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.

Assessment of stability of sulphated lactosyl archaeol archaeosomes for use as a vaccine adjuvant.

Archaeosomes, composed of sulphated lactosyl archaeol (SLA) glycolipids, have been proven to be an effective vaccine adjuvant in multiple preclinical models of infectious disease or cancer. In addition to efficacy, the stability of vaccine components including the adjuvant is an important parameter to consider when developing novel vaccine formulations. To properly evaluate the potential of SLA glycolipids to be used as vaccine adjuvants in a clinical setting, a comprehensive evaluation of their stability is required. Herein, we evaluated the long term stability of preformed empty SLA archaeosomes prior to admixing with antigen at 4 °C or 37 °C for up to 6 months. In addition, the stability of adjuvant and antigen was evaluated for up to 1 month following admixing. Multiple analytical parameters evaluating the molecular integrity of SLA and the liposomal profile were assessed. Following incubation at 4 °C or 37 °C, the SLA glycolipid did not show any pattern of degradation as determined by mass spectroscopy, nuclear magnetic resonance (NMR) and thin layer chromatography (TLC). In addition, SLA archaeosome vesicle characteristics, such as size, zeta potential, membrane fluidity and vesicular morphology, were largely consistent throughout the course of the study. Importantly, following storage for 6 months at both 4 °C and 37 °C, the adjuvant properties of empty SLA archaeosomes were unchanged, and following admixing with antigen, the immunogenicity of the vaccine formulations was also unchanged when stored at both 4 °C and 37 °C for up to 1 month. Overall this indicates that SLA archaeosomes are highly stable adjuvants that retain their activity over an extended period of time even when stored at high temperatures.

In vitro evaluation of archaeosome vehicles for transdermal vaccine delivery.

Archaeosomes composed of archaeal total polar lipids (TPL) or semi-synthetic analog vesicles have been used as vaccine adjuvants and delivery systems in animal models for many years. Typically administered by intramuscular or subcutaneous injections, archaeosomes can induce robust, long-lasting humoral and cell-mediated immune responses against entrapped antigens and provide protection in murine models of infectious disease and cancer. Herein, we evaluated various archaeosomes for transdermal delivery, since this route may help eliminate needle-stick injuries and needle re-use, and therefore increase patient compliance. Archaeosomes composed of TPL from different archaea (Halobacterium salinarum, Methanobrevibacter smithii, Haloferax volcanii) and various semi-synthetic glycolipid combinations were evaluated for their ability to diffuse across the skin barrier using an ex vivo pig skin model and the results were compared to conventional synthetic ester liposomes. Physicochemical characteristics were determined for selected formulations including vesicle size, size distribution, zeta potential, fluidity, antigen (ovalbumin) incorporation efficiency and release. Archaeosomes, in particular those composed of M. smithii TPL or the synthetic glycolipid sulfated S-lactosylarchaeol (SLA) mixed with uncharged glycolipid lactosyl archaeol (LA), appeared to be effective carriers for ovalbumin, achieving much better antigen distribution and vesicle accumulation in the skin epidermis than conventional liposomes. The enhanced skin permeation of archaeosomes may be attributed to their chemical structure and physicochemical properties such as particle size, surface charge, stability, and fluidity of their lipid bilayer.

Support for the revocation of general safety test regulations in biologics license applications.

The United States Food and Drug Administration recently removed the requirement for a General Safety Test (GST) for biologics in the Code of Federal Regulations (21 CFR 610.11). The GST, as well as abnormal toxicity (European Pharmacopeia) and innocuity tests (World Health Organization), were designed to test for extraneous toxic contaminants on each product lot intended for human use. Tests require one-week observations for general health and weight following injection of specified volumes of product batches into guinea pigs and mice. At the volumes specified, dose-related toxicity may result when the product is pharmacologically active in rodents. With vaccines, required doses may be > 3 logs higher than intended human dose on a weight-adjusted basis and if an immune modulatory adjuvant is included, systemic immune hyperactivation may cause toxicity. Herein, using the CpG/alum adjuvant combination we evaluated the different test protocols and showed their unsuitability for this adjuvant combination.

The effect of preexisting anti-carrier immunity on subsequent responses to CRM197 or Qb-VLP conjugate vaccines.

