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It has been almost two decades since the discovery of mucosal-associated invariant T (MAIT)-cells. Several advances in the field have been made such as the discovery of the antimicrobial activity of MAIT-cells, the abundance of these cells in human mucosa and in liver and the discovery of ligands able to bind MR1 and activate MAIT-cells. MAIT-cells are a unique subset of innate-like T-cells that express a canonical T-cell receptor with the alpha chain containing hAV7S2 and AJ33 in humans (TCRVα7.2Jα33) and respond to bacterial/fungus vitamin B2 metabolites by an MR1-dependent pathway. Indirect activation is also observed during chronic viral infections by and IL-12/IL-18 pathway. In this review, the mechanisms of activation, the timeline of MAIT-cell development in humans as well as their role in human infection are discussed. On the whole, we believe that harnessing the anti-microbial ability of MAIT-cells could contribute for the design of potent immunotherapies and vaccines against “hard-to-kill” infectious agents that remain as public health threats worldwide. [ABSTRACT FROM AUTHOR]

Withania somnifera (L.) Dunal (family Solanaceae) is a medicinal plant known for, among many pharmacological properties, an immune boosting effect. Our recent study revealed that its key immunostimulatory factor is lipopolysaccharide of plant-associated bacteria. This is peculiar, because, although LPS can elicit protective immunity, it is an extremely potent pro-inflammatory toxin (endotoxin). However, W. somnifera is not associated with such toxicity. In fact, despite the presence of LPS, it does not trigger massive inflammatory responses in macrophages. To gain insights into the safe immunostimulatory effect of W. somnifera, we conducted a mechanistic study on its major phytochemical constituent, withaferin A, which is known for anti-inflammatory activity. Endotoxin-triggered immunological responses in the presence and absence of withaferin A were characterized by both in vitro macrophage-based assay and in vivo cytokine profiling in mice. Collectively, our results demonstrate that withaferin A selectively attenuates the pro-inflammatory signaling triggered by endotoxin without impairing other immunological pathways. This finding provides a new conceptual framework to understand the safe immune-boosting effect of W. somnifera and possibly other medicinal plants. Furthermore, the finding opens a new opportunity to facilitate the development of safe immunotherapeutic agents, such as vaccine adjuvants. [ABSTRACT FROM AUTHOR]

Abstract: Due to the fact that the life cycle of malaria parasites is complex, undergoing both an extracellular and intracellular phases in its host, the human immune system has to mobilize both the humoral and cellular arms of immune responses to fight against this parasitic infection. Whereas humoral immunity is directed toward the extracellular stages which include sporozoites and merozoites, cell-mediated immunity (CMI), in which T cells play a major role, targets hepatic stages – liver stages – of the parasites. In this review, the role of T cells in protective immunity against liver stages of the malaria infection is being re-evaluated. Furthermore, this review intends to address how to translate the findings regarding the role of T cells obtained in experimental systems to actual development of malaria vaccine for humans. [Copyright &y& Elsevier]

Abstract: NKT cells are known to play a role against certain microbial infections, including malaria and HIV, two major global infectious diseases. NKT cells exhibit either protective or pathogenic role against malaria. They are depleted by HIV infection and have a direct pathogenic role against many opportunistic infections common in end-stage AIDS. This review discusses the various features of the interaction between NKT cells and malaria parasites and HIV, and the potential to harness this interaction for therapeutic and vaccine strategies. [Copyright &y& Elsevier]

T cells from different subsets play a major role in protective immunity against pre-erythrocytic stages of malaria parasites. Exposure of humans and animals to malaria sporozoites induces (αβ CD8+ and CD4+ T cells specific for antigens expressed in pre-erythrocytic stages of Plasmodium. These T cells inhibit parasite development in the liver, and immunization with subunit vaccines expressing the respective antigenic moieties confers protection against sporozoite challenge. γδ and natural killer T cells can also play a role in protective immunity. Recent studies with mice transgenic for the αβ T-cell receptor have revealed the existence of complex mechanisms regulating the induction and development of these responses. [Copyright &y& Elsevier]

Radiation-attenuated sporozoite (RAS) vaccines can completely prevent blood stage Plasmodium infection by inducing liver-resident memory CD8+ T cells to target parasites in the liver. Such T cells can be induced by 'Prime-and-trap' vaccination, which here combines DNA priming against the P. yoelii circumsporozoite protein (CSP) with a subsequent intravenous (IV) dose of liver-homing RAS to "trap" the activated and expanding T cells in the liver. Prime-and-trap confers durable protection in mice, and efforts are underway to translate this vaccine strategy to the clinic. However, it is unclear whether the RAS trapping dose must be strictly administered by the IV route. Here we show that intradermal (ID) RAS administration can be as effective as IV administration if RAS are co-administrated with the glycolipid adjuvant 7DW8-5 in an ultra-low inoculation volume. In mice, the co-administration of RAS and 7DW8-5 in ultra-low ID volumes (2.5 µL) was completely protective and dose sparing compared to standard volumes (10–50 µL) and induced protective levels of CSP-specific CD8+ T cells in the liver. Our finding that adjuvants and ultra-low volumes are required for ID RAS efficacy may explain why prior reports about higher volumes of unadjuvanted ID RAS proved less effective than IV RAS. The ID route may offer significant translational advantages over the IV route and could improve sporozoite vaccine development. [ABSTRACT FROM AUTHOR]

The present study sought to search for the immunodominance related to the N-terminal, Central and C-terminal regions of HTLV-1 Tax using novel, cutting-edge peptide microarray analysis. In addition, in silico predictions were performed to verify the presence of nine amino acid peptides present along Tax restricted to the human leukocyte antigen (HLA)-A2.02*01 haplotype, as well as to verify the ability to induce pro-inflammatory and regulatory cytokines, such as IFN-γ and IL-4, respectively. Our results indicated abundant dose-dependent reactivity for HLA-A*02:01 in all regions (N-terminal, Central and C-terminal), but with specific hotspots. Furthermore, the results of fold-change over the Tax11–19 reactivity obtained at lower concentrations of HLA-A*02:01 reveal that peptides from the three regions contain sequences that react 100 times more than Tax11–19. On the other hand, Tax11–19 has similar or superior HLA-A*02:01 reactivity at higher concentrations of this haplotype. The in silico analysis showed a higher frequency of IFN-γ-inducing peptides in the N-terminal portion, while the C-terminal portion showed a higher frequency of IL-4 inducers. Taken together, these results shed light on the search for new Tax immunodominant epitopes, in addition to the canonic Tax11–19, for the rational design of immunomodulatory strategies for HTLV-1 chronic diseases. [ABSTRACT FROM AUTHOR]

