Allergen-Encapsulating Nanoparticles Reprogram Pathogenic Allergen-Specific Th2 Cells to Suppress Food Allergy.

Food allergy is a prevalent, potentially deadly disease caused by inadvertent sensitization to benign food antigens. Pathogenic Th2 cells are a major driver for disease, and allergen-specific immunotherapies (AIT) aim to increase the allergen threshold required to elicit severe allergic symptoms. However, the majority of AIT approaches require lengthy treatments and convey transient disease suppression, likely due to insufficient targeting of pathogenic Th2 responses. Here, the ability of allergen-encapsulating nanoparticles to directly suppress pathogenic Th2 responses and reactivity is investigated in a mouse model of food allergy. NPs associate with pro-tolerogenic antigen presenting cells, provoking accumulation of antigen-specific, functionally suppressive regulatory T cells in the small intestine lamina propria. Two intravenous doses of allergen encapsulated in poly(lactide-co-glycolide) nanoparticles (NPs) significantly reduces oral food challenge (OFC)-induced anaphylaxis. Importantly, NP treatment alters the fates of pathogenic allergen-specific Th2 cells, reprogramming these cells toward CD25+FoxP3+ regulatory and CD73+FR4+ anergic phenotypes. NP-mediated reductions in the frequency of effector cells in the gut and mast cell degranulation following OFC are also demonstrated. These studies reveal mechanisms by which an allergen-encapsulating NP therapy and, more broadly, allergen-specific immunotherapies, can rapidly attenuate allergic responses by targeting pathogenic Th2 cells.

Enhanced mucosal B- and T-cell responses against SARS-CoV-2 after heterologous intramuscular mRNA prime/intranasal protein boost vaccination with a combination adjuvant.

Current COVID-19 mRNA vaccines delivered intramuscularly (IM) induce effective systemic immunity, but with suboptimal immunity at mucosal sites, limiting their ability to impart sterilizing immunity. There is strong interest in rerouting immune responses induced in the periphery by parenteral vaccination to the portal entry site of respiratory viruses, such as SARS-CoV-2, by mucosal vaccination. We previously demonstrated the combination adjuvant, NE/IVT, consisting of a nanoemulsion (NE) and an RNA-based RIG-I agonist (IVT) induces potent systemic and mucosal immune responses in protein-based SARS-CoV-2 vaccines administered intranasally (IN). Herein, we demonstrate priming IM with mRNA followed by heterologous IN boosting with NE/IVT adjuvanted recombinant antigen induces strong mucosal and systemic antibody responses and enhances antigen-specific T cell responses in mucosa-draining lymph nodes compared to IM/IM and IN/IN prime/boost regimens. While all regimens induced cross-neutralizing antibodies against divergent variants and sterilizing immunity in the lungs of challenged mice, mucosal vaccination, either as homologous prime/boost or heterologous IN boost after IM mRNA prime was required to impart sterilizing immunity in the upper respiratory tract. Our data demonstrate the benefit of hybrid regimens whereby strong immune responses primed via IM vaccination are rerouted by IN vaccination to mucosal sites to provide optimal protection to SARS-CoV-2.

Multicomponent intranasal adjuvant for mucosal and durable systemic SARS-CoV-2 immunity in young and aged mice.

Multiple FDA-approved SARS-CoV-2 vaccines currently provide excellent protection against severe disease. Despite this, immunity can wane relatively fast, particularly in the elderly and novel viral variants capable of evading infection- and vaccination-induced immunity continue to emerge. Intranasal (IN) vaccination more effectively induces mucosal immune responses than parenteral vaccines, which would improve protection and reduce viral transmission. Here, we developed a rationally designed IN adjuvant consisting of a combined nanoemulsion (NE)-based adjuvant and an RNA-based RIG-I agonist (IVT DI) to drive more robust, broadly protective antibody and T cell responses. We previously demonstrated this combination adjuvant (NE/IVT) potently induces protective immunity through synergistic activation of an array of innate receptors. We now demonstrate that NE/IVT with the SARS-CoV-2 receptor binding domain (RBD), induces robust and durable humoral, mucosal, and cellular immune responses of equivalent magnitude and quality in young and aged mice. This contrasted with the MF59-like intramuscular adjuvant, Addavax, which showed a decrease in immunogenicity with age. Robust antigen-specific IFN-γ/IL-2/TNF-α was induced in both young and aged NE/IVT-immunized animals, which is significant as their reduced production is associated with suboptimal protective immunity in the elderly. These findings highlight the potential of adjuvanted mucosal vaccines for improving protection against COVID-19.

