Intranasal administration of unadjuvanted SARS-CoV-2 spike antigen boosts antigen-specific immune responses induced by parenteral protein subunit vaccine prime in mice and hamsters.

With the continued transmission of SARS-CoV-2 across widely vaccinated populations, it remains important to develop new vaccines and vaccination strategies capable of providing protective immunity and limiting the spread of disease. Heterologous prime-boost vaccination based on the selection of different vaccine formulations and administration routes for priming and booster doses presents a promising strategy for inducing broader immune responses in key systemic and respiratory mucosal compartments. Intranasal vaccination can induce mucosal immune responses at the site of SARS-CoV-2 infection; however, the lack of clinically approved mucosal adjuvants makes it difficult to induce robust immune responses with protein subunit vaccines. Herein, we evaluated the immunogenicity of heterologous prime-boost regimens in mice and hamsters based on a parenteral vaccination of the antigen in combination with sulfated lactosylarchaeol (SLA) archaeosomes, a liposome adjuvant comprised of a single semisynthetic archaeal lipid, followed by an intranasally administered unadjuvanted SARS-CoV-2 spike antigen. Intranasal administration of unadjuvanted spike to mice and hamsters increased serum spike-specific IgG titers and spike-neutralizing activity compared with nonboosted animals. Spike-specific IgA responses were also detected in the bronchoalveolar lavage fluid in the lungs of mice that received an intranasal boost. In hamsters, the intranasal boost showed high efficacy against SARS-CoV-2 infection by protecting from body weight loss and reducing viral titers in the lungs and nasal turbinate. Overall, our heterologous intramuscular prime-intranasal boost with SLA-adjuvanted and unadjuvanted spike, respectively, demonstrated the potential of protein subunit formulations to promote antigen-specific systemic and mucosal immune responses.

Production, purification and immunogenicity of Gag virus-like particles carrying SARS-CoV-2 components.

The virus-like particle (VLP) platform is a robust inducer of humoral and cellular immune responses; hence, it has been used in vaccine development for several infectious diseases. In the current work, VLPs carrying SARS-CoV-2 Spike (S) protein (Wuhan strain) with an HIV-1 Gag core were produced using suspension HEK 293SF-3F6 cells by transient transfection. The Gag was fused with green fluorescent protein (GFP) for rapid quantification of the VLPs. Five different versions of Gag-Spike VLPs (Gag-S-VLPs) consisting of Gag-S alone or combined with other SARS-CoV-2 components, namely Gag-S-Nucleocapsid (N), Gag-S-Matrix (M), Gag-S-Envelope (E), Gag-S-MEN, along with Gag alone were produced and processed by clarification, nuclease treatment, concentration by tangential flow filtration (TFF) and diafiltration. A pilot mouse study was performed to evaluate the immunogenicity of the Gag-S-VLPs through the measurement of the humoral and/or cellular responses against all the mentioned SARS-CoV-2 components. Antibody response to Spike was observed in all variants. The highest number of Spike-specific IFN-γ + T cells was detected with Gag-S-VLPs. No induction of antigen-specific cellular responses to M, N or E proteins were detected with any of the Gag-S, M, E/or N VLPs tested. Therefore, the Gag-S-VLP, by reason of consistently eliciting strong antigen-specific cellular and antibody responses, was selected for further evaluation. The purification process was improved by replacing the conventional centrifugation by serial microfiltration in the clarification step, followed by Spike-affinity chromatography to get concentrated VLPs with higher purity. Three different doses of Gag-S-VLP in conjunction with two adjuvants (Quil-A or AddaVax) were used to assess the dose-dependent antigen-specific cellular and antibody responses in mice. The Gag-S-VLP adjuvanted with Quil-A resulted in a stronger Spike-specific cellular response compared to that adjuvanted with AddaVax. A strong spike neutralisation activity was observed for all doses, independent of the adjuvant combination.

