Nasal Immunization with a Recombinant HIV gp120 and Nanoemulsion Adjuvant Produces Th1 Polarized Responses and Neutralizing Antibodies to Primary HIV Type 1 Isolates

ABSTRACT Epidemiological and experimental data suggest that both robust neutralizing antibodies and potent cellular responses play important roles in controlling primary HIV-1 infection. In this study we have investigated the induction of systemic and mucosal immune responses to HIV gp120 monomer immunogen administered intranasally in a novel, oil-in-water nanoemulsion (NE) adjuvant. Mice and guinea pigs intranasally immunized by the application of recombinant HIV gp120 antigen mixed in NE demonstrated robust serum anti-gp120 IgG, as well as bronchial, vaginal, and serum anti-gp120 IgA in mice. The serum of these animals demonstrated antibodies that cross-reacted with heterologous serotypes of gp120 and had significant neutralizing activity against two clade-B laboratory strains of HIV (HIVBaL and HIVSF162) and five primary HIV-1 isolates. The analysis of gp120-specific CTL proliferation, INF-γ induction, and prevalence of anti-gp120 IgG2 subclass antibodies indicated that nasal vaccination in NE also induced systemic, Th1-polarized cellular immune responses. This study suggests that NE should be evaluated as a mucosal adjuvant for multivalent HIV vaccines.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63251/1/aid.2007.0148.pd

Intranasal nanoemulsion vaccine confers long‐lasting immunomodulation and sustained unresponsiveness in a murine model of milk allergy

BackgroundImmunotherapy for food allergy requires prolonged treatment protocols and, in most cases, does not lead to durable modulation of the allergic immune response. We have demonstrated an intranasal (IN) nanoemulsion adjuvant that redirects allergen‐specific Th2 responses toward Th1 and Th17 immunity, and protects from allergen challenge after only 2‐4 monthly administrations. Here, we investigate the ability of this technology to provide long‐term modulation of allergy in a murine model of cow’s milk allergy.MethodsSix weeks after sensitization to bovine casein, mice received four, monthly IN immunizations with nanoemulsion formulated with casein. Protection from casein challenge was assessed at 4 and 16 weeks after the final vaccine administration.ResultsThe NE vaccine significantly blunted the physiological responses to allergen challenge, and this effect persisted for at least 16 weeks. The protection from challenge was associated with the suppression of casein‐specific Th2 immunity and induced Th1 and Th17 cytokines as well as induction of IL‐10. Of interest, while immunized animals showed significantly decreased Th2 cytokine responses, cow’s milk‐specific IgE remained elevated in the serum at levels associated with reactivity in control sensitized animals. Protection was associated with suppressed mast cell activation and markedly reduced mast cell infiltration into the small intestine.ConclusionThe sustained unresponsiveness of at least 16 weeks after vaccination suggests that the nanoemulsion vaccine alters the allergic phenotype in a persistent manner different from traditional desensitization, and this leads to long‐term suppressive effects on allergic disease without eliminating serum IgE.This study evaluates the ability of an intranasal nanoemulsion‐based vaccine to induce long‐term modulation of allergic reactions in a mouse model of cow’s milk allergy. Intranasal immunization with nanoemulsion adjuvant suppresses Th2 responses and anaphylaxis. The sustained unresponsiveness of at least 16 weeks after vaccination suggests that the nanoemulsion vaccine alters the allergic phenotype.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154959/1/all14064_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154959/2/all14064.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154959/3/all14064-sup-0003-FigS3.pd

Pre-Clinical Evaluation of a Novel Nanoemulsion-Based Hepatitis B Mucosal Vaccine

