New and safe formulation for scorpion immunotherapy: Comparative study between saponin and FCA adjuvants associated to attenuated venom

Envenoming by scorpion is a major health problem in Maghreb regions as well as in several regions of the world. Immunotherapy is the only effective treatment for scorpion stings. The immune sera are obtained from hyper-immunized animals with a formulation of venom associated to Freund’s Complete Adjuvant (FCA). This formulation seems to protect against several alterations in immunized animals leading to worsening of their health due to added toxicity of native venom and FCA adjuvant. This study aims to provide a more efficient and non-toxic alternative to this formulation. Two formulations of saponin or FCA associated to irradiated venom of Androctonus australis hector (Aah) were used to compare their safety and their efficiency to better enhance the antibody titers against toxic antigens.Both of these formulations were used in immunization schedule of three months. Blood samples were collected every week, cell count, myeloperoxydase (MPO) and eosinophil peroxidase (EPO) activities and specific antibody titers were evaluated. Four months after the last immunization, rabbits were challenged with increased doses of native Aah venom.Results showed that immunization with saponin formulation induced lower inflammatory cell activation as well as reduced MPO and EPO activities compared to that using FCA. The formulation of irradiated venom with saponin seems also to be more efficient in the activation of lymphocytes resulting in higher titers of specific IgG. The immunoprotective effect evaluation showed that the formulation using saponin seems to protected animals until 3 LD50 of native venom compared to that using FCA which protected only until 2 LD50. These results indicate that saponin formulation with irradiated antigen could be more efficient and safe immunizing preparation for the production of sera against scorpion envenomation.     

Development and characterization of a new carrier for vaccine delivery based on calcium-alginate nanoparticles: Safe immunoprotective approach against scorpion envenoming

To enhance humoral defense against diseases, vaccine formulation is routinely prepared to improve immune response. Studies in nanomaterials as a carrier of vaccine delivery are promising and interesting. In this study, attenuated Androctonus australis hector (Aah) venom and its toxic fraction were encapsulated into different formulations inside calcium–alginate nanoparticles (Ca–Alg Nps), and used as a vaccine delivery system against scorpion envenomation. Ca–Alg Nps were prepared by ionic gelation and characterized. An immunization schedule was undertaken in rabbits in order to study how Aah venom entrapped in Ca–Alg Nps might induce protective immunity. Results showed the influence of different parameters on the suitable nanoparticle formation. They also showed no toxicity of free Ca–Alg Nps and a different inflammatory profile depending on the nanovaccine formulations. More interestingly, evaluation of specific IgG titer and IgG1/IgG2a isotype balance revealed a protective effect with the nanoparticles encapsulating the attenuated antigens. Challenge up to 6 LD 50 of native venom, allowed to an important immunoprotection of all immunized rabbits, with no recorded death. Taken together and with respect to the properties of nanoparticles and high immunogenicity, calcium–alginate nanoparticles could be considered as a new promising adjuvant system and a vaccine delivery against scorpion envenomation.     

Immunogenicity and protection-inducing ability of recombinant Plasmodium vivax rhoptry-associated protein 2 in Aotus monkeys: A potential vaccine candidate

Rhoptry proteins have been extensively shown to be important in invasion and parasitophorous vacuole (PV) formation. This work evaluates the immunogenicity and protective efficacy of Plasmodium vivax RAP2 in the non-human Aotus primate model, when expressed as a recombinant molecule in E. coli and formulated in Freund and Alum hydroxide adjuvants. Our results show that rPvRAP2 is immunogenic in both formulations, finding a trend of higher cytokine levels in immunized monkeys, specially in IL-4 levels (using Freund's adjuvant) and IL-5 (using Alum hydroxide). RAP2 is suggested as a P. vivax-vaccine candidate since immunized monkeys exhibited lower parasitemias than control groups after being experimentally challenged with the P. vivax VCG-I strain.     

