Polyelectrolyte-coated liposomes microfluidically assembled in one-step for enhancing cell endocytosis and in-vivo immune responses.

To enhance the cellular uptake of liposomes, we prepared conventional liposomes with targeting molecules and surface-charged liposomes and evaluated their potential as nano-carriers and vaccine adjuvants by comparing their endocytosis efficiencies using immune cells. Surface-charged liposomes were synthesized via a one-step microfluidic method, which provided a novel, simple, fast, and highly reproducible method for preparing liposomes. Flow cytometry revealed that cationic polyelectrolyte-coated liposomes exhibited higher endocytosis efficiencies (of up to a factor of 100) in A774A.1 cells and JAWs II cells compared with uncoated liposomes or those coated with anionic polyelectrolytes. Positively charged liposomes exhibited some cytotoxicity at quaternary-chitosan coating concentrations higher than 6 mg/mL; however, significantly lower cytotoxicities (by a factor of almost ten) were obtained by protein mixing. Furthermore, BALB/c mice vaccinated with a mixture of Anthrax vaccine adsorbed (AVA) and quaternary chitosan-coated liposomes showed faster and stronger anti-PA IgG inductions compared to those vaccinated with AVA alone, with titers positively correlating with the amount of cationic liposome used. This finding clearly reveals that quaternary chitosan-coated liposomes act as both nano-carriers and vaccine adjuvants that significantly enhance in-vivo immune responses to vaccines with low immunogenicities.

Nanoparticles assembled from fucoidan and trimethylchitosan as anthrax vaccine adjuvant: In vitro and in vivo efficacy in comparison to CpG.

Fucoidan/trimethylchitosan nanoparticles (FUC-TMC-NPs) have the potential to improve the immunostimulating efficiency of anthrax vaccine adsorbed (AVA). FUC-TMC-NPs with positive (+) or negative (-) surface charges were prepared via polyelectrolyte complexation, both charged NP types permitted high viability and presented no cytotoxicity on L929, A549 and JAWS II dendritic cells. Flow cytometry measurements indicated lower (+)-FUC-TMC-NPs internalization levels than (-)-FUC-TMC-NPs, yet produced high levels of pro-inflammatory cytokines IFN-γ, IL12p40, and IL-4. Moreover, fluorescence microscope images proved that both charged NP could deliver drugs into the nucleus. In vivo studies on A/J mice showed that (+)-FUC-TMC-NPs carrying AVA triggered an efficient response with a higher IgG anti-PA antibody titer than AVA with CpG oligodeoxynucleotides, and yielded 100 % protection when challenged with the anthracis spores. Furthermore, PA-specific IgG1 and IgG2a analysis confirmed that (+)-FUC-TMC-NPs strongly stimulated humoral immunity. In conclusion, (+)-FUC-TMC-NP is promising anthrax vaccine adjuvant as an alternative to CpG.

A fucoidan-quaternary chitosan nanoparticle adjuvant for anthrax vaccine as an alternative to CpG oligodeoxynucleotides.

We examined the efficacy of fucoidan-N-(2-hydroxy-3-trimethylammonium)propylchitosan nanoparticles (FUC-HTCC NPs) as adjuvants for anthrax vaccine adsorbed (AVA). Positively and negatively surface-charged FUC-HTCC NPs were prepared via polyelectrolyte complexation by varying the mass ratio of FUC and HTCC. When cultured with L929 cells or JAWS II dendritic cells, both charged NPs showed high cell viability and low cytotoxicity, observed via MTT assay and lactate dehydrogenase release assay, respectively. In addition, we have monitored excellent NPs uptake efficacy by dendritic cells and observed that combining FUC-HTCC NPs with AVA significantly increases the magnitude of IgG-anti-protective antigen titers in A/J mice compared to that by CpG oligodeoxynucleotides plus AVA or AVA alone, and PA-specific IgG1 and IgG2a analysis confirmed that FUC-HTCC NPs strongly stimulated humoral immunity. Furthermore, FUC-HTCC NPs plus AVA provided a superior survival rate (100%) of A/J mice compared to CpG oligodeoxynucleotides plus AVA (75%) or AVA alone (50%) following anthrax lethal toxin challenge. The findings support FUC-HTCC NPs as a potential adjuvant of AVA for rapid induction of protective immunity.

