Alginate coated chitosan nanoparticles are an effective subcutaneous adjuvant for hepatitis B surface antigen

We recently described a delivery system that is composed of a chitosan core to which the hepatitis B surface antigen (HBsAg) was adsorbed and subsequently coated with sodium alginate. In this present work, alginate coated chitosan nanoparticles were evaluated as a subcutaneous adjuvant for HBsAg. HBsAg loaded, alginate coated or uncoated chitosan nanoparticles, associated or not with CpGODN were subcutaneously administered to mice and several immunological parameters were evaluated. A high anti-HBsAg IgG titer (2271+/-120 mIU/ml), with the majority of antibodies being of Th2 type, was observed within group I, vaccinated with HBsAg loaded onto coated nanoparticles. However, regarding cellular immune response, no significant differences were observed for antigen-specific splenocyte proliferation or for the secretion of IFN-gamma and IL-4, when compared to the control group. The co-delivery of antigen-loaded nanoparticles in the presence of the immunopotentiator, CpG ODN 1826, resulted in an increase of anti-HBsAg IgG titers that was not statistically different from the first group; however, an increase of the IgG2a/IgG1 ratio from 0.1 to 1.0 and an increase (p<0.01) of the IFN-gamma production by the splenocytes stimulated with the HBV antigen was observed. The enhancement of the immune response observed with the antigen-loaded nanoparticles demonstrated that chitosan is a promising platform for parenteral HBsAg delivery and, when co-administered with the CpG ODN, resulted in a mixed Th1/Th2 type immune response.     

Evaluation of the immune response following a short oral vaccination schedule with hepatitis B antigen encapsulated into alginate-coated chitosan nanoparticles

The purpose of this work was to assess the ability of recombinant hepatitis B vaccine, encapsulated in alginate-coated chitosan nanoparticles, to induce local and systemic immune responses following oral vaccination. The antigen was administered either alone or in combination with the immunopotentiator, synthetic oligodeoxynucleotide containing immunostimulatory CpG motif (CpG ODN) as adjuvant, and associated or not with the alginate-coated chitosan nanoparticles. After two immunizations the group I (HBsAg associated with nanoparticles) and the group VI (HBsAg and CpG, both associated with nanoparticles) showed enhanced immune responses. Both groups showed significant higher values of the CD69 expression in CD4+ and CD8+ T-lymphocytes and lower values of this marker in B lymphocytes. Moreover, a strongest proliferative response of the splenocytes, ex vivo stimulated with concanavalin A, was observed in the same groups. Although with a presence of non-responder mice within the groups, only mice of the groups I and VI elicited the generation of anti-HBsAg antibodies detected in serum (IgG) and in the intestinal washings (sIgA). The results demonstrated that coated chitosan nanoparticles might have potential for being used as a deliver system for oral vaccination with the recombinant hepatitis B surface antigen.     

CpG ODN纳米粒增强免疫抑制小鼠对重组乙肝疫苗的免疫应答

目的：探讨CpG ODN壳聚糖纳米粒联合重组乙肝疫苗对免疫抑制小鼠的免疫增强作用。方法：选月环磷酰胺建立免疫抑制模型小鼠。将乙肝疫苗单独或和CpG ODN或CpG ODN纳米粒经后腿胫骨前肌注射到小鼠体内，ELISA方法检测抗HBsAg IgG抗体、IL-12水平；流式细胞仪检测外周血CD4^＋、CD8^＋T淋巴细胞亚群。结果：CpG ODN纳米粒联合疫苗组的CD4^＋细胞百分数，IL-12水平及抗HBsAg IgG抗体的含量显著高于CpG ODN联合疫苗组（P〈0．05）。结论：包裹在壳聚糖纳米粒中的CpG ODN较相同剂量的CpG ODN更能增强免疫抑制小鼠对乙肝疫苗的免疫应答。     

Intranasal administration of CpG oligonucleotides induces mucosal and systemic Type 1 immune responses and adjuvant activity to porcine reproductive and respiratory syndrome killed virus vaccine in piglets in vivo

