Immunostimulatory Peptide Research Articles

Thymosin α1 and Its Role in Viral Infectious Diseases: The Mechanism and Clinical Application.

Thymosin α1 (Tα1) is an immunostimulatory peptide that is commonly used as an immune enhancer in viral infectious diseases such as hepatitis B, hepatitis C, and acquired immune deficiency syndrome (AIDS). Tα1 can influence the functions of immune cells, such as T cells, B cells, macrophages, and natural killer cells, by interacting with various Toll-like receptors (TLRs). Generally, Tα1 can bind to TLR3/4/9 and activate downstream IRF3 and NF-κB signal pathways, thus promoting the proliferation and activation of target immune cells. Moreover, TLR2 and TLR7 are also associated with Tα1. TLR2/NF-κB, TLR2/p38MAPK, or TLR7/MyD88 signaling pathways are activated by Tα1 to promote the production of various cytokines, thereby enhancing the innate and adaptive immune responses. At present, there are many reports on the clinical application and pharmacological research of Tα1, but there is no systematic review to analyze its exact clinical efficacy in these viral infectious diseases via its modulation of immune function. This review offers an overview and discussion of the characteristics of Tα1, its immunomodulatory properties, the molecular mechanisms underlying its therapeutic effects, and its clinical applications in antiviral therapy.

Dynamic Imine Bonding Facilitates Mannan Release from a Nanofibrous Peptide Hydrogel.

Recently, there has been increased interest in using mannan as an immunomodulatory bioconjugate. Despite notable immunological and functional differences between the reduced (R-Man) and oxidized (O-Man) forms of mannan, little is known about the impact of mannan oxidation state on its in vivo persistence or its potential controlled release from biomaterials that may improve immunotherapeutic or prophylactic efficacy. Here, we investigate the impact of oxidation state on the in vitro and in vivo release of mannan from a biocompatible and immunostimulatory multidomain peptide hydrogel, K2(SL)6K2 (abbreviated as K2), that has been previously used for the controlled release of protein and small molecule payloads. We observed that O-Man released more slowly from K2 hydrogels in vitro than R-Man. In vivo, the clearance of O-Man from K2 hydrogels was slower than O-Man alone. We attributed the slower release rate to the formation of dynamic imine bonds between reactive aldehyde groups on O-Man and the lysine residues on K2. This imine interaction was also observed to improve K2 + O-Man hydrogel strength and shear recovery without significantly influencing secondary structure or peptide nanofiber formation. There were no observed differences in the in vivo release rates of O-Man loaded in K2, R-Man loaded in K2, and R-Man alone. These data suggest that, after subcutaneous injection, R-Man naturally persists longer in vivo than O-Man and minimally interacts with the peptide hydrogel. These results highlight a potentially critical, but previously unreported, difference in the in vivo behavior of O-Man and R-Man and demonstrate that K2 can be used to normalize the release of O-Man to that of R-Man. Further, since K2 itself is an adjuvant, a combination of O-Man and K2 could be used to enhance the immunostimulatory effects of O-Man for applications such as infectious disease vaccines and cancer immunotherapy.

PASylated Thymosin α1: A Long-Acting Immunostimulatory Peptide for Applications in Oncology and Virology.

Thymosin α1 (Tα1) is an immunostimulatory peptide for the treatment of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections and used as an immune enhancer, which also offers prospects in the context of COVID-19 infections and cancer. Manufacturing of this N-terminally acetylated 28-residue peptide is demanding, and its short plasma half-life limits in vivo efficacy and requires frequent dosing. Here, we combined the PASylation technology with enzymatic in situ N-acetylation by RimJ to produce a long-acting version of Tα1 in Escherichia coli at high yield. ESI-MS analysis of the purified fusion protein indicated the expected composition without any signs of proteolysis. SEC analysis revealed a 10-fold expanded hydrodynamic volume resulting from the fusion with a conformationally disordered Pro/Ala/Ser (PAS) polypeptide of 600 residues. This size effect led to a plasma half-life in rats extended by more than a factor 8 compared to the original synthetic peptide due to retarded kidney filtration. Our study provides the basis for therapeutic development of a next generation thymosin α1 with prolonged circulation. Generally, the strategy of producing an N-terminally protected PASylated peptide solves three major problems of peptide drugs: (i) instability in the expression host, (ii) rapid degradation by serum exopeptidases, and (iii) low bioactivity because of fast renal clearance.

