Vaccination has had a major impact on the control of infectious diseases. However, there are still many infectious diseases for which the development of an effective vaccine has been elusive. In many cases the failure to devise vaccines is a consequence of the inability of vaccine candidates to evoke appropriate immune responses. This is especially true where cellular immunity is required for protective immunity and this problem is compounded by the move toward devising sub-unit vaccines. Over the past decade nanoscale size (<1000 nm) materials such as virus-like particles, liposomes, ISCOMs, polymeric, and non-degradable nanospheres have received attention as potential delivery vehicles for vaccine antigens which can both stabilize vaccine antigens and act as adjuvants. Importantly, some of these nanoparticles (NPs) are able to enter antigen-presenting cells by different pathways, thereby modulating the immune response to the antigen. This may be critical for the induction of protective Th1-type immune responses to intracellular pathogens. Their properties also make them suitable for the delivery of antigens at mucosal surfaces and for intradermal administration. In this review we compare the utilities of different NP systems for the delivery of sub-unit vaccines and evaluate the potential of these delivery systems for the development of new vaccines against a range of pathogens.

Burkholderia mallei are Gram-negative bacteria, responsible for the disease glanders. B. mallei has recently been classified as a Tier 1 agent owing to the fact that this bacterial species can be weaponised for aerosol release, has a high mortality rate and demonstrates multi-drug resistance. Furthermore, there is no licensed vaccine available against this pathogen. Lipopolysaccharide (LPS) has previously been identified as playing an important role in generating host protection against Burkholderia infection. In this study, we present gold nanoparticles (AuNPs) functionalised with a glycoconjugate vaccine against glanders. AuNPs were covalently coupled with one of three different protein carriers (TetHc, Hcp1 and FliC) followed by conjugation to LPS purified from a non-virulent clonal relative, B. thailandensis. Glycoconjugated LPS generated significantly higher antibody titres and compared with LPS alone. Further, they improved protection against a lethal inhalation challenge of B. mallei in the murine model of infection.

The Gram-negative Burkholderia mallei is a zoonotic pathogen and the causative agent of glanders disease. Because the bacteria maintain the potential to be used as a biothreat agent, vaccine strategies are required for human glanders prophylaxis. A rhesus macaque (Macaca mulatta) model of pneumonic (inhalational) glanders was established and the protective properties of a nanoparticle glycoconjugate vaccine composed of B. thailandensis LPS conjugated to FliC was evaluated. An aerosol challenge dose of ~1×104 CFU B. mallei produced mortality in 50% of naïve animals (n = 2/4), 2–3 days post-exposure. Although survival benefit was not observed by vaccination with a glycoconjugate glanders vaccine (p=0.42), serum LPS-specific IgG titres were significantly higher on day 80 in 3 vaccinated animals who survived compared with 3 vaccinated animals who died. Furthermore, B. mallei was isolated from multiple organs of both non-vaccinated survivors, but not from any organs of 3 vaccinated survivors at 30 days post-challenge. Taken together, this is the first time a candidate vaccine has been evaluated in a non-human primate aerosol model of glanders and represents the initial step for consideration in pre-clinical studies.

Burkholderia pseudomallei is a Gram-negative, facultative intracellular pathogen that causes the disease melioidosis in humans and other mammals. Respiratory infection with B. pseudomallei leads to a fulminant and often fatal disease. It has previously been shown that glycoconjugate vaccines can provide significant protection against lethal challenge; however, the limited number of known Burkholderia antigens has slowed progress toward vaccine development. The objective of this study was to identify novel antigens and evaluate their protective capacity when incorporated into a nanoglycoconjugate vaccine platform. First, an in silico approach to identify antigens with strong predicted immunogenicity was developed. Protein candidates were screened and ranked according to predicted subcellular localization, transmembrane domains, adhesive properties, and ability to interact with major histocompatibility complex (MHC) class I and class II. From these in silico predictions, we identified seven "high priority" proteins that demonstrated seroreactivity with anti-B. pseudomallei murine sera and convalescent human melioidosis sera, providing validation of our methods. Two novel proteins, together with Hcp1, were linked to lipopolysaccharide (LPS) and incorporated with the surface of a gold nanoparticle (AuNP). Animals receiving AuNP glycoconjugate vaccines generated high proteinand polysaccharide-specific antibody titers. Importantly, immunized animals receiving the AuNP-FlgL-LPS alone or as a combination demonstrated up to 100% survival and reduced lung colonization following a lethal challenge with B. pseudomallei. Together, this study provides a rational approach to vaccine design that can be adapted for other complex pathogens and provides a rationale for further preclinical testing of AuNP glycoconjugate in animal models of infection.KEYWORDS Burkholderia pseudomallei, melioidosis, nanoglycoconjugate, nanovaccine, reverse vaccinology B urkholderia pseudomallei is a Gram-negative aerobic bacterium common to tropical and subtropical climates worldwide. This saprophytic bacterium can survive in soil and water and, upon transmission to humans or other susceptible mammals, cause the disease melioidosis. Human disease can present with a wide variety of clinical manifestations, including cutaneous and soft tissue abscesses, lymphadenopathy, and sepsis (1, 2). Clinical symptoms have been correlated with the route of infection; percutaneous infection often results in a purulent lesion at the site of inoculation, whereas respiratory

The efficacy of 15 nm gold nanoparticles (AuNP) coated with Yersinia pestis F1-antigen, as an immunogen in mice, has been assessed. The nanoparticles were decorated with F1-antigen using N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide coupling chemistry. Mice given AuNP-F1 in alhydrogel generated the greatest IgG antibody response to F1-antigen when compared with mice given AuNP-F1 in PBS or given unconjugated F1-antigen in PBS or alhydrogel. Compared with unconjugated F1-antigen, the IgG2a response was enhanced in mice dosed with AuNP-F1 in PBS (p < 0.05) but not in mice immunised with AuNP-F1 in alhydogel. All treatment groups developed a memory response to F1-antigen, the polarity of which was inflenced by formulation in alhydrogel. The sera raised against F1-antigen coupled to AuNPs was able to competitively bind to rF1-antigen, displacing protective macaque sera.