Antigen Conjugate Research Articles

Antigenic peptide-tobacco mosaic virus conjugates as a fungal vaccine candidate

Fungal infections are becoming an increasingly serious challenge in clinic due to the increase in drug resistance and the lack of anti-fungal drugs. Vaccination is a useful approach to prevent fungal infections. However, the balance between effectiveness and side effects presents a challenge in vaccine development. In this work, we designed a plant virus-based conjugate vaccine. The non-infectiveness and innate immunogenicity of plant viruses make this vaccine both safe and effective. By conjugating a fungal antigenic peptide to the tobacco mosaic virus (TMV), the resultant vaccine improved the uptake efficiency of antigenic peptides by antigen-presenting cells and enhanced the ability to target lymph nodes. The results of in vivo vaccination in mice showed a significant increase of antigen-specific IgG antibody levels induced by the TMV conjugate vaccine. This work suggests that TMV conjugate vaccines may become a potential vaccine candidate for preventing fungal infections.

Selection of trophoblast cell surface antigen 2-targeted aptamer for the development of cytotoxic aptamer-drug conjugate

Trophoblast cell surface antigen 2 expressed in several malignant cancers promotes tumor growth and metastasis via several signal transduction pathways. Trop2 is reputed as a prospective biomarker and therapeutic target. Trophoblast cell surface antigen 2-targeted agents, including antibodies, antibody conjugates and therapeutic combinations, could be utilized to fight cancers. To develop an effective drug targeting strategy, we resorted to a new trophoblast cell surface antigen 2-targeted anticancer treatment through aptamer conjugated with chemotherapeutic drug. This study identified trophoblast cell surface antigen 2-specific ssDNA aptamers using engineered trophoblast cell surface antigen 2 overexpression cells for cell-SELEX. The obtained ssDNA aptamer bound to trophoblast cell surface antigen 2 overexpressed cells with nanomolar affinity and was specific for several tumor cell types which express trophoblast cell surface antigen 2 abundantly. Significant cytotoxicity against HT29 cell by the conjugate of trophoblast cell surface antigen 2 aptamer and Emtansine was observed while resulting negligible therapeutic effect on human normal intestinal epithelial cell line HIEC in vitro, indicating that the conjugate shows potential as a promising therapeutic agent. Furthermore, the isolated aptamer demonstrated the ability for the targeted delivery, resulting excellent therapeutic effectiveness of aptamer-drug conjugate for xenograft tumor model of mice with human colorectal cancer.

Self-assembled monovalent lipidated mannose ligand as a standalone nanoadjuvant

Subunit vaccines require an immunostimulant (adjuvant) and/or delivery system to induce immunity. However, currently, available adjuvants are either too dangerous in terms of side effects for human use (experimental adjuvants) or have limited efficacy and applicability. In this study, we examined the capacity of mannose-lipopeptide ligands to enhance the immunogenicity of a vaccine consisting of polyleucine(L15)-antigen conjugates anchored to liposomes. The clinically tested Group A Streptococcus (GAS) B-cell epitope, J8, combined with universal T helper PADRE (P) was used as the antigen. Six distinct mannose ligands were incorporated into neutral liposomes carrying L15PJ8. While induced antibody titers were relatively low, the ligand carrying mannose, glycine/lysine spacer, and two palmitic acids as liposomal membrane anchoring moieties (ligand 3), induced significantly higher IgG titers than non-mannosylated liposomes. The IgG titers were significantly enhanced when positively charged liposomes were employed. Importantly, the produced antibodies were able to kill GAS bacteria. Unexpectedly, the physical mixture of only ligand 3 and PJ8 produced self-assembled nanorods that induced antibody titers as high as those elicited by the lead liposomal formulation and antigen adjuvanted with the potent, but toxic, complete Freund’s adjuvant (CFA). Antibodies produced upon immunization with PJ8 + 3 were even more opsonic than those induced by CFA + PJ8. Importantly, in contrast to CFA, ligand 3 did not induce observable adverse reactions or excessive inflammatory responses. Thus, we demonstrated that a mannose ligand, alone, can serve as an effective vaccine nanoadjuvant.