Synthetic Antibody Mimics Based on Cancer-Targeting Immunostimulatory Peptides.

De novo cancer-targeting immunostimulatory peptides have been designed and developed as synthetic antibody mimics. A series of bifunctional peptides incorporating NKp30-binding and NK-cell-activating domains were synthesized as linear dimers and then extended into branching trimeric peptides by the incorporation of GRP78-targeting and tumor-cell-binding sequences. A selected trimeric peptide from this small set of peptides displayed binding capabilities on GRP78+ HepG2 and A549 target cells. Cell binding diminished in the presence of an anti-GRP78 peptide blocker, thus suggesting GRP78-binding dependence. Similarly, the selected trimeric peptide was also found to exhibit NK cell binding in an NKp30-dependent manner, which translated into NK cell activation as indicated by cytokine secretion. In co-culture, fluorescence microscopy revealed that the target GFP-expressing A549 cells were visibly associated with the effector NK cells when pre-activated with lead trimeric peptide. Accordingly, A549 cells were found to be compromised, as evidenced by the loss of GFP signal and notable detection of early-/late-stage apoptosis. Investigation of the immunological markers related to toxicity revealed detectable secretion of pro-inflammatory cytokines and chemokines, including IFN-γ, TNF-α, and IL-8. Furthermore, administration of peptide-activated NK cells into A549-tumor-bearing mice resulted in a consistent decrease in tumor growth when compared to the untreated control group. Taken together, the identification of a lead trimeric peptide capable of targeting and activating NK cells' immunotoxicity directly towards GRP78+ /B7H6- tumors provides a novel proof-of-concept for the development of cancer-targeting immunostimulatory peptide ligands that mimic antibody-targeting and -activating functions related to cancer immunotherapy applications.

Biological activity of LALF32-51–E7, a vaccine candidate for the treatment of anogenital lesions associated to HPV16

Cervical cancer is the second leading cause of cancer death in women. It is well established that human papillomavirus (HPV) is the cause of virtually 100% of these cancers, 70% of which are attributable to infections with HPV types 16 or 18. We developed a fusion protein comprising a cell penetrating and immunostimulatory peptide corresponding to residues 32 to 51 of the Limulus polyphemus protein (LALF32-51) linked to the HPV16 E7 antigen (LALF32-51–E7) that is obtained from Escherichia coli. The development of new treatments as mentioned requires a robust and reproducible procedure for batch release. We used the combination of three batches of LALF32-51-E7 with Al(OH)3 in an experimental design to develop a biological activity test for batch release of this candidate, which uses prophylactic immunization in the TC-1 tumor mouse model. Two injections, spaced for 7 days with LALF31-52-E7+Al(OH)3 are sufficient for inducing a potent anti-tumor response in the TC-1 tumor model. LALF31-52-E7 vaccine candidates in combination with Al(OH)3, exhibited in C57BL/6 mice over 86% protection against tumor challenge and high titers of specific antibodies against LALF31-52-E7. The results obtained across three batches of LALF32-51–E7+Al(OH)3 with this simple and uncomplicated assay based on prophylactic immunization in the TC-1 mouse model suggest that it would be feasible to implement it as a biological activity assay for the release of production batches of LALF32-51–E7.

Immunological properties and protective efficacy of a single mycobacterial antigen displayed on polyhydroxybutyrate beads.

