Co-immunizing with PD-L1 induces CD8+ DCs-mediated anti-tumor immunity in multiple myeloma

Tumor therapeutic vaccines have faced a challenge for effective protection against malignant tumors by inducing tumor-specific CD8⁺ T cell responses. Here, we designed a DNA vaccine containing a tumor-specific antigen of Dickkopf-1 (DKK-1) and an immune checkpoint of programmed death ligand 1 (PD-L1) delivered by PLGA/PEI nanoparticle-mediated delivery system for multiple myeloma therapy. Murine subcutaneous tumor model established with human DKK1 (hDKK-1)-SP2/0 cells were intramuscularly immunized with PLGA/PEI-pPD-L1/pDDK-1 vaccine and equal amount of control 3 times at 10 day-intervals. Compared with PLGA/PEI-pDKK1 immunization group, PLGA/PEI-pPD-L1/pDKK-1 co-immunization enhanced the induction and mature of CD11c⁺ DCs and CD8⁺CD11c⁺ DCs, and promoted antigen-specific Th1 responses and cytotoxic T lymphocyte (CTL) responses. The reduced tumor volume and weight as well as increased tumor inhibition rate were observed in PLGA/PEI-pPD-L1/pDKK-1 vaccine co-immunization group, indicated that the vaccine could effectively inhibit the tumor growth of multiple myeloma. The anti-tumor activity of PLGA/PEI-pPD-L1/pDKK-1 vaccine was abrogated by CD8 cell depletion accompanied with the reduced percentages of CD8⁺CD11c⁺ DCs and CD8⁺ T cells in the spleen and TILs. These results indicated that the anti-tumor efficacy of PLGA/PEI-pPD-L1/pDKK-1 vaccine was required for CD8⁺CD11c⁺ DCs-mediated CD8⁺ T cell immunity responses. This vaccine strategy may represent a potential and promising approach for hematological malignancy treatment.     

Vaccine potential of cytosolic proteins loaded fibrin microspheres of Cryptococcus neoformans in BALB/c mice

Cryptococcosis is a leading mycological cause of mortality among immunologically compromised individuals. In order to develop an effective vaccine against Cryptococcus neoformans, the cytosolic proteins (Cp) of the pathogen have been used as an antigen in combination with different formulations. In the present study, we have demonstrated that Cp encapsulated poly-lactide co-glycolide (PLGA) microsphere further co-encapsulated into the biocompatible fibrin cross-linked plasma beads (Fib-PLGA-Cp) mediated cytosolic delivery elicited strong immune response in the BALB/c mice. In contrast, other formulations of Cp failed to impart significant level of protection. The immune response, involved with Fib-PLGA-Cp protection, appear to interact with the target cells by both endocytosis as well as membrane fusion mode, thus helping in the activation of both CD4⁺ and CD8⁺ T-cells. Analysis of cytokine profiles in immunized animals revealed that the protective response was associated with the Th1/Th2 polarization in favor of type-1 cytokine [interferons (IFN)-γ and interleukin (IL)-2] cells. Furthermore, vaccination with Fib-PLGA-Cp elicited high immunoglobulin (Ig) G_l and IgG_2a isotype response; successfully cleared fungal burden in vital organs and also increased the survival rate of immunized animals. Altogether the present study is a clear indicative of the possible use of fibrin microsphere-based targeted delivery of cytosolic proteins to induce protective immune responses against experimental murine cryptococcosis.     

Ferritin protein cage nanoparticles as versatile antigen delivery nanoplatforms for dendritic cell (DC)-based vaccine development

We utilized ferritin protein cage nanoparticles (FPCN) as antigen delivery nanoplatforms for DC-based vaccine development and investigated DC-mediated antigen-specific immune responses. Antigenic peptides, OT-1 (SIINFEKL) or OT-2 (ISQAVHAAHAEINEAGR) which are derived from ovalbumin, were genetically introduced either onto the exterior surface or into the interior cavity of FPCN. FPCN carrying antigenic peptides (OT-1-FPCN and OT-2-FPCN) were effectively delivered to DCs and processed within endosomes. Delivered antigenic peptides, OT-1 or OT-2, to DCs successfully induced antigen-specific CD8⁺ or CD4⁺ T cell proliferations both in vitro and in vivo. Naïve mice immunized with OT-1-FPCN efficiently differentiated OT-1 specific CD8⁺ T cells into functional effector cytotoxic T cells resulting in selective killing of antigen-specific target cells. Effective differentiation of proliferated OT-2 specific CD4⁺ T cells into functional CD4⁺ Th1 and Th2 cells was confirmed with the productions of IFN-γ/IL-2 and IL-10/IL-13 cytokines, respectively.From the Clinical EditorIn this study, the authors utilized ferritin protein cage nanoparticles as antigen delivery nanoplatforms for dendritic cell-based vaccine development and investigated DC-mediated antigen-specific immune responses using strong model antigens derived from ovalbumin, suggesting potential future clinical applicability of this or similar techniques.     

