A Single Dose, Thermostable, Trivalent Human Papillomavirus Vaccine Formulated Using Atomic Layer Deposition.

Formulations of human papillomavirus (HPV) 16, 18, and 31 L1 capsomere protein antigens were spray dried to obtain glassy microspheres that were then coated by atomic layer deposition (ALD) with nanometer-thin protective layers of alumina. Spray-drying was used to formulate human papillomavirus (HPV) 16, 18, and 31 L1 capsomere protein antigens within glassy microspheres to which nanoscopic protective layers of alumina were applied using ALD. Suspensions of alumina-coated, capsomere-containing microparticles were administered in a single dose to mice. ALD-deposited alumina coatings provided thermostability and a delayed in vivo release of capsomere antigens, incorporating both a prime and a boost dose in one injection. Total serotype-specific antibody titers as well as neutralizing titers determined from pseudovirus infectivity assays were unaffected by incubation of the ALD-coated vaccines for at 4, 50, or 70 °C for three months prior to administration. In addition, even after incubation for three months at 70 °C, single doses of ALD-coated vaccines produced both higher total antibody responses and higher neutralizing responses than control immunizations that used two doses of conventional liquid formulations stored at 4 °C.

Thermostability of a trivalent, capsomere-based vaccine for human papillomavirus infection.

Currently licensed vaccines require a cold-chain to maintain efficacy. This cold-chain requirement reduces the availability of vaccines in resource-poor areas of the world. Commercially available human papillomavirus (HPV) vaccines protect against the most common HPV types related to cervical cancer; however, their impact is limited in many regions due to cold-chain requirements. The goal of this study was to test the thermostability of an adjuvanted, trivalent HPV L1 capsomere-based vaccine (containing HPV types 16, 18, and 31) that was formulated by using lyophilization to embed the antigens within a solid, glassy matrix. Thermal stabilities were determined by storing the vaccine formulations for 3 months at 50 °C, followed by immunization of BALB/c mice and measurement of antibody responses. Antibody responses to capsomere vaccines formulated with alum were unchanged after storage for 3 months at 50 °C. Neutralizing responses to these vaccines were unchanged by high-temperature storage, and were equivalent to those generated after administration of the commercially available liquid HPV vaccine Gardasil®9.

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

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.

Development of a highly thermostable, adjuvanted human papillomavirus vaccine.

A major impediment to economical, worldwide vaccine distribution is the requirement for a "cold chain" to preserve antigenicity. We addressed this problem using a model human papillomavirus (HPV) vaccine stabilized by immobilizing HPV16 L1 capsomeres, i.e., pentameric subunits of the virus capsid, within organic glasses formed by lyophilization. Lyophilized glass and liquid vaccine formulations were incubated at 50°C for 12weeks, and then analyzed for retention of capsomere conformational integrity and the ability to elicit neutralizing antibody responses after immunization of BALB/c mice. Capsomeres in glassy-state vaccines retained tertiary and quaternary structure, and critical conformational epitopes. Moreover, glassy formulations adjuvanted with aluminum hydroxide or aluminum hydroxide and glycopyranoside lipid A were not only as immunogenic as the commercially available HPV vaccine Cervarix®, but also retained complete neutralizing immunogenicity after high-temperature storage. The thermal stability of such adjuvanted vaccine powder preparations may thus eliminate the need for the cold chain.

Common exposure to STL polyomavirus during childhood.

STL polyomavirus (STLPyV) was recently identified in human specimens. To determine seropositivity for STLPyV, we developed an ELISA and screened patient samples from 2 US cities (Denver, Colorado [500]; St. Louis, Missouri [419]). Overall seropositivity was 68%-70%. The age-stratified data suggest that STLPyV infection is widespread and commonly acquired during childhood.

Dynamics of urinary polyomavirus shedding in healthy adult women.

