Formulation and coating of microneedles with inactivated influenza virus to improve vaccine stability and immunogenicity.

Microneedle patches coated with solid-state influenza vaccine have been developed to improve vaccine efficacy and patient coverage. However, dip coating microneedles with influenza vaccine can reduce antigen activity. In this study, we sought to determine the experimental factors and mechanistic pathways by which inactivated influenza vaccine can lose activity, as well as develop and assess improved microneedle coating formulations that protect the antigen from activity loss. After coating microneedles using a standard vaccine formulation, the stability of influenza vaccine was reduced to just 2%, as measured by hemagglutination activity. The presence of carboxymethylcellulose, which was added to increase viscosity of the coating formulation, was shown to contribute to vaccine activity loss. After screening a panel of candidate stabilizers, the addition of trehalose to the coating formulation was found to protect the antigen and retain 48-82% antigen activity for all three major strains of seasonal influenza: H1N1, H3N2 and B. Influenza vaccine coated in this way also exhibited thermal stability, such that activity loss was independent of temperature over the range of 4-37 degrees C for 24h. Dynamic light scattering measurements showed that antigen activity loss was associated with virus particle aggregation, and that stabilization using trehalose largely blocked this aggregation. Finally, microneedles using an optimized vaccine coating formulation were applied to the skin to vaccinate mice. Microneedle vaccination induced robust systemic and functional antibodies and provided complete protection against lethal challenge infection similar to conventional intramuscular injection. Overall, these results show that antigen activity loss during microneedle coating can be largely prevented through optimized formulation and that stabilized microneedle patches can be used for effective vaccination.

[1]  Mark R Prausnitz,et al.  Coated microneedles for transdermal delivery. , 2007, Journal of controlled release : official journal of the Controlled Release Society.

[2]  P. Palese,et al.  Influenza: old and new threats , 2004, Nature Medicine.

[3]  T. Anchordoquy,et al.  Stabilization of lipid/DNA complexes during the freezing step of the lyophilization process: the particle isolation hypothesis. , 2000, Biochimica et biophysica acta.

[4]  R. Compans,et al.  Induction of Heterosubtypic Immunity to Influenza Virus by Intranasal Immunization , 2007, Journal of Virology.

[5]  Mark R. Prausnitz,et al.  Effect of Microneedle Design on Pain in Human Volunteers , 2008, The Clinical journal of pain.

[6]  H. Hull,et al.  Recommendations of the Advi sory Committee on Immunization Practices , 2008 .

[7]  J H Crowe,et al.  Trehalose and sucrose protect both membranes and proteins in intact bacteria during drying , 1995, Applied and environmental microbiology.

[8]  J. R. Morgan,et al.  Stabilization of Active Recombinant Retroviruses in an Amorphous Dry State with Trehalose , 1998, Biotechnology progress.

[9]  W. Hinrichs,et al.  Inulin sugar glasses preserve the structural integrity and biological activity of influenza virosomes during freeze-drying and storage. , 2007, European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences.

[10]  G. G. Stokes.  "J." , 1890, The New Yale Book of Quotations.

[11]  S. Sambhara,et al.  Vaccines against epidemic and pandemic influenza , 2008 .

[12]  H L Robinson,et al.  DNA vaccines: protective immunizations by parenteral, mucosal, and gene-gun inoculations. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[13]  P. Laurent,et al.  Evaluation of the clinical performance of a new intradermal vaccine administration technique and associated delivery system. , 2007, Vaccine.

[14]  R. Cox,et al.  Influenza Virus: Immunity and Vaccination Strategies. Comparison of the Immune Response to Inactivated and Live, Attenuated Influenza Vaccines , 2004, Scandinavian journal of immunology.

[15]  M. Prausnitz,et al.  Transdermal Influenza Immunization with Vaccine-Coated Microneedle Arrays , 2009, PLoS ONE.

[16]  N. Cox,et al.  Prevention and Control of Influenza: recommendations of the Advisory Committee on Immunization Practices (ACIP). , 2006, MMWR. Recommendations and reports : Morbidity and mortality weekly report. Recommendations and reports.

[17]  R. Compans,et al.  Transcutaneous immunization with inactivated influenza virus induces protective immune responses. , 2006, Vaccine.

[18]  F. de Looze,et al.  Intradermal influenza vaccine administered using a new microinjection system produces superior immunogenicity in elderly adults: a randomized controlled trial. , 2008, The Journal of infectious diseases.

[19]  Zhu Guo,et al.  Needle-Free Skin Patch Delivery of a Vaccine for a Potentially Pandemic Influenza Virus Provides Protection against Lethal Challenge in Mice , 2007, Clinical and Vaccine Immunology.

[20]  J. Crowe,et al.  Trehalose lowers membrane phase transitions in dry yeast cells. , 1994, Biochimica et biophysica acta.