Oligonucleotides containing CpG motifs (CpG ODN) are strong adjuvants for immune responses, particularly in mice. Recently, it has been showed that CpG ODN is a promising mucosal adjuvant in mice, but data on mucosal immune responses induced by CpG ODN in piglets are scarce. We have previously demonstrated that CpG ODN is a potent adjuvant to pseudorabies attenuated virus (PRV) vaccine when administered subcutaneously (SC) in newborn piglets. Herein, we evaluated intranasal (IN) delivery of CpG ODN with porcine reproductive and respiratory syndrome (PRRS) killed virus vaccine (PRRSV) to determine its potential as a mucosal adjuvant to a commercial vaccine. CpG ODN augmented systemic (IgG in serum, Peripheral blood mononuclear cells (PBMC) proliferation) and mucosal (IgA in feces, nasal and oral secretions) immune responses against antigen. CpG ODN stimulated both T-helper type1 (Type 1) (IgG2) and Type 2 (IgA) responses when delivered intranasally. Results from this study indicate that stimulatory CpG ODN may be effective as a mucosal adjuvant with commercial vaccine in husbandry animals.     

Plasmid DNA loaded chitosan nanoparticles for nasal mucosal immunization against hepatitis B

This work investigates the preparation and in vivo efficacy of plasmid DNA loaded chitosan nanoparticles for nasal mucosal immunization against hepatitis B. Chitosan pDNA nanoparticles were prepared using a complex coacervation process. Prepared nanoparticles were characterized for size, shape, surface charge, plasmid loading and ability of nanoparticles to protect DNA against nuclease digestion and for their transfection efficacy. Nasal administration of nanoparticles resulted in serum anti-HBsAg titre that was less compared to that elicited by naked DNA and alum adsorbed HBsAg, but the mice were seroprotective within 2 weeks and the immunoglobulin level was above the clinically protective level. However, intramuscular administration of naked DNA and alum adsorbed HBsAg did not elicit sIgA titre in mucosal secretions that was induced by nasal immunization with chitosan nanoparticles. Similarly, cellular responses (cytokine levels) were poor in case of alum adsorbed HBsAg. Chitosan nanoparticles thus produced humoral (both systemic and mucosal) and cellular immune responses upon nasal administration. The study signifies the potential of chitosan nanoparticles as DNA vaccine carrier and adjuvant for effective immunization through non-invasive nasal route.     

Evaluation of Mucoadhesive PLGA Microparticles for Nasal Immunization

In this study, hepatitis B surface antigen (HBsAg) loaded poly(lactic-co-glycolic acid) (PLGA) microparticles were prepared and coated with chitosan and trimethyl chitosan (TMC) to evaluate the effect of coating material for nasal vaccine delivery. The developed formulations were characterized for size, zeta potential, entrapment efficiency, and mucin adsorption ability. Plain PLGA microparticles demonstrated negative zeta potential. However, coated microparticles showed higher positive zeta potential. Results indicated that TMC microparticles demonstrated substantially higher mucin adsorption when compared to chitosan-coated microparticles and plain PLGA microparticles. The coated and uncoated microparticles showed deposition in nasal-associated lymphoid tissue under fluorescence microscopy. The coated and uncoated microparticles were then administered intranasally to mice. Immune-adjuvant effect was determined on the basis of specific antibody titer observed in serum and secretions using enzyme-linked immunosorbent assay. It was observed that coated particles showed a markedly increased anti-HBsAg titer as compared to plain PLGA microparticles, but the results were more pronounced with the TMC-coated PLGA microparticles.     