Blood-Based Immune Protein Markers of Disease Progression in Murine Models of Acute and Chronic Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD) is a chronic ailment afflicting millions of people worldwide, with the majority of recognized cases within industrialized countries. The impacts of IBD at the individual level are long-lasting with few effective treatments available, resulting in a large burden on the health care system. A number of existing animal models are utilized to evaluate novel treatment strategies. Two commonly used models are (1) acute colitis mediated by dextran sulphate sodium (DSS) treatment of wild-type mice and (2) chronic colitis mediated by the transfer of proinflammatory T cells into immunodeficient mice. Despite the wide use of these particular systems to evaluate IBD therapeutics, the typical readouts of clinical disease progression vary depending on the model used, which may be reflective of mechanistic differences of disease induction. The most reliable indicator of disease in both models remains intestinal damage which is typically evaluated upon experimental endpoint. Herein, we evaluated the expression profile of a panel of cytokines and chemokines in both DSS and T cell transfer models in an effort to identify a number of inflammatory markers in the blood that could serve as reliable indicators of the relative disease state. Out of the panel of 25 markers tested, 6 showed statistically significant shifts with the DSS model, compared to 11 in the T cell transfer model with IL-6, IL-13, IL-22, TNF-α and IFN-γ being common markers of disease in both models. Our data highlights biological differences between animal models of IBD and helps to guide future studies when selecting efficacy readouts during the evaluation of experimental IBD therapeutics.

Effectiveness of VSV vectored SARS-CoV-2 spike when administered through intranasal, intramuscular or a combination of both.

A critical feature of the VSV vector platform is the ability to pseudotype the virus with different glycoproteins from other viruses, thus altering cellular tropism of the recombinant virus. The route of administration is critical in triggering local and systemic immune response and protection. Most of the vaccine platforms used at the forefront are administered by intramuscular injection. However, it is not known at what level ACE2 is expressed on the surface of skeletal muscle cells, which will have a significant impact on the efficiency of a VSV-SARS-CoV-2 spike vaccine to mount a protective immune response when administered intramuscularly. In this study, we investigate the immunogenicity and efficacy of a prime-boost immunization regimen administered intranasally (IN), intramuscularly (IM), or combinations of the two. We determined that the prime-boost combinations of IM followed by IN immunization (IM + IN) or IN followed by IN immunization (IN + IN) exhibited strong spike-specific IgG, IgA and T cell response in vaccinated K18 knock-in mice. Hamsters vaccinated with two doses of VSV expressing SARS-CoV-2 spike, both delivered by IN or IM + IN, showed strong protection against SARS-CoV-2 variants of concern Alpha and Delta. This protection was also observed in aged hamsters. Our study underscores the highly crucial role immunization routes have with the VSV vector platform to elicit a strong and protective immune response.

Sulfated Lactosyl Archaeol Archaeosome-Adjuvanted Vaccine Formulations Targeting Rabbit Hemorrhagic Disease Virus Are Immunogenic and Efficacious.

Vaccines play an important role in maintaining human and animal health worldwide. There is continued demand for effective and safe adjuvants capable of enhancing antigen-specific responses to a target pathogen. Rabbit hemorrhagic disease virus (RHDV) is a highly contagious calicivirus that often induces high mortality rates in rabbits. Herein, we evaluated the activity of an experimental sulfated lactosyl archaeol (SLA) archaeosome adjuvant when incorporated in subunit vaccine formulations targeting RHDV. The subunit antigens consisted of RHDV-CRM197 peptide conjugates or recombinant RHDV2 VP60. SLA was able to enhance antigen-specific antibody titers and cellular responses in mice and rabbits. Three weeks following immunization, antigen-specific antibody levels in rabbits vaccinated with RHDV2 VP60 + SLA were significantly higher than those immunized with antigen alone, with geomean titers of 7393 vs. 117. In addition, the SLA-adjuvanted VP60-based formulations were highly efficacious in a rabbit RHDV2 challenge model with up to 87.5% animals surviving the viral challenge. These findings demonstrate the potential utility of SLA adjuvants in veterinary applications and highlight its activity in different types of mammalian species.

Roles of natural resistance-associated macrophage protein-1 in modulating bacterial distribution and immune responses during Salmonella enterica serovar Typhimurium infection in murine pregnancy.