Rapid and sensitive detection of Yersinia pestis by lateral-flow assay in simulated clinical samples.

BACKGROUND: Yersinia pestis is a contributing agent to the epidemic disease, plague, which killed an estimated 200 million people during historical times. In this study, a rapid, cheap, sensitive, and specific technique, the lateral flow assay (F1 strips), has been successfully developed to detect this pathogen, by using paired monoclonal antibodies (MAbs) against Y. pestis capsule like fraction 1 (F1) protein. Compared with the polyclonal antibody (PAb) based F1 strips, the Mab-based F1 strips have a remarkable increased detection limitation (10 to 100 folds). Furthermore, besides the limitation and specificity evaluation, the application of this F1 strip on simulated clinical samples indicate the LFA can be a good candidate to detect plague. METHODS: Recombinant F1 antigen was expressed and purified from a series of works. The various anti-F1 monoclonal antibodies generated from hybridoma cells were screened with the ELISA technique. To evaluate the feasibility of this Y. pestis F1 test strip, the F1 protein/Y. pestis was spiked into simulated clinical samples such as human serum, mouse bronchoalveolar lavage fluids, and mouse blood to mimic natural infection status. Additionally, this technique was applied to detect the Y. pestis in the environment-captured rats, to evaluate the practical usefulness of the strips. RESULTS: By using this MAb-based-LFA technique, 4 ng/ml of recombinant F1-protein and 103 CFU/ml of Y. pestis could be detected in less than 10 mins, which is at least 10-folds than that of the PAb format. On the other hand, although various Yersinia strains were applied to the strips, only Y. pestis strain showed a positive result; all other Yersinia species did not produce a positive signal, indicating the high efficiency and specificity of the MAb-based F1-strips. CONCLUSION: Based on our findings, we suggest that the MAb-format-LFA will be valuable as a diagnostic tool for the detection of Y. pestis. This report shows that the F1 strip is sufficient to support not only the detection of plague in simulated clinical samples, but also it may be a good candidate to meet the epidemiological surveillance during an outbreak of the biological warfare.

Characterization of cross protection of Swine-Origin Influenza Virus (S-OIV) H1N1 and reassortant H5N1 influenza vaccine in BALB/c mice given a single-dose vaccination.

BACKGROUND: Influenza virus has antigen drift and antigen shift effect, vaccination with some influenza vaccine might not induce sufficient immunity for host to the threat of other influenza virus strains. S-OIV H1N1 and H5N1 influenza vaccines in single-dose immunization were evaluated in mice for cross protection to the challenge of A/California/7/2009 H1N1 or NIBRG-14 H5N1 virus. RESULTS: Both H1N1 and H5N1 induced significant homologous IgG, HAI, and microneutralization antibody responses in the mice, while only vaccines plus adjuvant produced significant heterogeneous IgG and HAI antibody responses. Both alum and MPLA adjuvants significantly reduced the S-OIV H1N1 vaccine dose required to elicit protective HAI antibody titers from 0.05 µg to 0.001 µg. Vaccines alone did not protect mice from challenge with heterogeneous influenza virus, while H5N1 vaccine plus alum and MPLA adjuvants did. Mouse body weight loss was also less significant in the presence of adjuvant than in the vaccine without adjuvant. Furthermore, both H1N1 and H5N1 lung viral titers of immunized mice were significantly reduced post challenge with homologous viruses. CONCLUSION: Only in the presence of MPLA adjuvant could the H5N1 vaccine significantly reduce mouse lung viral titers post H1N1 virus challenge, and not vice versa. MPLA adjuvant induced cross protection with a single dose vaccination to the challenge of heterogeneous influenza virus in mice. Lung viral titer seemed to be a better indicator compared to IgG, neutralization antibody, and HAI titer to predict survival of mice infected with influenza virus.

Role of amphotericin B upon enhancement of protective immunity elicited by oral administration with liposome-encapsulated-Japanese encephalitis virus nonstructural protein 1 (NS1) in mice.

