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Abstract SpikoGen® COVID-19 vaccine is based on the spike protein extracellular domain of the ancestral Wuhan-Hu-1 strain modified by removal of the furin cleavage site and addition of stabilising mutations expressed as a recombinant protein in insect cells. It is formulated with Advax-CpG55.2™ adjuvant to ensure optimal immunogenicity. In this study, data from several SpikoGen® clinical trials was retrospectively analysed to assess for any effect of gender or age on seroconversion, neutralizing antibody levels or the incidence of adverse events. Following the 1st dose, older age was associated with a reduced rate of fatigue (RR 0.97, p < 0.001), headache (RR 0.98, p = 0.034) and myalgia (RR 0.97, p=0.016), following the 2nd dose, the rate of fatigue (RR 0.98, p = 0.017) but following the 3rd dose no effect of age on adverse events was evident. Similarly, following the 1st dose, men reported a 19% lower incidence of fatigue, 36% lower incidence of headache and 28% lower incidence of myalgia when compared to women. Interestingly, there was no relationship between age or gender and serum neutralizing antibody levels, although after each vaccine dose there was a consistent trend to women having a higher seroconversion rate. There was no correlation between neutralizing antibody levels and adverse events. Unlike what is seen with mRNA vaccines, reactogenicity trended lower after each subsequent SpikoGen® dose. Overall, SpikoGen® exhibited positive immunogenicity and low reactogenicity, indicating that a low incidence of adverse events does not equate to poor immunogenicity. SpikoGen® remains a promising protein-based vaccine platform for COVID-19 protection.

Recombinant protein vaccines represent a well-established, reliable and safe approach for pandemic vaccination. SpikoGen® is a recombinant spike protein trimer manufactured in insect cells and formulated with Advax-CpG55.2 adjuvant. In murine, hamster, ferret and non-human primate studies, SpikoGen® consistently provided protection against a range of SARS-CoV-2 variants. A pivotal Phase 3 placebo-controlled efficacy trial involving 16,876 participants confirmed the ability of SpikoGen® to prevent infection and severe disease caused by the virulent Delta strain. SpikoGen® subsequently received a marketing authorization from the Iranian FDA in early October 2021 for prevention of COVID-19 in adults. Following a successful pediatric study, its approval was extended to children 5 years and older. Eight million doses of SpikoGen® have been delivered, and a next-generation booster version is currently in development. This highlights the benefits of adjuvanted protein-based approaches which should not overlook when vaccine platforms are being selected for future pandemics.

Abstract We developed a novel intranasal SARS-CoV-2 subunit vaccine called NARUVAX-C19/Nano based on the spike protein receptor-binding domain (RBD) entrapped in mannose-conjugated chitosan nanoparticles (NP). A toll-like receptor 9 agonist, CpG55.2, was also added as an adjuvant to see if this would potentiate the cellular immune response to the NP vaccine. The NP vaccine was assessed for immunogenicity, protective efficacy, and ability to prevent virus transmission from vaccinated animals to naive cage-mates. The results were compared with a RBD protein vaccine mixed with alum adjuvant and administered intramuscularly. BALB/c mice vaccinated twice intranasally with the NP vaccines exhibited secretory IgA and a pronounced Th1-cell response, not seen with the intramuscular alum-adjuvanted RBD vaccine. NP vaccines protected Syrian hamsters against a wild-type SARS-CoV-2 infection challenge as indicated by significant reductions in weight loss, lung viral load and lung pathology. However, despite significantly reduced viral load in the nasal turbinates and oropharyngeal swabs from NP-vaccinated hamsters, virus transmission was not prevented to naïve cage-mates. In conclusion, intranasal RBD-based NP formulations induced mucosal and Th1-cell mediated immune responses in mice and protected Syrian hamsters against SARS-CoV-2 infection but not against viral transmission.

Within the last two decades, SARS-CoV-2 was the third zoonotic severe acute respiratory betacoronavirus (sarbecovirus) to infect humans, following SARS and MERS. The disruptions caused by the pandemic underscore the need for a universal vaccine against respiratory betacoronaviruses. Our group previously developed the universal platform for vaccine development, MultiTEP, which has been utilized in this study to generate a range of SARS-CoV-2 epitope vaccine candidates. We prepared and characterized 18 vaccines incorporating small peptide fragments from SARS-CoV-2 Spike protein fused with the MultiTEP sequence using overlapping PCR. Wild-type mice were immunized intramuscularly with the immunogen formulated in AdvaxCpG adjuvant. Serum antibodies were detected by ELISA, surrogate neutralization, and pseudovirus neutralization assays. Finally, the most promising vaccine candidate was administered to three non-human primates. All vaccines generated high titers of spike-binding IgG antibodies. However, only three vaccines generated antibodies that blocked RBD binding to the ACE2 receptor in a surrogate virus neutralization assay. However, none of the vaccines induced antibodies able to neutralize pseudotype viruses, including after the administration of the lead vaccine to NHPs. MultiTEP-based COVID-19 vaccines elicited robust, IgG-binding responses against the Spike protein in mice and non-human primates, but these antibodies were not neutralizing, underscoring the need to refine this approach further.

Adjuvants enhance immune responses stimulated by vaccines. To date, many seasonal influenza vaccines are not formulated with an adjuvant. In the present study, the adjuvant Advax-SM™ was combined with next generation, broadly reactive influenza hemagglutinin (HA) vaccines that were designed using a computationally optimized broadly reactive antigen (COBRA) methodology. Advax-SM™ is a novel adjuvant comprising inulin polysaccharide and CpG55.2, a TLR9 agonist. COBRA HA vaccines were combined with Advax-SM™ or a comparator squalene emulsion (SE) adjuvant and administered to mice intramuscularly. Mice vaccinated with Advax-SM™ adjuvanted COBRA HA vaccines had increased serum levels of anti-influenza IgG and IgA, high hemagglutination inhibition activity against a panel of H1N1 and H3N2 influenza viruses, and increased anti-influenza antibody secreting cells isolated from spleens. COBRA HA plus Advax-SM™ immunized mice were protected against both morbidity and mortality following viral challenge and, at postmortem, had no detectable lung viral titers or lung inflammation. Overall, the Advax-SM™-adjuvanted COBRA HA formulation provided effective protection against drifted H1N1 and H3N2 influenza viruses.

COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has significantly impacted public health and the economy worldwide. Most of the currently licensed COVID-19 vaccines act by inhibiting the receptor-binding function of the SARS-CoV-2 spike protein. The constant emergence of SARS-CoV-2 variants resulting from mutations in the receptor-binding domain (RBD) leads to vaccine immune evasion and underscores the importance of broadly acting COVID-19 vaccines. Inactivated whole virus vaccines can elicit broader immune responses to multiple epitopes of several antigens and help overcome such immune evasions. We prepared a psoralen-inactivated SARS-CoV-2 vaccine (SARS-CoV-2 PsIV) and evaluated its immunogenicity and efficacy in nonhuman primates (NHPs) when administered with the Advax-CpG adjuvant. We also evaluated the SARS-CoV-2 PsIV as a booster shot in animals vaccinated with a DNA vaccine that can express the full-length spike protein. The Advax-CpG-adjuvanted SARS-CoV-2 PsIV elicited a dose-dependent neutralizing antibody response in the NHPs, as measured using a serum microneutralization assay against the SARS-CoV-2 Washington strain and the Delta variant. The animals vaccinated with the DNA vaccine followed by a boosting dose of the SARS-CoV-2 PsIV exhibited the highest neutralizing antibody responses and were able to quickly clear infection after an intranasal challenge with the SARS-CoV-2 Delta variant. Overall, the data show that the Advax-CpG-adjuvanted SARS-CoV-2 PsIV, either by itself or as a booster shot following nucleic acid (NA) vaccines, has the potential to protect against emerging variants.