PROBLEM: Salmonella enterica serovar Typhimurium (S.Tm) infection in Nramp1+/+ mice during pregnancy can lead to profound bacterial growth in the feto-placental unit and adverse pregnancy outcomes, including fetal loss, maternal illness and death. The kinetics and mechanisms by which S.Tm gains entry within individual feto-placental unit, and disseminates through tissues leading to placental resorption and fetal demise remain unclear. METHOD OF STUDY: Mice were systemically infected with S.Tm. Bacterial burden within spleen and individual placentas, and placental/fetal resorptions were quantified. Flow cytometric analysis of immune cell types in the spleen and individual placentas was performed. Cytokine expression in maternal serum was determined through cytometric bead array. RESULTS: Systemic infection with S.Tm resulted in preferential bacterial proliferation in placentas compared to the spleen in Nramp1+/+ mice. At 24 h post-infection, the mean infection rate of individual placentas per mouse was ∼50%, increasing to >75% by 72 h post-infection, suggesting that initial infection in few sites progresses to rapid spread of infection through the uterine milieu. This correlated with a steady increase in placental/fetal resorption rates. Placental infection was associated with local increased neutrophil percentages, whereas numbers and percentages in the spleen remained unchanged, suggesting dichotomous modulation of inflammation between the systemic compartment and the feto-maternal interface. Reduced survival rates of pregnant mice during infection correlated with decreased serum IFN-γ but increased IL-10 levels relative to non-pregnant controls. CONCLUSION: Pregnancy compromises host resistance conferred by Nramp1 against S.Tm through compartment-specific regulation of maternal and placental cellular responses, and modulation of systemic cytokine expression.

Adjuvants: Engineering Protective Immune Responses in Human and Veterinary Vaccines.

Adjuvants are key components of many vaccines, used to enhance the level and breadth of the immune response to a target antigen, thereby enhancing protection from the associated disease. In recent years, advances in our understanding of the innate and adaptive immune systems have allowed for the development of a number of novel adjuvants with differing mechanisms of action. Herein, we review adjuvants currently approved for human and veterinary use, describing their use and proposed mechanisms of action. In addition, we will discuss additional promising adjuvants currently undergoing preclinical and/or clinical testing.

Effect of Chiral Purity on Adjuvanticity of Archaeol-Based Glycolipids.

Archaeosomes composed of sulfated lactosyl archaeol (SLA) glycolipids from stereoisomerically pure archaeol (1) are vaccine adjuvants that can boost immunogenicity and vaccine efficacy in preclinical models. Herein, we report a new synthesis of 2,3-bis((3,7,11,15-tetramethylhexadecyl)oxy) propan-1-ol (3) by treating (±)-3-benzyloxy-1,2-propanediol with a mesylated phytol derivative through a double nucleophilic substitution reaction, followed by reductive debenzylation. Three SLA archaeosomes from archaeols of different chiral purities were prepared, and the effect of stereochemistry on their adjuvanticity toward ovalbumin was investigated. It was found that all SLA archaeosomes induced strong humoral and cell-mediated antigen-specific immune responses following immunization of C57BL/6NCrl mice, with no significant differences, irrespective of the chiral purities. The responses were comparable or better than those obtained using mimetics of approved adjuvants. The performance of SLA archaeosomes during immunization and their lack of dependence on the stereochemistry of archaeol points toward a promising, safe, scalable, and economically viable vaccine adjuvant system.

Evaluation of Adjuvant Activity and Bio-Distribution of Archaeosomes Prepared Using Microfluidic Technology.

Archaeosomes, composed of sulfated lactosyl archaeol (SLA) glycolipids, have been proven to be an effective vaccine adjuvant in multiple preclinical models of infectious disease or cancer. They have classically been prepared using a thin-film hydration method with an average particle size of 100-200 nm. In this study, we developed methods to generate SLA archaeosomes at different sizes, i.e., 30 nm and 100 nm, via microfluidic mixing technology and evaluated their physicochemical characteristics, as well as adjuvant activity and in vivo biodistribution in mice. Archaeosomes, prepared using thin-film and microfluidic mixing techniques, had similar nanostructures and physicochemical characteristics, with both appearing stable during the course of this study when stored at 4 °C or 37 °C. They also demonstrated similar adjuvant activity when admixed with ovalbumin antigen and used to immunize mice, generating equivalent antigen-specific immune responses. Archaeosomes, labeled with CellVueTM NIR815, had an equivalent biodistribution with both sizes, namely the highest signal at the injection site at 24 h post injection, followed by liver, spleen and inguinal lymph node. The presence of SLA archaeosomes of either size helped to retain OVA antigen (OVA-Cy5.5) longer at the injection site than unadjuvanted OVA. Overall, archaeosomes of two sizes (30 nm and 100 nm) prepared using microfluidic mixing maintained similar physicochemical properties, adjuvant activity and biodistribution of antigen, in comparison to those compared by the conventional thin film hydration method. This suggests that microfluidics based approaches could be applied to generate consistently sized archaeosomes for use as a vaccine adjuvant.