Retinoic Acid Signaling Is Required for Dendritic Cell Maturation and the Induction of T Cell Immunity.

Vitamin A and its biologically active metabolites, all-trans and 9-cis retinoic acid (RA), are thought to be important in generating and modulating immune function. However, RA modulates the function of many types of immune cells, and its specific role in dendritic cell (DC) activation, Ag presentation, and T cell effector function has not been fully characterized. Because RA works primarily through RA receptor (RAR)α, we examined mice with a myeloid cell-specific defect in RA signaling. These transgenic mice have a CD11c-cre-driven expression of a truncated form of RARα that specifically blocks the signaling of all forms of RARs in myeloid cells. This defect results in abnormal DC function, with impaired DC maturation and activation, and reduced Ag uptake and processing. These DC abnormalities were associated with a reduced ability to mount Ag-specific T cell responses to immunization despite having normally functioning T cells. In contrast, the loss of DC-specific RA signaling did not significantly alter levels of Ag-specific Abs postimmunization and resulted in an increase in bronchial IgA. Our findings indicate that RA signaling in DCs is crucial for immune activation, and its absence impairs the development of Ag-specific effector functions of T cell immunity.

A multicomponent intranasal adjuvant drives durable humoral, cellular, and mucosal immune responses to SARS-CoV-2 in young and aged mice.

Multiple FDA-approved SARS-CoV-2 vaccines provide excellent protection against severe disease. Despite this, immunity can wane relatively fast, particularly in the elderly and novel viral variants capable of evading infection- and vaccination-induced immunity continue to emerge. Intranasal (IN) vaccination more effectively induces mucosal immune responses than parenteral vaccines, which would improve protection and reduce viral transmission. Here, we developed a rationally designed IN adjuvant consisting of a combined nanoemulsion (NE)-based adjuvant and an RNA-based RIG-I agonist (IVT DI) to drive more robust, broadly protective antibody and T cell responses. We previously demonstrated this combination adjuvant (NE/IVT) potently induces protective immunity through synergistic activation of an array of innate receptors. We now demonstrate that NE/IVT with the SARS-CoV-2 receptor binding domain (RBD), induces robust and durable humoral, mucosal, and cellular immune responses of equivalent magnitude and quality in young and aged mice. This contrasted with the MF59-like intramuscular adjuvant, Addavax, which showed a marked decrease in immunogenicity with age. Robust antigen-specific IFNγ/IL-2/TNF-α was induced in both young and aged NE/IVT-immunized animals, which is significant as their reduced production is associated with suboptimal protective immunity in the elderly. These findings highlight the potential of adjuvanted mucosal vaccines for improving protection against COVID-19.

Masked Delivery of Allergen in Nanoparticles Safely Attenuates Anaphylactic Response in Murine Models of Peanut Allergy.

Food allergy is a growing health concern worldwide. Current allergen-specific immunotherapy (AIT) approaches require frequent dosing over extended periods of time and may induce anaphylaxis due to allergen-effector cell interactions. A critical need remains to develop novel approaches that refine AIT for the treatment of food allergies. Previous studies show that poly(lactide-co-glycolide) (PLG) nanoscale particles (NP) effectively suppress Th1- and Th17-driven immune pathologies. However, their ability to suppress the distinct Th2-polarized immune responses driving food allergy are unknown. Herein, we describe the safety and efficacy of NPs containing encapsulated peanut allergen in desensitizing murine models of peanut allergy. Peanut extract encapsulation allowed for the safe intravenous delivery of allergen relative to non-encapsulated approaches. Application of 2-3 doses, without the need for dose escalation, was sufficient to achieve prophylactic and therapeutic efficacy, which correlated with suppression of Th2-mediated disease and reduced mast cell degranulation. Efficacy was associated with strong reductions in a broad panel of Th1, Th2, and Th17 cytokines. These results demonstrate the ability of PLG NPs to suppress allergen-specific immune responses to induce a more tolerogenic phenotype, conferring protection from intragastric allergen challenge. These promising studies represent a step forward in the development of improved immunotherapies for food allergy.

