Yoshikazu Honda-Okubo

A novel hepatitis B vaccine containing Advax™, a polysaccharide adjuvant derived from delta inulin, induces robust humoral and cellular immunity with minimal reactogenicity in preclinical testing

Although current HBV vaccines have an outstanding record of safety and efficacy, reduced immunogenicity is a problem in those of older age, or having renal impairment or diabetes mellitus. In this study, we tested the ability of Advax™ adjuvant, a novel polysaccharide adjuvant based on delta inulin, to enhance the immunogenicity of hepatitis B surface antigen (HBs) in mice and guinea pigs by comparison to the traditional alum adjuvant. Advax™ provided antigen-sparing, significantly enhanced both anti-HBs antibody titers, and anti-HBs CD4 and CD8 T-cells, with increases in Th1, Th2 and Th17 cytokine responses. Unlike alum, the adjuvant effect of Advax™ was seen even when injected 24h before the HBs antigen. Advax™ adjuvant similarly enhanced humoral and cellular immune responses in guinea pigs to a third generation preS-HBs antigen. Advax™ adjuvant when combined with HBs antigen could provide enhanced protection over current generation HBV vaccines for immunization of low responder populations.

Advax™, a polysaccharide adjuvant derived from delta inulin, provides improved influenza vaccine protection through broad-based enhancement of adaptive immune responses.

Advax™ adjuvant is derived from inulin, a natural plant-derived polysaccharide that when crystallized in the delta polymorphic form, becomes immunologically active. This study was performed to assess the ability of Advax™ adjuvant to enhance influenza vaccine immunogenicity and protection. Mice were immunized with influenza vaccine alone or combined with Advax™ adjuvant. Immuno-phenotyping of the anti-influenza response was performed including antibody isotypes, B-cell ELISPOT, CD4 and CD8 T-cell proliferation, influenza-stimulated cytokine secretion, DTH skin tests and challenge with live influenza virus. Advax™ adjuvant increased neutralizing antibody and memory B-cell responses to influenza. It similarly enhanced CD4 and CD8 T-cell proliferation and increased influenza-stimulated IL-2, IFN-γ, IL-5, IL-6, and GM-CSF responses. This translated into enhanced protection against mortality and morbidity in mice. Advax™ adjuvant provided significant antigen dose-sparing compared to influenza antigen alone. Protection could be transferred from mice that had received Advax™-adjuvanted vaccine to naïve mice by immune serum. Enhanced humoral and T-cell responses induced by Advax™-formulated vaccine were sustained 12months post-immunization. Advax™ adjuvant had low reactogenicity and no adverse events were identified. This suggests Advax™ adjuvant could be a useful influenza vaccine adjuvant.

Randomized clinical trial of immunogenicity and safety of a recombinant H1N1/2009 pandemic influenza vaccine containing Advax™ polysaccharide adjuvant.

BACKGROUNDnTimely vaccine supply is critical during influenza pandemics. A recombinant hemagglutinin (rHA)-based vaccine could overcome production hurdles of egg-based vaccines but has never previously been tested in a real-life pandemic setting. The primary aim was to determine the efficacy of a recombinant pandemic vaccine and whether its immunogenicity could be enhanced by a novel polysaccharide adjuvant (Advax™).nnnMETHODSn281 adults aged 18-70 years were recruited in a randomized, subject and observer blinded, parallel-group study of rHA H1N1/2009 vaccine with or without adjuvant. Immunizations were at 0 and 3 weeks with rHA 3, 11 or 45 μg. Serology and safety was followed for 6 months.nnnRESULTSnAt baseline, only 9.1% of subjects (95% CI: 6.0-13.2) had seroprotective H1N1/2009 titers. Seroconversion rates varied by rHA dose, presence of adjuvant, subject age and number of immunizations. Eighty percent (95% CI: 52-96) of 18-49 year olds who received rHA 45 μg with adjuvant were seroprotected at week 3, representing a 11.1-fold increase in antibody titers from baseline. Advax™ adjuvant increased seroprotection rates by 1.9 times after the first, and 2.5 times after the second, immunization when compared to rHA alone. Seroprotection was sustained at 26 weeks and the vaccine was well tolerated with no safety issues.nnnCONCLUSIONSnThe study confirmed the ability to design, manufacture, and release a recombinant vaccine within a short time from the start of an actual influenza pandemic. Advax™ adjuvant significantly enhanced rHA immunogenicity.