Intranasal immunization with a proteosome-adjuvanted SARS-CoV-2 spike protein-based vaccine is immunogenic and efficacious in mice and hamsters.

With the persistence of the SARS-CoV-2 pandemic and the emergence of novel variants, the development of novel vaccine formulations with enhanced immunogenicity profiles could help reduce disease burden in the future. Intranasally delivered vaccines offer a new modality to prevent SARS-CoV-2 infections through the induction of protective immune responses at the mucosal surface where viral entry occurs. Herein, we evaluated a novel protein subunit vaccine formulation containing a resistin-trimerized prefusion Spike antigen (SmT1v3) and a proteosome-based mucosal adjuvant (BDX301) formulated to enable intranasal immunization. In mice, the formulation induced robust antigen-specific IgG and IgA titers, in the blood and lungs, respectively. In addition, the formulations were highly efficacious in a hamster challenge model, reducing viral load and body weight loss. In both models, the serum antibodies had strong neutralizing activity, preventing the cellular binding of the viral Spike protein based on the ancestral reference strain, the Beta (B.1.351) and Delta (B.1.617.2) variants of concern. As such, this intranasal vaccine formulation warrants further development as a novel SARS-CoV-2 vaccine.

Methods to Evaluate Immune Cell Recruitment and Cellular Uptake and Distribution of Antigen Following Intramuscular Administration of Vaccine to Mice.

An effective vaccine depends on the stimulation of the immune system to generate effective antigen-specific immune responses capable of neutralizing mediators of disease long after vaccination. However, the ability of the vaccine to enhance immune parameters such as cell activation, cell recruitment and antigen uptake shortly following administration contributes to the development of long-term responses directed toward the antigen. Here, we describe a flow cytometry-based method to identify changes in immune cell profile and assess cellular uptake and distribution of antigen following vaccination.

A Method to Evaluate In Vivo CD8+ T Cell Cytotoxicity in a Murine Model.

Herein, a method to measure in vivo CD8+ T cell cytotoxicity in a murine model is presented. The activation of a strong CD8+ T cell response is paramount when designing vaccines to tackle intracellular infections and for cancer therapy. CD8+ T cells can directly kill infected and transformed cells and are directly associated with beneficial protection in many disease models. CD8+ T cell cytotoxicity can be measured using multiple methods including measuring IFNγ production by ELISPOT or measuring intracellular cytokines or cytotoxic granules by flow cytometry. However, to determine the ability of CD8+ T cells to kill their target in the context of its cognate receptor and in their native environment, the in vivo cytotoxic T cell assay (in vivo CTL) is ideal. The in vivo CTL assay provides a snapshot of the whole ability of the host to kill "Target" cells by measuring the loss of injected target cells relative to "Non-target" cells. The assay involves isolating splenocytes from donor mice, forming "Target" and "Non-target" cellular samples and injecting them intravenously into naïve and experimental mice at a chosen time-point in the experiment. Mice are humanely sacrificed 20 h later, and their spleens are excised and processed for flow cytometric analysis. The extent of "Target" cell killing relative to "Non-target" cells is determined by comparing the surviving proportions of these cells among experimental mice relative to naïve mice. The in vivo CTL assay is a rapid, sensitive, and reliable method to measure the potency of CD8+ T cells in their host to kill their target.

IFN-alpha receptor deficiency enhances host resistance to oral Salmonella enterica serovar Typhimurium infection during murine pregnancy.