Venom conjugated polylactide applied as biocompatible material for passive and active immunotherapy against scorpion envenomation

Scorpion envenoming represents a public health issue in subtropical regions of the world. Treatment and prevention need to promote antitoxin immunity. Preserving antigenic presentation while removing toxin effect remains a major challenge in toxin vaccine development. Among particulate adjuvant, particles prepared with poly (d,l-lactide) polymer are the most extensively investigated due to their excellent biocompatibility and biodegradability. The aim of this study is to develop surfactant-free PLA nanoparticles that safely deliver venom toxic fraction to enhance specific immune response. PLA nanoparticles are coated with AahG50 (AahG50/PLA) and BotG50 (BotG50/PLA): a toxic fraction purified from Androctonus australis hector and Buthus occitanus tunetanus venoms, respectively. Residual toxicities are evaluated following injections of PLA-containing high doses of AahG50 (or BotG50). Immunization trials are performed with the detoxified fraction administered alone without adjuvant. A comparative study of the effect of Freund is also included. The neutralizing capacity of sera is determined in naive mice. Six months later, immunized mice are challenged subcutaneously with increased doses of AahG50.Subcutaneous lethal dose 50 (LD50) of AahG50 and BotG50 is of 575 μg/kg and 1300 μg/kg respectively. By comparison, BotG50/PLA is totally innocuous while 50% of tested mice survive 2875 μg AahG50/kg. Alhydrogel and Freund are not able to detoxify such a high dose. Cross-antigenicity between particulate and soluble fraction is also, ensured. AahG50/PLA and BotG50/PLA induce high antibody levels in mice serum. The neutralizing capacity per mL of anti-venom was 258 μg/mL and 186 μg/mL calculated for anti-AahG50/PLA and anti-BotG50/PLA sera, respectively. Animals immunized with AahG50/PLA are protected against AahG50 injected dose of 3162 μg/kg as opposed all non-immunized mice died at this dose. We find that the detoxification approach based PLA nanoparticles, benefit the immunogenicity and protective efficacy of venom immunogen.     

Intranasal immunization with recombinant gD2 reduces disease severity and mortality following genital challenge with herpes simplex virus type 2 in guinea pigs.

The ability of a genetically detoxified mutant of heat labile enterotoxin (LTK63) to act as a mucosal adjuvant following intranasal immunization with recombinant gD2 has previously been reported in mice [Ugozzoli M, O'Hagan DT, Ott GS. Intranasal immunization of mice with herpes simplex virus type 2 recombinant gD2: the effect of adjuvants on mucosal and serum antibody responses. Immunol 1998;93:563-571.]. In the current studies, these observations were extended to the guinea pig model. Immunized guinea pigs were subsequently challenged intravaginally with HSV-2. Intranasal immunization with gD2 and LTK63 induced a significant reduction in disease severity and a reduction in mortality. However, only intramuscular immunization with a potent adjuvant (MF59) induced protection against the incidence of disease.     

Hemozoin is a potent adjuvant for hemagglutinin split vaccine without pyrogenicity in ferrets

Background

Synthetic hemozoin (sHZ, also known as β-hematin) from monomeric heme is a particle adjuvant which activates antigen-presenting cells (APCs), such as dendritic cells and macrophages, and enhances humoral immune responses to several antigens, including ovalbumin, human serum albumin, and serine repeat antigen 36 of Plasmodium falciparum. In the present study, we evaluated the adjuvanticity and pyrogenicity of sHZ as an adjuvant for seasonal trivalent hemagglutinin split vaccine (SV) for humans using the experimental ferret model.

Method

Ferrets were twice immunized with trivalent SV, SV with sHZ (SV/sHZ) or Fluad, composed of trivalent SV with MF59. Serum hemagglutination inhibition (HI) titers against three viral hemagglutinin (HA) antigens were measured at every week after the immunization. The pyrogenicity of SV/sHZ was examined by monitoring the body temperature of the immunized ferrets. To evaluate the protective efficacy of SV/sHZ, the immunized ferrets were challenged with influenza virus B infection, followed by measurement of viral titers in the nasal cavity and body temperature.

