Canister valve and actuator deposition in metered dose inhalers formulated with low-GWP propellants.

A challenge in pressurised metered-dose inhaler (pMDI) formulation design is management of adhesion of the drug to the canister wall, valve and actuator internal components and surfaces. Wall-material interactions differ between transparent vials used for visual inspection and metal canister pMDI systems. This is of particular concern for low greenhouse warming potential (GWP) formulations where propellant chemistry and solubility with many drugs are not well understood. In this study, we demonstrate a novel application of X-ray fluorescence spectroscopy using synchrotron radiation to assay the contents of surrogate solution and suspension pMDI formulations of potassium iodide and barium sulphate in propellants HFA134a, HFA152a and HFO1234ze(E) using aluminium canisters and standard components. Preliminary results indicate that through unit life drug distribution in the canister valve closure region and actuator can vary significantly with new propellants. For solution formulations HFO1234ze(E) propellant shows the greatest increase in local deposition inside the canister valve closure region as compared to HFA134a and HFA152a, with correspondingly reduced actuator deposition. This is likely driven by chemistry changes. For suspension formulations HFA152a shows the greatest differences, due to its low specific gravity. These changes must be taken into consideration in the development of products utilising low-GWP propellants.

New modular platform based on multi-adjuvanted amphiphilic chitosan nanoparticles for efficient lipopeptide vaccine delivery against group A streptococcus.

An effective vaccine against group A streptococcus (GAS) is highly desirable for definitive control of GAS infections. In the present study, two variants of amphiphilic chitosan nanoparticles-based GAS vaccines were developed. The vaccines were primarily composed of encapsulated KLH protein (a source of T helper cell epitopes) and lipidated M-protein derived B cell peptide epitope (lipoJ14) within the amphiphilic structure of nanoparticles. The only difference between them was one of the nanoparticles vaccines received additional surface coating with poly (I:C). The formulated vaccines exhibited nanosized particles within the range of 220-240 nm. Cellular uptake study showed that nanoparticles vaccine without additional poly (I:C) coating has greater uptake by dendritic cells and macrophages compared to nanoparticles vaccine that was functionalized with poly (I:C). Both vaccines were found to be safe in mice and showed negligible cytotoxicity against HEK293 cells. Upon immunization in mice, both nanoparticle vaccines produced high antigen-specific antibodies titres that were regulated by a balanced Th1 and Th2 response compared to physical mixture. These antibodies elicited high opsonic activity against the tested GAS strains. Overall, our data demonstrated that amphiphilic chitosan nanoparticles platform induced a potent immune response even without additional inclusion of poly (I:C).

Validation of a cell integrated next-generation impactor to assess in vitro drug transport of physiologically relevant aerosolised particles.

The development of novel inhaled formulations in the pre-clinical stage has been impeded by a lack of meaningful information related to drug dissolution and transport at the lung epithelia due to the absence of physiologically relevant in vitro respiratory models. The objective of the present study was to develop an in vitro experimental model, which combined the next generation impactor (NGI) and two respiratory epithelial cell lines, for examining the aerodynamic performance of dry powder inhalers and the fate of aerosolised drugs following lung deposition. The NGI impaction plates of stage 3 (i.e., a cut-off diameter of 2.82-4.46 µm) and stage 7 (i.e., a cut-off diameter of 0.34-0.55 µm) were modified to accommodate 3 cell cultures inserts. Specifically, Calu-3 cells and H441 cells, which are representative of the bronchial and alveolar epithelia in the lung, respectively, were cultivated at the air-liquid interface on SnapwellsTM with polycarbonate membranes. The aerodynamic particle size distribution of the modified NGI was investigated using resveratrol dry powder formulation (as a model drug). The suitability of such an in vitro model was confirmed by examining the in vitro aerodynamic performance of the model drug as compared to the conventional NGI setup (i.e., without the integrated Snapwell inserts), as well as the effect of experimental conditions (e.g., 60 L/min airflows) on the cells in the integrated Snapwell inserts. After deposition of the aerodynamically fractioned resveratrol, the permeation of the drug across the cell layer to the basolateral chamber of the Snapwell inserts was evaluated over 24 h. Results obtained from the drug transport study showed that the cell-integrated NGI provided realistic drug delivery conditions to the cells that can be used to assess the fate of fractionated aerosol particles. This system enables a better understanding of the in vitro drug deposition in the lungs and allows studies on both aerodynamic characterisation and drug transport (drug biological interactions with the cells) to be performed simultaneously.

