Activity Relationship of Poly(ethylenimine)-Based Liposomes as Group A Streptococcus Vaccine Delivery Systems.

Untreated group A Streptococcus (GAS) can lead to a range of life-threatening diseases, including rheumatic heart disease. To date, no therapeutic or prophylactic vaccines are commercially available to treat or prevent GAS infection. Development of a peptide-based subunit vaccine offers a promising solution, negating the safety issues of live-attenuated or inactive vaccines. Subunit vaccines administer small peptide fragments (antigens), which are typically poorly immunogenic. Therefore, these peptide antigens require formulation with an immune stimulant and/or vaccine delivery platform to improve their immunogenicity. We investigated polyelectrolyte complexes (PECs) and polymer-coated liposomes as self-adjuvanting delivery vehicles for a GAS B cell peptide epitope conjugated to a universal T-helper epitope and a synthetic toll-like receptor 2-targeting moiety lipid core peptide-1 (LCP-1). A structure-activity relationship of cationic PEC vaccines containing different external PEI-coatings (poly(ethylenimine); 10 kDa PEI, 25 kDa PEI, and a synthetic mannose-functionalized 25 kDa PEI) formed vaccines PEC-1, PEC-2, and PEC-3, respectively. All three PEC vaccines induced J8-specific systemic immunoglobulin G (IgG) antibodies when administered intranasally to female BALB/c mice without the use of additional adjuvants. Interestingly, PEC-3 induced the highest antibody titers among all tested vaccines, with the ability to effectively opsonize two clinically isolated GAS strains. A comparative study of PEC-2 and PEC-3 with liposome-based delivery systems was performed subcutaneously. LCP-1 was incorporated into a liposome formulation (DPPC, DPPG and cholesterol), and the liposomes were externally coated with PEI (25 kDa; Lip-2) or mannosylated PEI (25 kDa; Lip-3). All liposome vaccines induced stronger humoral immune responses compared to their PEC counterparts. Notably, sera of mice immunized with Lip-2 and Lip-3 produced significantly higher opsonic activity against clinically isolated GAS strains compared to the positive control, P25-J8 emulsified with the commercial adjuvant, complete Freund's adjuvant (CFA). This study highlights the capability of a PEI-liposome system to act as a self-adjuvanting vehicle for the delivery of GAS peptide antigens and protection against GAS infection.

Naïve antibody library derived monoclonal antibody against VP35 of Ebola virus.

Ebola virus is notorious for causing severe and even deadly haemorrhagic fever in infected humans and non-human primates. The high fatality rate of Ebola virus disease (EVD) has highlighted the need for effective diagnosis and treatment. Two monoclonal antibodies (mAbs) have been approved by USFDA for treatment of EVD. Virus surface glycoprotein is the common target for diagnostic and therapy including vaccines. Even so, VP35, a viral RNA polymerase cofactor and interferon inhibitor could be a potential target to curb EVD. The present work describes the isolation of three mAb clones from a phage-displayed human naïve scFv library against recombinant VP35. The clones showed binding against rVP35 in vitro and inhibition of VP35 in luciferase reporter gene assay. Structural modelling analysis was also carried out to identify the binding interactions involved in the antibody-antigen interaction model. This allows some insight into the "fitness" of the binding pocket between the paratope and target epitope which would be useful for the design of new mAbs through in silico means in the future. In conclusion, the information obtained from the 3 isolated mAbs could be potentially useful in the quest to improve VP35 targeting for therapeutic development in the future.

Development of a Platform for Producing Recombinant Protein Components of Epitope Vaccines for the Prevention of COVID-19.

A new platform for creating anti-coronavirus epitope vaccines has been developed. Two loop-like epitopes with lengths of 22 and 42 amino acid residues were selected from the receptor-binding motif of the Spike protein from the SARS-CoV-2 virus that participate in a large number of protein-protein interactions in the complexes with ACE2 and neutralizing antibodies. Two types of hybrid proteins, including one of the two selected epitopes, were constructed. To fix conformation of the selected epitopes, an approach using protein scaffolds was used. The homologue of Rop protein from the Escherichia coli ColE1 plasmid containing helix-turn-helix motif was used as an epitope scaffold for the convergence of C- and N-termini of the loop-like epitopes. Loop epitopes were inserted into the turn region. The conformation was additionally fixed by a disulfide bond formed between the cysteine residues present within the epitopes. For the purpose of multimerization, either aldolase from Thermotoga maritima, which forms a trimer in solution, or alpha-helical trimerizer of the Spike protein from SARS-CoV-2, was attached to the epitopes incorporated into the Rop-like protein. To enable purification on the heparin-containing sorbents, a short fragment from the heparin-binding hemagglutinin of Mycobacterium tuberculosis was inserted at the C-terminus of the hybrid proteins. All the obtained proteins demonstrated high level of immunogenicity after triplicate parenteral administration to mice. Sera from the mice immunized with both aldolase-based hybrid proteins and the Spike protein SARS-CoV-2 trimerizer-based protein with a longer epitope interacted with both the inactivated SARS-CoV-2 virus and the Spike protein receptor-binding domain at high titers.

