BALB/c mice were immunised with inactivated Francisella tularensis live vaccine strain (LVS) and the level of protection afforded against aerosol challenge with virulent strains of F. tularensis ascertained. Intramuscular (IM) injection of inactivated LVS with an aluminium-hydroxide-based adjuvant-stimulated IgG1-biased LVS-specific antibody responses and afforded no protection against aerosol challenge with subspecies holarctica (strain HN63). Conversely, IM injection of inactivated LVS adjuvanted with preformed immune-stimulating complexes (ISCOMS) admixed with immunostimulatory CpG oligonucleotides afforded robust protection against aerosol-initiated infection with HN63. However, despite a significantly extended time-to-death relative to naïve controls, the majority of mice immunised with the most potent vaccine formulation were not protected against a low-dose aerosol challenge with subspecies tularensis (strain Schu S4). These data indicate that parenterally administered non-living vaccines can be used for effective immunisation against aerosol challenges with subspecies holarctica, although not high virulence strains of F. tularensis.

Certain toll-like receptor (TLR) agonists, e.g. CpG DNA, can be used as potent vaccine 'adjuvants'. It is known that some sequences of single stranded (ss) RNA stimulate proinflammatory and antiviral responses following interaction with TLR 7 and 8. We have encapsulated ovalbumin (OVA) in the presence and absence of polyuridylic acid (poly-U) inside polylactide microparticles. In comparison to microparticles containing only OVA, bulk cultures of bone marrow-derived plasmacytoid and myeloid dendritic cells produced more (P<0.05) IL-12 and interferon (IFN)-alpha when stimulated with microparticles containing OVA and poly-U. Subcutaneous injection of comicroencapsulated OVA and poly-U resulted in statistically elevated levels of serum anti-OVA IgG1 (P<0.05 versus naïve mice). Conversely, anti-OVA IgG1 levels in C57 BL6 mice immunised with OVA loaded microparticles (without RNA) were statistically indifferent to naïve animals. Furthermore, injection of coencapsulated OVA and poly-U resulted in (P<0.05) greater numbers of OVA specific IFN-gamma secreting T-cells as compared with mice injected with OVA loaded microparticles. A similar trend was seen in mice immunised with OVA loaded microparticles decorated with CpG or solutions of admixed OVA and CpG (P<0.05). These data demonstrate, for the first time, that appropriately formulated ssRNA can act as a potent adjuvant and modulator of adaptive immunological responses.

Intratracheal delivery of aerosolized monoclonal antibodies with specificity for Yersinia pestis LcrV and F1 antigens protected mice in a model of pneumonic plague. These data support the utility of inhaled antibodies as a fast-acting postexposure treatment for plague.

ABSTRACT Burkholderia pseudomallei , the causative agent of melioidosis, is a gram-negative bacterium which can cause either chronic infections or acute lethal sepsis in infected individuals. The disease is endemic in Southeast Asia and northern Australia, but little is known about the mechanisms of protective immunity to the bacterium. In this study, we have developed a procedure to utilize dendritic cells in combination with CpG oligodeoxynucleotides as a vaccine delivery vector to induce protective immune responses to various strains of B. pseudomallei . Our results show that strong cell-mediated immune responses were generated, while antibody responses, although low, were detectable. Upon virulent challenge with B. pseudomallei strain K96243, NCTC 4845, or 576, animals immunized with dendritic cells that were pulsed with heat-killed K96243 and matured in the presence of CpG 1826 showed significant levels of protection. These results show that a vaccine strategy that actively targets dendritic cells can evoke protective immune responses.

Protection against virulent plague challenge by the parenteral and aerosol routes was afforded by a single administration of microencapsulated Caf1 and LcrV antigens from Yersinia pestis in BALB/c mice. Recombinant Caf1 and LcrV were individually encapsulated in polymeric microspheres, to the surface of which additional antigen was adsorbed. The microspheres containing either Caf1 or LcrV were blended and used to immunise mice on a single occasion, by either the intra-nasal or intra-muscular route. Both routes of immunisation induced systemic and local immune responses, with high levels of serum IgG being developed in response to both vaccine antigens. In Elispot assays, secretion of cytokines by spleen and draining lymph node cells was demonstrated, revealing activation of both Th1 and Th2 associated cytokines; and spleen cells from animals immunised by either route were found to proliferate in vitro in response to both vaccine antigens. Virulent challenge experiments demonstrated that non-invasive immunisation by intra-nasal instillation can provide strong systemic and local immune responses and protect against high level challenge. Microencapsulation of these vaccine antigens has the added advantage that controlled release of the antigens occurs in vivo, so that protective immunity can be induced after only a single immunising dose.