Mucoadhesive glycol chitosan nanoparticles for intranasal delivery of hepatitis B vaccine: enhancement of mucosal and systemic immune response

In this study, for the first time, glycol chitosan (GC) nanoparticles (NPs) were prepared and evaluated to obtain systemic and mucosal immune responses against nasally administered hepatitis B surface antigen (HBsAg). Size, zeta potential and morphology of the NPs were investigated as a function of preparation method. NPs with high loading efficacy (?>?95%) and positively charged surface were obtained with an average particle size of approximately 200?nm. The structural integrity of HBsAg in NPs was evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and further confirmed by measuring the in vitro antigenicity using an enzyme immunoassay. During in vivo studies, GC NPs showed the lowest nasal clearance rate and better mucosal uptake when compared with chitosan (CS) NPs. The immunogenicity of NPs-based delivery system(s) was assessed by measuring anti-HBsAg antibody titer in mice serum and secretions after intranasal administration. The alum-based HBsAg vaccine injected subcutaneously was used as positive control. Results indicated that alum-based HBsAg induced strong humoral but negligible mucosal immunity. However, GC NPs induced stronger immune response at both of the fronts as compared to generated by CS NPs. This study demonstrates that this newly developed system has potential for mucosal administration of vaccines.     

M-cell targeted biodegradable PLGA nanoparticles for oral immunization against hepatitis B

The transcytotic capability and expression of distinct carbohydrate receptors on the intestinal M-cells render it a potential portal for the targeted oral vaccine delivery. PLGA nanoparticles loaded with HBsAg were developed and antigen was stabilized by co-encapsulation of trehalose and Mg(OH)(2). Additionally, Ulex europaeus 1 (UEA-1) lectin was anchored to the nanoparticles to target them to M-cells of the peye's patches. The developed systems was characterized for shape, size, polydispersity index and loading efficiency. Bovine submaxillary mucin (BSM) was used as a biological model for the in vitro determination of lectin activity and specificity. The targeting potential of the lectinized nanoparticles were determined by Confocal Laser Scanning Microscopy (CLSM) using dual staining technique. The immune stimulating potential was determined by measuring the anti-HBsAg titre in the serum of Balb/c mice orally immunized with various lectinized formulations and immune response was compared with the alum-HBsAg given intramuscularly. Induction of the mucosal immunity was assessed by estimating secretary IgA (sIgA) level in the salivary, intestinal and vaginal secretion. Additionally, cytokines (interleukin-2; IL-2 and interferon-gamma; IFN-gamma) level in the spleen homogenates was also determined. The results suggest that HBsAg can be successfully stabilized by co-encapsulation of protein stabilizers. The lectinized nanoparticles have demonstrated approximately 4-fold increase in the degree of interaction with the BSM as compared to plain nanoparticles and sugar specificity of the lectinized nanoparticles was also maintained. CLSM showed that lectinized nanoparticles were predominantly associated to M-cells. The serum anti-HBsAg titre obtained after oral immunization with HBsAg loaded stabilized lectinized nanoparticles was comparable with the titre recorded after alum-HBsAg given intramuscularly. The stabilized UEA-1 coupled nanopartilces exhibited enhanced immune response as compared to stabilized non-lectinized nanoparticles. Furthermore, the stabilized lectinized nanoparticles elicited sIgA in the mucosal secretion and IL-2 and IFN-gamma in the spleen homogenates. These stabilized lectinized nanoparticles could be a promising carrier-adjuvant for the targeted oral-mucosal immunization.     

Dose sparing of CpG oligodeoxynucleotide vaccine adjuvants by nanoparticle delivery

The main objective of these studies was to investigate whether the nanoparticle delivery has any immunopotentiation effect at modest doses of a few micro- or nanograms of CpG oligodeoxynucleotide (CpG ODN) and what would be the influence on T cell responses at such low doses. Various doses (5 to 0.05 microg) of a model CpG ODN adjuvant (#1826) along with 2 Lf tetanus toxoid (TT) were formulated in either nanoparticles using poly(D,L-lactic-co-glycolic acid) (PLGA) 50:50 co-polymer, or saline. Strong antigen specific ex vivo T cell proliferation was observed for the Balb/c mice receiving immunogens in nanoparticles. At 5 microg dose of CpG ODN, the T cell stimulation index (SI) was 241 as compared with 74 for the same dose when given in saline. Comparable SI value of 78 was observed at 100-fold lower dose (0.05 microg) using nanoparticles. Similarly, significantly higher (P<0.01) cytokine secretion was observed for nanoparticles groups. A ten-fold lower dose (0.5 microg instead of 5 microg) of CpG ODN in nanoparticles was adequate to obtain levels of IFN-gamma, TNF-alpha, and IL-2 comparable to those observed following immunisations in saline. The immunopotentiation effect of the particulate delivery on antibody response (total IgG and subtypes) was not so marked. These studies emphasise that antigen delivery in biodegradable nanoparticles can facilitate induction of strong T cell responses, particularly of the Th1 type, at extremely lower doses of CpG ODN. Such reduction in the effective dose would be advantageous for minimising the potential side effects of these novel adjuvants.     

