Towards the Development of a Mucosally Active Vaccine against Group A Streptococcus (GAS)

Vaccination is the administration of material (a vaccine) that results in immunity to a disease or a pathogen. Vaccination in general is considered to be one of the most effective methods for prevention of infectious diseases. The evidence for the efficacy of many vaccines, such as the influenza vaccine, the human papilloma virus vaccine and the small pox and chicken pox vaccine among others highlight the contribution of vaccines to the betterment of public health. Streptococcus pyogenes or Group A Streptococcus (GAS) infection is a common cause of pharyngitis, scarlet fever and impetigo. GAS infection results in other more severe complications, namely acute rheumatic fever/rheumatic heart disease, streptococcal reactive arthritis, etc. It is the post-infectious, immune-mediated sequelae that cause severe morbidity and mortality in developing countries. The global prevalence of serious streptococcal infections and the continued endemicity of fatal post-streptococcal sequelae in developing countries have resulted in renewed interest in GAS, especially in developing a vaccine to prevent the associated diseases. Traditional vaccines (killed or live attenuated) have been effective against many bacterial and viral infectious diseases. However, application of these traditional techniques in many cases including GAS has not shown the same success. Risks associated with the use of live–attenuated pathogens for a GAS vaccine include the development of autoimmune diseases. Other vaccine strategy such as subunit vaccines (recombinant proteins or peptides) and carrier protein conjugated vaccine are hampered by the lack of suitable adjuvant, carriers and delivery systems. Mucosal surface of the upper respiratory tract is a primary site of GAS infections. As such an optimal GAS vaccine would induce both mucosal and systemic immune responses. This would prevent primary GAS infection and the development of GAS-associated diseases. The current thesis is focused on the design, synthesis and immunological evaluation of novel delivery systems against GAS. The first chapter reviews strategies utilized in the field of GAS vaccine development, basis of mucosal immunity and new findings in immunology and mechanism of action of particulate polymers and lipopeptides which represent the basis of our novel strategies for GAS vaccine development. In the chapters that follow, the immunological evaluation of a polyacrylate polymer based delivery system is described in chapter 2. This vaccine delivery system consisted of a target GAS B cell epitope (J14) covalently attached to a polyacrylic acid based polymer imparting adjuvanting activity. Chemical attachment of J14 was essential for immunogenicity and following systemic and intranasal administration of this nanoparticulate construct without additional adjuvant, J14-specific systemic IgG was induced. The antibodies elicited were also capable of in vitro opsonisation of a GAS strain found in endemic areas. The promising preliminary data shows the potential of this novel self-adjuvanting polyacrylate polymer based construct as a peptide vaccine delivery platform, which may afford promising opportunities for treating systemic GAS infection. It is also of relevance to other disease models where systemic immune responses are desired. A major aim of this thesis was to develop and optimize an intranasally administered, lipopeptide based mucosally active GAS vaccine candidate. The third chapter includes immunological assessment of a lipopeptide vaccine library composed of: (i) J14 and (ii) a lipid moiety based on lipoamino acids (LAA). Systemic J14-specific IgG antibodies were detected following intranasal immunization of inbred B10.Br (H-2k) and outbred Swiss mice without the need for an additional adjuvant. The effect of changing the position of J14, and lipid moiety attachment on antibody titer was assessed. The point of lipid moiety and J14 attachment had a significant influence on systemic J14-specific IgG antibody titer in outbred Swiss mice. Overall, the optimal lipopeptide GAS vaccine featured a C-terminal lipid moiety, conjugated through a central lysine residue and J14 on the lysine side-chain. The fourth chapter describes the design, synthesis and evaluation of a three component mucosally active GAS vaccine candidate, composed of: (i) a universal helper T cell epitope (P25), (ii) the target GAS B cell epitope (J14), and (iii) a LAA based lipid moiety. Structure activity relationship of the synthesized lipopeptides demonstrated that the choice of B and T helper epitope had a significant impact on mucosal IgA response. Replacement of P25 with another T helper epitope (TH2R) or replacing the B cell epitope with another GAS epitope (88/30) abolished IgA activity in B10.Br and Swiss mice. No significant differences were observed for the systemic IgG response. Our data would suggest that mucosal activity by the lipopeptide of interest is due to the sum of all of its three components rather than due to an individual component. Further investigation is needed to determine the contributing factors for mucosal immune response. Bactericidal assay showed the mucosally active lipopeptide was capable of in vitro opsonisation of a virulent GAS strain. IgG isotyping displayed the lipopeptide elicits a mixed T helper type 1(Th1)/Th2 response. Dynamic light scattering (DLS) measurements and transmission electron microscopy (TEM) images were used to analyse the particle size and the lipopeptide of interest formed particles in the nanometer range in aqueous solution. The fifth chapter identifies our lipopeptide vaccine strategy as a novel Toll-like receptor (TLR) 2 targeting synthetic ligand. A LAA based lipid moiety composed of two alkyl chains of 16 carbons was found to be optimal for TLR2 activation regardless of the position of lipid attachment

