Development of a self-boosting rabies vaccine using PULSED microparticles

Introduction: : Rabies is the most lethal viral infection known to mankind, with a fatality rate of nearly 100% in the absence of treatment. Fortunately, post-exposure prophylaxis (PEP), consisting of rabies immune globulin and a multi-dose vaccination regimen, reduces mortality to less than 1%. Nevertheless, rabies still causes approximately 60,000 deaths each year. These PEP-preventable deaths, which almost exclusively occur in low-resource and rural areas, are due to a lethal combination of late-onset symptoms that only appear only after the disease has progressed to the point where treatment is ineffective, an onerous dosing schedule requiring up to four injections administered over a period of four weeks, and a lack of convenient access to medical care. In this study, we present the development of a novel single-injection rabies vaccine, achieved by engineering antigen-loaded microparticles that release their cargo in time-controlled pulses that mimic a conventional PEP dosing schedule to confer protection without the need for frequent visits for medical care.

Materials and

Methods: : We leveraged the Particles Uniformly Liquified and Sealed to Encapsulate Drug (PULSED) technique that we have previously developed to fabricate microstructured particles composed of poly(lactic-co-glycolic acid) (PLGA) with varying molecular weight, terminal groups, and copolymer ratios. These particles were subsequently loaded with fluorescently labeled dextran (10 kDa) and their release kinetics were evaluated both in vitro and in vivo. PULSED particle compositions that released at times matching clinical rabies PEP vaccination schedules were then selected for further study. In parallel, we screened more than 400 excipient formulations within microparticles, assessing their ability to maintain the immunity-conferring structure of the inactivated rabies vaccine stable through particle fabrication and release at 37°C. To do this, microparticles were mechanically fractured after incubation in release media consisting of PBS, albumin, and glucose and analyzed using a conformation-specific enzyme-linked immunosorbent assay (ELISA) known to correlate with the ability to confer protection. Rat immunization studies were performed to compare the neutralizing antibody titers obtained from our single-injection vaccine to those achieved using a typical, multi-dose vaccination regimen using a Rapid Fluorescent Focus Inhibition Test (RFFIT).

Results, Conclusions, and Discussions:: The need for a controlled-release solution was confirmed by comparing the neutralizing antibody levels generated with a two-injection vaccination to a single injection of vaccine at higher doses. Even when the cumulative dose was increased more than 20-fold, a single dose was still unable to elicit comparable neutralizing antibody titers in rats (Fig. 1A). Equipped with this data, we aimed to develop a single-injection formulation that would provide a total of three doses—one initial soluble dose that was co-delivered with two additional doses encapsulated in PULSED microparticles engineered to release after 7 and 14 days (Fig. 1B). To achieve this, we first performed in vitro and in vivo release studies to identify a PLGA composition that would provide delayed pulsatile release at these time points, identifying several candidates appropriate for the desired timeline (Fig. 1C,D). We then screened a broad array of excipient candidates, including sugars, salts, amino acids, proteins, non-denaturing surfactants, and buffering agents with a focus on preventing deleterious changes in antigen conformation to preserve the ability to confer immunity despite stresses experienced during encapsulation (drying, heating) and release (acidification, sustained exposure to 37°C). Without the use of stabilizing excipients, the ELISA reactivity of encapsulated antigen was completed depleted within 2 days. Additionally, approximately two-thirds of the excipient formulations screened were unable to preserve any antigen in its immunity-conferring conformation for even 6 days. Nevertheless, we identified a number of formulations that were capable of preserving activity, with the top 50 formulations shown in Fig. 1E. Top formulations were also shown to release the rabies vaccine in its ELISA-reactive state, suggesting the ability to confer immunity. In conclusion, excipients addressing key environmental stresses were critical to reducing damage to the rabies antigen within PULSED microparticles. If successfully translated to humans, a single-injection rabies vaccine has the potential to enhance access to medical care and subsequently reduce the number of rabies deaths in low-resource settings.

Acknowledgements and/or References (Optional):: This work was supported by the National Institutes of Health (R21AI183245) and Particles for Humanity, Inc. We thank the One Health-Rabies Laboratory at the University of Missouri for their assistance in performing the RFFIT assay.