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.

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.

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.

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.

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.

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.

Mimotope mimicking epidermal growth factor receptor alleviates mononuclear cell infiltration in exocrine glands induced by muscarinic acetylcholine 3 receptor.

The muscarinic type 3 receptor (M3R) plays a pivotal role in the pathogenesis of Sjögren's syndrome (SS). Characterization of the crosstalk between M3R and EGFR has been investigated in some human malignancies. In the current study, we sought to investigate whether EGFR mimic immunization could alleviate the abnormal immune responses in an experimental SS-like model triggered by M3R peptides. After immunization with the combination of mimotope and M3R peptide, the active immunization targeting EGFR induced by the mimotope could reduce the marked infiltration of mononuclear cells, the high titer of antibodies against M3R and the accumulation of crucial pro-inflammatory cytokines in mice immunized with M3R peptide. Mechanistic analysis showed that mimotope immunization could alleviate the autoimmune response through inhibiting mitochondrion-mediated anti-apoptosis and up-regulating the FAS apoptosis pathway. These results may help to clarify the role of M3R in the pathogenesis of SS and suggested that transactivation of the EGFR signaling pathway may help M3R activate the autoimmune response in the pathogenesis of SS.

Identification of an Endogenously Generated Cryptic Collagen Epitope (XL313) That May Selectively Regulate Angiogenesis by an Integrin Yes-associated Protein (YAP) Mechano-transduction Pathway.

Extracellular matrix (ECM) remodeling regulates angiogenesis. However, the precise mechanisms by which structural changes in ECM proteins contribute to angiogenesis are not fully understood. Integrins are molecules with the ability to detect compositional and structural changes within the ECM and integrate this information into a network of signaling circuits that coordinate context-dependent cell behavior. The role of integrin αvß3 in angiogenesis is complex, as evidence exists for both positive and negative functions. The precise downstream signaling events initiated by αvß3 may depend on the molecular characteristics of its ligands. Here, we identified an RGD-containing cryptic collagen epitope that is generated in vivo. Surprisingly, rather than inhibiting αvß3 signaling, this collagen epitope promoted αvß3 activation and stimulated angiogenesis and inflammation. An antibody directed to this RGDKGE epitope but not other RGD collagen epitopes inhibited angiogenesis and inflammation in vivo. The selective ability of this RGD epitope to promote angiogenesis and inflammation depends in part on its flanking KGE motif. Interestingly, a subset of macrophages may represent a physiologically relevant source of this collagen epitope. Here, we define an endothelial cell mechano-signaling pathway in which a cryptic collagen epitope activates αvß3 leading to an Src and p38 MAPK-dependent cascade that leads to nuclear accumulation of Yes-associated protein (YAP) and stimulation of endothelial cell growth. Collectively, our findings not only provide evidence for a novel mechano-signaling pathway, but also define a possible therapeutic strategy to control αvß3 signaling by targeting a pro-angiogenic and inflammatory ligand of αvß3 rather than the receptor itself.

Synergistic anti-tumor therapy by a comb-like multifunctional antibody nanoarray with exceptionally potent activity.

Simultaneously blocking multiple mediators offers new hope for the treatment of complex diseases. However, the curative potential of current combination therapy by chronological administration of separate monoclonal antibodies (mAbs) or multi-specific mAbs is still moderate due to inconvenient manipulation, low cooperative effectors, poor pharmacokinetics and insufficient tumor accumulation. Here, we describe a facile strategy that arms distinct mAbs with cooperative effectors onto a long chain to form a multicomponent comb-like nano mAb. Unlike dissociative parental mAbs, the multifunctional mAb nanoarray (PL-RB) constructed from type I/II anti-CD20 mAbs shows good pharmacokinetics. This PL-RB simultaneously targets distinct epitopes on a single antigen (Ag) and neighboring Ags on different lymphocytes. This unique intra- and intercellular Ag cross-linking endows the multifunctional mAb nanoarray with potent apoptosis activity. The exceptional apoptosis, complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) that are synchronously evoked by the nano PL-RB are further synergistically promoted via enhanced permeability and retention (EPR), which resulted in high intratumor accumulation and excellent anti-lymphoma efficiency.

