Multiple Antigen Peptide Research Articles

Design and synthesis of HLA-A*02-restricted Hantaan virus multiple-antigenic peptide for CD8+ T cells

BackgroundHantaan virus (HTNV) can cause hemorrhagic fever with renal syndrome (HFRS) in humans with severe morbidity and high mortality. Although inactivated HFRS vaccines are given annually for prevention in populations, China still has the highest number of HFRS cases and deaths worldwide. Consequently, vaccination for HFRS requires the development of novel, more effective vaccines. Epitope peptide vaccines have been developed rapidly in recent years and are considered a novel approach for the prevention of infection. Specifically, the multiple antigenic peptide (MAP) design with preferable immunogenicity can arouse a satisfactory immune response for vaccination. However, there are few reports on the design and evaluation of MAP for HTNV.MethodsThree HLA-A*02-restricted 9-mer cytotoxic T lymphocyte (CTL) epitopes on HTNV glycoprotein and one HLA-A*02-restricted 9-mer CTL epitope on the HTNV nucleocapsid, which have been proven to be immunoprotective in our previous study, were selected for the design of HTNV MAP. A four-branched HTNV MAP was evaluated by the IFN-γ-secreting enzyme-linked immunospot assay and proliferation induction capacity of CD8+ T cells and compared with the single HTNV CTL epitope in 17 HLA-A*02+ patients with HFRS. The Mann–Whitney U test was used for comparison of parameters between different subject groups.ResultsThe macromolecular HTNV MAP was designed with a polylysine core and four radially branched single CTL epitope chains. Importantly, HTNV MAP could stimulate CD8+ T cell secretion of IFN-γ in HLA-A*02+ patients with HFRS. The frequency of IFN-γ-secreting CD8+ T cells in the MAP stimulation group was significantly higher than that in the single HTNV CTL epitope stimulation groups (P < 0.005). Meanwhile, the activity of IFN-γ-secreting CD8+ T cells in the HTNV MAP group was also higher than that of the single CTL epitope groups (P < 0.05). Moreover, there was a much stronger ability of HTNV MAP to stimulate CD8+ T cell proliferation compared with that of a single HTNV CTL epitope.ConclusionsThe designed HTNV MAP could induce CTL responses ex vivo and may be considered a candidate for the design and development of novel HTNV peptide vaccines.

A Multiple Antigen Peptide Vaccine Containing CD4+ T Cell Epitopes Enhances Humoral Immunity against Trichinella spiralis Infection in Mice.

Multiepitope peptide vaccine has some advantages over traditional recombinant protein vaccine due to its easy and fast production and possible inclusion of multiple protective epitopes of pathogens. However, it is usually poorly immunogenic and needs to conjugate to a large carrier protein. Peptides conjugated to a central lysine core to form multiple antigen peptides (MAPs) will increase the immunogenicity of peptide vaccine. In this study, we constructed a MAP consisting of CD4+ T cell and B cell epitopes of paramyosin (Pmy) of Trichinella spiralis (Ts-Pmy), which has been proved to be a good vaccine candidate in our previous work. The immunogenicity and induced protective immunity of MAP against Trichinella spiralis (T. spiralis) infection were evaluated in mice. We demonstrated that mice immunized with MAP containing CD4+ T cell and B cell epitopes (MAP-TB) induced significantly higher protection against the challenge of T. spiralis larvae (35.5% muscle larva reduction) compared to the MAP containing B cell epitope alone (MAP-B) with a 12.4% muscle larva reduction. The better protection induced by immunization of MAP-TB was correlated with boosted antibody titers (both IgG1 and IgG2a) and mixed Th1/Th2 cytokine production secreted by the splenocytes of immunized mice. Further flow cytometry analysis of lymphocytes in spleens and draining lymph nodes demonstrated that mice immunized with MAP-TB specifically enhanced the generation of T follicular helper (Tfh) cells and germinal center (GC) B cells, while inhibiting follicular regulatory CD4+ T (Tfr) cells and regulatory T (Treg) cells. Immunofluorescence staining of spleen sections also confirmed that MAP-TB vaccination enhanced the formation of GCs. Our results suggest that CD4+ T cell epitope of Ts-Pmy is crucial in vaccine component for inducing better protection against T. spiralis infection.