M-cell targeted biodegradable PLGA nanoparticles for oral immunization against hepatitis B

The transcytotic capability and expression of distinct carbohydrate receptors on the intestinal M-cells render it a potential portal for the targeted oral vaccine delivery. PLGA nanoparticles loaded with HBsAg were developed and antigen was stabilized by co-encapsulation of trehalose and Mg(OH)₂. Additionally, Ulex europaeus 1 (UEA-1) lectin was anchored to the nanoparticles to target them to M-cells of the peye's patches. The developed systems was characterized for shape, size, polydispersity index and loading efficiency. Bovine submaxillary mucin (BSM) was used as a biological model for the in vitro determination of lectin activity and specificity. The targeting potential of the lectinized nanoparticles were determined by Confocal Laser Scanning Microscopy (CLSM) using dual staining technique. The immune stimulating potential was determined by measuring the anti-HBsAg titre in the serum of Balb/c mice orally immunized with various lectinized formulations and immune response was compared with the alum-HBsAg given intramuscularly. Induction of the mucosal immunity was assessed by estimating secretary IgA (sIgA) level in the salivary, intestinal and vaginal secretion. Additionally, cytokines (interleukin-2; IL-2 and interferon-γ; IFN-γ) level in the spleen homogenates was also determined. The results suggest that HBsAg can be successfully stabilized by co-encapsulation of protein stabilizers. The lectinized nanoparticles have demonstrated approximately 4-fold increase in the degree of interaction with the BSM as compared to plain nanoparticles and sugar specificity of the lectinized nanoparticles was also maintained. CLSM showed that lectinized nanoparticles were predominantly associated to M-cells. The serum anti-HBsAg titre obtained after oral immunization with HBsAg loaded stabilized lectinized nanoparticles was comparable with the titre recorded after alum-HBsAg given intramuscularly. The stabilized UEA-1 coupled nanopartilces exhibited enhanced immune response as compared to stabilized non-lectinized nanoparticles. Furthermore, the stabilized lectinized nanoparticles elicited sIgA in the mucosal secretion and IL-2 and IFN-γ in the spleen homogenates. These stabilized lectinized nanoparticles could be a promising carrier-adjuvant for the targeted oral-mucosal immunization.     

Microfold-cell targeted surface engineered polymeric nanoparticles for oral immunization

Present work was envisaged to develop novel M-cell targeted polymeric particles that are capable of protecting the antigen from harsh gastric conditions. Ulex europaeus agglutinin (UEA-1) lectin was anchored for selective delivery of antigen to gut-associated lymphoid tissue (GALT). In the present investigation, chitosan nanoparticles were prepared by ionic gelation followed by antigen (bovine serum albumin, BSA) adsorption. Developed nanoparticles were further coated by UEA-1 lectin conjugated alginate and characterized for size, shape, zeta-potential, entrapment efficiency, and in vitro release. The immunological response of the developed system were performed in Balb/c mice and compared with aluminium hydroxide gel-based conventional vaccine. Results indicated that immunization with UEA-1 lectin conjugated alginate-coated particles induces efficient systemic as well as mucosal immune responses against BSA compared to other formulations. Aluminium-based vaccine dominated throughout the study, while failed in case of mucosal antibody. Additionally, IgG1 and IgG2a isotypes were determined to confirm the TH1/TH2 mixed immune response. The developed formulation exhibited superior systemic response along with dominating mucosal immunity. These data demonstrate the potential of UEA-alginate-coated nanoparticles as effective delivery system via oral route.     

