Influenza subunit vaccine coated microneedle patches elicit comparable immune responses to intramuscular injection in guinea pigs.

Delivery of influenza vaccine using innovative approaches such as microneedles has been researched extensively in the past decade. In this study we present concentration followed by formulation and coating of monobulks from 2008/2009 seasonal vaccine on to 3M's solid microstructured transdermal system (sMTS) by a GMP-scalable process. The hemagglutinin (HA) in monobulks was concentrated by tangential flow filtration (TFF) to achieve HA concentrations as high as 20mg/ml. The stability of the coated antigens was evaluated by the functional assay, single radial immunodiffusion (SRID). The data generated show stability of the coated antigen upon storage at 4°C and room temperature in the presence of desiccant for at least 8 weeks. Freeze-thaw stability data indicate the stability of the coated antigen in stressed conditions. The vaccine coated microstructures were evaluated in vivo in a guinea pig model, and resulted in immune titers comparable to the traditional trivalent vaccine administered intramuscularly. The data presented indicate the potential use of the technology in delivery of influenza vaccine. This paper also addresses the key issues of stability of coated antigen, reproducibility and scalability of the processes used in preparation of influenza vaccine coated microneedle patches that are important in developing a successful product.

The mechanism of action of MF59 - an innately attractive adjuvant formulation.

MF59 is a safe and effective vaccine adjuvant which was originally approved to be included in a licensed influenza vaccine to be used in the elderly in Europe in 1997. The MF59 adjuvanted influenza vaccine (Fluad™) is now licensed in more than 20 countries worldwide and more than 85 million doses have been administered. More recently the vaccine adjuvant has also been shown to be safe and effective in young children and resulted in a significant increase in influenza vaccine efficacy in a controlled clinical trial in Europe. Since the early days of its discovery we have explored the mechanism of action of MF59, using a variety of available techniques. In recent years we have explored more thoroughly the mechanism of action using new and more sophisticated techniques. It is remarkable how consistent the data has been, using a variety of different approaches both in several small animal models and also using human immune cells in vitro. Here we present a summary of all the work performed to date on the mechanism of action of MF59 and we present a unified theory based on the accumulated data of how it exerts its adjuvant effects. A key element of the mechanism of action appears to be the creation of a transient 'immunocompetent' local environment at the injection site, resulting in the recruitment of key immune cells, which are able to take up antigen and adjuvant and transport them to the local lymph nodes, where the immune response is induced. This recruitment appears to be triggered by the induction of a chemokine driven gradient by the impact of MF59 on local cells, which are activated to secrete further chemokines, which are recruitment factors for more immune cells.

MF59 is a safe and potent vaccine adjuvant for flu vaccines in humans: what did we learn during its development?

The MF59 adjuvant has been included in a licensed influenza vaccine for a decade. Hence, we have a significant amount of clinical data to establish its potency and safety. We can now reassess our early preclinical studies and determine whether or not they were useful to predict human responses. The main lesson learned is that mouse models can be valuable, but one must ask the right questions and the models must be used appropriately.

Delivery systems and adjuvants for oral vaccines.

The oral route is the ideal means of delivering prophylactic and therapeutic vaccines, offering significant advantages over systemic delivery. Most notably, oral delivery is associated with simple administration and improved safety. In addition, unlike systemic immunisation, oral delivery can induce mucosal immune responses. However, the oral route of vaccine delivery is the most difficult because of the numerous barriers posed by the gastrointestinal tract. To facilitate effective immunisation with peptide and protein vaccines, antigens must be protected, uptake enhanced and the innate immune response activated. Numerous delivery systems and adjuvants have been evaluated for oral vaccine delivery, including live vectors, inert particles and bacterial toxins. Although developments in oral vaccines have been disappointing so far, in terms of the generation of products, the availability of a range of novel delivery systems offers much greater hope for the future development of improved oral vaccines.

Immunological implications of the use of plant lectins for drug and vaccine targeting to the gastrointestinal tract.