SummaryIn 2015, there were an estimated 10.4 million new tuberculosis (TB) cases and 1.4 million deaths worldwide. Bacille Calmette–Guérin (BCG), an attenuated strain of Mycobacterium bovis, is the vaccine available against TB, but it is insufficient for global TB control. This study evaluated the immunogenicity of the Mycobacterium tuberculosis antigen Rv1626 in mice while assessing the effect of co‐delivering either Cpe30 (immunostimulatory peptide), CS.T3378–395 (promiscuous T helper epitope) or flagellin (TLR5 agonist) or a combination of all three immunostimulatory agents. Rv1626 and the respective immunostimulatory proteins/peptides were co‐displayed on polyhydroxybutyrate beads assembled inside an engineered endotoxin‐free mutant of Escherichia coli. Mice vaccinated with these beads produced immune responses biased towards Th1‐/Th17‐type responses, but inclusion of Cpe30, CS.T3378–395 and flagellin did not enhance immunogenicity of the Rv1626 protein. This was confirmed in a M. bovis challenge experiment in mice, where Rv1626 beads reduced bacterial cell counts in the lungs by 0.48 log10 compared with the adjuvant alone control group. Co‐delivery of immunostimulatory peptides did not further enhance protective immunity.

Effect of antihypertensive therapy with angiotensin-converting enzyme inhibitors on chronic periodontitis: a case-control study.

Angiotensin-converting enzyme (ACE) downregulates the activity of bradykinin, a potent proinflammatory and immunostimulatory peptide liberated from an internal portion of kininogens. Here, we asked whether periodontitis is worsened in patients under antihypertensive treatment with ACE inhibitors. Periodontal parameters were recorded from 30 individuals taking ACE inhibitors (case) and 35 taking a non-ACE inhibitor medication (control). Data were analyzed by nonparametric and parametric statistical tests. Most sociodemographic figures were similar in both groups. However, family income was statistically higher in the control group, and the percentage of sites with visible plaque (PL) was statistically higher in the case group (P=0.043 and P=0.005, respectively). The prevalence of individuals with chronic periodontitis varied from 31.5% in the control group to 63.4% in the case group (P=0.001). Patients in the case group presented a 3.2-fold higher risk of having sites with pocket depth ≥5mm and a 2.9-fold higher risk of having sites with clinical attachment loss ≥5mm in comparison with those in the control group (P=0.009 and P=0.001, respectively; adjusted for family income and visible PL). Angiotensin-converting enzyme inhibitors may increase the prevalence and extent of chronic periodontitis in Brazilian patients.

Immunostimulatory peptide biomaterials in wound healing contexts

Immunostimulatory peptide biomaterials in wound healing contexts

Enhanced mucosal immune responses induced by a combined candidate mucosal vaccine based on Hepatitis A virus and Hepatitis E virus structural proteins linked to tuftsin.

Hepatitis A virus (HAV) and Hepatitis E virus (HEV) are the most common causes of infectious hepatitis. These viruses are spread largely by the fecal-oral route and lead to clinically important disease in developing countries. To evaluate the potential of targeting hepatitis A and E infection simultaneously, a combined mucosal candidate vaccine was developed with the partial open reading frame 2 (ORF2) sequence (aa 368–607) of HEV (HE-ORF2) and partial virus protein 1 (VP1) sequence (aa 1–198) of HAV (HA-VP1), which included the viral neutralization epitopes. Tuftsin is an immunostimulatory peptide which can enhance the immunogenicity of a protein by targeting it to macrophages and dendritic cells. Here, we developed a novel combined protein vaccine by conjugating tuftsin to HE-ORF2 and HA-VP1 and used synthetic CpG oligodeoxynucleotides (ODNs) as the adjuvant. Subsequent experiments in BALB/c mice demonstrated that tuftsin enhanced the serum-specific IgG and IgA antibodies against HEV and HAV at the intestinal, vaginal and pulmonary interface when delivered intranasally. Moreover, mice from the intranasally immunized tuftsin group (HE-ORF2-tuftsin + HA-VP1-tuftsin + CpG) showed higher levels of IFN-γ-secreting splenocytes (Th1 response) and ratio of CD4+/CD8+ T cells than those of the no-tuftsin group (HE-ORF2 + HA-VP1 + CpG). Thus, the tuftsin group generated stronger humoral and cellular immune responses compared with the no-tuftsin group. Moreover, enhanced responses to the combined protein vaccine were obtained by intranasal immunization compared with intramuscular injection. By integrating HE-ORF2, HA-VP1 and tuftsin in a vaccine, this study validated an important concept for further development of a combined mucosal vaccine against hepatitis A and E infection.