Freeze-Dryable, Stable, and Click-Reactive Nanoparticles Composed of Cello-oligosaccharides for Biomolecular Sensing.

Nanoparticles have been widely used as platforms for biomolecular sensing because of their high specific surface area and attractive properties depending on their constituents and structures. Nevertheless, it remains challenging to develop nanoparticulate sensing platforms that are easily storable without aggregation and conjugatable with various ligands in a simple manner. Herein, we demonstrate that nanoparticulate assemblies of cello-oligosaccharides with terminal azido groups are promising candidates. Azidated cello-oligosaccharides can be readily synthesized via the enzyme-catalyzed oligomerization reaction. This study characterized the assembled structures of azidated cello-oligosaccharides produced during the enzymatic synthesis and revealed that the terminal azidated cello-oligosaccharides formed rectangular nanosheet-shaped lamellar crystals. The azido groups located on the nanosheet surfaces were successfully exploited for antigen conjugation via the click chemistry. The resultant antigen-conjugated nanosheets allowed for the quantitative and specific detection of a corresponding antibody, even in 10% serum, owing to the antifouling properties of cello-oligosaccharide assemblies against proteins. It was found that the functionalized nanosheets were redispersible in water after freeze-drying. This remarkable characteristic is attributed to the well-hydrated saccharide residues on the nanosheet surfaces. Moreover, the antibody detection capability did not decline after the thermal treatment of the functionalized nanosheets in a freeze-dried state. Our findings contribute to developing convenient nanoparticulate biomolecular sensing platforms.

The Papain-like Protease Domain of Severe Acute Respiratory Syndrome Coronavirus 2 Conjugated with Human Beta-Defensin 2 and Co1 Induces Mucosal and Systemic Immune Responses against the Virus.

Most of the licensed vaccines against SARS-CoV-2 target spike proteins to induce viral neutralizing antibodies. However, currently prevalent SARS-CoV-2 variants contain many mutations, especially in their spike proteins. The development of vaccine antigens with conserved sequences that cross-react with variants of SARS-CoV-2 is needed to effectively defend against SARS-CoV-2 infection. Given that viral infection is initiated in the respiratory mucosa, strengthening the mucosal immune response would provide effective protection. We constructed a mucosal vaccine antigen using the papain-like protease (PLpro) domain of non-structural protein 3 of SARS-CoV-2. To potentiate the mucosal immune response, PLpro was combined with human beta-defensin 2, an antimicrobial peptide with mucosal immune adjuvant activity, and Co1, an M-cell-targeting ligand. Intranasal administration of the recombinant PLpro antigen conjugate into C57BL/6 and hACE2 knock-in (KI) mice induced antigen-specific T-cell and antibody responses with complement-dependent cytotoxic activity. Viral challenge experiments using the Wuhan and Delta strains of SARS-CoV-2 provided further evidence that immunized hACE2 KI mice were protected against viral challenge infections. Our study shows that PLpro is a useful candidate vaccine antigen against SARS-CoV-2 infection and that the inclusion of human beta-defensin 2 and Co1 in the recombinant construct may enhance the efficacy of the vaccine.

Polymeric Nanoparticles as Oral and Intranasal Peptide Vaccine Delivery Systems: The Role of Shape and Conjugation.

Mucosal vaccines are highly attractive due to high patient compliance and their suitability for mass immunizations. However, all currently licensed mucosal vaccines are composed of attenuated/inactive whole microbes, which are associated with a variety of safety concerns. In contrast, modern subunit vaccines use minimal pathogenic components (antigens) that are safe but typically poorly immunogenic when delivered via mucosal administration. In this study, we demonstrated the utility of various functional polymer-based nanostructures as vaccine carriers. A Group A Streptococcus (GAS)-derived peptide antigen (PJ8) was selected in light of the recent global spread of invasive GAS infection. The vaccine candidates were prepared by either conjugation or physical mixing of PJ8 with rod-, sphere-, worm-, and tadpole-shaped polymeric nanoparticles. The roles of nanoparticle shape and antigen conjugation in vaccine immunogenicity were demonstrated through the comparison of three distinct immunization pathways (subcutaneous, intranasal, and oral). No additional adjuvant or carrier was required to induce bactericidal immune responses even upon oral vaccine administration.

