Activated iNKT cells promote memory CD8+ T cell differentiation during viral infection

α-Galactosylceramide (α-GalCer) is the prototypical lipid ligand for invariant NKT cells. Recent studies have proposed that α-GalCer is an effective adjuvant in vaccination against a range of immune challenges, however its mechanism of action has not been completely elucidated. A variety of delivery methods have been examined including pulsing dendritic cells with α-GalCer to optimize the potential of α-GalCer. These methods are currently being used in a variety of clinical trials in patients with advanced cancer but cannot be used in the context of vaccine development against pathogens due to their complexity. Using a simple delivery method, we evaluated α-GalCer adjuvant properties, using the mouse model for cytomegalovirus (MCMV). We measured several key parameters of the immune response to MCMV, including inflammation, effector, and central memory CD8(+) T cell responses. We found that α-GalCer injection at the time of the infection decreases viral titers, alters the kinetics of the inflammatory response, and promotes both increased frequencies and numbers of virus-specific memory CD8(+) T cells. Overall, our data suggest that iNKT cell activation by α-GalCer promotes the development of long-term protective immunity through increased fitness of central memory CD8(+) T cells, as a consequence of reduced inflammation.     

Endocine?, N3OA and N3OASq; Three Mucosal Adjuvants That Enhance the Immune Response to Nasal Influenza Vaccination

Annual outbreaks of seasonal influenza are controlled or prevented through vaccination in many countries. The seasonal vaccines used are either inactivated, currently administered parenterally, or live-attenuated given intranasally. In this study three mucosal adjuvants were examined for the influence on the humoral (mucosal and systemic) and cellular influenza A-specific immune responses induced by a nasally administered vaccine. We investigated in detail how the anionic Endocine™ and the cationic adjuvants N3OA and N3OASq mixed with a split inactivated influenza vaccine induced influenza A-specific immune responses as compared to the vaccine alone after intranasal immunization. The study showed that nasal administration of a split virus vaccine together with Endocine™ or N3OA induced significantly higher humoral and cell-mediated immune responses than the non-adjuvanted vaccine. N3OASq only significantly increased the cell-mediated immune response. Furthermore, nasal administration of the influenza vaccine in combination with any of the adjuvants; Endocine™, N3OA or N3OASq, significantly enhanced the mucosal immunity against influenza HA protein. Thus the addition of these mucosal adjuvants leads to enhanced immunity in the most relevant tissues, the upper respiratory tract and the systemic circulation. Nasal influenza vaccination with an inactivated split vaccine can therefore provide an important mucosal immune response, which is often low or absent after traditional parenteral vaccination.     

The effect of adjuvants on vaccine-induced antibody response against influenza in aged mice

While influenza remains a major threat to public health, researchers continue to search for a universal solution to improving the efficacy of the influenza vaccine. Even though influenza affects people of all different ages, it can be extremely hazardous to people of 65 years of age or older since that is the population that makes up the high majority of the death toll caused by influenza-related diseases. Elderly individuals suffer the effects of immunosenescence as they age, which is the diminishing of the overall immune response. Immunosenescence occurs by specifically affecting the adaptive immune response which controls the establishment of immunity after vaccination or infection. There are many studies under way that are trying to find a resolution to the problem of the influenza vaccine not providing enough protection in the elderly population. One of the possible strategies is to seek the use of an optimal adjuvant, an immunological agent that can enhance immune responses, with the current vaccine formulation. Here, we used the murine model to review the effects of adjuvants on the antibody response to influenza vaccines in aged mice. Since adjuvants can enhance the production of important inflammatory cytokines and activation of dendritic cells, the stimulation of these cells are boosted to increase the effectiveness of the influenza vaccine in aged mice which would hopefully translate to the elderly.     