CONTEXT: Certain antigens, such as haptens (small molecules), short peptides, and carbohydrates (e.g. bacterial polysaccharides) are non- or poorly immunogenic unless conjugated to a carrier molecule that provides a structural scaffold for antigen presentation as well as T cell help required for B-cell activation and maturation. However, the carriers themselves are immunogenic and resulting carrier-specific immune responses may impact the immunogenicity of other conjugate vaccines using the same carrier that are administered subsequently. OBJECTIVE: Herein, using two different carriers (cross-reactive material 197, CRM and Qb-VLP), we examined in mice the impact that preexisting anti-carrier antibodies (Ab) had on subsequent immune responses to conjugates with either the same or a different carrier. METHOD: For this purpose, we used two nicotine hapten conjugates (NIC7-CRM or NIC-Qb), two IgE peptide conjugates (Y-CRM or Y-Qb), and a pneumococcal polysaccharide conjugate (Prevnar 13(®)). RESULTS: Prior exposure to CRM or Qb-VLP significantly reduced subsequent responses to the conjugated antigen having the homologous carrier, with the exception of Prevnar 13® where anti-polysaccharide responses were similar to those in animals without preexisting anti-carrier Ab. CONCLUSION: Collectively, the data suggest that the relative sizes of the antigen and carrier, as well as the conjugation density for a given conjugate impact the extent of anti-carrier suppression. All animals developed anti-carrier responses with repeat vaccination and the differences in Ab titer between groups with and without preexisting anti-carrier responses became less apparent; however, anti-carrier effects were more durable for Ab function.

Vaccination with cancer- and HIV infection-associated endogenous retrotransposable elements is safe and immunogenic.

The expression of endogenous retrotransposable elements, including long interspersed nuclear element 1 (LINE-1 or L1) and human endogenous retrovirus, accompanies neoplastic transformation and infection with viruses such as HIV. The ability to engender immunity safely against such self-antigens would facilitate the development of novel vaccines and immunotherapies. In this article, we address the safety and immunogenicity of vaccination with these elements. We used immunohistochemical analysis and literature precedent to identify potential off-target tissues in humans and establish their translatability in preclinical species to guide safety assessments. Immunization of mice with murine L1 open reading frame 2 induced strong CD8 T cell responses without detectable tissue damage. Similarly, immunization of rhesus macaques with human LINE-1 open reading frame 2 (96% identity with macaque), as well as simian endogenous retrovirus-K Gag and Env, induced polyfunctional T cell responses to all Ags, and Ab responses to simian endogenous retrovirus-K Env. There were no adverse safety or pathological findings related to vaccination. These studies provide the first evidence, to our knowledge, that immune responses can be induced safely against this class of self-antigens and pave the way for investigation of them as HIV- or tumor-associated targets.

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.

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.

Preclinical evaluation of manufacturable SARS-CoV-2 spike virus-like particles produced in Chinese Hamster Ovary cells.

BACKGROUND: As the COVID-19 pandemic continues to evolve, novel vaccines need to be developed that are readily manufacturable and provide clinical efficacy against emerging SARS-CoV-2 variants. Virus-like particles (VLPs) presenting the spike antigen at their surface offer remarkable benefits over other vaccine antigen formats; however, current SARS-CoV-2 VLP vaccines candidates in clinical development suffer from challenges including low volumetric productivity, poor spike antigen density, expression platform-driven divergent protein glycosylation and complex upstream/downstream processing requirements. Despite their extensive use for therapeutic protein manufacturing and proven ability to produce enveloped VLPs, Chinese Hamster Ovary (CHO) cells are rarely used for the commercial production of VLP-based vaccines. METHODS: Using CHO cells, we aimed to produce VLPs displaying the full-length SARS-CoV-2 spike. Affinity chromatography was used to capture VLPs released in the culture medium from engineered CHO cells expressing spike. The structure, protein content, and glycosylation of spikes in VLPs were characterized by several biochemical and biophysical methods. In vivo, the generation of neutralizing antibodies and protection against SARS-CoV-2 infection was tested in mouse and hamster models. RESULTS: We demonstrate that spike overexpression in CHO cells is sufficient by itself to generate high VLP titers. These VLPs are evocative of the native virus but with at least three-fold higher spike density. In vivo, purified VLPs elicit strong humoral and cellular immunity at nanogram dose levels which grant protection against SARS-CoV-2 infection. CONCLUSIONS: Our results show that CHO cells are amenable to efficient manufacturing of high titers of a potently immunogenic spike protein-based VLP vaccine antigen.

Virus-like particles (VLPs) have a structure that is similar to viruses but they cannot cause infection or illness. If VLPs are injected into the body they produce an immune response similar to that seen following infection by a virus. This means that VLPs can be used as vaccines against viruses that cause illness in people. Many drugs, named biologics, are manufactured using living cells, including cells that were originally derived from Chinese Hamster Ovaries (CHO cells). We developed a simple method to produce VLPs similar to the SARS-CoV-2 virus in CHO cells. We show that vaccination of rodents with these VLPs prevents them from becoming ill following infection with SARS-CoV-2. These VLPs could become a part of an alternative, easily produced vaccine for the prevention of COVID-19 in humans.

Tuning the immune response: sulfated archaeal glycolipid archaeosomes as an effective vaccine adjuvant for induction of humoral and cell-mediated immunity towards the SARS-CoV-2 Omicron variant of concern.

