α-Galactosylceramide (α-GalCer) is a glycolipid that stimulates natural killer T cells to produce both T helper (Th) 1 and Th2 cytokines. This property enables α-GalCer to ameliorate a wide variety of infectious, neoplastic, and autoimmune diseases; however, its effectiveness against any one disease is limited by the opposing activities of the induced Th1 and Th2 cytokines. Here, we report that a synthetic C-glycoside analogue of α-GalCer, α-C-galactosylceramide (α-C-GalCer), acts as natural killer T cell ligand in vivo, and stimulates an enhanced Th1-type response in mice. In two disease models requiring Th1-type responses for control, namely malaria and melanoma metastases, α-C-GalCer exhibited a 1,000-fold more potent antimalaria activity and a 100-fold more potent antimetastatic activity than α-GalCer. Moreover, α-C-GalCer consistently stimulated prolonged production of the Th1 cytokines interferon-γ and interleukin (IL)-12, and decreased production of the Th2 cytokine IL-4 compared with α-GalCer. Finally, α-C-GalCer's enhanced therapeutic activity required the presence of IL-12, which was needed to stimulate natural killer cells for optimal interferon-γ production, but did not affect IL-4. Overall, our results suggest that α-C-GalCer may one day be an excellent therapeutic option for diseases resolved by Th1-type responses.
Abstract Glycoconjugate vaccines, which are currently made by covalently coupling a bacterial capsular polysaccharide to a carrier protein, have been used to immunize many millions of people and have enormously reduced disease burden, particularly among children. However, the immunogenicity and efficacy of these vaccines have been relatively low and heterogeneous in some high-risk populations. The classic antigen presentation hypothesis for glycoconjugates proposes that helper CD4+ T cells recognize a peptide derived from the carrier protein. We investigated mechanisms of processing and presentation of a prototypic glycoconjugate vaccine in which group B streptococcal type III polysaccharide is coupled to a carrier. Upon uptake into the endosome of an antigen-presenting cell, the polysaccharide undergoes oxidative depolymerization, which, in conjunction with acidic protease-mediated digestion of the carrier protein, creates a carbohydrate T cell epitope conjugated to an MHCII binding peptide. These epitopes formed in the endosome bind to MHCII through the peptide and are subsequently presented on the APC surface, with the hydrophilic carbohydrate positioned for easy recognition by the T-cell receptor. Recognition of the carbohydrate in the presence of MHCII drives T-cell help for antibody-producing B cells. An enhanced understanding of immune mechanisms such as T-cell recognition of carbohydrates may lead to markedly improved vaccines against infectious diseases.
Modern, subunit-based vaccines have so far failed to induce significant T cell responses, contributing to ineffective vaccination against many pathogens. Importantly, while today’s adjuvants are designed to trigger innate and non-specific immune responses, they fail to directly stimulate the adaptive immune compartment. Programmed cell death 1 (PD-1) partly regulates naïve-to-antigen-specific effector T cell transition and differentiation by suppressing the magnitude of activation. Indeed, we previously reported on a microbial-derived, peptide-based PD-1 checkpoint inhibitor, LD01, which showed potent T cell-stimulating activity when combined with a vaccine. Here we sought to improve the potency of LD01 by designing and testing new LD01 derivatives. Accordingly, we found that a modified version of an 18-amino acid metabolite of LD01, LD10da, improved T cell activation capability in a malaria vaccine model. Specifically, LD10da demonstrates improved antigen-specific CD8 + T cell expansion when combined prophylactically with an adenovirus-based malaria vaccine. A single dose of LD10da at the time of vaccination is sufficient to increase antigen-specific CD8 + T cell expansion in wild-type mice. Further, we show that LD10 can be encoded and delivered by a Modified Vaccinia Ankara viral vector and can enhance antigen-specific CD8 + T cell expansion comparable to that of synthetic peptide administration. Therefore, LD10da represents a promising biologic-based immunomodulator that can be genetically encoded and delivered, along with the antigen, by viral or other nucleic acid vectors to improve the efficacy and delivery of vaccines for ineradicable and emerging infectious diseases.
Broadly neutralizing antibodies (bnAbs) isolated from HIV-infected individuals delineate vulnerable sites on the HIV envelope glycoprotein that are potential vaccine targets. A linear epitope within the N-terminal region of the HIV-1 fusion peptide (FP8) is the primary target of VRC34.01, a bnAb that neutralizes ~50% of primary HIV isolates. FP8 has attracted attention as a potential HIV vaccine target because it is a simple linear epitope. Here, platform technologies based on RNA bacteriophage virus-like particles (VLPs) were used to develop multivalent vaccines targeting the FP8 epitope. Both recombinant MS2 VLPs displaying the FP8 peptide and Qβ VLPs displaying chemically conjugated FP8 peptide induced high titers of FP8-specific antibodies in mice. Moreover, a heterologous prime-boost-boost regimen employing the two FP8-VLP vaccines and native envelope trimer was the most effective approach for eliciting HIV-1 neutralizing antibodies. Given the potent immunogenicity of VLP-based vaccines, this vaccination strategy—inspired by bnAb-guided epitope mapping, VLP bioengineering, and prime-boost immunization approaches—may be a useful strategy for eliciting bnAb responses against HIV.
C-glycoside analogues of alpha-galactosylceramide were shown to activate both human and mouse invariant NKT (iNKT) cells. Among these analogues, GCK152, which has an aromatic ring in the acyl chain, exhibited a stronger stimulatory activity against human iNKT cells and a much weaker activity against murine iNKT cells than GCK127 that has an almost identical fatty acyl chain as alpha-galactosylceramide. In this study, we have found that invariant TCR (invTCR) expressed by iNKT cells, but not CD1d expressed by APCs, command the species-specific preferential activity of C-glycosides, and that their preferential activity against human vs murine iNKT cells correlate with the binding affinity of glycolipid-CD1d complex to invTCR of respective iNKT cells rather than that of glycolipid to human or murine CD1d molecules. Overall, the structural difference of invTCR appears to supersede those of CD1d molecule in shaping the strength of the biological activity of C-glycoside analogues.
Malaria is a severe infectious disease with relatively high mortality, thus having been a scourge of humanity. There are a few candidate malaria vaccines that have shown a protective efficacy in humans against malaria. One of the candidate human malaria vaccines, which is based on human malaria sporozoites and called PfSPZ Vaccine, has been shown to protect a significant proportion of vaccine recipients from getting malaria. PfSPZ Vaccine elicits a potent response of hepatic CD8+ T cells that are specific for malaria antigens in non-human primates. To further characterize hepatic CD8+ T cells induced by the sporozoite-based malaria vaccine in a mouse model, we have used a cutting-edge Single-cell Barcode (SCBC) assay, a recently emerged approach/method for investigating the nature of T-cells responses during infection or cancer. Using the SCBC technology, we have identified a population of hepatic CD8+ T cells that are polyfunctional at a single cell level only in a group of vaccinated mice upon malaria challenge. The cytokines/chemokines secreted by these polyfunctional CD8+ T-cell subsets include MIP-1α, RANTES, IFN-γ, and/or IL-17A, which have shown to be associated with protective T-cell responses against certain pathogens. Therefore, a successful induction of such polyfunctional hepatic CD8+ T cells may be a key to the development of effective human malaria vaccine. In addition, the SCBC technology could provide a new level of diagnostic that will allow for a more accurate determination of vaccine efficacy.
