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1.  Pulmonary Immunization of Guinea Pigs with Diphtheria CRM-197 Antigen as Nanoparticle Aggregate Dry Powders Enhance Local and Systemic Immune Responses 
The AAPS Journal  2010;12(4):699-707.
This study establishes the immune response elicited in guinea pigs after pulmonary and parenteral immunizations with diphtheria CRM-197 antigen (CrmAg). Several spray-dried powders of formalin-treated/untreated CrmAg nanoaggregates with L-leucine were delivered to the lungs of guinea pigs. A control group consisting of alum with adsorbed CrmAg in saline was administered by intramuscular injection. Animals received three doses of powder vaccines containing 20 or 40 μg of CrmAg. The serum IgG titers were measured for 16 weeks after the initial immunization; IgA titers were measured at the time of sacrifice in the broncho-alveolar lavage fluid. Further, toxin neutralization tests in naïve guinea pigs were performed for a few select serum samples. Histopathology of the lung tissues was conducted to evaluate inflammation or injury to the lung tissues. While the highest titer of serum IgG antibody was observed in guinea pigs immunized by the intramuscular route, those animals immunized with dry powder formulation by the pulmonary route, and without the adjuvant alum, exhibited high IgA titers. A pulmonary administered dry powder, compared to parenteral immunization, conferred complete protection in the toxin neutralization test. Mild inflammation was observed in lung tissues of animals receiving dry powder vaccines by the pulmonary route. Thus, administering novel CrmAg as dry powders to the lungs may be able to overcome some of the disadvantages observed with the existing diphtheria vaccine which is administered by the parenteral route. In addition, these powders will have the advantage of eliciting a high mucosal immune response in the lungs without using traditional adjuvants.
PMCID: PMC2976983  PMID: 20878294
CRM-197; diphtheria vaccine; dry powder formulation; guinea pigs; pulmonary delivery
2.  Pulmonary Immunization Using Antigen 85-B Polymeric Microparticles to Boost Tuberculosis Immunity 
The AAPS Journal  2010;12(3):338-347.
This study aims to evaluate immunization with polymeric microparticles containing recombinant antigen 85B (rAg85B) delivered directly to the lungs to protect against tuberculosis. rAg85B was expressed in Escherichia coli and encapsulated in poly(lactic-co-glycolic acid) microparticles (P-rAg85B). These were delivered as dry powders to the lungs of guinea pigs in single or multiple doses of homologous and heterologous antigens. Bacille Calmette–Guérin (BCG) delivered subcutaneously was employed as the positive control and as part of immunization strategies. Immunized animals were challenged with a low-dose aerosol of Mycobacterium tuberculosis (MTB) H37Rv to assess the extent of protection measured as reduction in bacterial burden (CFU) in the lungs and spleens of guinea pigs. Histopathological examination and morphometric analysis of these tissues were also performed. The heterologous strategy of BCG prime–P-rAg85B aerosol boosts appeared to enhance protection from bacterial infection, as indicated by a reduction in CFU in both the lungs and spleens compared with untreated controls. Although the CFU data were not statistically different from the BCG and BCG–BCG groups, the histopathological and morphometric analyses indicated the positive effect of BCG–P-rAg85B in terms of differences in area of tissue affected and number and size of granulomas observed in tissues. P-rAg85B microparticles appeared to be effective in boosting a primary BCG immunization against MTB infection, as indicated by histopathology and morphometric analysis. These encouraging observations are relevant to boosting adults previously immunized with BCG or exposed to MTB, commonly the case in the developing world, and should be followed by further assessment of an appropriate immunization protocol for maximum protection.
PMCID: PMC2895436  PMID: 20422340
antigen 85B; BCG; boost immunization; microparticles; pulmonary delivery
3.  Immunization of Guinea Pigs with Novel Hepatitis B Antigen as Nanoparticle Aggregate Powders Administered by the Pulmonary Route 
The AAPS Journal  2010;12(3):330-337.
