Intranasal vaccination with influenza HA/GO-PEI nanoparticles provides immune protection against homo- and heterologous strains.

Intranasal (i.n.) immunization is a promising vaccination route for infectious respiratory diseases such as influenza. Recombinant protein vaccines can overcome the safety concerns and long production phase of virus-based influenza vaccines. However, soluble protein vaccines are poorly immunogenic if administered by an i.n. route. Here, we report that polyethyleneimine-functionalized graphene oxide nanoparticles (GP nanoparticles) showed high antigen-loading capacities and superior immunoenhancing properties. Via a facile electrostatic adsorption approach, influenza hemagglutinin (HA) was incorporated into GP nanoparticles and maintained structural integrity and antigenicity. The resulting GP nanoparticles enhanced antigen internalization and promoted inflammatory cytokine production and JAWS II dendritic cell maturation. Compared with soluble HA, GP nanoparticle formulations induced significantly enhanced and cross-reactive immune responses at both systemic sites and mucosal surfaces in mice after i.n. immunization. In the absence of any additional adjuvant, the GP nanoparticle significantly boosted antigen-specific humoral and cellular immune responses, comparable to the acknowledged potent mucosal immunomodulator CpG. The robust immune responses conferred immune protection against challenges by homologous and heterologous viruses. Additionally, the solid self-adjuvant effect of GP nanoparticles may mask the role of CpG when coincorporated. In the absence of currently approved mucosal adjuvants, GP nanoparticles can be developed into potent i.n. influenza vaccines, providing broad protection. With versatility and flexibility, the GP nanoplatform can be easily adapted for constructing mucosal vaccines for different respiratory pathogens.

Optimization, Production, and Characterization of a CpG-Oligonucleotide-Ficoll Conjugate Nanoparticle Adjuvant for Enhanced Immunogenicity of Anthrax Protective Antigen.

We have synthesized and characterized a novel phosphorothioate CpG oligodeoxynucleotide (CpG ODN)-Ficoll conjugated nanoparticulate adjuvant, termed DV230-Ficoll. This adjuvant was constructed from an amine-functionalized-Ficoll, a heterobifunctional linker (succinimidyl-[(N-maleimidopropionamido)-hexaethylene glycol] ester) and the CpG-ODN DV230. Herein, we describe the evaluation of the purity and reactivity of linkers of different lengths for CpG-ODN-Ficoll conjugation, optimization of linker coupling, and conjugation of thiol-functionalized CpG to maleimide-functionalized Ficoll and process scale-up. Physicochemical characterization of independently produced lots of DV230-Ficoll reveal a bioconjugate with a particle size of approximately 50 nm and covalent attachment of more than 100 molecules of CpG per Ficoll. Solutions of purified DV230-Ficoll were stable for at least 12 months at frozen and refrigerated temperatures and stability was further enhanced in lyophilized form. Compared to nonconjugated monomeric DV230, the DV230-Ficoll conjugate demonstrated improved in vitro potency for induction of IFN-α from human peripheral blood mononuclear cells and induced higher titer neutralizing antibody responses against coadministered anthrax recombinant protective antigen in mice. The processes described here establish a reproducible and robust process for the synthesis of a novel, size-controlled, and stable CpG-ODN nanoparticle adjuvant suitable for manufacture and use in vaccines.

Reduction Sensitive PEG Hydrogels for Codelivery of Antigen and Adjuvant To Induce Potent CTLs.