Severe Acute Respiratory Syndrome-Associated Coronavirus Vaccines Formulated with Delta Inulin Adjuvants Provide Enhanced Protection while Ameliorating Lung Eosinophilic Immunopathology

ABSTRACT Although the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) epidemic was controlled by nonvaccine measures, coronaviruses remain a major threat to human health. The design of optimal coronavirus vaccines therefore remains a priority. Such vaccines present major challenges: coronavirus immunity often wanes rapidly, individuals needing to be protected include the elderly, and vaccines may exacerbate rather than prevent coronavirus lung immunopathology. To address these issues, we compared in a murine model a range of recombinant spike protein or inactivated whole-virus vaccine candidates alone or adjuvanted with either alum, CpG, or Advax, a new delta inulin-based polysaccharide adjuvant. While all vaccines protected against lethal infection, addition of adjuvant significantly increased serum neutralizing-antibody titers and reduced lung virus titers on day 3 postchallenge. Whereas unadjuvanted or alum-formulated vaccines were associated with significantly increased lung eosinophilic immunopathology on day 6 postchallenge, this was not seen in mice immunized with vaccines formulated with delta inulin adjuvant. Protection against eosinophilic immunopathology by vaccines containing delta inulin adjuvants correlated better with enhanced T-cell gamma interferon (IFN-γ) recall responses rather than reduced interleukin-4 (IL-4) responses, suggesting that immunopathology predominantly reflects an inadequate vaccine-induced Th1 response. This study highlights the critical importance for development of effective and safe coronavirus vaccines of selection of adjuvants based on the ability to induce durable IFN-γ responses. IMPORTANCE Coronaviruses such as SARS-CoV and Middle East respiratory syndrome-associated coronavirus (MERS-CoV) cause high case fatality rates and remain major human public health threats, creating a need for effective vaccines. While coronavirus antigens that induce protective neutralizing antibodies have been identified, coronavirus vaccines present a unique problem in that immunized individuals when infected by virus can develop lung eosinophilic pathology, a problem that is further exacerbated by the formulation of SARS-CoV vaccines with alum adjuvants. This study shows that formulation of SARS-CoV spike protein or inactivated whole-virus vaccines with novel delta inulin-based polysaccharide adjuvants enhances neutralizing-antibody titers and protection against clinical disease but at the same time also protects against development of lung eosinophilic immunopathology. It also shows that immunity achieved with delta inulin adjuvants is long-lived, thereby overcoming the natural tendency for rapidly waning coronavirus immunity. Thus, delta inulin adjuvants may offer a unique ability to develop safer and more effective coronavirus vaccines.

A single immunization with inactivated H1N1 influenza vaccine formulated with delta inulin adjuvant (Advax™) overcomes pregnancy-associated immune suppression and enhances passive neonatal protection.

Pregnant women and neonates represent high-risk groups for influenza infection, and in general have suppressed responses to standard influenza vaccines due to pregnancy-associated immune suppression and immune system immaturity, respectively. We therefore wished to test whether addition of Advax™, a polysaccharide adjuvant based on delta inulin, to an inactivated influenza vaccine (A/H1N1/PR8) administered during pregnancy would safely enhance vaccine immunogenicity and thereby provide improved protection of pregnant mothers and their newborns. Pregnant mice received a single intramuscular injection of β-propiolactone-inactivated H1N1 antigen alone or with Advax adjuvant. Pregnant dams receiving Advax-adjuvanted vaccine exhibited significantly increased serum and breast milk anti-influenza IgG titers. This translated into higher serum anti-influenza IgG titers in the pups of these dams. Complete protection was seen in pups of dams that received Advax-adjuvanted vaccine whereas no survival was seen in pups of control mothers or mothers immunized with unadjuvanted influenza vaccine. Cross-fostering studies confirmed that enhanced protection of pups of dams that received Advax-adjuvanted vaccine was mediated by enhanced transfer of maternal IgG to the pups via breast-feeding. The delta inulin adjuvant was not associated with any reproductive or developmental adverse effects. This study shows that Advax adjuvant was safe when administered with influenza vaccine during pregnancy and provided protection of pups via enhanced breast milk transfer of anti-influenza antibodies, not seen with administration of unadjuvanted vaccine.

Advax delta inulin adjuvant overcomes immune immaturity in neonatal mice thereby allowing single–dose influenza vaccine protection

n Abstractn n Neonates are at high risk for influenza morbidity and mortality due to immune immaturity and lack of priming by prior influenza virus exposure. Inactivated influenza vaccines are ineffective in infants under six months and to provide protection in older children generally require two doses given a month apart. This leaves few options for rapid protection of infants, e.g. during an influenza pandemic. We investigated whether Advax™, a novel polysaccharide adjuvant based on delta inulin microparticles could help overcome neonatal immune hypo-responsiveness. We first tested whether it was possible to use Advax to obtain single-dose vaccine protection of neonatal pups against lethal influenza infection. Inactivated influenza A/H1N1 vaccine (iH1N1) combined with Advax™ adjuvant administered as a single subcutaneous immunization to 7-day-old mouse pups significantly enhanced serum influenza-specific IgM, IgG1, IgG2a and IgG2b levels and was associated with a 3–4 fold increase in the frequency of splenic influenza-specific IgM and IgG antibody secreting cells. Pups immunized with Advax had significantly higher splenocyte influenza-stimulated IFN-γ, IL-2, IL-4, and IL-10 production by CBA and a 3–10 fold higher frequency of IFN-γ, IL-2, IL-4 or IL-17 secreting T cells by ELISPOT. Immunization with iH1N1+Advax induced robust protection of pups against virus challenge 3 weeks later, whereas pups immunized with iH1N1 antigen alone had no protection. Protection by Advax-adjuvanted iH1N1 was dependent on memory B cells rather than memory T cells, with no protection in neonatal μMT mice that are B-cell deficient. Hence, Advax adjuvant overcame neonatal immune hypo-responsiveness and enabled single-dose protection of pups against otherwise lethal influenza infection, thereby supporting ongoing development of Advax™ as a neonatal vaccine adjuvant.n n

