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.

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.

Plasmodium-infected erythrocytes induce secretion of IGFBP7 to form type II rosettes and escape phagocytosis.

In malaria, rosetting is described as a phenomenon where an infected erythrocyte (IRBC) is attached to uninfected erythrocytes (URBC). In some studies, rosetting has been associated with malaria pathogenesis. Here, we have identified a new type of rosetting. Using a step-by-step approach, we identified IGFBP7, a protein secreted by monocytes in response to parasite stimulation, as a rosette-stimulator for Plasmodium falciparum- and P. vivax-IRBC. IGFBP7-mediated rosette-stimulation was rapid yet reversible. Unlike type I rosetting that involves direct interaction of rosetting ligands on IRBC and receptors on URBC, the IGFBP7-mediated, type II rosetting requires two additional serum factors, namely von Willebrand factor and thrombospondin-1. These two factors interact with IGFBP7 to mediate rosette formation by the IRBC. Importantly, the IGFBP7-induced type II rosetting hampers phagocytosis of IRBC by host phagocytes.

Malaria is a life-threatening disease transmitted by mosquitoes infected with Plasmodium parasites. Part of the parasite life cycle happens inside human red blood cells. The surface of an infected red blood cell is coated with parasite proteins, which attract the attention of white blood cells called monocytes. These immune cells circulate in the bloodstream and use a process called phagocytosis to essentially 'eat' any infected cells they encounter. However, the monocytes cannot always reach the infected cells. Some of the proteins made by the parasites make the infected red blood cells stickier than normal. This allows the infected red blood cells to surround themselves in a protective cage of uninfected red blood cells. Known as "rosettes" because of their flower-like shape, these cages seem to protect the infected cells from attack by the immune system. Lee et al. noticed that adding white blood cells to parasite-infected red blood cells made them clump together more, but it was unclear exactly how and why this happened. To find out, Lee et al. took fluid from around monocytes grown in the laboratory and added it to red blood cells infected with Plasmodium parasites. This made the cells clump together, suggesting that something in the fluid may potentially be alerting the parasites to impending immune attack. The fluid contained almost 700 different molecules, and Lee et al. narrowed down their investigations to the five most likely candidates. Interfering with the activities of these five proteins revealed that one ­ a protein IGFBP7 ­ not only alerted the parasites but also helped them to form the rosettes. It turns out that the parasites appear to use IGFBP7 like a bridge, linking it to two other human proteins to stick red blood cells together. Once the rosettes had formed, the monocytes were unable to eat the infected blood cells by themselves. Instead several monocytes had to work together as a team to consume the whole rosette. Further research is now needed to shed light on this interaction between malaria parasites and human cells. Such research would be particularly relevant in the clinical setting, since some previous studies has linked the forming of rosettes to the severity of disease for malaria.

Fast Tracks and Roadblocks for Zika Vaccines.

In early 2014, a relatively obscure virus, the Zika virus, made headlines worldwide following an increase in the number of congenital malformations. Since then, research on Zika virus, treatment and vaccines have progressed swiftly with various drugs being repurposed and vaccines heading into clinical trials. Nonetheless, the need for a vaccine is crucial in order to eradicate this re-emerging arthropod-borne virus which remained silent since its first discovery in 1947. In this review, we focused on how the inconspicuous virus managed to spread, the key immunological factors required for a vaccine and the various vaccine platforms that are currently being studied.

Plasmodium co-infection protects against chikungunya virus-induced pathologies.

Co-infection with Plasmodium and chikungunya virus (CHIKV) has been reported in humans, but the impact of co-infection on pathogenesis remains unclear. Here, we show that prior exposure to Plasmodium suppresses CHIKV-associated pathologies in mice. Mechanistically, Plasmodium infection induces IFNγ, which reduces viraemia of a subsequent CHIKV infection and suppresses tissue viral load and joint inflammation. Conversely, concomitant infection with both pathogens limits the peak of joint inflammation with no effect on CHIKV viraemia. Reduced peak joint inflammation is regulated by elevated apoptosis of CD4+ T-cells in the lymph nodes and disrupted CXCR3-mediated CD4+ T-cell migration that abolishes their infiltration into the joints. Virus clearance from tissues is delayed in both infection scenarios, and is associated with a disruption of B cell affinity-maturation in the spleen that reduces CHIKV-neutralizing antibody production.

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.

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).

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.

Multilayer engineered nanoliposomes as a novel tool for oral delivery of lipopeptide-based vaccines against group A Streptococcus.

AIM: To develop an oral nanovaccine delivery system for lipopeptide-based vaccine candidate against group A Streptococcus. MATERIALS & METHODS: Lipid-core peptide-1-loaded nanoliposomes were prepared as a template and coated with opposite-charged polyelectrolytes to produce particles with size <200 nm. Efficacy of this oral nanovaccine delivery system was evaluated in mice model. RESULTS: Polymer-coated liposomes produced significantly higher antigen-specific mucosal IgA and systemic IgG titers in comparison to vaccine formulated with a strong mucosal adjuvant upon oral immunization in mice. Moreover, high levels of systemic antibody titers were retained even at day 185 postprimary immunization. CONCLUSION: Efficient oral delivery platform for lipopeptide-based vaccines has been developed.