Tumour neoantigen mimicry by microbial species in cancer immunotherapy.

Tumour neoantigens arising from cancer-specific mutations generate a molecular fingerprint that has a definite specificity for cancer. Although this fingerprint perfectly discriminates cancer from healthy somatic and germline cells, and is therefore therapeutically exploitable using immune checkpoint blockade, gut and extra-gut microbial species can independently produce epitopes that resemble tumour neoantigens as part of their natural gene expression programmes. Such tumour molecular mimicry is likely not only to influence the quality and strength of the body's anti-cancer immune response, but could also explain why certain patients show favourable long-term responses to immune checkpoint blockade while others do not benefit at all from this treatment. This article outlines the requirement for tumour neoantigens in successful cancer immunotherapy and draws attention to the emerging role of microbiome-mediated tumour neoantigen mimicry in determining checkpoint immunotherapy outcome, with far-reaching implications for the future of cancer immunotherapy.

pH-Dependent Grafting of Cancer Cells with Antigenic Epitopes Promotes Selective Antibody-Mediated Cytotoxicity.

A growing class of immunotherapeutics work by redirecting components of the immune system to recognize markers on the surface of cancer cells. However, such modalities will remain confined to a relatively small subgroup of patients because of the lack of universal targetable tumor biomarkers among all patients. Here, we designed a unique class of agents that exploit the inherent acidity of solid tumors to selectively graft cancer cells with immuno-engager epitopes. Our targeting approach is based on pHLIP, a unique peptide that selectively targets tumors in vivo by anchoring to cancer cell surfaces in a pH-dependent manner. We established that pHLIP-antigen conjugates trigger the recruitment of antibodies to the surface of cancer cells and induce cytotoxicity by peripheral blood mononuclear and engineered NK cells. These results indicate that these agents have the potential to be applicable to treating a wide range of solid tumors and to circumvent problems associated with narrow windows of selectivity.

Targeted imaging and targeted therapy of breast cancer cells via fluorescent double template-imprinted polymer coated silicon nanoparticles by an epitope approach.

Targeting is vital for precise positioning and efficient therapy, and integrated platforms for diagnosis and therapy have attracted more and more attention. Herein, we established dual-template molecularly imprinted polymer (MIP) coated fluorescent silicon nanoparticles (Si NPs) by using the linear peptide of the extracellular region of human epidermal growth factor receptor-2 (HER2) and adopting doxorubicin (DOX) as templates for targeted imaging and targeted therapy. Benefiting from the epitope imprinting approach, the imprinted sites generated by peptides on the MIP surface can be employed for recognizing the corresponding protein, which allowed the MIP to specifically and actively target HER2-positive breast cancer cells. Because of its ability to identify breast cancer cells, the MIP was applied for targeted fluorescence imaging by taking advantage of the excellent fluorescence properties of Si NPs, and the DOX-loaded MIP (MIP@DOX) can act as a therapeutic probe to effectively target and kill breast cancer cells. In fluorescence images, the targeting of the MIP promoted more uptake of the nanoparticles by cells than the non-imprinted polymer (NIP), so HER2-positive breast cancer cells incubated with the MIP exhibited stronger fluorescence, and there was no significant difference in fluorescence when HER2-negative cells and normal cells were respectively hatched with the MIP and NIP. Importantly, the cell viability was evaluated to demonstrate targeted accumulation and therapy of MIP@DOX for breast cancer cells. The nanoplatform for diagnosis and therapy combined the high sensitivity of fluorescence with the high selectivity of the molecular imprinting technique, which holds vital potential in targeted imaging and targeted therapy in vitro.

