Search

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.

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 (strain A/Puerto Rico/8/1934 H1N1) 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-β-D-Galp-(1,4)-β-D-Glcp-(1,1)-archaeol) can give equal or better protection compared to AddaVax™ or the traditional antigen-encapsulated archaeosome formulations.

Archaeosomes are liposomes composed of natural or synthetic archaeal lipids that can be used as adjuvants to induce strong long-lasting humoral and cell-mediated immune responses against entrapped antigen. However, the entrapment efficiency of antigen within archaeosomes constituted using standard liposome forming methodology is often only 5-40%. In this study, we evaluated different formulation methods using a simple semi-synthetic archaeal lipid (SLA, sulfated lactosyl archaeol) and two different antigens, ovalbumin (OVA) and hepatitis B surface antigen (HBsAg). Antigen was entrapped within archaeosomes using the conventional thin film hydration-rehydration method with or without removal of non-entrapped antigen, or pre-formed empty archaeosomes were simply admixed with an antigen solution. Physicochemical characteristics were determined (size distribution, zeta potential, vesicle morphology and lamellarity), as well as location of antigen relative to bilayer using cryogenic transmission electron microscopy (TEM). We demonstrate that antigen (OVA or HBsAg) formulated with SLA lipid adjuvants using all the different methodologies resulted in a strong antigen-specific immune response. Nevertheless, the advantage of using a drug substance process that comprises of simply admixing antigen with pre-formed empty archaeosomes, represents a simple, efficient and antigenic dose-sparing formulation for adjuvanting and delivering vaccine antigens.

Hepatitis C virus (HCV) chronically infects 2%-3% of the world's population, causing liver disease and cancer with prolonged infection. The narrow host range of the virus, being restricted largely to human hepatocytes, has made the development of relevant models to evaluate the efficacy of vaccines a challenge. We have developed a novel approach to accomplish this by generating a murine hepatoma cell line stably expressing nonstructural HCV antigens which can be used in vitro or in vivo to test HCV vaccine efficacies. These HCV-recombinant hepatoma cells formed large solid-mass tumours when implanted into syngeneic mice, allowing us to test candidate HCV vaccines to demonstrate the development of an HCV-specific immune response that limited tumour growth. Using this model, we tested the therapeutic potential of recombinant anti-HCV-specific vaccines based on two fundamentally different attenuated pathogen vaccine systems-attenuated Salmonella and recombinant adenoviral vector based vaccine. While attenuated Salmonella that secreted HCV antigens limited growth of the HCV-recombinant tumours when used in a therapeutic vaccination trial, replication-competent but noninfectious adenovirus expressing nonstructural HCV antigens showed overall greater survival and reduced weight loss compared to non-replicating nondisseminating adenovirus. Our results demonstrate a model with anti-tumour responses to HCV nonstructural (NS) protein antigens and suggest that recombinant vaccine vectors should be explored as a therapeutic strategy for controlling HCV and HCV-associated cancers.

Archaeosomes are liposomes traditionally comprised of total polar lipids (TPL) or semi-synthetic glycerolipids of ether-linked isoprenoid phytanyl cores with varied glyco- and amino-head groups. As adjuvants, they induce robust, long-lasting humoral and cell-mediated immune responses and enhance protection in murine models of infectious disease and cancer. Traditional total polar lipid (TPL) archaeosome formulations are relatively complex and first generation semi-synthetic archaeosomes involve many synthetic steps to arrive at the final desired glycolipid composition. We have developed a novel archaeosome formulation comprising a sulfated disaccharide group covalently linked to the free sn-1 hydroxyl backbone of an archaeal core lipid (sulfated S-lactosylarchaeol, SLA) that can be more readily synthesized yet retains strong immunostimulatory activity for induction of cell-mediated immunity following systemic immunization. Herein, we have evaluated the immunostimulatory effects of SLA archaeosomes when used as adjuvant with ovalbumin (OVA) and hepatitis B surface antigen (HBsAg) and compared this to various other adjuvants including TLR3/4/9 agonists, oil-in-water and water-in-oil emulsions and aluminum hydroxide. Overall, we found that semi-synthetic sulfated glycolipid archaeosomes induce strong Ag-specific IgG titers and CD8 T cells to both antigens. In addition, they induce the expression of a number of cytokines/chemokines including IL-6, G-CSF, KC & MIP-2. SLA archaeosome formulations demonstrated strong adjuvant activity, superior to many of the other tested adjuvants.