Abstract Pathological forms of Tau protein are directly associated with neurodegeneration and correlate with Alzheimer’s Disease (AD) symptoms, progression, and severity. Previously, using various mouse models of Tauopathies and AD, we have demonstrated the immunogenicity and efficacy of the MultiTEP-based adjuvanted vaccine targeting the phosphatase activating domain (PAD) of Tau, AV-1980R/A. Here, we analyzed its immunogenicity in non-human primates (NHP), the closest phylogenic relatives to humans with a similar immune system, to initiate the transition of this vaccine into clinical trials. We have demonstrated that AV-1980R/A is highly immunogenic in these NHPs, activating a broad but unique to each monkey repertoire of MultiTEP-specific T helper (Th) cells that, in turn, activate B cells specific to PAD. The resulting anti-PAD IgG antibodies recognize pathological Tau tangles and Tau-positive neuritis in AD case brain sections with no staining in control non-AD cases. These published data and efficacy results support the AV-1980R/A vaccine progression to first-in-human clinical trials.

COVID-19 vaccines have played an important role in reducing the impact of the current pandemic. Previously, we developed NARUVAX-C19 vaccine based on a recombinant Wuhan spike protein extracellular domain expressed in insect cells and formulated with a squalene emulsion adjuvant (Sepivac SWE™). The current study assessed the immunogenicity, efficacy, and safety of NARUVAX-C19 vaccine in rhesus macaques and hamsters. Macaques immunized intramuscularly with two doses of NARUVAX-C19 vaccine showed no adverse effects and demonstrated cellular immunity as assessed by T cell IFN-γ responses against spike protein, in addition to inducing a humoral response. Serum from immunized animals neutralized the homologous wild-type SARS-CoV-2 virus as well as the Alpha and Delta variants. In hamsters, immunization with NARUVAX-C19 vaccine protected against a heterologous challenge with the Delta virus, as reflected by reduced lung and nasal viral loads and lung pathology in immunized animals. Nevertheless, some NARUVAX-C19 vaccinated animals were still shown to transmit infection to naïve sentinel animals. Overall, NARUVAX-C19 vaccine induced broadly cross-neutralizing antibody and T cell IFN-γ responses in rhesus macaques and provided heterologous protection of hamsters against infection by the Delta virus variant. This data supports the utility of squalene emulsion-based adjuvanted recombinant vaccine in protection against SARS-CoV-2 and supports their continued clinical development.

ABSTRACT: A Campylobacter species was first described as the etiological agent of Spotty Liver Disease (SLD) in 2015 and subsequently named as Campylobacter hepaticus in 2016. The bacterium predominantly affects barn and/or free-range hens at peak lay, is fastidious and difficult to isolate, which has impeded elucidation of its sources, means of persistence and transmission. Ten farms from South-Eastern Australia, of which 7 were free range entities participated in the study. A total of 1,404 specimens from layers and 201 from environmental sources, were examined for the presence of C. hepaticus. In this study, our principal findings included the continuing detection of C. hepaticus infection in a flock following an outbreak, indicating a possible transition of infected hens to asymptomatic carriers, that was also characterized by no further occurrence of SLD in the flock. We also report that the first outbreaks of SLD on newly commissioned free-range farms affected layers ranging from 23 to 74 wk of age, while subsequent outbreaks in replacement flocks on these farms occurred during the more conventional peak lay period (23–32 wk of age). Finally, we report that in the on-farm environment, C. hepaticus DNA was detected in layer feces, inert elements such as stormwater, mud, soil, as well as in fauna such as flies, red mites, Darkling beetles, and rats. While in off-farm locations, the bacterium was detected in feces from a variety of wild birds and a canine.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, results in approximately 1.6 million deaths annually. BCG is the only TB vaccine currently in use and offers only variable protection; however, the development of more effective vaccines is hindered by a lack of defined correlates of protection (CoP) against M. tuberculosis. Pulmonary vaccine delivery is a promising strategy since it may promote lung-resident immune memory that can respond rapidly to respiratory infection. In this study, CysVac2, a subunit protein previously shown to be protective against M. tuberculosis in mouse models, was combined with either Advax® adjuvant or a mixture of alum plus MPLA and administered intratracheally into mice. Peripheral immune responses were tracked longitudinally, and lung-local immune responses were measured after challenge. Both readouts were then correlated with protection after M. tuberculosis infection. Although considered essential for the control of mycobacteria, induction of IFN-γ-expressing CD4+ T cells in the blood or lungs did not correlate with protection. Instead, CD4+ T cells in the lungs expressing IL-17A correlated with reduced bacterial burden. This study identified pulmonary IL-17A-expressing CD4+ T cells as a CoP against M. tuberculosis and suggests that mucosal immune profiles should be explored for novel CoP.

Abstract Pre-erythrocytic malaria vaccines that induce high-titer, durable antibody responses can potentially provide protection from infection. Here, we engineered a virus-like particle (VLP)-based vaccine targeting a recently described vulnerable epitope at the N-terminus of the central repeat region of the Plasmodium falciparum circumsporozoite protein that is recognized by the potently inhibitory monoclonal antibody L9 and show that immunization with L9 VLPs induces strong antibody responses that provide protection from blood-stage malaria in a mouse infection model.

Abstract Recombinant protein approaches offer major promise for safe and effective vaccine prevention of SARS-CoV-2 infection. We developed a recombinant spike protein vaccine (called NARUVAX-C19) and characterized its ability when formulated with a nanoemulsion adjuvant to induce anti-spike antibody and T-cell responses and provide protection including against viral transmission in rodent. In mice, NARUVAX-C19 vaccine administered intramuscularly twice at 21-day interval elicited balanced Th1/Th2 humoral and T-cell responses with high titers of neutralizing antibodies against wild-type (D614G) and delta (B.1.617.2) variants. In Syrian hamsters, NARUVAX-C19 provided complete protection against wild-type (D614G) infection and prevented its transmission to naïve animals (n = 2/group) placed in the same cage as challenged animals (n = 6/group). The results contrasted with only weak protection seen with a monomeric spike receptor-binding domain (RBD) vaccine even when formulated with the same adjuvant. These encouraging results warrant the ongoing development of this COVID-19 vaccine candidate.

Allergic rhinitis is an important risk factor for bronchial asthma. Allergen-specific immunotherapy (ASIT) is the gold standard for treatment of allergic rhinitis, conjunctivitis, and asthma. A disadvantage of current ASIT methods is the length of therapy which requires numerous allergen administrations. The success of ASIT is determined by its schedule, which, depending on the vaccine and type of allergy, can be pre-seasonal (before the allergy season begins), combined pre/co-seasonal (during the allergy season) etc. The aim of the present study was to evaluate a vaccine based on recombinant Artemisia vulgaris pollen major Art v 1 protein formulated with ISA-51 adjuvant for therapy of allergic rhinitis and bronchial asthma in Artemisia-sensitized mice in an ultrashort (4 subcutaneous injections at weekly intervals) pre- and co-seasonal ASIT regimen.To simulate co-seasonal ASIT in mice, mice were regularly challenged with intranasal and nebulized Artemisia vulgaris pollen extract at the same time as receiving subcutaneous ASIT. For comparison, we used a previous Art v 1 protein vaccine formulated with SWE adjuvant, which in this study was modified by adding CpG oligonucleotide (Th1-biasing synthetic toll-like receptor 9 agonist), and a commercial vaccine containing a modified Artemisia vulgaris extract with aluminum hydroxide adjuvant. The therapeutic potential of Art v 1 based vaccine formulations with different ASIT regimens was evaluated in high and low (10 times lower) dose regimens.The ISA-51-adjuvanted vaccine formulations were the only ones among those studied in the ultrashort pre- and co-seasonal ASIT regimens to provide significant reduction in both signs of allergic rhinitis and bronchial asthma in sensitized mice (vs. positive control). In the ISA-51 adjuvanted group, immune response polarization toward Th1/Treg was observed in pre-seasonal ASIT, as reflected in a significant decrease in the serum level of total and Art v 1-specific IgE and increased ratios of allergen-specific IgG2a/IgG1 and IFN-γ/IL-4. The high dose SWE-CpG-adjuvanted vaccine had similar efficacy to the ISA-51 adjuvanted groups whereas the commercial vaccine showed significantly less effectiveness.The findings support further preclinical safety studies of the Art v 1-based vaccine formulated with ISA-51 adjuvant.