PROBLEM: Maternal tolerance during pregnancy increases the risk of infection with certain intracellular pathogens. Systemic Salmonella enterica serovar Typhimurium (S.Tm) infection during pregnancy in normally resistant 129X1/SvJ mice leads to severe placental infection, as well as fetal and maternal deaths. However, the effect of oral infection with S.Tm in pregnant mice and the roles of infection-induced inflammation and cell death pathways in contributing to susceptibility to infection are unclear. METHOD OF STUDY: Non-pregnant and pregnant C57BL/6J wild-type (WT) and cell death pathway-altered mice (IFNAR1-/- , Caspase-1, 11-/- , RIP3-/- ) were infected orally with S.Tm. Host survival and fetal resorption were determined. Bacterial burden in mesenteric lymph nodes (MLNs), spleen, liver, and placentas was enumerated at various time points post-infection. Serum cytokine expression was measured through cytometric bead array. RESULTS: Oral infection of WT mice with S.Tm on days 9-10 of gestation resulted in systemic dissemination of the bacteria, substantial placental colonization, and fetal loss 5 days post-infection. Histopathological examination of the placentas indicated that infection-induced widespread focal necrosis and neutrophil infiltration throughout the spongiotrophoblast (SpT) layer. In the non-pregnant state, IFNAR1-/- mice exhibited increased survival following oral S.Tm infection relative to Caspase-1, 11-/- , RIP3-/- , and WT mice. The increased resistance to S.Tm infection in IFNAR1-/- mice was seen during pregnancy as well, with decreased bacterial burden within MLNs, spleen, and placenta, which correlated with the decreased resorptions relative to WT and Caspase-1, 11-/- mice. CONCLUSION: Oral S.Tm exposure leads to placental infection, inflammation, and resorption, whereas IFNAR1 deficiency enhances host resistance both in the non-pregnant and pregnant states.

The Synergistic Effects of Sulfated Lactosyl Archaeol Archaeosomes When Combined with Different Adjuvants in a Murine Model.

Archaeosomes, composed of sulfated lactosyl archaeol (SLA) glycolipids, have been proven to be an effective vaccine adjuvant in multiple preclinical models of infectious disease or cancer. SLA archaeosomes are a promising adjuvant candidate due to their ability to strongly stimulate both humoral and cytotoxic immune responses when simply admixed with an antigen. In the present study, we evaluated whether the adjuvant effects of SLA archaeosomes could be further enhanced when combined with other adjuvants. SLA archaeosomes were co-administered with five different Toll-like Receptor (TLR) agonists or the saponin QS-21 using ovalbumin as a model antigen in mice. Both humoral and cellular immune responses were greatly enhanced compared to either adjuvant alone when SLA archaeosomes were combined with either the TLR3 agonist poly(I:C) or the TLR9 agonist CpG. These results were also confirmed in a separate study using Hepatitis B surface antigen (HBsAg) and support the further evaluation of these adjuvant combinations.

Sulfated Lactosyl Archaeol Archaeosomes Synergize with Poly(I:C) to Enhance the Immunogenicity and Efficacy of a Synthetic Long Peptide-Based Vaccine in a Melanoma Tumor Model.

Cancer remains a leading cause of morbidity and mortality worldwide. While novel treatments have improved survival outcomes for some patients, new treatment modalities/platforms are needed to combat a wider variety of tumor types. Cancer vaccines harness the power of the immune system to generate targeted tumor-specific immune responses. Liposomes composed of glycolipids derived from archaea (i.e., archaeosomes) have been shown to be potent adjuvants, inducing robust, long-lasting humoral and cell-mediated immune responses to a variety of antigens. Herein, we evaluated the ability of archaeosomes composed of sulfated lactosyl archaeol (SLA), a semi-synthetic archaeal glycolipid, to enhance the immunogenicity of a synthetic long peptide-based vaccine formulation containing the dominant CD8+ T cell epitope, SIINFEKL, from the weakly immunogenic model antigen ovalbumin. One advantage of immunizing with long peptides is the ability to include multiple epitopes, for example, the long peptide antigen was also designed to include the immediately adjacent CD4+ epitope, TEWTSSNVMEER. SLA archaeosomes were tested alone or in combination with the toll-like receptor 3 (TLR3) agonist Poly(I:C). Overall, SLA archaeosomes synergized strongly with Poly(I:C) to induce robust antigen-specific CD8+ T cell responses, which were highly functional in an in vivo cytolytic assay. Furthermore, immunization with this vaccine formulation suppressed tumor growth and extended mouse survival in a mouse melanoma tumor model. Overall, the combination of SLA archaeosomes and Poly(I:C) appears to be a promising adjuvant system when used along with long peptide-based antigens targeting cancer.