Invariant NKT Cells: Regulation and Function during Viral Infection

Natural killer T cells (NKT cells) represent a subset of T lymphocytes that express natural killer (NK) cell surface markers. A subset of NKT cells, termed invariant NKT cells (iNKT), express a highly restricted T cell receptor (TCR) and respond to CD1d-restricted lipid ligands. iNKT cells are now appreciated to play an important role in linking innate and adaptive immune responses and have been implicated in infectious disease, allergy, asthma, autoimmunity, and tumor surveillance. Advances in iNKT identification and purification have allowed for the detailed study of iNKT activity in both humans and mice during a variety of chronic and acute infections. Comparison of iNKT function between non-pathogenic simian immunodeficiency virus (SIV) infection models and chronic HIV-infected patients implies a role for iNKT activity in controlling immune activation. In vitro studies of influenza infection have revealed novel effector functions of iNKT cells including IL-22 production and modulation of myeloid-derived suppressor cells, but ex vivo characterization of human iNKT cells during influenza infection are lacking. Similarly, as recent evidence suggests iNKT involvement in dengue virus pathogenesis, iNKT cells may modulate responses to a number of emerging pathogens. This Review will summarize current knowledge of iNKT involvement in responses to viral infections in both human and mouse models and will identify critical gaps in knowledge and opportunities for future study. We will also highlight recent efforts to harness iNKT ligands as vaccine adjuvants capable of improving vaccination-induced cellular immune responses.     

Baculovirus-infected insect cells expressing peptide-MHC complexes elicit protective antitumor immunity

Evaluation of T cell responses to tumor- and pathogen-derived peptides in preclinical models is necessary to define the characteristics of efficacious peptide vaccines. We show in this study that vaccination with insect cells infected with baculoviruses expressing MHC class I linked to tumor peptide mimotopes results in expansion of functional peptide-specific CD8+ T cells that protect mice from tumor challenge. Specific peptide mimotopes selected from peptide-MHC libraries encoded by baculoviruses can be tested using this vaccine approach. Unlike other vaccine strategies, this vaccine has the following advantages: peptides that are difficult to solublize can be easily characterized, bona fide peptides without synthesis artifacts are presented, and additional adjuvants are not required to generate peptide-specific responses. Priming of antitumor responses occurs within 3 days of vaccination and is optimal 1 wk after a second injection. After vaccination, the Ag-specific T cell response is similar in animals primed with either soluble or membrane-bound Ag, and CD11c+ dendritic cells increase expression of maturation markers and stimulate proliferation of specific T cells ex vivo. Thus, the mechanism of Ag presentation induced by this vaccine is consistent with cross-priming by dendritic cells. This straightforward approach will facilitate future analyses of T cells elicited by peptide mimotopes.     

Effect of adjuvants on responses to skin immunization by microneedles coated with influenza subunit vaccine

Recent studies have demonstrated the effectiveness of vaccine delivery to the skin by vaccine-coated microneedles; however there is little information on the effects of adjuvants using this approach for vaccination. Here we investigate the use of TLR ligands as adjuvants with skin-based delivery of influenza subunit vaccine. BALB/c mice received 1 μg of monovalent H1N1 subunit vaccine alone or with 1 μg of imiquimod or poly(I:C) individually or in combination via coated microneedle patches inserted into the skin. Poly(I:C) adjuvanted subunit influenza vaccine induced similar antigen-specific immune responses compared to vaccine alone when delivered to the skin by microneedles. However, imiquimod-adjuvanted vaccine elicited higher levels of serum IgG2a antibodies and increased hemagglutination inhibition titers compared to vaccine alone, suggesting enhanced induction of functional antibodies. In addition, imiquimod-adjuvanted vaccine induced a robust IFN-γ cellular response. These responses correlated with improved protection compared to influenza subunit vaccine alone, as well as reduced viral replication and production of pro-inflammatory cytokines in the lungs. The finding that microneedle delivery of imiquimod with influenza subunit vaccine induces improved immune responses compared to vaccine alone supports the use of TLR7 ligands as adjuvants for skin-based influenza vaccines.     

Near-Infrared Laser Adjuvant for Influenza Vaccine

Safe and effective immunologic adjuvants are often essential for vaccines. However, the choice of adjuvant for licensed vaccines is limited, especially for those that are administered intradermally. We show that non-tissue damaging, near-infrared (NIR) laser light given in short exposures to small areas of skin, without the use of additional chemical or biological agents, significantly increases immune responses to intradermal influenza vaccination without augmenting IgE. The NIR laser-adjuvanted vaccine confers increased protection in a murine influenza lethal challenge model as compared to unadjuvanted vaccine. We show that NIR laser treatment induces the expression of specific chemokines in the skin resulting in recruitment and activation of dendritic cells and is safe to use in both mice and humans. The NIR laser adjuvant technology provides a novel, safe, low-cost, simple-to-use, potentially broadly applicable and clinically feasible approach to enhancing vaccine efficacy as an alternative to chemical and biological adjuvants.     