Cancer immunotherapy by a recombinant phage vaccine displaying EGFR mimotope: an in vivo study.

To date, several small molecule inhibitors and monoclonal-antibodies (like ICR-62) have been used to treat tumors over-expressing epidermal growth factor receptor (EGFR). However, the limitations associated with these conventional applications accentuate the necessity of alternative approaches. Mimotopes as compelling molecular tools could rationally be employed to circumvent these drawbacks. In the present study, an M13 phage displaying ICR-62 binding peptide mimotope is exploited as a vaccine candidate. It exhibited high affinity towards ICR62 and polyclonal anti-P-BSA antibodies. Following the mice immunization, phage-based mimotope vaccine induced humoral immunity. Elicited anti-EGFR mimotope antibodies were detected using ELISA method. Moreover, the phage vaccine was tested on the Lewis lung carcinoma mice model to investigate the prophylactic and therapeutic effects. The tumor volume was measured and recorded in different animal groups to evaluate the anti-tumor effects of the vaccine. Our data indicate that the reported phage-based mimotope could potentially elicit specific antibodies resulting in low titers of EGFR-specific antibodies and reduced tumor growth. However, in vivo experiments of prophylactic or therapeutic vaccination showed no specific advantage. Furthermore, phage-mimotope vaccine might be a promising approach in the field of cancer immunotherapy.

Immunological activity difference between native calreticulin monomers and oligomers.

We have recently demonstrated that the greatly increased immunological activities of recombinant murine calreticulin (rCRT) are largely attributed to its self-oligomerization. Although native CRT (nCRT) can also oligomerize under stress conditions in vitro, whether this phenomenon could occur inside cells and the immunological activity difference between nCRT monomers and oligomers remained unclear. In this study, we illustrated the formation of CRT oligomers in tranfectant cells under "heat & low pH" (42°C/pH 6.5) condition. The mixture of nCRT oligomers and monomers (OnCRT) was obtained after 3 hr treatment of murine monomeric nCRT (MnCRT) under similar condition (42°C/pH 5.0) in vitro. The OnCRT thus obtained was better recognized by 2 monoclonal Abs from mice that had been immunized with oligomeric rCRT. Unlike MnCRT, OnCRT was able to elicit CRT-specific IgG production in mice. OnCRT also stimulated bone-marrow derived dendritic cells (BMDCs) to secrete significantly higher levels of TNF-α, IL-6 and IL-12p40 than did MnCRT in vitro. We postulate that oligomerization of soluble CRT may occur under certain pathophysiological conditions (e.g. ultrahyperpyrexia) and the resultant oligomers may exhibit exaggerated immunostimulating activities, thereby affiliating the inflammatory responses in vivo.

Immunization against a saccharide epitope accelerates clearance of experimental gonococcal infection.

The emergence of ceftriaxone-resistant strains of Neisseria gonorrhoeae may herald an era of untreatable gonorrhea. Vaccines against this infection are urgently needed. The 2C7 epitope is a conserved oligosaccharide (OS) structure, a part of lipooligosaccharide (LOS) on N gonorrhoeae. The epitope is expressed by 94% of gonococci that reside in the human genital tract (in vivo) and by 95% of first passaged isolates. Absence of the 2C7 epitope shortens the time of gonococcal carriage in a mouse model of genital infection. To circumvent the limitations of saccharide immunogens in producing long lived immune responses, previously we developed a peptide mimic (called PEP1) as an immunologic surrogate of the 2C7-OS epitope and reconfigured it into a multi-antigenic peptide, (MAP1). To test vaccine efficacy of MAP1, female BALB/c mice were passively immunized with a complement-dependent bactericidal monoclonal antibody specific for the 2C7 epitope or were actively immunized with MAP1. Mice immunized with MAP1 developed a TH1-biased anti-LOS IgG antibody response that was also bactericidal. Length of carriage was shortened in immune mice; clearance occurred in 4 days in mice passively administered 2C7 antibody vs. 6 days in mice administered control IgG3λ mAb in one experiment (p = 0.03) and 6 vs. 9 days in a replicate experiment (p = 0.008). Mice vaccinated with MAP1 cleared infection in 5 days vs. 9 days in mice immunized with control peptide (p = 0.0001 and p = 0.0002, respectively in two replicate experiments). Bacterial burden was lower over the course of infection in passively immunized vs. control mice in both experiments (p = 0.008 and p = 0.0005); burdens were also lower in MAP1 immunized mice vs. controls (p<0.0001) and were inversely related to vaccine antibodies induced in the vagina (p = 0.043). The OS epitope defined by mAb 2C7 may represent an effective vaccine target against gonorrhea, which is rapidly becoming incurable with currently available antibiotics.