7DW8-5 is a potent glycolipid adjuvant that improves malaria vaccine efficacy in mice by inducing IFN-γ and increasing protective CD8+ T cell responses. The addition of 7DW8-5 was previously shown to improve the efficacy of a CD8+ T cell-mediated heterologous 'prime-and-trap' malaria vaccine against Plasmodium yoelii sporozoite challenge in inbred female mice. Here, we report significant differential sex-specific responses to 7DW8-5 in inbred and outbred mice. Male mice express significantly less IFN-γ and IL-4 compared to females following intravenous 7DW8-5 administration. Additionally, unlike in female mice, 7DW8-5 did not improve the vaccine efficacy against sporozoite challenge in prime-and-trap vaccinated male mice. Our findings highlight the importance of including both female and male sexes in experimental adjuvant studies. [ABSTRACT FROM AUTHOR]

Highlights: [•] ZPSs elicit immunomodulatory activities in T cells after presentation by MHCII. [•] Carbohydrate epitopes generated from glycoconjugates are presented to T cells. [•] Tcarbs are monoclonal T-cell populations that recognize only carbohydrates. [•] Understanding immune mechanisms is essential for designing new-generation vaccines. [Copyright &y& Elsevier]

Glycoconjugate vaccines have provided enormous health benefits globally, but they have been less successful in some populations at high risk for developing disease. To identify new approaches to enhancing glycoconjugate effectiveness, we investigated molecular and cellular mechanisms governing the immune response to a prototypical glycoconjugate vaccine. We found that in antigen-presenting cells a carbohydrate epitope is generated upon endolysosomal processing of group B streptococcal type III polysaccharide coupled to a carrier protein. In conjunction with a carrier protein-derived peptide, this carbohydrate epitope binds major histocompatibility class II (MHCII) and stimulates carbohydrate-specific CD4+ T cell clones to produce interleukins 2 and 4-cytokines essential for providing T cell help to antibody-producing B cells. An archetypical glycoconjugate vaccine that we constructed to maximize the presentation of carbohydrate-specific T cell epitopes is 50-100 times more potent and substantially more protective in a neonatal mouse model of group B Streptococcus infection than a vaccine constructed by methods currently used by the vaccine industry. Our discovery of how glycoconjugates are processed resulting in presentation of carbohydrate epitopes that stimulate CD4+ T cells has key implications for glycoconjugate vaccine design that could result in greatly enhanced vaccine efficacy. [ABSTRACT FROM AUTHOR]

Abstract: Natural killer T (NKT) cells are known to play a role against certain microbial infections, including malaria and HIV, two major global infectious diseases. Strategies that can harness and amplify the immunotherapeutic potential of NKT cells can serve as powerful tools in the fight against such diseases. 7DW8-5, a novel glycolipid, may be one such tool. The interaction of 7DW8-5 with CD1d molecules induces activation of NKT cells, thereby activating various immune-competent cells including dendritic cells (DCs) to provide a significant adjuvant effect for several vaccines. This review discusses the discovery and characterization of 7DW8-5 and the practical considerations of its preclinical and clinical development as a potential glycolipid adjuvant for candidate malaria and HIV vaccines. [Copyright &y& Elsevier]

A major malaria vaccine candidate, the circumsporozoite (CS) protein of Plasmodium, is a pre-erythrocytic stage antigen that is attached to the surface of the sporozoites through a glycosylphosphatidylinositol (GPI) anchor. However, here we show that the motif that signals for glycosylphosphatidylinositol anchor addition interferes with the immunogenicity of this protein and reduces protection in mice upon immunization with a recombinant adenovirus. The presence of the glycosylphosphatidylinositol-anchoring motif sequentially affected total circumsporozoite protein production, cellular distribution, antigen processing and secretion, leading to less effective antigen presentation. Consistently, vaccination with an adenovirus recombinant carrying the anchoring motif-disrupted circumsporozoite gene, resulted in significant increase of the number of interferon-γ (IFN-γ) producing T cells and specific IgG2a isotype antibodies, ensuing more effective vaccination. Given that the anchoring motif is highly conserved among different species of Plasmodium, anti-malaria subunit vaccines encoded by recombinant vectors that aim at the induction of strong cellular immunity could maximize immunogenicity by removing anchoring motifs. [Copyright &y& Elsevier]

T cells are critical mediators of immunity to the pre-erythrocytic stages of malaria parasites. In this review, we survey the role of the various T cell subsets in combating the pre-erythrocytic stages; in particular, the role of NK T cells. Moreover, we show how studies using malaria models have revealed a unique behavior of NK T cells, namely the bridging of innate and adaptive immunity. [Copyright &y& Elsevier]

The Vγ5/Vδ1+ -T-cell receptor (TCR)-bearing T-cell clone, 2CBET-3, was generated from C57BL/6 mice. Upon stimulation, 2CBET-3 cells produce interleukin (IL)-3, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor-α, but not IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, macrophage colony-stimulating factor, or interferon-γ. These cells were evaluated for their ability to be stimulated by a variety of murine cell lines, including fibroblasts, trophoblasts, melanoma cells, embryonic carcinomas, B-cell lymphomas, mastocytoma cells, and keratinocytes. The human B-lymphoma cell and line, Daudi, also was included in these studies. We found that 2CBET-3 cells produced cytokines up to several hundredfold above the control levels in response to the B-cell line, Daudi, and A20/2J, but not to the B-cell line 439.4.2. After fixation with glutaraldehyde, Daudi and A20/2J continued to stimulate this γδ T-cell line. 2CBET-3 cells also responded to the keratinocyte line PAM212, but not to another, XB-2. When lipopolysaccharides (LPS) from Escherichia coli or S. typhimurium were added to 2CBET-3 calls in the presence of A20/2J cells, 2CBET-3 cells responded with increased cytokine production compared with the cytokine production in the presence of A20/2J cells alone. 2CBET-3 cells by themselves did not respond to LPS alone or to supernatants from A20/2J cells incubated with LPS. Unlike 2CBET-3, the epidermal T-cell hybridoma 70BET-49, expressing a Vγ5/Vδ1-TCR identical to that of 2CBET-3, did not respond to A20/2J cells in the presence or absence of LPS, suggesting a requirement for molecules other than the TCR for Vγ5/Vδ1-TCR+ T-cell stimulation by the B-cell lines and by LPS. This unique reactivity of γδ-TCR+ cells is defferent from that of γβ-TCR+ cells and may reflect a functional specialization of γδ-TCR+ cells in the response to bacterial infections. [ABSTRACT FROM AUTHOR]

α‐Galactosylceramides are glycosphingolipids that show promise in cancer immunotherapy. After presentation by CD1d, they activate natural killer T cells (NKT), which results in the production of a variety of pro‐inflammatory and immunomodulatory cytokines. Herein, we report the synthesis and biological evaluation of photochromic derivatives of KRN‐7000, the activity of which can be modulated with light. Based on established structure–activity relationships, we designed photoswitchable analogues of this glycolipid that control the production of pro‐inflammatory cytokines, such as IFN‐γ. The azobenzene derivative α‐GalACer‐4 proved to be more potent than KRN‐7000 itself when activated with 370 nm light. Photolipids of this type could improve our mechanistic understanding of cytokine production and could open new directions in photoimmunotherapy. [ABSTRACT FROM AUTHOR]