[21]  G. Leroux-Roels,et al.  Seasonal influenza vaccine delivered by intradermal microinjection: A randomised controlled safety and immunogenicity trial in adults. , 2008, Vaccine.

[22]  P. Furth,et al.  Low-volume jet injection for intradermal immunization in rabbits , 2002, BMC biotechnology.

[23]  L. Babiuk,et al.  Cutaneous vaccination: the skin as an immunologically active tissue and the challenge of antigen delivery. , 2000, Journal of controlled release : official journal of the Controlled Release Society.

[24]  Diane E. Sutter,et al.  Improved genetic immunization via micromechanical disruption of skin-barrier function and targeted epidermal delivery , 2002, Nature Medicine.

[25]  J. Matriano,et al.  Macroflux® Microprojection Array Patch Technology: A New and Efficient Approach for Intracutaneous Immunization , 2004, Pharmaceutical Research.

[26]  L. Falo,et al.  Epidermal dendritic cells induce potent antigen-specific CTL-mediated immunity. , 1997, The Journal of investigative dermatology.

[27]  P. van Damme,et al.  Safety and efficacy of a novel microneedle device for dose sparing intradermal influenza vaccination in healthy adults. , 2009, Vaccine.

[28]  W. Hinrichs,et al.  Development of Stable Influenza Vaccine Powder Formulations: Challenges and Possibilities , 2008, Pharmaceutical Research.

[29]  G. Kersten,et al.  Bioneedles as vaccine carriers. , 2008, Vaccine.

[30]  W. Hinrichs,et al.  The development of stable influenza vaccine powder formulations for new needle-free dosage forms , 2017 .

[31]  G. Glenn,et al.  Dose sparing with intradermal injection of influenza vaccine. , 2004, The New England journal of medicine.

[32]  Keiji Fukuda,et al.  Influenza-associated hospitalizations in the United States. , 2004, JAMA.

[33]  J. Carpenter,et al.  An infrared spectroscopic study of the interactions of carbohydrates with dried proteins. , 1989, Biochemistry.

[34]  A. S. Beare,et al.  The role of serum haemagglutination-inhibiting antibody in protection against challenge infection with influenza A2 and B viruses , 1972, Epidemiology and Infection.

[35]  M. Cormier,et al.  Effect of delivery parameters on immunization to ovalbumin following intracutaneous administration by a coated microneedle array patch system. , 2006, Vaccine.

[36]  A. Smith,et al.  Enabling topical immunization via microporation: a novel method for pain-free and needle-free delivery of adenovirus-based vaccines , 2003, Gene Therapy.

[37]  F. Yao,et al.  In vivo gene transfer to skin and wound by microseeding. , 1998, The Journal of surgical research.

[38]  G. Glenn,et al.  Transcutaneous immunization with heat-labile enterotoxin: development of a needle-free vaccine patch , 2007, Expert review of vaccines.

[39]  J. Carpenter,et al.  Stabilization of dry phospholipid bilayers and proteins by sugars. , 1987, The Biochemical journal.

[40]  P. Mercer,et al.  Bevel-down superior to bevel-up in intradermal skin testing. , 1997, Annals of allergy, asthma & immunology : official publication of the American College of Allergy, Asthma, & Immunology.

[41]  G. Vyas,et al.  Hemagglutination Assay for Antigen and Antibody Associated with Viral Hepatitis , 1970, Science.

[42]  Michel Cormier,et al.  Microneedle-based vaccines. , 2009, Current topics in microbiology and immunology.

[43]  J. Carpenter,et al.  Stabilization of phosphofructokinase during air-drying with sugars and sugar/transition metal mixtures. , 1987, Cryobiology.

[44]  M. Prausnitz,et al.  Immunization by vaccine-coated microneedle arrays protects against lethal influenza virus challenge , 2009, Proceedings of the National Academy of Sciences.

[45]  A. Herrmann,et al.  Analysis of delay times of hemagglutinin-mediated fusion between influenza virus and cell membranes , 2004, European Biophysics Journal.

[46]  Fernand Labrie,et al.  Gender differences in mouse skin morphology and specific effects of sex steroids and dehydroepiandrosterone. , 2005, The Journal of investigative dermatology.

[47]  R. Lamb,et al.  Paramyxovirus fusion: real-time measurement of parainfluenza virus 5 virus-cell fusion. , 2006, Virology.

[48]  F. Newman,et al.  Serum antibody responses after intradermal vaccination against influenza. , 2004, The New England journal of medicine.

[49]  J. Ulmer,et al.  Increased DNA Vaccine Delivery and Immunogenicity by Electroporation In Vivo , 2000, The Journal of Immunology.

[50]  Bhupal Singh.  Influenza , 1916, Nature Reviews Disease Primers.

[51]  Wendell Q. Sun,et al.  Stability of dry liposomes in sugar glasses. , 1996, Biophysical journal.

[52]  D. Steinhauer,et al.  Role of hemagglutinin cleavage for the pathogenicity of influenza virus. , 1999, Virology.