Educating our immune system via vaccination is an attractive approach to combat infectious diseases. Eliciting antigen specific cytotoxic T cells (CTLs), CD8+ effector T cells, is essential in controlling intracellular infectious diseases such as influenza (Flu), tuberculosis (TB), hepatitis, and HIV/AIDS, as well as tumors. However, vaccination utilizing subunit peptides to elicit a potent CD8+ T cell response with antigenic peptides is typically ineffective due to poor immunogenicity. Here we have engineered a reduction sensitive nanoparticle (NP) based subunit vaccine for intracellular delivery of an antigenic peptide and immunostimulatory adjuvant. We have co-conjugated an antigenic peptide (ovalbumin-derived CTL epitope [OVA257-264: SIINFEKL]) and an immunostimulatory adjuvant (CpG ODNs, TLR9 agonist) to PEG hydrogel NPs via a reduction sensitive linker. Bone-marrow derived dendritic cells (BMDCs) treated with the SIINFEKL conjugated NPs efficiently cross-presented the antigenic peptide via MHC-I surface receptor and induced proliferation of OT-I T cells. CpG ODN-conjugated NPs induced maturation of BMDCs as evidenced by the overexpression of CD80 and CD40 costimulatory receptors. Moreover, codelivery of NP conjugated SIINFEKL and CpG ODN significantly increased the frequency of IFN-γ producing CD8+ effector T cells in mice (∼6-fold improvement over soluble antigen and adjuvant). Furthermore, the NP subunit vaccine-induced effector T cells were able to kill up to 90% of the adoptively transferred antigenic peptide-loaded target cell. These results demonstrate that the reduction sensitive NP subunit vaccine elicits a potent CTL response and provide compelling evidence that this approach could be utilized to engineer particulate vaccines to deliver tumor or pathogen associated antigenic peptides to harness the immune system to fight against cancer and infectious diseases.

Incorporation of CpG into a liposomal vaccine formulation increases the maturation of antigen-loaded dendritic cells and monocytes to improve local and systemic immunity.

Liposomal vaccine formulations incorporating stimulants that target innate immune receptors have been shown to significantly increase vaccine immunity. Following vaccination, innate cell populations respond to immune stimuli, phagocytose and process Ag, and migrate from the injection site, via the afferent lymphatic vessels, into the local lymph node. In this study, the signals received in the periphery promote and sculpt the adaptive immune response. Effector lymphocytes then leave the lymph node via the efferent lymphatic vessel to perform their systemic function. We have directly cannulated the ovine lymphatic vessels to detail the in vivo innate and adaptive immune responses occurring in the local draining lymphatic network following vaccination with a liposome-based delivery system incorporating CpG. We show that CpG induces the rapid recruitment of neutrophils, enhances dendritic cell-associated Ag transport, and influences the maturation of innate cells entering the afferent lymph. This translated into an extended period of lymph node shutdown, the induction of IFN-γ-positive T cells, and enhanced production of Ag-specific Abs. Taken together, the results of this study quantify the real-time in vivo kinetics of the immune response in a large animal model after vaccination of a dose comparable to that administered to humans. This study details enhancement of numerous immune mechanisms that provide an explanation for the immunogenic function of CpG when employed as an adjuvant within vaccines.

Polyethyleneimine is a potent systemic adjuvant for glycoprotein antigens.

Polyethyleneimine (PEI) is an organic polycation used extensively as a gene and DNA vaccine delivery reagent. Although the DNA targeting activity of PEI is well documented, its immune activating activity is not. We recently reported that PEI has robust mucosal adjuvanticity when administered intranasally with glycoprotein antigens. Here, we show that PEI has strong immune activating activity after systemic delivery. PEI administered subcutaneously with viral glycoprotein (HIV-1 gp140) enhanced antigen-specific serum IgG production in the context of mixed Th1/Th2-type immunity. PEI elicited higher titers of both antigen binding and neutralizing antibodies than alum in mice and rabbits and induced an increased proportion of antibodies reactive with native antigen. In an intraperitoneal model, PEI recruited neutrophils followed by monocytes to the site of administration and enhanced antigen uptake by antigen-presenting cells. The Th bias was modulated by PEI activation of the Nlrp3 inflammasome; however its global adjuvanticity was unchanged in Nlrp3-deficient mice. When coformulated with CpG oligodeoxynucleotides, PEI adjuvant potency was synergistically increased and biased toward a Th1-type immune profile. Taken together, these data support the use of PEI as a versatile systemic adjuvant platform with particular utility for induction of secondary structure-reactive antibodies against glycoprotein antigens.

