, particularly antibody responses, are key for protection against many endemic infectious diseases. Antibody responses are often accompanied by T helper (Th) cell immunity. Herein we study small biodegradable poly (ethylene glycol)-b-poly (lactic-co-glycolic acid) nanoparticles (PEG-b-PLGA NPs, 25-50 nm) as antigen- or adjuvant-carriers. The antigen carrier function of PEG-b-PLGA NPs was compared against an experimental benchmark polystyrene nanoparticles (PS NPs, 40-50 nm), both conjugated with the model antigen ovalbumin (OVA-PS NPs, and OVA-PEG-b-PLGA NPs). The OVA-PEG-b-PLGA NPs induced sustained antibody responses to Day 120 after two immunizations. The OVA-PEG-b-PLGA NPs as a self-adjuvanting vaccine further induced IL-4 producing T-helper cells (Th2), but not IFN-γ producing T-cells (Th1). The PEG-b-PLGA NPs as a carrier for CpG adjuvant (CpG-PEG-b-PLGA NPs) were also tested as mix-in vaccine adjuvants comparatively for protein antigens, or for protein-conjugated to PS NPs or to PEG-b-PLGA NPs. While the addition of this adjuvant NP did not further increase T-cell responses, it improved the consistency of antibody responses across all immunization groups. Together these data support further development of PEG-b-PLGA NPs as a vaccine carrier, particularly where it is desired to induce Th2 immunity and achieve sustained antibody titers in the absence of affecting Th1 immunity.

A Synthetic Nanoparticle Based Vaccine Approach Targeting MSP4/5 Is Immunogenic and Induces Moderate Protection Against Murine Blood-Stage Malaria.

Malaria remains a significant health problem in many tropical and sub-tropical regions. The development of vaccines against the clinically active blood-stage of infection needs to consider variability and polymorphism in target antigens, and an adjuvant system able to induce broad spectrum immunity comprising both antibodies and helper T cells. Moreover, recent studies have shown some conventional pro-inflammatory adjuvants can also promote expansion of immunosuppressive regulatory T cells (Treg) and myeloid derived suppressor cells (MDSC), both of which could negatively impact malaria disease progression. Herein, we explore the ability of a model nanoparticle delivery system (polystyrene nanoparticles; PSNPs), previously proven to not induce conventional inflammation, Treg or MDSC, to induce immunity to MSP4/5 from Plasmodium yoelii, a member of the MSP4 and MSP5 family of proteins which are highly conserved across diverse malaria species including P. falciparum. The results show PSNPs-MSP4/5 conjugates are highly immunogenic, inducing immune responses comprising both T helper 1 (Th1) and Th2 cellular immunity, and a spectrum of antibody subclasses including IgG1, IgG2a, and IgG2b. Benchmarked against Alum and Complete Freund's Adjuvant (CFA), the immune responses that were induced were of comparable or higher magnitude, for both T cell frequencies by ELISpot and antibody responses in terms of ELISA end titer. Importantly, immunization with PSNPs-MSP4/5 induced partial protection against malaria blood-stage infection (50-80%) shown to be mechanistically dependent on interferon gamma (IFN-γ) production. These results expand the scope of adjuvants considered for malaria blood-stage vaccine development to those that do not use conventional adjuvant pathways and emphasizes the critical role of cellular immunity and specifically IFN-γ producing cells in providing moderate protection against blood-stage malaria comparable to Freunds adjuvant.

Cyrene as a bio-based solvent for HATU mediated amide coupling.

Amide bonds are one of the underpinning linkages in all living systems and are fundamental within drug discovery. Current methods towards their synthesis frequently rely on the use of dipolar aprotic solvents; however, due to increasingly stringent regulations and growing societal pressures, safe and more sustainable alternatives are highly sought after. Herein, we evaluate the application of the bio-based solvent Cyrene™ in the HATU mediated synthesis of amides and peptides. We found that Cyrene functioned as a competent replacement for DMF in the synthesis of a series of lead-like compounds and dipeptides (25 examples, 63-100%).