Dendritic cell targeted chitosan nanoparticles for nasal DNA immunization against SARS CoV nucleocapsid protein

This work investigates the formulation and in vivo efficacy of dendritic cell (DC) targeted plasmid DNA loaded biotinylated chitosan nanoparticles for nasal immunization against nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) as antigen. The induction of antigen-specific mucosal and systemic immune response at the site of virus entry is a major challenge for vaccine design. Here, we designed a strategy for noninvasive receptor mediated gene delivery to nasal resident DCs. The pDNA loaded biotinylated chitosan nanoparticles were prepared using a complex coacervation process and characterized for size, shape, surface charge, plasmid DNA loading and protection against nuclease digestion. The pDNA loaded biotinylated chitosan nanoparticles were targeted with bifunctional fusion protein (bfFp) vector for achieving DC selective targeting. The bfFp is a recombinant fusion protein consisting of truncated core-streptavidin fused with anti-DEC-205 single chain antibody (scFv). The core-streptavidin arm of fusion protein binds with biotinylated nanoparticles, while anti-DEC-205 scFv imparts targeting specificity to DC DEC-205 receptor. We demonstrate that intranasal administration of bfFp targeted formulations along with anti-CD40 DC maturation stimuli enhanced magnitude of mucosal IgA as well as systemic IgG against N protein. The strategy led to the detection of augmented levels of N protein specific systemic IgG and nasal IgA antibodies. However, following intranasal delivery of naked pDNA no mucosal and systemic immune responses were detected. A parallel comparison of targeted formulations using intramuscular and intranasal routes showed that the intramuscular route is superior for induction of systemic IgG responses compared with the intranasal route. Our results suggest that targeted pDNA delivery through a noninvasive intranasal route can be a strategy for designing low-dose vaccines.     

A comparative study of the antigen-specific immune response induced by co-delivery of CpG ODN and antigen using fusion molecules or biodegradable microparticles

CpG ODN are toll-like receptor 9 (TLR9) agonists that can enhance antigen presentation by antigen presenting cells (APCs) such as dendritic cells (DCs). The most potent antigen-specific responses are seen when CpG ODN and the antigen are co-localized in the same APC. CpG ODN-antigen fusion molecules and biodegradable microparticles entrapping CpG ODN and antigen can ensure both components are delivered to the same APC. In this study, we compared the efficacy of the CpG-ODN fusion molecules against biodegradable microparticles entrapping antigen and CpG ODN. Microparticles were prepared using a double emulsion solvent evaporation methodology. CpG ODN-OVA fusion molecules were prepared by mixing maleimide-activated protein with thiolated CpG ODN. Both CpG ODN-OVA fusion molecules and microparticles co-entrapping CpG ODN and OVA generated stronger IgG2a and interferon-gamma (IFN-gamma) responses than delivery of soluble CpG ODN and OVA. The microparticles generated stronger IgG2a and IFN-gamma immune responses than did CpG ODN-antigen fusion molecules.     

CpG oligodeoxynucleotide synergizes innate defense regulator peptide for enhancing the systemic and mucosal immune responses to pseudorabies attenuated virus vaccine in piglets in vivo

Oligonucleotides containing CpG motifs (CpG ODN) are strong adjuvants for humoral and cellular immune responses in mice, and innate defense-regulator peptides (IDRs) are known to facilitate the uptake of antigens into antigen presenting cells (APCs), but data on synergistic effects of CpG and IDRs in piglets are scarce. In this report, the combination of porcine-specific CpG ODN and HH2 (a kind of IDR which was selected for its better synergy with CpG ODN) was used as immunoadjuvant to enhance the immune responses of the newborn piglets to Pseudorabies attenuated virus (PRV) vaccine. The titers of specific antibodies and serum IgG1/IgG2 subtypes to PRV vaccine, interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), IL-12 and IL-4 were examined to identify the immune responses of the newborn piglets. The results showed that piglets immunized intranasally (IN) and subcutaneously (SC) with PRV vaccine and CpG-HH2 complex both presented high titers of PRV-specific antibodies and IgG2 isotype, a Th1-dominated (IFN-γ and IL-12) cytokine profiles, high levels of IgA in saliva, broncheoalveolar lavage (BAL) and intestinal washings. The results suggested that, CpG-HH2 complex augmented systemic (IgG in serum) and mucosal (IgA in saliva, BAL and intestinal washings) immune responses against antigen. CpG-HH2 complex stimulated both T-helper type1 (Th1) (IgG2) and Th2 (IgA) responses when delivered IN, and IN route could induce stronger mucosal immune responses than SC route. All these data indicate that CpG-HH2 complex is a potential effective adjuvant for the PRV vaccine in newborn piglets.     