We have carried out an in vitro investigation into the mechanism by which microencapsulation enhances the immunogenicity of recombinant protective antigen (rPA) from Bacillus anthracis. Murine bone marrow derived dendritic cells (DC) were cocultured with soluble and microencapsulated rPA and the activation status of the cells monitored using FACS. As compared with soluble rPA, it was found that coculture of DC with rPA-loaded microparticles stimulated higher levels of MHC II, CD54, CD80 and CD86 expression (p<0.05). To investigate the longevity of antigen presentation, splenocytes from naïve mice were pulsed overnight with (3)H-thymidine following 1, 3 or 6 days coculture with DC transiently exposed to soluble or microencapsulated rPA. Splenocyte proliferation was more pronounced, and continued for a more protracted period, if the 'feeder' cells were exposed to microencapsulated antigen as compared with soluble antigen or 'empty' microspheres. To this end, our findings indicate that microsphere uptake increases the surface expression of MHC and co-stimulatory molecules on DC and can facilitate prolonged presentation of antigen to T-cells, possibly by acting as an intracellular depot.

ESAT-6 from Mycobacterium tuberculosis is an important T-cell antigen for cell-mediated immunity in the early phase of tuberculosis infection. Since the lung is the organ in which infection is initiated, immune responses in the lung play a significant role in restricting the initial infection with M. tuberculosis. The aim of the present study was to assess whether efficient cell-meditated immune responses in the lung and draining mediastinal lymph nodes could be stimulated by pulmonary administration of ESAT-6 encapsulated in poly(lactide) (PLA) microspheres. BALB/c mice were immunised intranasally on days 1, 28 and 56 with 2 μg microencapsulated ESAT-6. Cellular responses in the lungs, spleen and mediastinal lymph nodes (MLN) were characterised using ELISPOT and proliferation assays. Fluorescence activated cell sorting (FACS) was used to assess the expression of CD44 on CD4+ and CD8+ cells derived from the MLN of immunised animals. For comparison, groups of mice were immunised intranasally with soluble ‘free’ ESAT-6 or intramuscularly with ESAT-6 in Alhydrogel™. Intranasal instillation of microencapsulated ESAT-6 induced greatest numbers of ESAT-6 specific IFN-γ and IL-4 secreting cells in the lung and MLN (P<0.05). Similarly, ESAT-6 specific recall responses were strongest following intranasal immunisation of mice with microsphere encapsulated antigen (P<0.05). FACS demonstrated a higher proportion of T cells expressing CD44 in the MLN from mice immunised intranasally with microencapsulated ESAT-6. These data support the notion that the immune system is compartmentalised and responses are often strongest in compartments proximal to the site of vaccine application. Furthermore, our data indicate that, for efficient activation of cell-mediated responses, antigens must be presented to the immune system in an appropriate formulation.

We have investigated immunological responses in BALB/c mice following transcutaneous (TC) delivery of fraction 1 (F1) and V subunits from Yersinia pestis in conjunction with an enterotoxin-derived adjuvant (cholera toxin, CT). It was found that two or more TC applications of F1 and V subunits (admixed with cholera toxin) served to elicit significant levels of anti-F1 and V antibodies in the serum of immunised mice. IL-6 secretion from cultured splenocytes derived from immunised mice indicated that a single TC application of F1 and V subunits (admixed with cholera toxin) conferred a cell-mediated response. As compared with intranasal or direct intradermal injection of F1 and V, the numbers of F1/V-specific antibody-forming cells in the spleens of animals immunised by TC application of F1 and V (admixed with CT) was relatively low. It was noted that TC application of F1 and V admixed with CT was very effective for priming responses that were boosted by intranasal or intradermal routes. Similarly, it was found that TC application of F1 and V admixed with CT could be used to efficiently boost pre-existing responses engendered by intradermal injection or intranasal instillation of F1 and V. In order to assess if TC application of F1 and V admixed with CT could protect experimental animals from plague, immunised mice were injected with a virulent strain of Y. pestis. It was found that two TC applications of F1 and V admixed with CT conferred only limited protection against 102MLDs. However, three TC applications of F1 and V admixed with CT conferred solid protection against 102MLDs. Hence we have shown, for the first time, that TC application of F1 and V admixed with CT can protect animals against challenge with a virulent strain of plague causing bacteria. These data suggest that transcutaneous immunisation may be a simple and non-invasive method for immunising individuals against plague.