After vaccination, memory CD8+ T cells migrate to different organs to mediate immune surveillance. In most nonlymphoid organs, following an infection, CD8+ T cells differentiate to become long-lived effector-memory cells, thereby providing long-term protection against a secondary infection. In this study, we demonstrated that Ag-specific CD8+ T cells that migrate to the mouse brain following a systemic Listeria infection do not display markers reminiscent of long-term memory cells. In contrast to spleen and other nonlymphoid organs, none of the CD8+ T cells in the brain reverted to a memory phenotype, and all of the cells were gradually eliminated. These nonmemory phenotype CD8+ T cells were found primarily within the choroid plexus, as well as in the cerebrospinal fluid-filled spaces. Entry of these CD8+ T cells into the brain was governed primarily by CD49d/VCAM-1, with the majority of entry occurring in the first week postinfection. When CD8+ T cells were injected directly into the brain parenchyma, cells that remained in the brain retained a highly activated (CD69hi) phenotype and were gradually lost, whereas those that migrated out to the spleen were CD69low and persisted long-term. These results revealed a mechanism of time-bound immune surveillance to the brain by CD8+ T cells that do not reside in the parenchyma.

Fas death pathway is important for lymphocyte homeostasis, but the role of Fas pathway in T cell memory development is not clear. We show that whereas the expansion and contraction of CD8+ T cell response against Listeria monocytogenes were similar for wild-type (WT) and Fas ligand (FasL) mutant mice, the majority of memory CD8+ T cells in FasL mutant mice displayed an effector memory phenotype in the long-term in comparison with the mainly central memory phenotype displayed by memory CD8+ T cells in WT mice. Memory CD8+ T cells in FasL mutant mice expressed reduced levels of IFN-γ and displayed poor homeostatic and Ag-induced proliferation. Impairment in CD8+ T cell memory in FasL mutant hosts was not due to defective programming or the expression of mutant FasL on CD8+ T cells, but was caused by perturbed cytokine environment in FasL mutant mice. Although adoptively transferred WT memory CD8+ T cells mediated protection against L. monocytogenes in either the WT or FasL mutant hosts, FasL mutant memory CD8+ T cells failed to mediate protection even in WT hosts. Thus, in individuals with mutation in Fas pathway, impairment in the function of the memory CD8+ T cells may increase their susceptibility to recurrent/latent infections. The importance of T cell memory for the control of intracellular pathogens is well documented (1, 2, 3); however, the mechanisms that govern the induction and maintenance of memory T cells are not clear. During T cell priming, CD8+ T cells undergo massive proliferation (>1000-fold), which is followed by a contraction phase wherein the majority (>90%) of activated cells die by apoptosis (4). Engagement of Fas, with its ligand (FasL),3 is thought to play a key role in the regulation of T cells (5, 6) because the interaction between activated T cells expressing Fas and FasL provides a mechanism for deleting T cells. This view is supported by the observation that lpr mice that bear mutation in Fas gene develop lymphadenopathy (7), and T cells from lpr mice are resistant to activation-induced cell death (8, 9, 10). Furthermore, T cell tolerance induction to superantigen is impaired or delayed in lpr mice (11, 12, 13, 14). The role of Fas in regulating CD8+ T cell homeostasis has been controversial because autoreactive CD8+ T cells induced by cross-presentation of self Ags are deleted by a Fas-dependent mechanism (15), whereas the expansion and contraction of Ag-specific CD8+ T cell response induced after viral infection in vivo have been shown to occur by Fas-independent mechanisms (16, 17). We evaluated the development and maintenance of OVA-specific CD8+ T cells in normal and FasL mutant mice using recombinant intracellular bacterium, Listeria monocytogenes (LM)-expressing OVA as an immunogen. Our results reveal that although the expansion and contraction of CD8+ T cell response proceed normally in FasL mutant mice, memory CD8+ T cells in FasL mutant mice display a skewed phenotype and poor homeostatic proliferation and fail to mediate effective protection against pathogen rechallenge.