Amphotericin B (AmB) is an antifungal antibiotic the activity of which has been associated with modulation of pro-inflammatory cytokines expression in cultured cells. Herein we reveal that co-administration with AmB enhances the immunogenicity of oral Lip-JENS1 vaccine which derived from liposomes functionalized with DSPC (distearoylphosphatidylcholine) and cholesterol (2:1, molar ratio)-bearing JE virus NS1 protein (600 microg ml(-1)). Oral single dose of Lip-JENS1 elicited a detectable serum NS1-specific IgG antibody response from a mouse model. Remarkably, the addition of AmB (125 microg per mouse), particularly, 2 h prior to, but not simultaneously with, the administration of Lip-JENS1 significantly enhanced the systemic antigen-specific antibody response, providing superior protection against lethal JEV challenges. Further, we observed AmB-induced the transcription of cytokine expression and translocation of transcriptional factor NF-kappaB from the cytoplasm to the nucleus for the murine macrophage J774A.1. Moreover, Peyer's-patch lymphocytes (PPL) from AmB-treated mice produced high levels of IL-1beta, IL-6 and TNF-alpha expression compared to the corresponding control of cells from non-treated mice. Taken together, the results suggest that AmB exerts a profound influence upon mucosal vaccination with Lip-JENS1, possibly playing an adjuvant-augmented role to "fine-tune" humoral as well as cellular immune response, thus conferring enhanced protective immunity for immunising individuals against JE infection.

A comparison of immunogenicity and protective immunity against experimental plague by intranasal and/or combined with oral immunization of mice with attenuated Salmonella serovar Typhimurium expressing secreted Yersinia pestis F1 and V antigen.

We investigated the relative immunogenicity and protective efficacy of recombinant X85MF1 and X85V strains of DeltacyaDeltacrpDeltaasd-attenuated Salmonella Typhimurium expressing, respectively, secreted Yersinia pestis F1 and V antigens, following intranasal (i.n.) or i.n. combined with oral immunization for a mouse model. A single i.n. dose of 10(8) CFU of X85MF1 or X85V induced appreciable serum F1- or V-specific IgG titres, although oral immunization did not. Mice i.n. immunized three times (i.n. x 3) with Salmonella achieved the most substantial F1/V-specific IgG titres, as compared with corresponding titres for an oral-primed, i.n.-boosted (twice; oral-i.n. x 2) immunization regimen. The level of V-specific IgG was significantly greater than that of F1-specific IgG (P<0.001). Analysis of the IgG antibodies subclasses revealed comparable levels of V-specific Th-2-type IgG1 and Th-1-type IgG2a, and a predominance of F1-specific Th-1-type IgG2a antibodies. In mice immunized intranasally, X85V stimulated a greater IL-10-secreting-cell response in the lungs than did X85MF1, but impaired the induction of gamma-interferon-secreting cells. A program of i.n. x 3 and/or oral-i.n. x 2 immunization with X85V provided levels of protection against a subsequent lethal challenge with Y. pestis, of, respectively, 60% and 20%, whereas 80% protection was provided following the same immunization but with X85MF1.

Enhanced immune response by amphotericin B following NS1 protein prime-oral recombinant Salmonella vaccine boost vaccination protects mice from dengue virus challenge.

A recombinant vaccine strain SL3261/pLT105 of attenuated aroA Salmonella enterica serovar Typhimurium SL3261 strain expressing a secreted dengue virus type 2 non-structural NS1 and Yersinia pestis F1 (Caf1) fusion protein, rNS1:Caf1, was generated. Immunological evaluation was performed by prime-boost vaccine regimen. Oral immunization of mice with 1 x 10(9)cfu of SL3261/pLT105 only induced low levels of NS1-specific antibody response and protective immunity following dengue virus challenge. The parenteral NS1 protein priming-oral Salmonella boosting protocol enhanced both NS1-specific serum IgG response and protective efficacy as compared to mice immunized with each type vaccine alone. Addition of an antifungal antibiotic amphotericin B (AmB) to Salmonella vaccine further enhanced the synergic effects of prime-boost vaccine regimen on the elicited NS1-specific serum IgG response and the protective efficacy. Together, the results demonstrated that the rNS1:Caf1 producing Salmonella SL3261/pLT105 strain fails to provide effective protection as an oral vaccine alone despite co-administration of AmB as an adjuvant capable of enhancing the immune responses, and moreover, the protein priming-oral Salmonella vaccine boosting approach in combination with AmB as an immunization regimen may have the potential to be further explored as an alternative approach for dengue vaccine development.