Co-encapsulated CpG oligodeoxynucleotides and ovalbumin in PLGA microparticles; an in vitro and in vivo study.

PURPOSE: The objective of this work was to evaluate the effect in the immune response produced by CpG oligodeoxynucleotides (ODN) co-encapsulated with the antigen ovalbumin (OVA) within poly(lactic-co-glycolic) acid (PLGA) 502 and 752 microparticles (MP). METHODS: MP were prepared by blending 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) with PLGA and Total Recirculation One Machine System (TROMS) technology and contained OVA along with CpG sequences associated to DOTAP. After confirming the integrity of both encapsulated molecules, BALB/c mice were immunized with the resulting MP and OVA-specific antibodies and cytokine production were assessed in order to determine the immunological profile induced in mice. RESULTS: One m near non-charged MP co-encapsulated very efficiently both OVA and CpG ODN. The release of both OVA and CpG was slow and incomplete irrespective of polymer. The results of the immune response induced in BALB/c mice indicated that, depending on the PLGA polymer used, co-encapsulation did not improve the immunogenicity of the antigen, compared either with the simply co-administration of both antigen and CpG, or with the microencapsulated antigen. Thus, mice immunized with OVA associated to PLGA 756 displayed an IgG2a characterized response which was biased to an IgG1 profile in case of CpG co-encapsulation. On the contrary, the co-encapsulation of CpG with OVA into PLGA 502 significantly improved the isotype shifting in comparison with the one showed by mice immunized with OVA loaded PLGA 502. CONCLUSION: This study underlines the importance of MP characteristics to fully exploit simultaneous antigen and CpG ODN particulate delivery as effective vaccine construct.This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Properties regulating the nature of the plasmacytoid dendritic cell response to Toll-like receptor 9 activation.

Human plasmacytoid dendritic cells (PDCs) can produce interferon (IFN)-alpha and/or mature and participate in the adaptive immune response. Three classes of CpG oligonucleotide ligands for Toll-like receptor (TLR)9 can be distinguished by different sequence motifs and different abilities to stimulate IFN-alpha production and maturation of PDCs. We show that the nature of the PDC response is determined by the higher order structure and endosomal location of the CpG oligonucleotide. Activation of TLR9 by the multimeric CpG-A occurs in transferrin receptor (TfR)-positive endosomes and leads exclusively to IFN-alpha production, whereas monomeric CpG-B oligonucleotides localize to lysosome-associated membrane protein (LAMP)-1-positive endosomes and promote maturation of PDCs. However, CpG-B, when complexed into microparticles, localizes in TfR-positive endosomes and induces IFN-alpha from PDCs, whereas monomeric forms of CpG-A localize to LAMP-1-positive endosomes accompanied by the loss of IFN-alpha production and a gain in PDC maturation activity. CpG-C sequences, which induce both IFN-alpha and maturation of PDCs, are distributed in both type of endosomes. Encapsulation of CpG-C in liposomes stable above pH 5.75 completely abrogated the IFN-alpha response while increasing PDC maturation. This establishes that the primary determinant of TLR9 signaling is not valency but endosomal location and demonstrates a strict compartmentalization of the biological response to TLR9 activation in PDCs.

TLR9 agonist CpG enhances protective nasal HSP60 peptide vaccine efficacy in experimental autoimmune arthritis.