Results

sHZ enhanced HI titers against three viral HA antigens in a dose-dependent manner, to an extent comparable to that of Fluad. The highest dose of sHZ (800 μg) immunized with SV conferred sterile protection against infection with heterologous Influenza B virus, without causing any pyrogenic reaction such as high fever.

Conclusion

In the present study, sHZ enhanced the protective efficacy of SV against influenza infection without inducing pyrogenic reaction, suggesting sHZ to be a promising adjuvant candidate for human SV.     

Formulation of HIV-1 Tat and p24 antigens by PLA nanoparticles or MF59 impacts the breadth, but not the magnitude, of serum and faecal antibody responses in rabbits

Biodegradable nanoparticles coated with proteins represent a promising method for in vivo delivery of vaccines. Here we used a rabbit model to compare quantitatively and qualitatively the antibody responses induced by poly(D,L-lactide) nanoparticles (PLA) and by emulsion adjuvant MF59 using three HIV-1 antigens: p24gag, WT Tat and a mutated, detoxified form of Tat. We could show that all antigens and adjuvants lead to the induction of similar level of IgG titres in serum when injected subcutaneously. p24, but not Tat, could also induce faecal IgG in rabbits when formulated with PLA or MF59. The nature of the adjuvant had consequences on the spectrum of specificity induced, depending on the antigen: PLA adjuvant focussed the anti-p24 response to an immunodominant domain when compared to MF59. With wild-type Tat, no difference between adjuvants was observed in the spectrum of specificity induced. On the opposite, detoxified Tat coated on PLA increased the number of epitopes recognized by serum IgG compared to MF59 adjuvantation. The impact of these qualitative differences depending on the antigen/adjuvant associations will be important to take into account for further designs of vaccinal formulation using particulate adjuvants.     

Recombinant lipidated dengue-3 envelope protein domain III stimulates broad immune responses in mice

The linkage of an immunogen with a toll-like receptor ligand has great potential to induce highly potent immune responses with the initial features of antigen-presenting cell activation. In the current study, we expressed recombinant dengue-3 envelope protein domain III (D3ED III) in lipidated form using an Escherichia coli-based system. The recombinant lipidated dengue-3 envelope protein domain III (LD3ED III) augments the expression levels of IL-12 family cytokines. LD3ED III-immunized mice enhance wide ranges of T cell responses as indicated by IFN-γ, IL-17, IL-21 production. Additionally, LD3ED III-immunized mice increase the frequencies of anti-D3ED III antibody producing cells. The boosted antibody titers cover various IgG isotypes, including IgG1, IgG2a, IgG2b, and IgG3. Importantly, LD3ED III-immunized mice induce neutralizing antibody capacity associated with a reduction of viremia levels after challenges. In contrast, mice that are immunized with D3ED III formulated with aluminum phosphate (D3ED III/Alum) only enhance Th2 responses and boost IgG1 antibody titers. Neither neutralizing antibody responses nor the inhibition of viremia levels after challenge is observed in mice that are immunized with D3ED III/Alum. These results suggest that LD3ED III can induce broad profiles of cellular and humoral immune responses.     

Candidate HIV-1 gp140DeltaV2, Gag and Tat vaccines protect against experimental HIV-1/MuLV challenge

Pre-clinical HIV-1 vaccine protocols, using multiple vaccine modalities and a potent adjuvant were assessed for vaccine efficacy in an experimental HIV-1 challenge model. C57Bl/6 mice were immunized with DNA plasmids encoding HIV-1 gp140, Gag and Tat alone or in combination with the corresponding recombinant proteins formulated in the adjuvant MF59. HIV-1 DNA alone or a DNA prime protein boost schedule resulted in complete protection against challenge with HIV-1/MuLV-infected murine cells. Although HIV-1 protein immunization in combination with MF59 resulted in partial protection, the DNA priming seemed to be crucial for obtaining full protection against the challenge. It is likely that the partial protection seen after immunization with protein alone is, to a certain extent, due to effects of the adjuvant since some animals that received the adjuvant MF59 alone were protected from the challenge. For the most part, antigen-specific cell-mediated immune responses as detected in the spleen (in contrast to responses detected in peripheral blood) of immunized animals appeared to be associated with protection in this study.     