A novel nucleolin-binding peptide for Cancer Theranostics.

Background: Cancer-specific ligands have been of great interest as pharmaceutical carriers due to the potential for site-specific delivery. In particular, cancer-specific peptides have many advantages over nanoparticles and antibodies, including high biocompatibility, low immunogenicity, and the formation of nontoxic metabolites. The goal of the present study was the development of a novel cancer-specific ligand.Methods: Cancer-specific peptide ligands were screened using a one-bead-one-compound (OBOC) combinatorial method combined with a multiple-antigen-peptide (MAP) synthesis method. The specificity of the peptide ligands toward cancer cells was tested in vitro using a whole-cell binding assay, flow cytometry, and fluorescence confocal microscopy. The tissue distribution profile and therapeutic efficacy of a paclitaxel (PTX)-conjugated peptide ligand was assessed in vivo using xenograft mouse models.Results: We discovered that AGM-330 specifically bound to cancer cells in vitro and in vivo. Treatment with PTX-conjugated AGM-330 dramatically inhibited cancer cell growth in vitro and in vivo compared to treatment with PTX alone. The results of pull-down assay and LC-MS/MS analyses showed that membrane nucleolin (NCL) was the target protein of AGM-330. Although NCL is known as a nuclear protein, we observed that it was overexpressed on the membranes of cancer cells. In particular, membrane NCL neutralization inhibited growth in cancer cells in vitro.Conclusions: In summary, our findings indicated that NCL-targeting AGM-330 has great potential for use in cancer diagnosis and targeted drug delivery in cancer therapy.

Abstract B062: Enabling the rapid generation of allogeneic artificial antigen presenting cell (aAPC) Red Cell Therapeutics with a loadable MHC system

Abstract Next generation sequencing technologies, coupled with personal neoantigen identification approaches, have significantly improved the capability to develop patient-specific T-cell-based therapies targeting tumors. Current peptide neoantigen vaccine approaches are promising, but do not adequately stimulate and expand patient T cells to the levels required to achieve robust efficacy. To address these limitations, Rubius Therapeutics has developed allogeneic artificial antigen presenting cells (aAPCs), which express the required signals for complete T cell activation: antigenic peptide-MHC complex, costimulatory ligand and cytokine. By engineering red cells to express immunomodulatory signals 1, 2 and 3, these aAPCs dramatically expand antigen-specific T cells in vivo and promote T cell memory and effector function. To use the aAPC approach with personal neoantigens, Rubius Therapeutics has developed a loadable MHC system that enables the rapid generation of aAPCs. Unloaded MHC class I molecules typically do not express robustly on the cell surface, as innate biophysical instability and internal cellular quality control mechanisms prevent MHC molecules that lack a loaded peptide from displaying on the cell surface. We demonstrate here that the empty MHC class I complex can be stably presented on the red cell surface. This was achieved by fusing wild-type MHC class I [human] HLA-A2 and β2 microglobulin to the glycophorin A transmembrane domain. Of note, disulfide engineering of HLA-A2 did not change expression when compared to wild-type constructs on the red cells. Fluorescently labelled peptides were used to measure the kinetics of peptide loading, which demonstrated that disulfide-engineering dramatically increased binding rates. Further, peptide competition experiments indicated slightly increased affinity for peptide with disulfide engineered HLA-A2. Functional testing revealed that addition of exogenous HPV E7 peptide and co-incubation of loaded cells with Jurkat-Lucia NFAT cells expressing HPV E7-specific TCR cells demonstrated TCR-specific activity. Additional TCR activity assays showed that peptide loaded onto empty wild-type HLA-A2 was stable up to 3 days, while disulfide-engineered HLA-A2 activity was abrogated after 1 day. Finally, this concept was extended to other HLA genes, demonstrating that our approach could achieve expression of a variety of HLA alleles on the red cell surface, including MHC class II alleles in the HLA-DR and HLA-DP gene families. Collectively, these results demonstrate that Rubius’ loadable aAPC system is highly generalizable and can be applied to produce aAPC populations presenting multiple antigenic peptides across a range of functional alleles on the red cell surface. Rubius’ allogeneic aAPC system represents a novel approach to generate effective personal neoantigen-specific therapies in a wide patient population with requisite ease of manufacturability Citation Format: Christopher L Moore, Sneha Pawar, Mellissa Nixon, Timothy J Lyford, Douglas C McLaughlin, Shamael R Dastagir, Abigail Bracha, Lori Melancon, Christopher L Carpenter, Thomas J Wickham, Tiffany F Chen. Enabling the rapid generation of allogeneic artificial antigen presenting cell (aAPC) Red Cell Therapeutics with a loadable MHC system [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B062. doi:10.1158/1535-7163.TARG-19-B062