There are many immune‐boosting medicinal plants that can potently activate innate immune cells. Recent studies indicate that the active factors of some immune‐boosting plants are lipopolysaccharides (LPSs) of plant‐associated bacteria. However, little is currently known about the potential risk and benefit of LPSs in medicinal plants. To facilitate their characterization, we established a simple cell‐line‐based assay that can be used to screen the toxicity and benefit of LPSs in medicinal plants. The assay can distinguish endotoxic diphosphoryl lipid A (DPL) from beneficial monophosphoryl lipid A (MPL), which is a clinically used immunological adjuvant for vaccines. The established assay was used to characterize commercial supplements of Ashwagandha, which was shown to contain immunostimulatory LPSs. The study revealed that Ashwagandha activates macrophages in a manner similar to MPL. The current finding underscores the importance of further studies to characterize the LPSs in immune‐boosting medicinal plants. [ABSTRACT FROM AUTHOR]

Background: For the purpose of studying functional human dendritic cells (DCs) in a humanized mouse model that mimics the human immune system (HIS), a model referred to as HIS mice was established.Methods: Human immune system mice were made by engrafting NOD/SCID/IL2Rgammanull (NSG) mice with human hematopoietic stem cells (HSCs) following the transduction of genes encoding human cytokines and human leukocyte antigen (HLA)-A2.1 by adeno-associated virus serotype 9 (AAV9) vectors.Results: Our results indicate that human DC subsets, such as CD141+CD11c+ and CD1c+CD11c+ myeloid DCs, distribute throughout several organs in HIS mice including blood, bone marrow, spleen, and draining lymph nodes. The CD141+CD11c+ and CD1c+CD11c+ human DCs isolated from HIS mice immunized with adenoviruses expressing malaria/human immunodeficiency virus (HIV) epitopes were able to induce the proliferation of malaria/HIV epitopes-specific human CD8+ T cells in vitro. Upregulation of CD1c was also observed in human CD141+ DCs 1 day after immunization with the adenovirus-based vaccines.Conclusions: Establishment of such a humanized mouse model that mounts functional human DCs enables preclinical assessment of the immunogenicity of human vaccines in vivo. [ABSTRACT FROM AUTHOR]

Vaccination with radiation-attenuated sporozoites has been shown to induce CD8+ T cell-mediated protection against pre-erythrocytic stages of malaria. Empirical evidence suggests that successive inoculations often improve the efficacy of this type of vaccines. An initial dose (prime) triggers a specific cellular response, and subsequent inoculations (boost) amplify this response to create a robust CD8+ T cell memory. In this work we propose a model to analyze the effect of T cell dynamics on the performance of prime-boost vaccines. This model suggests that boost doses and timings should be selected according to the T cell response elicited by priming. Specifically, boosting during late stages of clonal contraction would maximize T cell memory production for vaccines using lower doses of irradiated sporozoites. In contrast, single-dose inoculations would be indicated for higher vaccine doses. Experimental data have been obtained that support theoretical predictions of the model. [ABSTRACT FROM AUTHOR]

Malaria is a mosquito-borne infectious disease caused by the parasite Plasmodium spp. Malaria continues to have a devastating impact on human health. Sporozoites are the infective forms of the parasite inside mosquito salivary glands. Circumsporozoite protein (CSP) is a major and immunodominant protective antigen on the surface of Plasmodium sporozoites. Here, we report a generation of specific monoclonal antibodies that recognize the central repeat and C-terminal regions of P. falciparum CSP. The monoclonal antibodies 3C1, 3C2, and 3D3--specific for the central repeat region--have higher titers and protective efficacies against challenge with sporozoites compared with 2A10, a gold standard monoclonal antibody that was generated in early 1980s. [ABSTRACT FROM AUTHOR]

Humanized mouse models present an important tool for preclinical evaluation of new vaccines and therapeutics. Here we show the human variable repertoire of antibody sequences cloned from a previously described human immune system (HIS) mouse model that possesses functional human CD4+ T cells and B cells, namely HIS-CD4/B mice. We sequenced variable IgG genes from single memory B-cell and plasma-cell sorted from splenocytes or whole blood lymphocytes of HIS-CD4/B mice that were vaccinated with a human plasmodial antigen, a recombinant Plasmodium falciparum circumsporozoite protein (rPfCSP). We demonstrate that rPfCSP immunization triggers a diverse B-cell IgG repertoire composed of various human VH family genes and distinct V(D)J recombinations that constitute diverse CDR3 sequences similar to humans, although low hypermutated sequences were generated. These results demonstrate the substantial genetic diversity of responding human B cells of HIS-CD4/B mice and their capacity to mount human IgG class-switched antibody response upon vaccination. [ABSTRACT FROM AUTHOR]

A CD1d-binding, invariant ( i ) natural killer T (NKT)-cell stimulatory glycolipid, α-Galactosylceramide (αGalCer), has been shown to act as an adjuvant. We previously identified a fluorinated phenyl ring-modified αGalCer analog, 7DW8-5, displaying a higher binding affinity for CD1d molecule and more potent adjuvant activity than αGalCer. In the present study, 7DW8-5 co-administered intramuscularly (i.m.) with a recombinant adenovirus expressing a Plasmodium yoelii circumsporozoite protein (PyCSP), AdPyCS, has led to a co-localization of 7DW8-5 and a PyCSP in draining lymph nodes (dLNs), particularly in dendritic cells (DCs). This occurrence initiates a cascade of events, such as the recruitment of DCs to dLNs and their activation and maturation, and the enhancement of the ability of DCs to prime CD8+ T cells induced by AdPyCS and ultimately leading to a potent adjuvant effect and protection against malaria. [ABSTRACT FROM AUTHOR]

A number of studies have shown that CD8+ T cells mediate protective anti-malaria immunity in a mouse model. However, whether human CD8+ T cells play a role in protection against malaria remains unknown. We recently established human immune system (HIS) mice harboring functional human CD8+ T cells (HIS-CD8 mice) by transduction with HLA-A∗0201 and certain human cytokines using recombinant adeno-associated virus-based gene transfer technologies. These HIS-CD8 mice mount a potent, antigen-specific HLA-A∗0201-restricted human CD8+ T-cell response upon immunization with a recombinant adenovirus expressing a human malaria antigen, the Plasmodium falciparum circumsporozoite protein (PfCSP), termed AdPfCSP. In the present study, we challenged AdPfCSP-immunized HIS-CD8 mice with transgenic Plasmodium berghei sporozoites expressing full-length PfCSP and found that AdPfCSP-immunized (but not naïve) mice were protected against subsequent malaria challenge. The level of the HLA-A∗0201-restricted, PfCSP-specific human CD8+ T-cell response was closely correlated with the level of malaria protection. Furthermore, depletion of human CD8+ T cells from AdPfCSP-immunized HIS-CD8 mice almost completely abolished the anti-malaria immune response. Taken together, our data show that human CD8+ T cells mediate protective anti-malaria immunity in vivo . [ABSTRACT FROM AUTHOR]

