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The development and use of vaccines and their ability to prevent infection/disease is a shining example of the benefit of biomedical research. Modern vaccines often utilize subunit immunogens that exhibit minimal immunogenicity and require the use of adjuvants to maximize the induction of protective immune responses. We recently described a novel class of vaccine adjuvants, mast cell (MC) activators, that exhibit safe and effective vaccine adjuvant activity when administered by intranasal or intradermal routes. A compound library containing 580 functionalized benzopyrans, a structural motif found in a diverse array of natural and designed bioactive compounds, was screened using a MC degranulation assay to identify novel MC activating compounds for future evaluation as novel vaccine adjuvants. This approach identified 12 novel MC degranulating compounds. Therefore, MC degranulation can be used to reliably detect novel compounds for evaluation as adjuvants for use in mucosal vaccine strategies.

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Development of nasal immunization for human use is hindered by the lack of acceptable adjuvants. Although CT is an effective adjuvant, its toxicity will likely prevent its use in nasal vaccines. This study compared non-toxin adjuvants to CT for their ability to induce protective antibody responses with nasal immunization. C3H/HeN and C57BL/6 mice were immunized with rPA formulated with the following adjuvants: CT, IL-1α, LPS, CpG, Pam3CSK4, 3M-019, resiquimod/R848 or c48/80. Serum and nasal wash cytokine concentrations were monitored 6h post-vaccination as biomarkers for acute activation of the innate immune system. Not all of the adjuvants induced significant changes in innate serum or nasal wash cytokines, but when changes were observed, the cytokine signatures were unique for each adjuvant. All adjuvants except Pam3CSK4 induced significantly increased anti-rPA serum IgG titers in both strains of mice, while only IL-1α, c48/80 and CpG enhanced mucosal anti-rPA IgA. Pam3CSK4 was the only adjuvant unable to enhance the induction of serum LeTx-neutralizing antibodies in C3H/HeN mice while c48/80 was the only adjuvant to induce increased serum LeTx-neutralizing antibodies in C57BL/6 mice. Only CT enhanced total serum IgE in C3H/HeN mice while IL-1α enhanced total serum IgE in C57BL/6 mice. The adjuvant influenced antigen-specific serum IgG subclass and T cell cytokine profiles, but these responses did not correlate with the induction of LeTx-neutralizing activity. Our results demonstrate the induction of diverse innate and adaptive immune responses by non-toxin nasal vaccine adjuvants that lead to protective humoral immunity comparable to CT and that these responses may be influenced by the host strain.

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There is a current biodefense interest in protection against anthrax. Here, we developed a new generation of stable and effective anthrax vaccine. We studied the immune response elicited by recombinant protective antigen (rPA) delivered intranasally with a novel mucosal adjuvant, a mast cell activator compound 48/80 (C48/80). The vaccine formulation was prepared in a powder form by spray-freeze-drying (SFD) under optimized conditions to produce particles with a target size of D(50) = 25 µm, suitable for delivery to the rabbit nasal cavity. Physicochemical properties of the powder vaccines were characterized to assess their delivery and storage potential. Structural stability of rPA was confirmed by circular dichroism and attenuated total reflectance-Fourier transform infrared spectroscopy, whereas functional stability of rPA and C48/80 was monitored by cell-based assays. Animal study was performed using a unit-dose powder device for direct nasal application. Results showed that C48/80 provided effective mucosal adjuvant activity in rabbits. Freshly prepared SFD powder vaccine formulations or powders stored for over 2 years at room temperature elicited significantly elevated serum PA-specific and lethal toxin neutralization antibody titers that were comparable to that induced by intramuscular immunization with rPA. Nasal delivery of this vaccine formulation may be a viable alternative to the currently licensed vaccine or an attractive vaccine platform for other mucosally transmitted diseases.

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IL-1α and IL-1ß were evaluated for their ability to provide adjuvant activity for the induction of serum antibody responses when nasally administered with protein antigens in mice and rabbits. In mice, intranasal (i.n.) immunization with pneumococcal surface protein A (PspA) or tetanus toxoid (TT) combined with IL-1ß induced protective immunity that was equivalent to that induced by parenteral immunization. Nasal immunization of awake (i.e., not anesthetized) rabbits with IL-1-adjuvanted vaccines induced highly variable serum antibody responses and was not as effective as parenteral immunization for the induction of antigen-specific serum IgG. However, i.n. immunization of deeply anesthetized rabbits with rPA+IL-1α consistently induced rPA-specific serum IgG ELISA titers that were not significantly different than those induced by intramuscular (IM) immunization with rPA+alum although lethal toxin-neutralizing titers induced by nasal immunization were lower than those induced by IM immunization. Gamma scintigraphy demonstrated that the enhanced immunogenicity of nasal immunization in anesthetized rabbits correlated with an increased nasal retention of i.n. delivered non-permeable radio-labeled colloidal particles. Our results demonstrate that, in mice, IL-1 is an effective adjuvant for nasally administered vaccines for the induction of protective systemic immunity and that in non-rodent species, effective induction of systemic immunity with nasally administered vaccines may require formulations that ensure adequate retention of the vaccine within the nasal cavity.