BACKGROUND Transcutaneous immunization (TCI) is a needle-free technique that delivers antigens and adjuvants to potent epidermal immune cells. To address critical unmet needs in biodefense against anthrax, we have designed a novel vaccine delivery system using a dry adhesive patch that simplifies administration and improves tolerability of a subunit anthrax vaccine. METHODS Mice and rabbits were vaccinated with recombinant protective antigen of Bacillus anthracis and the heat-labile toxin of Escherichia coli. Serologic changes, levels of toxin-neutralizing antibodies (TNAs), and pulmonary and nodal responses were monitored in the mice. A lethal aerosolized B. anthracis challenge model was used in A/J mice, to demonstrate efficacy. RESULTS The level of systemic immunity and protection induced by TCI was comparable to that induced by intramuscular vaccination, and peak immunity could be achieved with only 2 doses. The addition of adjuvant in the patch induced superior TNA levels, compared with injected vaccination. CONCLUSIONS Anthrax vaccine patches stimulated robust and functional immune responses that protected against lethal challenge. Demonstration of responses in the lung suggests that a mechanism exists for protection against challenge with aerosolized anthrax spores. A formulated, pressure-sensitive, dry adhesive patch, which is stable and can be manufactured in large scale, elicited comparable immunoglobulin G and TNA responses, suggesting that an anthrax vaccine patch is feasible and should advance into clinical evaluation.

In vitro studies using dendritic cells have identified that microencapsulated antigens are taken up and processed differently as compared with soluble proteins, and these findings have been reviewed. Similarly, in vivo, it is evident that microencapsulated materials have different properties in terms of uptake and trafficking. Intranasal (IN) instillation of encapsulated protective antigen resulted in a significant increase in the percentage of activated CD4+ and B-cells in the spleens of immunised mice, whereas IN instillation of soluble antigen failed to do so. This corroborates earlier findings concerning the uptake and trafficking of microparticles following bronchopulmonary administration. These data support the tenet that microencapsulation serves to modify the uptake, trafficking and processing of antigens.

Activation of cells, in primary culture, by nanospheres containing antigen has been investigated. Single cell suspensions of spleen cells from primed and naıïve animals were cocultured with escalating quantities of soluble tetanus toxoid (TT) or TT encapsulated within nanospheres fabricated from poly(lactide-co-glycolide) (PLGA). Concomitantly, spleen cells were also cultured in the presence of ‘empty’ PLGA nanospheres that contained no TT. Nanospheres loaded with antigen were found to elicit increased proliferation of splenocytes from preimmunised mice in comparison to free antigen during coculture at equivalent doses of immunogen (at low and intermediate doses). Interestingly, cellular proliferation was abolished if B-cells were removed from the splenocyte cultures. Production of IFN-γ and IL-6 was increased, for formulated as compared to free antigen, in microcultures from both naıïve and pre-immunised animals. Secretion of IFN-γ or IL-6 was not observed when primed or naıïve spleen cells were stimulated with ‘empty’ polymeric spheres. Some unspecific cytotoxicity was detected if cells were cocultured with high concentrations of PLGA particles, although toxic effects were not seen at concentrations where maximum levels of cytokine secretion and cellular proliferation were recorded. These cell culture data indicate that, at least in this in vitro model, nanoparticulate TT is able to elicit cytokine production that is probably consistent with increased stimulation. This mechanism is likely to be distinct from non-specific effects caused by components of the delivery vehicle itself.