Non-Invasive Fluorescent Monitoring of Ovarian Cancer in an Immunocompetent Mouse Model.

Ovarian cancers (OCs) are the most lethal gynaecological malignancy, with high levels of relapse and acquired chemo-resistance. Whilst the tumour⁻immune nexus controls both cancer progression and regression, the lack of an appropriate system to accurately model tumour stage and immune status has hampered the validation of clinically relevant immunotherapies and therapeutic vaccines to date. To address this need, we stably integrated the near-infrared phytochrome iRFP720 at the ROSA26 genomic locus of ID8 mouse OC cells. Intrabursal ovarian implantation into C57BL/6 mice, followed by regular, non-invasive fluorescence imaging, permitted the direct visualization of tumour mass and distribution over the course of progression. Four distinct phases of tumour growth and dissemination were detectable over time that closely mimicked clinical OC progression. Progression-related changes in immune cells also paralleled typical immune profiles observed in human OCs. Specifically, we observed changes in both the CD8+ T cell effector (Teff):regulatory (Treg) ratio, as well as the dendritic cell (DC)-to-myeloid derived suppressor cell (MDSC) ratio over time across multiple immune cell compartments and in peritoneal ascites. Importantly, iRFP720 expression had no detectible influence over immune profiles. This new model permits non-invasive, longitudinal tumour monitoring whilst preserving host⁻tumour immune interactions, and allows for the pre-clinical assessment of immune profiles throughout disease progression as well as the direct visualization of therapeutic responses. This simple fluorescence-based approach provides a useful new tool for the validation of novel immuno-therapeutics against OC.

Design of Peptide-Based Nanovaccines Targeting Leading Antigens From Gynecological Cancers to Induce HLA-A2.1 Restricted CD8+ T Cell Responses.

Gynecological cancers are a leading cause of mortality in women. CD8+ T cell immunity largely correlates with enhanced survival, whereas inflammation is associated with poor prognosis. Previous studies have shown polystyrene nanoparticles (PSNPs) are biocompatible, do not induce inflammation and when used as vaccine carriers for model peptides induce CD8+ T cell responses. Herein we test the immunogenicity of 24 different peptides, from three leading vaccine target proteins in gynecological cancers: the E7 protein of human papilloma virus (HPV); Wilms Tumor antigen 1 (WT1) and survivin (SV), in PSNP conjugate vaccines. Of relevance to vaccine development was the finding that a minimal CD8+ T cell peptide epitope from HPV was not able to induce HLA-A2.1 specific CD8+ T cell responses in transgenic humanized mice using conventional adjuvants such as CpG, but was nevertheless able to generate strong immunity when delivered as part of a specific longer peptide conjugated to PSNPs vaccines. Conversely, in most cases, when the minimal CD8+ T cell epitopes were able to induce immune responses (with WT1 or SV super agonists) in CpG, they also induced responses when conjugated to PSNPs. In this case, extending the sequence around the CD8+ T cell epitope, using the natural protein context, or engineering linker sequences proposed to enhance antigen processing, had minimal effects in enhancing or changing the cross-reactivity pattern induced by the super agonists. Nanoparticle approaches, such as PSNPs, therefore may offer an alternative vaccination strategy when conventional adjuvants are unable to elicit the desired CD8+ T cell specificity. The findings herein also offer sequence specific insights into peptide vaccine design for nanoparticle-based vaccine carriers.

Scope and limitations of a DMF bio-alternative within Sonogashira cross-coupling and Cacchi-type annulation.

Pd-catalysed C-C bond formation is an essential tool within the pharmaceutical and agrochemical industries. Many of these reactions rely heavily on polar aprotic solvents; however, despite their utility, these solvents are incompatible with the drive towards more sustainable chemical synthesis. Herein, we describe the scope and limitations of an alternative to DMF derived from renewable sources (CyreneTM) in Sonogashira cross-coupling and Cacchi-type annulations.