Cyclic Peptide Conjugate Vaccines and Physically Mixed Cyclic Peptide Vaccines for Subcutaneous Immunization.

Immune stimulants (adjuvants) enhance immune system recognition to provide an effective and individualized immune response when delivered with an antigen. Synthetic cyclic deca-peptides, co-administered with a toll-like receptor targeting lipopeptide, have shown self-adjuvant properties, dramatically boosting the immune response in a murine model as a subunit peptide-based vaccine containing group A Streptococcus peptide antigens.Here, we designed a novel peptide and lipid adjuvant system for the delivery of group A Streptococcus peptide antigen and a T helper peptide epitope. Following linear peptide synthesis on 2-chlorotrityl chloride resin, the linear peptide was cleaved and head-to-tail cyclized in solution. The selective arrangement of amino acids in the deca-peptide allowed for selective conjugation of lipids and/or peptide antigens following cyclisation. Using both solution-phase peptide chemistry and copper-catalyzed azide-alkyne cycloaddition reaction were covalently (and selectively) ligated lipid and/or peptide antigens onto the cyclic deca-peptide core. Subcutaneous administration of the vaccine design to mice resulted in the generation of a large number of serum immunoglobulin (Ig) G antibodies.

Conjugation of HIV-1 envelope to hepatitis B surface antigen alters vaccine responses in rhesus macaques

An effective HIV-1 vaccine remains a critical unmet need for ending the AIDS epidemic. Vaccine trials conducted to date have suggested the need to increase the durability and functionality of vaccine-elicited antibodies to improve efficacy. We hypothesized that a conjugate vaccine based on the learned response to immunization with hepatitis B virus could be utilized to expand T cell help and improve antibody production against HIV-1. To test this, we developed an innovative conjugate vaccine regimen that used a modified vaccinia virus Ankara (MVA) co-expressing HIV-1 envelope (Env) and the hepatitis B virus surface antigen (HBsAg) as a prime, followed by two Env–HBsAg conjugate protein boosts. We compared the immunogenicity of this conjugate regimen to matched HIV-1 Env-only vaccines in two groups of 5 juvenile rhesus macaques previously immunized with hepatitis B vaccines in infancy. We found expansion of both HIV-1 and HBsAg-specific circulating T follicular helper cells and elevated serum levels of CXCL13, a marker for germinal center activity, after boosting with HBsAg–Env conjugate antigens in comparison to Env alone. The conjugate vaccine elicited higher levels of antibodies binding to select HIV Env antigens, but we did not observe significant improvement in antibody functionality, durability, maturation, or B cell clonal expansion. These data suggests that conjugate vaccination can engage both HIV-1 Env and HBsAg specific T cell help and modify antibody responses at early time points, but more research is needed to understand how to leverage this strategy to improve the durability and efficacy of next-generation HIV vaccines.

Application of Highly Sensitive Immunosensor Based on Optical Waveguide Light-Mode Spectroscopy (OWLS) Technique for the Detection of the Herbicide Active Ingredient Glyphosate.