Respiratory syncytial virus (RSV) remains the most common cause of lower respiratory tract infections in children worldwide. Development of a vaccine has been hindered due the risk of enhanced respiratory disease (ERD) following natural RSV exposure and the young age (

Abstract Repurposing of existing drugs and drug candidates is an ideal approach to identify new potential therapies for SARS-CoV-2 that can be tested without delay in human trials of infected patients. Here we applied a virtual screening approach using Autodock Vina and molecular dynamics simulation in tandem to calculate binding energies for repurposed drugs against the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). We thereby identified 80 promising compounds with potential activity against SARS-Cov2, consisting of a mixture of antiviral drugs, natural products and drugs with diverse modes of action. A substantial proportion of the top 80 compounds identified in this study had been shown by others to have SARS-CoV-2 antiviral effects in vitro or in vivo, thereby validating our approach. Amongst our top hits not previously reported to have SARS-CoV-2 activity, were eribulin, a macrocyclic ketone analogue of the marine compound halichondrin B and an anticancer drug, the AXL receptor tyrosine kinase inhibitor bemcentinib. Our top hits from our RdRp drug screen may not only have utility in treating COVID-19 but may provide a useful starting point for therapeutics against other coronaviruses. Hence, our modelling approach successfully identified multiple drugs with potential activity against SARS-CoV-2 RdRp.

Onchocerciasis remains a debilitating neglected tropical disease. Due to the many challenges of current control methods, an effective vaccine against the causative agent Onchocerca volvulus is urgently needed. Mice and cynomolgus macaque non-human primates (NHPs) were immunized with a vaccine consisting of a fusion of two O. volvulus protein antigens, Ov-103 and Ov-RAL-2 (Ov-FUS-1), and three different adjuvants: Advax-CpG, alum, and AlT4. All vaccine formulations induced high antigen-specific IgG titers in both mice and NHPs. Challenging mice with O. volvulus L3 contained within subcutaneous diffusion chambers demonstrated that Ov-FUS-1/Advax-CpG-immunized animals developed protective immunity, durable for at least 11 weeks. Passive transfer of sera, collected at several time points, from both mice and NHPs immunized with Ov-FUS-1/Advax-CpG transferred protection to naïve mice. These results demonstrate that Ov-FUS-1 with the adjuvant Advax-CpG induces durable protective immunity against O. volvulus in mice and NHPs that is mediated by vaccine-induced humoral factors.

Abstract The devastating impact of the COVID-19 pandemic caused by SARS–coronavirus 2 (SARS-CoV-2) has raised important questions about its origins and the mechanism of its transfer to humans. A further question was whether companion or commercial animals could act as SARS-CoV-2 vectors, with early data suggesting susceptibility is species specific. To better understand SARS-CoV-2 species susceptibility, we undertook an in silico structural homology modelling, protein–protein docking, and molecular dynamics simulation study of SARS-CoV-2 spike protein’s ability to bind angiotensin converting enzyme 2 (ACE2) from relevant species. Spike protein exhibited the highest binding to human (h)ACE2 of all the species tested, forming the highest number of hydrogen bonds with hACE2. Interestingly, pangolin ACE2 showed the next highest binding affinity despite having a relatively low sequence homology, whereas the affinity of monkey ACE2 was much lower despite its high sequence similarity to hACE2. These differences highlight the power of a structural versus a sequence-based approach to cross-species analyses. ACE2 species in the upper half of the predicted affinity range (monkey, hamster, dog, ferret, cat) have been shown to be permissive to SARS-CoV-2 infection, supporting a correlation between binding affinity and infection susceptibility. These findings show that the earliest known SARS-CoV-2 isolates were surprisingly well adapted to bind strongly to human ACE2, helping explain its efficient human to human respiratory transmission. This study highlights how in silico structural modelling methods can be used to rapidly generate information on novel viruses to help predict their behaviour and aid in countermeasure development.

Abstract Influenza viruses cause annual seasonal epidemics and sporadic pandemics; vaccination is the most effective countermeasure. Intranasal live attenuated influenza vaccines (LAIVs) are needle-free, mimic the natural route of infection, and elicit robust immunity. However, some LAIVs require reconstitution and cold-chain requirements restrict storage and distribution of all influenza vaccines. We generated a dry-powder, thermostable LAIV (T-LAIV) using Preservation by Vaporization technology and assessed the stability, immunogenicity, and efficacy of T-LAIV alone or combined with delta inulin adjuvant (Advax™) in ferrets. Stability assays demonstrated minimal loss of T-LAIV titer when stored at 25 °C for 1 year. Vaccination of ferrets with T-LAIV alone or with delta inulin adjuvant elicited mucosal antibody and robust serum HI responses in ferrets, and was protective against homologous challenge. These results suggest that the Preservation by Vaporization-generated dry-powder vaccines could be distributed without refrigeration and administered without reconstitution or injection. Given these significant advantages for vaccine distribution and delivery, further research is warranted.

Abstract This study tests the hypothesis that an Onchocerca volvulus vaccine, consisting of two recombinant antigens (Ov-103 and Ov-RAL-2) formulated with the combination-adjuvant Advax-2, can induce protective immunity in genetically diverse Collaborative Cross recombinant inbred intercross mice (CC-RIX). CC-RIX lines were immunized with the O. volvulus vaccine and challenged with third-stage larvae. Equal and significant reductions in parasite survival were observed in 7 of 8 CC-RIX lines. Innate protective immunity was seen in the single CC-RIX line that did not demonstrate protective adaptive immunity. Analysis of a wide array of immune factors showed that each line of mice have a unique set of immune responses to vaccination and challenge suggesting that the vaccine is polyfunctional, inducing different equally-protective sets of immune responses based on the genetic background of the immunized host. Vaccine efficacy in genetically diverse mice suggests that it will also be effective in genetically complex human populations.

Abstract A malaria vaccine that elicits long-lasting protection and is suitable for use in endemic areas remains urgently needed. Here, we assessed the immunogenicity and prophylactic efficacy of a vaccine targeting a recently described epitope on the major surface antigen on Plasmodium falciparum sporozoites, circumsporozoite protein (CSP). Using a virus-like particle (VLP)-based vaccine platform technology, we developed a vaccine that targets the junctional region between the N-terminal and central repeat regions of CSP. This region is recognized by monoclonal antibodies, including mAb CIS43, that have been shown to potently prevent liver invasion in animal models. We show that CIS43 VLPs elicit high-titer and long-lived anti-CSP antibody responses in mice and is immunogenic in non-human primates. In mice, vaccine immunogenicity was enhanced by using mixed adjuvant formulations. Immunization with CIS43 VLPs conferred partial protection from malaria infection in a mouse model, and passive transfer of serum from immunized macaques also inhibited parasite liver invasion in the mouse infection model. Our findings demonstrate that a Qβ VLP-based vaccine targeting the CIS43 epitope combined with various adjuvants is highly immunogenic in mice and macaques, elicits long-lasting anti-CSP antibodies, and inhibits parasite infection in a mouse model. Thus, the CIS43 VLP vaccine is a promising pre-erythrocytic malaria vaccine candidate.

