Dual dye in-vivo imaging of differentially charged PLGA carriers reveals antigen-depot effect, leading to improved immune responses in preclinical models.

The present in-vivo study investigated the behavior and performance of differently charged poly(lactic­co­glycolic) acid microparticles (PLGA MP) as vaccination platform. For this purpose, particles loaded with ovalbumin (OVA) as model antigen were subcutaneously (s.c.) injected in SKH1 mice. The utilized SKH1 hairless mice exhibit a fully operative immune system and allow parallel imaging investigations due to the lack of hair. Usage of this species enabled the combination of two investigations within a single study protocol, namely noninvasive in-vivo imaging and immune responses directed towards the antigen. All treatments were well tolerated, no safety drop-outs occurred. The fate of the model antigen OVA as well as the PLGA particles was monitored using a dual dye approach (CF660C & DiR) by multispectral fluorescence imaging (msFI). A depot effect for the OVA antigen adsorbed to the MP surface could be observed for the positively charged MPs. The immune response against OVA was then analyzed. OVA alone did not induce an immune response, whereas the positively charged as well as the neutral MP induced a strong and consistent humoral immune response with a clear favor of IgG1 over IgG2a subclass antibodies. In contrast, negatively charged MP were not able to induce measurable antibody responses. Cellular immune response was weak and inconsistent for all treated groups, which verifies previous in-vitro results conducted with the herein described microparticulate antigen platform. In conclusion, the characterization of the in-vivo performance yielded valuable information about antigen and carrier fate after application. The presented adjuvant platform is capable of inducing strong TH2 dominated immune responses characterized by enhanced IgG1 subclass titers which are critical for vaccines aimed at promoting induction of neutralizing antibodies.

Rodents as pre-clinical models for predicting vaccine performance in humans.

Vaccines represent a key building block for establishing a successful and sustainable control strategy against infectious diseases. Vaccine development often depends on the availability of correlates for protection and reliable animal models for the screening, selection and prioritization of potential vaccine candidates. This is performed according to their immunogenicity, efficacy and safety profiles in pre-clinical studies, which are also critical for identification of candidate antigens, selection of an optimal delivery system and design of appropriate vaccine formulations. Thus, pre-clinical studies in animal models are a prerequisite for addressing crucial issues and generating a solid pre-clinical package for the approval of clinical trials. This review addresses the strengths, limitations and perspectives of rodents as a vaccine development and pre-clinical validation tool.

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.

The synthetic TLR2 agonist BPPcysMPEG leads to efficient cross-priming against co-administered and linked antigens.

The property of DC to generate effective CTL responses is influenced by TLR signaling. TLR ligands contain molecular signatures associated with pathogens, have an impact on the antigen processing and presentation by DC, and are being exploited as potential adjuvants. We hypothesized that the TLR2/6 heterodimer agonist S-[2,3-bispalmitoyiloxy-(2R)-propyl]-R-cysteinyl-amido-monomethoxyl polyethylene glycol (BPP), a synthetic derivative of the Mycoplasma macrophage activating lipopeptide-2, is a potent adjuvant for cross-priming against cellular antigens. Systemic administration of BPP-induced maturation of CD8alpha+ DC and CD8alpha- DC in the spleen and resulted in enhanced cross-presentation of intravenously co-administered antigen in mice. In addition, administration of BPP and cell-associated OVA generated an effective CTL response against OVA in vivo in a CD4+ T helper cell-dependent manner, but independent of IFN-alpha. Delivering antigenic peptides directly linked to BPP led to superior CTL immunity as compared to giving antigens and adjuvants admixed. In contrast to other TLR ligands, such as CpG, systemic activation of DC with BPP did not result in shut-down of antigen presentation by splenic DC subsets, although cross-priming against subsequently encountered antigens was reduced. Together, our data provide evidence that BPP is a potent stimulus to generate CTL via cross-priming.

A Toll-like receptor 2/6 agonist reduces allergic airway inflammation in chronic respiratory sensitisation to Timothy grass pollen antigens.