Development of excipients free inhalable co-spray-dried tobramycin and diclofenac formulations for cystic fibrosis using two and three fluid nozzles.

This study aims to investigate the effect of physicochemical properties and aerosol performance of two (2FN) and three-fluid nozzles (3FN) on the inhalable co-formulation of tobramycin and diclofenac dry powders. Combination formulations of tobramycin and diclofenac at 2:1 and 4:1 w/w ratios were prepared at a laboratory scale using a spray dryer in conjunction with a 2FN or 3FN. Powder size, morphology, solid-state characteristics, and aerodynamic and dissolution properties were characterised. The nozzle types and the formulation composition influenced the yield, particle size, solid-state properties, aerosolization behaviour and dissolution of the co-spray dried formulations. In particular, using the 2FN the co-spray dried formulation of tobramycin and diclofenac at 2:1 w/w showed smaller particle size (D50, 3.01 ± 0.06 µm), high fine particle fractions (FPF) (61.1 ± 3.6% for tobramycin and 65.92 ± 3 for diclofenac) and faster dissolution with approx. 70% diclofenac released within 3 h and approx. 90% tobramycin was released within 45 min. However, the 3FN for the co-spray dried formulation of tobramycin and diclofenac at a 2:1 w/w ratio showed a larger particle size (D50, 3.42 ± 0.02 µm), lower FPF (40.6 ± 3.4% for tobramycin and 36.9 ± 0.84 for diclofenac) and comparative slower dissolution with approx. 60% diclofenac was released within 3 h and 80% tobramycin was released within 45 min. A similar trend was observed when the tobramycin to diclofenac ratio was increased to 4:1 w/w. Overall results suggest that spray drying with 2FN showed a superior and viable approach to producing excipients-free inhalable co-spray dried formulations of tobramycin and diclofenac. However, the formulation produced using the 3FN showed higher enrichment of hydrophobic diclofenac and an ability to control the tobramycin drug release in vitro.

Development of a hyperbranched polymer-based methotrexate nanomedicine for rheumatoid arthritis.

Methotrexate (MTX) is an effective disease modifying anti-rheumatic drug, but can cause significant hepatotoxicity and liver failure in some individuals. The goal of this work was to develop a MTX-conjugated hyperbranched polymeric nanoparticle based on oligo(ethylene glycol) methyl ether methacrylate (OEGMA) and examine its ability to selectively deliver MTX to rheumatic joints while sparing the liver. MTX was conjugated to the hyperbranched polymer via a matrix metalloproteinase-13 cleavable peptide linker. Two populations of nanoparticles were produced, with sizes averaging 20 and 200nm. Tri-peptide (FFK)-modified MTX was liberated in the presence of matrix metalloproteinase 13 (MMP-13)and showed 100 to 1000-fold lower antiproliferative capacity in monocytic THP-1 cells compared to unmodified MTX, depending on whether the gamma-carboxylate of MTX was functionalized with O-tert-butyl. Nanoparticles showed prolonged plasma exposure after intravenous injection with a terminal half-life of approximately 1 day, but incomplete (50%) absorption after subcutaneous administration. Nanoparticles selectively accumulated in inflamed joints in a rat model of rheumatoid arthritis and showed less than 5% biodistribution in the liver after 5 days. MTX-OtBu nanoparticles also showed no hepatocellular toxicity at 500 µM MTX equivalents. This work provides support for the further development of OEGMA-based hyperbranched polymers as MTX drug delivery systems for rheumatoid arthritis. STATEMENT OF SIGNIFICANCE: Nanomedicines containing covalently conjugated methotrexate offer the potential for selective accumulation of the potent hepatotoxic drug in rheumatic joints and limited liver exposure. One limitation of the high surface presentation of methotrexate on a nanoparticle surface, however, is the potential for enhanced liver uptake. We developed several OEGMA-based hyperbranched polymers containing alpha-carboxyl modified and unmodified methotrexate conjugated via an MMP-13 cleavable hexapeptide linker. The modified methotrexate polymer showed promising in vitro and in vivo behavior warranting further development and optimization as an anti-rheumatic nanomedicine. This work presents a new avenue for further research into the development of hyperbranched polymers for rheumatoid arthritis and suggests interesting approaches that may overcome some limitations associated with the translation of anti-rheumatic nanomedicines into patients.