Immunization of woodchucks with adjuvanted sHDAg (p24): immune response and outcome following challenge

The immunogenicity and the protection induced by an hepatitis delta virus (HDV) vaccine consisting of the small nucleoprotein (HDAg) (p24) and adjuvanted with MF59 or Freund's adjuvant (FA) were evaluated in woodchucks chronically infected with woodchuck hepatitis virus (WHV) and challenged with hepatitis delta virus. Humoral and T-cell-mediated responses to HDAg were measured. Anti-HD antibodies appeared earlier in the FA/p24 animals. After challenge, all MF59/p24 vaccinated animals showed a response to HDAg-derived peptides, compared to two of the five FA/p24 animals and one of the control animals. Serum HDV-RNA peak values and persistence were considerably reduced in immunized animals, in comparison to controls. Furthermore, HDV-RNA was absent in autopsy liver tissues of 50% of the MF59/p24 animals, whereas high levels were present in all of the FA/p24 animals and controls. Histological liver analysis performed before and after challenge revealed the presence of acute hepatitis-like lesions only in the controls. Overall, the results suggest that the MF59/p24 vaccine better controls the infection in terms of viral replication and survival.     

Dynamics of APC recruitment at the site of injection following injection of vaccine adjuvants

Vaccines often contain adjuvants to strengthen the response to the vaccine antigen. However, their modes of action at the site of injection (SOI) are poorly understood. Therefore, we assessed the local effects of adjuvant on the innate immune system in mice. We investigated the safe, widely used adjuvants MF59 and aluminum hydroxide (alum), as well as trehalose-6,6′-dibehenate (TDB), Complete Freund’s Adjuvant (CFA) and the Toll-Like-Receptor-ligands lipopolysaccharide (LPS) and Pam3CysSerLys4 (Pam₃CSK₄). We assessed muscle immune cell infiltration after adjuvant injection and observed 16 h post immunization (hpi) an increased influx with CFA, MF59 and TDB, but not with alum, LPS or Pam₃CSK₄. An elevated influx with the latter three became visible only 72 hpi. Contribution of granulocytes, macrophages and dendritic cells to the influx differed per adjuvant and in time. Adjuvants generally induced a local pro-inflammatory micro-milieu that was transient except for CFA and TDB. The gene expression of CXCL-1, CCL-2 and CCL-5, involved in recruitment of immune cells, varied per adjuvant and corresponded grossly with the observed influx of granulocytes and monocytes/macrophages. Muscles injected with CFA or MF59 (when co-injected with peptide) resulted in APC ex vivo capable to induce proliferation of peptide-specific T-cells. By adding in vitro an excess of peptide to the APC/T cell co-cultures, we observed an adjuvant-enhanced co-stimulation or antigen presentation by APC after CFA- but not MF59-injection. After TDB-injection this effect was observed only at 72 hpi, but not 24 hpi. Thus the cellular influx profile and the local cytokine and chemokine micro-milieu in the muscle were strongly influenced by the type of adjuvant. Additionally, the capacity of muscle APC to load and present antigen was affected by the adjuvant. These findings may assist the development of novel adjuvanted vaccines in a more rational manner.     