A multiepitope peptide vaccine against HCV stimulates neutralizing humoral and persistent cellular responses in mice

BackgroundAlthough DAAs hold promise to significantly reduce rates of chronic HCV infections, its eradication still requires development of an effective vaccine. Prolonged T cell responses and cross neutralizing antibodies are ideal for vaccination against the infection. We aimed to design and synthesize a 6 multi epitope peptide vaccine candidate and provide evidence for production of extended cellular and neutralizing Abs in mice.MethodsSix peptides derived from conserved epitopes in E1, E2 (n = 2),NS4B, NS5A and NS5B were designed, synthesized in a multiple antigenic peptide (MAP) form and administered w/o adjuvant to BALB/c mice as HCVp6-MAP at doses ranging from 800 ng to 16 μg. Humoral responses to structural epitopes were assayed by ELISA at different times after injection. ELISpot assay was used to evaluate IFN ɣ producing CD4+/ CD8+ T- lymphocytes at extended durations i.e. > 20 weeks. Viral neutralization by mice sera was tested for genotypes 2a (JFH1) and a chimeric 2a/4a virus (ED43/JFH1) in HCVcc culture.ResultsHCVp6-MAP confers potent viral neutralization and specific cellular responses at > 1600 ng/ animal for at least 20 weeks.ConclusionWe report on a promising anti HCV vaccine for future studies on permissive hosts and in clinical trials.

Immunization with a peptide mimicking Lipoteichoic acid protects mice against Staphylococcus aureus infection

Lipoteichoic acid (LTA), a major component of the cell wall of Staphylococcus aureus (S. aureus), is not generally considered as an ideal vaccine candidate since it is a thymus-independent antigen. In this study, we screened a 12-mer phage peptide library and identified a series of peptide sequences that can mimic the epitope of LTA. A tetra-branched multiple antigenic peptide, named MAP2-3, comprising one of the positive peptide sequences (GHKEDRQWCQHS), was synthesized. Immunization with MAP2-3 induced LTA-specific IgG antibodies, prolonged the survival time, and decreased the bacterial burden in organs of mice infected with S. aureus. Moreover, passive immunization with polyclonal anti-MAP2-3 sera reduced bacterial load in organs of mice with bacteremia, alleviated acute lung injury in mice with pneumonia, and decreased the size of lesions in mice with skin infection. The number of LTA-specific antibody-secreting cells in the spleen of MAP2-3 immunized mice were significantly higher than that in the control mice. In summary, as a surrogate of LTA, vaccination with MAP2-3 elicited humoral immune response and protected mice from S. aureus infection. This study provides a new option to design vaccines against S. aureus.

Multiple Antigenic Peptide System Coupled with Amyloid Beta Protein Epitopes As An Immunization Approach to Treat Alzheimer's Disease.

Latest studies suggest that Alzheimer's disease (AD) is one of the "four big killers" that threaten the health of the elderly. AD affects about 46 million people across the world, and there is a critical need for research on new therapies for treating AD. The purpose of the present study was to develop and evaluate immunogens to elicit antibodies against the formation of amyloid beta protein (Aβ), a pathological hallmark of AD, as a therapeutic strategy in AD. In this study, serial potential immunogenic epitopes were screened according to the Aβ sequence. The screened linear epitopes on the Aβ C-terminal fragment were coupled with either the carrier protein keyhole limpet hemocyanin (KLH) or the synthesized 4-branch peptide (MAP4). MAP4 immunogens could effectively elicit immunogenicity against Aβ1-42 monomer and fiber in Balb/C mice. Furthermore, MAP4 sera could also effectively inhibit the formation of the Aβ1-42 fiber. Interestingly, one of the MAP4 sera was able to depolymerize the Aβ1-42 fibers that had aggregated. The monoclonal antibody, 1H7, was shown to inhibit the formation of Aβ1-42 fiber. MAP4 carrier may provide benefits over current immunization strategies, as it does not induce inflammation. In conclusion, the MAP4-Aβ conjugates offer a promising approach for the development of a safe and effective AD vaccine.