Nanovaccines and their mode of action

Nanosized particles including nanovaccines are a novel approach to the development of vaccines to combat diseases. Nanovaccines have the promise to utilize the immune system to cure infections and to prevent infections and diseases from spreading. Rational vaccine development requires an understanding of vaccine mediated stimulation of the immune system. We review here immunostimulatory properties of nanovaccines including their immunogenicity, adjuvant properties, inflammatory responses and the mechanisms of uptake and stimulation of immune cells. Examples of various nanoparticles currently being developed as vaccines are also provided

Toll-like receptor 2 mediated dendritic cell activation: Key target for lipopeptide vaccines design

Toll-like receptors (TLRs) which recognise pathogen associated molecular patterns (PAMPs) are a vital link between the innate and adaptive (or learned) immunity. TLRmediated activation of dendritic cells (DCs) is a crucial step in triggering adaptive immunity as DCs are the principal initiator and modulator of the immune response. With the advent of well-defined synthetic lipid moieties that structurally represent PAMPs, it is now possible to design synthetic vaccines containing disease-specific epitopes. This review provides a brief overview of why DCs represent a crucial target and tool for vaccination and TLR mediated DC activation efficiently stimulates immune responses. Particular emphasis is given to our recent understanding of the lipid component of various lipidated peptides (lipopeptides) targeting TLR2 mediated DC activation

Simple synthetic toll-like receptor 2 ligands

Dimeric/oligomeric states of G-protein coupled receptors have been difficult to target. We report here bivalent ligands consisting of two identical oxytocin-mimetics that induce a three order magnitude boost in G-protein signaling of oxytocin receptors (OTRs) in vitro and a 100- and 40-fold gain in potency in vivo in the social behavior of mice and zebrafish. Through receptor mutagenesis and interference experiments with synthetic peptides mimicking transmembrane helices (TMH), we show that such superpotent behavior follows from the binding of the bivalent ligands to dimeric receptors based on a TMH1-TMH2 interface. Moreover, in this arrangement, only the analogues with a well-defined spacer length (∼25 Å) precisely fit inside a channel-like passage between the two protomers of the dimer. The newly discovered oxytocin bivalent ligands represent a powerful tool for targeting dimeric OTR in neurodevelopmental and psychiatric disorders and, in general, provide a framework to untangle specific arrangements of G-protein coupled receptor dimers

Design of fully synthetic, self-adjuvanting vaccine incorporating the tumor-associated carbohydrate Tn antigen and lipoamino acid-based toll-like receptor 2 ligand

Overexpression of certain tumor-associated carbohydrate antigens (TACA) caused by malignant transformation offers promising targets to develop novel antitumor vaccines, provided the ability to break their inherent low immunogenicity and overcome the tolerance of the immune system. We designed, synthesized, and immunologically evaluated a number of fully synthetic new chimeric constructs incorporating a cluster of the most common TACA (known as Tn antigen) covalently attached to T-cell peptide epitopes derived from polio virus and ovalbumin and included a synthetic built-in adjuvant consisting of two 16-carbon lipoamino acids. Vaccine candidates were able to induce significantly strong antibody responses in mice without the need for any additional adjuvant, carrier protein, or special pharmaceutical preparation (e.g., liposomes). Vaccine constructs were assembled either in a linear or in a branched architecture, which demonstrated the intervening effects of the incorporation and arrangement of T-cell epitopes on antibody recognition

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

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

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

Design of three-component vaccines against group A streptococcal infections: Importance of spatial arrangement of vaccine components

Immunological assessment of group A streptococcal (GAS) branched lipopeptides demonstrated the impact of spatial arrangement of vaccine components on both the quality and quantity of their immune responses. Each lipopeptide was composed of three components: a GAS B-cell epitope (J14), a universal CD4+ T-cell helper epitope (P25), and an immunostimulant lipid moiety that differs only in its spatial arrangement. The best systemic immune responses were demonstrated by a lipopeptide featuring the lipid moiety at the lipopeptide C-terminus. However, this candidate did not achieve protection against bacterial challenge. The best protection (100%) was shown by a lipopeptide featuring a C-terminal J14, conjugated through a lysine residue to P25 at the N-terminus, and a lipid moiety on the lysine side chain. The former candidate features α-helical conformation required to produce protective J14-specific antibodies. Our results highlight the importance of epitope orientation and lipid position in the design of three-component synthetic vaccines.© 2010 American Chemical Society