The herbicide active ingredient glyphosate is the most widely applied herbicidal substance worldwide. Currently it is the market-leading pesticide, and its use is projected to further grow 4.5-fold between 2022 and 2029. Today, glyphosate use exceeds one megaton per year worldwide, which represents a serious environmental burden. A factor in the overall boost in the global use of glyphosate has been the spread of glyphosate-tolerant genetically modified (GM) crops that allow post-emergence applications of the herbicide on these transgenic crops. In turn, cultivation of glyphosate-tolerant GM crops represented 56% of the glyphosate use in 2019. Due to its extremely high application rate, xenobiotic behaviour and a water solubility (11.6 mg/mL at 25 °C) unusually high among pesticide active ingredients, glyphosate has become a ubiquitous water pollutant and a primary drinking water contaminant worldwide, presenting a threat to water quality. The goal of our research was to develop a rapid and sensitive method for detecting this herbicide active ingredient. For this purpose, we applied the novel analytical biosensor technique optical waveguide light-mode spectroscopy (OWLS) to the label-free detection of glyphosate in a competitive immunoassay format using glyphosate-specific polyclonal antibodies. After immobilising the antigen conjugate in the form of a glyphosate conjugated to human serum albumin for indirect measurement, the sensor chip was used in a flow-injection analyser system. For the measurements, an antibody stock solution was diluted to 2.5 µg/mL. During the measurement, standard solutions were mixed with the appropriate concentration of antibodies and incubated for 1 min before injection. The linear detection range and the EC50 value of the competitive detection method were between 0.01 and 100 ng/mL and 0.60 ng/mL, respectively. After investigating the indirect method, we tested the cross-reactivity of the antibody with glyphosate and structurally related compounds.

SpyTag/SpyCatcher display of influenza M2e peptide on norovirus-like particle provides stronger immunization than direct genetic fusion.

Virus-like particles (VLPs) are similar in size and shape to their respective viruses, but free of viral genetic material. This makes VLP-based vaccines incapable of causing infection, but still effective in mounting immune responses. Noro-VLPs consist of 180 copies of the VP1 capsid protein. The particle tolerates C-terminal fusion partners, and VP1 fused with a C-terminal SpyTag self-assembles into a VLP with SpyTag protruding from its surface, enabling conjugation of antigens via SpyCatcher. To compare SpyCatcher-mediated coupling and direct peptide fusion in experimental vaccination, we genetically fused the ectodomain of influenza matrix-2 protein (M2e) directly on the C-terminus of norovirus VP1 capsid protein. VLPs decorated with SpyCatcher-M2e and VLPs with direct M2 efusion were used to immunize mice. We found that direct genetic fusion of M2e on noro-VLP raised few M2e antibodies in the mouse model, presumably because the short linker positions the peptide between the protruding domains of noro-VLP, limiting its accessibility. On the other hand, adding aluminum hydroxide adjuvant to the previously described SpyCatcher-M2e-decorated noro-VLP vaccine gave a strong response against M2e. Surprisingly, simple SpyCatcher-fused M2e without VLP display also functioned as a potent immunogen, which suggests that the commonly used protein linker SpyCatcher-SpyTag may serve a second role as an activator of the immune system in vaccine preparations. Based on the measured anti-M2e antibodies and cellular responses, both SpyCatcher-M2e as well as M2e presented on the noro-VLP via SpyTag/Catcher show potential for the development of universal influenza vaccines.

Abstract CT213: Biomarker analysis from Phase 1/2 study of tusamitamab ravtansine (SAR408701) in patients with advanced non-small cell lung cancer (NSCLC)