Oligodeoxynucleotides containing synthetic immunostimulatory motifs augment potent Th1 immune responses to HBsAg in mice

Toll-like receptor 9 (TLR9) modulators have potent Th1-adjuvant activity. We recently reported the development of immunomodulatory oligonucleotides (IMOs) containing novel structures (immunomers) and synthetic immunostimulatory CpR (R=2'-deoxy-7-deazguanosine) or R'pG (R'=1-(2'-deoxy-beta-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine) motifs. IMOs activate TLR9 pathways, resulting in cytokine secretion profiles different from those induced by CpG DNA. In the present study we evaluated the adjuvant activity of IMOs containing CpG, CpR, or R'pG motifs in combination with hepatitis B surface antigen (HBsAg) in a mouse model. Mice immunized with HBsAg plus IMO produced higher levels of IgG2a and lower levels of IgG1 than did mice immunized with HBsAg alone or with alum. High IgG2a responses were found at week 4 and remained high until 14 weeks after immunization. Adoptive transfer of splenocytes from HBsAg/IMO-immunized mice to na?ve mice resulted in strong IgG2a production in response to antigen boost. Splenocytes of mice immunized with HBsAg/IMO produced high levels of IFN-gamma, but not Th2 cytokines IL-4 and IL-5, in antigen-recall experiments in vitro. The use of IMOs as adjuvants to HBsAg resulted in the production of strong anti-HBsAg antibodies at antigen doses as low as 0.2 microg. These data demonstrate that IMOs enhance the immunogenicity of HBsAg through potent Th1 immune responses, which may allow lower doses of antigen in vaccination.     

Mucosal delivery of bacterial antigens and CpG oligonucleotides formulated in biphasic lipid vesicles in pigs

The ineffectiveness of simple delivery of soluble antigens to mucosal membranes for immunization has stimulated extensive studies of strategies for appropriate delivery systems and adjuvants. Biphasic lipid vesicles are formulations suitable for the delivery of proteins, peptides, and oligo/polynucleotides. The purpose of these studies was to investigate the ability of biphasic lipid vesicles (as vaccine-targeting adjuvants) containing a bacterial antigen and unmethylated oligonucleotides containing CGdinucleotides - CpG motifs (CpG ODNs) to induce systemic and mucosal immune responses in pigs. Results showed that while the protein, either alone or with CpG ODNs, did not induce mucosal immune responses, administration of antigen and CpG ODNs in biphasic lipid vesicles resulted in induction of both systemic and local antibody responses after immunization using a combined mucosal/systemic approach.     

Treatment of intravaginal HSV-2 infection in mice: a comparison of CpG oligodeoxynucleotides and resiquimod (R-848)

The mammalian innate immune system recognizes pathogens via a series of pattern-recognition receptors such as the toll-like receptors (TLR) that interact with pathogen-associated molecular patterns (PAMPs) and lead to the rapid activation of innate immune cells. In this study, we compared the efficacy of CpG ODN (a TLR9 agonist) and resiquimod (R-848; a TLR7/8 agonist) for topical immunoprophylaxis or immunotherapy of vaginal herpes simplex virus type 2 (HSV-2) infection in mice. Efficacy against HSV infection was observed with CpG ODN but less so with R-848, even after repeated administrations. Intravaginal (IVAG) administration of CpG ODN resulted in strong local but relatively weak systemic immune activation, as determined by levels of the chemokines IP-10, MIG and I-TAC in vaginal tissue and plasma, respectively. In contrast, IVAG administration of R-848 resulted in high levels of plasma IP-10, similar to those seen after parenteral administration, but overall, weaker or shorter-lived local immune responses than obtained with CpG ODN. These findings suggest that differences in biodistribution and sites of immune activation between CpG ODN and R-848 after IVAG delivery account for differences in efficacy, and demonstrate the need for local mucosal innate activation for protection against HSV-2.     