We expressed the CTL epitope of OVA (OVA257–264) in an acute (Listeria monocytogenes (LM)-OVA) and a chronic intracellular pathogen (Mycobacterium bovis (BCG)-OVA), to evaluate the kinetics of Ag presentation. LM-OVA proliferated rapidly in vivo, resulting in profound LM-OVA expansion within the first 24 h of infection, culminating in the generation of a potent CD8+ T cell response, which peaked on day 7 but underwent a rapid attrition subsequently. In contrast, BCG-OVA exhibited reduced growth in vivo, resulting in a delayed CD8+ T cell response that increased progressively with time. Relative to LM-OVA, BCG-OVA induced persistently increased numbers of apoptotic (annexin V+) CD8+ T cells. Ag presentation in vivo was evaluated by transferring Thy1.2+ carboxyfluorescein-labeled OT1 transgenic CD8+ T cells into infected Thy1.1+ congeneic recipient mice. LM-OVA induced rapid Ag presentation that was profound in magnitude, with most of the transferred cells getting activated within 4 days and resulting in a massive accumulation of activated donor CD8+ T cells. In contrast, Ag presentation induced by BCG-OVA was delayed, weaker in magnitude, which peaked around the second week of infection and declined to a low level subsequently. Increasing the dose of BCG-OVA while enhancing the magnitude of Ag presentation did not change the kinetics. Furthermore, a higher dose of BCG-OVA also accelerated the attrition of OVA257–264-specific CD8+ T cells. Relative to LM-OVA, the dendritic cells in BCG-OVA-infected mice were apoptotic for prolonged periods, suggesting that the rapid death of APCs may limit the magnitude of Ag presentation during chronic stages of mycobacterial infection.

We evaluated CD8+ T cell responses against the dominant CTL epitope, OVA257–264, expressed by an acute (Listeria monocytogenes (LM) OVA) vs a chronic pathogen (Mycobacterium bovis bacillus Calmette-Guérin (BCG) OVA) to reveal the influence on CD8+ T cell memory and consequent protection against a challenge with OVA-expressing tumor cells. Infection with lower doses of both pathogens resulted in stronger bacterial growth but weaker T cell memory indicating that memory correlates with pathogen dose but not with bacterial expansion. The CD8+ T cell response induced by LM-OVA was helper T cell-independent and was characterized by a rapid effector response followed by a rapid, but massive, attrition. In contrast, BCG-OVA induced a delayed and weak response that was compensated for by a longer effector phase and reduced attrition. This response was partly dependent on CD4+ T cells. CD8+ T cell response induced by BCG-OVA, but not LM-OVA, was highly dependent on pathogen persistence to compensate for the weak initial CD8+ T cell priming. Despite a stronger initial T cell response with LM-OVA, BCG-OVA provided more effective tumor (B16OVA) control at both local and distal sites due to the induction of a persistently activated acquired, and a more potent innate, immunity.

Induction of T-cell memory by vaccination ensures long-term protection against pathogens. We determined whether on-going inflammatory responses during vaccination influenced T-cell priming. A preexposure of mice toMycobacterium bovisBCG impaired their subsequent ability to prime T cells againstListeria monocytogenes. This was characterized by a decrease inL. monocytogenes-specific gamma interferon (IFN-γ)-secreting CD4+and CD8+T cells. The intensity of T-cell priming towardsL. monocytogenesdepended on the extent ofL. monocytogenesexpansion, and a cessation of this expansion caused byM. bovisBCG-induced inflammation resulted in impairment in T-cell priming. A challenge ofM. bovisBCG-infected mice with a higherL. monocytogenesdose increasedL. monocytogenessurvival and restored T-cell priming towardsL. monocytogenes. Impairment in T-cell priming towardsL. monocytogenesdue toM. bovisBCG-induced inflammation resulted in a compromised protective efficacy in the long term after mice were rechallenged withL. monocytogenes. Preexisting inflammation selectively impaired T-cell priming for replicating immunogens as CD8+T-cell response to ovalbumin administered as an inert antigen (ovalbumin-archaeosomes) was enhanced byM. bovisBCG preimmunization, whereas priming towards ovalbumin administered as a live immunogen (L. monocytogenes-ovalbumin) was impaired. Thus, depending on the nature of the immunogen, the presence of prior inflammatory responses may either impede or boost vaccine efficacy.