Better adjuvants are needed for vaccines against seasonal influenza. TLR7 agonists are potent activators of innate immune responses and thereby may be promising adjuvants. Among the imidazoquinoline compounds, 1-benzyl-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine (BBIQ) was reported to be a highly active TLR7 agonist but has remained relatively unexplored because of its commercial unavailability. Indeed, in silico molecular modeling studies predicted that BBIQ had a higher TLR7 docking score and binding free energy than imiquimod, the gold standard TLR7 agonist. To circumvent the availability issue, we developed an improved and higher yield method to synthesize BBIQ. Testing BBIQ on human and mouse TLR7 reporter cell lines confirmed it to be TLR7 specific with significantly higher potency than imiquimod. To test its adjuvant potential, BBIQ or imiquimod were admixed with recombinant influenza hemagglutinin protein and administered to mice as two intramuscular immunizations 2 weeks apart. Serum anti-influenza IgG responses assessed by ELISA 2 weeks after the second immunization confirmed that the mice that received vaccine admixed with BBIQ had significantly higher anti-influenza IgG1 and IgG2c responses than mice immunized with antigen alone or admixed with imiquimod. This confirmed BBIQ to be a TLR7-specific adjuvant able to enhance humoral immune responses.

We urgently need to identify drugs to treat patients suffering from COVID-19 infection. Drugs rarely act at single molecular targets. Off-target effects are responsible for undesirable side effects and beneficial synergy between targets for specific illnesses. They have provided blockbuster drugs, e.g., Viagra for erectile dysfunction and Minoxidil for male pattern baldness. Existing drugs, those in clinical trials, and approved natural products constitute a rich resource of therapeutic agents that can be quickly repurposed, as they have already been assessed for safety in man. A key question is how to screen such compounds rapidly and efficiently for activity against new pandemic pathogens such as SARS-CoV-2. Here, we show how a fast and robust computational process can be used to screen large libraries of drugs and natural compounds to identify those that may inhibit the main protease of SARS-CoV-2. We show that the shortlist of 84 candidates with the strongest predicted binding affinities is highly enriched (≥25%) in compounds experimentally validated in vivo or in vitro to have activity in SARS-CoV-2. The top candidates also include drugs and natural products not previously identified as having COVID-19 activity, thereby providing leads for experimental validation. This predictive in silico screening pipeline will be valuable for repurposing existing drugs and discovering new drug candidates against other medically important pathogens relevant to future pandemics.

Wormwood (Artemisia) pollen is among the top 10 aeroallergens globally that cause allergic rhinitis and bronchial asthma. Allergen-specific immunotherapy (ASIT) is the gold standard for treating patients with allergic rhinitis, conjunctivitis, and asthma. A significant disadvantage of today’s ASIT methods is the long duration of therapy and multiplicity of allergen administrations. The goal of this study was to undertake a pilot study in mice of a novel ultrashort vaccine immunotherapy regimen incorporating various adjuvants to assess its ability to treat allergic bronchial asthma caused by wormwood pollen.We evaluated in a mouse model of wormwood pollen allergy candidates comprising recombinant Art v 1 wormwood pollen protein formulated with either newer (Advax, Advax-CpG, ISA-51) or more traditional [aluminum hydroxide, squalene water emulsion (SWE)] adjuvants administered by the intramuscular or subcutaneous route vs. intranasal administration of a mucosal vaccine formulation using chitosan-mannose nanoparticle entrapped with Art v 1 protein. The vaccine formulations were administered to previously wormwood pollen-sensitized animals, four times at weekly intervals. Desensitization was determined by measuring decreases in immunoglobulin E (IgE), cellular immunity, ear swelling test, and pathological changes in the lungs of animals after aeroallergen challenge. Art v 1 protein formulation with Advax, Advax-CpG, SWE, or ISA-51 adjuvants induced a significant decrease in both total and Art v 1-specific IgE with a concurrent increase in Art v 1-specific IgG compared to the positive control group. There was a shift in T-cell cytokine secretion toward a Th1 (Advax-CpG, ISA-51, and Advax) or a balanced Th1/Th2 (SWE) pattern. Protection against lung inflammatory reaction after challenge was seen with ISA-51, Advax, and SWE Art v 1 formulations. Overall, the ISA-51-adjuvanted vaccine group induced the largest reduction of allergic ear swelling and protection against type 2 and non-type 2 lung inflammation in challenged animals. This pilot study shows the potential to develop an ultrashort ASIT regimen for wormwood pollen-induced bronchial asthma using appropriately adjuvanted recombinant Art v 1 protein. The data support further preclinical studies with the ultimate goal of advancing this therapy to human clinical trials.

Whereas, multiple vaccine types have been developed to curb the spread of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) among humans, there are very few vaccines being developed for animals including pets. To combat the threat of human-to-animal, animal-to-animal, and animal-to-human transmission and the generation of new virus variants, we developed a subunit SARS-CoV-2 vaccine which is based on the recombinant spike protein extracellular domain expressed in insect cells and then formulated with appropriate adjuvants. Sixteen 8–12-week-old outbred female and male kittens (n = 4 per group) were randomly assigned into four treatment groups: spike protein alone; spike plus ESSAI oil-in-water (O/W) 1849102 adjuvant; spike plus aluminum hydroxide adjuvant; and a PBS control. All animals were vaccinated intramuscularly twice, 2 weeks apart, with 5 μg of spike protein in a volume of 0.5 ml. On days 0 and 28, serum samples were collected to evaluate anti-spike IgG, antibody inhibition of spike binding to angiotensin-converting enzyme 2 (ACE-2), neutralizing antibodies against wild-type and delta variant viruses, and hematology studies. At day 28, all groups were challenged with SARS-CoV-2 wild-type virus 106 TCID50 intranasally. On day 31, tissue samples (lung, heart, and nasal turbinates) were collected for viral RNA detection, and virus titration. After two immunizations, both vaccines induced high titers of serum anti-spike IgG that inhibited spike ACE-2 binding and neutralized both wild-type and delta variant virus. Both adjuvanted vaccine formulations protected juvenile cats against virus shedding from the upper respiratory tract and viral replication in the lower respiratory tract and hearts. These promising data warrant ongoing evaluation of the vaccine's ability to protect cats against SARS-CoV-2 infection and in particular to prevent transmission.

Abstract Background Alzheimer disease (AD) is characterized by the accumulation of beta-amyloid (Aβ) plaques and neurofibrillary tangles composed of hyperphosphorylated tau, which together lead to neurodegeneration and cognitive decline. Current therapeutic approaches have primarily aimed to reduce pathological aggregates of either Aβ or tau, yet phase 3 clinical trials of these approaches have thus far failed to delay disease progression in humans. Strong preclinical evidence indicates that these two abnormally aggregated proteins interact synergistically to drive downstream neurodegeneration. Therefore, combinatorial therapies that concurrently target both Aβ and tau might be needed for effective disease modification. Methods A combinatorial vaccination approach was designed to concurrently target both Aβ and tau pathologies. Tau22/5xFAD (T5x) bigenic mice that develop both pathological Aβ and tau aggregates were injected intramuscularly with a mixture of two MultiTEP epitope vaccines: AV-1959R and AV-1980R, targeting Aβ and tau, respectively, and formulated in AdvaxCpG, a potent polysaccharide adjuvant. Antibody responses of vaccinated animals were measured by ELISA, and neuropathological changes were determined in brain homogenates of vaccinated and control mice using ELISA and Meso Scale Discovery (MSD) multiplex assays. Results T5x mice immunized with a mixture of Aβ- and tau-targeting vaccines generated high Aβ- and tau-specific antibody titers that recognized senile plaques and neurofibrillary tangles/neuropil threads in human AD brain sections. Production of these antibodies in turn led to significant reductions in the levels of soluble and insoluble total tau, and hyperphosphorylated tau as well as insoluble Aβ42, within the brains of bigenic T5x mice. Conclusions AV-1959R and AV-1980R formulated with AdvaxCpG adjuvant are immunogenic and therapeutically potent vaccines that in combination can effectively reduce both of the hallmark pathologies of AD in bigenic mice. Taken together, these findings warrant further development of this vaccine technology for ultimate testing in human AD.