Abstract Background Tusamitamab ravtansine is an antibody-drug conjugate of a humanized carcinoembryonic antigen (CEA)-related cell adhesion molecule 5 (CEACAM5)-specific monoclonal antibody linked to DM4. A Phase 1/2 study (NCT02187848) showed tusamitamab ravtansine antitumor activity in pretreated patients (pts) with advanced nonsquamous NSCLC and high CEACAM5 expression. Here, we explore biomarker associations with tumor CEACAM5 expression by immunohistochemistry (IHC), and whether biomarkers predict objective response rate (ORR). Methods We assessed CEACAM5 expression by IHC, RNA sequencing, and whole exome sequencing (WES) on latest archival tumor samples; and circulating CEACAM5 (cCEACAM5) and CEA (cCEA). We enrolled 2 cohorts of pts with IHC CEACAM5 membrane expression at ≥2+ intensity: in ≥50% of tumor cells (high expressors, HEs, n = 64); and in ≥1% to <50% of tumor cells (moderate expressors, MEs, n = 28). Pts received tusamitamab ravtansine 100 mg/m2 IV every 2 weeks. Results cCEA and cCEACAM5 were strongly associated (Spearman rho, 0.9), with weak associations between IHC CEACAM5 and cCEA or cCEACAM5 (Spearman rho, 0.3 and 0.4, respectively). Higher levels of CEACAM5 mRNA were observed in CEACAM5 HEs vs MEs (P=0.0027). EGFR and KRAS genetic alterations by WES were present in 44.8% and 65.5% of CEACAM5 HEs, respectively, and 21.4% and 78.6% of CEACAM5 MEs, respectively. Confirmed partial responses were seen in 13/64 HEs (ORR 20.3%) and 2/28 MEs (ORR 7.1%). In CEACAM5 HEs with available baseline (BL) cCEA data, 25/62 (40.3%) had a cCEA level ≥100 µg/L, with a median value of 71.6 µg/L (range 1-8809); corresponding values in CEACAM5 MEs were 7/28 (25.0%) and 12.4 µg/L (range 0.5-684). In response evaluable CEACAM5 HEs with available BL cCEA data (n = 61), ORR was 10/24 (41.7%) in pts with high cCEA (≥100 µg/L) and 3/37 (8.1%) in pts with low cCEA (<100 µg/L); corresponding ORRs in CEACAM5 MEs were 0/7 and 2/21 (9.5%). Conclusions In CEACAM5 HEs, high cCEA was associated with numerically greater ORR vs low cCEA (41.7% vs 8.1%). Associations were also observed between: cCEA and cCEACAM5; IHC CEACAM5, cCEA, and cCEACAM5; and IHC CEACAM5 and CEACAM5 tumor mRNA levels, but not between IHC CEACAM5 and actionable oncogenic drivers. Clinical Trials Registration: ClinicalTrials.gov NCT02187848 Citation Format: Anas Gazzah, Joon Sang Lee, Emma Wang, Nils Ternès, Hong Wang, Eric Boitier, Aude Lartigau, Mustapha Chadjaa, Colette Dib, Gaëlle Muzard, Sandrine Valence, Anne Remaury, Cintia C. Palu, Anne-Laure Bauchet. Biomarker analysis from Phase 1/2 study of tusamitamab ravtansine (SAR408701) in patients with advanced non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT213.

Carbon Dots and Tumor Antigen Conjugates as Nanovaccines for Elevated Cancer Immunotherapy.

Cancer immunotherapy has become one of the current research hotspots. However, the deficiencies including restricted immunogenicity, insufficient antigen presentation, and low responsive rate limited their therapeutic applications. Own to the small size and excellent biocompatibility, carbon dots (CDs) can serve as nanovectors to improve the efficacy of cancer immunotherapy. Herein, a tumor antigen-based nanovaccines (GMal+B16F10-Ag and GMal+CT26-Ag) by the conjugation of CDs with the tumor cell-derived antigens (B16F10-Ag and CT26-Ag) is constructed. These nanovaccines can be effectively taken up by dendritic cells (DC2.4), promote DC cell maturation, cross-present the antigen to T cells, specifically target B16F10 melanoma or CT26 colon cancers, and inhibit tumor growth distinctly. This work illustrates the promise of CDs acting as versatile carriers for antigen delivery to achieve the optimal immunotherapeutic outcomes.

Liposomal Formulations of a Polyleucine-Antigen Conjugate as Therapeutic Vaccines against Cervical Cancer.