DENV-Mimetic Polymersome Nanoparticles Bearing Multi-Epitope Lipopeptides Antigen as the Next-Generation Dengue Vaccine.

Dengue remains a severe threat to public health. The safety and efficacy of the licensed dengue vaccine is not clinically satisfactory, which necessitate the need of new approach in designing an effective dengue vaccine without eliciting adverse reaction. Herein, we have designed a lipidated multi-epitope peptide vaccine (LipoDV) that can elicit highly targeted humoral and cell-mediated immune responses. To improve its immunogenicity, LipoDV was presented on the surface of MPLA-functionalized polymersome nanoparticles (PNs-LipoDV-MPLA). The as-constructed vaccine delivery platform resembles the structural morphology of DENV owing to its spherical nanoscale particle size and surface immunostimulatory properties given by LipoDV and MPLA that emulating the functional role of DENV E and prM/M proteins respectively. A proof-of-concept study demonstrated that BALB/c mice immunized with PNs-LipoDV-MPLA induced a stronger antigen-specific antibody response with an enhanced cell-mediated immunity as characterized by the elevated IFN-γ secretion in comparison to other tested vaccine candidates which possess a lesser structural trait of DENV. The DENV-mimicking nanoparticles vaccine exhibited negligible toxicity as analyzed by hemolytic test, MTT assay, histopathological examination and abnormal toxicity test on immunized mice. Collectively, our study provides a strong foundation in designing an effective peptide-based vaccine delivery platform against DENV infection.

Development of Inhalable Spray Dried Nitrofurantoin Formulations for the Treatment of Emphysema.

A central characteristic of emphysematous progression is the continuous destruction of the lung extracellular matrix (ECM). Current treatments for emphysema have only addressed symptoms rather than preventing or reversing the loss of lung ECM. Nitrofurantoin (NF) is an antibiotic that has the potential to induce lung fibrosis as a side effect upon oral administration. Our study aims to repurpose NF as an inhalable therapeutic strategy to upregulate ECM expression, thereby reversing the disease progression within the emphysematous lung. Spray-dried (SD) formulations of NF were prepared in conjunction with a two-fluid nozzle (2FN) and three-fluid nozzle (3FN) using hydroxypropyl methylcellulose (HPMC) and NF at 1:1 w/w. The formulations were characterized for their physicochemical properties (particle size, morphology, solid-state characteristics, aerodynamic behaviour, and dissolution properties) and characterized in vitro with efficacy studies on human lung fibroblasts. The 2FN formulation displayed a mass mean aerodynamic diameter (MMAD) of 1.8 ± 0.05 µm and fine particle fraction (FPF) of 87.4 ± 2.8% with significantly greater deposition predicted in the lower lung region compared to the 3FN formulation (MMAD: 4.4 ± 0.4 µm; FPF: 40 ± 5.8%). Furthermore, drug dissolution studies showed that NF released from the 2FN formulation after 3 h was significantly higher (55.7%) as compared to the 3FN formulation (42.4%). Importantly, efficacy studies in human lung fibroblasts showed that the 2FN formulation induced significantly enhanced ECM protein expression levels of periostin and Type IV Collagen (203.2% and 84.2% increase, respectively) compared to untreated cells, while 3FN formulations induced only a 172.5% increase in periostin and a 38.1% increase in type IV collagen. In conclusion, our study highlights the influence of nozzle choice in inhalable spray-dried formulations and supports the feasibility of using SD NF prepared using 2FN as a potential inhalable therapeutic agent to upregulate ECM protein production.