Hepatitis B virus infection remains an important global health concern despite the availability of safe and effective prophylactic vaccines. Limitations to these vaccines include requirement for refrigeration and three immunizations thereby restricting use in the developing world. A new nasal hepatitis B vaccine composed of recombinant hepatitis B surface antigen (HBsAg) in a novel nanoemulsion (NE) adjuvant (HBsAg-NE) could be effective with fewer administrations.Physical characterization indicated that HBsAg-NE consists of uniform lipid droplets (349+/-17 nm) associated with HBsAg through electrostatic and hydrophobic interactions. Immunogenicity of HBsAg-NE vaccine was evaluated in mice, rats and guinea pigs. Animals immunized intranasally developed robust and sustained systemic IgG, mucosal IgA and strong antigen-specific cellular immune responses. Serum IgG reached > or = 10(6) titers and was comparable to intramuscular vaccination with alum-adjuvanted vaccine (HBsAg-Alu). Normalization showed that HBsAg-NE vaccination correlates with a protective immunity equivalent or greater than 1000 IU/ml. Th1 polarized immune response was indicated by IFN-gamma and TNF-alpha cytokine production and elevated levels of IgG(2) subclass of HBsAg-specific antibodies. The vaccine retains full immunogenicity for a year at 4 degrees C, 6 months at 25 degrees C and 6 weeks at 40 degrees C. Comprehensive pre-clinical toxicology evaluation demonstrated that HBsAg-NE vaccine is safe and well tolerated in multiple animal models.Our results suggest that needle-free nasal immunization with HBsAg-NE could be a safe and effective hepatitis B vaccine, or provide an alternative booster administration for the parenteral hepatitis B vaccines. This vaccine induces a Th1 associated cellular immunity and also may provide therapeutic benefit to patients with chronic hepatitis B infection who lack cellular immune responses to adequately control viral replication. Long-term stability of this vaccine formulation at elevated temperatures suggests a direct advantage in the field, since potential excursions from cold chain maintenance could be tolerated without a loss in therapeutic efficacy

Intranasal delivery of allergen in a nanoemulsion adjuvant inhibits allergen- specific reactions in mouse models of allergic airway disease

BackgroundAtopic diseases are an increasing problem that involve both immediate hypersensitivity reactions mediated by IgE and unique cellular inflammation. Many forms of specific immunotherapy involve the administration of allergen to suppress allergic immune responses but are focused on IgE- mediated reactions. In contrast, the effect of allergen- specific immunotherapy on allergic inflammation is complex, not entirely consistent and not well understood. We have previously demonstrated the ability of allergen administered in a nanoemulsion (NE) mucosal adjuvant to suppress IgE- mediated allergic responses and protect from allergen challenge in murine food allergy models. This activity was associated with decreases in allergen- specific IL- 10 and reductions in allergic cytokines and increases in regulatory T cells.ObjectiveHere, we extend these studies to using 2 distinct models, the ovalbumin (OVA) and cockroach (CRA) models of allergic airway disease, which are based predominantly on allergic inflammation.MethodsAcute or chronic allergic airway disease was induced in mice using ovalbumin and cockroach allergen models. Mice received three therapeutic immunizations with allergen in NE, and reactivity to airway challenge was determined.ResultsTherapeutic immunization with cockroach or OVA allergen in NE markedly reduced pathology after airway challenge. The 2 models demonstrated protection from allergen challenge- induced pathology that was associated with suppression of Th2- polarized immune responses in the lung. In addition, the reduction in ILC2 numbers in the lungs of allergic mice along with reduction in epithelial cell alarmins, IL- 25 and IL- 33, suggests an overall change in the lung immune environment induced by the NE immunization protocol.Conclusions and Clinical RelevanceThese results demonstrate that suppression of allergic airway inflammation and bronchial hyper- reactivity can be achieved using allergen- specific immunotherapy without significant reductions in allergen- specific IgE and suggest that ILC2 cells may be critical targets for this activity.The ability of intranasal vaccines to modulate allergic immune responses and inflammation in the lung was studied in two distinct models of allergic airway disease. Mice were sensitized to ovalbumin or cockroach allergen and subsequently received three immunizations with allergen in nanoemulsion adjuvant. Nanoemulsion vaccines modulated the allergen- specific cytokine milieu in the lungs to suppress Th2 cytokine production, alarmin expression and recruitment of ILC2s. The immune modulation in the lungs was associated with protection from allergen challenge- induced inflammation and reactivity.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/170274/1/cea13903.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/170274/2/cea13903_am.pd

Mucosal Immunization with a Novel Nanoemulsion-Based Recombinant Anthrax Protective Antigen Vaccine Protects against Bacillus anthracis Spore Challenge▿