TMC–MCC (N-trimethyl chitosan–mono-N-carboxymethyl chitosan) nanocomplexes for mucosal delivery of vaccines

In this study, for the first time, TMC/MCC complex nanoparticles as a delivery system and as an adjuvant were developed and evaluated to obtain systemic and mucosal immune responses against nasally administered tetanus toxoid (TT). Nanoparticles were developed by complexation between the oppositely charged chitosan derivatives, N-trimethyl chitosan (TMC, polycationic) and mono-N-carboxymethyl chitosan (MCC, polyampholytic) without using any crosslinker for mucosal vaccination. The cellular viability was found to be higher with TMC/MCC complex compared to that of MCC and TMC alone. Size, zeta potential and morphology of the nanoparticles were investigated as a function of preparation method. Nanoparticles with high loading efficacy (95%) and positively charged surface were obtained with an average particle size of 283 ± 2.5 nm. The structural integrity of the TT in the nanoparticles was confirmed by SDS–PAGE electrophoresis analysis. Cellular uptake studies indicated that FITC-BSA loaded nanoparticles were effectively taken up into the mouse Balb/c monocyte macrophages. Mice were nasally immunized with TT loaded TMC/MCC complex nanoparticles and compared to that of TMC and MCC nanoparticles. TMC/MCC complex nanoparticles were shown to induce both the mucosal and systemic immune response indicating that this newly developed system has potential for mucosal administration of vaccines.     

Biodegradable Particles as Vaccine Delivery Systems: Size Matters

Poly(lactide-co-glycolide) (PLGA) particles have strong potential as antigen delivery systems. The size of PLGA particles used to vaccinate mice can affect the magnitude of the antigen-specific immune response stimulated. In this study, we fabricated and characterized 17 μm, 7 μm, 1 μm, and 300 nm PLGA particles coloaded with a model antigen ovalbumin (OVA) and CpG oligodeoxynucleotides (CpG ODN). PLGA particles demonstrated a size-dependent burst release followed by a more sustained release of encapsulated molecules. PLGA particles that were 300 nm in size showed the highest internalization by, and maximum activation of, dendritic cells. The systemic antigen-specific immune response to vaccination was measured after administration of two intraperitoneal injections, 7 days apart, of 100 μg OVA and 50 μg CpG ODN in C57BL/6 mice. In vivo studies showed that 300 nm sized PLGA particles generated the highest antigen-specific cytotoxic T cell responses by days  14 and 21. These mice also showed the highest IgG2a:IgG1 ratio of OVA-specific antibodies on day  28. This study suggests that the smaller the PLGA particle used to deliver antigen and adjuvants the stronger the antigen-specific cytotoxic T cell response generated.KEY WORDS: CpG ODN, cytotoxic T lymphocytes, dendritic cells, nanoparticles, poly (lactide-co-glycolide), vaccine     

Intranasal administration of plasmid DNA-coated nanoparticles results in enhanced immune responses

Intranasal immunization offers potential forthe elicitation of effective mucosal and systemic immune responses. In this study, a previously reported novel cationic nanoparticle engineered from a microemulsion precursor was further modified, optimized and applied intranasally to mice to explore its potential as a plasmid DNA (pDNA) vaccine delivery system. To this end, more uniform nanoparticles (around 100 nm) containing less cationic surfactant were developed. The pDNA-coated nanoparticles significantly enhanced the specific serum IgG and IgA titres to an expressed model antigen, beta-galactosidase, by 18-28 and 25-30 fold, respectively, when compared with naked pDNA alone. An enhanced splenocyte proliferative response was also observed after immunization with the pDNA-coated nanoparticles. It was concluded that these plasmid DNA-coated nanoparticles may have potential for immunization via the nasal route.