Abstract Background Toll-like receptor 9 is a key innate immune receptor involved in detecting infectious diseases and cancer. TLR9 activates the innate immune system following the recognition of single-stranded DNA oligonucleotides (ODN) containing unmethylated cytosine-guanine (CpG) motifs. Due to the considerable number of rotatable bonds in ODNs, high-throughput in silico screening for potential TLR9 activity via traditional structure-based virtual screening approaches of CpG ODNs is challenging. In the current study, we present a machine learning based method for predicting novel mouse TLR9 (mTLR9) agonists based on features including count and position of motifs, the distance between the motifs and graphically derived features such as the radius of gyration and moment of Inertia. We employed an in-house experimentally validated dataset of 396 single-stranded synthetic ODNs, to compare the results of five machine learning algorithms. Since the dataset was highly imbalanced, we used an ensemble learning approach based on repeated random down-sampling. Results Using in-house experimental TLR9 activity data we found that random forest algorithm outperformed other algorithms for our dataset for TLR9 activity prediction. Therefore, we developed a cross-validated ensemble classifier of 20 random forest models. The average Matthews correlation coefficient and balanced accuracy of our ensemble classifier in test samples was 0.61 and 80.0%, respectively, with the maximum balanced accuracy and Matthews correlation coefficient of 87.0% and 0.75, respectively. We confirmed common sequence motifs including ‘CC’, ‘GG’,‘AG’, ‘CCCG’ and ‘CGGC’ were overrepresented in mTLR9 agonists. Predictions on 6000 randomly generated ODNs were ranked and the top 100 ODNs were synthesized and experimentally tested for activity in a mTLR9 reporter cell assay, with 91 of the 100 selected ODNs showing high activity, confirming the accuracy of the model in predicting mTLR9 activity. Conclusion We combined repeated random down-sampling with random forest to overcome the class imbalance problem and achieved promising results. Overall, we showed that the random forest algorithm outperformed other machine learning algorithms including support vector machines, shrinkage discriminant analysis, gradient boosting machine and neural networks. Due to its predictive performance and simplicity, the random forest technique is a useful method for prediction of mTLR9 ODN agonists.

ABSTRACT The development of effective vaccines against fungal infections requires the induction of protective, pathogen-specific cell-mediated immune responses. Here, we asked whether combination adjuvants based on delta inulin (Advax) formulated with Toll-like receptor (TLR) agonists could improve vaccine protection mediated by a fungal recombinant protein, Bl-Eng2 (i.e., Blastomyces endoglucanase 2), which itself harbors an immunodominant antigen and dectin-2 agonist/adjuvant. We found that Bl-Eng2 formulated with Advax3 containing TLR9 agonist or Advax8 containing TLR4 agonist provided the best protection against pulmonary infection with Blastomyces dermatitidis, being more effective than complete Freund’s adjuvant or Adjuplex. Advax3 was most efficient in inducing gamma interferon (IFN-γ)- and interleukin-17 (IL-17)-producing antigen-specific T cells that migrated to the lung upon Blastomyces dermatitidis infection. Mechanistic studies revealed Bl-Eng2/Advax3 protection was tempered by neutralization of IL-17 and particularly IFN-γ. Likewise, greater numbers of lung-resident T cells producing IFN-γ, IL-17, or both IFN-γ and IL-17 correlated with fewer fungi recovered from lung. Protection was maintained after depletion of CD4+ T cells, partially reduced by depletion of CD8+ T cells, and completely eliminated after depletion of both CD4+ and CD8+ T cells. We conclude that Bl-Eng2 formulated with Advax3 is promising for eliciting vaccine-induced antifungal immunity, through a previously uncharacterized mechanism involving CD8+ and also CD4+ T cells producing IFN-γ and/or IL-17. Although no licensed vaccine exists as yet against any fungal disease, these findings indicate the importance of adjuvant selection for the development of effective fungal vaccines. IMPORTANCE Fungal disease remains a challenging clinical and public health problem. Despite medical advances, invasive fungal infections have skyrocketed over the last decade and pose a mounting health threat in immunocompetent and -deficient hosts, with worldwide mortality rates ranking 7th, even ahead of tuberculosis. The development of safe, effective vaccines remains a major hurdle for fungi. Critical barriers to progress include the lack of defined fungal antigens and suitable adjuvants. Our research is significant in identifying adjuvant combinations that elicit optimal vaccine-induced protection when formulated with a recombinant protective antigen and uncovering the mechanistic bases of the underlaying vaccine protection, which will foster the strategic development of antifungal vaccines.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in children worldwide, with the most severe disease occurring in very young infants. Despite half a century of research there still are no licensed RSV vaccines. Difficulties in RSV vaccine development stem from a number of factors, including: (a) a very short time frame between birth and first RSV exposure; (b) interfering effects of maternal antibodies; and (c) differentially regulated immune responses in infants causing a marked T helper 2 (Th2) immune bias. This review seeks to provide an age-specific understanding of RSV immunity critical to the development of a successful pediatric RSV vaccine. Historical and future approaches to the prevention of infant RSV are reviewed, including passive protection using monoclonal antibodies or maternal immunization strategies versus active infant immunization using pre-fusion forms of RSV F protein antigens formulated with novel adjuvants such as Advax that avoid excess Th2 immune polarization.

Respiratory syncytial virus (RSV) remains the most common cause of lower respiratory tract infections in children worldwide. Development of a vaccine has been hindered by the risk of developing enhanced respiratory disease (ERD) upon natural exposure to the virus. Generation of higher quality neutralizing antibodies with stabilized pre-fusion F protein antigens has been proposed as a strategy to prevent ERD. We sought to test whether there was evidence of ERD in naïve BALB/c mice immunized with an unadjuvanted, stabilized pre-fusion F protein, and challenged with RSV line 19. We further sought to determine the extent to which formulation with a Th2-biased (alum) or a more Th1/Th2-balanced (Advax-SM) adjuvant influenced cellular responses and lung pathology. When exposed to RSV, mice immunized with pre-fusion F protein alone (PreF) exhibited increased airway eosinophilia and mucus accumulation. This was further exacerbated by formulation of PreF with Alum (aluminum hydroxide). Conversely, formulation of PreF with a Th1/Th2-balanced adjuvant, Advax-SM, not only suppressed RSV viral replication, but also inhibited airway eosinophilia and mucus accumulation. This was associated with lower numbers of lung innate lymphocyte cells (ILC2s) and CD4+ T cells producing IL-5+ or IL-13+ and increased IFNγ+ CD4+ and CD8+ T cells, in addition to RSV F-specific CD8+ T cells. These data suggest that in the absence of preimmunity, stabilized PreF antigens may still be associated with aberrant Th2 responses that induce lung pathology in response to RSV infection, and can be prevented by formulation with more Th1/Th2-balanced adjuvants that enhance CD4+ and CD8+ IFNγ+ T cell responses. This may support the use of stabilized PreF antigens with Th1/Th2-balanced adjuvants like, Advax-SM, as safer alternatives to alum in RSV vaccine candidates.