CD8+ T cells undergo rapid expansion during infection with intracellular pathogens, which is followed by swift and massive culling of primed CD8+ T cells. The mechanisms that govern the massive contraction and maintenance of primed CD8+ T cells are not clear. We show in this study that the transcription factor, FoxO3a, does not influence Ag presentation and the consequent expansion of CD8+ T cell response during Listeria monocytogenes infection, but plays a key role in the maintenance of memory CD8+ T cells. The effector function of primed CD8+ T cells as revealed by cytokine secretion and CD107a degranulation was not influenced by inactivation of FoxO3a. Interestingly, FoxO3a-deficient CD8+ T cells displayed reduced expression of proapoptotic molecules BIM and PUMA during the various phases of response, and underwent reduced apoptosis in comparison with wild-type cells. A higher number of memory precursor effector cells and memory subsets was detectable in FoxO3a-deficient mice compared with wild-type mice. Furthermore, FoxO3a-deficient memory CD8+ T cells upon transfer into normal or RAG1-deficient mice displayed enhanced survival. These results suggest that FoxO3a acts in a cell-intrinsic manner to regulate the survival of primed CD8+ T cells. Copyright © 2013 by The American Association of Immunologists, Inc.

Ag presentation to T cells orchestrates the development of acquired immune response. Although it is considered that Ag presentation may persist at high levels during chronic infections, we have previously reported that in mice infected with bacillus Calmette-Guérin, Ag presentation gets drastically curtailed during the chronic stage of infection despite antigenic persistence. In this report we evaluated the mechanism of this curtailment. Ag presentation declined precipitously as the T cell response developed, and Ag presentation was not curtailed in mice that were deficient in CD8+ T cells or MHC class II, suggesting that T cells regulate Ag presentation. Curtailment of Ag presentation was reduced in IFN-γ-deficient mice, but not in mice with a deficiency/mutation in inducible NOS2, perforin, or Fas ligand. In hosts with no T cells (Rag1−/−), Ag presentation was not curtailed during the chronic stage of infection. However, adoptive transfer of wild-type, but not IFN-γ−/−, CD4+ and CD8+ T cells into Rag1-deficient hosts strongly curtailed Ag presentation. Increased persistence of Ag presentation in IFN-γ-deficient hosts correlated to increased survival of dendritic cells, but not of macrophages, and was not due to increased stimulatory capacity of IFN-γ-deficient dendritic cells. These results reveal a novel mechanism indicating how IFN-γ prevents the persistence of Ag presentation, thereby preventing memory T cells from going into exhaustion.

International audience; Ag presentation to CD8(+) T cells often commences immediately after infection, which facilitates their rapid expansion and control of infection. Subsequently, the primed cells undergo rapid contraction. We report that this paradigm is not followed during infection with virulent Salmonella enterica, serovar Typhimurium (ST), an intracellular bacterium that replicates within phagosomes of infected cells. Although susceptible mice die rapidly (approximately 7 days), resistant mice (129 x 1SvJ) harbor a chronic infection lasting approximately 60-90 days. Using rOVA-expressing ST (ST-OVA), we show that T cell priming is considerably delayed in the resistant mice. CD8(+) T cells that are induced during ST-OVA infection undergo delayed expansion, which peaks around day 21, and is followed by protracted contraction. Initially, ST-OVA induces a small population of cycling central phenotype (CD62L(high)IL-7Ralpha(high)CD44(high)) CD8(+) T cells. However, by day 14-21, majority of the primed CD8(+) T cells display an effector phenotype (CD62L(low)IL-7Ralpha(low)CD44(high)). Subsequently, a progressive increase in the numbers of effector memory phenotype cells (CD62L(low)IL-7Ralpha(high)CD44(high)) occurs. This differentiation program remained unchanged after accelerated removal of the pathogen with antibiotics, as majority of the primed cells displayed an effector memory phenotype even at 6 mo postinfection. Despite the chronic infection, CD8(+) T cells induced by ST-OVA were functional as they exhibited killing ability and cytokine production. Importantly, even memory CD8(+) T cells failed to undergo rapid expansion in response to ST-OVA infection, suggesting a delay in T cell priming during infection with virulent ST-OVA. Thus, phagosomal lifestyle may allow escape from host CD8(+) T cell recognition, conferring a survival advantage to the pathogen.

