PLGA (85:15) nanoparticle based delivery of rL7/L12 ribosomal protein in mice protects against Brucella abortus 544 infection: A promising alternate to traditional adjuvants.

There is a compelling need for the development of suitable adjuvants for human use to enhance the efficacy of the upcoming vaccines for the prevention of life threatening infections. In the current study, we have tried to explore the immunogenic potential of nanoparticles (NPs) made of PLGA (poly lactic-co-glycolic acid), a biodegradable and biocompatible polymer approved by FDA for human use after entrapping rL7/L12 protein, an immunodominant antigen of Brucella. Adjuvant properties were exhibited by the formulation as it elicited high IgG antibody titers just after first immunization which increased significantly after the booster administration. A good elicitation of the Th1 cytokines especially IFN-γ was recorded. Amongst the IgG antibody subclasses, IgG1 remained the predominant subclass to be elicited in mice serum after immunization; however IgG1/2a ratio showed a mixed profile of Th1/Th2 response. Lymphocyte proliferation assay as a marker of amplification in cellular immunity demonstrated that the splenocytes of the immunized mice had a high proliferation index with reference to the control, revealing that L7/L12 entrapping PLGA nanoparticles are potent inducer of inflammatory cell response indispensable to combat Brucella infection. Enumeration of splenic CFU after 14 days of infection with Brucella abortus 544 showed a significant reduction in log CFU of splenic bacteria in the vaccinated mice as compared to the control group. Therefore it is evident that PLGA nano formulations delivering the entrapped vaccine candidate in mice elicit specific humoral as well as cellular responses specific to the entrapped Brucella antigen. So there is much promise in this approach and this work by highlighting the adjuvant properties of the PLGA nanospheres will accelerate the development of improved vaccines safe for human as well as veterinary use.

Use of S-[2,3-bispalmitoyiloxy-(2R)-propyl]-R-cysteinyl-amido-monomethoxy polyethylene glycol as an adjuvant improved protective immunity associated with a DNA vaccine encoding Cu,Zn superoxide dismutase of Brucella abortus in mice.

This study was conducted to evaluate the immunogenicity and protective efficacy of a DNA vaccine encoding Brucella abortus Cu,Zn superoxide dismutase (SOD) using the Toll-like receptor 2/6 agonist S-[2,3-bispalmitoyiloxy-(2R)-propyl]-R-cysteinyl-amido-monomethoxy polyethylene glycol (BPPcysMPEG) as an adjuvant. Intranasal coadministration of BPPcysMPEG with a plasmid carrying the SOD-encoding gene (pcDNA-SOD) into BALB/c mice elicited antigen-specific humoral and cellular immune responses. Humoral responses were characterized by the stimulation of IgG2a and IgG1 and by the presence of SOD-specific secretory IgA in nasal and bronchoalveolar lavage fluids. Furthermore, T-cell proliferative responses and increased production of gamma interferon were also observed upon splenocyte restimulation with recombinant SOD. Cytotoxic responses were also stimulated, as demonstrated by the lysis of RB51-SOD-infected J774.A1 macrophages by cells recovered from immunized mice. The pcDNA-SOD/BPPcysMPEG formulation induced improved protection against challenge with the virulent strain B. abortus 2308 in BALB/c mice over that provided by pcDNA-SOD, suggesting the potential of this vaccination strategy against Brucella infection.

Co-immunization with interlukin-18 enhances the protective efficacy of liposomes encapsulated recombinant Cu-Zn superoxide dismutase protein against Brucella abortus.

Brucellosis is a worldwide zoonotic disease caused by Brucella abortus and a number of closely related species. Brucellosis has severe impact on the health and economic prosperity of the developing countries due to the persistent nature of infection and unavailability of effective control measures. The Cu-Zn superoxide dismuatse (SOD) protein of Brucella have been extensively studied as a major antigen involved in bacterial evading mechanism of host defence. Being a critical pro-inflammatory cytokine interleukin-18 (IL-18) plays key role in induction of immune mediated protection against intracellular pathogens. In the present study, we aimed to investigate the immunogenic potential of fusogenic liposomes (escheriosomes) encapsulated recombinant Cu-Zn SOD (rSOD) protein alone or in combination with recombinant IL-18 (rIL-18). Escheriosomes encapsulated rSOD mediated immune responses were further increased upon co-immunization with rIL-18. Furthermore, immunization with escheriosomes encapsulated rSOD alone or in combination with rIL-18, increased resistance in mice against challenge with B. abortus 544.

Liposomised recombinant ribosomal L7/L12 protein protects BALB/c mice against Brucella abortus 544 infection.

Brucella abortus, a facultative intracellular pathogen, is of tremendous zoonotic importance because of its ability to induce spontaneous abortion in cattle and other livestock. It is also known to cause persistent undulant fever, endocarditis, arthritis, osteomyelitis and meningitis in humans. The available vaccines against this dreadful infection suffer from limitations like short-term immunity, increased risk of hypersensitivity and low prophylactic index in the recipients. In the present study, we have demonstrated that liposomal form of a recombinant ribosomal L7/L12 protein, a B-T cell antigen of B. abortus, activates strong immune response in the host. In contrast, free antigen generates moderate immune response in the immunised animals. The liposomisation of rL7/L12 protein causes tremendous increase in cell-mediated immune response in terms of delayed type hypersensitivity, T-cell proliferation and up-regulation in type I cytokine expression, etc. Moreover, the liposome encapsulated antigen elicited stronger humoral immune response as compared to standard vaccine (S-19) or IFA-L7/L12 combination in the immunised animals. The effectiveness of liposome-based vaccine was also substantiated by better systemic clearance of bacterial load after challenging the animals with B. abortus 544 pathogen. The results of the present study suggest the potential of liposome-based rL7/L12 antigen as prospective and efficient candidate vaccine capable of eliciting both cell mediated as well as humoral immune responses against experimental murine brucellosis.

Escheriosome-mediated delivery of recombinant ribosomal L7/L12 protein confers protection against murine brucellosis.

Brucellosis is an important zoonotic disease that causes abortion in cattle and undulant fever, arthritis, endocarditis and meningitis in human. In spite of the fact that immunization could be an efficient measure to control brucellosis, not a single ideal vaccine against this important disease has been developed so far. In order to develop an effective vaccine against Brucella abortus (B. abortus), various protective immunodominant gene/protein products of the pathogen have been studied in combination with different adjuvants. For example, recombinant ribosomal protein L7/L12 (rL7/L12) although an interesting T-cell antigen, normally failed to evoke protective immune response when used in free form. In the present study we have demonstrated that Escherischia coli (E. coli) lipid liposome (escheriosome)-mediated cytosolic delivery of recombinant rL7/L12 protein can elicit strong immunological responses in the Balb/c mice. In contrast, egg PC/Chol liposome entrapped rL7/L12, in a manner similar to its free form, was found to impart relatively poor immune response. Furthermore, escheriosome entrapped rL7/L12 protein elicited high IgG2a isotype response suggestive of its relevance in imparting protection against brucellosis in mice. Altogether the present study is a clear indicative of the possible use of escheriosome-based delivery of rL7/L12 protein to induce protective immune responses against experimental murine brucellosis.