Enhanced anti-tumor immune responses and delay of tumor development in human epidermal growth factor receptor 2 mice immunized with an immunostimulatory peptide in poly(D,L-lactic-co-glycolic) acid nanoparticles.

IntroductionCancer vaccines have the potential to induce curative anti-tumor immune responses and better adjuvants may improve vaccine efficacy. We have previously shown that Hp91, a peptide derived from the B box domain in high-mobility group box protein 1 (HMGB1), acts as a potent immune adjuvant.MethodIn this study, Hp91 was tested as part of a therapeutic vaccine against human epidermal growth factor receptor 2 (HER2)-positive breast cancer.ResultsFree peptide did not significantly augment immune responses but, when delivered in poly(D,L-lactic-co-glycolic) acid nanoparticles (PLGA-NPs), robust activation of dendritic cells (DCs) and increased activation of HER2-specific T cells was observed in vitro. Vaccination of HER2/neu transgenic mice, a mouse breast cancer model that closely mimics the immune modulation and tolerance in some breast cancer patients, with Hp91-loaded PLGA-NPs enhanced the activation of HER2-specific cytotoxic T lymphocyte (CTL) responses, delayed tumor development, and prolonged survival.ConclusionsTaken together these findings demonstrate that the delivery of the immunostimulatory peptide Hp91 inside PLGA-NPs enhances the potency of the peptide and efficacy of a breast cancer vaccine.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-015-0552-9) contains supplementary material, which is available to authorized users.

A Novel Strategy to Improve Antigen Presentation for Active Immunotherapy in Cancer. Fusion of the Human Papillomavirus Type 16 E7 Antigen to a Cell Penetrating Peptide

ABSTRACT Aim: Fundamental for an effective therapeutic cancer vaccine is facilitating the delivery of exogenous antigens to antigen presenting cells, ensuing processing and presentation via the MHC-class I and induction of an effective immune response. In this regard, we propose the use of cell-penetrating peptides fused to a tumor antigen. To demonstrate this concept we designed a fusion protein comprising a novel our proprietary cell-penetrating and immunostimulatory peptide (CIGB 550) linked to the human papillomavirus 16 (HPV 16) -E7 antigen (CIGB550-E7). The E7 viral protein alone is often poorly recognized by the immune system. HPV 16 infection has been linked to the development of cervical, anal and head and neck cancers. Methods: Mice: Female C57BL/6 mice, 6–8 weeks old. Immunizations: The immunizations with CIGB550-E7 were given subcutaneously in the flank, in a final volume of 0.2 mL/dose. Tumor cells: The TC-1 cells were injected subcutaneously in the leg of C57BL/6 mice at 5 × 104 or 2 × 105 doses. Starting 7–10 days later and every 3–4 days thereafter, the area was observed and palpated for the presence of a tumor nodule. Tumor diameters were measured. Results: 1. Coupling E7 to CIGB550 takes advantage that allows it to enter to the cells and thereby significantly improves the presentation of E7-derived peptides to T-cells 2. Therapeutic immunization with CIGB550-E7 vaccine induces regression of palpable tumors and survival increase. 3. In vitro analyses indicated that immunizations with this vaccine leads to the induction of a Th1- like cell mediated immune response based IFN-gamma secretion T cells and significant IFN-gamma increase following antigenic recall, using the ELISPOT assay. Conclusions: The relevance of this result is the design of an original vaccine with promissory perspectives to treat HPV16 related malignancies, based on the covalent linkage of CIGB550 and HPV16 E7 antigen. Our data underline the efficacy of this approach at inducing broad immune responses in vivo, and offer a new strategy that could improve subunit cancer vaccine in a clinical setting. Disclosure: All authors have declared no conflicts of interest.