Preparation of Dual-Template Epitope Imprinted Polymers for Targeted Fluorescence Imaging and Targeted Drug Delivery to Pancreatic Cancer BxPC-3 Cells.

Molecularly imprinted polymers were commonly used for drug delivery. However, single-template molecularly imprinted polymers often fail to achieve both drug delivery and precise targeting. To address this issue, a dual-template molecularly imprinted polymer nanoparticle used for targeted diagnosis and drug delivery for pancreatic cancer BxPC-3 cells (FH-MIPNPs) was prepared. In the FH-MIPNPs, the 71-80 peptide of human fibroblast growth-factor-inducible 14 modified with glucose (Glu-FH) and bleomycin (BLM) were used as templates simultaneously, so that the FH-MIPNPs could load BLM and bind to the BxPC-3 cells, which overexpress human fibroblast growth-factor-inducible 14 (FN14). Targeted imaging experiments in vitro show that the FH-MIPNPs could specifically target BxPC-3 cells and that there is no targeting effect on cells without expression of FN14. In vivo antitumor experiment results demonstrated that the FH-MIPNP-loaded BLM (FH-MIPNPs/BLM) could inhibit the growth of xenografts tumor of BxPC-3 (tumor volume increased to 1.05×), which shows that FH-MIPNPs/BLM had obvious targeted therapeutic effect compared to the other three control groups of BLM, FH-NIPNPs/BLM, and physiological saline (tumor volume increased to 1.5×, 1.6×, and 2.4×, respectively). What is more, FH-MIPNPs have low biotoxicity through toxicity experiments in vitro and in vivo, which is favorable toward making molecularly imprinted polymers an effective platform for tumor-targeted imaging and therapy.

HIV-1 Membrane-Proximal External Region Fused to Diphtheria Toxin Domain-A Elicits 4E10-Like Antibodies in Mice.

The production of broadly neutralizing antibodies (bNAbs) is a major goal in the development of an HIV-1 vaccine. The membrane-proximal external region (MPER) of gp41, which plays a critical role in the virus membrane fusion process, is highly conserved and targeted by bNAbs 2F5, 4E10, and 10E8. As such, MPER could be a promising epitope for vaccine design. In this study, diphtheria toxin domain A (CRM197, amino acids 1-191) was used as a scaffold to display the 2F5 and 4E10 epitopes of MPER, named CRM197-A-2F5 and CRM197-A-4E10. Modest neutralizing activities were detected against HIV-1 clade B and D viruses in the sera from mice immunized with CRM197-A-4E10. Monoclonal antibodies raised from CRM197-A-4E10 could neutralize several HIV-1 strains, and epitope-mapping analysis indicated that some antibodies recognized the same amino acids as 4E10. Collectively, we show that 4E10-like antibodies can be induced by displaying MPER epitopes using an appropriate scaffold. These results provide insights for HIV-1 MPER-based immunogens design.

A Case Study on the Keap1 Interaction with Peptide Sequence Epitopes Selected by the Peptidomic mRNA Display.

Many protein-protein and peptide-protein interactions (PPIs) play key roles in the regulation of biological functions, and therefore, the modulation of PPIs has become an attractive target of new drug development. Although a number of PPIs have already been identified, over 100 000 unknown PPIs are predicted to exist. To uncover such unknown PPIs, it is important to devise a conceptually distinct method from that of currently available methods. Herein, an mRNA display by using a total RNA library derived from various human tissues, which serves as a unique method to physically isolate peptide epitopes that potentially bind to a target protein of interest, is reported. In this study, selection was performed against Kelch-like ECH-associated protein (Keap1) as a model target protein, leading to a peptide epitope originating from astrotactin-1 (ASTN1). It turned out that this ASTN1 peptide was able to interact with Keap1 more strongly than that with a known peptide derived from Nrf2; a well-known, naturally occurring Keap1 binder. This case study demonstrates the applicability of peptidomic mRNA display for the rapid exploration of consensus binding peptide motifs and the potential for the discovery of unknown PPIs with other proteins of interest.

Naturally presented HLA class I-restricted epitopes from the neurotrophic factor S100-ß are targets of the autoimmune response in type 1 diabetes.