The hypothesis was examined that physiologic variation of estrogen concentrations during the menstrual cycle can provoke BK virus (BKV) excretion. BKV and JCV viral loads were determined in urine specimens obtained almost daily from 20 healthy, non-pregnant women over 2 months. Asymptomatic urinary shedding of BKV was observed in 123 (12.0%) of 1,021 specimens from 11 (55%) study subjects. Two subjects excreted JCV in their urine, with one subject excreting detectable JCV in all urine specimens. Analysis of 36 complete menstrual cycles revealed no difference in the prevalence of BKV excretion between pre-ovulatory and post-ovulatory phases of the menstrual cycle. The unexpected day-to-day variability in BKV excretion suggests that as yet unidentified factors may contribute to the periodic shedding of BKV by healthy women.

Dynamics of pregnancy-associated polyomavirus urinary excretion: a prospective longitudinal study.

Asymptomatic polyomaviruria of pregnancy has been documented in point prevalence studies, but little attention has been given to the dynamics of polyomavirus excretion during pregnancy because of its benign course. We tested the hypothesis that the frequency and/or magnitude of polyomavirus excretion would increase as pregnancy progresses. Urine specimens were obtained prospectively from 179 healthy women during uncomplicated pregnancies and 37 healthy non-pregnant women. Real-time polymerase chain reaction was used to determine BK virus (BKV) and JC virus (JCV) viral loads in urine, blood, and rectal and vaginal swabs collected during routine obstetric and gynecologic clinic visits. Asymptomatic urinary shedding of BKV and/or JCV was observed in 384 (48.0%) of 800 specimens from 100 (55.8%) pregnant women. BKV excretion was more common in pregnant than non-pregnant women (41.3% vs. 13.5%, P = 0.0026). The frequency of JCV excretion was no different in pregnant compared to non-pregnant women. The frequency and magnitude of polyomavirus shedding did not vary with gestational age. Post-partum shedding of BKV, but not JCV, rapidly decreased to undetectable levels. Pregnancy-associated BKV excretion begins early in pregnancy and terminates rapidly post-partum. Neither the frequency nor magnitude of BKV or JCV shedding increased with pregnancy progression. Further study into the host factors that regulate pregnancy-associated BKV excretion may allow identification of the host factors that predict susceptibility to BKV-associated diseases in immune compromised patients.

86Rb+ efflux mediated by alpha4beta2*-nicotinic acetylcholine receptors with high and low-sensitivity to stimulation by acetylcholine display similar agonist-induced desensitization.

The nicotinic acetylcholine receptors (nAChR) assembled from alpha4 and beta2 subunits are the most densely expressed subtype in the brain. Concentration-effect curves for agonist activation of alpha4beta2*-nAChR are biphasic. This biphasic agonist sensitivity is ascribed to differences in subunit stoichiometry. The studies described here evaluated desensitization elicited by low concentrations of epibatidine, nicotine, cytisine or methylcarbachol of brain alpha4beta2-nAChR function measured with acetylcholine-stimulated (86)Rb(+) efflux from mouse thalamic synaptosomes. Each agonist elicited concentration-dependent desensitization. The agonists differed in potency. However, IC(50) values for each agonist for desensitization of (86)Rb(+) efflux both with high (EC(50) approximately 3 microM) and low (EC(50) approximately 150 microM) acetylcholine sensitivity were not significantly different. Concentrations required to elicit desensitization were higher that their respective K(D) values for receptor binding. Even though the two components of alpha4beta2*-nAChR-mediated (86)Rb(+) efflux from mouse brain differ markedly in EC(50) values for agonist activation, they are equally sensitive to desensitization by exposure to low agonist concentrations. Mice were also chronically treated with nicotine by continuous infusion of 0, 0.5 or 4.0mg/kg/h and desensitization induced by nicotine was evaluated. Consistent with previous results, chronic nicotine treatment increased the density of epibatidine binding sites. Acute exposure to nicotine also elicited concentration-dependent desensitization of both high-sensitivity and low-sensitivity acetylcholine-stimulated (86)Rb(+) efflux from cortical and thalamic synaptosomes. Although chronic nicotine treatment reduced maximal (86)Rb(+) efflux from thalamus, IC(50) values in both brain regions were unaffected by chronic nicotine treatment.