ABSTRACT Recent global advocacy efforts have highlighted the importance of development of a vaccine against group A Streptococcus (GAS). Combo5 is a non-M protein-based vaccine that provides protection against GAS skin infection in mice and reduces the severity of pharyngitis in nonhuman primates. However, Combo5 with the addition of aluminum hydroxide (alum) as an adjuvant failed to protect against invasive GAS infection of mice. Here, we show that formulation of Combo5 with adjuvants containing saponin QS21 significantly improves protective efficacy, even though all 7 adjuvants tested generated high antigen-specific IgG antibody titers, including alum. Detailed characterization of Combo5 formulated with SMQ adjuvant, a squalene-in-water emulsion containing a TLR4 agonist and QS21, showed significant differences from the results obtained with alum in IgG subclasses generated following immunization, with an absence of GAS opsonizing antibodies. SMQ, but not alum, generated strong interleukin-6 (IL-6), gamma interferon (IFN-γ), and tumor necrosis alpha (TNF-α) responses. This work highlights the importance of adjuvant selection for non-M protein-based GAS vaccines to optimize immune responses and protective efficacy. IMPORTANCE Availability of a group A Streptococcus vaccine remains an unmet public health need. Here, we tested different adjuvant formulations to improve the protective efficacy of non-M protein vaccine Combo5 in an invasive disease model. We show that novel adjuvants can dramatically shape the type of immune response developed following immunization with Combo5 and significantly improve protection. In addition, protection afforded by Combo5 is not mediated by opsonizing antibodies, believed to be the main correlate of protection against GAS infections. Overall, this report highlights the importance of adjuvant selection in raising protective immune responses against GAS invasive infection. Adjuvants that can provide a more balanced Th1/Th2-type response may be required to optimize protection of GAS vaccines, particularly those based on non-M protein antigens.

Dengue fever, caused by any of four dengue viruses (DENV1-4), is a major global burden. Currently, there is no effective vaccine that prevents infection in dengue naïve populations. We tested the ability of two novel adjuvants (Advax-PEI and Advax-2), using aluminum hydroxide (alum) as control, to enhance the immunogenicity of formalin- or psoralen-inactivated (PIV or PsIV) DENV2 vaccines in mice. Mice were vaccinated on days 0 and 30, and serum samples were collected on days 30, 60, 90, and 101. Neutralizing antibodies were determined by microneutralization (MN) assays, and the geometric mean 50% MN (MN50) titers were calculated. For the PIV groups, after one dose MN50 titers were higher in the novel adjuvant groups compared to the alum control, while MN50 titers were comparable between the adjuvant groups after the second dose. For the PsIV groups, both novel adjuvants induced higher MN50 titers than the alum control after the second dose. Spleen cells were collected on days 45 and 101 for enzyme-linked immunospot (ELISPOT) for IFNγ and IL4. Both PIV and PsIV groups elicited different degrees of IFNγ and IL4 responses. Overall, Advax-2 gave the best responses just ahead of Advax-PEI. Given Advax-2’s extensive human experience in other vaccine applications, it will be pursued for further development.

Onchocerciasis (river blindness), caused by the filarial nematode Onchocerca volvulus, is a neglected tropical disease mainly of sub-Saharan Africa. Worldwide, an estimated 20.9 million individuals live with infection and a further 205 million are at risk of disease. Current control methods rely on mass drug administration of ivermectin to kill microfilariae and inhibit female worm fecundity. The identification and development of efficacious vaccines as complementary preventive tools to support ongoing elimination efforts are therefore an important objective of onchocerciasis research. We evaluated the protective effects of co-administering leading O. volvulus-derived recombinant vaccine candidates (Ov-103 and Ov-RAL-2) with subsequent natural exposure to the closely related cattle parasite Onchocerca ochengi. Over a 24-month exposure period, vaccinated calves (n = 11) were shown to acquire infection and microfilaridermia at a significantly lower rate compared to unvaccinated control animals (n = 10). Furthermore, adult female worm burdens were negatively correlated with anti-Ov-103 and Ov-RAL-2 IgG1 and IgG2 responses. Peptide arrays identified several Ov-103 and Ov-RAL-2-specific epitopes homologous to those identified as human B-cell and helper T-cell epitope candidates and by naturally-infected human subjects in previous studies. Overall, this study demonstrates co-administration of Ov-103 and Ov-RAL-2 with Montanide™ ISA 206 VG is highly immunogenic in cattle, conferring partial protection against natural challenge with O. ochengi. The strong, antigen-specific IgG1 and IgG2 responses associated with vaccine-induced protection are highly suggestive of a mixed Th1/Th2 associated antibody responses. Collectively, this evidence suggests vaccine formulations for human onchocerciasis should aim to elicit similarly balanced Th1/Th2 immune responses.

Abstract Background Diabetic macular edema (DME) and proliferative diabetic retinopathy (PDR) are sight-threatening complications of diabetes mellitus and leading causes of adult-onset blindness worldwide. Genetic risk factors for diabetic retinopathy (DR) have been described previously, but have been difficult to replicate between studies, which have often used composite phenotypes and been conducted in different populations. This study aims to identify genetic risk factors for DME and PDR as separate complications in Australians of European descent with type 2 diabetes. Methods Caucasian Australians with type 2 diabetes were evaluated in a genome-wide association study (GWAS) to compare 270 DME cases and 176 PDR cases with 435 non-retinopathy controls. All participants were genotyped by SNP array and after data cleaning, cases were compared to controls using logistic regression adjusting for relevant covariates. Results The top ranked SNP for DME was rs1990145 (p = 4.10 × 10− 6, OR = 2.02 95%CI [1.50, 2.72]) on chromosome 2. The top-ranked SNP for PDR was rs918519 (p = 3.87 × 10− 6, OR = 0.35 95%CI [0.22, 0.54]) on chromosome 5. A trend towards association was also detected at two SNPs reported in the only other reported GWAS of DR in Caucasians; rs12267418 near MALRD1 (p = 0.008) in the DME cohort and rs16999051 in the diabetes gene PCSK2 (p = 0.007) in the PDR cohort. Conclusion This study has identified loci of interest for DME and PDR, two common ocular complications of diabetes. These findings require replication in other Caucasian cohorts with type 2 diabetes and larger cohorts will be required to identify genetic loci with statistical confidence. There is considerable overlap in the patient cohorts with each retinopathy subtype, complicating the search for genes that contribute to PDR and DME biology.

The authors wish to make the following corrections to this paper [...]

ccJE+Advax is an inactivated cell culture Japanese encephalitis (JE) vaccine formulated with Advax, a novel polysaccharide adjuvant based on delta inulin. This vaccine has previously shown promise in murine and equine studies and the current study sought to better understand its mechanism of action and assess the feasibility of single dose vaccine protection. Mice immunised with ccJE+Advax had higher serum neutralisation titres than those immunised with ccJE alone or with alum adjuvant. ccJE+Advax induced extraordinarily broad cross-neutralising antibodies against multiple flaviviruses including West Nile virus (WNV), Murray Valley encephalitis virus (MVEV), St Louis encephalitis virus (SLEV) and Dengue virus-1 and -2 (DENV-1 and -2). Notably, the DENV-2 cross-neutralising antibodies from ccJE+Advax immunised mice uniquely had no DENV-2 antibody-dependent infection enhancement (ADIE) activity, in contrast to high ADIE activity seen with DENV-1 cross-reactive antibodies induced by mbJE or ccJE alone or with alum adjuvant. JEV-stimulated splenocytes from ccJE+Advax immunised mice showed increased IL-17 and IFN-γ production, consistent with a mixed Th1 and Th17 response, whereas ccJE-alum was associated with production of mainly Th2 cytokines. In a mouse lethal challenge study against highly virulent JaTH160 JEV strain, ccJE+Advax conferred complete protection in a two-dose schedule with 50 ng of vaccine antigen and near complete protection after a single 200 ng dose of vaccine antigen. There is an ongoing lack of human vaccines against particular flaviviruses, including WNV, SLEV and MVEV. Given its ability to provide single-dose JEV protection and induce broadly neutralising antibodies devoid of ADIE activity, ccJE+Advax vaccine could be useful in situations where rapid protection is desirable, e.g., during a local outbreak or for use in travellers or armies requiring rapid deployment to JEV endemic regions.