Targeted allergen-specific immunotherapy within the skin improves allergen delivery to induce desensitization to peanut.

Aim: Epicutaneous immunotherapy (EPIT) with peanut has been demonstrated to be safe but efficacy may be limited by allergen uptake through the skin barrier. To enhance allergen uptake into the skin, the authors used peanut-coated microneedles and compared them with EPIT in a peanut allergy mouse model. Methods: Sensitized mice were treated with peanut-coated microneedles or peanut-EPIT and then challenged with peanut to determine protection. Results: Treatment with peanut-coated microneedles was safe and showed enhanced desensitization to peanut compared with peanut-EPIT administered via a similar schedule. Protection was associated with reduced Th2 immune responses and mast cell accumulation in the intestine. Conclusion: Peanut-coated microneedles have the potential to present a safe method of improving allergen delivery for cutaneous immunotherapy.

Epicutaneous immunotherapy (EPIT) with peanut has been demonstrated to be safe but efficacy has been varied. The tight barrier provided by the skin may limit the amount of allergen taken up through the skin and thus reduce efficacy. The authors evaluated a microneedle-based approach to improve the amount of allergen deposited into the skin to improve efficacy. Mice were made allergic to peanut and then treated with peanut-coated microneedles or peanut-EPIT. Mice were challenged with peanut to determine suppression of allergic reactivity. In mice, treatment with peanut-coated microneedles was safe and enhanced desensitization to peanut compared with peanut-EPIT administered via a similar schedule. Peanut-coated microneedles may present a novel method of improving allergen immunotherapy delivered through the skin.

A Combination Adjuvant for the Induction of Potent Antiviral Immune Responses for a Recombinant SARS-CoV-2 Protein Vaccine.

Several SARS-CoV-2 vaccines have received EUAs, but many issues remain unresolved, including duration of conferred immunity and breadth of cross-protection. Adjuvants that enhance and shape adaptive immune responses that confer broad protection against SARS-CoV-2 variants will be pivotal for long-term protection as drift variants continue to emerge. We developed an intranasal, rationally designed adjuvant integrating a nanoemulsion (NE) that activates TLRs and NLRP3 with an RNA agonist of RIG-I (IVT DI). The combination adjuvant with spike protein antigen elicited robust responses to SARS-CoV-2 in mice, with markedly enhanced TH1-biased cellular responses and high virus-neutralizing antibody titers towards both homologous SARS-CoV-2 and a variant harboring the N501Y mutation shared by B1.1.7, B.1.351 and P.1 variants. Furthermore, passive transfer of vaccination-induced antibodies protected naive mice against heterologous viral challenge. NE/IVT DI enables mucosal vaccination, and has the potential to improve the immune profile of a variety of SARS-CoV-2 vaccine candidates to provide effective cross-protection against future drift variants.

Vaccines as therapies for food allergies.

Food allergy is a growing public health epidemic with few available treatments beyond allergen avoidance and rescue medications for accidental exposures. A major focus of therapeutic development for food allergies is allergen-specific immunotherapy (AIT) in which patients are exposed to increasing amounts of allergen in controlled dosing to induce desensitization or tolerance. The work of the past few decades has culminated in the recent FDA approval of a peanut product for oral AIT for peanut allergies. Despite these advances, current AIT protocols are cumbersome, take a long time to reach clinical benefit and often have significant side effects. Therefore, there is a great need to develop new therapeutics for food allergy. One area of research aims to improve AIT through the use of adjuvants which are substances traditionally added to vaccines to stimulate or direct a specific immune response. Adjuvants that induce Th1-polarized and regulatory immune responses while suppressing Th2 immunity have shown the most promise in animal models. The addition of adjuvants to AIT may reduce the amount and frequency of allergen required to achieve clinical benefit and may induce more long-lasting immune responses. In this chapter, we highlight examples of adjuvanted AIT and vaccines in development to treat food allergies.