Partial deletion of the nicotinic cholinergic receptor alpha 4 or beta 2 subunit genes changes the acetylcholine sensitivity of receptor-mediated 86Rb+ efflux in cortex and thalamus and alters relative expression of alpha 4 and beta 2 subunits.

Alpha4 and beta2 nicotinic cholinergic receptor (nAChR) subunits can assemble in heterologous expression systems as pentameric receptors with different subunit stoichiometries that exhibit differential sensitivity to activation by acetylcholine, yielding biphasic concentration-effect curves. nAChR-mediated (86)Rb(+) efflux in mouse brain synaptosomes also displays biphasic acetylcholine (ACh) concentration-response curves. Both phases are mediated primarily by alpha4beta2(*)-nAChR, because deletion of either the alpha4 or beta2 subunit reduces response at least 90%. A relatively larger decrease in the component of (86)Rb(+) efflux with lower ACh sensitivity occurred with partial deletion of alpha4 (alpha4(+/-)), whereas a larger decrease in the component with higher ACh sensitivity was elicited by partial deletion of beta2 (beta2(+/-)). Immunoprecipitation with selective antibodies demonstrated that more than 70% of [(3)H]epibatidine binding sites in both regions contained only alpha4 and beta2 subunits. Subsequently, alpha4 and beta2 subunit content in the cortex and thalamus of alpha4 and beta2 wild types and heterozygotes was analyzed with Western blots. Partial deletion of alpha4 decreased and partial deletion of beta2 increased the relative proportion of the alpha4 subunit in assembled receptors. Although these methods do not allow exact identification of stoichiometry of the subtypes present in wild-type cortex and thalamus, they do demonstrate that cortical and thalamic nAChRs of the alpha4(+/-) and beta2(+/-) genotypes differ in relative expression of alpha4 and beta2 subunits a result that corresponds to the relative functional changes observed after partial gene deletion. These results strongly suggest that alpha4beta2-nAChR with different stoichiometry are expressed in native tissue.

Gene targeting demonstrates that alpha4 nicotinic acetylcholine receptor subunits contribute to expression of diverse [3H]epibatidine binding sites and components of biphasic 86Rb+ efflux with high and low sensitivity to stimulation by acetylcholine.

[3H]Epibatidine binds to nAChR subtypes in mouse brain with higher (KD approximately 0.02 nM) and lower affinity (KD approximately 7 nM), which can be further subdivided through inhibition by selected agonists and antagonists. These subsets are differentially affected by targeted deletion of alpha7, beta2 or beta4 subunits. Most, but not all, higher and lower affinity binding sites require beta2 (Marks, M.J., Whiteaker, P., Collins, A.C., 2006. Deletion of the alpha7, beta2 or beta4 nicotinic receptor subunit genes identifies highly expressed subtypes with relatively low affinity for [3H]epibatidine. Mol. Pharmacol. 70, 947-959). Effects of functional alpha4 gene deletion are reported here. Deletion of alpha4 virtually eliminated cytisine-sensitive, higher-affinity [3H]epibatidine binding as did beta2 deletion, confirming that these sites are alpha4beta2*-nAChR. Cytisine-resistant, higher-affinity [3H]epibatidine binding sites are diverse and some of these sites require alpha4 expression. Lower affinity [3H]epibatidine binding sites are also heterogeneous and can be subdivided into alpha-bungarotoxin-sensitive and -resistant components. Deleting alpha4 did not affect the alpha-bungarotoxin-sensitive component, but markedly reduced the alpha-bungarotoxin-resistant component. This effect was similar, but not quite identical, to the effect of beta2 deletion. This provides the first evidence that lower-affinity epibatidine binding sites in the brain require expression of alpha4 subunits. The effects of alpha4 gene targeting on receptor function were measured using a 86Rb+ efflux assay. Concentration-effect curves for ACh-stimulated 86Rb+ efflux are biphasic (EC50 values=3.3 microM and 300 microM). Targeting alpha4 produced substantial gene-dose dependent reductions in both phases in whole brain and in most of the 14 brain regions assayed. These effects are very similar to those following deletion of beta2. Thus, alpha4beta2*-nAChRs mediate a significant fraction of both phases of ACh stimulated 86Rb+ efflux.