Immunogenicity and Efficacy of a Novel Multi-Antigenic Peptide Vaccine Based on Cross-Reactivity between Feline and Human Immunodeficiency Viruses

For the development of an effective HIV-1 vaccine, evolutionarily conserved epitopes between feline and human immunodeficiency viruses (FIV and HIV-1) were determined by analyzing overlapping peptides from retroviral genomes that induced both anti-FIV/HIV T cell-immunity in the peripheral blood mononuclear cells from the FIV-vaccinated cats and the HIV-infected humans. The conserved T-cell epitopes on p24 and reverse transcriptase were selected based on their robust FIV/HIV-specific CD8+ cytotoxic T lymphocyte (CTL), CD4+ CTL, and polyfunctional T-cell activities. Four such evolutionarily conserved epitopes were formulated into four multiple antigen peptides (MAPs), mixed with an adjuvant, to be tested as FIV vaccine in cats. The immunogenicity and protective efficacy were evaluated against a pathogenic FIV. More MAP/peptide-specific CD4+ than CD8+ T-cell responses were initially observed. By post-third vaccination, half of the MAP/peptide-specific CD8+ T-cell responses were higher or equivalent to those of CD4+ T-cell responses. Upon challenge, 15/19 (78.9%) vaccinated cats were protected, whereas 6/16 (37.5%) control cats remained uninfected, resulting in a protection rate of 66.3% preventable fraction (p = 0.0180). Thus, the selection method used to identify the protective FIV peptides should be useful in identifying protective HIV-1 peptides needed for a highly protective HIV-1 vaccine in humans.

Broad cross-reactivity of the T-cell repertoire achieves specific and sufficiently rapid target searching

The molecular recognition of T-cell receptors is the hallmark of the adaptive immunity. Given the finiteness of the T-cell repertoire, individual T-cell receptors are necessary to be cross-reactive to multiple antigenic peptides. In this study, we quantify the variability of the cross-reactivity by using a string model that estimates the binding affinity between two sequences of amino acids. We examine sequences of 10,000 human T-cell receptors and 10,000 antigenic peptides, and obtain a full spectrum of cross-reactivity of the receptor-peptide binding. Then, we find that the cross-reactivity spectrum is broad. Some T-cells are reactive to 1000 peptides, but some T-cells are reactive to only one or two peptides. Since the degree of cross-reactivity has a correlation with the (un)binding affinity of receptors, we further investigate how the broad cross-reactivity affects the target searching of T-cells. High cross-reactive T-cells may not require many trials for searching correct targets, but they may spend long time to unbind from incorrect targets. In contrast, low cross-reactive T-cells may not spend long time to ignore incorrect targets, but they require many trials for screening correct targets. We evaluate this hypothesis, and show that the broad cross-reactivity of the natural T-cell repertoire can balance the trade-off between the rapid screening and unbinding penalty.

Active Vaccination With EMMPRIN-Derived Multiple Antigenic Peptide (161-MAP) Reduces Angiogenesis in a Dextran Sodium Sulfate (DSS)-Induced Colitis Model.

