The efficacy of poly-ICLC against Ebola-Zaire virus (EBOV) infection in mice and cynomolgus monkeys.

The potential protection of poly-ICLC (Hiltonol®) a double stranded RNA (dsRNA) against EBOV infection was assessed with prophylactic and therapeutic administration to wild type and TLR3-negative mice, and in non-human primates (NHPs) by measuring EBOL serum titers, survival extension, and serum liver and kidney function markers. Various doses of aqueous and liposomal poly-ICLC monotherapy provided robust protection in otherwise lethal murine EBOV challenge models, when treatment is started on the day 0 or one day after virus challenge. There was no advantage of liposomal vs. the aqueous poly-ICLC form. Protection appeared to be independent of TLR-3. NHPs treated with poly-ICLC and challenged with EBOV survived longer but eventually succumbed to Ebola infection. Nevertheless, the liver and kidney serum markers were markedly reduced in the infected and treated NHPs. In the two longest surviving poly-ICLC- treated NHPs, the day 10 serum EBOV titer was reduced 2.1 and 30 fold respectively.

Enhancement of intranasal vaccination with recombinant chain A ricin vaccine (rRV) in mice by the mucosal adjuvants LTK63 and LTR72.

Intranasal (i.n.) vaccination of mice with three doses of 40 microg of rRV stimulated low anti-ricin ELISA and neutralizing antibody responses, which were only marginally protective against aerosol-delivered 5-10 LD(50) of ricin toxin. To enhance the protection, and to reduce the lung injury of vaccinated mice that survived ricin toxin challenge, the mucosal adjuvant LTK63 or LTR72, two mutants of Escherichia coli LT enterotoxin adjuvant was administered with rRV. The safety of intranasally administered LTR63 was assessed as well. With 4, 2, or l microg of LTR63, the anti-ricin ELISA serum immunoglobulin geometric mean titer (GMT) increased up to 147-, 356-, 493-, and 17-fold for IgG, IgG1, IgG2a, and IgA, respectively. The comparable increases for GMTs of IgG and IgG1 in the presence LTR72 were up to 147-, and 617-fold, respectively. All three dose levels of LTK63 enhanced the ELISA GMTs in the lung lavage up to 192-, 22-, 4-, and 5-fold for IgG, IgG1, IgG2a, and IgA, respectively. Compared to GMT of rRV alone, the serum-neutralizing antibody GMTs for the three dose levels were enhanced up to 11-fold with LTK63. LTK63 augmented the ricin-related lymphoproliferative response of the cultured spleen lymphocytes and of the isolated CD4+ T lymphocytes. In the cultured lymphocytes, LTK63 stimulated predominantly TH1 cytokines. While only 10% of the mice that were vaccinated with rRV survived lethal challenge, in the presence of LTK63 or LTR72, the respective survival rates were augmented to 100%. Compared to the surviving mice vaccinated with rRV alone, the vaccine with LTK63 or LTR72 did not attenuate the extent of the ricin-related lung injury at a single or two time-points, respectively. Safety of LTK63 administration was indicated by the absence of histopathological changes in every organ, including the lungs and in the central nervous systems (CNS) of the mice during the entire 92 days of the study. In the nasal passages of the mice that received LTK63, a transient inflammation occurred without permanent epithelial changes. Administration of three dose levels of the adjuvant in the presence of rRV caused no additional changes. LTK63 and LTR72 both were very effective and safe mucosal adjuvants at all three dose levels employed in these studies. Both significantly enhanced the protection of a marginally effective dose of rRV against aerosol-delivered ricin challenge. LTK63 stimulated cytokines, which could be surrogate markers of efficacy, with human relevance potential. In spite of the better efficacy, rRV with LTK63, or with LTR72, failed to reduce the ricin-related lung injury. Most likely, a larger than suboptimal dose could resolve the lung injury of the vaccinated mice in the presence of a larger dose of the mucosal adjuvant.

Pectin/zein beads for potential colon-specific drug delivery: synthesis and in vitro evaluation.

Novel complex hydrogel beads were prepared from two edible polymers: pectin, a carbohydrate from citrus fruits, and zein, a protein from corn. The pectin/zein complex hydrogels did not swell in physiological environments, but hydrolyzed in the presence of pectinases. An in vitro study showed the capacity of the hydrogels to endure protease attack and residence time variation. The physical and biological properties of the new hydrogels were attributed to molecular entanglement of the two polymers. The pectin networks were stabilized by the bound zein molecules. In turn, the pectin networks shielded the bound zein from protease digestion.

Enhancement of intranasal vaccination in mice with deglycosylated chain A ricin by LTR72, a novel mucosal adjuvant.