Mechanistic insight into the induction of cellular immune responses by encapsulated and admixed archaeosome-based vaccine formulations.

Archaeosomes are liposomes formulated using total polar lipids (TPLs) or semi-synthetic glycolipids derived from archaea. Conventional archaeosomes with entrapped antigen exhibit robust adjuvant activity as demonstrated by increased antigen-specific humoral and cell-mediated responses and enhanced protective immunity in various murine infection and cancer models. However, antigen entrapment efficiency can vary greatly resulting in antigen loss during formulation and variable antigen:lipid ratios. In order to circumvent this, we recently developed an admixed archaeosome formulation composed of a single semi-synthetic archaeal lipid (SLA, sulfated lactosylarchaeol) which can induce similarly robust adjuvant activity as an encapsulated formulation. Herein, we evaluate and compare the mechanisms involved in the induction of early innate and antigen-specific responses by both admixed (Adm) and encapsulated (Enc) SLA archaeosomes. We demonstrate that both archaeosome formulations result in increased immune cell infiltration, enhanced antigen retention at injection site and increased antigen uptake by antigen-presenting cells and other immune cell types, including neutrophils and monocytes following intramuscular injection to mice using ovalbumin as a model antigen. In vitro studies demonstrate SLA in either formulation is preferentially taken up by macrophages. Although the encapsulated formulation was better able to induce antigen-specific CD8+ T cell activation by dendritic cells in vitro, both encapsulated and admixed formulations gave equivalently enhanced protection from tumor challenge when tested in vivo using a B16-OVA melanoma model. Despite some differences in the immunostimulatory profile relative to the SLA (Enc) formulation, SLA (Adm) induces strong in vivo immunogenicity and efficacy, while offering an ease of formulation.

Type I interferons differentially modulate maternal host immunity to infection by Listeria monocytogenes and Salmonella enterica serovar Typhimurium during pregnancy.

PROBLEM: IFN-alpha receptor deficiency (IFNAR-/- ) enhances immunity to Listeria monocytogenes (LM) and Salmonella enterica serovar Typhimurium (ST) in the non-pregnant state by inhibiting pathogen-induced immune cell death. However, the roles of IFNAR signaling in modulating immunity to infection during pregnancy are not well understood. METHOD OF STUDY: C57BL/6J wild-type (WT) and IFNAR-/- mice were infected systemically with LM or ST. Bacterial burden in spleen and individual placentas was enumerated at day 3 post-infection. Immune cell numbers and percentages were quantified in spleen and individual placentas, respectively, through flow cytometry. Cytokine expression in serum, spleen, and individual placentas was measured through cytometric bead array. RESULTS: IFNAR-/- mice exhibited decreased splenic monocyte numbers in non-pregnant and pregnant state, and an altered distribution of placental immune cell types in the non-infected state. IFNAR-/- mice controlled LM infection more effectively than WT mice even during pregnancy. This correlated with enhanced serum IL-12 expression, despite reduced splenic monocyte numbers relative to WT controls. In contrast, pregnant IFNAR-/- mice unlike their non-pregnant counterparts exhibited increased susceptibility to ST infection, which was associated with decreased serum IL-12 expression. CONCLUSION: Type I IFN responses differentially impact host resistance to LM and ST infection during pregnancy through modulation of immune cell distribution and cytokine responses.

Simplified Admix Archaeal Glycolipid Adjuvanted Vaccine and Checkpoint Inhibitor Therapy Combination Enhances Protection from Murine Melanoma.