TLR4-dependent activation of dendritic cells by an HMGB1-derived peptide adjuvant.

High mobility group box protein 1 (HMGB1) acts as an endogenous danger molecule that is released from necrotic cells and activated macrophages. We have previously shown that peptide Hp91, whose sequence corresponds to an area within the B-Box domain of HMGB1, activates dendritic cells (DCs) and acts as an adjuvant in vivo. Here we investigated the underlying mechanisms of Hp91-mediated DC activation. Hp91-induced secretion of IL-6 was dependent on clathrin- and dynamin-driven endocytosis of Hp91 and mediated through a MyD88- and TLR4-dependent pathway involving p38 MAPK and NFκB. Endosomal TLR4 has been shown to activate the MyD88-independent interferon pathway. Hp91-induced activation of pIRF3 and IL-6 secretion was reduced in IFNαβR knockout DCs, suggesting an amplification loop via the IFNαβR. These findings elucidate the mechanisms by which Hp91 acts as immunostimulatory peptide and may serve as a guide for the future development of synthetic Th1-type peptide adjuvants for vaccines.Electronic supplementary materialThe online version of this article (doi:10.1186/1479-5876-12-211) contains supplementary material, which is available to authorized users.

Vaccination with the RSV fusion protein formulated with a combination adjuvant induces long-lasting protective immunity

Respiratory syncytial virus (RSV) is one of the primary causative agents of upper and lower respiratory tract infections in young children, in particular infants. Recently, we reported the protective efficacy of a RSV vaccine formulation consisting of a truncated version of the fusion (F) protein formulated with a Toll-like receptor (TLR) agonist and an immunostimulatory peptide in a carrier system (ΔF/TriAdj). To evaluate the duration of immunity induced by this vaccine candidate, we carried out long-term trials. The ΔF was formulated with triple adjuvant (TriAdj) containing either polyinosinic : polycytidylic acid (polyI : C) or cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODNs) and administered intranasally to mice. One year after the second vaccination all mice were challenged with RSV. Both ΔF/TriAdj formulations mediated the induction of high levels of IgG1, IgG2a and virus-neutralizing antibodies, and IgA in the lungs. Based on the numbers of IFN-γ- and IL-5-secreting cells in the spleen, the immune response was slightly T-helper cell type 1 (Th1)-biased. This was confirmed by the presence of F85-93-specific CD8(+) effector T cells in the lungs of both ΔF/TriAdj(polyI : C)- and ΔF/TriAdj(CpG)-immunized mice. Both ΔF/TriAdj formulations induced RSV-specific CD8(+) T cells. However, ΔF/TriAdj(polyI : C) generated significantly higher IgG affinity maturation and higher numbers of RSV-specific CD8(+) effector memory T cells in lungs and CD8(+) central memory T cells in spleen and lymph nodes than ΔF/TriAdj(CpG). After RSV challenge, no virus replication and no evidence of vaccine-induced pathology were detected in mice immunized with either of the ΔF/TriAdj formulations, demonstrating that the duration of immunity induced with these vaccines is at least one year.

Activity of the HMGB1-derived immunostimulatory peptide Hp91 resides in the helical C-terminal portion and is enhanced by dimerization

