Single-administration, thermostable human papillomavirus vaccines prepared with atomic layer deposition technology

Abstract

Cold-chain requirements affect worldwide distribution of many vaccines. In addition, vaccines requiring multiple doses impose logistical and financial burdens, as well as patient compliance barriers. To address such limitations, we have developed new technologies to prepare thermostable, single-shot, prime-boost microparticle vaccines. Antigen/adjuvant formulations containing glass-forming polymers and trehalose first are spray-dried to form glassy microparticles that confer thermostability. Atomic layer deposition (ALD) reactions conducted in fluidized beds are then used to coat the microparticles with defined numbers of molecular layers of alumina that modulate the timed release of the internalized antigen and act as adjuvants. We have used a model HPV16 L1 capsomere antigen to evaluate the properties of these technologies. Thermostabilized powders containing HPV16 L1 capsomeres were prepared by spray-drying, coated by ALD with up to 500 molecular layers of alumina, and injected into mice. Antigen distribution was assessed by live-animal IR dye tracking of injected labeled antigen. Antibody responses were measured weekly by ELISA, and neutralizing antibodies were measured by pseudovirus neutralization assays at selected time points. Thermostability was evaluated by measuring antibody responses after incubating ALD-coated antigen powders for one month at 50 °C. Single doses of the ALD-coated vaccine formulations elicited a prime-boost immune response, and produced neutralizing responses and antibody titers that were equivalent or superior to conventional prime-boost doses of liquid formulations. Antibody titers were unaffected by month-long incubation of the formulations at 50 °C. Single-dose, thermostable antigen preparations may overcome current limitations in HPV vaccine delivery as well as being widely applicable to other antigens.

Keywords: Biotechnology; Immunology.

Fig. 1. Particles visualized by various imaging technologies.

a Spray-dried, uncoated particles imaged by SEM show a narrow particle size distribution and surface dimpling characteristics that were optimized by adjusting formulations to aid with fluidization during ALD. b Particles coated with 250 coats of alumina show no significant change in single particle morphology. c TEM image of particles with 250 ALD-alumina coats. Measurements confirm the value of 2.3 Å/ALD cycle as measured by ellipsometry. d Measurements using SEM after FIB milling of a particle coated with 250 coats of alumina confirm the value of 2.3 Å/ALD cycle.

Fig. 2. Particle size distribution of spray-dried and ALD-coated spray-dried particles as measured by flow imaging microscopy for particles with varying numbers of ALD-alumina coats.

Left to right in each group of bars: Spray-dried, no coating (blue), 100 ALD cycles (red); 250 cycles, (green); 500 cycles (purple). Size distribution of the particles were estimated using the estimated spherical diameter (ESD) values returned by VisualSpreadsheet for each particle.

Fig. 3. In vivo release of IR-dye labeled HPV16 L1 protein relative to the number of alumina layers applied to thermally stabilized microparticles.

Fluorescent images of SKH1 mice recorded at weeks 1, 4, 10 and 14 following injection into their right dorsal thigh with a 5µg of HPV16 L1 that was labeled with IRDye 800CW and adsorbed to alum prior to spray-drying but not coated. b 5µg HPV16 L1 that was labeled with IRDye 800CW, adsorbed on alum, spray-dried and coated with 100 ALD-alumina layers. c 5µg HPV16 L1 that was labeled with IRDye 800CW, adsorbed to alum, spray-dried and coated with 250 ALD-alumina layers, and d 5µg HPV16 L1 that was labeled with IRDye 800CW, adsorbed to alum, spray-dried and coated with 500 ALD-alumina layers.

Fig. 4. Antibody responses to vaccine formulations after immunization of BALB/c mice.

Total anti-HPV16 antibody titers were measured by ELISA. Plots show geometric average (n = 10) of responses at each time point. a 5 µg prime/boost on days 0/21 with a suspension of HPV16 L1 capsomeres adsorbed on alum (indicated by black arrow; black squares) compared to similar spray-dried and reconstituted formulations (red circles). b 5 µg prime/boost on days 0/21 with a suspension of HPV16 L1 capsomeres adsorbed on alum (indicated by black arrow; black squares) compared to a single 10 µg immunization with alum-adsorbed spray-dried capsomeres with 250 coats of ALD-alumina on day 0 (red triangles). c Single 10 µg immunization with spray-dried capsomeres with 250 coats of ALD-alumina with (red triangles) and without (black diamonds) included alum. d Single 10 µg immunization with spray-dried capsomeres with 250 coats of ALD-alumina (black diamonds) and after incubation at 50 °C for 1 month (red squares).

Fig. 5. Comparison of neutralizing antibody responses for uncoated versus alumina-coated HPV16 L1 vaccine preparations.

Neutralizing antibody responses (day 195 post prime) measured by percent neutralization of HPV16 pseudovirus (n=10) after a 5µg prime/boost immunization with liquid HPV16 L1 capsomeres plus alum (black squares, IC₅₀ = 1600) or equivalent spray-dried formulations (red circles, IC₅₀ = 4300), b 5µg prime/boost immunization with liquid HPV16 L1 capsomeres (black squares, IC₅₀ = 1600) or a single 10µg dose of spray-dried HPV16 L1 capsomeres with 250 coats of alumina (red triangles, red triangles, IC₅₀ = 13,800), and c single 10µg immunization with spray-dried HPV16 L1 capsomeres coated with 250 coats of alumina with (red triangles, IC₅₀ = 12,300) and without (black diamonds, IC₅₀ = 13,800) internal alum.