Biodegradable Nanoparticle Mediated Antigen Delivery to Human Cord Blood Derived Dendritic Cells for Induction of Primary T Cell Responses

Dendritic cells (DCs) in the peripheral tissues act as sentinels of the immune system. They detect and capture pathogens entering the body and present their antigens to T cells to trigger responses directed towards elimination of the pathogen. The induction of peripheral tolerance against self and certain foreign antigens is also believed to be mediated through DCs. The outcome of any immune response is largely controlled by the microenvironment of antigen capture, processing and presentation by DCs. The “context” of antigen delivery to DCs will directly influence the microenvironment of antigen presentation and hence the regulation of immune responses. We report here preliminary investigations describing the formulation of a pharmaceutically acceptable, biodegradable, and strategic nanoparticulate delivery system, and its application for efficient antigen loading of DCs to achieve antigen specific T cell activation. “Pathogen-mimicking” nanoparticles capable of interacting with DCs were fabricated by incorporating monophosphoryl lipid A (MPLA; toll-like receptor (TLR) 4 ligand) or CpG ODN (seq #2006; TLR9 ligand) in biodegradable copolymer, poly(_d,_l-lactic-co-glycolic acid) (PLGA). The uptake of PLGA nanoparticles by human umbilical cord blood derived DCs (DCs propagated from CD34₊ progenitors) was conclusively demonstrated by scanning electron microscopy (SEM), fluorescence activated cell sorting (FACS) and confocal laser scanning microscopy (CLSM). Cell phenotype at day 12 of cultures was determined as immature DC using specific cell surface markers, i.e. CD11c_hi (～90%), MHC-II₊ (～70%), CD86_low (～20%), CD83_low (～5%), CD80₊ (～40%), CD40₊ (～40%), and CCR7_low (～5%). Tetanus toxoid (TT), a model antigen, was encapsulated in nanoparticles along with an immunomodulator, i.e. either MPLA or CpG ODN. DCs pulsed with various antigen formulations were co-cultured with autologous naïve T cells at various cell ratios (DC: T cells were 1:5–20). The DCs pulsed with TT and MPLA together in nanoparticles induced significantly higher T cell proliferation (P<0.05) as compared to when DCs pulsed with TT and MPLA in solution were employed. A similar trend was observed when CpG ODN was used instead of MPLA in the TT nanoparticles. This strategy of antigen delivery to DCs was then tested with a cancer vaccine candidate, a MUC1 lipopeptide. The T cell proliferation observed in the presence of nanoparticulate MUC1 and MPLA pulsed-DCs was much higher than DCs pulsed with soluble antigen (P<0.0005). These results indicate that PLGA nanoparticles mimicking certain features of pathogens are efficient delivery systems for targeting vaccine antigens to DCs and activation of potent T cell responses.     

RETRACTED: Pierisformoside B exhibits neuroprotective and anti-inflammatory effects in murine hippocampal and microglial cells via the HO-1/Nrf2-mediated pathway

Ideal potential vaccine adjuvants to stimulate a Th1 immune response are urgently needed to control intracellular infections in clinical applications. Telocinobufagin (TBG), an active component of Venenum bufonis, exhibits immunomodulatory activity. Therefore, we investigated whether TBG enhances the Th1 immune response to ovalbumin (OVA) and formalin-inactivated Salmonella typhimurium (FIST) in mice. TBG augmented serum OVA- and FIST-specific IgG and IgG2a and the production of IFNγ by antigen-restimulated splenocytes. TBG also dramatically enhanced splenocyte proliferative responses to concanavalin A, lipopolysaccharide, and OVA and substantially increased T-bet mRNA levels and the CD3⁺/CD3⁺CD4⁺/CD3⁺CD8⁺ phenotype in splenocytes from OVA-immunized mice. In in vivo protection studies, TBG significantly decreased the bacterial burdens in the spleen and prolonged the survival time of FIST-immunized mice challenged with live S. typhimurium. In vivo neutralization of IFNγ with anti-IFNγ mAbs led to a significant reduction in FIST-specific IgG2a and IFNγ levels and in anti-Salmonella effect in TBG/FIST-immunized mice. In conclusion, these results suggest that TBG enhances a Th1 immune response to control intracellular infections.     