We have previously shown that an 18 amino acid long peptide, named Hp91, whose sequence corresponds to a region within the endogenous protein HMGB1, activates dendritic cells (DCs) and acts as adjuvant in vivo by potentiating Th1-type antigen-specific immune responses. We analyzed the structure–function relationship of the Hp91 peptide to investigate the amino acids and structure responsible for immune responses. We found that the cysteine at position 16 of Hp91 enabled formation of reversible peptide dimmers, monomer and dimmer were compared for DC binding and activation. Stable monomers and dimers were generated using a maleimide conjugation reaction. The dimer showed enhanced ability to bind to and activate DCs. Furthermore, the C-terminal 9 amino acids of Hp91, named UC1018 were sufficient for DC binding and Circular dichroism showed that UC1018 assumes an alpha-helical structure. The ninemer peptide UC1018 induced more potent antigen-specific CTL responses in vivo as compared to Hp91 and it protected mice from tumor development when used in a prophylactic vaccine setting. We have identified a short alpha helical peptide that acts as potent adjuvant inducing protective immune responses in vivo.

Polymerisation of a T Cell Epitope with an Immunostimulatory C3d Peptide Sequence Enhances Antigen Specific T Cell Responses

The complement protein C3d and C3d derived peptides that bind CD21 are known to enhance immunity to co-immunised antigens. In this study we have synthesised the minimal CD21 binding sequence of C3d (1227LYNVEA1232) as mono, di and tri tandem repeats and derivatised the N-terminus with an acryloyl moiety. These acryloyl-(C3d) n peptides were co-polymerised with a acryloyl-T cell epitope (PAS1K) from the Porphyromonas gingivalis antigen the RgpA–Kgp proteinase–adhesin complex. The ability of C3d containing polymers to enhance T cell immunity in vitro and in vivo was evaluated. When used to stimulate in vitro PAS1K-primed or RgpA–Kgp complex-primed T cells the C3d containing PAS1K polymers induced a mixed and significantly (p < 0.05) higher IL-4 and IFNγ T cell response compared to that induced by the PAS1K peptide or polymer. PAS1K polymers containing tandem repeats of C3d induced a significantly (p < 0.05) stronger maximal proliferative response, at the same antigenic dose, compared to that induced by the PAS1K peptide or polymer. When used as immunogens to prime T cells all of the C3d containing PAS1K polymers induced a dominant IFNγ T cell response and reduced the antigen dose required for maximal proliferation 150-fold compared to that required for the PAS1K-peptide or polymer primed T cells. In conclusion, the 6 residue sequence LYNVEA from C3d is sufficient to enhance immunity to an antigen and that the effect is more pronounced when C3d is part of the immunising antigen rather than an in vitro stimulating antigen.

Expression, purification and characterization of a recombinant fusion protein based on the human papillomavirus-16 E7 antigen

A fusion protein comprising a cell penetrating and immunostimulatory peptide corresponding to residues 32 to 51 of the Limulus polyphemus protein linked to human papillomavirus (HPV)-16 E7 antigen (LALF32-51-E7) was expressed in E. coli BL21 (DE3) cells. The recombinant protein in E. coli accounted for approximately 18% of the total cellular protein and purified with a single affinity chromatographic step. Yields of approximately 38 mg purified LALF32-51-E7 per liter of induced culture was obtained with an overall 52% recovery and constitutes a promising setting for the future production and scaling-up. Purified protein was characterized as soluble aggregates with molecular weight larger than 670 kDa, which is considered an important property to increase the immunogenicity of an antigen preparation. The recombinant fusion protein LALF32-51-E7 will be a promising vaccine candidate for the treatment of HPV-16 related malignancies.

Lysine-tagged peptide coupling onto polylactide nanoparticles coated with activated ester-based amphiphilic copolymer: A route to highly peptide-functionalized biodegradable carriers