NKT TCR recognition of CD1d-α-C-galactosylceramide

NKT cells respond to a variety of CD1d-restricted glycolipid Ags that are structurally related to the prototypic Ag α-galactosylceramide (α-GalCer). A modified analog of α-GalCer with a carbon-based glycosidic linkage (α-C-GalCer) has generated great interest because of its apparent ability to promote prolonged, Th1-biased immune responses. In this study, we report the activation of spleen NKT cells to α-C-GalCer, and related C-glycoside ligands, is weaker than that of α-GalCer. Furthermore, the Vβ8.2 and Vβ7 NKT TCR affinity for CD1d-α-C-GalCer, and some related analogs, is ～10-fold lower than that for the NKT TCR-CD1d-α-GalCer interaction. Nevertheless, the crystal structure of the Vβ8.2 NKT TCR-CD1d-α-C-GalCer complex is similar to that of the corresponding NKT TCR-CD1d-α-GalCer complex, although subtle differences at the interface provide a basis for understanding the lower affinity of the NKT TCR-CD1d-α-C-GalCer interaction. Our findings support the concept that for CD1d-restricted NKT cells, altered glycolipid ligands can promote markedly different responses while adopting similar TCR-docking topologies.     

Immunostimulant patch containing heat-labile enterotoxin from Escherichia coli enhances immune responses to injected influenza virus vaccine through activation of skin dendritic cells

Vaccine strategies, such as influenza virus vaccination of the elderly, are highly effective at preventing disease but provide protection for only the responding portion of the vaccinees. Adjuvants improve the magnitude and rates of responses, but their potency must be attenuated to minimize side effects. Topical delivery of strong adjuvants such as heat-labile enterotoxin from Escherichia coli (LT) induces potent immune responses. We hypothesized that LT delivered alone in an immunostimulating (LT-IS) patch placed on the skin at the site of injection could augment the immune response to injected vaccines. This was based on the observation that topically applied LT induces migration of activated antigen-presenting cells (APCs) from the skin to the proximal draining lymph node (DLN), and that APCs loaded with antigen by injection in the same anatomical region also migrate to the same DLN. We observed that when influenza virus vaccine is injected and an LT-IS patch is placed to target the same DLN, the influenza virus antibody response is enhanced. Similarly, influenza virus-specific T cells isolated from the lungs show increased levels of gamma interferon and interleukin-4 production. An LT-IS patch placed near an injected vaccine also leads to increased levels of hemagglutination inhibition titers, enhanced mucosal immunoglobulin A responses, and enhanced antigen presentation. Although the mechanisms by which an LT-IS patch exerts its enhancing effects need further study, the enhanced immune responses, ability to safely use potent adjuvants, and simplicity of LT-IS patch application address an important unmet need and provide a new immune enhancement strategy.     

Therapeutic vaccination with simian immunodeficiency virus (SIV)-DNA + IL-12 or IL-15 induces distinct CD8 memory subsets in SIV-infected macaques

DNA vaccination is an invaluable approach for immune therapy in that it lacks vector interference and thus permits repeated vaccination boosts. However, by themselves, DNA-based vaccines are typically poor inducers of Ag-specific immunity in humans and non-human primates. Cytokines, such as IL-12 and IL-15, have been shown to be potent adjuvants for the induction and maintenance of cellular immune responses, in particular during HIV infection. In this study, we examined the ability of therapeutic vaccination with SIV-DNA+IL-12 or IL-15 as molecular adjuvants to improve DNA vaccine potency and to enhance memory immune responses in SIV-infected macaques. Our results demonstrate that incorporating IL-12 into the vaccine induces SIV-specific CD8 effector memory T cell (T(EM)) functional responses and enhances the capacity of IFN-gamma-producing CD8 T(EM) cells to produce TNF. Lower levels of PD-1 were expressed on T cells acquiring dual function upon vaccination as compared with mono-functional CD8 T(EM) cells. Finally, a boost with SIV-DNA+IL-15 triggered most T cell memory subsets in macaques primed with either DNA-SIV or placebo but only CD8 T(EM) in macaques primed with SIV-DNA+IL-12. These results indicate that plasmid IL-12 and IL-15 cytokines represent a significant addition to enhance the ability of therapeutic DNA vaccines to induce better immunity.     