Poly(hydrophobic amino acid) Conjugates for the Delivery of Multiepitope Vaccine against Group A Streptococcus.

Peptide-based vaccines are composed of small, defined, antigenic peptide epitopes. They are designed to induce well-controlled immune responses. Multiple epitopes are often employed in these vaccines to cover strain variability of a pathogen. However, peptide epitopes cannot stimulate adequate immune responses on their own and require an adjuvant (immune stimulant) and/or delivery system. Here, we designed and synthesized a multiepitope vaccine candidate against Group A Streptococcus (GAS) composed of several B-cell epitopes (J8, PL1, and 88/30) derived from GAS M-protein, universal PADRE T-helper cell epitope, and a polyleucine self-adjuvanting unit. The vaccine components were conjugated together (using mercapto-maleimide and azide-alkyne Huisgen cycloaddition reactions) or delivered as a mixture. The conjugated multiepitope vaccine candidate self-assembled into small nanoparticles and chain-like aggregated nanoparticles (CLANs) that were able to induce the production of J8-, PL1-, and 88/30-specific antibodies in mice. The multiepitope conjugate and the physical mixture of conjugates bearing the individual epitopes produced similar nanoparticles and induced comparable immune responses. Hence, simple physical mixing can replace complex chemical conjugation to produce multiepitope nanoparticles with equivalent morphology and immunological efficacy. This greatly simplifies vaccine production.

Cell-Penetrating Peptides-Based Liposomal Delivery System Enhanced Immunogenicity of Peptide-Based Vaccine against Group A Streptococcus.

Peptide-based vaccine development represents a highly promising strategy for preventing Group A Streptococcus (GAS) infection. However, these vaccines need to be administered with the help of a delivery system and/or immune adjuvant. Cell-penetrating peptides (CPPs) have been used as a powerful tool for delivering various therapeutic agents, including peptides, as they can overcome the permeability barrier of cell membranes. Here, we used CPPs to deliver our lead lipopeptide-based vaccine (LCP-1). CPPs were anchored through a spacer to LCP-1-bearing multilamellar and unilamellar liposomes and administered to Swiss outbred mice. Tat47-57 conjugated to two palmitic acids via a (Gly)6 spacer (to form a liposome-anchoring moiety) was the most efficient system for triggering immune responses when combined with multilamellar liposomes bearing LCP-1. The immunostimulatory potential of a variety of other CPPs was examined following intranasal administration in mice. Among them, LCP-1/liposomes/Tat47-57 and LCP-1/liposomes/KALA induced the highest antibody titers. The antibodies produced showed high opsonic activity against clinically isolated GAS strains D3840 and GC2 203. The use of the CPP-liposome delivery system is a promising strategy for liposome-based GAS vaccine development.

Recent advances in nano/microparticle-based oral vaccines.

Background: Vaccines are often recognized as one of the most cost-effective public health interventions in controlling infectious diseases. Most pathogens infiltrate the body from mucosal sites, primarily from the oral and pulmonary region and reach the systemic circulation where disease manifestation starts. Traditional needle-based vaccines are usually not capable of inducing immunity at the mucosal sites where pathogen infiltrates start, but induces systemic immunity. In contrast to needle-based vaccines, mucosally administered vaccines induce immunity at both the mucosal sites and systemically. The oral route of immunization is the most convenient way to administer the vaccines. However, due to the complicated and hostile gastrointestinal structure and environment, vaccines need to overcome major hurdles while retaining their stability and immunogenicity. Area covered: This review will briefly discuss different barriers to oral vaccine development. It gives a brief overview of different types of nano/microparticle-based oral vaccines and discusses how physicochemical characteristics of the particles influence overall immunity after oral immunization. Expert opinion: Formulation strategies using novel lipid and polymer-based nano/microparticle platforms retain stability and antigenicity of vaccines against the harsh gastrointestinal condition. The physicochemical properties of particles can be uniquely tailored to prolong the release of antigens, and attached ligands (M-cells and APC-ligands) can precisely target uptake by immune cells. These represent viable strategies for efficient delivery of oral vaccines.