Human papilloma virus (HPV) is responsible for all cases of cervical cancer. While prophylactic vaccines are available, the development of peptide-based vaccines as a therapeutic strategy is still under investigation. In comparison with the traditional and currently used treatment strategies of chemotherapy and surgery, vaccination against HPV is a promising therapeutic option with fewer side effects. A peptide derived from the HPV-16 E7 protein, called 8Qm, in combination with adjuvants showed promise as a therapeutic vaccine. Here, the ability of polymerized natural amino acids to act as a self-adjuvating delivery system as a therapeutic vaccine was investigated for the first time. Thus, 8Qm was conjugated to polyleucine by standard solid-phase peptide synthesis and self-assembled into nanoparticles or incorporated in liposomes. The liposome bearing the 8Qm conjugate significantly increased mice survival and decreased tumor growth after a single immunization. Further, these liposomes eradicated seven-day-old well-established tumors in mice. Dendritic cell (DC)-targeting moieties were introduced to further enhance vaccine efficacy, and the newly designed liposomal vaccine was tested in mice bearing 11-day-old tumors. Interestingly, these DCs-targeting moieties did not significantly improve vaccine efficacy, whereas the simple liposomal formulation of 8Qm-polyleucine conjugate was still effective in tumor eradication. In summary, a peptide-based anticancer vaccine was developed that stimulated strong cellular immune responses without the help of a classical adjuvant.

Polymeric Nanoparticles as a Self‐Adjuvanting Peptide Vaccine Delivery System: The Role of Shape

AbstractAntigens incorporated in subunit vaccines are typically poorly immunogenic, so a strong immunostimulant (adjuvant) and/or delivery system is required to boost immunogenicity. In this work, the various functional polymer nanostructures, that is, rods, worms, spheres, and tadpoles are used to develop potent peptide antigen delivery systems. The antigen PADRE‐J8 (PJ8), derived from Group A Streptococcus (GAS) M‐protein, is either physically mixed or chemically conjugated to polymeric nanoparticles of different shapes. The physical mixture of polymeric nanoparticles and antigen is more effective in inducing antibody production than their chemical conjugates. Moreover, rod‐shaped polymeric nanoparticles in physical mixture with PJ8 elicited higher and more opsonic antibody titers than powerful complete Freund's adjuvant (CFA)‐adjuvanted antigen. Herein, for the first time it is demonstrated that a) the block copolymer, in nanoparticle form, can act as an immune adjuvant, b) nanoparticle shape plays a crucial role in their immunogenicity, and c) antigen conjugation is not required, nor is antigen encapsulation or absorption.

Highly stable gold nanoparticle-antigen conjugates with self-adjuvanting property for induction of robust antigen-specific immune responses

Poor long-term stability and formation of irreversible aggregates when subjected to a freeze-drying process greatly limits the clinical application of gold nanoparticles (GNPs) as a vaccine carrier. In this study, we synthesized a GNP–antigen conjugate with high colloidal stability by using a thiolated polyethylene glycol (PEG) linker to conjugate a model antigen (ovalbumin; OVA) onto the GNP surface (i.e. GNP-OVA) and demonstrated this conjugate had self-adjuvanting properties to augment antigen-specific immune responses. The synthesized GNP had an average hydrodynamic size of 13.8 ± 2.1 nm (n = 3); after conjugation of OVA, the diameter increased to 28.6 ± 7.3 nm (n = 3). The obtained GNP-OVA can maintain a stable dispersion state in aqueous solutions for at least 12 months and withstand stresses during lyophilization without creating irreversible aggregates. Compared with OVA alone or a mixture of PEG-functionalized GNP (GNP-PEG) and OVA (i.e. GNP-PEG/OVA), the chemical conjugation of OVA onto GNP-PEG substantially increased antigen uptake and upregulated major histocompatibility complex class II expression in macrophages. This indicated that the GNP can function as not only an adjuvant to promote the phagocytic activity of macrophages but also a carrier to deliver the conjugated antigen into the immune cells for the enhancement of its antigen presentation capability. Importantly, OVA-specific immunoglobulin G levels in the mice immunized with GNP-OVA were 4.1 and 2.9 times higher than those in the mice injected with OVA and GNP-PEG/OVA, respectively. These results demonstrated that the GNP-antigen conjugate exhibited remarkable stability either in liquid or freeze-dried form, which makes it attractive for further pharmaceutical applications. Moreover, covalently linking antigens onto the GNP surface was enabled to enhance the immunogenicity of antigens and boost immune responses, showing the potential of the GNP conjugation strategy for vaccine development.

