Cooperative Institute for Research in Environmental Sciences

A winning proposal for the Innovative Research Program, 2010:

New needle-free methods of HPV vaccine delivery of aerosols and dry powders

Investigator(s): Bob Sievers and Steve Cape

Objective(s): In this collaboration of a chemist, engineer and physician (R. Garcea, M.D., from MCDB), we aim to prepare fine dry powders of a new vaccine against human papillomavirus (HPV) that can be packaged in foil laminate blisters for delivery to the respiratory tract using a simple, inexpensive dry powder inhaler (DPI), such as the PuffHaler, which was invented by CIRES scientists and students to deliver aerosols. Additionally, these powders will be subsequently processed into thin wafers for sublingual delivery. Development of these HPV vaccine dosage forms will position our group to pursue additional major funding for further development of alternative HPV vaccines and tests of immunogenicity.

Figure 1. Distribution of Women’s Cancers in Relation to Economic Status [3].

Background and importance:

Infection with HPV is now accepted as the primary etiologic cause of cervical cancer. Although over 20 HPVs are designated as "high risk", types 16 and 18 are associated with 70% of all cervical cancers. Cervical cancer is the second leading cause of cancer-related death in women worldwide. Strikingly, the death rate from cervical cancer is concentrated in resource-poor areas where Pap smear early detection and other preventive medical care are unavailable (Figure 1). It is estimated that >300,000 women die every year from cervical cancer in these areas, in comparison with 3-5,000 women in the USA (e.g., approx. 100,000 women in India alone die every year from cervical cancer, more than from breast cancer). The recent development of prophylactic vaccines protective against "high risk" HPVs is a landmark in medicine and offers the hope of reducing the incidence of cervical cancer. The current vaccines, Gardasil™ and Cervarix™, are composed of recombinant virus-like particles (VLPs) of the major viral capsid protein L1 for the HPV types 16 and 18. Although they have >95% efficacy in preventing HPV infection, these vaccines are expensive and require refrigeration. Previous characterization of the immunogenic properties of VLP subunits and L1 capsomeres showed that capsomeres are near equivalent to VLPs in protecting against infection in animal models [1,2]. Capsomeres are purified after recombinant expression of L1 in E. coli, which will likely reduce cost, and then can be precipitated, resuspended, and stored at room temperature without loss of immunogenicity.

What makes this innovative:

Despite the remarkable efficacy of the present VLP HPV vaccines, their cost (approx. $360 for the initial three dose series) is prohibitive in lowresource settings. Furthermore, the current vaccines are injectable and require refrigeration, which also severely complicate effective delivery in resource-poor areas. The current imperative for cervical cancer care is the development of a low-cost, needle-free, thermally stable vaccine that is accessible to underdeveloped areas of the world. This project addresses this imperative by exploring the development of HPV vaccine dry dosage forms that should be cheaper to manufacture, do not require refrigeration, are needle-free, and can be delivered in a simple and inexpensive manner. Some recent preliminary studies with liquid solutions of HPV VLP or 16 L1 protein indicate that immunizations against HPV via the respiratory or sublingual mucosa are viable approaches [4,5].

Expected outcome and impact:

We expect to prepare two dosage forms of the HPV 16 L1 protein capsomeres (inhalable dry powders and sublingual dry wafers) that will be ready for immunogenicity testing. We expect that there will not be any unacceptable degradation of the protein capsomeres during powder/wafer processing and storage at room temperature. Achieving these goals will help position our group to pursue major funding for further development of this concept and vaccine product that may lead to a dramatic decrease in the incidence of cervical cancer worldwide. We hope to get funding from the NIH, the Gates Foundation, and/or private sources.

Research plan:

The HPV 16 L1 capsomere protein will be provided by Prof. Bob Garcea in the Dept. of Molecular, Cellular, and Developmental Biology, who has developed proprietary methods for preparing this material from E. coli bacteria. We will formulate the protein capsomeres with stabilizing and particle-forming excipients (sugars, amino acids, buffers, etc.). The formulated capsomere solutions will then be processed into dry powders by the CO2-Assisted Nebulization with the Bubble Dryer® (CAN-BD) technique [6] invented and developed in the Sievers’ laboratory. These powders will be characterized in terms of protein integrity (e.g., gel electrophoresis) and physical properties important for pulmonary delivery (e.g., aerodynamic particle size, aerosol properties, emitted dose, moisture content, etc.). The powders will then be packaged into foil laminate blisters for the PuffHaler DPI.

For sublingual dosage forms, the CAN-BD dry powders will be processed into thin dry wafers by adding appropriate excipients and applying direct compression. Excipients traditionally used by the pharmaceutical industry and Generally Regarded As Safe (GRAS) in the formulation of oral tablets will be investigated. These excipients will be dry blended with the CAN-BD powders to achieve the desired wafer properties: i.e., binding cohesiveness to consolidate the wafer ingredients, targeted disintegration rates (to cause the wafer to break apart within the desired timeframe when rehydrated sublingually), and reduced adhesion to the mold during direct compression wafer formation. Many of the wafer-forming excipients also have mucoadhesive properties that will likely be advantageous for a sublingual dosage form; once placed under the tongue the vaccine wafer should remain in place and not be swallowed, spit out, or otherwise prematurely removed from contact with the sublingual mucosa. Sugar additives may improve user acceptability.

Storage stability of the two dosage forms will be tested, with the goal of preserving protein integrity and physical properties of the inhalable powders or sublingual wafers over time. Stability testing will be conducted under real-time (room temperature) and accelerated (40°C) storage conditions.

References

[1] Rose RC, White W, Li M, Suzich J, Lane C, Garcea, RL. Human papillomavirus type 11 (HPV-11) recombinant L1 capsomeres induce virus-neutralizing antibodies. J Virol 1998; 72:6151-54.

[2] Yuan H, Estes PA, Chen Y, Newsome J, Olcese VA, Garcea RL, Schlegel R. Immunization with a pentameric L1 fusion protein protects against papillomavirus infection. J Virol 2001; 75:7848-53.

[3] Cohen J. High hopes and dilemmas for a cervical cancer vaccine. Science 2005; 308:618-21

[4] Nardelli-Haefliger D, Lurati F, Wirthner D, Spertini F, Schiller JT, Lowy DR, Ponci F, De Grandi P. Immune responses induced by lower airway mucosal immunisation with a human papillomavirus type 16 virus-like particle vaccine. Vaccine 2005; 23:3634-41.

[5] Cho H-J, Kim J-Y, Lee Y, Kim JM, Kim YB, Chun T, Oh Y-K. Enhanced humoral and cellular immune responses after sublingual immunization against human papillomavirus 16 L1 protein with adjuvants. Vaccine 2010; 28:2598-606.

[6] Cape SP, Villa JA, Huang ET, Yang TH, Carpenter JF, Sievers RE. Preparation of active proteins, vaccines and pharmaceuticals as fine powders using supercritical or near-critical fluids. Pharm Res 2008; 25:1967-90.