Triptolide regulates T cell-mediated immunity via induction of CD11c dendritic cell differentiation

Triptolide(TPT) isolated from one of the Chinese herbs, Tripterygium wilfordii Hook. F. (TWHF), are known to have a variety of immunomodulatory activities. This study was performed to investigate the effect of TPT on the differentiation of splenic DCs and its influence on T cell-mediated immunity regarding to DC subsets CD11c^lowI-a/e^lowCD45RB⁺(CD11c^low DCs) and CD11c^highI-a/e^highCD45RB^- (CD11c^high DCs) in male C57BL/6 mice spleens in vitro. The percentage of CD11c^low DCs was significantly increased after treatment with TPT compared to their counterparts (CD11c^high DCs). It was found that unlike the gradually decreasing interleukin (IL)-12 secretion of CD11c^high DCs induced by TPT, CD11c^low DCs showed a obvious dose-dependent response between the increasing of IL-10 production and TPT stimulation. After treatment with anti-IL-12R or anti-IL-10 monoclonal antibody in CD4⁺ T cells + CD11c^high DCs or CD11c^low DCs mixed lymphocyte reaction, the induction of these DCs on T cells was inhibited dramatically. These data demonstrated that TPT might induce the differentiation of splenic DCs to CD11c^low DCs followed by shifting of Th1 to Th2 with enhancement of T lymphocyte immune function in vitro.     

Enhanced humoral and CD8 + T cell immunity in mice vaccinated by DNA vaccine against human respiratory syncytial virus through targeting the encoded F protein to dendritic cells

Human respiratory syncytial virus (RSV) is the most important cause of serious lower respiratory tract infection in infants, the elderly, and the immunocompromised population. There is no licensed vaccine against RSV until now. It has been reported that targeting antigen to DEC205, a phagocytosis receptor on dendritic cells (DCs), could induce enhanced CD4 + and CD8 + T cell responses in mice. To develop RSV DNA vaccine and target the encoded antigen protein to DCs, the ectodomain of fusion glycoprotein (sF, amino acids: 23–524) of RSV was fused with anti-DEC205 single-chain Fv fragment (scDEC) and designated scDECF. Following successful expression from the recombinant plasmid of pVAX1/scDECF, the recombinant protein of scDECF was found capable of specifically binding to DEC205 receptor on CHOmDEC205 cells, and facilitating uptake of RSV F by DC2.4 cells in vitro. Furthermore, the higher levels of RSV-specific IgG antibody responses and neutralization antibody titers, as well as RSV F-specific CD8 + T cell responses were induced in mice immunized intramuscularly by pVAX1/scDECF than by the control plasmid of pVAX1/scISOF encoding sF protein fused with isotype matched control single-chain Fv fragment (scISO). Compared with pVAX1/scISOF, both the ratio of IgG2a/IgG1, > 1, and the enhanced IFN-γ cytokine were induced in mice following pVAX1/scDECF immunization, which exhibited a Th1 dominant response in pVAX1/scDECF vaccinated mice. Notably, the elevated efficiency of RSV F protein bound by DCs in vivo could also be observed in mice inoculated by pVAX1/scDECF. Collectively, these results demonstrate the enhanced IgG and CD8⁺ T cell immune responses have been induced successfully by DNA vaccine against RSV by targeting F antigen to DCs via the DEC205 receptor, and this DC-targeting vaccine strategy merits further investigation.     

Dendritic cells treated with a prostaglandin I2 analog,iloprost, promote antigen-specific regulatory T cell differentiation in mice