Ulcerative colitis (UC) is an autoimmune disease that affects the colon and shares many clinical and histological features with the dextran sulfate sodium (DSS)-induced colitis model in mice. Angiogenesis is a critical component in many autoimmune diseases, as well as in the DSS-induced colitis model, and is it partially mediated by EMMPRIN, a multifunctional protein that can induce the expression of both the potent pro-angiogenic vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs). We asked whether targeting EMMPRIN by active vaccination, using a novel, specific epitope in the protein, synthesized as a multiple antigenic peptide (MAP), could trigger beneficial effects in the DSS-induced colitic C57BL/6J mice. Mice were vaccinated with four boost injections (50 μg each) of either 161-MAP coding for the EMMPRIN epitope or the scrambled control peptide (Scr-MAP) emulsified in Freund's adjuvant. We show that male mice that were vaccinated with 161-MAP lost less weight, demonstrated improved disease activity indices (DAI), had reduced colitis histological score, and their colons were longer in comparison to mice vaccinated with the Scr-MAP. The 161-MAP vaccination also reduced serum and colon levels of EMMPRIN, colon concentrations of VEGF, MMP-9, and TGFβ, and vessel density assessed by CD31 staining. A similar effect was observed in female mice vaccinated with 161-MAP, including weight loss, colitis histological score, colon length, colon levels of EMMPRIN and colon concentrations of VEGF. However, for female mice, the changes in DAI values, EMMPRIN serum levels, and MMP-9 and TGFβ colon concentrations did not reach significance. We conclude that our strategy of alleviating autoimmunity in this model through targeting angiogenesis by actively vaccinating against EMMPRIN was successful and efficient in reducing angiogenesis.

Immunolocalization of VirB11 protein in the Anaplasma marginale outer membrane and its reaction with bovine immune sera

Type IV secretion system is composed by a group of proteins associated with substrate transfer between bacteria and eukaryotic organisms. VirB11 is a protein of this system and, in A. marginale it induces partial protection in cattle immunized with external membrane fragments of the rickettsia. The objectives of this study were: to determine the conservation rate of VirB11 among different Mexican isolates and to corroborate its cellular localization on the outer membrane of A. marginale. Nucleotide and amino acid sequence alignment of virB11 from nine Mexican isolates showed 100 % identity. Bovine sera from both naturally and experimentally infected cattle reacted against a multiple antigenic peptide (MAP‑VirB11) designed based on a B-type epitope present in VirB11. In addition, MAP‑VirB11‑rabbit immune sera reacted with native VirB11 by ELISA and western blot in rickettsial extracts and on the surface of bovine infected erythrocytes by indirect immunofluorescence and the localization of VirB11 protein was determined on the outer surface of A. marginale initial bodies.

Immunogenicity of a Tripartite Cell Penetrating Peptide Containing a MUC1 Variable Number of Tandem Repeat (VNTR) and A T Helper Epitope.

Peptide-based vaccines for cancer have many advantages however, for optimization these immunogens should incorporate peptide epitopes that induce CD8, as well as CD4 responses, antibody and long term immunity. Cell penetrating peptides (CPP) with a capacity of cytosolic delivery have been used to deliver antigenic peptides and proteins to antigen presenting cells to induce cytotoxic T cell, helper T cell and humoral responses in mice. For this study, a tripartite CPP including a mucin 1 (MUC1) variable number of tandem repeat (VNTR) containing multiple T cell epitopes and tetanus toxoid universal T helper epitope peptide (tetCD4) was synthesised (AntpMAPMUC1tet) and immune responses investigated in mice. Mice vaccinated with AntpMAPMUC1tet + CpG show enhanced antigen-specific interferon-gamma (IFN-γ) and IL-4 T cell responses compared with AntpMAPMUC1tet vaccination alone and induced a Th1 response, characterised by a higher ratio of IgG2a antibody/IgG1 antibodies. Furthermore, vaccination generated long term MUC1-specific antibody and T cell responses and delayed growth of MUC1+ve tumours in mice. This data demonstrates the efficient delivery of branched multiple antigen peptides incorporating CPP and that the addition of CpG augments immune responses.

Abstract 2554: To clone tumor-specific TCRs from a cell-based immunotherapy-benefit cervical cancer patient