Efficient biomolecule conjugation to the surface of biodegradable colloidal carriers is crucial for their targeting efficiency in drug/vaccine delivery applications. We here propose a potent strategy to drastically improve peptide immobilization on biodegradable polylactide (PLA) nanoparticles (NPs). Our approach particularly relies on the use of an amphiphilic block copolymer PLA-b-poly(N-acryloxysuccinimide-co-N-vinylpyrrolidone) (PLA-b-P(NAS-co-NVP)) as NP surface modifier, whose the N-succinimidyl (NS) ester functions of the NAS units along the polymer chain ensure N-terminal amine peptide coupling. The well-known immunostimulatory peptide sequence derived from the human interleukin 1β (IL-1β), VQGEESNDK, was coupled on the NPs of 169nm mean diameter in phosphate buffer (pH 8, 10mM). A maximum amount of 2mg immobilized per gram of NPs (i.e. 0.042peptidenm−2) was obtained. Introduction of a three lysine tag at the peptide N-terminus (KKKVQGEESNDK) resulted in a dramatic improvement of the immobilized peptide amounts (27.5mg/g NP, i.e. 0.417peptidenm−2). As a comparison, the density of tagged peptide achievable on surfactant free PLA NPs of similar size (140nm), through classical EDC or EDC/NHS activation of the surface PLA carboxylic end-groups, was found to be 6mg/g NP (i.e. 0.075peptidenm−2), showing the decisive impact of the P(NAS-co-NVP)-based hairy corona for high peptide coupling. These results demonstrate that combined use of lysine tag and PLA-b-P(NAS-co-NVP) surfactant represents a valuable platform to tune and optimize surface bio-functionalization of PLA-based biodegradable carriers.

A novel fusion protein-based vaccine comprising a cell penetrating and immunostimulatory peptide linked to human papillomavirus (HPV) type 16 E7 antigen generates potent immunologic and anti-tumor responses in mice

The ultimate success of cancer vaccination is dependent upon the generation of tumor-specific CTLs. In this study, we designed and evaluated a novel fusion protein comprising a cell penetrating and immunostimulatory peptide corresponding to residues 32–51 of the Limulus polyphemus protein (LALF32–51) linked to human papillomavirus (HPV) 16 E7 antigen (LALF32–51–E7). We demonstrated that LALF32–51 penetrates the cell membrane and delivers E7 into cells. In a preclinical model of HPV16-induced cervical carcinoma we showed that vaccination with adjuvant-free LALF32–51–E7 fusion protein significantly improves the presentation of E7-derived peptides to T-cells in vitro and induces suppression of tumor growth.

Antimicrobial and immunostimulatory peptide, KLK, induces an increase in cytosolic Ca2+concentration by mobilizing Ca2+from intracellular stores

The cationic antimicrobial immunomodulatory peptide, KLK (KLKL₅KLK), exerts profound membrane interacting properties, impacting on ultrastructure and fluidity. KLK-membrane interactions that lead to these alterations require the ability of the peptide to move into an α-helical conformation. We show that KLK induces an increase of the intracellular Ca²(+) concentration in human T24 cells. The effect of KLK is buffer-sensitive, as it is detected when HBSS buffer is used, but not with PBS. This, together with the lack of effect of the middle leucine-to-proline-substituted peptide derivative [KPK (KLKLLPLLKLK)], indicates that it is the conformational propensity rather than the net positive charge that contributes to the effect of KLK on intracellular Ca²(+) level of T24 cells. We show that, although KLK slightly stimulates Ca²(+) influx into the cell, the bulk increase of Ca²(+) levels is due to KLK-induced depletion of intracellular Ca²(+) stores. Finally, we demonstrate a KLK-induced switch of PS (phosphatidylserine) from the inner to the outer plasma membrane leaflet that contributes to the onset of early apoptotic changes in these cells.

HMGB1-derived peptide acts as adjuvant inducing immune responses to peptide and protein antigen

There is a need for new adjuvants that will induce immune responses to subunit vaccines. We show that a short peptide, named Hp91, whose sequence corresponds to an area within the endogenous molecule high mobility group box (HMGB1) protein 1 potentiates cellular immune responses to peptide antigen and cellular and humoral immune responses to protein antigen in vivo. Hp91 promoted the in vivo production of the immunomodulatory cytokines, IFN-γ, TNF-α, IL-6, and IL-12 (p70), as well as antigen-specific activation of CD8+ T cells. These results demonstrate the ability of a short immunostimulatory peptide to serve as an adjuvant for subunit vaccines.