Iloprost, a stable prostaglandin I₂ (PGI₂) analog, can inhibit allergic inflammation in an ovalbumin (OVA)-induced asthma model via inhibition of airway dendritic cell (DC) function. However, the underlying mechanism of PGI₂ signaling-mediated immunosuppression remains unclear. This study explored whether iloprost-treated DCs can suppress inflammation by promoting antigen-specific regulatory T cell (Treg) differentiation through PGI₂-G-protein-coupled receptor (IP). We established an allergic lung inflammation model using a hydrogel biomaterial delivery system and observed that iloprost significantly suppressed OVA-induced Th2 lung inflammation and increased the frequency of OVA-specific Tregs in vivo. We further observed that iloprost-treated DCs displayed tolerogenic characteristics, including low inflammatory cytokine (IL-12, TNF-α, IL-6, IL-23) expression levels, high anti-inflammatory cytokine (IL-10) production, and a semimature phenotype. In addition, iloprost-treated DCs increased OVA-specific CD4⁺Foxp3⁺ T cell differentiation from naïve T cells in an IP-dependent pathway in vitro and in vivo. Blocking experiments showed that iloprost-treated DCs promoted Treg differentiation, at least in part, through programmed death ligand 1 (PD-L1), whereas iloprost-induced PD-L1 expression in DCs was through the IP receptor. Furthermore, iloprost treatment suppressed DC-mediated airway inflammation and increased the frequency of OVA-specific Tregs through PD-L1 in vivo. Taken together, these results show that PGI₂-IP signaling mediated by iloprost in DCs may lead to immune tolerance, suggesting that the PGI₂ analog has the potential to be applied therapeutically for tolerogenic DC immunotherapy in autoimmune diseases or allergic asthma.     

Vaccine potential of cytosolic proteins loaded fibrin microspheres of Cryptococcus neoformans in BALB/c mice

Cryptococcosis is a leading mycological cause of mortality among immunologically compromised individuals. In order to develop an effective vaccine against Cryptococcus neoformans, the cytosolic proteins (Cp) of the pathogen have been used as an antigen in combination with different formulations. In the present study, we have demonstrated that Cp encapsulated poly-lactide co-glycolide (PLGA) microsphere further co-encapsulated into the biocompatible fibrin cross-linked plasma beads (Fib-PLGA-Cp) mediated cytosolic delivery elicited strong immune response in the BALB/c mice. In contrast, other formulations of Cp failed to impart significant level of protection. The immune response, involved with Fib-PLGA-Cp protection, appear to interact with the target cells by both endocytosis as well as membrane fusion mode, thus helping in the activation of both CD4(+) and CD8(+) T-cells. Analysis of cytokine profiles in immunized animals revealed that the protective response was associated with the Th1/Th2 polarization in favor of type-1 cytokine [interferons (IFN)-γ and interleukin (IL)-2] cells. Furthermore, vaccination with Fib-PLGA-Cp elicited high immunoglobulin (Ig) G(l) and IgG(2a) isotype response; successfully cleared fungal burden in vital organs and also increased the survival rate of immunized animals. Altogether the present study is a clear indicative of the possible use of fibrin microsphere-based targeted delivery of cytosolic proteins to induce protective immune responses against experimental murine cryptococcosis.     

Dendritic cells transfected with indoleamine 2,3-dioxygenase gene suppressed acute rejection of cardiac allograft

BackgroundImmunomodulation by indoleamine 2,3-dioxygenase (IDO) has been documented in many studies yet its underlying mechanisms remain undefined, especially in solid organ transplantation. Recent research demonstrated that the active expression of IDO in dendritic cells (DCs) regulates immune reaction. This study assessed whether DCs transfected with IDO gene inhibit T cells responses and suppress cardiac allograft rejection.MethodsAdenovirus vector containing IDO gene was transfected into DCs to obtain IDO-positive DCs (IDO⁺ DCs). To evaluate the effect of IDO⁺ DCs on T cells in vitro, CD4⁺ T cell proliferation and apoptosis was assessed in mixed lymphocyte reactions and measured by flow cytometry, respectively. IDO⁺ DCs from C57BL/6 mice were injected into BALB/c recipients before heterotopic cardiac transplantation.ResultsSupernatant fluids from cultures of IDO⁺ DCs had decreased tryptophan and increased kynurenine levels, reflecting IDO activity. IDO⁺ DCs suppressed CD4⁺ T cell responses in vitro, as reflected by decreased proliferation and increased apoptosis. In the transplant model, IDO⁺ DCs prolonged survival and alleviated rejection of cardiac allograft in recipients injected with IDO⁺ DCs. In vivo, IDO⁺ DCs also significantly impaired CD4⁺ T cell responses promoting increased apoptosis and a Th2-dominant cytokine shift.ConclusionsIDO overexpression in DCs suppressed T cells alloresponses in vitro, and IDO⁺ DCs attenuated acute allograft rejection in vivo. Regulation of tryptophan catabolism by means of IDO overexpression in DCs may be a useful approach in cardiac transplantation and immunological tolerance.     