Liposomes composed of sulfated lactosyl archaeol (SLA) have been shown to be a safe and effective vaccine adjuvant with a multitude of antigens in preclinical studies. In particular, SLA-adjuvanted SARS-CoV-2 subunit vaccines based on trimeric spike protein antigens were shown to be immunogenic and efficacious in mice and hamsters. With the continued emergence of SARS-CoV-2 variants, we sought to evaluate next-generation vaccine formulations with an updated antigenic identity. This was of particular interest for the widespread Omicron variant, given the abundance of mutations and structural changes observed within its spike protein compared to other variants. An updated version of our resistin-trimerized SmT1 corresponding to the B.1.1.529 variant was successfully generated in our Chinese Hamster Ovary (CHO) cell-based antigen production platform and characterized, revealing some differences in protein profile and ACE2 binding affinity as compared to reference strain-based SmT1. We next evaluated this Omicron-based spike antigen for its immunogenicity and ability to generate robust antigen-specific immune responses when paired with SLA liposomes or AddaS03 (a mimetic of the AS03 oil-in-water emulsion adjuvant system found in commercialized SARS-CoV-2 protein vaccines). Immunization of mice with vaccine formulations containing this updated antigen with either adjuvant stimulated neutralizing antibody responses favouring Omicron over the reference strain. Cell-mediated responses, which play an important role in the neutralization of intracellular infections, were induced to a much higher degree with the SLA adjuvant relative to the AddaS03-adjuvanted formulations. As such, updated vaccines that are better capable of targeting towards SARS-CoV-2 variants can be generated through an optimized combination of antigen and adjuvant components.

Generation of a Liposomal Vaccine Adjuvant Based on Sulfated S-Lactosylarchaeol (SLA) Glycolipids.

Vaccine formulations utilize adjuvants to enhance the level and breadth of the immune response to a target antigen. Liposomes composed of sulfated S-lactosylarchaeol (SLA) glycolipids can induce strong humoral and cell-mediated antigen-specific immune responses to co-administered antigens in mice. This has been demonstrated with a variety of protein antigens, where the protein is either encapsulated within or simply admixed with the archaeal liposomes (archaeosomes). In this process, a dried film of SLA glycolipid is hydrated in water or antigen solution to generate a large multilamellar (ML) liposomal suspension which is then size reduced by sonication to form unilamellar vesicles (UL) with a narrower size distribution. Herein, we describe the generation of liposomes based on the archaeal-based lipid SLA for use as an adjuvant in vaccine formulations.

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.

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.

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.

Immunogenicity of SARS-CoV-2 spike antigens derived from Beta & Delta variants of concern.

Using our strongly immunogenic SmT1 SARS-CoV-2 spike antigen platform, we developed antigens based on the Beta & Delta variants of concern (VOC). These antigens elicited higher neutralizing antibody activity to the corresponding variant than comparable vaccine formulations based on the original reference strain, while a multivalent vaccine generated cross-neutralizing activity in all three variants. This suggests that while current vaccines may be effective at reducing severe disease to existing VOC, variant-specific antigens, whether in a mono- or multivalent vaccine, may be required to induce optimal immune responses and reduce infection against arising variants.

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.

The Quantification of Antigen-Specific T Cells by IFN-γ ELISpot.

The enzyme-linked immune absorbent spot (ELISpot) assay allows for the quantification of the number of cells producing a particular secreted analyte. As T lymphocytes secrete cytokines such as interferon (IFN)-γ upon binding of the T cell receptor with its cognate antigen epitope, IFN-γ ELISpot allows for the measurement of antigen-specific T cells in an immune sample. Immune cells are isolated from the vaccinated subject and incubated with the epitope/antigen of interest on polyvinylidene difluoride (PVDF)-lined microplates precoated with a capture antibody to IFN-γ. Cytokine spots are then detected utilizing an IFN-γ-specific detection antibody and an enzyme-linked conjugate. Here, we describe the quantification of OVA-specific CD8 and CD4 T cells from mouse splenocytes to measure vaccine-induced cellular responses.

Application of Cryogenic Transmission Electron Microscopy for Evaluation of Vaccine Delivery Carriers.

Cryogenic transmission electron microscopy (Cryo-TEM) enables visualizing the physicochemical structure of nanocarriers in solution. Here, we demonstrate the typical applications of Cryo-TEM in characterizing archaeosome-based vesicles as antigen carriers, including the morphology and size of vaccine carriers. Cryo-TEM tomography, incorporated with immunogold labeling for identifying and localizing the antigens, reveals the antigen distribution within archaeosomes in three dimensions (3D).

Measurement of Antigen-Specific IgG Titers by Direct ELISA.

Direct ELISA allows for the measurement of antibody levels to a particular antigen. Serum or plasma from the vaccinated subject are incubated on high-binding capacity microplates precoated with the antigen of interest and detected utilizing an enzyme-linked secondary antibody. Herein, using influenza hemagglutinin as model antigen, we describe the quantification of antigen-specific IgG titers in mouse serum to measure vaccine-induced humoral responses.

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.