Tuberculosis (TB) remains a main public health concern and 10.4 million new cases occurred in 2015 around the world. BCG is the only approved vaccine against TB, but has variable efficacy and new vaccines are needed. We developed two new mTB vaccine candidates based on the recombinant fusion proteins, rCMX and rECMX formulated with Advax4, a new combination adjuvant combining delta inulin, CpG oligonucleotide and murabutide. BALB/c mice were immunized three times intramuscularly with these vaccine formulations. Injection of Advax4 alone increased the percentage of lymphatic endothelial cells and activated macrophages (F480/CD11b+) in the draining lymph nodes consistent with a chemotactic adjuvant effect. Advax4+CMX and Advax4+ECMX induced the highest levels of IgG1 and IgG2a antibodies against rCMX and rECMX, respectively. Immunized mice challenged with Mycobacterium tuberculosis (Mtb) had increased vaccine-specific Th1 responses in the lungs together with reduced Mtb – associated alveolar damage, although only the Advax4+ECMX vaccine demonstrated significant reduction of lung bacterial load. This study confirmed Advax4+ECMX as a potential TB vaccine candidate, with potential for further optimization and clinical development.

Abstract There is an urgent need for the rational design of safe and effective vaccines to protect against chronic bacterial pathogens such as Mycobacterium tuberculosis. Advax™ is a novel adjuvant based on delta inulin microparticles that enhances immunity with a minimal inflammatory profile and has entered human trials to protect against viral pathogens. In this report we determined if Advax displays broad applicability against important human pathogens by assessing protective immunity against infection with M. tuberculosis. The fusion protein CysVac2, comprising the M. tuberculosis antigens Ag85B (Rv1886c) and CysD (Rv1285) formulated with Advax provided significant protection in the lungs of M. tuberculosis-infected mice. Protection was associated with the generation of CysVac2-specific multifunctional CD4+ T cells (IFN-γ+TNF+IL-2+). Addition to Advax of the TLR9 agonist, CpG oligonucleotide (AdvaxCpG), improved both the immunogenicity and protective efficacy of CysVac2. Immunisation with CysVac2/AdvaxCpG resulted in heightened release of the chemoattractants, CXCL1, CCL3, and TNF, and rapid influx of monocytes and neutrophils to the site of vaccination, with pronounced early priming of CysVac2-specific CD4+ T cells. As delta inulin adjuvants have shown an excellent safety and tolerability profile in humans, CysVac2/AdvaxCpG is a strong candidate for further preclinical evaluation for progression to human trials.

Timely vaccine supply is critical during influenza pandemics but is impeded by current virus-based manufacturing methods. The 2009 H1N1/2009pdm ‘swine flu’ pandemic reinforced the need for innovation in pandemic vaccine design. We report on insights gained during rapid development of a pandemic vaccine based on recombinant haemagglutinin (rHA) formulated with Advax™ delta inulin adjuvant (Panblok-H1/Advax). Panblok-H1/Advax was designed and manufactured within 1 month of the pandemic declaration by WHO and successfully entered human clinical testing in under 3 months from first isolation and sequencing of the novel pandemic virus, requiring several major challenges to be overcome. Panblok-H1/Advax successfully induced neutralising antibodies against the pandemic strain, but also induced cross-neutralising antibodies in a subset of subjects against an H1N1 strain (A/Puerto Rico/8/34) derived from the 1918 Spanish flu, highlighting the possibility to use Advax to induce more broadly cross-protective antibody responses. Interestingly, the rHA from H1N1/2009pdm exhibited variants in the receptor binding domain that had a major impact on receptor binding and hemagglutination ability. We used an in silico structural modeling approach to better understand the unusual behavior of the novel hemagglutinin, thereby demonstrating the power of computational modeling approaches for rapid characterization of new pandemic viruses. While challenges remain in ensuring ultrafast vaccine access for the entire population in response to future pandemics, the adjuvanted recombinant Panblok-H1/Advax vaccine proved its utility during a real-life pandemic situation.

The development of a safe and effective vaccine to protect against COVID-19 is a global priority due to the current high SARS-CoV-2 infection rate. Currently, there are over 160 SARS-CoV-2 vaccine candidates at the clinical or pre-clinical stages of development. Of these, there are only three whole-virus vaccine candidates produced using β-propiolactone or formalin inactivation. Here, we prepared a whole-virus SARS-CoV-2 vaccine (SARS-CoV-2 PsIV) using a novel psoralen inactivation method and evaluated its immunogenicity in mice using two different adjuvants, alum and Advax-2. We compared the immunogenicity of SARS-CoV-2 PsIV against SARS-CoV-2 DNA vaccines expressing either full-length or truncated spike proteins. We also compared the psoralen-inactivated vaccine against a DNA prime, psoralen-inactivated vaccine boost regimen. After two doses, the psoralen-inactivated vaccine, when administered with alum or Advax-2 adjuvants, generated a dose-dependent neutralizing antibody responses in mice. Overall, the pattern of cytokine ELISPOT responses to antigen-stimulation observed in this study indicates that SARS-CoV-2 PsIV with the alum adjuvant promotes a Th2-type response, while SARS-CoV-2 PsIV with the Advax-2 adjuvant promotes a Th1-type response.

Global immunization campaigns have resulted in a major decline in the global incidence of polio cases, with wild-type poliovirus remaining endemic in only two countries. Live oral polio vaccine (OPV) played a role in the reduction in polio case numbers; however, the risk of OPV developing into circulating vaccine-derived poliovirus makes it unsuitable for eradication programs. Trivalent inactivated polio virus (TIPV) vaccines which contain formalin-inactivated antigens produced from virulent types 1, 2 and 3 reference polio strains grown in Vero monkey kidney cells have been advocated as a replacement for OPV; however, TIPVs have weak immunogenicity and multiple boosts are required before peak neutralizing titers are reached. This study examined whether the incorporation of the novel polysaccharide adjuvant, Advax-CpG, could boost the immunogenicity of two TIPV vaccines, (i) a commercially available polio vaccine (IPOL®, Sanofi Pasteur) and (ii) a new TIPV formulation developed by Statens Serum Institut (SSI). Mice were immunized intramuscularly based on recommended vaccine dosage schedules and serum antibody titers were followed for 12 months post-immunization. Advax-CpG significantly enhanced the long-term immunogenicity of both TIPV vaccines and had at least a 10-fold antigen dose-sparing effect. An exception was the poor ability of the SSI TIPV to induce serotype type 1 neutralizing antibodies. Immunization with monovalent IPVs suggested that the low type 1 response to TIPV may be due to antigen competition when the type 1 antigen was co-formulated with the type 2 and 3 antigens. This study provides valuable insights into the complexity of the formulation of multivalent polio vaccines and supports the further development of adjuvanted antigen-sparing TIPV vaccines in the fight to eradicate polio.

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.

