Jim E. Eyles

Intra nasal administration of poly-lactic acid microsphere co-encapsulated Yersinia pestis subunits confers protection from pneumonic plague in the mouse

Equivocal doses of soluble, or high molecular weight poly (lactic acid) microsphere co-encapsulated, F1 and V subunit antigens of Yersinia pestis were used to immunize mice intra-nasally. Animals were dosed on day 1 and 7 with 2.724 micrograms V plus 0.956 micrograms F1. Co-encapsulated antigens induced superior systemic and mucosal immunity in comparison with free F1 and V. All of the mice immunized with soluble antigens died shortly after an aerosol challenge consisting of 1 x 10(5) colony-forming units of plague bacteria. In contrast, 66% of the co-encapsulated subunit vaccinees survived this lethal challenge. Humoral immunity to plague was improved further, resulting in 80% protection from challenge, if a relatively high dose (10 micrograms) of cholera toxin B subunit was added to the microsphere suspension prior to intra-nasal delivery. Significantly, by adding 10 micrograms cholera toxin B subunit to the free antigen solution, a 100% post-challenge survival rate was attained. We conclude that in this animal model of pneumonic plague, intra-nasal administration of microgram quantities of Yersinia pestis subunits confers protective immunity, provided the vaccines are microencapsulated or admixed with a strong mucosal adjuvant, such as the cholera toxin B subunit.

Analysis of local and systemic immunological responses after intra-tracheal, intra-nasal and intra-muscular administration of microsphere co-encapsulated Yersinia pestis sub-unit vaccines

Intra-tracheal, intra-nasal and intra-muscular immunisation with admixed Y. pestis sub-units (3 micrograms V, 0.47 microgram F1) or equivalent doses of poly-L-lactide microsphere co-encapsulated antigens was done. Systemic and mucosal responses to F1 and V differed according to immunisation route, and encapsulated status of the sub-units. Irrespective of immunisation site, particulated sub-units stimulated statistically superior primary systemic reactions, with intra-tracheal and nasal microsphere immunisations eliciting superior serum anti-V IgG titres in comparison to intra-muscular injection of free vaccines (p < 0.001 beyond day 8). Pulmonary and nasal delivery of microspheres induced primary serum anti-V IgG titres which were greater (p < 0.039) or equal to (p > 0.056) those after intra-muscular injection of spheres. In terms of serum anti-F1 titres, mice responded best to intra-muscular, and comparatively poorly to intra-nasal immunisations. Intra-tracheal administration of microspheres induced strongest responses in the respiratory tract, dominated by the IgG rather than IgA isotype. An intra-nasal booster immunisation on day 63 potentiated strong local and circulating anti-V IgG titres in microsphere vaccinees. Priming and boosting with free vaccines induced significantly depressed secondary serum anti-F1 titres relative to microsphere immunisations (p < 0.024 at days 78 and 120). In contrast to other priming sites, intra-tracheal instillation of encapsulated vaccines facilitated the induction of IgG antibody to both F1 and V in day 146 broncho-alveolal washings. With the exception of primary responses to F1 in mice immunised intra-tracheally with microspheres, IgG1 was the dominant subclass of anti-F1/V IgG in serum. We conclude that introduction of biodegradable microspheres containing the F1 and V sub-units into to the upper or lower respiratory tract engenders immune responses of a magnitude comparable with that induced by parenteral immunisation, and may present a means of protecting individuals from plague.

Immunological responses to nasal delivery of free and encapsulated tetanus toxoid : studies on the effect of vehicle volume

