Ashwini Kumar Giddam

Liposome-based delivery system for vaccine candidates: constructing an effective formulation

The discovery of liposomes in 1965 by Bangham and coworkers changed the prospects of drug delivery systems. Since then, the application of liposomes as vaccine delivery systems has been studied extensively. Liposomal vaccine delivery systems are made up of nano- or micro-sized vesicles consisting of phospholipid bilayers, in which the bioactive molecule is encapsulated/entrapped, adsorbed or surface coupled. In general, liposomes are not immunogenic on their own; thus, liposomes combined with immunostimulating ligands (adjuvants) or various other formulations have been used as vaccine delivery systems. A thorough understanding of formulation parameters allows the design of effective liposomal vaccine delivery systems. This article provides an overview of various factors that influence liposomal immunogenicity. In particular, the effects of vesicle size, surface charge, bilayer composition, lamellarity, pegylation and targeting of liposomes are described.

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.

Self-adjuvanting vaccine against group A streptococcus: Application of fibrillized peptide and immunostimulatory lipid as adjuvant

Peptides are of great interest to be used as vaccine antigens due to their safety, ease of manufacturing and specificity in generating immune response. There have been massive discoveries of peptide antigens over the past decade. However, peptides alone are poorly immunogenic, which demand co-administration with strong adjuvant to enhance their immunogenicity. Recently, fibril-forming peptides such as Q11 and lipoamino acid-based carrier have been identified to induce substantial immune responses when covalently linked to peptide epitope. In this study, we have incorporated either Q11 or lipoamino acids to a peptide epitope (J14) derived from M protein of group A streptococcus to develop self-adjuvanting vaccines. J14, Q11 and lipoamino acids were also conjugated together in a single vaccine construct in an attempt to evaluate the synergy effect of combining multiple adjuvants. Physicochemical characterization demonstrated that the vaccine constructs folded differently and self-assembled into nanoparticles. Significantly, only vaccine constructs containing double copies of lipoamino acids (regardless in conjugation with Q11 or not) were capable to induce significant dendritic cells uptake and subsequent J14-specific antibody responses in non-sizes dependent manners. Q11 had minimal impact in enhancing the immunogenicity of J14 even when it was used in combination with lipoamino acids. These findings highlight the impact of lipoamino acids moiety as a promising immunostimulant carrier and its number of attachment to peptide epitope was found to have a profound effect on the vaccine immunogenicity.

Group A Streptococcal vaccine candidate: contribution of epitope to size, antigen presenting cell interaction and immunogenicity

AIM Utilize lipopeptide vaccine delivery system to develop a vaccine candidate against Group A Streptococcus. MATERIALS & METHODS Lipopeptides synthesized by solid-phase peptide synthesis-bearing carboxyl (C)-terminal and amino (N)-terminal Group A Streptococcus peptide epitopes. Nanoparticles formed were evaluated in vivo. RESULTS Immune responses were induced in mice without additional adjuvant. We demonstrated for the first time that incorporation of the C-terminal epitope significantly enhanced the N-terminal epitope-specific antibody response and correlated with forming smaller nanoparticles. Antigen-presenting cells had increased uptake and maturation by smaller, more immunogenic nanoparticles. Antibodies raised by vaccination recognized isolates. CONCLUSION Demonstrated the lipopeptidic nanoparticles to induce an immune response which can be influenced by the combined effect of epitope choice and size.

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.

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.

Self-Adjuvanting Therapeutic Peptide-Based Vaccine Induce CD8 + Cytotoxic T Lymphocyte Responses in a Murine Human Papillomavirus Tumor Model

Vaccine candidates for the treatment of human papillomavirus (HPV)-associated cancers are aimed to activate T-cells and induce development of cytotoxic anti-tumor specific responses. Peptide epitopes derived from HPV-16 E7 oncogenic protein have been identified as promising antigens for vaccine development. However, peptide-based antigens alone elicit poor cytotoxic T lymphocyte (CTL) responses and need to be formulated with an adjuvant (immunostimulant) to achieve the desired immune responses. We have reported the ability of polyacrylate 4-arm star-polymer (S4) conjugated with HPV-16 E744-57 (8Qmin) epitope to reduce and eradicate TC-1 tumor in the mouse model. Herein, we have studied the mechanism of induction of immune responses by this polymer-peptide conjugate and found prompt uptake of conjugate by antigen presenting cells, stimulating stronger CD8(+) rather than CD4(+) or NK cell responses.

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.

A semi-synthetic whole parasite vaccine designed to protect against blood stage malaria

UNLABELLED Although attenuated malaria parasitized red blood cells (pRBCs) are promising vaccine candidates, their application in humans may be restricted for ethical and regulatory reasons. Therefore, we developed an organic microparticle-based delivery platform as a whole parasite malaria-antigen carrier to mimic pRBCs. Killed blood stage parasites were encapsulated within liposomes that are targeted to antigen presenting cells (APCs). Mannosylated lipid core peptides (MLCPs) were used as targeting ligands for the liposome-encapsulated parasite antigens. MLCP-liposomes, but not unmannosylated liposomes, were taken-up efficiently by APCs which then significantly upregulated expression of MHC-ll and costimulatory molecules, CD80 and CD86. Two such vaccines using rodent model systems were constructed - one with Plasmodium chabaudi and the other with P. yoelii. MLCP-liposome vaccines were able to control the parasite burden and extended the survival of mice. Thus, we have demonstrated an alternative delivery system to attenuated pRBCs with similar vaccine efficacy and added clinical advantages. Such liposomes are promising candidates for a human malaria vaccine. STATEMENT OF SIGNIFICANCE Attenuated whole parasite-based vaccines, by incorporating all parasite antigens, are very promising candidates, but issues relating to production, storage and safety concerns are significantly slowing their development. We therefore developed a semi-synthetic whole parasite malaria vaccine that is easily manufactured and stored. Two such prototype vaccines (a P. chabaudi and a P. yoelii vaccine) have been constructed. They are non-infectious, highly immunogenic and give good protection profiles. This semi-synthetic delivery platform is an exciting strategy to accelerate the development of a licensed malaria vaccine. Moreover, this strategy can be potentially applied to a wide range of pathogens.

Investigation of structure-activity relationships of synthetic anti-gonadotropin releasing hormone vaccine candidates.

The immunoneutralization of gonadotropin‐releasing hormone (GnRH) can be used for the treatment of human hormone‐dependent male and female cancers or as immunocontraceptives in animals. Vaccine candidates 1 [Th(K‐LP)GnRH], 2 [GnRH(K‐LP)Th], 3 [GnRH(K‐Th)LP], and 4 [Th(K‐GnRH)LP] (for which K=lysine, LP=lipopeptide Ser‐Ser‐C16‐C16, and Th=T helper cell epitope influenza HA2), were synthesized by assembling a CD4+ T helper cell epitope (Th), GnRH, and an adjuvanting lipid moiety (LP) in various spatial arrangements. All compounds were efficiently taken up by antigen‐presenting cells with significant immunogenicity without an external adjuvant. Compounds 2, 3, and 4, in which GnRH is conjugated through its C terminus, produced higher GnRH‐specific antibody responses than construct 1, in which the GnRH moiety is conjugated through its N terminus. All four constructs induced a significant antiproliferative effect (up to 55 %) on GnRH‐receptor‐positive LNCaP cells, but showed weaker activity in the GnRH‐receptor‐negative SKOV‐3 cell line. Marked degenerative changes were observed in morphology and follicular development in the ovaries of immunized mice, with approximately 30 % higher degenerative antral and atretic follicles.