Plant lectins are under consideration as targeting agents to enhance the efficacy of orally administered drugs and vaccines. A significant issue that must be considered is the immunogenicity of these molecules since an immune response to the targeting agent may interfere with its ability to interact with the epithelium. In contrast, the ability of certain lectins to activate the immune system may be exploited in the delivery of vaccines. We previously demonstrated that plant lectins vary widely in their immunogenicity and in particular that mistletoe lectins (ML) I, II and II (MLI, MLII, MLIII) are potent immunogens when administered nasotracheally. Here, we measured immune responses following oral delivery of the MLs and assessed their ability to enhance responses to a co-administered antigen to determine if the molecules possess adjuvant activity. Oral administration of the lectins induced potent lectin-specific systemic and mucosal antibody responses. In addition, each of the three lectins possessed adjuvant activity when delivered orally together with ovalbumin (OVA). The lectins enhanced both serum and mucosal antibody responses to the co-delivered antigen. This shows for the first time that MLI, MLII and MLIII possess adjuvant activity when administered orally and may provide a platform for the generation of effective mucosal adjuvants.

Mistletoe lectins enhance immune responses to intranasally co-administered herpes simplex virus glycoprotein D2.

The mucosal adjuvant properties of the three type 2 ribosome-inactivating proteins (RIPs) from the European mistletoe, Viscum album L., were investigated. Mistletoe lectins were compared with cholera toxin (CT) as adjuvants when delivered nasotracheally together with herpes simplex virus glycoprotein D2 (gD2). All three mistletoe lectins (MLI, MLII, MLIII) were potent mucosal adjuvants. Co-administration of MLI, MLII or MLIII with gD2 led to significantly higher levels of gD2-specific mucosal immunoglobulin A (IgA) and systemic immunoglobulin G (IgG) antibody than when the antigen was delivered alone. The levels of antibodies induced were similar to those generated in mice immunized with gD2 and the potent mucosal adjuvant CT. Administration of ML1 with gD2 enhanced the antigen-specific splenic T-cell proliferative response. Interleukin-5 (IL-5), but not interferon-gamma (IFN-gamma), was detected in supernatants from splenocytes stimulated in vitro with gD2. This indicates that MLI enhanced type 2 T-helper cell (Th2) responses to the bystander antigen, gD2. Analysis of the gD2- and lectin-specific IgG subclass titres in mice immunized with gD2 and MLI, MLII or MLIII revealed a high ratio of IgG1 : IgG2a, which is compatible with the selective induction of Th2-type immune responses.

Synergistic adjuvant activity of immunostimulatory DNA and oil/water emulsions for immunization with HIV p55 gag antigen.

A synthetic oligonucleotide containing a previously identified adjuvant active CpG DNA sequence was evaluated for its ability to augment antibody and CTL responses to p55 gag from HIV-1 in mice. Surprisingly, the CpG oligonucleotide, although, it had previously been described as the most potent adjuvant sequence in mice for the particulate HbsAg, was ineffective when used in a simple combination with urea-solubilized p55 antigen. However, a potent adjuvant effect was observed with the CpG sequence when it was formulated with emulsions. Enhancement of antibody titer by CpG emulsion formulations was observed with urea-solubilized p55 antigen, however, significantly higher titers were obtained with p55 bound to polylactide-co-glycolide microparticles. In both cases IgG2a was enhanced in the presence of CpG. It appears likely that presentation of CpG with emulsions and particulate antigens enhances their delivery into antigen presenting cells (APC) and results in more effective presentation of antigen and adjuvant. To support this hypothesis, preliminary in vitro studies were undertaken to show upregulation of CD86 on mouse bone marrow-derived dendritic cells (BMDC) in vitro, following incubation with CpG formulations.

Recent developments in adjuvants for vaccines against infectious diseases.

New generation vaccines, particularly those based on recombinant proteins and DNA, are likely to be less reactogenic than traditional vaccines, but are also less immunogenic. Therefore, there is an urgent need for the development of new and improved vaccine adjuvants. Adjuvants can be broadly separated into two classes, based on their principal mechanisms of action; vaccine delivery systems and 'immunostimulatory adjuvants'. Vaccine delivery systems are generally particulate e.g. emulsions, microparticles, iscoms and liposomes, and mainly function to target associated antigens into antigen presenting cells (APC). In contrast, immunostimulatory adjuvants are predominantly derived from pathogens and often represent pathogen associated molecular patterns (PAMP) e.g. LPS, MPL, CpG DNA, which activate cells of the innate immune system. Once activated, cells of innate immunity drive and focus the acquired immune response. In some studies, delivery systems and immunostimulatory agents have been combined to prepare adjuvant delivery systems, which are designed for more effective delivery of the immunostimulatory adjuvant into APC. Recent progress in innate immunity is beginning to yield insight into the initiation of immune responses and the ways in which immunostimulatory adjuvants may enhance this process. However, a rational approach to the development of new and more effective vaccine adjuvants will require much further work to better define the mechanisms of action of existing adjuvants. The discovery of more potent adjuvants may allow the development of vaccines against infectious agents such as HIV which do not naturally elicit protective immunity. New adjuvants may also allow vaccines to be delivered mucosally.