Intranasal (i.n.) vaccination with two suboptimal doses of 8 microg of deglycosylated chain A ricin (DGCA) stimulated low anti-ricin ELISA IgG and neutralizing antibody responses and the vaccine was only marginally protective against a lethal ricin toxin aerosol challenge. However, in the presence of 4, 2, or 1 microg of the mucosal adjuvant LTR72, a mutant of the heat-labile enterotoxin of Escherichia coli, the low antibody response and protection were substantially enhanced. In comparison to the vaccination with DGCA alone, vaccination with DGCA in the presence of three dose levels of LTR72, the anti-ricin ELISA serum IgG geometric mean titer (GMT) was increased, respectively, 191-, 572-, and 51-fold for IgG; 91-, 93-, and 60-fold for IgG1; nine-, six-, and two-fold for IgG2a; zero-, two-, and zero-fold for IgA. The three dose levels of the adjuvant enhanced the anti-ricin ELISA immunoglobulin GMTs in the lung lavage 4-, 14-, and 7-fold for IgG; two-, five-, and six-fold for IgG1; two-, six-, and two-fold IgG2a; and zero-, three-, and zero-fold for IgA, respectively. Compared to GMT obtained with the aqueous vaccine (1:2), the 10% serum neutralizing antibody GMT for the three dose levels was enhanced 25-, 60-, and 62-fold, respectively while the 50% neutralizing antibody GMT was enhanced more than 3-, 19- and 10-fold. Only 20% of the mice immunized with DGCA survived the lethal whole body aerosol challenge with 5-10 LD50 ricin toxin, while in the presence of 4, 2, and 1 microg LTR72, 100, 100 and 90% of the vaccinated mice survived, respectively. Safety of administration of two doses of LTR72 is indicated by the absence of histopathological changes in every organ including the lung and the CNS of the mice during the vaccination and during 57 days of the study. In the nasal passages of the mice in the absence of DGCA, LTR72 caused a transient inflammation for less than 7 weeks without permanent epithelial changes. Administration of the adjuvant in the presence of DGCA did not cause additional changes. Compared to the surviving mice vaccinated with DGCA alone, administration of the mucosal adjuvant with DGCA in spite of the better efficacy did not attenuate the lung injury at a single time point (16 days post-challenge). In mice treated with high(er) dose of vaccine, histological examinations during longer observation period rather than at one time point could reveal a different pattern.

Interaction of various pectin formulations with porcine colonic tissues.

Pectins of low and high degrees of esterification, as well as pectin derivatives carrying primary amines, were investigate for gel forming ability with mucosal tissues. The combination of scanning electronic microscopy and small deformation dynamic mechanical studies revealed that pectins with higher net electrical charges are more bioadhesive than the less charged ones. Both the negatively charged pectin formulation, P-25, and the positively charged formulation, P-N, were able to synergize with the mucus to produce rheologically strengthened gels. The highly esterified pectin, P-94, also synergized with the mucosal glycoproteins to form a gel structure via coil entanglements. The ex vivo studies further confirmed the microstructures of mucus gel networks with adsorbed pectins. When incubated with porcine intestinal mucus membrane, P-94 gels were found generally bound to the lumen area, P-25 gels were able to penetrate deeply near the wall area, P-N gels interacted with mucins via electrostatic bonding and dispersed into the whole area from the lumen to the wall. Hence, both P-N and P-94, by enhancing the protective barrier properties of mucus systems, may be useful alternatives for the treatment of mucus related irritation and infection. In drug-delivery systems, P-N and P-25 would deliver incorporated drugs mainly by pectin dissolution, while a diffusion mechanism would release drugs from P-94 gels.

Oral immunization of mice with ricin toxoid vaccine encapsulated in polymeric microspheres against aerosol challenge.

Mucosal (oral) immunization of mice with carrier-delivered ricin toxoid (RT) vaccine was accomplished by one long (7 weeks) or two short (4 weeks) immunization schedules. For the long and short immunization schedule two lots of vaccine were administered prepared with the same procedure but at different occasions. The long schedule consisted of a total of seven doses of 50 microg of vaccine in microencapsulated (lot #108) or aqueous form administered on days 1, 2, 3, 28, 29, 30 and 49. With the short schedule a total of seven or six doses of 25 microg (lot #111) were administered on days 1, 2, 3, 14, 15, 16 and 30, or on 1, 2, 14, 15, 30, 31 and 32, respectively. Mice immunized orally with the long schedule, 50 microg of RT vaccine incorporated into poly-DL-lactide-co-glycolyde (DL-PLG) microspheres (MS) produced serum IgG, IgG2a and IgA ELISA antibodies. All mice immunized with RT in DL-PLG MS (RT-MS) were protected against a lethal ricin aerosol challenge. In contrast, with the same schedule and with the same dose, the aqueous vaccine (RT) failed to stimulate IgG, IgG2a and IgA antibodies, and these mice were not protected against an aerosol ricin toxin challenge. With the shorter immunization scheme, seven doses of 25 microg RT-MS stimulated a significant, though reduced, protection with the microencapsulated, but not with the aqueous vaccine. When the first and second 3-day cycles of the short immunization schedule was reduced to two doses, and the 3-day cycle was administered at the end of the schedule, neither RT-MS nor RT stimulated protection against the challenge. These results indicated that successful oral immunization with RT-MS depended on both the dose and the schedule, consisting of three consecutive days of administration in two cycles, 4 weeks apart. Altering this schedule and the dose, resulted in a reduced protection or no protection at all. Furthermore, under the conditions of this study, the advantage of the microencapsulated RT vaccine over the aqueous vaccine for effective oral immunization was well demonstrated.