Cutting edge: nonglycosidic CD1d lipid ligands activate human and murine invariant NKT cells

Invariant NKT cells (iNKT cells) recognize CD1d/glycolipid complexes. We demonstrate that the nonglycosidic compound threitolceramide efficiently activates iNKT cells, resulting in dendritic cell (DC) maturation and the priming of Ag-specific T and B cells. Threitolceramide-pulsed DCs are more resistant to iNKT cell-dependent lysis than alpha-galactosylceramide-pulsed DCs due to the weaker affinity of the human iNKT TCR for CD1d/ threitolceramide than CD1d/alpha-galactosylceramide complexes. iNKT cells stimulated with threitolceramide also recover more quickly from activation-induced anergy. Kinetic and functional experiments showed that shortening or lengthening the threitol moiety by one hydroxymethylene group modulates ligand recognition, as human and murine iNKT cells recognize glycerolceramide and arabinitolceramide differentially. Our data broaden the range of potential iNKT cell agonists. The ability of these compounds to assist the priming of Ag-specific immune responses while minimizing iNKT cell-dependent DC lysis makes them attractive adjuvants for vaccination strategies.     

提高黏膜免疫的方法及研究进展

黏膜是很多病原体入侵机体的重要入口,黏膜疫苗能诱导产生黏膜保护性免疫应答和系统性免疫应答,阻止病原微生物黏附、入侵和繁殖。但多数候选黏膜疫苗的安全性、稳定性、免疫效力及保护作用还无法达到理想的效果,佐剂或载体的使用改善了黏膜疫苗存在的不足,使黏膜疫苗有了广阔的发展前景。文章综述了提高黏膜免疫的方法及研究进展。     

Immunization with HIV-1 Gag protein conjugated to a TLR7/8 agonist results in the generation of HIV-1 Gag-specific Th1 and CD8+ T cell responses

One strategy to induce optimal cellular and humoral immune responses following immunization is to use vaccines or adjuvants that target dendritic cells and B cells. Activation of both cell types can be achieved using specific TLR ligands or agonists directed against their cognate receptor. In this study, we compared the ability of the TLR7/8 agonist R-848, which signals only via TLR7 in mice, with CpG oligodeoxynucleotides for their capacity to induce HIV-1 Gag-specific T cell and Ab responses when used as vaccine adjuvants with HIV-1 Gag protein in mice. Injection of R-848 and CpG oligodeoxynucleotides alone enhanced the innate immune responses in vivo as demonstrated by high serum levels of inflammatory cytokines, including IL-12p70 and IFN-alpha, and increased expression of CD80, CD86, and CD40 on CD11c(+) dendritic cells. By contrast, R-848 was a relatively poor adjuvant for inducing primary Th1 or CD8(+) T cell responses when administered with HIV-1 Gag protein. However, when a TLR7/8 agonist structurally and functionally similar to R-848 was conjugated to HIV-1 Gag protein both Th1 and CD8(+) T cells responses were elicited as determined by intracellular cytokine and tetramer staining. Moreover, within the population of HIV-1 Gag-specific CD8(+) CD62(low) cells, approximately 50% of cells expressed CD127, a marker shown to correlate with the capacity to develop into long-term memory cells. Overall, these data provide evidence that TLR7/8 agonists can be effective vaccine adjuvants for eliciting strong primary immune responses with a viral protein in vivo, provided vaccine delivery is optimized.     