In the present study, the combined adjuvant effect of 7DW8-5, a potent α-GalCer-analog, and monophosphoryl lipid A (MPLA), a TLR4 agonist, on the induction of vaccine-induced CD8 + T-cell responses and protective immunity was evaluated. Mice were immunized with peptides corresponding to the CD8 + T-cell epitopes of a malaria antigen, a circumsporozoite protein of Plasmodium yoelii , and a tumor antigen, a Wilms Tumor antigen-1 (WT-1), together with 7DW8-5 and MPLA, as an adjuvant. These immunization regimens were able to induce higher levels of CD8 + T-cell responses and, ultimately, enhanced levels of protection against malaria and tumor challenges compared to the levels induced by immunization with peptides mixed with 7DW8-5 or MPLA alone. Co-administration of 7DW8-5 and MPLA induces activation of memory-like effector natural killer T (NKT) cells, i.e. CD44 + CD62L − NKT cells. Our study indicates that 7DW8-5 greatly enhances important synergistic pathways associated to memory immune responses when co-administered with MPLA, thus rendering this combination of adjuvants a novel vaccine adjuvant formulation. [ABSTRACT FROM AUTHOR]

Background: Plasmodium circumsporozoite protein (CSP) is a major surface antigen present in the sporozoite (Spz) stage of a malaria parasite. RTS, S vaccine, the most clinically advanced malaria vaccine, consists of a large portion of Plasmodium falciparum CSP (PfCSP). A highly infectious, recombinant rodent malaria, Plasmodium yoelii parasite bearing a full-length PfCSP, PfCSP/Py Spz, was needed as a tool to evaluate the role of PfCSP in mediating, protective, anti-malaria immunity in a mouse model. Methods: A transgenic parasite, PfCSP/Py Spz, was generated by inserting a construct expressing the PfCSP at the locus of the P. yoelii CSP gene by double cross-over homologous recombination. Then the biological and protective properties of PfCSP/Py Spz were determined. Results: This PfCSP/Py parasite produced up to 30,000 Spz in mosquito salivary glands, which is equal or even higher than the number of Spz produced by wild-type P. yoelii parasites. Five bites of PfCSP/Py-infected mosquitoes could induce blood infection in BALB/c mice. Conclusions: The current study has demonstrated a successful establishment of a transgenic P. yoelii parasite clone that is able to express a full-length PfCSP, PfCSP/Py parasite. Importantly, this PfCSP/Py parasite can be as infectious as the wild-type P. yoelii parasite both in mosquito vector and in mouse, a mammalian host. A new transgenic parasite that expresses a full-length PfCSP may become a useful tool for researchers to investigate immunity against PfCSP in a mouse model. [ABSTRACT FROM AUTHOR]

In this study, we developed human immune system (HIS) mice that possess functional human CD4 + T cells and B cells, named HIS-CD4/B mice. HIS-CD4/B mice were generated by first introducing HLA class II genes, including DR1 and DR4, along with genes encoding various human cytokines and human B cell activation factor (BAFF) to NSG mice by adeno-associated virus serotype 9 (AAV9) vectors, followed by engrafting human hematopoietic stem cells (HSCs). HIS-CD4/B mice, in which the reconstitution of human CD4 + T and B cells resembles to that of humans, produced a significant level of human IgG against Plasmodium falciparum circumsporozoite (PfCS) protein upon immunization. CD4 + T cells in HIS-CD4/B mice, which possess central and effector memory phenotypes like those in humans, are functional, since PfCS protein-specific human CD4 + T cells secreting IFN-γ and IL-2 were detected in immunized HIS-CD4/B mice. Lastly, PfCS protein-immunized HIS-CD4/B mice were protected from in vivo challenge with transgenic P. berghei sporozoites expressing the PfCS protein. The immune sera collected from protected HIS-CD4/B mice reacted against transgenic P. berghei sporozoites expressing the PfCS protein and also inhibited the parasite invasion into hepatocytes in vitro. Taken together, these studies show that our HIS-CD4/B mice could mount protective human anti-malaria immunity, consisting of human IgG and human CD4 + T cell responses both specific for a human malaria antigen. [ABSTRACT FROM AUTHOR]

A CDld-binding glycolipid, a-Galactosylceramide (aGalCer), activates invariant NK T cells and acts as an adjuvant. We previously identified a fluorinated phenyl ring-modified aGalCer analog, 7DW8-5, displaying nearly 100-fold stronger CDld binding affinity. In the current study, 7DW8-5 was found to exert a more potent adjuvant effect than aGalCer for a vaccine based on radiation-attenuated sporozoites of a rodent malaria parasite, Plasmodium yoelii, also referred to as irradiated P. yoelii sporozoites (IrPySpz). 7DW8-5 had a superb adjuvant effect only when the glycolipid and IrPySpz were conjointly administered i.m. Therefore, we evaluated the effect of distinctly different biodistribution patterns of aGalCer and 7DW8-5 on their respective adjuvant activities. Although both glycolipids induce a similar cytokine response in sera of mice injected i.v., after i.m. injection, aGalCer induces a systemic cytokine response, whereas 7DW8-5 is locally trapped by CDld expressed by dendritic cells (DCs) in draining lymph nodes (dLNs). Moreover, the i.m. coadministration of 7DW8-5 with IrPySpz results in the recruitment of DCs to dLNs and the activation and maturation of DCs. These events cause the potent adjuvant effect of 7DW8-5, resulting in the enhancement of the CD8+ T cell response induced by IrPySpz and, ultimately, improved protection against malaria. Our study is the first to show that the colocalization of a CDld-binding invariant NK T cell-stimulatory glycolipid and a vaccine, like radiation-attenuated sporozoites, in dLN-resident DCs upon i.m. conjoint administration governs the potency of the adjuvant effect of the glycolipid. [ABSTRACT FROM AUTHOR]

The ability of different glycosphingolipids (GSLs) to activate type I natural killer T cells (NKT cells) has been known for 2 decades. The possible therapeutic use of these GSLs has been studied in many ways; however, studies are needed in which the efficacy of promising GSLs is compared under identical conditions. Here, we compare five unique GSLs structurally derived from α-galactosylceramide. We employed biophysical and biological assays, as well as x-ray crystallography to study the impact of the chemical modifications of the antigen on type I NKT cell activation. Although all glycolipids are bound by the T cell receptor of type I NKT cells in real time binding assays with high affinity, only a few activate type INKT cells in invivo or invitro experiments. The differences in biological responses are likely a result of different pharmacokinetic properties of each lipid, which carry modifications at different parts of the molecule. Our results indicate a need to perform a variety of assays to ascertain the therapeutic potential of type I NKT cell GSL activators. [ABSTRACT FROM AUTHOR]

