Development of a single-shot subunit vaccine for HIV-1 .4. Optimizing microencapsulation and pulsatile release of MN rgp120 from biodegradable microspheres

The administration of a subunit vaccine (e.g. gp120) for acquired immunodeficiency syndrome (AIDS) can be facilitated by a single shot vaccine that mimics repeated immunizations. To achieve this type of vaccine, we developed poly(lactic-coglycolic acid) (PLGA) microspheres that provide a pulsatile release of gp120. Studies of a water-in-oil-in-water microencapsulation process with either methylene chloride or ethyl acetate as the polymer solvent revealed that the encapsulation efficiency was greatly enhanced by increasing the kinematic viscosity of the polymer phase via a reduction in temperature (similar to 0 degrees C) and an increase in polymer concentration. Excess methylene chloride (1.5% v/v) in the second emulsion reduced the encapsulation efficiency while excess ethyl acetate (9% v/v) in the second emulsion was necessary for microsphere formation. The optimization of process variables allowed for complete encapsulation of the gp120 (100% efficiency), Drying of the microspheres had a significant impact on the initial burst of protein, and nitrogen drying provided the lowest burst in each case. By maintaining a low aqueous phase content in the initial emulsion, the density of the inner emulsion water droplets in the microspheres was reduced requiring significant polymer erosion to occur prior to protein release, The protein was released under physiological conditions in two discrete phases: an initial burst released over the first day and after several weeks or months, a second burst of protein was released. The second burst of protein was dependent upon the PLGA inherent viscosity and lactide/glycolide ratio (bulk erosion). The initial gp120 released under physiological conditions as well as the gp120 extracted from the microspheres maintained its native conformation as measured by a variety of physicochemical methods. Thus, PLGA or PLA microsphere formulations were developed to provide a second burst of gp120 to mimic immunizations at 1, 2, 3 or 6 months. A single administration Vaccine for gp120 may then include two or more microsphere formulations resuspended with soluble gp120 (primary immunization) to provide three immunizations or more over the course of 1 year.