CpG or IFN-α are more potent adjuvants than GM-CSF to promote anti-tumor immunity following idiotype vaccine in multiple myeloma

Idiotype (Id) protein in combination with GM-CSF has been used as vaccines for immunotherapy of patients with myeloma and B-cell tumors and the results have been disappointing. To search for better immune adjuvants to improve the efficacy of Id-based immunotherapy in myeloma, we evaluated and compared the efficacy of vaccination of Id protein in combination with CpG or IFN-α, or GM-CSF as a control, in the 5TGM1 myeloma mouse model. Our results showed that Id vaccine combined with CpG or IFN-α, but not GM-CSF, not only efficiently protected mice from developing myeloma but also eradicated established myeloma. The therapeutic responses were associated with an induction of strong humoral immune responses including anti-Id antibodies, and cellular immune responses including Id- and myeloma-specific CD8⁺ cytotoxic T lymphocytes (CTLs), CD4⁺ type-1 T-helper (Th1) cells and memory T cells in mice receiving Id vaccine combined with CpG or IFN-α. Furthermore, Id vaccine combined with CpG or IFN-α induced Id- and tumor-specific memory immune responses that protected surviving mice from tumor rechallenge. Thus, our study clearly shows that CpG or IFN-α are better immune adjuvants than GM-CSF. This information will be important for improving the strategies of Id-based immunotherapy for patients with myeloma and other B-cell tumors.     

The TLR7 agonist imiquimod enhances the anti-melanoma effects of a recombinant Listeria monocytogenes vaccine

Activation of innate immune cells through TLR triggers immunomodulating events that enhance cell-mediated immunity, raising the possibility that ligands to these receptors might act as adjuvants in conjunction with T cell activating vaccines. In this report, topical imiquimod, a synthetic TLR7 agonist, significantly enhanced the protective antitumor effects of a live, recombinant listeria vaccine against murine melanoma. This tumor protective effect was not dependent on direct application to the tumor and was associated with an increase in tumor-associated and splenic dendritic cells. Additionally, the combination of imiquimod treatment with prior vaccination led to development of localized vitiligo. These findings indicate that activation of the innate immune system with TLR ligands stimulates dendritic cell activity resulting in a bypass of peripheral tolerance and enhanced antitumor activity. The results of these studies have broad implications for future designs of immunotherapeutic vaccines against tumors and the treatment of metastatic melanoma.     

Transcutaneous anti-influenza vaccination promotes both CD4 and CD8 T cell immune responses in humans

Induction of T cell responses has become one of the major goals in therapeutic vaccination against viral diseases and cancer. The use of the skin as target organ for vaccine has been spurred by recent implication of epithelial dendritic cells in CD8 cell cross-priming and suggests that vaccination via the transcutaneous (TC) route may be relevant in the induction of cellular immune responses. We have previously shown that TC application of nanoparticles, on human skin explants, allows targeting of epidermal dendritic cells, possibly via hair follicles. In this study, we have investigated cellular immune responses against an influenza protein-based vaccine by TC vaccination, compared with i.m. vaccination in humans. In this study on 11 healthy volunteers, we found that a newly developed protocol based on cyanoacrylate skin surface stripping induced a significant increase in IFN-gamma-producing T cells specific for influenza vaccine by ELISPOT assays. Interestingly, TC vaccination induced both effector CD4 and CD8 T cell responses, whereas i.m. injection induced strong effector CD4 in the absence of CD8 T cells, as assessed by intracellular cytokine staining and tetramer analyses. This study proposes new perspectives for the development of vaccination strategies that trigger T cell immune responses in humans.     

Structural basis of Blastomyces Endoglucanase-2 adjuvancy in anti-fungal and -viral immunity

The development of safe subunit vaccines requires adjuvants that augment immunogenicity of non-replicating protein-based antigens. Current vaccines against infectious diseases preferentially induce protective antibodies driven by adjuvants such as alum. However, the contribution of antibody to host defense is limited for certain classes of infectious diseases such as fungi, whereas animal studies and clinical observations implicate cellular immunity as an essential component of the resolution of fungal pathogens. Here, we decipher the structural bases of a newly identified glycoprotein ligand of Dectin-2 with potent adjuvancy, Blastomyces endoglucanase-2 (Bl-Eng2). We also pinpoint the developmental steps of antigen-specific CD4⁺ and CD8⁺ T responses augmented by Bl-Eng2 including expansion, differentiation and tissue residency. Dectin-2 ligation led to successful systemic and mucosal vaccination against invasive fungal infection and Influenza A infection, respectively. O-linked glycans on Bl-Eng2 applied at the skin and respiratory mucosa greatly augment vaccine subunit- induced protective immunity against lethal influenza and fungal pulmonary challenge.     