The circumsporozoite protein (CSP) of Plasmodium falciparum is a major surface protein, which forms a dense coat on the sporozoite's surface. Preclinical research on CSP and clinical evaluation of a CSP fragment-based RTS, S/AS01 vaccine have demonstrated a modest degree of protection against P. falciparum, mediated in part by humoral immunity and in part by cell-mediated immunity. Given the partial protective efficacy of the RTS, S/AS01 vaccine in a recent Phase 3 trial, further improvement of CSP-based vaccines is crucial. In this report, we describe the preclinical development of a full-length, recombinant CSP (rCSP)-based vaccine candidate against P. falciparum malaria suitable for current Good Manufacturing Practice (cGMP) production. Utilizing a novel high-throughput Pseudomonas fluorescens expression platform, we demonstrated greater efficacy of full-length rCSP as compared to N-terminally truncated versions, rapidly down-selected a promising lead vaccine candidate, and developed a high-yield purification process to express immunologically active, intact antigen for clinical trial material production. The rCSP, when formulated with various adjuvants, induced antigen-specific antibody responses as measured by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay (IFA), as well as CD4+ T-cell responses as determined by ELISpot. The adjuvanted rCSP vaccine conferred protection in mice when challenged with transgenic P. berghei sporozoites containing the P. falciparum repeat region of CSP. Furthermore, heterologous prime/boost regimens with adjuvanted rCSP and an adenovirus type 35-vectored CSP (Ad35CS) showed modest improvements in eliciting CSP-specific T-cell responses and anti-malarial protection, depending on the order of vaccine delivery. Collectively, these data support the importance of further clinical development of adjuvanted rCSP, either as a stand-alone product or as one of the components in a heterologous prime/boost strategy, ultimately acting as an effective vaccine candidate for the mitigation of P. falciparum-induced malaria. [ABSTRACT FROM AUTHOR]

Recombinant Mycobacterium bovis bacillus Calmette-Guèrin (rBCG) has been explored as a vector for vaccines against HIV because of its ability to induce long lasting humoral and cell mediated immune responses. To maximize the potential for rBCG vaccines to induce effective immunity against HIV, various strategies are being employed to improve its ability to prime CD8+ T cells, which play an important role in the control of HIV infections. In this study we adopted a previously described approach of incorporating glycolipids that activate CD1d-restricted natural killer T (NKT) cells to enhance priming of CD8+ T cells by rBCG strains expressing an SIV Gag antigen (rBCG-SIV gag). We found that the incorporation of the synthetic NKT activating glycolipid α-galactosylceramide (α-GC) into rBCG-SIV gag significantly enhanced CD8+ T cell responses against an immunodominant Gag epitope, compared to responses primed by unmodified rBCG-SIV gag. The abilities of structural analogues of α-GC to enhance CD8+ T cell responses to rBCG were compared in both wild type and partially humanized mice that express human CD1d molecules in place of mouse CD1d. These studies identified an α-GC analogue known as 7DW8-5, which has previously been used successfully as an adjuvant in non-human primates, as a promising compound for enhancing immunogenicity of antigens delivered by rBCG.vectors. Our findings support the incorporation of synthetic glycolipid activators of NKT cells as a novel approach to enhance the immunogenicity of rBCG-vectored antigens for induction of CD8+ T cell responses. The glycolipid adjuvant 7DW8-5 may be a promising candidate for advancing to non-human primate and human clinical studies for the development of HIV vaccines based on rBCG vectors. [ABSTRACT FROM AUTHOR]

Immunological memory has been regarded as a unique feature of the adaptive immune response mediated in an antigen-specific manner by T and B lymphocytes. However, natural killer (NK) cells and yδT cells, which traditionally are classified as innate immune cells, have been shown in recent studies to have hallmark features of memory cells. Invariant NKT cell (iNKT cell)-mediated antitumor effects indicate that iNKT cells are activated in vivo by vaccination with iNKT cell ligand-loaded CD1d+ cells, but not by vaccination with unbound NKT cell ligand. In such models, it previously was thought that the numbers of IFN-γ-producing cells in the spleen returned to the basal level around 1 wk after the vaccination. In the current study, we demonstrate the surprising presence of effector memory-like iNKT cells in the lung. We found long-term antitumor activity in the lungs of mice was enhanced after vaccination with iNKT cell ligand-loaded dendritic cells. Further analyses showed that the KLRG1+ (Killer cell lectin-like receptor subfamily G, member 1-positive) iNKT cells coexpressing CD49d and granzyme A persisted for several months and displayed a potent secondary response to cognate antigen. Finally, analyses of CDR3β by RNA deep sequencing demonstrated that some particular KLRG1+ iNKT-cell clones accumulated, suggesting the selection of certain T-cell receptor repertoires by an antigen. The current findings identifying effector memory-like KLRG1+ iNKT cells in the lung could result in a paradigm shift regarding the basis of newly developed extrathymic iNKT cells and could contribute to the future development of antitumor immunotherapy by uniquely energizing iNKT cells. [ABSTRACT FROM AUTHOR]

In the present study, a novel adeno-associated virus (AAV) vector-mediated gene delivery approach was taken to improve the reconstitution of functional CD8+ T cells in humanized mice, thereby mimicking the human immune system (HIS). Human genes encoding HLA-A2 and selected human cytokines (A2/hucytokines) were introduced to an immune-deficient mouse model [NOD/SCID/IL2rγnull (NSG) mice] using AAV serotype 9 (AAV9) vectors, followed by transplantation of human hematopoietic stem cells. NSG mice transduced with AAV9 encoding A2/hucytokines resulted in higher levels of reconstitution of human CD45+ cells compared to NSG mice transduced with AAV9 encoding HLA-A2 alone or HLA-A2-transgenic NSG mice. Furthermore, this group of HIS mice also mounted the highest level of antigen-specific A2-restricted human CD8+ T-cell response upon vaccination with recombinant adenoviruses expressing human malaria and HIV antigens. Finally, the human CD8+ T-cell response induced in human malaria vaccine-immunized HIS mice was shown to be functional by displaying cytotoxic activity against hepatocytes that express the human malaria antigen in the context of A2 molecules. Taken together, our data show that AAV vector-mediated gene delivery is a simple and efficient method to transfer multiple human genes to immune-deficient mice, thus facilitating successful reconstitution of HIS in mice. The HIS mice generated in this study should ultimately allow us to swiftly evaluate the T-cell immunogenicity of various human vaccine candidates in a pre-clinical setting. [ABSTRACT FROM AUTHOR]