The recombinant gut-associated M17 leucine aminopeptidase in combination with different adjuvants confers a high level of protection against Fasciola hepatica infection in sheep

Fasciola hepatica M17 leucine aminopeptidase (FhLAP) is thought to play a role in catabolizing peptides generated by the concerted activity of gut-associated endopeptidases on host polypeptides, thus releasing amino acids to be used in protein anabolism. In this study, a recombinant functional form of this homo hexameric metallopeptidase produced in Escherichia coli was used in combination with adjuvants of different types in a vaccination trial in Corriedale sheep against experimental challenge with F. hepatica metacercariae. The experimental assay consisted of 6 groups of 10 animals; 5 of the groups (1-5) were subcutaneously inoculated at weeks 0 and 4 with100 μg of rFhLAP mixed with Freund's complete plus incomplete adjuvant (group 1), Alum (group 2), Adyuvac 50 (group 3), DEAE-D (group 4) and Ribi (group 5); the adjuvant control group (group 6) received Freund's adjuvant. Two weeks after the booster, the sheep were orally challenged with 200 metacercariae. Immunization with rFhLAP induced significant reduction in fluke burdens in all vaccinated groups: 83.8% in the Freund's group, 86.7% in the Alum group, 74.4% in the Adyuvac 50 group, 49.8% in the Ribi group and 49.5% in the DEAE-D group compared to the adjuvant control group. Morphometric analysis of recovered liver flukes showed no significant size modifications in the different vaccination groups. All vaccine preparations elicited specific IgG, IgG1 and IgG2 responses. This study shows that a liver fluke vaccine based on rFhLAP combined with different adjuvants significantly reduced worm burden in a ruminant species that was high in animals that received the enzyme along with the commercially approved adjuvants Alum and Adyuvac 50.     

Induction of protection in mice against a respiratory challenge by a vaccine formulated with the Chlamydia major outer membrane protein adjuvanted with IC31®

IC31^®, a novel adjuvant, has been shown to be effective by increasing the levels of IFN-γ in animal models when delivered with several antigens. Here, we tested the ability of IC31^®, to enhance the protective ability of the Chlamydia trachomatis native major outer membrane protein (nMOMP). BALB/c mice were immunized by the intramuscular (i.m.) and subcutaneous (s.c.) routes with nMOMP + IC31^®. Another group of animals was immunized with nMOMP + Alum and as a negative control mice were immunized with ovalbumin (Ova) + IC31^®. Animals immunized with nMOMP + IC31^® developed high Chlamydia-specific IgG titers. The serum levels of IgG1 were higher than those of the IgG2a. T cells, from the spleens of mice immunized with IC31^®-adjuvanted nMOMP demonstrated a strong lymphoproliferative reaction to Chlamydia elementary bodies (EB) compared with the groups immunized with nMOMP + Alum or Ova + IC31^®. A similar comparison between these groups of mice revealed that the levels of IFN-γ in the supernatants from stimulated T-cells were significantly higher in animals immunized with nMOMP + IC31^®. Following an intranasal challenge with C. trachomatis, the mice immunized with IC31^®-adjuvanted nMOMP showed significant protection. The change in body weight, an indication of the severity of the infection, was significantly less reduced in mice immunized with nMOMP + IC31^®. Furthermore, the weight of the lungs, as well as the pulmonary Chlamydia burden, was significantly lower in the animals immunized with nMOMP + IC31^® when compared with the groups immunized with nMOMP + Alum or with Ova + IC31^®. In conclusion, these results provide the rationale for further preclinical testing of IC31^® using other chlamydial antigens, and support the potential evaluation of this adjuvant in human vaccines against Chlamydia.     