Delta inulin, also known as microparticulate inulin (MPI), was modified by covalently attaching doxorubicin to its nanostructured surface for use as a targeted drug delivery vehicle. MPI is readily endocytosed by monocytes, macrophages, and dendritic cells and in this study, we sought to utilize this property to develop a system to target anti-cancer drugs to lymphoid organs. We investigated, therefore, whether MPI could be used as a vehicle to deliver doxorubicin selectively, thereby reducing the toxicity of this antibiotic anthracycline drug. Doxorubicin was covalently attached to the surface of MPI using an acid−labile linkage to enable pH-controlled release. The MPI-doxorubicin conjugate was characterized using FTIR and SEM, confirming covalent attachment and indicating doxorubicin coupling had no obvious impact on the physical nanostructure, integrity, and cellular uptake of the MPI particles. To simulate the stability of the MPI-doxorubicin in vivo, it was stored in artificial lysosomal fluid (ALF, pH 4.5). Although the MPI-doxorubicin particles were still visible after 165 days in ALF, 53% of glycosidic bonds in the inulin particles were hydrolyzed within 12 days in ALF, reflected by the release of free glucose into solution. By contrast, the fructosidic bonds were much more stable. Drug release studies of the MPI-doxorubicin in vitro, demonstrated a successful pH-dependent controlled release effect. Confocal laser scanning microscopy studies and flow cytometric analysis confirmed that when incubated with live cells, MPI-doxorubicin was efficiently internalized by immune cells. An assay of cell metabolic activity demonstrated that the MPI carrier alone had no toxic effects on RAW 264.7 murine monocyte/macrophage-like cells, but exhibited anti-cancer effects against HCT116 human colon cancer cells. MPI-doxorubicin had a greater anti-cancer cell effect than free doxorubicin, particularly when at lower concentrations, suggesting a drug-sparing effect. This study establishes that MPI can be successfully modified with doxorubicin for chemotherapeutic drug delivery.

The use of particles for monocyte-mediated delivery could be a more efficient strategy and approach to achieve intracellular targeting and delivery of antitubercular drugs to host macrophages. In this study, the potential of inulin microparticles to serve as a drug vehicle in the treatment of chronic tuberculosis using a monocytes-mediated drug targeting approach was evaluated. Isoniazid (INH) was conjugated to inulin via hydrazone linkage in order to obtain a pH-sensitive inulin-INH conjugate. The conjugate was then characterized using proton nuclear magnetic resonance (1HNMR), Fourier transform infrared spectroscopy (FTIR) as well as in vitro, cellular uptake and intracellular Mycobacterium tuberculosis (Mtb) antibacterial efficacy. The acid-labile hydrazone linkage conferred pH sensitivity to the inulin-INH conjugate with ~95, 77 and 65% of the drug released after 5 h at pH 4.5, 5.2, and 6.0 respectively. Cellular uptake studies confirm that RAW 264.7 monocytic cells efficiently internalized the inulin conjugates into endocytic compartments through endocytosis. The intracellular efficacy studies demonstrate that the inulin conjugates possess a dose-dependent targeting effect against Mtb-infected monocytes. This was through efficient internalization and cleavage of the hydrazone bond by the acidic environment of the lysosome, which subsequently released the isoniazid intracellularly to the Mtb reservoir. These results clearly suggest that inulin conjugates can serve as a pH-sensitive intracellular drug delivery system for TB treatment.

Tuberculosis (TB) caused by Mycobacterium tuberculosis infection is responsible for the most deaths by a single infectious agent worldwide, with 1.6 million deaths in 2017 alone. The World Health Organization, through its “End TB” strategy, aims to reduce TB deaths by 95% by 2035. In order to reach this goal, a more effective vaccine than the Bacillus Calmette-Guerin (BCG) vaccine currently in use is needed. Subunit TB vaccines are ideal candidates, because they can be used as booster vaccinations for individuals who have already received BCG and would also be safer for use in immunocompromised individuals in whom BCG is contraindicated. However, subunit TB vaccines will almost certainly require formulation with a potent adjuvant. As the correlates of vaccine protection against TB are currently unclear, there are a variety of adjuvants currently being used in TB vaccines in preclinical and clinical development. This review describes the various adjuvants in use in TB vaccines, their effectiveness, and their proposed mechanisms of action. Notably, adjuvants with less inflammatory and reactogenic profiles that can be administered safely via mucosal routes, may have the biggest impact on future directions in TB vaccine design.

The propensity of monocytes to migrate into sites of mycobacterium tuberculosis (TB) infection and then become infected themselves makes them potential targets for delivery of drugs intracellularly to the tubercle bacilli reservoir. Conventional TB drugs are less effective because of poor intracellular delivery to this bacterial sanctuary. This study highlights the potential of using semicrystalline delta inulin particles that are readily internalised by monocytes for a monocyte-based drug delivery system. Pyrazinoic acid was successfully attached covalently to the delta inulin particles via a labile linker. The formation of new conjugate and amide bond was confirmed using zeta potential, Proton Nuclear Magnetic Resonance (1HNMR) and Fourier transform infrared spectroscopy (FTIR). Scanning electron microscopy (SEM) confirmed that no significant change in size after conjugation which is an important parameter for monocyte targeting. Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) were used to establish the change in thermal properties. The analysis of in-vitro release demonstrated pH-triggered drug cleavage off the delta inulin particles that followed a first-order kinetic process. The efficient targeting ability of the conjugate for RAW 264.7 monocytic cells was supported by cellular uptake studies. Overall, our finding confirmed that semicrystalline delta inulin particles (MPI) can be modified covalently with drugs and such conjugates allow intracellular drug delivery and uptake into monocytes, making this system potentially useful for the treatment of TB.

We previously developed a potent candidate vaccine against bovine brucellosis caused by Brucella abortus using the influenza viral vector expressing Brucella Omp16 and L7/L12 proteins (Flu-BA). Our success in the Flu-BA vaccine trial in cattle and results of a pilot study in non-pregnant small ruminants prompted us in the current study to test its efficacy against B. melitensis infection in pregnant sheep and goats. In this study, we improved the Flu-BA vaccine formulation and immunization method to achieve maximum efficacy and safety. The Flu-BA vaccine formulation had two additional proteins Omp19 and SOD, and administered thrice with 20% Montanide Gel01 adjuvant, simultaneously by both subcutaneous and conjunctival routes at 21 days intervals in pregnant sheep and goats. At 42 days post-vaccination (DPV) we detected antigen-specific IgG antibodies predominantly of IgG2a isotype but also IgG1, and also detected a strong lymphocyte recall response with IFN-γ production. Importantly, our candidate vaccine prevented abortion in 66.7% and 77.8% of pregnant sheep and goats, respectively. Furthermore, complete protection (absence of live B. melitensis 16M) was observed in 55.6% and 66.7% of challenged sheep and goats, and 72.7% and 90.0% of their fetuses (lambs/yeanlings), respectively. The severity of B. melitensis 16M infection in vaccinated sheep and goats and their fetuses (index of infection and rates of Brucella colonization in tissues) was significantly lower than in control groups. None of the protection parameters after vaccination with Flu-BA vaccine were statistically inferior to protection seen with the commercial B. melitensis Rev.1 vaccine (protection against abortion and vaccination efficacy, alpha = 0.18-0.34, infection index, P = 0.37-0.77, Brucella colonization, P = 0.16 to P > 0.99). In conclusion, our improved Flu-BA vaccine formulation and delivery method were found safe and effective in protecting pregnant sheep and goats against adverse consequences of B. melitensis infection.

Fructose is a significant component in unprocessed food and has become one of the most commonly sweeteners used in food manufacturing. Fructose is also a useful pharmaceutical excipient and derivatives of fructose are exploited as renewable chemical building blocks. Fructose based polysaccharides have extensive pharmaceutical and dietary functions. We discuss here the chemistry and physical behaviours of this saccharide and how these factors affect the utility and health implications of fructose.

alpha-D-glucopyranosyl-[beta-D-fructofuranosyl](n-1)-D-fructofuranoside, commonly referred to as inulin, is a natural plant-derived polysaccharide with a diverse range of food and pharmaceutical applications. It is used by the food industry as a soluble dietary fibre and fat or sugar replacement, and in the pharmaceutical industry as a stabiliser and excipient. It can also be used as a precursor in the synthesis of a wide range of compounds. New uses for inulin are constantly being discovered, with recent research into its use for slow-release drug delivery. Inulin, when in a particulate form, possesses anti-cancer and immune enhancing properties. Given its increasing importance to industry, this review explains how inulin's unique physico-chemical properties bestow it with many useful pharmaceutical applications.