Polydopamine-mediated quantity-based magnetic relaxation sensing for the rapid and sensitive detection of chloramphenicol in fish samples

Chloramphenicol is an antibiotic that cause adverse effects in humans. In this work, a novel quantity-based magnetic relaxation switching (sMRS) sensor using polydopamine (PDA) for signal amplification was successfully developed for the rapid and sensitive detection of chloramphenicol in fish samples. We first prepared the conjugation of large magnetic nanoparticles (MNPs) and CAP antigen (MNP-antigen), which performed a competitive immune reaction with chloramphenicol and chloramphenicol antibody. Horseradish peroxidase, that can bind to antibody at the surface of large MNPs, was employed to catalyze the rapid polymerization of dopamine to PDA, which was easily deposited onto the surface of large MNPs. The concentration of chloramphenicol is inversely proportional to the content of HRP after reaction, in other words, it is inversely proportional to the content of PDA. PDA then reacted with 30 nm-diameter amine-functionalized MNPs (NH2-MNP30), the consumption of which resulted in a decrease in the concentration of free NH2-MNP30 particles in solution. After magnetic separation, the remaining free NH2-MNP30 served as the magnetic probe for signal readout. The limit of detection of the sMRS assay for detecting chloramphenicol was 16.6 pg/mL, which was 49-fold lower than MRS sensor without signal amplification. This quantity-based MRS sensor can provide a powerful platform for enabling the rapid and sensitive detection of chloramphenicol in food samples.

Cell-penetrating peptides enhance peptide vaccine accumulation and persistence in lymph nodes to drive immunogenicity

Peptide-based cancer vaccines are widely investigated in the clinic but exhibit modest immunogenicity. One approach that has been explored to enhance peptide vaccine potency is covalent conjugation of antigens with cell-penetrating peptides (CPPs), linear cationic and amphiphilic peptide sequences designed to promote intracellular delivery of associated cargos. Antigen-CPPs have been reported to exhibit enhanced immunogenicity compared to free peptides, but their mechanisms of action invivo are poorly understood. We tested eight previously described CPPs conjugated to antigens from multiple syngeneic murine tumor models and found that linkage to CPPs enhanced peptide vaccine potency invivo by as much as 25-fold. Linkage of antigens to CPPs did not impact dendritic cell activation but did promote uptake of linked antigens by dendritic cells both invitro and invivo. However, T cell priming invivo required Batf3-dependent dendritic cells, suggesting that antigens delivered by CPP peptides were predominantly presented via the process of cross-presentation and not through CPP-mediated cytosolic delivery of peptide to the classical MHC class I antigen processing pathway. Unexpectedly, we observed that many CPPs significantly enhanced antigen accumulation in draining lymph nodes. This effect was associated with the ability of CPPs to bind to lymph-trafficking lipoproteins and protection of CPP-antigens from proteolytic degradation in serum. These two effects resulted in prolonged presentation of CPP-peptides in draining lymph nodes, leading to robust T cell priming and expansion. Thus, CPPs can act through multiple unappreciated mechanisms to enhance T cell priming that can be exploited for cancer vaccines with enhanced potency.

Assessing the Immunomodulatory Effect of Size on the Uptake and Immunogenicity of Influenza- and Hepatitis B Subunit Vaccines In Vitro.

