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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.
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Administración Intranasal , Anticuerpos Antivirales , Vacunas contra la COVID-19 , COVID-19 , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Vacunas de Subunidad , Animales , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/administración & dosificación , Ratones , SARS-CoV-2/inmunología , Vacunas contra la COVID-19/inmunología , Vacunas contra la COVID-19/administración & dosificación , Vacunas de Subunidad/inmunología , Vacunas de Subunidad/administración & dosificación , Cricetinae , COVID-19/prevención & control , COVID-19/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Femenino , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/sangre , Inmunización Secundaria , Adyuvantes Inmunológicos/administración & dosificación , Ratones Endogámicos BALB C , Inmunidad Mucosa/inmunología , Humanos , Vacunación/métodosRESUMEN
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.
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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.
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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.
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Adyuvantes de Vacunas , COVID-19 , Cricetinae , Animales , Ratones , SARS-CoV-2 , Glucolípidos , Sulfatos , Células CHO , Liposomas , Glicoproteína de la Espiga del Coronavirus/genética , COVID-19/prevención & control , Cricetulus , Inmunidad Celular , Adyuvantes Inmunológicos , Adyuvantes Farmacéuticos , Archaea , Vacunas contra la COVID-19RESUMEN
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.
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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.
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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.
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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.
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COVID-19 , Glicoproteína de la Espiga del Coronavirus , Adyuvantes Inmunológicos , Animales , Anticuerpos Neutralizantes , Anticuerpos Antivirales , COVID-19/prevención & control , Vacunas contra la COVID-19 , Cricetinae , Humanos , Inmunización , Ratones , SARS-CoV-2RESUMEN
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.
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Glucolípidos , Liposomas , Adyuvantes Inmunológicos/farmacología , Animales , Glucolípidos/farmacología , Ratones , Ratones Endogámicos C57BL , OvalbúminaRESUMEN
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.
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Liposomas , Vacunas , Adyuvantes Inmunológicos , Adyuvantes de Vacunas , Animales , Archaea , Glucolípidos , Ratones , SulfatosRESUMEN
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.
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Vacunas , Adyuvantes Inmunológicos , Adyuvantes Farmacéuticos , Antígenos , Humanos , Inmunidad InnataRESUMEN
The huge worldwide demand for vaccines targeting SARS-CoV-2 has necessitated the continued development of novel improved formulations capable of reducing the burden of the COVID-19 pandemic. Herein, we evaluated novel protein subunit vaccine formulations containing a resistin-trimerized spike antigen, SmT1. When combined with sulfated lactosyl archaeol (SLA) archaeosome adjuvant, formulations induced robust antigen-specific humoral and cellular immune responses in mice. Antibodies had strong neutralizing activity, preventing viral spike binding and viral infection. In addition, the formulations were highly efficacious in a hamster challenge model reducing viral load and body weight loss even after a single vaccination. The antigen-specific antibodies generated by our vaccine formulations had stronger neutralizing activity than human convalescent plasma, neutralizing the spike proteins of the B.1.1.7 and B.1.351 variants of concern. As such, our SmT1 antigen along with SLA archaeosome adjuvant comprise a promising platform for the development of efficacious protein subunit vaccine formulations for SARS-CoV-2.
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Adyuvantes Inmunológicos/química , Antígenos Arqueales/química , Vacunas contra la COVID-19/uso terapéutico , Lípidos/química , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Peso Corporal , COVID-19/terapia , Chlorocebus aethiops , Cricetinae , Citocinas/metabolismo , Femenino , Humanos , Inmunidad Celular , Inmunidad Humoral , Inmunización Pasiva , Mesocricetus , Ratones , Ratones Endogámicos C57BL , Pruebas de Neutralización , Péptidos/química , Dominios Proteicos , SARS-CoV-2 , Receptores Toll-Like/inmunología , Células Vero , Carga Viral , Sueroterapia para COVID-19RESUMEN
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.
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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.
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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).
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Microscopía por Crioelectrón , Portadores de Fármacos , Sistemas de Liberación de Medicamentos , Microscopía Electrónica de Transmisión , Vacunas/administración & dosificación , Microscopía Electrónica de Transmisión/métodos , Programas Informáticos , Vacunas de Partículas Similares a VirusRESUMEN
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.
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Antígenos/inmunología , Vacunas/administración & dosificación , Vacunas/inmunología , Animales , Citometría de Flujo , Inmunidad , Inmunización , Inyecciones Intramusculares , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Ratones , Vacunación/métodos , Vacunas/químicaRESUMEN
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.
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Antígenos/inmunología , Ensayo de Immunospot Ligado a Enzimas , Interferón gamma , Linfocitos T/inmunología , Linfocitos T/metabolismo , Animales , Ensayo de Immunospot Ligado a Enzimas/métodos , Humanos , Ratones , Bazo/inmunología , Bazo/metabolismoRESUMEN
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.
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Especificidad de Anticuerpos/inmunología , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Inmunoglobulina G/inmunología , Animales , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Ratones , Vacunas/inmunologíaRESUMEN
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.
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Linfocitos T CD8-positivos/inmunología , Citotoxicidad Inmunológica , Animales , Biomarcadores , Linfocitos T CD8-positivos/metabolismo , Inmunidad Celular , Inmunización , Inmunofenotipificación , Ratones , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo , Vacunas/inmunologíaRESUMEN
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.