Although the roles of CD8+ T cells and a major preerythrocytic antigen, the circumsporozoite (CS) protein, in contributing protective antimalaria immunity induced by radiation-attenuated sporozoites, have been shown by a number of studies, the extent to which these players contribute to antimalaria immunity is still unknown. To address this question, we have generated C57BL/6 (B6) transgenic (Tg) mice, expressing Kd molecules under the MHC-I promoter, called MHC-I-Kd-Tg mice. In this study, we first determined that a single immunizing dose of IrPySpz induced a significant level of antimalaria protective immunity in MHC-IKd- Tg mice but not in B6 mice. Then, by depleting various T-cell subsets in vivo, we determined that CD8+ T cells are the main mediator of the protective immunity induced by IrPySpz. Furthermore, when we immunized (MHC-I-Kd-Tg × CS-Tg) F1 mice with IrPySpz after crossing MHC-I-Kd-Tg mice with PyCS-transgenic mice (CS-Tg), which are unable to mount PyCS-specific immunity, we found that IrPySpz immunization failed to induce protective antimalaria immunity in (MHC-I-Kd-Tg × CS-Tg) F1 mice, thus indicating the absence of PyCS antigen-dependent immunity in these mice. These results indicate that protective antimalaria immunity induced by IrPySpz in MHC-I-Kd-Tg mice is mediated by CS protein-specific, Kd-restricted CD8+ T cells. [ABSTRACT FROM AUTHOR]

Background: It has been shown that human immunodeficiency virus (HIV)-1 infection induces the production of endogenous lipids required for effective viral production, and the cluster of differentiation (CD)1 molecule CD1d is downregulated by HIV-1 infection. However, the role of endogenous lipid presentation and the implications of CD1 downregulation by HIV-1 infection have not yet been characterized. Results: In this study, we observed downregulation of both CD1c and CD1d expression through a Vpu-dependent and Nef-independent mechanism, and the concomitant HIV-1-induced production of host cholesterol decreased the extent of CD1c and CD1d modulation. While the modest downregulation of CD1c by HIV-1 infection decreased the ability of CD1c-restricted T cells to respond and secrete interferon-γ, the cholesterol upregulation in the same cells by HIV-1 infection appears to limit the downregulation of CD1c. Conclusions: The two conflicting HIV-1-mediated changes in CD1c expression appear to minimize the modulation of CD1c expression, thus leading the host to maintain a CD1c-restricted T-cell response against HIV-1. [ABSTRACT FROM AUTHOR]

Abstract: CD8+ T cells are critical for the control of various intracellular infections and cancers. To date, however, effective T cell-based vaccines remain elusive, due, in part, to the lack of in vivo models that facilitate the dissection of antigen-specific CD8+ T-cell responses primed by different antigen-presenting cells (APCs). In this study, we generated four lines of H-2Kd transgenic (Kd Tg) mice that differed in their expression of H-2Kd: dendritic cells (DCs) only (CD11c-Kd), macrophages only (huCD68-Kd), hepatocytes only (Alb-Kd), or all nucleated cells (major histocompatibility complex-I-Kd). Immunization of each of these Kd Tg mouse strains with a synthetic peptide or a recombinant adenovirus expressing a well-known immunodominant, H-2Kd-restricted CD8+ T-cell epitope, SYVPSAEQI, which was derived from the circumsporozoite protein of Plasmodium yoelii, promoted distinct SYVPSAEQI-specific CD8+ T-cell responses. The route of immunization also greatly influenced the magnitude of the epitope-specific CD8+ T-cell response. These tissue-specific Kd Tg mice may be valuable tools for determining the mode of induction of CD8+ T-cell responses by different APCs in vivo and for characterizing the CD8+ T-cell responses promoted in response to various microbial infections and/or different types of vaccines. [Copyright &y& Elsevier]

In humans, invariant natural killer T (iNKT) cells represent a small but significant population of peripheral blood mononuclear cells (PBMCs) with a high degree of variability. In this study, pursuant to our goal of identifying an appropriate non-human primate model suitable for pre-clinical glycolipid testing, we evaluated the percentage and function of iNKT cells in the peripheral blood of pig-tailed macaques. First, using a human CD1d-tetramer loaded with α-GalCer (α-GalCer- CD1d-Tet), we found that α-GalCer-CD1d-Tet+ CD3+ iNKT cells make up 0.13% to 0.4% of pig-tailed macaque PBMCs, which are comparable to the percentage of iNKT cells found in human PBMCs. Second, we observed that a large proportion of Vα24+ CD3+ cells are α-GalCer-CD1d-Tet+ CD3+ iNKT cells, which primarily consist of either the CD4+ or CD8+ subpopulation. Third, we found that pig-tailed macaque iNKT cells produce IFN-γ in response to α-GalCer, as shown by ELISpot assay and intracellular cytokine staining (ICCS), as well as TNF-a, as shown by ICCS, indicating that these iNKT cells are fully functional. Interestingly, the majority of pig-tailed macaque iNKT cells that secrete IFN-γ are CD8+ iNKT cells. Based on these findings, we conclude that the pig-tailed macaques exhibit potential as a non-human animal model for the pre-clinical testing of iNKT-stimulating glycolipids. [ABSTRACT FROM AUTHOR]

Tumors often display mechanisms to avoid or suppress immune recognition. One such mechanism is the shedding of gangliosides into the local tumor microenvironment, and a high concentration of circulating gangliosides is associated with poor prognosis. In this study, we identify ganglioside GD3, which was isolated from the polar lipid fraction of ovarian cancer-associated ascites, as an inhibitory factor that prevents innate immune activation of natural killer T (NKT) cells. Purified GD3 displayed a high affinity for both human and mouse CD1d, a molecule involved in the presentation of lipid antigens to T cells. Purified GD3, as well as substances within the ascites, bound to the CD1d antigenic-binding site and did not require additional processing for its inhibitory effect on NKT cells. Importantly, in vivo administration of GD3 inhibited α-galactosylceramide (α-GalCer)-induced NKT cell activation in a dose-dependent manner. These data therefore indicate that ovarian cancer tumors may use GD3 to inhibit the antitumor NKT cell response as an early mechanism of tumor immune evasion. [ABSTRACT FROM AUTHOR]

Abstract: Immunization of BALB/c mice with irradiated sporozoites (IrSp) of Plasmodium yoelii can lead to sterile immunity. The circumsporozoite protein (CSP) plays a dominant role in protection. Nevertheless after hyper-immunization with IrSp, complete protection is obtained in CSP-transgenic BALB/c mice that are T-cell tolerant to the CSP and cannot produce antibodies [CSP-Tg/JhT(−/−)]. This protection is mediated exclusively by CD8+ T cells . To identify the non-CSP protective T cell antigens, we studied the properties of 34 P. yoelii sporozoite antigens that are predicted to be secreted and to contain strong Kd-restricted CD8+ T cell epitopes. The synthetic peptides corresponding to the epitopes were used to screen for the presence of peptide-specific CD8+ T cells secreting interferon-γ (IFN-γ) in splenocytes from CSP-Tg/JhT(−/−) BALB/c mice hyper immunized with IrSp. However, the numbers of IFN-γ-secreting splenocytes specific for the non-CSP antigen-derived peptides were 20–100 times lower than those specific for the CSP-specific peptide. When mice were immunized with recombinant adenoviruses expressing selected non-CSP antigens, the animals were not protected against challenge with P. yoelii sporozoites although large numbers of CD8+ specific T cells were generated. [Copyright &y& Elsevier]