ABSTRACT Existing licensed anthrax vaccines are administered parenterally and require multiple doses to induce protective immunity. This requires trained personnel and is not the optimum route for stimulating a mucosal immune response. Microencapsulation of vaccine antigens offers a number of advantages over traditional vaccine formulations, including stability without refrigeration and the potential for utilizing less invasive routes of administration. Recombinant protective antigen (rPA), the dominant antigen for protection against anthrax infection, was encapsulated in poly- l -lactide 100-kDa microspheres. Alternatively, rPA was loosely attached to the surfaces of microspheres by lyophilization. All of the microspheric formulations were administered to A/J mice with a two-dose schedule by either the intramuscular route, the intranasal route, or a combination of these two routes, and immunogenicity and protective efficacy were assessed. An intramuscular priming immunization followed by either an intramuscular or intranasal boost gave optimum anti-rPA immunoglobulin G titers. Despite differences in rPA-specific antibody titers, all immunized mice survived an injected challenge consisting of 10 3 median lethal doses of Bacillus anthracis STI spores. Immunization with microencapsulated and microsphere-associated formulations of rPA also protected against aerosol challenge with 30 median lethal doses of STI spores. These results show that rPA can be encapsulated and surface bound to polymeric microspheres without impairing its immunogenicity and also that mucosal or parenteral administration of microspheric formulations of rPA efficiently protects mice against both injected and aerosol challenges with B. anthracis spores. Microspheric formulations of rPA could represent the next generation of anthrax vaccines, which could require fewer doses because they are more potent, are less reactogenic than currently available human anthrax vaccines, and could be self-administered without injection.

We have investigated intranasal delivery of novel vaccines for plague, based on poly-L-lactide (PLLA) microencapsulated recombinant V antigen (rV) of Yersinia pestis. Microspheres containing rV alone or co-encapsulated with the cytokines IFN-gamma, IL-4 or IL-6 were administered in a two-dose regimen and antibody responses and protective efficacy were monitored. All treatment groups stimulated high rV-specific antibody titres in serum, predominantly of the IgG1 isotype, which were maintained over several months. There was evidence of both IgG and IgA responses in lung samples from all groups. Formulations based on rV antigen alone or rV co-encapsulated with IL-6 provided complete protection against systemic challenge with Y. pestis strain GB; however protective efficacy was impaired by co-encapsulating either IFN-gamma or IL-4 with rV.

Summary We hypothesized that the addition of polymer to the surface of liposome/DNA complexes may potentially enhance in vivo delivery of plasmid DNA to antigen‐presenting cells and thereby facilitate enhanced immune responses to encoded protein. BALB/c mice were immunized subcutaneously or intramuscularly three times with a total of 50 µg of the plasmid pRc/CMV‐HBs(S) (ayw subtype) encoding for the hepatitis B surface antigen. We measured transgene‐specific total immunoglobulin G (IgG), IgG2a, IgG2b and IgG1 antibody responses as well as splenocyte and T‐cell proliferation and cytokine production upon re‐stimulation following immunization. Modification of lipid/DNA complexes by the polymer precipitation method used here for the addition of poly( d , l ‐lactic acid) was found to be consistently and significantly more effective than either unmodified liposomal DNA or naked DNA in eliciting transgene‐specific immune responses to plasmid‐encoded antigen when administered by the subcutaneous route. In addition, the polymer‐modified formulations delivered by this route were more effective than naked DNA delivered by the intramuscular route in inducing antibody responses ( n =5, P <0·03). Our observations provide ‘proof of principle’ for the use of these multicomponent formulations, which offer potential for manipulation and increased transfection efficiency in vivo for the purposes of genetic immunization.