Type 1 diabetes (T1D) results from the destruction of pancreatic ß-cells by the immune system, and CD8+ T lymphocytes are critical actors in this autoimmune response. Pancreatic islets are surrounded by a mesh of nervous cells, the peri-insular Schwann cells, which are also targeted by autoreactive T lymphocytes and express specific antigens, such as the neurotrophic factor S100-ß. Previous work has shown increased proliferative responses to whole S100-ß in both human T1D patients and the nonobese diabetic (NOD) mouse model. We describe for the first time naturally processed and presented epitopes (NPPEs) presented by class I human leukocyte antigen-A*02:01 (A2.1) molecules derived from S100-ß. These NPPEs triggered IFN-γ responses more frequently in both newly diagnosed and long-term T1D patients compared with healthy donors. Furthermore, the same NPPEs are recognized during the autoimmune response leading to diabetes in A2.1-transgenic NOD mice as early as 4 wk of age. Interestingly, when these NPPEs are used to prevent diabetes in this animal model, an acceleration of the disease is observed together with an exacerbation in insulitis and an increase in S100-ß-specific cytotoxicity in vaccinated animals. Whether these can be used in diabetes prevention needs to be carefully evaluated in animal models before use in future clinical assays.-Calviño-Sampedro, C., Gomez-Tourino, I., Cordero, O. J., Reche, P. A., Gómez-Perosanz, M., Sánchez-Trincado, J. L., Rodríguez, M. Á., Sueiro, A. M., Viñuela, J. E., Calviño, R. V. Naturally presented HLA class I-restricted epitopes from the neurotrophic factor S100-ß are targets of the autoimmune response in type 1 diabetes.

Glycation of hemoglobin leads to the immunogenicity as a result of neo-epitope generation.

Non-enzymatic glycation occurs rapidly which ultimately leads to the formation of advanced glycation endproducts (AGEs). These AGEs have shown to associated with the development of many diseases such as diabetes-mellitus. This study is focused on immunological characterization of glycated-Hb induced by d-ribose. Here, we analysed the immunogenicity of glycated-Hb by direct binding and competitive inhibition ELISA. Direct binding ELISA confirmed that glycated-Hb was highly immunogenic and induced high titre antibodies as compared to native-Hb. The antigen binding specificity and cross reactivity of these antibodies were also screened by competitive inhibition ELISA. The IgG from rabbit sera showed enhanced binding of glycated-Hb than native-Hb. Thus, it is possible that alterations in Hb induced by d-ribose could have generated highly immunogenic neoepitopes. Moreover, induced antibodies were also found to cross-react with other modified/native proteins. On the basis of the results of this study, we presume that this type of structural perturbations in Hb in vivo by d-ribose might take place in untreated diabetic condition that could induce such type of immunogenic auto-antibodies. Furthermore, increased level of these auto-antibodies could serve as a biomarker in diabetes and its progression.

Neoantigen-specific immunity in low mutation burden colorectal cancers of the consensus molecular subtype 4.

BACKGROUND: The efficacy of checkpoint blockade immunotherapies in colorectal cancer is currently restricted to a minority of patients diagnosed with mismatch repair-deficient tumors having high mutation burden. However, this observation does not exclude the existence of neoantigen-specific T cells in colorectal cancers with low mutation burden and the exploitation of their anti-cancer potential for immunotherapy. Therefore, we investigated whether autologous neoantigen-specific T cell responses could also be observed in patients diagnosed with mismatch repair-proficient colorectal cancers. METHODS: Whole-exome and transcriptome sequencing were performed on cancer and normal tissues from seven colorectal cancer patients diagnosed with mismatch repair-proficient tumors to detect putative neoantigens. Corresponding neo-epitopes were synthesized and tested for recognition by in vitro expanded T cells that were isolated from tumor tissues (tumor-infiltrating lymphocytes) and from peripheral mononuclear blood cells stimulated with tumor material. RESULTS: Neoantigen-specific T cell reactivity was detected to several neo-epitopes in the tumor-infiltrating lymphocytes of three patients while their respective cancers expressed 15, 21, and 30 non-synonymous variants. Cell sorting of tumor-infiltrating lymphocytes based on the co-expression of CD39 and CD103 pinpointed the presence of neoantigen-specific T cells in the CD39+CD103+ T cell subset. Strikingly, the tumors containing neoantigen-reactive TIL were classified as consensus molecular subtype 4 (CMS4), which is associated with TGF-ß pathway activation and worse clinical outcome. CONCLUSIONS: We have detected neoantigen-targeted reactivity by autologous T cells in mismatch repair-proficient colorectal cancers of the CMS4 subtype. These findings warrant the development of specific immunotherapeutic strategies that selectively boost the activity of neoantigen-specific T cells and target the TGF-ß pathway to reinforce T cell reactivity in this patient group.