Abstract Adoptive transfer of tumor-specific T cell receptor (TCR)-engineered T cells, TCR-T therapy, has shown great efficacy in clinical trials against solid tumors. A cervical cancer patient with bone metastasis was treated with a cell-based immunotherapy, named Multiple-Antigens Stimulating Cellular Therapy (MASCT), which is a combination of multiple tumor antigen peptides loaded dendritic cells (DCs) and autologous T cells stimulated by these DCs. After repeated treatment, the patient showed partially response and remained stable disease for 20 months. Moreover, boosted specific immune responses were detected in patient's periphery blood by IFNγ-Elispot assay against various tumor antigens, such as CEA, RGS5 and HPV18/58. The clinical benefits of this patient indicated that tumor-specific T cells were expanded in vivo and played an important role to control tumor progression. These T cells may be the good sources to isolate tumor-specific TCRs for TCR-T therapies. Given that, patient's PBMCs were stimulated in vitro with selected tumor peptides respectively, such as CEA, RGS5 and HPV18 E7. Tumor-specific T cells were defined as IFNγ-secreting T cells upon peptide stimulation and were further enriched by using magnetic beads. Part of the enriched T cells were analyzed for TCR alpha/beta repertoire via NGS, the other part were sorted into single T cells and analyzed for TCR alpha/beta pairing. The TCR repertoire NGS data revealed that the tumor-specific T cells harbor several hundred unique TCR alpha/beta clonotypes. Some unique TCR alpha/beta clonotypes were considered as tumor-specific TCR candidates, since the copy numbersof these clonotypes were significantly and repeatedly increased after stimulation with CEA, RGS5 or HPV18 E7, and further increased after IFNγ enrichment. After TCR alpha/beta pairing confirmation by single T cell sequencing, alpha and beta chains of these TCRs were sent to synthesis for further investigation. In conclusion we have successfully identified specific TCR clonotypes targeting selected tumor antigens from a MASCT-benefited cervical cancer patient. After functional validations, including epitope specificity, HLA restriction as well as tumor recognition, these tumor-specific TCRs may be good candidates for developing safer and more effective TCR-T therapy targeting multiple tumor antigens. Citation Format: Xiaoling Liang, Shudan Ou, Minjun Ma, Xihe Chen, Xiangjun Zhou, Yanyan Han. To clone tumor-specific TCRs from a cell-based immunotherapy-benefit cervical cancer patient [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2554.

Chitosan Microsphere Used as an Effective System to Deliver a Linked Antigenic Peptides Vaccine Protect Mice Against Acute and Chronic Toxoplasmosis.

Multiple antigenic peptide (MAP) vaccines have advantages over traditional Toxoplasma gondii vaccines, but are more susceptible to enzymatic degradation. As an effective delivery system, chitosan microspheres (CS) can overcome this obstacle and act as a natural adjuvant to promote T helper 1 (Th1) cellular immune responses. In this study, we use chitosan microparticles to deliver multiple antigenic epitopes from GRA10 (G10E), containing three dominant epitopes. When G10E was entrapped within chitosan microparticles (G10E-CS), adequate peptides for eliciting immune response were loaded in the microsphere core and this complex released G10E peptides stably. The efficiency of G10E-CS was detected both in vitro, via cell culture, and through in vivo mouse immunization. In vitro, G10E-CS activated Dendritic Cells (DC) and T lymphocytes by upregulating the secretion of costimulatory molecules (CD40 and CD86). In vivo, Th1 biased cellular and humoral immune responses were activated in mice vaccinated with G10E-CS, accompanied by significantly increased production of IFN-γ, IL-2, and IgG, and decreases in IL-4, IL-10, and IgG1. Immunization with G10E-CS conferred significant protection with prolonged survival in mice model of acute toxoplasmosis and statistically significant decreases in cyst burden in murine chronic toxoplasmosis. The results from this study indicate that chitosan microspheres used as an effective system to deliver a linked antigenic peptides is a promising strategy for the development of efficient vaccine against T. gondii.

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.

Gastric cancer vaccines synthesized using a TLR7 agonist and their synergistic antitumor effects with 5-fluorouracil