Microparticles for intranasal immunization.

Of the several routes available for mucosal immunization, the nasal route is particularly attractive because of ease of administration and the induction of potent immune responses, particularly in the respiratory and genitourinary tracts. However, adjuvants and delivery systems are required to enhance immune responses following nasal immunization. This review focuses on the use of microparticles as adjuvants and delivery systems for protein and DNA vaccines for nasal immunization. In particular we discuss our own work on poly(lactide co-glycolide) (PLG) microparticles with entrapped protein or adsorbed DNA as a vaccine delivery system. The possible mechanisms involved in the enhancement of immune responses through the use of DNA adsorbed onto PLG microparticles are also discussed.

Oral administration of influenza vaccine in combination with the adjuvants LT-K63 and LT-R72 induces potent immune responses comparable to or stronger than traditional intramuscular immunization.

Mucosal immunization strategies are actively being pursued in the hopes of improving the efficacy of vaccines against the influenza virus. Our group investigated the oral immunization of mice via intragastric gavage with influenza hemagglutinin (HA) combined with mutant Escherichia coli heat-labile enterotoxins K63 (LT-K63) and R72 (LT-R72). These oral immunizations resulted in potent serum antibody and HA inhibition titers, in some cases stronger than those obtained with traditional intramuscular administration, in addition to HA-specific immunoglobulin A in the saliva and nasal secretions. This study demonstrates that it may be possible to develop effective oral influenza vaccines.

A novel bioadhesive intranasal delivery system for inactivated influenza vaccines.

The aim of the current studies was to evaluate a bioadhesive delivery system for intranasal administration of a flu vaccine, in combination with a mucosal adjuvant (LTK63). A commercially available influenza vaccine, containing hemagglutinin (HA) from influenza/A Johannesberg H1N1 1996, and LTK63 or LTR72 adjuvants, which are genetically detoxified derivatives of heat labile enterotoxin from Escherichia coli, were administered IN in a bioadhesive delivery system, which comprised esterified hyaluronic acid (HYAFF) microspheres, to mice, rabbits and micro-pigs at days 0 and 28. For comparison, additional groups of animals were immunized intranasally with the HA vaccine alone, with soluble HA+LTK63, or IM with HA. In all three species, the groups of animals receiving IN immunization with the bioadhesive microsphere formulations, including LT mutants, showed significantly enhanced serum IgG responses (P<0.05) and higher hemagglutination inhibition (HI) titers in comparison to the other groups. In addition, the bioadhesive formulation also showed a significantly enhanced nasal wash IgA response (P<0.05). Most encouragingly, in pigs, the bioadhesive microsphere vaccine delivery system induced serum immune responses following IN immunization, which were significantly more potent than those induced by traditional IM immunization at the same vaccine dose (P<0.05).

Characterization of hepatitis C virus core-specific immune responses primed in rhesus macaques by a nonclassical ISCOM vaccine.

Current therapies for the treatment of hepatitis C virus (HCV) infection are only effective in a restricted number of patients. Cellular immune responses, particularly those mediated by CD8(+) CTLs, are thought to play a role in the control of infection and the response to antiviral therapies. Because the Core protein is the most conserved HCV protein among genotypes, we evaluated the ability of a Core prototype vaccine to prime cellular immune responses in rhesus macaques. Since there are serious concerns about using a genetic vaccine encoding for Core, this vaccine was a nonclassical ISCOM formulation in which the Core protein was adsorbed onto (not entrapped within) the ISCOMATRIX, resulting in approximately 1-microm particulates (as opposed to 40 nm for classical ISCOM formulations). We report that this Core-ISCOM prototype vaccine primed strong CD4(+) and CD8(+) T cell responses. Using intracellular staining for cytokines, we show that in immunized animals 0.30-0.71 and 0.32-2.21% of the circulating CD8(+) and CD4(+) T cells, respectively, were specific for naturally processed HCV Core peptides. Furthermore, this vaccine elicited a Th0-type response and induced a high titer of Abs against Core and long-lived cellular immune responses. Finally, we provide evidence that Core-ISCOM could serve as an adjuvant for the HCV envelope protein E1E2. Thus, these data provide evidence that Core-ISCOM is effective at inducing cellular and humoral immune responses in nonhuman primates.