A listeriolysin O subunit vaccine is protective against Listeria monocytogenes

Listeria monocytogenes is a facultative intracellular pathogen responsible for the life-threatening disease listeriosis. The pore-forming toxin listeriolysin O (LLO) is a critical virulence factor that plays a major role in the L. monocytogenes intracellular lifecycle and is indispensable for pathogenesis. LLO is also a dominant antigen for T cells involved in sterilizing immunity and it was proposed that LLO acts as a T cell adjuvant. In this work, we generated a novel full-length LLO toxoid (LLO^T) in which the cholesterol-recognition motif, a threonine-leucine pair located at the tip of the LLO C-terminal domain, was substituted with two glycine residues. We showed that LLO^T lost its ability to bind cholesterol and to form pores. Importantly, LLO^T retained binding to the surface of epithelial cells and macrophages, suggesting that it could efficiently be captured by antigen-presenting cells. We then determined if LLO^T can be used as an antigen and adjuvant to protect mice from L. monocytogenes infection. Mice were immunized with LLO^T alone or together with cholera toxin or Alum as adjuvants. We found that mice immunized with LLO^T alone or in combination with the Th2-inducing adjuvant Alum were not protected against L. monocytogenes. On the other hand, mice immunized with LLO^T along with the experimental adjuvant cholera toxin, were protected against L. monocytogenes, as evidenced by a significant decrease in bacterial burden in the liver and spleen three days post-infection. This immunization regimen elicited mixed Th1, Th2, and Th17 responses, as well as the generation of LLO-neutralizing antibodies. Further, we identified T cells as being required for immunization-induced reductions in bacterial burden, whereas B cells were dispensable in our model of non-pregnant young mice. Overall, this work establishes that LLO^T is a promising vaccine antigen for the induction of protective immunity against L. monocytogenes by subunit vaccines containing Th1-driving adjuvants.     

Analysis of the immunogenicity and bioactivities of a split influenza A/H7N9 vaccine mixed with MF59 adjuvant in BALB/c mice

The H7N9 influenza virus caused significant mortality and morbidity in humans during an outbreak in China in 2013. A recombinant H7N9 influenza seed with hemagglutinin (HA) and neuraminidase (NA) gene segments from A/Zhejiang/DTID-ZJU01/2013(H7N9) and six internal protein gene segments from A/Puerto Rico/8/34(H1N1; PR8) were generated using reverse genetics. We sought to determine the immunogenic, protective properties, and mechanisms of a split avian influenza A/H7N9 vaccine mixed with MF59 adjuvant in comparison to vaccines that included other adjuvant. BALB/c mice were vaccinated with two doses of different amounts and combinations of this novel A/ZJU01/PR8/2013 split vaccine with adjuvant. Mice were subsequently challenged with A/Zhejiang/DTID-ZJU01/2013(H7N9) by intranasal inoculation. We verified that MF59 enhanced the HI, MN, and IgG antibody titers to influenza antigens. Compared with alum, MF59 could more potentially induce humoral immune responses and Th2 cytokine production after virus infection, while both MF59 and alum can slightly increase NK cell activity. This split H7N9 influenza vaccine with MF59 adjuvant could effectively induce antibody production and protect mice from H7N9 virus challenge. We have selected this vaccine for manufacture and future clinical studies to protect humans from H7N9 virus infection.     

Recombinant Erns-E2 protein vaccine formulated with MF59 and CPG-ODN promotes T cell immunity against bovine viral diarrhea virus infection

To obtain an effective vaccine candidate against bovine viral diarrhea virus (BVDV) disease which causes great economical loss in cattle industries, recombinant E^rns-E2 protein vaccine containing MF59 and CPG-ODN adjuvants was prepared and assessed in this study. The recombinant plasmid (pET32a-E^rns-E2) was constructed and transformed into BL21 (DE3) cells to produce E^rns-E2 protein. We immunized mice with the MF59–and CPG-ODN–adjuvanted recombinant E^rns-E2 protein, E2 protein, or E^rns protein, respectively. To evaluate immunogenicity and efficacy of a vaccine-adjuvant combination, mice were challenged with BVDV BJ175170 strain after immunization. All adjuvanted vaccines elicited detectable humoral and cellular immune responses, the BVDV-specific antibody titers as well as interleukin 4 (IL-4) levels in sera of mice immunized with the recombinant E^rns-E2 protein were higher than in those of mice immunized with either the recombinant E^rns or E2 protein. Besides, immunization with the E^rns-E2 vaccines induced higher percentage of CD4⁺IFN-γ⁺, CD8⁺IFN-γ⁺ T cells and CD3⁺TNF-α⁺ T cells compared with the other vaccines. More protective efficacy against BVDV infection was acquired in the mice treated with the recombinant E^rns-E2 protein, as shown by a reduction of viremia and slight pathological changes compared with both the control mice and the other vaccinated mice. Our findings suggest that the use of the recombinant E^rns-E2 protein vaccine formulated with MF59 and CPG-ODN adjuvants enhances T cell responses and viral control, which warrants the E^rns-E2 protein vaccine-adjuvant combination could be as a vaccine strategy to against BVDV.     