Activation of Antigen-Specific CD8<Superscript>+</Superscript> T Cells by Poly-DL-Lactide/Glycolide (PLGA) Nanoparticle-Primed Gr-1<Superscript>high</Superscript> Cells

Purpose

The aim of this study was to investigate the induction of antigen-specific T cell activation and cell cycle modulation by a poly-DL-lactide/glycolide (PLGA) nanoparticle (NP)-primed CD11b⁺Gr-1^high subset isolated from mouse bone marrow.

Methods

PLGA NPs containing the ovalbumin (OVA) antigen were prepared using the double emulsion and solvent evaporation method, and protein release rate and cell viability were determined. The Lin2^¯CD11b⁺Gr-1^highLy6c^low (Gr-1^high) subset was sorted from the bone marrow of C57BL/6 J mice by fluorescence-activated cell sorting (FACS) and co-cultured with OT-I CD8⁺ splenic T cells. Proliferation of OT-I CD8⁺ T cells was monitored, and cell cycles were determined by 5-bromo-2′-deoxyuridine (BrdU) labeling.

Results

Treatment of Gr-1^high cells with PLGA/OVA NPs upregulated expression of the SIINFEKL-H2K^b complex in the context of MHC I. Co-cultures of OT-I CD8⁺ T cells with the PLGA/OVA NP-primed Gr-1^high cells induced the proliferation of T cells in vitro and modulated cell division and morphology. Treatment of Gr-1^high cells with PLGA/OVA NPs also induced cell apoptosis and necrosis.

Conclusion

This study demonstrated the function of PLGA/OVA NPs in the activation of OT-I CD8⁺ T cells and the capability of cross-presentation via the Gr-1^high polymorphonuclear subset from mouse bone marrow.

     

Encapsulating immunostimulatory CpG oligonucleotides in listeriolysin O-liposomes promotes a Th1-type response and CTL activity

Immunostimulatory sequences (ISS) are short DNA sequences containing unmethylated CpG dimers that have multiple effects on the host immune system, including the ability to stimulate antigen-specific cytotoxic T lymphocytes (CTLs) and drive Th1-type immune responses. Listeriolysin O (LLO)-containing pH-sensitive liposomes have been shown to efficiently deliver macromolecules to the cytosol of APCs and efficiently stimulate CTLs. We hypothesized that encapsulating ISS-oligodeoxyribonucleotides (ODNs) in this delivery system would enhance the cell-mediated immune response and skew Th1-type responses in protein antigen-based vaccination utilizing LLO-liposomes. In vitro studies indicated that coencapsulation of ISS in LLO-liposomes engendered activation of the NF-κB pathway while maintaining the efficient cytosolic delivery of antigen mediated by the coencapsulated LLO. Antigen-specific CTL responses monitored by using the model antigen ovalbumin (OVA) in mice were enhanced when mice were immunized with OVA and ISS-ODN-containing LLO-liposomes compared with those immunized with OVA-containing LLO-liposomes. The enhanced immune responses were of the Th1-type as monitored by the robust OVA-specific IgG2a induction and the OVA CD8 peptide-stimulated IFN-γ secretion. Our study suggests that including ISS-ODN in LLO-containing pH-sensitive liposomes yields a vaccine delivery system that enhances the cell-mediated immune response and skews this response toward the Th1-type.     

Composition and Surface Charge of DNA-Loaded Microparticles Determine Maturation and Cytokine Secretion in Human Dendritic Cells

PURPOSE: Biodegradable microparticles prepared from poly(lactide) (PLA) and poly(lactide-co-glycolide) (PLGA) have been shown to be promising carrier systems for vaccine delivery. Here, we have investigated the capacity of different PLA and PLGA microparticle formulations to induce stimulation of human blood monocyte-derived dendritic cells (DCs). METHODS: Stimulation of human derived dendritic cells by plain microparticles were compared with microparticles loaded with plasmid DNA or double-stranded salmon DNA either by encapsulation or adsorption to the surface of cationic microparticles. Stimulation of DCs was monitored by the up-regulation of surface maturation markers CD83 and CD86 and the secretion of IL-12 and TNF-alpha. RESULTS: Slowly degrading PLA microparticles did not induce any detectable stimulation or activation of DCs. In contrast, fast degrading PLGA microparticles were able to influence DC maturation and cytokine secretion dependent on their surface charge. Anionic PLGA microparticles induced an up-regulation of CD83 and high TNF-alpha secretion, which was further enhanced up to the level of the potent stimulator lipopolysaccharide (LPS) when plasmid DNA was encapsulated. Moreover, the secretion of significant amounts of IL-12 was observed. Cationic PLGA microparticles induced an up-regulation of CD86 and moderate TNF-alpha secretion, but no IL-12 secretion, with no additional effects in the presence of plasmid DNA. CONCLUSIONS: The data suggest that the composition and charge of biodegradable DNA-loaded microparticles profoundly influences maturation and cytokine secretion in DCs. Thus, the individual formulation of microparticles used as a vaccine carrier system might considerably influence the profile of the immune response.     