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Vi polysaccharide from Salmonella enterica serotype Typhi is used as one of the available vaccines to prevent typhoid fever. Measurement of Vi-specific serum antibodies after vaccination with Vi polysaccharide by enzyme-linked immunosorbent assay (ELISA) may be complicated due to poor binding of the Vi polysaccharide to ELISA plates resulting in poor reproducibility of measured antibody responses. We chemically conjugated Vi polysaccharide to fluorescent beads and performed studies to determine if a bead-based immunoassay provided a reproducible method to measure vaccine-induced anti-Vi serum IgG antibodies. Compared to ELISA, the Vi bead immunoassay had a lower background and therefore a greater signal-to-noise ratio. The Vi bead immunoassay was used to evaluate serum anti-Vi IgG in 996 subjects from the city of Kolkata, India, before and after vaccination. Due to the location being one where Salmonella serotype Typhi is endemic, approximately 45% of the subjects had protective levels of anti-Vi serum IgG (i.e., 1 microg/ml anti-Vi IgG) before vaccination, and nearly 98% of the subjects had protective levels of anti-Vi serum IgG after vaccination. Our results demonstrate that a bead-based immunoassay provides an effective, reproducible method to measure serum anti-Vi IgG responses before and after vaccination with the Vi polysaccharide vaccine.

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Passive transfer of antibody may be useful for preexposure prophylaxis against biological agents used as weapons of terror, such as Bacillus anthracis. Studies were performed to evaluate the ability of anthrax antiprotective antigen (anti-PA) and antilethal factor (anti-LF) neutralizing monoclonal antibodies (mAbs) to protect against an anthrax lethal toxin (LeTx) challenge in a mouse model and to identify correlates of immunity to LeTx challenge. Despite having similar affinities for their respective antigens, anti-PA (3F11) and anti-LF (9A11), passive transfer of up to 1.5 mg of anti-PA 3F11 mAb did not provide significant protection when transferred to mice 24 h before LeTx challenge, while passive transfer of as low as 0.375 mg of anti-LF 9A11 did provide significant protection. Serum collected 24 h after passive transfer had LeTx-neutralizing activity when tested using a standard LeTx neutralization assay, but neutralization titers measured using this assay did not correlate with protection against LeTx challenge. However, measurement of LeTx-neutralizing serum responses with an LeTx neutralization assay in vitro employing the addition of LeTx to J774A.1 cells 15 min before the addition of the serum did result in neutralization titers that correlated with protection against LeTx challenge. Our results demonstrate that only the LeTx neutralization titers measured utilizing the addition of LeTx to J774A.1 cells 15 min before the addition of sample correlated with protection in vivo. Thus, this LeTx neutralization assay may be a more biologically relevant neutralization assay to predict the in vivo protective capacity of LeTx-neutralizing antibodies.

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Immunization by the nasal route is an established method for the induction of mucosal and systemic humoral and cell-mediated antigen-specific responses. However, the effectiveness of nasal immunization is often hampered by the need for increased doses of antigen. Bioadhesives and absorption enhancers were investigated for their ability to enhance immune responses in mice after nasal immunization with model HIV-1 peptide and protein immunogens. Two additives, hydroxypropylmethylcellulose (HPMC) and capric acid, consistently enhanced antigen-specific serum IgG endpoint titers under conditions in which antigen dose was limiting. Nasal immunization of mice with 20 microg of an HIV-1 peptide immunogen plus cholera toxin (CT) as adjuvant induced serum antipeptide IgG titers of 1:9.5log2 after four immunizations while the addition of CA or HPMC to the vaccine formulation increased serum antipeptide IgG titers to 1:15.4log2 and 1:17.6log2, respectively. When 5 microg recombinant HIV-1 gp41 was used as the immunogen, the addition of CA or HPMC to the vaccine formulation increased serum anti-gp41 IgG titers to 1:11.6log2 and 1:8.8log2, respectively, compared to 1:5.2log2 after three nasal immunizations with 5 microg gp41 + CT alone. Thus, HPMC and capric acid may be useful additives that increase the immunogenicity of nasally administered vaccines and permit less antigen to be used with each immunization.

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