BackgroundVaccines play increasingly important roles in cancer treatment due to their advantages of effective targeting and few side effects. Our laboratory has attempted to construct vaccines by conjugating TLR7 agonists with tumor-associated antigens. Furthermore, immunochemotherapy has recently become an appealing approach to cancer therapy. 5-fluorouracil (5-FU), a commonly used chemotherapeutic agent, can reportedly potently and selectively kill tumor-associated MDSCs in vivo.MethodsGastric cancer vaccines were synthesized by the covalent attachment of our TLR7 agonist with the gastric cancer antigen MG7-Ag tetra-epitope, leading to T7 − ML (linear tetra-epitope) and T7 − MB (branched tetra-epitope). Cytokines induced by the vaccines in vitro were assessed by ELISA. A tumor challenge model was created by treating BALB/c mice on either a prophylactic or therapeutic vaccination schedule. 5-FU was simultaneously applied to mice in the combination treatment group. CTL and ADCC activities were determined by the LDH method, while CD3+/CD8+, CD3+/CD4+ T cells and MDSCs were evaluated by flow cytometry.ResultsIn vitro, rapid TNF-α and IL-12 inductions occurred in BMDCs treated with the vaccines. In vivo, among all the vaccines tested, T7 − MB most effectively reduced EAC tumor burdens and induced CTLs, antibodies and ADCC activity in BALB/c mice. Immunization with T7 − MB in combination with 5-FU chemotherapy reduced tumor sizes and extended long-term survival rates, mainly by improving T cell responses, including CTLs, CD3+/CD8+ and CD3+/CD4+ T cells. 5-FU also enhanced the T7 − MB efficiency by reversing immunosuppressive factors, i.e., MDSCs, which could not be validly inhibited by the vaccines alone. In addition, T7 − MB repressed tumor growth and immune tolerance when the therapeutic schedule was used, although the effects were weaker than those achieved with either T7 − MB alone or in combination with 5-FU on the prophylactic schedule.ConclusionsA novel effective gastric cancer vaccine was constructed, and the importance of branched multiple antigen peptides and chemical conjugation to vaccine design were confirmed. The synergistic effects and mechanisms of T7 − MB and 5-FU were also established, observing mainly T cell activation and MDSC inhibition.

DNA‐binding peptide dendrimer for efficient and selective intracellular delivery

Dendrimeric peptides or peptide dendrimers are branched peptides with multiple peptidyl units. Multiple antigen peptides (MAPs) are examples of dendrimeric peptides first introduced in 1988 by our laboratory as immunogens for producing monoclonal and polyclonal antibodies. We also designed antimicrobial dendrimeric peptides, such as D4R, using a tetrapeptide RLYR which contains a ChC‐motif (cationic‐hydrophobic‐cationic) found in the naturally‐occurring antimicrobial peptides such as tachyplesins and protegrins. Advantages of D4R as a short‐chain, tetra‐branched peptide dendrimer include its short preparation time (a few hours), its polycationic, low membranolytic activity to human red blood cells, and high proteolytic stability. Here, we show that D4R is also a cell‐penetrating and an intracellular DNA‐binding peptide dendrimer. Using solid‐phase synthesis, we exploited different chemistries to attach tags or small molecules to the α‐ or ɛ‐amino groups of lysine to form a tetra‐branched dendrimer with four chains of RLYR. An example is D4R‐f, a C‐terminal 5‐FAM‐labeled D4R which we showed that it can enter living cells through endocytosis‐independent mechanisms as determined by using epi‐fluorescence microscopy. Furthermore, we showed that D4R‐f is distributed in different intracellular organelles during different stages of the cell cycles and colocalizes with nuclear DNA in cells undergoing mitosis. Using agarose gel shift assay, we showed that D4R binds to double‐stranded DNA. Co‐incubation of D4R with DNA showed resistance against DNase‐mediated digestion. The control linear 16‐residue peptide with four tandemly repeating RLYR units was not cell‐penetrating like D4R. Modifying the ChC motifs of the short‐chain peptide dendrimers produced different results, suggesting that the RLYR motif is tunable. Taken together, this study highlights D4R as a prototypic DNA‐binding peptide dendrimer which penetrates into living cells through endocytosis‐independent uptake mechanisms. Our work also suggests the potentials of short‐chain DNA‐binding peptide dendrimers as a lead to design anti‐mitotic agents and a convenient new tool for non‐viral gene delivery.Support or Funding InformationThis research was supported in part by Nanyang Technological University Internal Funding ‐Synzyme and Natural Products (SYNC) and the AcRF Tier 3 funding (MOE2016‐T3‐1‐003).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Different Bioactive Neuropeptides are Expressed in Two Sub-Classes of GABAergic RME Nerve Ring Motorneurons in Ascaris suum