Alcoholic extract of Cicer microphyllum augments Th1 immune response in normal and chronically stressed Swiss albino mice

Objective The purpose of this study was to observe the effect of an alcoholic extract of Cicer microphyllum (I³M/38/A001) (whole plant without seeds and flowers) on the immunological parameters of sheep red blood cell immunized normal and chronically stressed Swiss albino mice. Methods Estimation of T‐cell subsets (CD3⁺, CD4⁺/CD8⁺), CD80/CD86, CD28, CD 69, costimulatory molecules and Th1/Th2 cytokines was carried out using a flow cytometer. This was followed by study of the delayed type hypersensitivity response, in‐vitro lymphocyte proliferation assay and measurement of Th1/Th2 cytokines in isolated peripheral blood mononuclear cells by flow cytometry. An enzyme immune assay was used to analyse corticosterone levels in the serum of chronically stressed animals. Key findings We found that oral administration of I³M/38/A001once daily at the graded doses of 6.25, 12.5, 25, 50, 100 and 200 mg/kg p.o. enhanced the proliferation and differentiation of T lymphocytes in sheep red blood cell normal and chronically stressed mice, as shown by flow cytometric analysis. The extract selectively induced type 1 immunity: it guided enhanced expression of Th1 cytokines, interferon‐γ and interleukin‐2, while no significant change in interleukin‐4 (Th2 cytokine) levels was observed. Confirmation of Th1 polarization was confirmed by the augmented levels of interferon‐γ and interleukin‐2 in isolated peripheral blood mononuclear cells. A significant suppression of raised corticosterone levels was also observed in stressed animals, which suggests the extract's normalizing effect on the hypothalamic–pituitary–adrenal axis. Co‐stimulatory molecules, CD28, CD69, CD80 and CD86, which are important secondary signals for the activation of the immune system, elicited significant expression in I³M/38/A001 treated mice. Conclusion Our studies show the immune potentiating and immune recuperative effect of the test drug in sheep red blood cell‐immunized normal and chronically stressed mice.     

Enhancement of Nasal HIV Vaccination with Adenoviral Vector-Based Nanocomplexes Using Mucoadhesive and DC-Targeting Adjuvants

Purpose

To investigate the vaccine effect of a replication-defective recombinant adenovirus 5 (rAd5)-based nanocomplex with chitooligosaccharides (Oligo) and mannosylated polyethyleneimine-triethyleneglycol (mPEI) as adjuvants for human immunodeficiency virus (HIV) infection.

Methods

Physical characteristics were determined through detecting the size, zeta potential and morphology of Oligo-mPEI-rAd5 nanocomplex, and in vitro vaccine uptake and transduction efficiency were estimated. Nanocomplexes were then administered intranasally to Balb/c mice to evaluate in vivo rAd5 residence in nasal cavity and HIVgag-specific immune responses using cytotoxic T lymphocyte (CTL), intracellular cytokine staining (ICS) and ELISA assay.

Results

The mucoadhesivity of Oligo prolonged nasal residence time, while the dendritic cell (DC) specificity of mPEI improved vaccine uptake. These two adjuvants jointly enhanced transduction efficiency of rAd5. Oligo-mPEI-rAd5 nanocomplex elicited potent HIVgag-specific CTL response and increased IFN-γ positive CD8⁺T and IL-4 positive CD4⁺T cells, indicating high cellular immune responses. This vaccine candidate also led to strong humoral immune responses (IgG/IgG1/IgG2a) with balanced Th1/Th2 CD4⁺T cell activity. Moreover, mice nasally immunized with Oligo-mPEI-rAd5 showed higher levels of SIgA in nasal washes than did mice immunized with rAd5.

Conclusions

Intranasal delivery of Oligo-mPEI-rAd5 with a prime-boost regimen is a potential immunization for HIV infection, inducing HIVgag-specific cellular, humoral and mucosal immune responses.     