The aim of this study was to increase understanding of the kinetics of microparticle distribution and elimination following intranasal application. To do this we investigated the in-vivo distribution of radioactivity following intranasal instillation of scandium-46 labelled styrene-divinyl benzene 7-microm-diameter microspheres. Groups of BALB/c mice received 0.250 mg (47.5 kBq) particles suspended in either 50-microL or 10-microL volumes of phosphate buffered saline. The in-vivo distribution of radioactivity was influenced by the volume of liquid that was used to instil the microsphere suspension. Comparatively large (50 microL) administration vehicle volumes resulted in substantial bronchopulmonary deposition (approximately 50% of administered dose). Intranasal instillation of microspheres suspended in 10-microL volumes tended to restrict particle deposition initially to the nasal cavity. For both administration vehicle volumes tested, the radioactivity per unit mass of excised nasal-associated lymphoid tissue (NALT) was found to be consistently elevated relative to other tissues. This corroborates the findings of other workers who have previously identified NALT as an active site of microparticle accumulation following intranasal application. Elimination via the alimentary canal was the principal fate of intranasally applied radiolabelled material. No significant concentration of radioactivity within excised gut-associated lymphoid tissue (GALT) (Peyer's patches) was noted. At latter time points we observed, in mice that received the 50-microL volume particle suspension nasally, accumulation of potentially relevant quantities of radioactivity in the liver (0.3% after 576 h) and spleen (0.04% after 576 h). Thus, our data corroborate the notion that epithelial membranes in the lung are probably less exclusive to the entry of microparticulates into systemic compartments than are those mucosae in the gastrointestinal tract or nasopharynx. This effect may contribute to the effectiveness of pulmonary delivered antigen-loaded microparticles as humoral immunogens.

With a view to developing improved mucosal immunisation strategies, we have quantitatively investigated the uptake of fluorescent polystyrene carboxylate microspheres (1.1 microm diameter), using histology and fluorescence-activated cell sorting, following intranasal delivery to BALB/c mice. To qualify these biodistribution data, antigen specific memory and effector responses in the spleens of mice immunised nasally with Yersinia pestis V antigen loaded poly(lactide) (PLA) microspheres (1.5 microm diameter) were assessed at 4, 7 and 11 days. Irrespective of administration vehicle volume (10 or 50 microl), appreciable numbers of fluorescent microspheres were detected within nasal associated lymphoid tissues (NALT) and draining cervical lymph nodes. Nasal administration of the particles suspended in 50 microl volumes of phosphate-buffered saline (PBS) served to deposit the fluorescent microspheres throughout the respiratory tract (P<0.05). In these animals, appreciable particle uptake into the mediastinal lymph node was noted (P<0.05). Also, spleens removed from mice 10 days after fluorescent particle application contained significantly more microspheres if the suspension had been nasally instilled using a 50 microl volume (P<0.05). Appreciable memory (and effector from day 7) responses were detected in mediastinal lymph nodes removed from mice immunised nasally with 50 microl volumes of microparticulated or soluble V antigen. Immunological responses in splenic tissue removed 7 days after intranasal immunisation corroborated the thesis that the spleen can act as an inductive site following bronchopulmonary deposition of particulated antigen: upon exposure to V in vitro, splenic T-cells from mice nasally immunised with 50 microl volumes of microspheres incorporated statistically greater (P<0.05) quantities of [3H]thymidine into newly synthesised DNA than did T-cells from cohorts nasally immunised with 50 microl volumes of V in solution. Similarly, significant numbers of anti-V IgG secreting cells were only detected in spleens from mice immunised intramuscularly or nasally with microparticles. These immunological and biodistribution data support the tenet that, following an appropriate method of mucosal delivery, microparticles can translocate to tissues in the systemic compartment of the immune system and thence provoke immunological reactions therein.