Viral subunit vaccines are a safer and more tolerable alternative to whole inactivated virus vaccines. However, they often come with limited efficacy, necessitating the use of adjuvants. Using free and particle-bound viral antigens, we assessed whether size affects the uptake of those antigens by human monocyte-derived dendritic cells (Mo-DCs) and whether differences in uptake affect their capacity to stimulate cytokine production by T cells. To this end, influenza antigens and hepatitis B surface antigen (HBsAg) were covalently conjugated to polystyrene particles of 500 nm and 3 μm. Cellular uptake of the antigens, either unconjugated or conjugated, and their capacity to stimulate T cells within a population of human peripheral blood mononuclear cells (PBMCs) were measured by flow cytometry. Conjugation of both antigens to particles significantly increased their uptake by Mo-DCs. Moreover, both the 500 nm and 3 μm influenza conjugates induced significantly higher numbers of cytokine-producing CD4+ T cells and induced increased production of the pro-inflammatory cytokines IFNγ and TNFα. In contrast, conjugation of HBsAg to particles did not notably affect the T cell response. In conclusion, conjugation of antigen to 500 nm and 3 μm particles leads to increased antigen uptake by human Mo-DCs, although the capacity of such conjugates to induce T cell stimulation likely depends on the immunological status of the PBMC donor.

Synthesis and immunogenicity of the linear conjugates of polyacrylic acid and antigenic peptide of human papillomavirus

Polyelectrolytes (polymers carrying positively or negatively charged groups) can be used as adjuvants in subunit vaccine formulations to increase the immunogenicity of antigenic molecules. A vaccine prototype can be obtained by covalently bonding antigenic molecules to polyelectrolytes or using their physical mixtures with polyelectrolytes. Covalent conjugates of polyelectrolytes with antigens are known to exhibit stronger immunogenicity compared to physical mixtures of antigens and polyelectrolytes. In this study, linear conjugates of polyacrylic acid (PAA) with an antigenic peptide sequence (STHVDIRTLEDLLMGW) of the HPV-16 E7 oncoprotein were produced. PAAs of different molecular weights with narrow dispersity were synthesized by RAFT polymerization. The linear conjugates of PAA and the peptide were obtained by covalent bonding via the chain-end thiol group of PAA and N-terminal maleimide group of the peptide using the maleimide-thiol reaction. For this purpose, the trithiocarbonate end group of the polymer was reduced with excess ethylenediamine to the thiol group. The produced PAA-peptide conjugates were characterized by GPC, fluorescence, and NMR spectroscopies. Conjugation efficiency was above 80% for all PAAs. Antigenic peptide conjugates of PAAs of different molecular weights were investigated in MTT and nitric oxide (NO) assays in which the linear PAA-peptide conjugates exhibit high biocompatibility and stimulated the macrophages to release high levels of NO.

Designing a novel E2-IFN-γ fusion protein against CSFV by immunoinformatics and structural vaccinology approaches

Subunit vaccines with high purity and safety are gradually becoming a main trend in vaccinology. However, adjuvants such as interferon-gamma (IFN-γ) are required to enhance immune responses of subunit vaccines due to their poor immunogenicity. The conjugation of antigen with adjuvant can induce more potent immune responses compared to the mixture of antigen and adjuvant. At the same time, the selection of linker, indispensable in the construction of the stable and bioactive fusion proteins, is complicated and time-consuming. The development of immunoinformatics and structural vaccinology approaches provides a means to address the abovementioned problem. Therefore, in this study, a E2-IFN-γ fusion protein with an optimal linker (E2-R2-PIFN) was designed by bioinformatics approaches to improve the immunogenicity of the classical swine fever virus (CSFV) E2 subunit vaccine. Moreover, the E2-R2-PIFN fusion protein was expressed in HEK293T cells and the biological effects of IFN-γ in E2-R2-PIFN were confirmed in vitro via Western blotting. Here, an alternative method is utilized to simplify the design and validation of the antigen-adjuvant fusion protein, providing a potential subunit vaccine candidate against CSFV. KEY POINTS: • An effective and simple workflow of antigen-adjuvant fusion protein design and validation was established by immunoinformatics and structural vaccinology. • A novel E2-IFN-γ fusion protein with an optimal linker was designed as a potential CSFV vaccine. • The bioactivity of the newly designed fusion protein was preliminarily validated through in vitro experiments.