BACKGROUND: The hygiene hypothesis negatively correlates the microbial burden of the environment with the prevalence of T helper type 2 (Th2)-related disorders, e.g. allergy and asthma. This is explained by Th1 triggering through pathogen-associated molecular patterns via Toll-like receptors (TLRs). In this study, the biological effects of a TLR2/6 agonist as a potential treatment of allergic inflammation are explored. METHODS: In a model of chronic allergic airway inflammation induced by intranasal administration of Timothy grass pollen allergen extract, early TLR agonism and/or interferon (IFN)-gamma administration was compared to the therapeutic and immune-modulating effects of dexamethasone with regard to the cellular inflammation and cytokine profiles. RESULTS: Eosinophilic inflammation was clearly reduced by TLR2/6 agonism. This effect was also seen without simultaneous administration of IFN-gamma. However, lymphocyte counts were not affected among the different treatment groups. More precise determination of the lymphocyte-mediated immune reaction showed that TLR2/6 agonism induced neither CD4+foxp3+ regulatory T cells in draining lymph nodes nor a pronounced Th1 immune response. In contrast, dexamethasone reduced both sensitisation as well as allergic inflammation and, in addition, CD11c+ antigen-presenting cells in lymph nodes. Our data clearly point to the potential to rebalance Th2-skewed allergic immune responses by therapeutic TLR2/6 agonist administration. CONCLUSION: The use of the TLR2/6 agonist is a promising therapeutic approach in diseases with an imbalance in T cell responses, such as allergy and asthma.

Subchronic toxicological evaluation of brea gum (Parkinsonia preacox) as a food additive in BALB/c mice.

Brea gum is a phloematic exudate from Parkinsonia praecox, an autoctonous tree that grows in the arid areas of Argentina. In this work, we propose its potential as a food additive. However, as no toxicological safety evaluation of brea gum has yet been reported, this preliminary study was conducted to evaluate its long-term toxicity over a 120-day period in BALB/c mice fed with brea gum at various levels in the diet. The results showed that animals on diets containing up to 5% brea gum were healthy, exhibiting growth curves similar to controls for both males (P = 0.9138) and females (P = 0.9459), thereby indicating that feed intake and utilization was not affected. A histopathological examination and weight recording of liver, kidneys, and intestine did not reveal any microscopic abnormalities or adverse toxicological effect (weights respect to control: P > 0.1). Moreover, hematological parameters and enzyme activities were within the normal values previously reported for mice. Our findings suggest that feeding brea gum at levels up to 5% to BALB/c mice do not exert any toxicological effects, supporting its potential use as a food additive for human consumption.

Pidotimod promotes functional maturation of dendritic cells and displays adjuvant properties at the nasal mucosa level.

Mucosal dendritic cells (DCs) are very important in the process of antigen presentation to T cells, playing a key role in the induction of primary and secondary immune responses. Pidotimod is a synthetic substance capable of modulating immune cell functions, but the effect of pidotimod on human DCs has not been investigated yet. Here we demonstrate the ability of pidotimod to induce DC maturation and up-regulate the expression of HLA-DR and co-stimulatory molecules CD83 and CD86, which are fundamental for communication with adaptative immunity cells. Pidotimod also stimulated DCs to release high amounts of pro-inflammatory molecules such as MCP-1 and TNF-alpha cytokines and to drive T cell proliferation and differentiation towards a Th1 phenotype. Moreover, we demonstrate that pidotimod in vivo promotes strong and specific humoral and cellular immune response when co-administered intranasally with a model antigen. Taken together our data suggest the possibility to use pidotimod as adjuvant molecule to facilitate the activation of the innate immune system as well as to promote an effective mucosal and systemic immune response.

Effects of omega-3 and -6 fatty acids on Mycobacterium tuberculosis in macrophages and in mice.

We recently showed that treatment of macrophages prior to Mycobacterium tuberculosis infection with the pro-inflammatory omega-6 lipid, arachidonic acid (AA) enhanced bacterial killing whereas the anti-inflammatory, omega-3 lipid eicosapentaenoic acid (EPA) stimulated bacterial growth. Here we tested if these effects were depending on when lipids were added to macrophages: before or during Mycobacterium smegmatis or M. tuberculosis infection. Collectively, our data suggested that a high omega-6 diet might be beneficial against mycobacteriosis, while a high omega-3 diet might be detrimental. AA also stimulated TNF-alpha secretion in M. tuberculosis-infected macrophages whereas EPA inhibited this process. AA strongly activated the MAP kinase p38 in uninfected cells but M. tuberculosis infected cells blocked the ability of AA to activate p38; AA-dependent killing is therefore independent of p38. We therefore tested diets enriched in omega-3 and omega-6 lipids on a mouse model of tuberculosis. In contrast to the in vitro results, the omega-6 tended to increase survival of M. tuberculosis in mice, while omega-3- tended to increase pathogen killing. Overall our results together with those previously reported in the literature suggest that it is almost impossible to predict, at the whole organism level, if a diet enriched in omega-3 or -6 will be beneficial or detrimental to intracellular pathogens.