Liposome-based intranasal delivery of lipopeptide vaccine candidates against group A streptococcus.

UNLABELLED: Group A streptococcus (GAS), an exclusively human pathogen, causes a wide range of diseases ranging from trivial to life threatening. Treatment of infection is often ineffective following entry of bacteria into the bloodstream. To date, there is no vaccine available against GAS. In this study, cationic liposomes encapsulating lipopeptide-based vaccine candidates against GAS have been employed for intranasal vaccine delivery. Cationic liposomes were prepared with dimethyldioctadecylammonium bromide (DDAB) using the film hydration method. Female Swiss mice were immunized intranasally with the liposomes. In contrast to unmodified peptides, lipopeptides entrapped by liposomes induced both mucosal and systemic immunity, IgA and IgG (IgG1 and IgG2a) production in mice, respectively. High levels of antibody (IgA and IgG) titres were detected even five months post immunization. Thus, the combination of lipopeptides and liposomes generates a very promising delivery system for intranasal vaccines. STATEMENT OF SIGNIFICANCE: Group A streptococcus, causing rheumatic heart diseases, kills approximately half a million people annually. There is no vaccine available against the infection. Mucosal immunity is vital in ensuring an individual is protected as this gram positive bacteria initially colonizes at the throat. Herein, we demonstrated that lipopeptides entrapped by liposomes induced both mucosal and systemic immunity. High levels of antibody (IgA and IgG) titres were detected even five months post immunization and lead vaccine candidate was able to induce humoral immune responses even after single immunization. Thus, the combination of lipopeptides and liposomes generates a very promising delivery system for intranasal vaccines.

Differing Efficacies of Lead Group A Streptococcal Vaccine Candidates and Full-Length M Protein in Cutaneous and Invasive Disease Models.

UNLABELLED: Group A Streptococcus (GAS) is an important human pathogen responsible for both superficial infections and invasive diseases. Autoimmune sequelae may occur upon repeated infection. For this reason, development of a vaccine against GAS represents a major challenge, since certain GAS components may trigger autoimmunity. We formulated three combination vaccines containing the following: (i) streptolysin O (SLO), interleukin 8 (IL-8) protease (Streptococcus pyogenes cell envelope proteinase [SpyCEP]), group A streptococcal C5a peptidase (SCPA), arginine deiminase (ADI), and trigger factor (TF); (ii) the conserved M-protein-derived J8 peptide conjugated to ADI; and (iii) group A carbohydrate lacking the N-acetylglucosamine side chain conjugated to ADI. We compared these combination vaccines to a "gold standard" for immunogenicity, full-length M1 protein. Vaccines were adjuvanted with alum, and mice were immunized on days 0, 21, and 28. On day 42, mice were challenged via cutaneous or subcutaneous routes. High-titer antigen-specific antibody responses with bactericidal activity were detected in mouse serum samples for all vaccine candidates. In comparison with sham-immunized mice, all vaccines afforded protection against cutaneous challenge. However, only full-length M1 protein provided protection in the subcutaneous invasive disease model. IMPORTANCE: This set of experiments demonstrates the inherent variability of mouse models for the characterization of GAS vaccine candidate protective efficacy. Such variability poses an important challenge for GAS vaccine development, as advancement of candidates to human clinical trials requires strong evidence of efficacy. This study highlights the need for an open discussion within the field regarding standardization of animal models for GAS vaccine development.

Levofloxacin and indolicidin for combination antimicrobial therapy.

Despite the increasing need for antibiotics to fight infectious diseases, fewer new antibiotics are available on the market. Unfortunately, developing a new class of antibiotics is associated with high commercial risk. Therefore, modification or combination of existing antibiotics to improve their efficacy is a promising strategy. Herein, we conjugated the antibiotic, levofloxacin, with two peptides, i.e. an antimicrobial peptide indolicidin and a cell penetrating peptide (TAT). Glycolic acid and glycine linkers were used between levofloxacin and peptides. We developed an optimized condition for coupling of levofloxacin via its carboxylic group to glycolic acid using solid phase peptide synthesis (SPPS). Antibacterial and haemolytic assays were carried out on the conjugates and only the levofloxacin-indolicidin conjugate demonstrated moderate antibacterial activity. Interestingly, physical mixture of levofloxacin and indolicidin showed improvement in the activity against Gram-positive bacteria.

Liposomes as nanovaccine delivery systems.

Since the discovery of liposomes by Alec Bangham in mid-1960s, these phospholipid vesicles have been widely used as pharmaceutical carriers. Liposomes have been extensively studied in the vaccine delivery field as a carrier and an immune stimulating agent. Liposomes are usually formulated as nanoparticles, mimicking the properties of pathogens, and have the ability to induce humoral and cell-mediated immune responses. In this review, we focused on modern nanotechnology-based approaches for the improvement of liposomal vaccine delivery systems. Topics such as size-dependent uptake, processing and activation of antigen presenting cells, targeting liposomes and route of administration are discussed.