Activation of inflammatory cells and cytokines by peptide epitopes in vitro: a simple in-vitro screening assay for prioritizing them for in-vivo studies.

OBJECTIVE: Antigen-specific immune modulation is an attractive approach to atherosclerosis treatment. The aim of this study was to develop an in-vitro assay to screen peptide molecules for their inflammatory propensity. MATERIALS: Human dendritic cells derived from CD14(+) monocytes were activated using peptides derived from apolipoprotein B100 (ApoB), heat shock protein 60 (HSP60) and complement cascade (peptide A) in vitro, and used for priming autologous T cells. Proliferation of T cells, their differentiation to regulatory cells (Treg) and their cytokine profile were studied. The efficacy of the peptides in preventing atherosclerosis was studied in ApoB(tm2Sgy)/Ldlr(tm1Her/J) knockout mice. RESULTS AND CONCLUSION: ApoB and HSP60 peptides induced T-cell proliferation and expansion of regulatory T cells with interleukin-10 and transforming growth factor-ß secretion. In comparison, peptide A was a poor stimulator of T cells and was found to induce tumor necrosis factor-α secretion by activated T cells. ApoB and HSP60 peptides were found to reduce early atherosclerotic lesion formation in mice by 32.1 and 33.5 %, respectively, while the reduction with peptide A was 5.7 %. Thus the in-vitro assay shows an apparent correlation with in-vivo activity and can be developed as a screening assay to prioritize the candidate molecules for animal efficacy testing.

Is epitope recognition of shrimp allergens useful to predict clinical reactivity?

BACKGROUND: Shrimp is a frequent cause of severe allergic reactions world-wide. Due to issues such as cross-reactivity, diagnosis of shrimp allergy is still inaccurate, requiring the need for double-blind, placebo-controlled food challenges (DBPCFC). A better understanding of the relationship between laboratory findings and clinical reactivity is needed. OBJECTIVE: To determine whether sensitization to certain shrimp allergens or recognition of particular IgE epitopes of those allergens are good biomarkers of clinical reactivity to shrimp. METHODS: Thirty-seven consecutive patients were selected with clinical histories of shrimp allergy. Skin prick test, specific IgE determinations, DBPCFC and immunoblot assays to shrimp extract were performed. IgE binding to synthetic overlapping peptides representing the sequence of the four allergens from the Pacific white shrimp (Litopenaeus vannamei) identified to date (Lit v1, Lit v2, Lit v3 and Lit v4) was analysed. RESULTS: Of 37 (46%) patients, 17 had a positive challenge to shrimp (11 children and 6 adults). By microarray, patients with positive challenges showed more intense binding to shrimp peptides than those with negative challenges. Statistically significant differences in terms of the frequency and intensity of IgE binding to some epitopes were observed between the two groups. Diagnostic efficiency was higher for individual epitopes than for proteins. Particularly, efficiency was highest for certain Lit v 1 and Lit v 2 epitopes, followed by Lit v 3 and Lit v 4 epitopes. CONCLUSION AND CLINICAL RELEVANCE: Patients with positive shrimp challenges present in general more intense and diverse epitope recognition to all four shrimp allergens. IgE antibodies to these shrimp epitopes could be used as biomarkers for prediction of clinical reactivity in subjects with sensitization to shrimp. Patients with positive shrimp challenges show more intense sensitization and more diverse epitope recognition. Several IgE-binding shrimp epitopes could be used as biomarkers for predicting clinical reactivity in subjects with sensitization to shrimp.