Important roles played by invariant natural killer T (iNKT) cells in asthma pathogenesis have been demonstrated. We identified functional iNKT cells and CD1d molecules in pig lungs. Pig iNKT cells cultured in the presence of α-GalCer proliferated and secreted Th1 and Th2 cytokines. Like in other animal models, direct activation of pig lung iNKT cells using α-GalCer resulted in acute airway hyperreactivity (AHR). Clinically, acute AHR-induced pigs had increased respiratory rate, enhanced mucus secretion in the airways, fever, etc. In addition, we observed petechial hemorrhages, infiltration of CD4 cells, and increased Th2 cytokines in AHR-induced pig lungs. Ex vivo proliferated iNKT cells of asthma induced pigs in the presence of C-glycoside analogs of α-GalCer had predominant Th2 phenotype and secreted more of Th2 cytokine, IL-4. Thus, baby pigs may serve as a useful animal model to study iNKT cell-mediated AHR caused by various environmental and microbial CD1d-specific glycolipid antigens. [ABSTRACT FROM AUTHOR]

Although adenovirus (Ad) has been regarded as an excellent vaccine vector, there are 2 major drawbacks to using this platform: (a) Ad-based vaccines induce a relatively weak humoral response against encoded transgenes, and (b) preexisting immunity to Ad is highly prevalent among the general population. To overcome these obstacles, we constructed an Ad-based malaria vaccine by inserting a B cell epitope derived from a Plasmodium yoelii circumsporozoite (CS) protein (referred to as the PyCS-B epitope) into the capsid proteins of WT/CS-GFP, a recombinant Ad expressing P. yoelii CS protein and GFP as its transgene. Multiple vaccinations with the capsid-modified Ad induced a substantially increased level of protection against subsequent malaria challenge in mice when compared with that of unmodified WT/CS-GFP. Increased protection correlated with augmented antibody responses against the PyCS-B epitope expressed in the capsid. Furthermore, replacement of hypervariable region 1 (HVR1) of the Ad capsid proteins with the PyCS-B epitope circumvented neutralization of the modified Ad by preexisting Ad-specific antibody, both in vivo and in vitro. Importantly, the immunogenicity of the Ad-containing PyCS-B epitope in the HVR1 and a P. yoelii CS transgene was maintained. Overall, this study demonstrates that the HVR1-modifed Ad vastly improves upon Ad as a promising malaria vaccine platform candidate. [ABSTRACT FROM AUTHOR]

The glycolipid a-galactosylceramide (α-GalCer) has been shown to bind CD1d molecules to activate invariant natural killerl(1NKT) cells, and subsequently induce activation of various immune-competent cells, including dendritic cells, thereby providing a significant adjuvant effect for various vaccines. However, in phase I clinical trials, α-GalCer was shown to display only marginal biological activity. In our search for a glycolipid that can exert more potent stimulatory activity against 1NKT cells and dendritic cells and produce an adjuvant effect superior to α-GalCer, we performed step-wise screening assays on a focused library of 25 α-GalCer analogues. Assays included quantification of the magnitude of stimulatory activity against human 1NKT cells in vitro, binding affinity to human and murine CD1d molecules, and binding affinity to the invariant t cell receptor of human 1NKT cells. Through this rigorous and iterative screening process, we have identified a lead candidate glycolipid, 7DW8-5, that exhibits a superior adjuvant effect than a-GalCer on HIV and malaria vaccines in mice. [ABSTRACT FROM AUTHOR]

We have previously shown that the C-glycoside analog of a-galactosylceramide (α-GalCer), α-C-GalCer, displays a superior inhibitory activity against the liver stages of the rodent malaria parasite Plasmodium yoelii than its parental glycolipid, α-GalCer. In this study, we demonstrate that NK cells, as well as IL-12, are a key contributor for the superior activity displayed by α-C-GalCer. Surprisingly, the diminished production of Th2 cytokines, including IL-4, by a-C-GalCer has no affect on its superior therapeutic activity relative to α-GalCer. Finally, we show that the in vivo administration of α-C-GalCer induces prolonged maturation of dendritic cells (DCs), as well as an enhanced proliferative response of mouse invariant Vα14 (Vα14i) NKT cells, both of which may also contribute to some degree to the superior activity of α-C-GalCer in vivo. [ABSTRACT FROM AUTHOR]

Abstract: Antigen presenting molecules play an important role in both innate and adoptive immune responses by priming and activating T cells. Among them, CD1 molecules have been identified to present both exogenous and endogenous lipid antigens to CD1-restricted T cells. The involvement of CD1-restricted T cells in autoimmune diseases and in defense against infectious diseases, however, remains largely unknown. Identifying novel antigenic lipids that bind to CD1 molecules and understanding the role of CD1-restricted T cells should lead to the successful development of vaccines, because the lipids can be used as antigens and also as adjuvants. In this paper, we have constructed functional recombinant human CD1 dimeric proteins and established a competitive ELISA assay to measure the lipid binding to CD1 molecules using the CD1 dimers. By using the competitive ELISA assay, we were able to show that the lipid extracts from murine malaria parasites can actually be loaded onto CD1 molecules. In addition, we have demonstrated that artificial antigen-presenting cells, which consist of magnetic beads coated with CD1d dimer and anti-CD28 antibody, stimulated and expanded human invariant NKT cells as efficiently as autologous immature DCs. A set of the tools presented in the current study should be valuable for screening various CD1 molecule-binding lipid antigens and for isolating CD1-restricted T cells. [Copyright &y& Elsevier]

Abstract: There is a substantial need to develop better influenza virus vaccines that can protect populations that are not adequately protected by the currently licensed vaccines. While live attenuated influenza virus vaccines induce superior immune responses compared to inactivated vaccines, the manufacturing process of both types of influenza virus vaccines is time consuming and may not be adequate during a pandemic. Adjuvants would be particularly useful if they could enhance the immune response to live attenuated influenza virus vaccines so that the amount of vaccine needed for a protective dose could be reduced. The glycolipid, alpha-galactosylceramide (alpha-GalCer), has recently been shown to have adjuvant activity for both inactivated and replicating recombinant vaccines. The goal of these experiments was to determine whether a derivative of alpha-GalCer, alpha-C-galactosylceramide (alpha-C-GalCer) can enhance the immune response elicited by a live attenuated influenza virus vaccine containing an NS1 protein truncation and reduce the amount of vaccine required to provide protection after challenge. Our results indicated that the adjuvant reduced both morbidity and mortality in BALB/c mice after challenge with wild type influenza virus. The adjuvant also increased the amount of influenza virus specific total IgG, IgG1, and IgG2a antibodies as well as IFN-γ secreting CD8+ T cells. By using knockout mice that are not able to generate NKT cells, we were able to demonstrate that the mechanism of adjuvant activity is dependent on NKT cells. Thus, our data indicate that stimulators of NKT cells represent a new avenue of adjuvants to pursue for live attenuated virus vaccines. [Copyright &y& Elsevier]