In light of growing interest in the intranasal route as a non-invasive mode of immunisation, we have investigated the relationship between the volume of liquid instilled into the nasal passages and the development of subsequent immunological responses. Groups of six mice were intranasally immunised with soluble or microsphere encapsulated tetanus toxoid on days 1, 14 and 28 of the experiment. Microsphere suspensions and tetanus toxoid solutions were nasally instilled in two different volumes of buffer (10 or 50 microl). Nasal instillation of microspheres in 10 microl of buffer generated statistically depressed (P<0.001) tertiary serum anti-toxoid IgG responses in comparison to animals immunised with 10 or 50 microl of soluble vaccine, or 50 microl of microsphere suspension. Relative to other treatments, nasal inoculation of encapsulated toxoid suspended in 50 microl generated statistically (P<0.05) superior levels of specific IgG and IgA antibodies in day 49 lung wash samples. When radiolabelled microspheres were nasally instilled into mouse nares in 50-microl volumes of buffer, a significant portion of the dose (48%) entered the lungs (P<0.001), whereas more particles remained in the nasal passages when a smaller (10 microl) volume of suspension was given (P<0.001). These biodistribution and immunological data indicate that to generate optimal bronchopulmonary and systemic responses in concert following nasal administration, microparticulated vaccines should be administered with a delivery device that targets the formulation to distal regions of the nasal passages and the lower respiratory tract.

Generation of protective immune responses to plague by mucosal administration of microsphere coencapsulated recombinant subunits

We have investigated noninvasive immunization to plague. Recombinant subunit antigens, F1 and V from Yersinia pestis, were coencapsulated in biodegradable poly(L100 LD(50s) inhalational challenge with virulent Y. pestis. These data expand on previous findings from our laboratories, providing further insight into the mechanics of safeguarding mice from plague through nasal immunization. Further, these results demonstrate that in a murine model, solid protection from pneumonic plague can be engendered by two intranasal administrations of appropriately formulated recombinant proteins.

Protection studies following bronchopulmonary and intramuscular immunisation with Yersinia pestis F1 and V subunit vaccines coencapsulated in biodegradable microspheres: a comparison of efficacy

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 microg V plus 5 microg 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) MLD(50) 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.

Tissue distribution of radioactivity following intranasal administration of radioactive microspheres

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‐μm‐diameter microspheres. Groups of BALB/c mice received 0.250 mg (47.5 kBq) particles suspended in either 50‐μl or 10‐μl 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 μl) administration vehicle volumes resulted in substantial bronchopulmonary deposition (∼ 50% of administered dose). Intranasal instillation of microspheres suspended in 10‐μl 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 radiolabeled material. No significant concentration of radioactivity within excised gut‐associated lymphoid tissue (GALT) (Peyers patches) was noted. At latter time points we observed, in mice that received the 50‐μl 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.

Biodegradable microparticles with different release profiles: effect on the immune response after a single administration via intranasal and intramuscular routes.

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.

Oral delivery and fate of poly(lactic acid) microsphere-encapsulated interferon in rats.

In the light of previous findings which suggest that particulate material can be absorbed and thence systemically disseminated from the gastrointestinal tract, we have investigated the oral uptake and distribution of soluble and microsphere‐encapsulated radiolabelled interferon‐γ.

The transfer of polystyrene microspheres from the gastrointestinal tract to the circulation after oral administration in the rat.

Factors relating to the transfer of latex microspheres of 0·87 μm mean diameter from the gastrointestinal tract (GIT) to the circulation have been investigated.

Studies on the co-encapsulation, release and integrity of two subunit antigens: rV and rF1 from Yersinia pestis.

In the development of combination or multiple sub‐unit vaccines, determination of the encapsulation, release and integrity of two or more proteins co‐encapsulated within microspheres is an important issue. A new extraction method, which exhibits excellent protein recovery, has been developed which enables samples to be used for sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) and subsequent measurement of individual antigens encapsulated within microspheres. Using the new method, the protein loading of poly‐(L‐lactide) microspheres co‐encapsulating two plague sub‐unit antigens was found to be 1.22% (w/w) for recombinant V antigen (rV) and 1.24% (w/w) for recombinant F1 (rF1) by SDS‐PAGE. The total protein loading was 2.49% (w/w) by bicinchoninic acid assay. The individual release of the two subunit antigens from the co‐encapsulated microspheres was determined by SDS‐PAGE analysis and rF1 was found to have a higher burst release than rV. The integrity and immunological activity of both rF1 and rV antigens was shown to be unaffected by the microencapsulation process.