Neuropeptides can have significant effects on neurons and synapses, but among the ∼250 predicted peptides in nematodes, few have been characterized functionally. Here, we report new neuropeptides in the 4 RME nerve ring motorneurons of the nematode Ascaris suum. These GABAergic neurons are involved in three-dimensional head movement. Mass spectrometry (MS) of single dissected RMEs detected a total of 12 neuropeptides (encoded by five genes), nine of which are novel. None of these are expressed in the DI/VI inhibitory GABAergic motorneurons that synapse onto body wall muscle. Using peptide sequences obtained by tandem MS, we cloned the peptide-encoding transcripts and synthesized riboprobes for in situ hybridization (ISH). This complementary technique corroborated the results from single-cell MS, showing that the dissections were not contaminated with adhering tissue from other cells. We also synthesized a multiple antigenic peptide to raise a highly specific antibody against one of the endogenous peptides, which labeled the same cells detected by MS and ISH. Our results show that the RMEs can be divided into two subsets: RMED/V (expressing afp-2, afp-15, Asu-nlp-58, and high levels of afp-16) and RMEL/R (expressing afp-15 and low levels of afp-4 and afp-16). Almost all of these peptides are bioactive in A. suum.

A single dose polyanhydride-based vaccine platform promotes and maintains anti-GnRH antibody titers

Traditionally, vaccination strategies require an initial priming vaccination followed by an antigen boost to generate adequate immunity. Here we describe vaccination against a self-peptide for reproductive sterilization utilizing a three-stage vaccine platform consisting of gonadotropin releasing hormone multiple antigenic peptide (GnRH-MAP) as a soluble injection coupled with subcutaneous administration of polyanhydride-immobilized GnRH-MAP and a cyto-exclusive implant containing GnRH-MAP dendrimer-loaded polyanhydride. This strategy generated and maintained cell-mediated and humoral immunity for up to 41 weeks after a single vaccination in mice with enhanced antibody avidity over time. All intact implants had a grossly visible tissue interface with neovascularization and lymphocytic aggregates. Despite detectable immunity, sterility was not achieved and the immune response did not lead to azoospermia in male mice nor prevent estrus and ovulation in female mice. However, the vaccine delivery device is tunable and the immunogen, adjuvants and release rates can all be modified to enhance immunity. This technology has broad implications for the development of long-term vaccination schemes.

Utilization of Feline ELISpot to Evaluate the Immunogenicity of a T Cell-Based FIV MAP Vaccine.

The prototype and the commercial dual-subtype feline immunodeficiency virus (FIV) vaccines conferred protection against homologous FIV strains as well as heterologous FIV strains from the vaccine subtypes with closely related envelope (Env) sequences. Such protection was mediated by the FIV neutralizing antibodies (NAbs) induced by the vaccines. Remarkably, both prototype and commercial FIV vaccines also conferred protection against heterologous FIV subtypes with highly divergent Env sequences from the vaccine strains. Such protection was not mediated by the vaccine-induced NAbs but was mediated by a potent FIV-specific T-cell immunity generated by the vaccines (Aranyos et al., Vaccine 34: 1480-1488, 2016). The protective epitopes on the FIV vaccine antigen were identified using feline interleukin-2 (IL-2) and interferon-γ (IFNγ) ELISpot assays with overlapping FIV peptide stimulation of the peripheral blood mononuclear cells (PBMC) from cats immunized with prototype FIV vaccine. Two of the protective FIV peptide epitopes were identified on FIV p24 protein and another two protective peptide epitopes were found on FIV reverse transcriptase. In the current study, the multiple antigenic peptides (MAPs) of the four protective FIV peptides were combined with an adjuvant as the FIV MAP vaccine. The laboratory cats were immunized with the MAP vaccine to evaluate whether significant levels of vaccine-specific cytokine responses can be generated to the FIV MAPs and their peptides at post-second and post-third vaccinations. The PBMC from vaccinated cats and non-vaccinated control cats were tested for IL-2, IFNγ, and IL-10 ELISpot responses to the FIV MAPs and peptides. These results were compared to the results from CD4+ and CD8+ T-cell proliferation to the FIV MAPs and peptides. Current study demonstrates that IL-2 and IFNγ ELISpot responses can be used to detect memory responses of the T cells from vaccinated cats after the second and third vaccinations.