Immunogenicity of the Plasmodium falciparum Pf332-DBL domain in combination with different adjuvants

The Plasmodium falciparum antigen 332 (Pf332) is a conserved blood-stage antigen, which has been suggested to play a role in parasite invasion. In the present study, we have investigated the immunogenicity of the Duffy-binding like (DBL)-domain of the Pf332 molecule in combination with different adjuvants in four animal species. Three of the adjuvants are applicable for human use (Montanide ISA 720, alum and levamisole), whilst Freund's adjuvant served as a positive control adjuvant. Montanide ISA 720 was able to generate a significant and Th2-biased IgG response in BALB/c and C57BL/6 mice. Alum was a strong inducer of a Th2-type immune response only in BALB/c mice, whereas it was a poor adjuvant together with Pf332-DBL in C57BL/6 mice, rabbits and rats. Levamisole did not show any obvious adjuvant effect in any of the immunized animals. Thus in the case with Pf332-DBL, Montanide ISA 720 may be an adjuvant to further explore in the development of a vaccine against malaria.     

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.     

Interferon-γ inductive effect of liposomes as an immunoadjuvant

Adjuvant effect of liposomes was compared to that of aluminium hydroxide (Alum) using ovalbumin (OVA) as a model antigen, and the difference in adjuvanticity of liposomes from Alum has been evaluated on the basis of cytokine production. Both adjuvants enhanced the IgG levels, but a remarkable difference was observed in the production of IgG subclass; Alum enhanced IgG1 levels, and liposomes enhanced IgG2a, IG2b, and IG3 levels. Further, Alum enhanced antigen-specific IgE levels, whereas liposomes did not. To clarify the difference in adjuvant effect of these adjuvants, secretion of cytokines, especially interferon-γ (IFN-γ) and interleukin-4 (IL-4) which regulate IgE production, was estimated ex vivo. Overnight culture of splenic cells obtained from mice immunized with liposomes encapsulating OVA elicits IFN-γ secretion, but not IL-4 secretion. On the other hand, the production of both cytokines was elevated by the immunization with OVA-Alum complex. The results indicate that the difference of adjuvant activity between liposomes and Alum may come from the difference in the secretion of IL-4 and that, consequently, a different class of antibody response is developed. More importantly, negatively charged liposomes containing phosphatidylserine remarkably promoted IFN-γ secretion compared to neutral liposomes.     

Use of a polyanionic carbomer, Carbopol971P, in combination with MF59, improves antibody responses to HIV-1 envelope glycoprotein

Identification of optimal antigen(s) and adjuvant combination(s) to elicit potent, protective, and long-lasting immunity has been a major challenge for the development of effective vaccines against chronic viral pathogens, such as HIV-1, for which there are not yet any licensed vaccines. Here we describe the use of a novel adjuvant approach employing Carbopol 971P^® NF (hereafter referred to as Carbopol971P), a cross-linked polyanionic carbomer, in combination with the Novartis proprietary oil-in-water adjuvant, MF59, as a potentially safe and effective adjuvant to augment humoral immune responses to the HIV-1 envelope glycoprotein (Env). Intramuscular immunization of small animals with recombinant Env glycoprotein (gp140) formulated in Carbopol971P plus MF59 gave significantly higher titers of binding and virus neutralizing antibodies as compared to immunization using gp140 with either MF59 or Carbopol971P alone. In addition, the antibodies generated were of higher avidity. Importantly, the use of Carbopol971P plus MF59 did not cause any serious adverse reactions or any obvious health problems in animals upon intramuscular administration. Hence, the Carbopol971P plus MF59 adjuvant formulation may provide a benefit for future vaccine applications.