We have recently shown that α-C-galactosylceramide (α-C-GalCer) stimulates invariant natural killer T (iNKT) cells and preferentially induces a T helper 1 (Th1)-type response in mice. However, α-C-GalCer was found to be a rather weak ligand against human iNKT cells in vitro. Therefore, in this study, we sought to identify a compound that displays a strong stimulatory activity against human iNKT cells, by determining the biological activities of several C-glycoside analogues. From the in vitro screening assays, we found that almost all C-glycoside analogues, which have an E-alkene linker between sugar and lipid moieties, are able to activate human iNKT cells and to induce the maturation and activation of human dendritic cells through iNKT-cell activation. In summary, although α-galactosylceramide (α-GalCer) remains the strongest iNKT-cell ligand, our study identified E-alkene-linked C-glycoside analogues as potent human iNKT-cell stimulants, and indicated that these analogues could be used as a therapeutic agent in the future for diseases resolved by Th1-type responses. [ABSTRACT FROM AUTHOR]

Cerebral malaria complicated by cognitive sequelae is a major cause of morbidity in humans infected with Plasmodium falciparum. To model cognitive function after malaria, we created a rodent model of cerebral malaria by infecting C57BL/6 mice with Plasmodium berghei strain ANKA. After 7 days, an object-recognition test of working memory revealed a significant impairment in the visual memory of infected mice. This impairment was observed in the absence of confounding effects of infection. The cognitive dysfunction correlated with hemorrhage and inflammation. Furthermore, microglial activity and morphological changes detected throughout the brains of infected mice were absent from the brains of control mice, and this correlated with the measured cognitive defects. Similar testing methods in human studies could help identify subjects at risk for an adverse cognitive outcome. This murine model should facilitate the study of adjunctive methods to ameliorate adverse neurological outcomes in cerebral malaria. [ABSTRACT FROM AUTHOR]

Summary: A number of studies have shown that the natural killer T cell (NKT) ligand α-galactosylceramide (α-GalCer) serves as an adjuvant for various vaccines, including viral vaccines, parasite vaccines and protein vaccines. In this report, we investigated the adjuvant activity of α-GalCer on HIV-1 DNA vaccines in mice. This is a first study to show that α-GalCer can enhance the immunogenicity of DNA vaccines, since co-administration of α-GalCer with suboptimal doses of DNA vaccines greatly enhanced antigen-specific CD4+ T-cell and CD8+ T-cell responses. Differently from other vaccines, α-GalCer was also able to enhance HIV-specific antibody response 10-fold. It is of practical importance to find out that, in a DNA prime-DNA boost regimen, the adjuvant activity of α-GalCer was most profound when co-administered at the priming, but not at the boosting phase. In a dose-sparing experiment, we found that the level of cell-mediated immune responses in mice vaccinated with 5μg of DNA in the presence of α-GalCer was equivalent to that of mice vaccinated with 50μg of DNA in the absence of α-GalCer. Finally, results from CD1d and interferon-γ receptor knockout mice confirm our previous data and determine the mechanistic dependence upon these molecules. These results illustrate that α-GalCer enhances the immunogenicity of DNA vaccines in a mechanism-based fashion. Since both mice and humans share the CD1d molecule, this information may aid in designing more effective DNA vaccines and vaccine adjuvants against HIV-1. [Copyright &y& Elsevier]

Natural killer T (NKT) cells recognize glycosphingolipids presented by CD1d molecules and have been linked to defense against microbial infections. Previously defined foreign glycosphingolipids recognized by NKT cells are uniquely found in nonpathogenic sphingomonas bacteria. Here we show that mouse and human NKT cells also recognized glycolipids, specifically a diacylglycerol, from Borrelia burgdorferi, which causes Lyme disease. The B. burgdorferi–derived, glycolipid-induced NKT cell proliferation and cytokine production and the antigenic potency of this glycolipid was dependent on acyl chain length and saturation. These data indicate that NKT cells recognize categories of glycolipids beyond those in sphingomonas and suggest that NKT cell responses driven by T cell receptor–mediated glycolipid recognition may provide protection against diverse pathogens. [ABSTRACT FROM AUTHOR]

α-Galactosylceramide (α-GalCer) is the prototype compound for studying the presentation of glycolipids on CD1d molecules to natural killer T (NKT) lymphocytes. A single i.v. dose of glycolipid triggers a cascade of events involving the production of several cytokines over the course of a day, a short-lived activation of NKT and natural killer (NK) cells, and a more prolonged adaptive T cell immune response if certain antigens are given together with α-GalCer. We find that a recently described analogue, α-C-galactosylceramide (α-C-GalCer), more potently induces these innate and adaptive immune responses in mice. α-C-GalCer acts as a more effective trigger for IL-12 and IFN-γ production, although it minimally elicits IL-4 and TNF-α release into the serum. Also, α-C-GalCer better mobilizes NKT and natural killer cells to resist B16 melanoma. To help understand these effects, we find that α-C-GalCer binds more stably to dendritic cells than α-GalCer and that dendritic cells loaded with α-C-GalCer induce larger and more long lasting NKT cell responses in vivo. When glycolipid is targeted to dendritic cells in spleen together with antigens in dying cells, such as irradiated tumor cells, α-C-GalCer is active as an adjuvant for T cell-mediated immunity at lower doses, just 20 ng per mouse, where it is also able to up-regulate the required CD40L costimulatory molecule on NKT cells. Therefore, α-C-GalCer represents a glycolipid that binds more stably to dendritic cells and acts as a more effective link between innate and adaptive immunity in vivo. [ABSTRACT FROM AUTHOR]

Abstract: Two novel hybrid molecules 3-O-sulfo-α/β-galactosylceramide 3 and 4, which are derived from an immunostimulatory agent α-GalCer 1 and self-glycolipid ligand sulfatide 2, were designed and synthesized. Compound 3 was shown to efficiently stimulate human NKT cells to secret IL-4 and IFN-γ, with activities similar to 1, suggesting that modification of the 3″-OH position of the galactose moiety with sulfate has no significant effect on NKT cell stimulation. As a comparison, the β-isomer 4 has no affinity to NKT cells, which demonstrates that the α-glycosidic bond of galactosylceramide is crucial to the NKT cells activation. [Copyright &y& Elsevier]