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

BACKGROUND: Atopic 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. OBJECTIVE: Here, 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. METHODS: Acute 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. RESULTS: Therapeutic 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 RELEVANCE: These 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.

Mucosal Nanoemulsion Allergy Vaccine Suppresses Alarmin Expression and Induces Bystander Suppression of Reactivity to Multiple Food Allergens.

We have demonstrated that intranasal immunotherapy with allergens formulated in a nanoemulsion (NE) mucosal adjuvant suppresses Th2/IgE-mediated allergic responses and protects from allergen challenge in murine food allergy models. Protection conferred by this therapy is associated with strong suppression of allergen specific Th2 cellular immunity and increased Th1 cytokines. Here we extend these studies to examine the effect of NE-allergen immunization in mice sensitized to multiple foods. Mice were sensitized to both egg and peanut and then received NE vaccine formulated with either one or both of these allergens. The animals were then subjected to oral challenges with either egg or peanut to assess reactivity. Immunization with NE formulations containing both egg and peanut markedly reduced reactivity after oral allergen challenge with either allergen. Interestingly, mice that received the vaccine containing only peanut also had reduced reactivity to challenge with egg. Protection from oral allergen challenge was achieved despite the persistence of allergen-specific IgE and was associated with strong suppression of both Th2-polarized immune responses, alarmins and type 2 innate lymphoid cells (ILC2). NE-induced bystander suppression of reactivity required IFN-γ and the presence of an allergen in the NE vaccine. These results demonstrate that anaphylactic reactions to food allergens can be suppressed using allergen-specific immunotherapy without having to eliminate allergen-specific IgE and suggests that modulation of Th2 immunity towards one allergen may induce bystander effects that suppress reactivity to other allergens through the induction of IFN-γ and suppression of alarmins in the intestine. In addition, these data suggest that a NE vaccine for a single food allergen may lead to a global suppression of allergic responses to multiple foods.

A Combination Adjuvant for the Induction of Potent Antiviral Immune Responses for a Recombinant SARS-CoV-2 Protein Vaccine.

Several SARS-CoV-2 vaccines have received EUAs, but many issues remain unresolved, including duration of conferred immunity and breadth of cross-protection. Adjuvants that enhance and shape adaptive immune responses that confer broad protection against SARS-CoV-2 variants will be pivotal for long-term protection. We developed an intranasal, rationally designed adjuvant integrating a nanoemulsion (NE) that activates TLRs and NLRP3 with an RNA agonist of RIG-I (IVT DI). The combination adjuvant with spike protein antigen elicited robust responses to SARS-CoV-2 in mice, with markedly enhanced T H 1-biased cellular responses and high virus-neutralizing antibody titers towards both homologous SARS-CoV-2 and a variant harboring the N501Y mutation shared by B1.1.7, B.1.351 and P.1 variants. Furthermore, passive transfer of vaccination-induced antibodies protected naive mice against heterologous viral challenge. NE/IVT DI enables mucosal vaccination, and has the potential to improve the immune profile of a variety of SARS-CoV-2 vaccine candidates to provide effective cross-protection against future drift variants.

Combined Intranasal Nanoemulsion and RIG-I Activating RNA Adjuvants Enhance Mucosal, Humoral, and Cellular Immunity to Influenza Virus.