During infection with lymphocytic choriomeningitis virus, CD8+ T cells differentiate rapidly into effectors (CD62LlowCD44high) that differentiate further into the central memory phenotype (CD62LhighCD44high) gradually. To evaluate whether this CD8+ T cell differentiation program operates in all infection models, we evaluated CD8+ T cell differentiation during infection of mice with recombinant intracellular bacteria, Listeria monocytogenes (LM) and Mycobacterium bovis (BCG), expressing OVA. We report that CD8+ T cells primed during infection with the attenuated pathogen BCG-OVA differentiated primarily into the central subset that correlated to reduced attrition of the primed cells subsequently. CD8+ T cells induced by LM-OVA also differentiated into central phenotype cells first, but the cells rapidly converted into effectors in contrast to BCG-OVA. Memory CD8+ T cells induced by both LM-OVA as well as BCG-OVA were functional in that they produced cytokines and proliferated extensively in response to antigenic stimulation after adoptive transfer. During LM-OVA infection, if CD8+ T cells were guided to compete for access to APCs, then they received reduced stimulation that was associated with increased differentiation into the central subset and reduced attrition subsequently. Similar effect was observed when CD8+ T cells encountered APCs selectively during the waning phase of LM-OVA infection. Taken together, our results indicate that the potency of the pathogen can influence the differentiation and fate of CD8+ T cells enormously, and the extent of attrition of primed CD8+ T cells correlates inversely to the early differentiation of CD8+ T cells primarily into the central CD8+ T cell subset.

Induction and maintenance of T cell memory is critical for the control of intracellular pathogens and tumors. Memory T cells seem to require few “maintenance signals,” though often such studies are done in the absence of competing immune challenges. Conversely, although attrition of CD8+ T cell memory has been characterized in heterologous viral models, this is not the case for bacterial infections. In this study, we demonstrate attrition of T cell responses to the intracellular pathogen Listeria monocytogenes (LM) following an immune challenge with a second intracellular bacterium, Mycobacterium bovis (bacillus Calmette-Guérin, BCG). Mice immunized with either LM or recombinant LM (expressing OVA; LM-OVA), develop a potent T cell memory response. This is reflected by peptide-specific CTL, IFN-γ production, and frequency of IFN-γ-secreting T cells to native or recombinant LM Ags. However, when the LM-infected mice are subsequently challenged with BCG, there is a marked reduction in the LM-specific T cell responses. These reductions are directly attributable to the effects on CD4+ and CD8+ T cells and the data are consistent with a loss of LM-specific T cells, not anergy. Attrition of the Ag (OVA)-specific T cell response is prevented when LM-OVA-immunized mice are challenged with a subsequent heterologous pathogen (BCG) expressing OVA, demonstrating memory T cell dependence on Ag. Although the reduction of the LM-specific T cell response did not impair protection against a subsequent LM rechallenge, for the first time, we show that T cell attrition can result in the reduction of Ag-specific antitumor (B16-OVA) immunity previously established with LM-OVA immunization.

Type 1 T-cell responses against intracellular pathogens play a crucial role in mediating protection. We examined whether the induction of a strong type 1 T-cell response during a chronic bacterial infection influences responses to superantigens capable of inducing acute shock. Intravenous infection of mice withMycobacterium bovisBCG appeared to induce a progressive anergy towards staphylococcal enterotoxin B (SEB) and towards antigen preparation of BCG (BCG-Ag) itself, based on diminished gamma interferon (IFN-γ) production by SEB- and BCG-Ag-stimulated splenocytes from infected mice. In contrast to these in vitro results, injection of SEB into BCG-infected mice led to a dramatic increase in the serum IFN-γ levels and the death of infected but not of control mice. In vitro hyporesponsiveness towards SEB and BCG-Ag occurred only with unfractionated splenocyte cultures, as purified T cells from infected mice produced higher levels of IFN-γ. Hyporesponsiveness towards SEB and BCG-Ag in unfractionated splenocyte cultures was not due to suppressive antigen-presenting cells (APCs), as APCs from infected mice stimulated higher levels of IFN-γ from purified T cells. The diminished IFN-γ levels observed with bulk splenocytes appear to be due to changes in the T-cell-to-APC ratio that result in a decreased proportion of T cells, coupled to reduced proliferative responses and an increased susceptibility of effector T cells to activation-induced cell death in vitro. Our results indicate that the reported phenomena of T-cell anergy during mycobacterial infection may be an in vitro consequence of the development of a strong type 1 response in vivo.