The currently available commercial human anthrax vaccine requires multiple injections for efficacy and has side effects due to its alum adjuvant. These factors limit its utility when immunizing exposed populations in emergent situations. We evaluated a novel mucosal adjuvant that consists of a nontoxic, water-in-oil nanoemulsion (NE). This material does not contain a proinflammatory component but penetrates mucosal surfaces to load antigens into dendritic cells. Mice and guinea pigs were intranasally immunized with recombinant Bacillus anthracis protective antigen (rPA) mixed in NE as an adjuvant. rPA-NE immunization was effective in inducing both serum anti-PA immunoglobulin G (IgG) and bronchial anti-PA IgA and IgG antibodies after either one or two mucosal administrations. Serum anti-PA IgG2a and IgG2b antibodies and PA-specific cytokine induction after immunization indicate a Th1-polarized immune response. rPA-NE immunization also produced high titers of lethal-toxin-neutralizing serum antibodies in both mice and guinea pigs. Guinea pigs nasally immunized with rPA-NE vaccine were protected against an intradermal challenge with ∼1,000 times the 50% lethal dose (∼1,000× LD50) of B. anthracis Ames strain spores (1.38 × 103 spores), which killed control animals within 96 h. Nasal immunization also resulted in 70% and 40% survival rates against intranasal challenge with 10× LD50 and 100× LD50 (1.2 × 106 and 1.2 × 107) Ames strain spores. Our results indicate that NE can effectively adjuvant rPA for intranasal immunization. This potentially could lead to a needle-free anthrax vaccine requiring fewer doses and having fewer side effects than the currently available human vaccine

Formulation, high throughput in vitro screening and in vivo functional characterization of nanoemulsion-based intranasal vaccine adjuvants.

Vaccine adjuvants have been reported to induce both mucosal and systemic immunity when applied to mucosal surfaces and this dual response appears important for protection against certain pathogens. Despite the potential advantages, however, no mucosal adjuvants are currently approved for human use. Evaluating compounds as mucosal adjuvants is a slow and costly process due to the need for lengthy animal immunogenicity studies. We have constructed a library of 112 intranasal adjuvant candidate formulations consisting of oil-in-water nanoemulsions that contain various cationic and nonionic surfactants. To facilitate adjuvant development we first evaluated this library in a series of high-throughput, in vitro assays for activities associated with innate and adaptive immune activation in vivo. These in vitro assays screened for the ability of the adjuvant to bind to mucin, induce cytotoxicity, facilitate antigen uptake in epithelial and dendritic cells, and activate cellular pathways. We then sought to determine how these parameters related to adjuvant activity in vivo. While the in vitro assays alone were not enough to predict the in vivo adjuvant activity completely, several interesting relationships were found with immune responses in mice. Furthermore, by varying the physicochemical properties of the surfactant components (charge, surfactant polar head size and hydrophobicity) and the surfactant blend ratio of the formulations, the strength and type of the immune response generated (TH1, TH2, TH17) could be modulated. These findings suggest the possibility of using high-throughput screens to aid in the design of custom adjuvants with unique immunological profiles to match specific mucosal vaccine applications

In vivo analyses of HBsAg-NE stability.

<p>HBsAg specific antibody responses to freshly prepared HBsAg-NE or HBsAg-NE stored under real-time (4°C), accelerated (25°C) and stressed (40°C) temperature conditions are depicted. CD-1 mice were vaccinated with either freshly prepared or stored HBsAg-NE and boosted at 6 weeks. Serum anti-HBsAg IgG antibody concentrations are presented as a mean of endpoint titers in individual sera +/− SD. Comparison of serum IgG elicited by freshly prepared HBsAg-NE to formulation stored for (A) 6 weeks, (B) 3 months, (C) 6 months or (D) 1 year at indicated temperatures. * indicates a statistical difference (<i>p value<0.05</i>) in the anti-HBsAg IgG titers between freshly mixed and stored formulation. Arrows indicate vaccine administration.</p

Pre-clinical toxicology evaluation.

a<p>- The number of animals used for analysis: CD-1 (n = 10), BALB/c, Wistar rats and Hartley guinea pigs (n = 5) and Beagles (n = 1) per group.</p>b<p>- Histological lesions were evaluated on a scale from 0 to 10 with +1 single microscopic focus, +2 at least 2 microscopic foci, +3 more than 3 foci or multiple locally extensive areas of pathology, +4 to +6 were associated increasing severity and more extensive distribution (these lesions could be associated with morbidity), +7 and above had increasing degrees of inflammation (+10 associated with mortality).</p>c<p>- Other tissues evaluated include heart, liver, kidneys, spleen, esophagus, trachea, stomach, intestines, pancreas, and adrenals.</p>d<p>- Metabolic analysis evaluated by standard biochemical serum profile analysis on a IDEXX Vet Test Analyzer™ and performed at the Animal Diagnostic Laboratory through the Unit for Laboratory Animal Medicine at the University of Michigan. Normal indicates all analytes fell within normal expected distributions per species.</p>e<p>- Administered every 2 weeks.</p>f<p>- Administered every 15 minutes.</p>g<p>- Administered every 4 hours.</p>h<p>- Administered every 4 weeks.</p