In drug-induced, immune-mediated hepatitis, interleukin-33 reduces hepatitis and improves survival independently and as a consequence of FoxP3+ T-cell activity.

Immune-mediated, drug-induced hepatitis is a rare complication of halogenated volatile anesthetic administration. IL-4-regulated Th2-polarized reactions initiate this type and other types of hepatitis, while the mechanisms that regulate the severity remain elusive. IL-33 is an innate, IL-4-inducing, Th2-polarizing cytokine that has been detected in patients with liver failure and has been associated with upregulated ST2+Foxp3+CD4+CD25+ T cells; however, roles for IL-33 in drug-induced hepatitis are unclear. We investigated IL-33 in an anesthetic, immune-mediated hepatitis modeled in BALB/c, IL-33-/- and ST2-/- mice, as well as in patients with anesthetic hepatitis. The hepatic IL-33 and ST2 levels were elevated in BALB/c mice (p < 0.05) with hepatitis, and anti-IL-33 diminished hepatitis (p < 0.05) without reducing IL-33 levels. The complete absence of IL-33 reduced IL-10 (p < 0.05) and ST2+Foxp3+CD4+CD25+ T cells (p < 0.05), as well as reduced the overall survival (p < 0.05), suggesting suppressive roles for IL-33 in anesthetic, immune-mediated hepatitis. All of the mice demonstrated similar levels of CD4+ T-cell proliferation following direct T-cell receptor stimulation, but we detected splenic IL-33 and ST2-negative Foxp3+CD4+CD25+ T cells in ST2-/- mice that developed less hepatitis than BALB/c mice (p < 0.05), suggesting that ST2-negative Foxp3+CD4+CD25+ T cells reduced hepatitis. In patients, serum IL-33 and IPEX levels were correlated in controls (r2 = 0.5, p < 0.05), similar to the levels in mice, but not in anesthetic hepatitis patients (r2 = 0.01), who had elevated IL-33 (p < 0.001) and decreased IPEX (p < 0.01). Our results suggest that, in anesthetic, immune-mediated hepatitis, IL-33 does not regulate the CD4+ T-cell proliferation that initiates hepatitis, but IL-33, likely independent of ST2, reduces hepatitis via upregulation of Foxp3+CD4+CD25+ T cells. Further studies are needed to translate the role of IL-33 to human liver disease.

Combination of M2e peptide with stalk HA epitopes of influenza A virus enhances protective properties of recombinant vaccine.

BACKGROUND: Influenza infection could be more effectively controlled if a multi-purpose vaccine with the ability to induce responses against most, or all, influenza A subtypes could be generated. Conserved viral proteins are a promising basis for the creation of a broadly protective vaccine. In the present study, the immunogenicity and protective properties of three recombinant proteins (vaccine candidates), comprising conserved viral proteins fused with bacterial flagellin, were compared. METHODS: Balb/c mice were immunized intranasally with recombinant proteins comprising either one viral protein (the ectodomain of the M2 protein, 'M2e') or two viral proteins (M2e and the hemagglutinin second subunit 'HA2' epitope) genetically fused with flagellin. Further, two different consensus variants of HA2 were used. Therefore, three experimental positives were used in addition to the negative control (Flg-his). The mucosal, humoral, and T-cell immune responses to these constructs were evaluated. RESULT: We have demonstrated that insertion of the HA2 consensus polypeptide (aa 76-130), derived from either the first (HA2-1) or second (HA2-2) virus phylogenetic group, into the recombinant Flg4M2e protein significantly enhanced its immunogenicity and protective properties. Intranasal administration of the vaccine candidates (Flg-HA2-2-4M2e or Flg-HA2-1-4M2e) induced considerable mucosal and systemic responses directed at both the M2e-protein and, in general, the influenza A virus. However, the immune response elicited by the Flg-HA2-1-4M2e protein was weaker than the one generated by Flg-HA2-2-4M2e. These recombinant proteins containing both viral peptides provide complete protection from lethal challenge with various influenza viruses: A/H3N2; A/H2N2; and A/H5N1. CONCLUSION: This study demonstrates that the intranasal administration of Flg-HA2-2-4M2e recombinant protein induces a strong immune response which provides broad protection against various influenza viruses. This construct is therefore a strong candidate for development as a universal vaccine.