The identification of plant lectins with mucosal adjuvant activity.

To date, the most potent mucosal vaccine adjuvants to be identified have been bacterial toxins. The present data demonstrate that the type 2 ribosome-inactivating protein (type 2 RIP), mistletoe lectin I (ML-I) is a strong mucosal adjuvant of plant origin. A number of plant lectins were investigated as intranasal (i.n.) coadjuvants for a bystander protein, ovalbumin (OVA). As a positive control, a potent mucosal adjuvant, cholera toxin (CT), was used. Co-administration of ML-I or CT with OVA stimulated high titres of OVA-specific serum immunoglobulin G (IgG) in addition to OVA-specific IgA in mucosal secretions. CT and ML-I were also strongly immunogenic, inducing high titres of specific serum IgG and specific IgA at mucosal sites. None of the other plant lectins investigated significantly boosted the response to co-administered OVA. Immunization with phytohaemagglutinin (PHA) plus OVA elicited a lectin-specific response but did not stimulate an enhanced response to OVA compared with the antigen alone. Intranasal delivery of tomato lectin (LEA) elicited a strong lectin-specific systemic and mucosal antibody response but only weakly potentiated the response to co-delivered OVA. In contrast, administration of wheatgerm agglutinin (WGA) or Ulex europaeus lectin 1 (UEA-I) with OVA stimulated a serum IgG response to OVA while the lectin-specific responses (particularly for WGA) were relatively low. Thus, there was not a direct correlation between immunogenicity and adjuvanticity although the strongest adjuvants (CT, ML-I) were also highly immunogenic.

Potency of a genetically detoxified mucosal adjuvant derived from the heat-labile enterotoxin of Escherichia coli (LTK63) is not adversely affected by the presence of preexisting immunity to the adjuvant.

The objective of the current studies was to evaluate whether the potency of a genetically detoxified mucosal adjuvant, derived from heat-labile enterotoxin of Escherichia coli (LTK63), was adversely affected by preexisting immunity. Studies of mice and pigs have involved consecutive intranasal immunization with LTK63 and 2 different vaccines (influenza virus hemagglutinin and a protein-polysaccharide conjugate of Neisseria meningitidis group C). The antibody responses to the vaccines plus LTK63 in naive animals were compared with the responses achieved in animals that previously had been immunized with the alternate vaccine plus LTK63. The data showed that the responses of both animal models to intranasal immunization were not adversely affected by the presence of preexisting immunity to the LTK63 adjuvant.

Recent developments in vaccine delivery systems.

New generation vaccines, particularly those based on recombinant proteins and DNA, are likely to be less reactogenic than traditional vaccines, but are also less immunogenic. Therefore, there is an urgent need for the development of new and improved vaccine adjuvants. Adjuvants can be broadly separated into two classes, based on their principal mechanisms of action; vaccine delivery systems and 'immunostimulatory adjuvants'. Vaccine delivery systems are generally particulate e.g. emulsions, microparticles, iscoms and liposomes, and mainly function to target associated antigens into antigen presenting cells (APC), including macrophages and dendritic cells. This review will focus on recent developments in vaccine delivery systems. Immunostimulatory adjuvants are predominantly derived from pathogens and often represent pathogen associated molecular patterns (PAMP) e.g. LPS, MPL, CpG DNA, which activate cells of the innate immune system. Once activated, cells of innate immunity drive and focus the acquired immune response. In some studies, delivery systems and immunostimulatory agents have been combined for more effective delivery of the immunostimulatory adjuvant into APC. A rational approach to the development of new and more effective vaccine adjuvants will require much further work to better define the mechanisms of action of existing adjuvants. The discovery of more potent adjuvants may allow the development of vaccines against infectious agents such as HIV which do not naturally elicit protective immunity. New adjuvants and delivery system combinations may also allow vaccines to be delivered mucosally.