Minireview: Divergent roles of α7 nicotinic acetylcholine receptors expressed on antigen-presenting cells and CD4+ T cells in the regulation of T cell differentiation

α7 nAChRs expressed on immune cells regulate antigen-specific antibody and proinflammatory cytokine production. Using spleen cells from ovalbumin (OVA)-specific T cell receptor transgenic DO11.10 mice and the α7 nAChR agonist GTS-21, investigation of (1) antigen processing-dependent and (2) -independent, antigen presenting cell (APC)-dependent, naïve CD4⁺ T cell differentiation, as well as (3) non-specific APC-independent, anti-CD3/CD28 mAbs-induced CD4⁺ T cell differentiation, revealed the differential roles of α7 nAChRs expressed on T cells and APCs in the regulation of CD4⁺ T cell differentiation. GTS-21 suppressed OVA-induced antigen processing- and APC-dependent differentiation into regulatory T cells (Tregs) and effector T cells (Th1, Th2 and Th17) without affecting OVA uptake or cell viability. By contrast, GTS-21 upregulated OVA peptide-induced antigen processing-independent T cell differentiation into all lineages. During anti-CD3/CD28 mAbs-induced T cell differentiation in the presence of polarizing cytokines, GTS-21 promoted wild-type T cell differentiation into all lineages, but did not affect α7 nAChR-deficient T cell differentiation. These results demonstrate (1) that α7 nAChRs on APCs downregulate T cell differentiation by inhibiting antigen processing and thereby interfering with antigen presentation; and (2) that α7 nAChRs on T cells upregulate differentiation into Tregs and effector T cells. Thus, the divergent roles of α7 nAChRs on APCs and T cells likely regulate the intensity of immune responses. These findings suggest the possibility of using α7 nAChR agonists to harvest greater numbers of Tregs and Th1 and Th2 cells for adoptive immune therapies for treatment of autoimmune diseases and cancers.     

Maturation of dendritic cells by maitake α-glucan enhances anti-cancer effect of dendritic cell vaccination

Dendritic cells (DCs) play a primary role in antigen presentation to CD4⁺ and CD8⁺ T cells and induce acquired immune response against cancer cells. Therefore, determining positive modulators of DC activation to improve therapeutic approaches for cancer immunotherapy is greatly needed. In this study, we investigated the effect of maitake α-glucan YM-2A, isolated from Grifola frondosa, on the maturation and function of DCs and its adjuvant effect on a tumor-associated antigen (TAA)-loaded DC vaccine against murine tumor. We showed that YM-2A induced morphological changes and increased cell-surface maturation markers and cytokine production in DCs. In a mixed lymphocyte reactions assay, YM-2A-treated DCs increased proliferation and production of IFN-γ by allogeneic CD4⁺ and CD8⁺ T cells. These results indicate that YM-2A phenotypically and functionally activates DCs. Furthermore, YM-2A-treated TAA-loaded DC vaccine significantly reduced tumor growth and improved survival in two murine tumor models, CT-26 tumor-bearing BALB/c mice and B16 melanoma-bearing C57BL/6 mice. YM-2A-treated TAA-loaded DC vaccine increased splenic IFN-γ producing CD4⁺ and CD8⁺ T cells in CT-26 tumor-bearing BALB/c mice. Antibody neutralization studies indicated that YM-2A-induced DC maturation is mediated, in part, by the Dectin-1-dependent pathway. Overall, YM-2A-treatment with a TAA-loaded DC vaccine could be an excellent candidate for immunotherapy against cancer.     

Polyactin A is a novel and potent immunological adjuvant for peptide-based cancer vaccine

Synthetic peptide cancer vaccines are poorly immunogenic sub-unit vaccines and thus essentially need adjuvants in their formulations to increase the efficacy by enhancing the peptide-specific immune response. However, aluminum-based compounds are almost dependent for clinical use at present. Therefore, the increasing use of peptide-based vaccines makes the need for novel and potent adjuvants. Polyactin A (PAA) has been used for the clinical treatment of impaired immunity in China for over 30 years. To figure out the adjuvant effects of PAA for E75 peptide breast cancer vaccine (Her2 p369-p377), the generation of mature dendritic cells (DCs) from peripheral blood monocytes (PBMCs) cultured with PAA, IL-4 and TNF-α was assessed by morphologic features and the expressions of special surface markers using flow cytometry. Then the functional features of PBMCs-derived DCs cultured with PAA, IL-4 and TNF-α were investigated by inducing E75-specific cytotoxicity. Finally, C57BL/6-Tg(HLA-A2.1)1Enge/J transgenic mice were immunized with E75 and various amounts of PAA, and splenic lymphocyte proliferation and the IFN-γ level were determined. The results showed that PAA, just like granulocyte-macrophage colony stimulating factor, with IL-4 and TNF-α efficiently induced mature DCs from PBMCs, and these DCs could trigger a potent E75 peptide-specific CD8⁺ T-cell response in vitro. Immunization with E75 and PAA significantly increased positive rates of CD4⁺ and CD8⁺ T lymphocytes, and enhanced splenocytes proliferation and levels of IFN-γ in splenocytes when induced by E75. Our results indicated that PAA could efficiently induce E75-specific immunologic responses in vitro and in vivo. Therefore, PAA possesses potent adjuvant effect on peptide-based cancer vaccine. Our study provides a safe, effective and novel adjuvant for clinical use.     