Serological responses following influenza A H1N1 2009 infection in adults

OBJECTIVEnThe aim of this study was to determine the humoral immune response to influenza A H1N1 2009 and cross reactivity against seasonal H1N1 and H3N2 strains.nnnMETHODSnBaseline and convalescent sera from 88 subjects with confirmed H1N1 2009 were screened for serological responses by HAI assay.nnnRESULTSnProtective antibody titres to H1N1 2009 were present in 87% post-infection, but varied with age, sex and pregnancy. Some cross reactivity with seasonal influenza strains was observed.nnnCONCLUSIONSnFemales and pregnant subjects had an attenuated immune response to H1N1 2009 in comparison to the rest of the study population. Antibodies from the serum of H1N1 2009 infected subjects cross reacted with seasonal H1N1 and H3N2 influenza viruses.

Delta Inulin Adjuvant Enhances Plasmablast Generation, Expression of Activation-Induced Cytidine Deaminase and B-Cell Affinity Maturation in Human Subjects Receiving Seasonal Influenza Vaccine

There is a major need for new adjuvants to improve the efficacy of seasonal and pandemic influenza vaccines. Advax is a novel polysaccharide adjuvant based on delta inulin that has been shown to enhance the immunogenicity of influenza vaccine in animal models and human clinical trials. To better understand the mechanism for this enhancement, we sought to assess its effect on the plasmablast response in human subjects. This pilot study utilised cryopreserved 7 day post-vaccination (7dpv) peripheral blood mononuclear cell samples obtained from a subset of 25 adult subjects from the FLU006-12 trial who had been immunized intramuscularly with a standard dose of 2012 trivalent inactivated influenza vaccine (TIV) alone (n=9 subjects) or combined with 5mg (n=8) or 10mg (n=8) of Advax adjuvant. Subjects receiving Advax adjuvant had increased 7dpv plasmablasts, which in turn exhibited a 2-3 fold higher rate of non-silent mutations in the B-cell receptor CDR3 region associated with higher expression of activation-induced cytidine deaminase (AID), the major enzyme controlling BCR affinity maturation. Together, these data suggest that Advax adjuvant enhances influenza immunity in immunized subjects via multiple mechanisms including increased plasmablast generation, AID expression and CDR3 mutagenesis resulting in enhanced BCR affinity maturation and increased production of high avidity antibody. How Advax adjuvant achieves these beneficial effects on plasmablasts remains the subject of ongoing investigation. Trial Registration Australia New Zealand Clinical Trials Register ACTRN12612000709842 https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=362709

Development of a SARS Coronavirus Vaccine from Recombinant Spike Protein Plus Delta Inulin Adjuvant

Given periodic outbreaks of fatal human infections caused by coronaviruses, development of an optimal coronavirus vaccine platform capable of rapid production is an ongoing priority. This chapter describes the use of an insect cell expression system for rapid production of a recombinant vaccine against severe acute respiratory syndrome coronavirus (SARS). Detailed methods are presented for expression, purification, and release testing of SARS recombinant spike protein antigen, followed by adjuvant formulation and animal testing. The methods herein described for rapid development of a highly protective SARS vaccine are equally suited to rapid development of vaccines against other fatal human coronavirus infections, e.g., the MERS coronavirus.

Advax, a Delta Inulin Microparticle, Potentiates In-built Adjuvant Property of Co-administered Vaccines

Advax, a delta inulin-derived microparticle, has been developed as an adjuvant for several vaccines. However, its immunological characteristics and potential mechanism of action are yet to be elucidated. Here, we show that Advax behaves as a type-2 adjuvant when combined with influenza split vaccine, a T helper (Th)2-type antigen, but behaves as a type-1 adjuvant when combined with influenza inactivated whole virion (WV), a Th1-type antigen. In addition, an adjuvant effect was not observed when Advax-adjuvanted WV vaccine was used to immunize toll-like receptor (TLR) 7 knockout mice which are unable to respond to RNA contained in WV antigen. Similarly, no adjuvant effect was seen when Advax was combined with endotoxin-free ovalbumin, a neutral Th0-type antigen. An adjuvant effect was also not seen in tumor necrosis factor (TNF)-α knockout mice, and the adjuvant effect required the presences of dendritic cells (DCs) and phagocytic macrophages. Therefore, unlike other adjuvants, Advax potentiates the intrinsic or in-built adjuvant property of co-administered antigens. Hence, Advax is a unique class of adjuvant which can potentiate the intrinsic adjuvant feature of the vaccine antigens through a yet to be determined mechanism.