Structural and mechanistic insights into starch microgel/anthocyanin complex assembly and controlled release performance.

We report a self-assembly method for the fabrication of multilayer-starch-based microgels used for anthocyanin encapsulation. Alcohol-heating treatment and ionization reactions were employed to reduce the crystallinity of starch and introduce ionic groups on the molecule to further cross-link it with sodium trimetaphosphate and produce a starch microgel. The molecular interactions between the starch and the anthocyanins facilitated the anthocyanin encapsulation and the starch-microgel/anthocyanin complexes with one, two, and three self-assembled layers were obtained. The Lay-1 microgel exhibited an encapsulation efficiency of 50.1% when the anthocyanin concentration, cross-linking starch concentration, contact time, and temperature were 0.25 mg/mL, 1.5 mg/mL, 3 h, and 40 °C, respectively. An increase in the number of layers resulted in a more compact microgel structure with the zeta potential presenting variations upon structural changes. Furthermore, the encapsulated anthocyanins presented a slow release from Lay-1, while the multilayered microgels (Lay-2 and Lay-3) displayed outstanding encapsulation stability. This study gives an insight on the encapsulation and release of anthocyanins by starch microgels, and provides a novel strategy for the design of starch-based encapsulation materials presenting great stability.

Optimization strategy for plasmid DNAs containing multiple-epitopes of foot-and-mouth disease virus by cis-expression with IL-2.

To optimize previous candidate DNA vaccine, a cis-expression plasmid DNA encoding two genes, human IL-2 and multiple-epitopes genes of foot-and-mouth disease virus (FMDV) was constructed with internal ribosome entry site (IRES) from encephalomyocarditis virus (EMCV) and intramuscularly inoculated into mice at 1-week interval. Specific antibodies in serum and cytokines (IL-2, IL-4 and IFN-gamma) from splenocytes were detected by indirect ELISA. Splenocytes proliferation rate was determined by a standard 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) method. The results showed that higher specific antibody, proliferate rate and cytokines were induced by plasmid DNA cis-expression with IL-2 compared to non-cis-expression plasmid DNA. Another series of mice were inoculated with plasmid DNA and boosted with antigenic protein. Specific antibody, proliferation rate and cytokines were induced significantly higher than those of mice immunized with protein or plasmid DNA only. However, only the cis-expression plasmid DNA elicited higher neutralization antibody in mice and provided one third protection against homologous virus in guinea pigs. In conclusion, cis-expression strategy with IL-2 up-regulated specific immunological response and provide protection against homologous virus.

Effects of epitopes combination and adjuvants on immune responses to anti-Alzheimer disease DNA vaccines in mice.

Alzheimer disease (AD) is a neurodegenerative disorder characterized by neuropathological hallmarks including deposits of the beta-amyloid peptide (AssP). Studies have shown that immunization with Abeta42 peptide reduces both the spatial memory impairments and Alzheimer disease-like neuropathologic changes in Alzheimer disease transgenic mice, but can cause side effect of a cell-mediated autoimmune meningoencephalitis. Recently, some studies showed that DNA vaccination could be used to generate an antibody response to Abeta without the adverse cell-mediated immune effect. In the current study, we generate four DNA vaccine plasmids (pV-GE1, pV-GE2, pV-GE3, and pV-GE4) against Alzheimer disease by separately fusing Abeta epitope sequences (coding for EFGH, DAEFGH, EFGH+EFGH, and EFGH+DAEFGH) with IgG heavy chain coding region of mouse. Meanwhile, the full-length gene Abeta encoding plasmid (pV-Abeta), empty vector (pVAX) and synthetic AssP were also included as control. The sera of BALB/c mice immunized via intramuscular with plasmids and peptide were tested by indirect ELISA for auto-AssP immunoreactivity. The results showed that all the DNA vaccine plasmids induced AssP-specific antibodies; moreover pV-GE2 and pV-Abeta constructs elicited higher antibody titers than other constructs (P < 0.05). To further enhance the immune response, GM-CSF encoding plasmid (pGM-CSF) and purified BCG-DNA were used as molecular adjuvants. BCG-DNA could enhance humoral and cellular immune responses simultaneously and did not alter the phenotype of the immune responses, whereas pGM-CSF showed no obvious effect on immune response. These results suggest that this immunization strategy of using Abeta epitope encoding plasmid plus BCG-DNA adjuvant may serve as the basis for developing anti-Alzheimer disease vaccines.

Tissue distribution of a plasmid DNA containing epitopes of foot-and-mouth disease virus in mice.

It is known that only the minority of plasmid DNAs effect a cure or prevention after intramuscular injection into host. But what is the fate of the majority? And indeed how many of the injected DNAs work? Till now, little is known about it. To answer these questions, two methods including PCR and autoradiography were used in distribution study in mice that had received a single muscular inoculation of plasmid DNA containing antigenic epitopes of foot-and-mouth disease virus. The results showed that the plasmid DNAs were distributed by blood circulation and degraded soon. The degradation ratio of super coiled plasmid DNA was 20.9% in 10 min, 34.1% in 1h, 86.8% in 1 day and 97.8% in 1 week in sera in vivo. And over a half of the whole were output in urine and faeces. The rest resided most in muscles as 'antigen pool', next in immune organs, kidney, liver, heart, lung and little in brain or gonad. About 40% or 0.5% of total plasmid DNAs, inferring to be effective, resided in muscles or immune organs, respectively. Collective results suggested that 'nude' DNA, as water injection, was characterized as quick absorbent, extensive distribution, but low utilization rate. Finally, the immune mechanism for the DNA vaccine was discussed.

Protection of pigs against Taenia solium cysticercosis using recombinant antigen or in combination with DNA vaccine.

In the present study, we investigated the duration of protection afforded to pigs immunized in two different prime-boost regimens: one is homologus priming and boosting with a protein vaccine, and the other is priming with a DNA vaccine and boosting with the protein vaccine. Groups of pigs that received the same vaccination regimen were then challenged with Taenia solium eggs at 6, 12 or 20 weeks post-immunization (wpi), respectively. The results showed that all vaccinated pigs challenged at 6 or 12 wpi showed significant (P < 0.05) reduction in the development of cysts. When challenged at 20 wpi, pigs primed with the DNA vaccine (pcDNA3-cC1) followed by two boosters of the protein vaccine (GST-cC1) showed significant (P < 0.05) protection against the challenge of T. solium eggs, whereas pigs receiving three injections of the protein vaccine showed no significant protection compared to non-vaccinated controls (P > 0.05). Antibody isotype assays showed that DNA prime-protein boost regimen induced a predominantly IgG2 response, compared to an IgG1 biased response for the protein prime-protein boost regimen. In addition, peripheral blood mononuclear cells (PBMC) obtained from the DNA prime-protein boost group proliferated strongly in response to GST-cC1 protein, and this responsiveness persisted until 20 wpi. Taken together, our data suggest that the use of a prime-boost strategy combining DNA and protein vaccines may be better than protein alone for the longevity of protection against the challenge of T. solium eggs.