Archaeosomes are liposomes composed of natural or synthetic archaeal lipids that when used as adjuvants induce strong long-lasting humoral and cell-mediated immune responses against entrapped antigens. However, traditional entrapped archaeosome formulations have only low entrapment efficiency, therefore we have developed a novel admixed formulation which offers many advantages, including reduced loss of antigen, consistency of batch-to-batch production as well as providing the option to formulate the vaccine immediately before use, which is beneficial for next generation cancer therapy platforms that include patient specific neo-antigens or for use with antigens that are less stable. Herein, we demonstrate that, when used in combination with anti-CTLA-4 and anti-PD-1 checkpoint therapy, this novel admixed archaeosome formulation, comprised of preformed sulfated lactosyl archaeol (SLA) archaeosomes admixed with OVA antigen (SLA-OVA (adm)), was as effective at inducing strong CD8+ T cell responses and protection from a B16-OVA melanoma tumor challenge as the traditionally formulated archaeosomes with encapsulated OVA protein. Furthermore, archaeosome vaccine formulations combined with anti-CTLA-4 and anti-PD-1 therapy, induced OVA-CD8+ T cells within the tumor and immunohistochemical analysis revealed the presence of CD8+ T cells associated with dying or dead tumor cells as well as within or around tumor blood vessels. Overall, archaeosomes constitute an attractive option for use with combinatorial checkpoint inhibitor cancer therapy platforms.

Archaeal glycolipid adjuvanted vaccines induce strong influenza-specific immune responses through direct immunization in young and aged mice or through passive maternal immunization.

Vaccine induced responses are often weaker in those individuals most susceptible to infection, namely the very young and the elderly, highlighting the need for safe and effective vaccine adjuvants. Herein we evaluated different archaeosome formulations as an adjuvant to the H1N1 influenza hemagglutinin protein and compared immune responses (anti-HA IgG and hemagglutination inhibition assay titers) as well as protection to an influenza A virus (strainA/PuertoRico/8/1934H1N1)homologous challenge to those generated using a squalene-based oil-in-water nano-emulsion, AddaVax™ in a murine model. The impact of age (young adult vs aged) on vaccine induced immune responses as well as the protection in pups due to the transfer of maternal antibodies was measured. Overall, we show that archaeal lipid based adjuvants can induce potent anti-HA responses in young and aged mice that can also be passed from vaccinated mothers to pups. Furthermore, young and aged mice immunized with archaeal lipid adjuvants as well as pups from immunized mothers were protected from challenge with influenza. In addition, we show that a simple admixed archaeosome formulation composed of a single sulfated glycolipid namely sulfated lactosylarchaeol (SLA; 6'-sulfate-ß-D-Galp-(1,4)-ß-D-Glcp-(1,1)-archaeol) can give equal or better protection compared to AddaVax™ or the traditional antigen-encapsulated archaeosome formulations.

Lack of functional selectin-ligand interactions enhances innate immune resistance to systemic Listeria monocytogenes infection.

Selectin-ligand interactions are important for leukocyte homing and functionality. The roles of selectin-ligand interactions in modulating immunity to intracellular infections are not completely understood. Mice lacking the expression of fucosyltransferase-IV and -VII (Fucosyltransferase-IV and -VII double knockout, FtDKO) exhibit deficient functionality of selectin-ligand interactions. We addressed the kinetics of infection and immunity to Listeria monocytogenes (LM), an intracellular pathogen, in FtDKO mice. These mice exhibited enhanced ability to clear infection and increased survival to a lethal dose of LM infection relative to wild-type (WT) C57BL/6J controls. This was associated with increased levels of neutrophils, monocytes, and dendritic cells (DCs) in the blood and/or infected organs. Adoptive transfer of bone marrow (BM) cells from FtDKO mice to WT mice resulted in enhanced neutrophil numbers and improved clearance of LM bacteria in recipients. In vivo depletion of myeloid innate immune cells, particularly neutrophils, monocytes, macrophages, and DCs, using anti-Ly-6G (RB6-8C5) monoclonal antibody, reduced the ability of FtDKO mice to curtail LM infection. Nevertheless, depletion using anti-Ly-6G (1A8) known to exclusively deplete neutrophils did not abrogate increased resistance of FtDKO mice to LM infection, suggesting a role for other myeloid innate immune cells in this model. Examination of BM hematopoietic progenitors through flow cytometry and cell culture colony-forming unit assay showed increased frequencies of granulocyte-macrophage progenitors in FtDKO relative to WT mice, Overall, our results indicate that functional selectin ligand deficiency enhances innate immune-mediated resistance to systemic LM infection despite defective leukocyte migration and lymphocyte homing.