The Impact of Polymer Size and Cleavability on the Intravenous Pharmacokinetics of PEG-Based Hyperbranched Polymers in Rats.

A better understanding of the impact of molecular size and linkers is important for PEG-based hyperbranched polymers (HBPs) intended as tailored drug delivery vehicles. This study aimed to evaluate the effects of crosslinker chemistry (cleavable disulphide versus non-cleavable ethylene glycol methacrylate (EGDMA) linkers) and molecular weight within the expected size range for efficient renal elimination (22 vs. 48 kDa) on the intravenous pharmacokinetic and biodistribution properties of 89Zr-labelled HBPs in rats. All HBPs showed similar plasma pharmacokinetics over 72 h, despite differences in linker chemistry and size. A larger proportion of HBP with the cleavable linker was eliminated via the urine and faeces compared to a similar-sized HBP with the non-cleavable linker, while size had no impact on the proportion of the dose excreted. The higher molecular weight HBPs accumulated in organs of the mononuclear phagocyte system (liver and spleen) more avidly than the smaller HBP. These results suggest that HBPs within the 22 to 48 kDa size range show no differences in plasma pharmacokinetics, but distinct patterns of organ biodistribution and elimination are evident.

Development of Polyelectrolyte Complexes for the Delivery of Peptide-Based Subunit Vaccines against Group A Streptococcus.

Peptide subunit vaccines hold great potential compared to traditional vaccines. However, peptides alone are poorly immunogenic. Therefore, it is of great importance that a vaccine delivery platform and/or adjuvant that enhances the immunogenicity of peptide antigens is developed. Here, we report the development of two different systems for the delivery of lipopeptide subunit vaccine (LCP-1) against group A streptococcus: polymer-coated liposomes and polyelectrolyte complexes (PECs). First, LCP-1-loaded and alginate/trimethyl chitosan (TMC)-coated liposomes (Lip-1) and LCP-1/alginate/TMC PECs (PEC-1) were examined for their ability to trigger required immune responses in outbred Swiss mice; PEC-1 induced stronger humoral immune responses than Lip-1. To further assess the adjuvanting effect of anionic polymers in PECs, a series of PECs (PEC-1 to PEC-5) were prepared by mixing LCP-1 with different anionic polymers, namely alginate, chondroitin sulfate, dextran, hyaluronic acid, and heparin, then coated with TMC. All produced PECs had similar particle sizes (around 200 nm) and surface charges (around + 30 mV). Notably, PEC-5, which contained heparin, induced higher antigen-specific systemic IgG and mucosal IgA titers than all other PECs. PEC systems, especially when containing heparin and TMC, could function as a promising platform for peptide-based subunit vaccine delivery for intranasal administration.

Lipopeptide-Based Oral Vaccine Against Hookworm Infection.

BACKGROUND: The human hookworm, Necator americanus, is a parasite that infects almost half a billion people worldwide. Although treatment is available, vaccination is favorable to combat the spread of this parasite due to its wide distribution and continuous reinfection cycle in endemic communities. METHODS: We have designed a lipopeptide oral delivery system using a B-cell epitope derived from the aspartic protease Na-APR-1 from N americanus, attached to a T-helper epitope. Lipopeptides were self-assembled into nanoparticles or entrapped in liposomes that were electrostatically coated with alginate and trimethyl chitosan polymer shields. The adjuvant-free vaccine candidates were orally administered to mice and generated a humoral immune response against both peptide antigen, and the parent protein in the hookworm gut. RESULTS: The vaccine candidates were evaluated in a rodent hookworm challenge model, resulting in up to 98% and 99% decreases in mean intestinal worm and egg burdens in immunized mice, respectively. CONCLUSIONS: Lipopeptide survived the gastrointestinal conditions, induced humoral immune responses and drived protection against parasite challenge infection.