Synthesis of 2-BMIDA 6,5-bicyclic heterocycles by Cu(i)/Pd(0)/Cu(ii) cascade catalysis of 2-iodoaniline/phenols.

A one-pot cascade reaction for the synthesis of 2-BMIDA 6,5-bicyclic heterocycles has been developed using Cu(i)/Pd(0)/Cu(ii) catalysis. 2-Iodoanilines and phenols undergo a Cu(i)/Pd(0)-catalyzed Sonogashira reaction with ethynyl BMIDA followed by in situ Cu(ii)-catalyzed 5-endo-dig cyclization to generate heterocyclic scaffolds with a BMIDA functional group in the 2-position. The method provides efficient access to borylated indoles, benzofurans, and aza-derivatives, which can be difficult to access through alternative methods.

A Model to Study the Impact of Polymorphism Driven Liver-Stage Immune Evasion by Malaria Parasites, to Help Design Effective Cross-Reactive Vaccines.

Malaria parasites engage a multitude of strategies to evade the immune system of the host, including the generation of polymorphic T cell epitope sequences, termed altered peptide ligands (APLs). Herein we use an animal model to study how single amino acid changes in the sequence of the circumsporozoite protein (CSP), a major target antigen of pre-erythrocytic malaria vaccines, can lead to a reduction of cross reactivity by T cells. For the first time in any APL model, we further compare different inflammatory adjuvants (Montanide, Poly I:C), non-inflammatory adjuvants (nanoparticles), and peptide pulsed dendritic cells (DCs) for their potential capacity to induce broadly cross reactive immune responses. Results show that the capacity to induce a cross reactive response is primarily controlled by the T cell epitope sequence and cannot be modified by the use of different adjuvants. Moreover, we identify how specific amino acid changes lead to a one-way cross reactivity: where variant-x induced responses are re-elicited by variant-x and not variant-y, but variant-y induced responses can be re-elicited by variant-y and variant-x. We discuss the consequences of the existence of this one-way cross reactivity phenomenon for parasite immune evasion in the field, as well as the use of variant epitopes as a potential tool for optimized vaccine design.

Polymorphism in liver-stage malaria vaccine candidate proteins: immune evasion and implications for vaccine design.

The pre-erythrocytic stage of infection by malaria parasites represents a key target for vaccines that aim to eradicate malaria. Two important broad immune evasion strategies that can interfere with vaccine efficacy include the induction of dendritic cell (DC) dysfunction and regulatory T cells (Tregs) by blood-stage malaria parasites, leading to inefficient priming of T cells targeting liver-stage infections. The parasite also uses 'surgical strike' strategies, whereby polymorphism in pre-erythrocytic antigens can interfere with host immunity. Specifically, we review how even single amino acid changes in T cell epitopes can lead to loss of binding to major histocompatibility complex (MHC), lack of cross-reactivity, or antagonism and immune interference, where simultaneous or sequential stimulation with related variants of the same T cell epitope can cause T cell anergy or the conversion of effector to immunosuppressive T cell phenotypes.

A Nanoparticle Based Sp17 Peptide Vaccine Exposes New Immuno-Dominant and Species Cross-reactive B Cell Epitopes.

Sperm protein antigen 17 (Sp17), expressed in primary as well as in metastatic lesions in >83% of patients with ovarian cancer, is a promising ovarian cancer vaccine candidate. Herein we describe the formulation of nanoparticle based vaccines based on human Sp17 (hSp17) sequence derived peptides, and map the immuno-dominant T cell and antibody epitopes induced using such formulations. The primary T and B cell immuno-dominant region within Sp17 was found to be the same when using biocompatible nanoparticle carriers or the conventional "mix-in" pro-inflammatory adjuvant CpG, both mapping to amino acids (aa) 111-142. However, delivery of hSp17111-142 as a nanoparticle conjugate promoted a number of new properties, changing the dominant antibody isotype induced from IgG2a to IgG1 and the fine specificity of the B cell epitopes within hSp17111-142, from an immuno-dominant region 134-142 aa for CpG, to region 121-138 aa for nanoparticles. Associated with this change in specificity was a substantial increase in antibody cross-reactivity between mouse and human Sp17. These results indicate conjugation of antigen to nanoparticles can have major effects on fine antigen specificity, which surprisingly could be beneficially used to increase the cross-reactivity of antibody responses.