Synthetic Immunotherapeutics against Gram-negative Pathogens.

While traditional drug discovery continues to be an important platform for the search of new antibiotics, alternative approaches should also be pursued to complement these efforts. We herein designed a class of molecules that decorate bacterial cell surfaces with the goal of re-engaging components of the immune system toward Escherichia coli and Pseudomonas aeruginosa. More specifically, conjugates were assembled using polymyxin B (an antibiotic that inherently attaches to the surface of Gram-negative pathogens) and antigenic epitopes that recruit antibodies found in human serum. We established that the spacer length played a significant role in hapten display within the bacterial cell surface, a result that was confirmed both experimentally and via molecular dynamics simulations. Most importantly, we demonstrated the specific killing of bacteria by our agent in the presence of human serum. By enlisting the immune system, these agents have the potential to pave the way for a potent antimicrobial modality.

Efficient co-delivery of neo-epitopes using dispersion-stable layered double hydroxide nanoparticles for enhanced melanoma immunotherapy.

Cancer immunotherapy has shown tremendous progresses in recent years for various cancers and layered double hydroxide (LDH) nanoparticles are demonstrated as effective adjuvants for protein-based vaccines. This research further shows that the colloidal stability of LDH-based vaccines significantly influences the therapeutic efficacy and LDH nanoparticles are able to adjuvant multiple tumor-associated antigen peptides to provoke strong cell-mediated immune responses for effective inhibition of cancer growth. The LDH-based multi-target therapeutic vaccines were constructed by assembling epitope peptides and CpG onto LDH nanoparticles. Using melanoma as the model cancer and Tyrosinase-related protein 2 (Trp2) peptide as the model antigen, we demonstrated that dispersion-stable LDH-based vaccine induced stronger cytotoxic T-lymphocyte (CTL) responses and significantly inhibited tumor growth in comparison with aggregated LDH-based vaccine. We further constructed multi-target dispersion-stable LDH-based vaccine by co-loading Trp2, two mutated epitopes (M27 and M30) and CpG, which showed remarkable inhibition of melanoma growth. These results suggest that dispersion-stable LDH nanoparticles are an ideal platform to load multi-antigens and immune stimulants as effective personalized therapeutic cancer vaccines.

Design, synthesis and evaluation of an anthraquinone derivative conjugated to myelin basic protein immunodominant (MBP85-99) epitope: Towards selective immunosuppression.

Anthraquinone type compounds, especially di-substituted amino alkylamino anthraquinones have been widely studied as immunosuppressants. The anthraquinone ring is part of mitoxandrone that has been used for the treatment of multiple sclerosis (MS) and several types of tumors. A desired approach for the treatment of MS would be the immunosuppression and elimination of specific T cells that are responsible for the induction of the disease. Herein, the development of a peptide compound bearing an anthraquinone derivative with the potential to specifically destroy the encephalitogenic T cells responsible for the onset of MS is described. The compound consists of the myelin basic protein (MBP) 85-99 immunodominant epitope (MBP85-99) coupled to an anthraquinone type molecule (AQ) via a disulfide (S-S) and 6 amino hexanoic acid (Ahx) residues (AQ-S-S-(Ahx)6MBP85-99). AQ-S-S-(Ahx)6MBP85-99 could bind to HLA II DRB1*-1501 antigen with reasonable affinity (IC50 of 56 nM) The compound was localized to the nucleus of Jurkat cells (an immortalized line of human T lymphocytes) 10 min after its addition to the medium and resulted in lowered Bcl-2 levels (apoptosis). Entrance of the compound was abolished when cells were pre-treated with cisplatin, an inhibitor of thioredoxin reductase. Accordingly, levels of free thiols were elevated in the culture supernatants of Jurkat cells exposed to N-succinimidyl 3-(2-pyridyldithio) propionate coupled to (Ahx)6MBP85-99 via a disulphide (SPDP-S-S-(Ahx)6MBP85-99) but returned to normal after exposure to cisplatin. These results raise the possibility of AQ-S-S-(Ahx)6MBP85-99 being used as an eliminator of encephalitogenic T cells via implication of the thioredoxin system for the generation of the toxic, thiol-containing moiety (AQ-SH). Future experiments would ideally determine whether SPDP-S-S-(Ahx)6MBP85-99 could incorporate into HLA II DRB1*-1501 tetramers and neutralize encephalitogenic T cell lines sensitized to MBP85-99.