Subunit-based mucosal vaccine delivery systems for pulmonary delivery - Are they feasible?

Pulmonary infections are the most common cause of death globally. However, the development of mucosal vaccines that provide protective immunity against respiratory pathogens are limited. In contrast to needle-based vaccines, efficient vaccines that are delivered via noninvasive mucosal routes (such as via the lungs and nasal passage) produce both antigen-specific local mucosal IgA and systemic IgG protective antibodies. One major challenge in the development of pulmonary vaccines using subunit antigens however, is the production of optimal immune responses. Subunit vaccines therefore rely upon use of adjuvants to potentiate immune responses. While the lack of suitable mucosal adjuvants has hindered progress in the development of efficient pulmonary vaccines, particle-based systems can provide an alternative approach for the safe and efficient delivery of subunit vaccines. In particular, the rational engineering of particulate vaccines with optimal physicochemical characteristics can produce long-term protective immunity. These protect antigens against enzymatic degradation, target antigen presenting cells and initiate optimal humoral and cellular immunity. This review will discuss our current understanding of pulmonary immunology and developments in fabricating particle characteristics that may evoke potent and durable pulmonary immunity.

Intranasal delivery of nanoparticle-based vaccines.

Most pathogens gain access to the human body and initiate systemic infections through mucosal sites. A large number of currently marketed licensed vaccines are parenterally administered; they generate strong systemic immunity but not mucosal immunity. Nasal vaccination is an appealing strategy for the induction of mucosal-specific immunity; however, its development is mostly challenged by several factors, such as inefficient antigen uptake, its rapid mucociliary clearance, size-restricted permeation across epithelial barriers and absence of safe human mucosal adjuvants. Therefore, a safer mucosal-adjuvanting strategy or efficient mucosal delivery platform is much warranted. This review summarizes challenges and the rationale for nasal vaccine development with a special focus on the use of nanoparticles based on polymers and lipids for mucosal vaccine delivery.

Highly Immunogenic Trimethyl Chitosan-based Delivery System for Intranasal Lipopeptide Vaccines against Group A Streptococcus.

BACKGROUND: Group A streptococcus (GAS) primarily colonizes the mucosal region of the upper respiratory tract, slowly leading to systemic infections. Thus, GAS-specific antibody responses are desirable at mucosal sites for early prevention against GAS colonization. METHODS: Herein, we developed a potent nanoliposomes-based delivery system for mucosally active lipid core peptide (LCP)-based vaccines. RESULTS: Trimethyl chitosan (TMC)-coated liposomes that bore a B-cell epitope derived from GAS Mprotein, stimulated potent epitope-specific mucosal and systemic antibody titres after only one boost following intranasal immunization in Swiss outbred mice. The immune responses were durable even at day 139 post-primary immunization. CONCLUSION: The enhanced vaccine efficacy, lowered dose, and simple and cost-effective process of producing the coated nanoliposomes should be particularly useful in developing potent peptide-based vaccines to prevent infections at the mucosal sites.

Double adjuvanting strategy for peptide-based vaccines: trimethyl chitosan nanoparticles for lipopeptide delivery.

AIM: To develop novel polymer-based nanoscale delivery system for lipopeptide-based vaccine against group A Streptococcus (GAS). MATERIALS & METHODS: Four types of lipopeptide antigen-loaded polymeric nanoparticles (NP) were prepared. NP were accessed for their capacity to be taken up by dendritic cells; effect on dendritic cell maturation; ability to induce mucosal and systemic immunity; and capability to induce antibody responses that opsonize GAS bacteria. RESULTS & DISCUSSION: The combination of adjuvanting properties of lipopeptides and dextran/trimethyl chitosan-based NP had a synergistic effect on humoral immunity, and the produced antibodies showed high opsonic activity against clinical GAS isolates. CONCLUSION: Biocompatible NP-bearing trimethyl chitosan and dextran are efficient as mucosal adjuvants for the intranasal delivery of lipopeptide-based vaccines.