Effect of AcHERV-GmCSF as an Influenza Virus Vaccine Adjuvant

Introduction

The first identification of swine-originated influenza A/CA/04/2009 (pH1N1) as the cause of an outbreak of human influenza accelerated efforts to develop vaccines to prevent and control influenza viruses. The current norm in many countries is to prepare influenza vaccines using cell-based or egg-based killed vaccines, but it is difficult to elicit a sufficient immune response using this approach. To improve immune responses, researchers have examined the use of cytokines as vaccine adjuvants, and extensively investigated their functions as chemoattractants of immune cells and boosters of vaccine-mediated protection. Here, we evaluated the effect of Granulocyte-macrophage Colony-Stimulating Factor (GmCSF) as an influenza vaccine adjuvant in BALB/c mice.

Method and Results

Female BALB/c mice were immunized with killed vaccine together with a murine GmCSF gene delivered by human endogenous retrovirus (HERV) envelope coated baculovirus (1×10⁷ FFU AcHERV-GmCSF, i.m.) and were compared with mice immunized with the killed vaccine alone. On day 14, immunized mice were challenged with 10 median lethal dose of mouse adapted pH1N1 virus. The vaccination together with GmCSF treatment exerted a strong adjuvant effect on humoral and cellular immune responses. In addition, the vaccinated mice together with GmCSF were fully protected against infection by the lethal influenza pH1N1 virus.

Conclusion

Thus, these results indicate that AcHERV-GmCSF is an effective molecular adjuvant that augments immune responses against influenza virus.     

Small cationic DDA:TDB liposomes as protein vaccine adjuvants obviate the need for TLR agonists in inducing cellular and humoral responses

Most subunit vaccines require adjuvants in order to induce protective immune responses to the targeted pathogen. However, many of the potent immunogenic adjuvants display unacceptable local or systemic reactogenicity. Liposomes are spherical vesicles consisting of single (unilamellar) or multiple (multilamellar) phospholipid bi-layers. The lipid membranes are interleaved with an aqueous buffer, which can be utilised to deliver hydrophilic vaccine components, such as protein antigens or ligands for immune receptors. Liposomes, in particular cationic DDA:TDB vesicles, have been shown in animal models to induce strong humoral responses to the associated antigen without increased reactogenicity, and are currently being tested in Phase I human clinical trials. We explored several modifications of DDA:TDB liposomes--including size, antigen association and addition of TLR agonists--to assess their immunogenic capacity as vaccine adjuvants, using Ovalbumin (OVA) protein as a model protein vaccine. Following triple homologous immunisation, small unilamellar vesicles (SUVs) with no TLR agonists showed a significantly higher capacity for inducing spleen CD8 IFNγ responses against OVA in comparison with the larger multilamellar vesicles (MLVs). Antigen-specific antibody reponses were also higher with SUVs. Addition of the TLR3 and TLR9 agonists significantly increased the adjuvanting capacity of MLVs and OVA-encapsulating dehydration-rehydration vesicles (DRVs), but not of SUVs. Our findings lend further support to the use of liposomes as protein vaccine adjuvants. Importantly, the ability of DDA:TDB SUVs to induce potent CD8 T cell responses without the need for adding immunostimulators would avoid the potential safety risks associated with the clinical use of TLR agonists in vaccines adjuvanted with liposomes.     

Introduction of aromatic group on 4'-OH of α-GalCer manipulated NKT cell cytokine production

The glycosphingolipid ??-GalCer has been found to influence mammalian immune system significantly through the natural killer T cells. Unfortunately, the pre-clinical and clinical studies revealed several critical disadvantages that prevented the therapeutic application of ??-GalCer in treating cancer and other diseases. Recently, the detailed illustration of the CD1d/??-GalCer/NKT TCR complex crystal structural, together with other latest structural and biological understanding on glycolipid ligands and NKT cells, provided a new platform for developing novel glycolipid ligands with optimized therapeutic effects. Here, we designed a series of novel aromatic group substituted ??-GalCer analogues. The biological activity of these analogues was characterized and the results showed the unique substitution group manipulated the immune responses of NKT cells. Computer modeling and simulation study indicated the analogues had unique binding mode when forming CD1d/glycolipid/NKT TCR complex, comparing to original ??-GalCer.