Mapping T and B cell epitopes in sperm protein 17 to support the development of an ovarian cancer vaccine.

Ovarian cancer (OC) is the seventh most common cancer in women worldwide, and the leading cause of death from gynaecological malignancy. Immunotherapeutic strategies including cancer vaccines are considered less toxic and more specific than current treatments. Sperm surface protein (Sp17) is a protein aberrantly expressed in primary as well as in metastatic lesions in >83% of ovarian cancer patients. Vaccines based on the Sp17 protein are immunogenic and protective in animal models. To map the immunogenic regions and support the development of human Sp17 peptide based vaccines, we used 6 overlapping peptides of the human Sp17 sequence adjuvanted with CpG to immunise humanised HLA-A2.1 transgenic C57BL/6 mice, and assessed immunogenicity by ELISPOT and ELISA. No CD8 T cells were found to be induced to a comprehensive panel of 10 HLA-A2.1 or H-2K(b) binding predicted epitopes. However, one of the 6 peptides, hSp17111-142, induced high levels of antibodies and IFN-γ producing T cells (but not IL-17 or IL-4) both in C57BL/6 and in C57BL/6-HLA-A2.1 transgenic mice. C57BL/6 mice immunised with CpG adjuvanted hSp17111-142 significantly prolonged the life-span of the mice bearing the ovarian carcinoma ID8 cell line. We further mapped the immuno-dominant B and T cell epitope regions within hSp17111-142 using ELISPOT and competition ELISA. Herein, we report the identification of a single immuno-dominant B cell (134-142 aa) epitope and 2 T helper 1 (Th1) cell epitopes (111-124 aa and 124-138 aa). These result together support further exploration of hSp17111-142 peptide formulations as vaccines against ovarian cancer.

Montanide, Poly I:C and nanoparticle based vaccines promote differential suppressor and effector cell expansion: a study of induction of CD8 T cells to a minimal Plasmodium berghei epitope.

The development of practical and flexible vaccines to target liver stage malaria parasites would benefit from an ability to induce high levels of CD8 T cells to minimal peptide epitopes. Herein we compare different adjuvant and carrier systems in a murine model for induction of interferon gamma (IFN-γ) producing CD8 T cells to the minimal immuno-dominant peptide epitope from the circumsporozoite protein (CSP) of Plasmodium berghei, pb9 (SYIPSAEKI, referred to as KI). Two pro-inflammatory adjuvants, Montanide and Poly I:C, and a non-classical, non-inflammatory nanoparticle based carrier (polystyrene nanoparticles, PSNPs), were compared side-by-side for their ability to induce potentially protective CD8 T cell responses after two immunizations. KI in Montanide (Montanide + KI) or covalently conjugated to PSNPs (PSNPs-KI) induced such high responses, whereas adjuvanting with Poly I:C or PSNPs without conjugation was ineffective. This result was consistent with an observed induction of an immunosuppressed environment by Poly I:C in the draining lymph node (dLN) 48 h post injection, which was reflected by increased frequencies of myeloid derived suppressor cells (MDSCs) and a proportion of inflammation reactive regulatory T cells (Treg) expressing the tumor necrosis factor receptor 2 (TNFR2), as well as decreased dendritic cell (DC) maturation. The other inflammatory adjuvant, Montanide, also promoted proportional increases in the TNFR2(+) Treg subpopulation, but not MDSCs, in the dLN. By contrast, injection with non-inflammatory PSNPs did not cause these changes. Induction of high CD8 T cell responses, using minimal peptide epitopes, can be achieved by non-inflammatory carrier nanoparticles, which in contrast to some conventional inflammatory adjuvants, do not expand either MDSCs or inflammation reactive Tregs at the site of priming.