Designer vaccine nanodiscs for personalized cancer immunotherapy.

Despite the tremendous potential of peptide-based cancer vaccines, their efficacy has been limited in humans. Recent innovations in tumour exome sequencing have signalled the new era of personalized immunotherapy with patient-specific neoantigens, but a general methodology for stimulating strong CD8α+ cytotoxic T-lymphocyte (CTL) responses remains lacking. Here we demonstrate that high-density lipoprotein-mimicking nanodiscs coupled with antigen (Ag) peptides and adjuvants can markedly improve Ag/adjuvant co-delivery to lymphoid organs and sustain Ag presentation on dendritic cells. Strikingly, nanodiscs elicited up to 47-fold greater frequencies of neoantigen-specific CTLs than soluble vaccines and even 31-fold greater than perhaps the strongest adjuvant in clinical trials (that is, CpG in Montanide). Moreover, multi-epitope vaccination generated broad-spectrum T-cell responses that potently inhibited tumour growth. Nanodiscs eliminated established MC-38 and B16F10 tumours when combined with anti-PD-1 and anti-CTLA-4 therapy. These findings represent a new powerful approach for cancer immunotherapy and suggest a general strategy for personalized nanomedicine.

A Novel Chemical Biology Approach for Mapping of Polymyxin Lipopeptide Antibody Binding Epitopes.

Polymyxins B and E (i.e., colistin) are a family of naturally occurring lipopeptide antibiotics that are our last line of defense against multidrug resistant (MDR) Gram-negative pathogens. Unfortunately, nephrotoxicity is a dose-limiting factor for polymyxins that limits their clinical utility. Our recent studies demonstrate that polymyxin-induced nephrotoxicity is a result of their extensive accumulation in renal tubular cells. The design and development of safer, novel polymyxin lipopeptides is hampered by our limited understanding of their complex structure-nephrotoxicity relationships. This is the first study to employ a novel targeted chemical biology approach to map the polymyxin recognition epitope of a commercially available polymyxin mAb and demonstrate its utility for mapping the kidney distribution of a novel, less nephrotoxic polymyxin lipopeptide. Eighteen novel polymyxin lipopeptide analogues were synthesized with modifications in the polymyxin core domains, namely, the N-terminal fatty acyl region, tripeptide linear segment, and cyclic heptapeptide. Surface plasmon resonance epitope mapping revealed that the monoclonal antibody (mAb) recognition epitope consisted of the hydrophobic domain (N-terminal fatty acyl and position 6/7) and diaminobutyric acid (Dab) residues at positions 3, 5, 8, and 9 of the polymyxin molecule. Structural diversity within the hydrophobic domains and Dab 3 position are tolerated. Enlightened with an understating of the structure-binding relationships between the polymyxin mAb and the core polymyxin scaffold, we can now rationally employ the mAb to probe the kidney distribution of novel polymyxin lipopeptides. This information will be vital in the design of novel, safer polymyxins through chemical tailoring of the core scaffold and exploration of the elusive/complex polymyxin structure-nephrotoxicity relationships.

Structural characterization of low molecular weight polysaccharide from Astragalus membranaceus and its immunologic enhancement in recombinant protein vaccine against systemic candidiasis.

Structure and immunologic enhancement of low molecular weight polysaccharide (LMW-ASP) isolated from the root of Astragalus membranaceus (Fisch) Bge. Were detected in recombinant protein vaccine. Structure analysis of LMW-ASP revealed that LMW-ASP (Mw=5.6kDa) was an acid heteropolysaccharide, which consisted of Glc, Gal, Ara, Xyl and GalA in ratio of 10.0：1.3：1.7：1.0：0.9. Recombinant protein (rP-HSP90C) contained epitope C (LKVIRK) from heat shock protein 90 (HSP90) of Candida albicans was used as a vaccine. The results indicated that LMW-ASP significantly promoted specific antibody titers IgG, IgG1, IgG2b, and IL-2, IL-4, IL-10, IL-12 in sera of mice immunized with rP-HSP90C (p<0.05). It was also found LMW-ASP improved DTH response in HSP90C-injceted mice. More importantly, the mice immunized with rP-HSP90C/LMW-ASP had fewer CFU (colony forming unites) in the kidneys compared to the mice immunized with rP-HSP90C (p<0.05). Therefore, LMW-ASP could be exploited into the novel adjuvant to enhance the efficacy of recombinant protein vaccine.