Plague is an extremely virulent and potentially lethal infection caused by the bacterium Y. pestis. The current vaccine used to immunise against plague often fails to engender solid (100%) protection against inhalational infection with Y. pestis. Similarly, logistical factors favour the development of non-parenteral immunisation protocols to counter plague. Recently an improved parenteral vaccination strategy for plague, based on the recombinant subunit approach, has entered clinical trails. The Yersinia pestis subunit antigens (F1 and V) have been successfully incorporated into novel vaccine delivery systems such as biodegradable microspheres composed of poly-L-(lactide) (PLLA). Intranasal and intratracheal administration of PLLA microencapsulated F1 and V serves to protect experimental animals from inhalational and subcutaneous challenge with virulent Y. pestis bacilli. Liposomes have also been used to improve the immunogenicity of intranasally administered Y. pestis antigens, and the effectiveness of this approach to plague immunisation has been evaluated. Tetanus and diphtheria still cause many deaths worldwide. The maintenance of protective immunity to diphtheria and tetanus requires booster injections of the currently licensed toxoid vaccines. Consequently, many people remain unprotected. Improved coverage may well result from the development of effective non-invasive vaccines that could be readily distributed and potentially self-administered. To this end, the intranasal and inhalational routes of administration have been extensively investigated. Tetanus and diphtheria toxoids have been delivered intranasally to experimental animals using a wide variety of adjuvants (enterotoxin derivatives), penetration enhancers (cyclodextrins, bile salts, surfactants, cationic polymers) and delivery systems (microspheres and liposomes). As compared with parenteral vaccination, nasal immunisation has been shown favourably effective in small animal models, and a limited number of early phase clinical trails. As a caveat to this, adjuvantisation of toxoid/subunit molecules appears to be a requisite for elicitation of appreciable immunological responses, following nasal administration of acellular immunogens. Testing in larger animal models and humans is needed to ascertain if the promising results obtained in rodents can be reciprocated without compromising safety.

We have compared the ability of intramuscularly and intratracheally administered recombinant F1 and V subunit antigens to safeguard mice from a lethal systemic challenge with plague. The combined subunits (1 mu g V plus 5 mu g F1) were inoculated either in the 'free' state as a solution, or entrapped within microspheres composed of a biodegradable polyester (Poly-L-lactide), on day 1 and 60 of the experiment. In comparison to the other regimens, introduction of microsphere suspensions into the respiratory tract resulted in statistically elevated levels of specific immunoglobulins in day 82 lung wash samples. A subcutaneous challenge with virulent Yersinia pestis bacteria on day 137, equivalent to more than 10(5) mouse LD(50)s, was comparatively well tolerated by all subunit treatment groups (with survival rates between 66 and 90%). In contrast, 80% of the mice injected intramuscularly with soluble F1 and V were defeated by a 10(7) MLD50 subcutaneous challenge, whereas the group immunised intramuscularly with microparticles were significantly better protected (p < 0.1) with 50% survival. Similarly, mice immunised intratracheally with microparticles were significantly better safeguarded (56% survival) compared with the group immunised with soluble subunits intramuscularly (p < 0.01). Soluble sub-units delivered intratracheally afforded 33% protection against 10(7) MLD(50)s These data indicate that bronchopulmonary administration of microsphere co-encapsulated recombinant F1 and V antigens elicits a similar level of protective immunity against systemic plague infection as that evoked by injecting co-encapsulated subunits into the muscle. Such findings corroborate the thesis that introduction of appropriately formulated F1 and V subunits into the respiratory tract may be an alternative to parenteral immunisation schedules for protecting individuals from plague. (C) 2000 Elsevier Science Ltd. All rights reserved.

In the development of single-dose microparticulate vaccines, identification of the type of protein release profile required to elicit high and sustainable immune responses is important. Microparticles exhibiting different protein release profiles (continuous, pulsatile and plateau) were made by solvent evaporation or solvent extraction methods from biodegradable polymers encapsulating the model antigen, bovine serum albumin (BSA). The immune responses obtained after a single intranasal or intramuscular administration of microparticles were determined, and also after a subcutaneous boost after 11 months. Microparticles were manufactured with acceptable protein loading and average volume size ranging from 1 to 10 microm. The integrity of BSA extracted and released from microparticles after 2 months incubation was retained. Microparticulate preparations administered by either intranasal or intramuscular routes, evoked rapid, high titre and long-lived (up to 11 months after priming) specific serum IgG responses which were significantly greater than for free BSA. The type of protein release from microparticles had no significant effect on the systemic immune responses. Interestingly, a formulation exhibiting a plateau-release profile was the only microparticulate system capable of inducing significantly greater IgA responses than free BSA after intranasal immunization. This study shows the benefit of microencapsulation in inducing high and long-lasting systemic immune responses after a single dose by both parenteral and mucosal delivery. We conclude that of the microparticles tested, the longevity and magnitude of humoral responses was not effected by the type of in-vitro protein release profile.