Novel nanoparticle-aggregate formulations containing recombinant hepatitis B surface antigen (rHBsAg) were administered to the lungs of guinea pigs and antibodies generated to this antigen evaluated. Preparations of dry powders of: (a) rHBsAg encapsulated within poly(lactic-co-glycolic acid) (PLGA)/polyethylene glycol (PEG) nanoparticles (antigen nanoparticles, AgNSD), (b) rHBsAg in a physical mixture with blank PLGA/PEG nanoparticles (antigen nanoparticle admixture (AgNASD), and (c) rHBsAg encapsulated in PLGA/PEG nanoparticles plus free rHBsAg (antigen nanoparticles and free antigen), were generated by spray drying with leucine. Control groups consisted of alum with adsorbed rHBsAg (AlumAg); reconstituted suspensions of spray-dried rHBsAg-loaded PLGA/PEG nanoparticles with leucine; and rHBsAg-loaded PLGA/PEG nanoparticles (AgN). Control preparations were administered by intramuscular injection; AgN was also spray instilled into the lungs. The IgG titers were measured in the serum for 24 weeks after the initial immunization; IgA titers were measured in the bronchio-alveolar lavage fluid. While the highest titer of serum IgG antibody was observed in guinea pigs immunized with AlumAg administered by the IM route, animals immunized with powder formulations via the pulmonary route exhibited high IgA titers. In addition, guinea pigs immunized with AgNASD via the pulmonary route exhibited IgG titers above 1,000 mIU/ml in the serum (IgG titers above 10 mIU/ml is considered protective). Thus, the disadvantages observed with the existing hepatitis B vaccine administered by the parenteral route may be overcome by administering them as novel dry powders to the lungs. In addition, these powders have the advantage of eliciting a high mucosal immune response in the lungs without traditional adjuvants.
PMCID: PMC2895445  PMID: 20419360
antibody titer; dry powder formulation; hepatitis B vaccine; pulmonary delivery
4.  Screening for Potential Adjuvants Administered by the Pulmonary Route for Tuberculosis Vaccines 
The AAPS Journal  2009;11(1):139-147.
Tuberculosis (TB) infects one third of the world’s population, and new infections occur at a rate of 1/s. Better vaccines are needed than the live mycobacterium Bacille Calmette-Guérin (BCG). Alveolar macrophages (AMΦs) play a central role in pulmonary manifestations of TB. Targeting immunomodulators to AMΦs, the first line of defense against Mycobacterium tuberculosis (Mtb), may initiate a potent cell-mediated immune response. Muramyl dipeptide (MDP) and trehalose dibehenate (TDB) have elicited strong immune response when delivered to the lungs as aerosols. AMΦs show toxicity in response to some immunomodulators. The objective of this work was to screen the immunomodulators MDP and/or TDB encapsulated in microparticles (MPs) and to evaluate certain indicators of toxicity in human AMΦ-like cells. Poly(lactide-co-glycolide) (PLGA) MPs containing MDP and/or TDB were prepared by spray-drying. The morphology, particle size distribution, and immunomodulator encapsulation efficiency of MPs were examined. THP-1 cells were exposed to these MPs for 24 h and characteristics of cell morphology, tumor necrosis factor-alpha (TNF-α) release, lactate dehydrogenase (LDH), N-acetyl-β-d-glucosaminidase (NAG) and alkaline phosphatase (ALP) activity in AMΦ culture supernatants were measured. MTT assay was used to assess the viability of cells. Spray-drying produced low-density MPs having volume median diameters between 4 and 6 μm as measured by laser diffraction and projected area diameter between 3 and 5 μm calculated by microscopy. More TNF-α was produced by THP-1 cells exposed to MPs composed of PLGA-MDP or PLGA alone than PLGA-TDB. LDH release following exposure to MPs of PLGA-MDP and PLGA alone was greater than controls. NAG release was higher following exposure to MPs of PLGA alone or PLGA-MDP 0.1% than PLGA-TDB (0.1% and 1.0%). Cells remained viable after exposure to MPs as per MTT assay. PLGA-MDP MPs demonstrated statistically elevated indicators of biochemical responses in cell culture compared to PLGA-TDB MPs, but the extent of their potential to elicit adverse effects in vivo must be studied independently.
PMCID: PMC2664889  PMID: 19277872
adjuvants; cytotoxicity; immunostimulant; microparticles; pulmonary

Results 1-4 (4)