Current influenza virus vaccines are focused on humoral immunity and are limited by the short duration of protection, narrow cross-strain efficacy, and suboptimal immunogenicity. Here, we combined two chemically and biologically distinct adjuvants, an oil-in-water nanoemulsion (NE) and RNA-based agonists of RIG-I, to determine whether the diverse mechanisms of these adjuvants could lead to improved immunogenicity and breadth of protection against the influenza virus. NE activates TLRs, stimulates immunogenic apoptosis, and enhances cellular antigen uptake, leading to a balanced TH1/TH2/TH17 response when administered intranasally. RIG-I agonists included RNAs derived from Sendai and influenza viral defective interfering RNAs (IVT DI, 3php, respectively) and RIG-I/TLR3 agonist, poly(I:C) (pIC), which induce IFN-Is and TH1-polarized responses. NE/RNA combined adjuvants potentially allow for costimulation of multiple innate immune receptor pathways, more closely mimicking patterns of activation occurring during natural viral infection. Mice intranasally immunized with inactivated A/Puerto Rico/8/1934 (H1N1) (PR/8) adjuvanted with NE/IVT DI or NE/3php (but not NE/pIC) showed synergistic enhancement of systemic PR/8-specific IgG with significantly greater avidity and virus neutralization activity than the individual adjuvants. Notably, NE/IVT DI induced protective neutralizing titers after a single immunization. Hemagglutinin stem-specific antibodies were also improved, allowing recognition of heterologous and heterosubtypic hemagglutinins. All NE/RNAs elicited substantial PR/8-specific sIgA. Finally, a unique cellular response with enhanced TH1/TH17 immunity was induced with the NE/RNAs. These results demonstrate that the enhanced immunogenicity of the adjuvant combinations was synergistic and not simply additive, highlighting the potential value of a combined adjuvant approach for improving the efficacy of vaccination against the influenza virus.

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

BACKGROUND: Immunotherapy 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. METHODS: Six 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. RESULTS: The 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. CONCLUSION: The 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.

Nanoemulsion Adjuvant Augments Retinaldehyde Dehydrogenase Activity in Dendritic Cells via MyD88 Pathway.

Mucosal surfaces are the primary point of entry for many infectious agents and mucosal immune responses serve as the primary defense to these pathogens. In order to mount an effective mucosal immune response, it is important to induce T cell homing to mucosal surfaces. Conventional vaccine adjuvants induce strong systemic immunity but often fail to produce mucosal immunity. We have developed an oil-in-water nanoemulsion (NE) adjuvant that provides mucosal immunity and efficient protection against mucosal pathogens when administered as part of an intranasal vaccine. In the present study, we demonstrate that intranasal immunization with NE indirectly activates the retinaldehyde dehydrogenase (RALDH) activity in dendritic cells through epithelial cell activity leading to SIgA as well as potent cellular responses and expression of α4ß7 and CCR9 gut homing receptors on T cells. Confirming these findings, ex-vivo stimulation of splenocytes from NE nasally immunized animals showed increase in Th1/Th17 cytokines while suppressing Th2 responses. In examining mechanisms underlying this activation NE activated RALDH via MyD88 dependent pathways in DCs but did not activate the retinoic acid receptor directly. These results suggest that RALDH immune activities can be achieved by epithelial activation without direct RAR activation, which has significant implications for understanding mucosal immunity and the design of mucosal vaccines.

Nanoemulsion adjuvant-driven redirection of TH2 immunity inhibits allergic reactions in murine models of peanut allergy.

BACKGROUND: Immunotherapy for food allergies involves progressive increased exposures to food that result in desensitization to food allergens in some subjects but not tolerance to the food. Therefore new approaches to suppress allergic immunity to food are necessary. Previously, we demonstrated that intranasal immunization with a nanoemulsion (NE) adjuvant induces robust mucosal antibody and TH17-polarized immunity, as well as systemic TH1-biased cellular immunity with suppression of pre-existing TH2-biased immunity. OBJECTIVE: We hypothesized that immunization with food in conjunction with the nanoemulsion adjuvant could lead to modulation of allergic reactions in food allergy by altering pre-existing allergic immunity and enhancing mucosal immunity. METHODS: Mice were sensitized to peanut with aluminum hydroxide or cholera toxin. The animals were then administered 3 monthly intranasal immunizations with peanut in the nanoemulsion adjuvant or saline. Mice were then challenged with peanut to examine allergen reactivity. RESULTS: The NE intranasal immunizations resulted in marked decreases in TH2 cytokine, IgG1, and IgE levels, whereas TH1 and mucosal TH17 immune responses were increased. After allergen challenge, these mice showed significant reductions in allergic hypersensitivity. Additionally, the NE immunizations significantly increased antigen-specific IL-10 production and regulatory T-cell counts, and the protection induced by NE was dependent in part on IL-10. Control animals immunized with intranasal peanut in saline had no modulation of their allergic response. CONCLUSIONS: NE adjuvant-mediated induction of mucosal TH17 and systemic TH1-biased immunity can suppress TH2-mediated allergy through multiple mechanisms and protect against anaphylaxis. These results suggest the potential therapeutic utility of this approach in the setting of food allergy.