Lipid core peptide/poly(lactic-co-glycolic acid) as a highly potent intranasal vaccine delivery system against Group A streptococcus.

Rheumatic heart disease represents a leading cause of mortality caused by Group A Streptococcus (GAS) infections transmitted through the respiratory route. Although GAS infections can be treated with antibiotics these are often inadequate. An efficacious GAS vaccine holds more promise, with intranasal vaccination especially attractive, as it mimics the natural route of infections and should be able to induce mucosal IgA and systemic IgG immunity. Nanoparticles were prepared by either encapsulating or coating lipopeptide-based vaccine candidate (LCP-1) on the surface of poly(lactic-co-glycolic acid) (PLGA). In vitro study showed that encapsulation of LCP-1 vaccine into nanoparticles improved uptake and maturations of antigen-presenting cells. The immunogenicity of lipopeptide incorporated PLGA-based nanoparticles was compared with peptides co-administered with mucosal adjuvant cholera toxin B in mice upon intranasal administration. Higher levels of J14-specific salivary mucosal IgA and systemic antibody IgG titres were observed for groups immunized with encapsulated LCP-1 compared to LCP-1 coated nanoparticles or free LCP-1. Systemic antibodies obtained from LCP-1 encapsulated PLGA NPs inhibited the growth of bacteria in six different GAS strains. Our results show that PLGA-based lipopeptide delivery is a promising approach for rational design of a simple, effective and patient friendly intranasal GAS vaccine resulting in mucosal IgA response.

The Role of Size in Development of Mucosal Liposome-Lipopeptide Vaccine Candidates Against Group A Streptococcus.

BACKGROUND: Group A streptococcus (GAS) is an exclusively human pathogenic bacteria. A delay in treatment of GAS infection often lead to severe diseases such as rheumatic heart disease which attributes to hundreds of thousands deaths annually. For the past few decades, the quest for a commercial GAS vaccine has been futile. Currently one of the most investigated strategies to develop vaccine against GAS includes the use of conserved epitopes from major virulent factor of GAS, M-protein. METHODS: In this study, cationic liposomes of various sizes (70 nm to 1000 nm) were prepared with dimethyldioctadecylammonium bromide (DDAB) encapsulating lipopeptide bearing M-protein derived B-cell epitope (J14). RESULTS: Smaller liposomes induced higher antibody titres, though the differences between groups were not statistically significant. CONCLUSION: Nonetheless, all mice which were immunized with liposome-lipopeptide delivery system elicited high levels of systemic (IgG) and mucosal antibodies (IgA), which were discernably higher than those induced with the help of commercial adjuvant (cholera toxin B subunit).

Structure-activity relationship of lipid core peptide-based Group A Streptococcus vaccine candidates.

Infection with Group A Streptococcus (GAS) can result in a range of different illnesses, some of which are fatal. Currently, our efforts to develop a vaccine against GAS focuses on the lipid core peptide (LCP) system, a subunit vaccine containing a lipoamino acid (LAA) moiety which allows the stimulation of systemic antibody activity. In the present study, a peptide (J14) representing the B-cell epitope from the GAS M protein was incorporated alongside a universal T-helper epitope (P25) in four LCP constructs of different spatial orientation or LAA lengths. Through structure-activity studies, it was discovered that while the alteration of the LCP orientation had a weaker effect on immunostimulation, increasing the LAA side chain length within the construct increased antibody responses in murine models. Furthermore, the mice immunised with the lead LCP construct were also able to maintain antibody activity throughout the course of five months. These findings highlight the importance of LAA moieties in the development of intranasal peptide vaccines and confirmed that its side chain length has an effect on the immunogenicity of the structure.