Novel anionic microparticles are a potent adjuvant for the induction of cytotoxic T lymphocytes against recombinant p55 gag from HIV-1.

Microparticles with entrapped antigens have recently been shown to possess significant potential as vaccine delivery systems and adjuvants. However, the potential of microparticles as adjuvants has been seriously limited by the common problem of degradation and denaturation of antigens following encapsulation and release. To overcome these problems, we have developed a novel way to use microparticles as adjuvants, by the adsorption of proteins onto their surface. Anionic microparticles were prepared through the inclusion of an anionic detergent, sodium dodecyl sulphate (SDS), in the microparticle preparation process. The anionic microparticles were capable of the efficient and reproducible adsorption of recombinant p55 gag protein from HIV-1. Microparticles with adsorbed p55 were capable of inducing potent cytotoxic T lymphocyte responses in mice following intramuscular immunization. In addition, the microparticles also exhibited a potent adjuvant effect for antibody induction against p55.

Microparticles in MF59, a potent adjuvant combination for a recombinant protein vaccine against HIV-1.

Novel adjuvant formulations involving PLG microparticles with entrapped recombinant protein antigens (env gp120 and p24 gag) from human immunodeficiency virus type-1 (HIV-1), dispersed in the emulsion adjuvant MF59 were evaluated as potential HIV-1 vaccine candidates in mice and baboons. In mice, the adjuvant combination induced significantly enhanced antibody responses in comparison to either adjuvant used alone. In addition, the polylactide co-glycolide polymer (PLG) microparticles and MF59 combination induced CTL activity against HIV-1 p24 gag. In baboons, the adjuvant combination induced significantly enhanced antibody titers after a single dose of gp120, but the responses were comparable to gp120 in MF59 alone after boosting. Both MF59+gp120 alone and PLG/gp120 in MF59 induced neutralizing antibodies against a T cell line-adapted (TCLA) strain and a primary isolate of HIV-1. In contrast to the observations with gp120, immunization in baboons with PLG/p24 in MF59 induced significantly enhanced antibody responses after boosting, in comparison to immunization with MF59 alone + p24.

Mucosal immunogenicity of plant lectins in mice.

The mucosal immunogenicity of a number of plant lectins with different sugar specificities was investigated in mice. Following intranasal (i.n.) or oral administration, the systemic and mucosal antibody responses elicited were compared with those induced by a potent mucosal immunogen (cholera toxin; CT) and a poorly immunogenic protein (ovalbumin; OVA). After three oral or i.n. doses of CT, high levels of specific serum antibodies were measured and specific IgA was detected in the serum, saliva, vaginal wash, nasal wash and gut wash of mice. Immunization with OVA elicited low titres of serum IgG but specific IgA was not detected in mucosal secretions. Both oral and i.n. delivery of all five plant lectins investigated ¿Viscum album (mistletoe lectin 1; ML-1), Lycospersicum esculentum (tomato lectin; LEA), Phaseolus vulgaris (PHA), Triticum vulgaris (wheat germ agglutinin (WGA), Ulex europaeus I (UEA-1) stimulated the production of specific serum IgG and IgA antibody after three i. n. or oral doses. Immunization with ML-1 induced high titres of serum IgG and IgA in addition to specific IgA in mucosal secretions. The response to orally delivered ML-1 was comparable to that induced by CT, although a 10-fold higher dose was administered. Immunization with LEA also induced high titres of serum IgG, particularly after i. n. delivery. Low specific IgA titres were also detected to LEA in mucosal secretions. Responses to PHA, WGA and UEA-1 were measured at a relatively low level in the serum, and little or no specific mucosal IgA was detected.

Intranasal immunization of mice with influenza vaccine in combination with the adjuvant LT-R72 induces potent mucosal and serum immunity which is stronger than that with traditional intramuscular immunization.

Immunization of mice by the intranasal route with influenza virus hemagglutinin in combination with the mutant Escherichia coli heat-labile enterotoxin R72 (LT-R72) induced significantly enhanced serum and mucosal antibodies, surpassing, in most cases, responses achieved by traditional intramuscular immunization using inactivated split influenza vaccine. Furthermore, intranasal immunization with LT-R72 induced a potent serum immunoglobulin G2a response, indicating that this adjuvant has Th1 character.