Intranasal nanoemulsion-based inactivated respiratory syncytial virus vaccines protect against viral challenge in cotton rats.

Respiratory Syncytial Virus is a leading cause of bronchiolitis and pneumonia in infants, the elderly and individuals with compromised immune systems. Despite decades of research, there is currently no available vaccine for RSV. Our group has previously demonstrated that intranasal immunization of mice with RSV inactivated by and adjuvanted with W805EC nanoemulsion elicits robust humoral and cellular immune responses, resulting in protection against RSV infection. This protection was achieved without the induction of airway hyper-reactivity or a Th2-skewed immune response. The cotton rat Sigmodon hispidus has been used for years as an excellent small animal model of RSV disease. Thus, we extended these rodent studies to the more permissive cotton rat model. Intranasal immunization of the nanoemulsion-adjuvanted RSV vaccines induced high antibody titers and a robust Th1-skewed cellular response. Importantly, vaccination provided sterilizing cross-protective immunity against a heterologous RSV challenge and did not induce marked or severe histological effects or eosinophilia in the lung after viral challenge. Overall, these data demonstrate that nanoemulsion-formulated whole RSV vaccines are both safe and effective for immunization in multiple animal models.

Migrating human neutrophils exhibit dynamic spatiotemporal variation in membrane lipid organization.

Highly ordered sphingolipid-enriched lipid raft microdomains (LRMs) within plasma membranes purportedly function as specialized signaling platforms. Leukocyte migration is believed to entail LRM redistribution, but progress in studying LRMs in situ during cell movement has been limited. By using an improved method for imaging the spectral shift of the environmentally sensitive probe, laurdan (expressed as a generalized polarization function), the plasma membrane order (i.e., tight packing of membrane bilayer lipids) of human polymorphonuclear neutrophils (PMNs) was mapped in real time during migration. Morphologically polarized PMNs exhibited prominent LRM clusters at the uropod, where in every instance membrane order was found to oscillate with mean periodicities of 37.0 ± 1.46 and 149.9 ± 9.0 seconds (P < 0.01). LRM aggregates were also demonstrated in punctate and clustered distributions of nonpolarized cells and transiently at the lamellipodia of polarized PMNs. Cellular polarization was not accompanied by an overall increase in membrane order. LRM disorganization with methyl-ß-cyclodextrin had small negative effects on cell velocity, but it abrogated directionally biased migration toward chemotactic gradients of FMLP or leukotriene B(4). LRMs disruption also caused redistribution of Rac 1/2 GTPase and GM3 ganglioside away from the lamellipodium, as well as extension of multiple pseudopods simultaneously or in rapid succession, rather than formation of a defined leading edge. Thus, we demonstrate that the plasma membrane order of migrating PMNs changes dynamically, with prominent oscillations consistently seen at the uropod. These findings solidify the existence of rapidly reorganizing LRMs in situ and support a role for LRMs in chemotaxin responsiveness.

Pre-clinical evaluation of a novel nanoemulsion-based hepatitis B mucosal vaccine.

BACKGROUND: 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. METHODOLOGY AND PRINCIPAL FINDINGS: 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. CONCLUSIONS: 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.

Nasal immunization with a recombinant HIV gp120 and nanoemulsion adjuvant produces Th1 polarized responses and neutralizing antibodies to primary HIV type 1 isolates.

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-gamma 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.