Biodegradable nanoparticle mediated antigen delivery to human cord blood derived dendritic cells for induction of primary T cell responses

Dendritic cells (DCs) in the peripheral tissues act as sentinels of the immune system. They detect and capture pathogens entering the body and present their antigens to T cells to trigger responses directed towards elimination of the pathogen. The induction of peripheral tolerance against self and certain foreign antigens is also believed to be mediated through DCs. The outcome of any immune response is largely controlled by the microenvironment of antigen capture, processing and presentation by DCs. The "context" of antigen delivery to DCs will directly influence the microenvironment of antigen presentation and hence the regulation of immune responses. We report here preliminary investigations describing the formulation of a pharmaceutically acceptable, biodegradable, and strategic nanoparticulate delivery system, and its application for efficient antigen loading of DCs to achieve antigen specific T cell activation. "Pathogen-mimicking" nanoparticles capable of interacting with DCs were fabricated by incorporating monophosphoryl lipid A (MPLA; toll-like receptor (TLR) 4 ligand) or CpG ODN (seq #2006; TLR9 ligand) in biodegradable copolymer, poly(D,L,-lactic-co-glycolic acid) (PLGA). The uptake of PLGA nanoparticles by human umbilical cord blood derived DCs (DCs propagated from CD34 progenitors) was conclusively demonstrated by scanning electron microscopy (SEM), fluorescence activated cell sorting (FACS) and confocal laser scanning microscopy (CLSM). Cell phenotype at day 12 of cultures was determined as immature DC using specific cell surface markers, i.e. CD11c (approximately 90%), MHC-II (approximately 70%), CD86 (approximately 20%), CD83 (approximately 5%), CD80 (approximately 40%), CD40 (approximately 40%), and CCR7 (approximately 5%). Tetanus toxoid (TT), a model antigen, was encapsulated in nanoparticles along with an immunomodulator, i.e. either MPLA or CpG ODN. DCs pulsed with various antigen formulations were co-cultured with autologous na?ve T cells at various cell ratios (DC: T cells were 1:5-20). The DCs pulsed with TT and MPLA together in nanoparticles induced significantly higher T cell proliferation (P<0.05) as compared to when DCs pulsed with TT and MPLA in solution were employed. A similar trend was observed when CpG ODN was used instead of MPLA in the TT nanoparticles. This strategy of antigen delivery to DCs was then tested with a cancer vaccine candidate, a MUC1 lipopeptide. The T cell proliferation observed in the presence of nanoparticulate MUC1 and MPLA pulsed-DCs was much higher than DCs pulsed with soluble antigen (P<0.0005). These results indicate that PLGA nanoparticles mimicking certain features of pathogens are efficient delivery systems for targeting vaccine antigens to DCs and activation of potent T cell responses.     

Nasal delivery of H5N1 avian influenza vaccine formulated with GenJet™ or in vivo-jetPEI® induces enhanced serological,cellular and protective immune responses

Avian influenza virus infection is a serious public health threat and preventive vaccination is the most cost-effective public health intervention strategy. Unfortunately, currently available unadjuvanted avian influenza vaccines are poorly immunogenic and alternative vaccine formulations and delivery strategies are in urgent need to reduce the high risk of avian influenza pandemics. Cationic polymers have been widely used as vectors for gene delivery in vitro and in vivo. In this study, we formulated H5N1 influenza vaccines with GenJet? or in vivo-jetPEI^®, and showed that these formulations significantly enhanced the immunogenicity of H5N1 vaccines and conferred protective immunity in a mouse model. Detailed analyses of adaptive immune responses revealed that both formulations induced mixed T_H1/T_H2 antigen-specific CD4 T-cell responses, antigen-specific cytotoxic CD8 T-cell and memory B-cell responses. Our findings suggest that cationic polymers merit future development as potential adjuvants for mucosal delivery of poorly immunogenic vaccines.