OBJECTIVES: Peptide-based immune tolerance induction is considered an attractive treatment option for autoimmune diseases. The authors have developed a novel method that can enhance the induction of protective peptide-specific T-cell responses, using a rat arthritis model. The authors focused on the Toll-like receptor 9 ligand CpG, which was shown to stimulate regulatory T-cell proliferation when added to plasmacytoid dendritic cells (pDC) using in-vitro cultures. METHODS: The peptide used is a heat shock protein 60 epitope (p1) that elicits tolerogenic peptide-specific immune responses in human arthritis patients and was recently shown to have protective capacity as a bystander antigen in the rat adjuvant arthritis model. Rats were treated with three nasal doses of p1, CpG or a combination of p1 and CpG. Antigen-presenting cells were studied in nose-draining lymph nodes (mandibular lymph nodes; MLN) after nasal treatment, and T-cell responses were analysed in joint-draining lymph nodes after arthritis induction. RESULTS: Nasal co-administration of p1/CpG significantly augmented the arthritis-protective effect of p1, while CpG treatment alone did not. Co-treatment of p1/CpG increased both the number and activation status of pDC in draining MLN, which was accompanied by amplified p1-specific T-cell proliferation and interleukin (IL)-10 production. During early arthritis, p1-specific IL-10 production was identified at the site of inflammation. P1 and p1/CpG-treated rats showed a greater amount of CD4+FoxP3+ regulatory T cells in the joint-draining lymph nodes, which correlated with lower arthritis scores. CONCLUSIONS: These clinical and immunological data suggest the use of CpG as a potent adjuvant for mucosal peptide-specific immune therapy in arthritis.

Comparison of liposome-polycation-DNA(LPD) and monophosphoryl lipid A(MPL) adjuvant formulations in BALB/c mice models.

It is of fundamental importance to use an appropriate adjuvant to generate a potent immune response for immunotherapy. In this study, we had a comparative investigation on the effectiveness of two adjuvant formulations, liposome-polycation-DNA (LPD) and monophosphoryl lipid A(MPL) in combination with a truncated peptide of bFGF(tbFGF) as antigen. LPD/tbFGF induced continuously increasing antibodies expression during the whole immunization period. In contrast, the level of antibodies was variable in MPL/tbFGF-immunized mice, MPL/tbFGF elicited potent antibodies response in the early-phase of immunization (during the first 3 immunizations), but the later immunizations did not produce a significant increase in the level of antibodies. Evaluation of IFN-γ and IL-4 responses revealed that both LPD/tbFGF and MPL/tbFGF demonstrated generation of higher level of IFN-γ, whereas no significant increase in IL-4 levels was detected in the two groups. In addition, histological analysis exhibited obvious germinal centers in the spleen tissues of LPD/tbFGF mice. The data suggested that LPD would be a promising long-effective adjuvant due to its potent and persistent immunostimulation and MPL could play an appropriate role in short-acting immunization.

The immunostimulatory activity of unmethylated and methylated CpG oligodeoxynucleotide is dependent on their ability to colocalize with TLR9 in late endosomes.

TLR9 recognizes CpG motifs present in pathogenic DNA and triggers potent immune responses. It is generally accepted that TLR9 distinguishes pathogenic DNA based, in part, on methylation status, where TLR9 binds unmethylated but not methylated CpG. However, we showed that methylated CpG induces potent TLR9-mediated responses when delivered in lipid nanoparticles. In this article, we report that methylation dictates the ability of free CpG DNA to colocalize with TLR9 in late endosomes. However, when delivered in lipid nanoparticles, CpG DNA and TLR9 colocalize, regardless of methylation status. Therefore, it is proposed that the ability of immune cells to distinguish unmethylated pathogenic from methylated mammalian DNA is controlled by a mechanism that regulates TLR9 mobilization and colocalization rather than a differential binding affinity.

Production of antibodies with peptide-CpG-DNA-liposome complex without carriers.

BACKGROUND: The screening of peptide-based epitopes has been studied extensively for the purpose of developing therapeutic antibodies and prophylactic vaccines that can be potentially useful for treating cancer and infectious diseases such as influenza virus, malaria, hepatitis B, and HIV. To improve the efficacy of antibody production by epitope-based immunization, researchers evaluated liposomes as a means of delivering vaccines; they also formulated adjuvants such as flagella and CpG-DNA to enhance the magnitude of immune responses. Here, we provide a potent method for peptide-based epitope screening and antibody production without conventional carriers. RESULTS: We present that a particular form of natural phosphodiester bond CpG-DNA encapsulated in a specific liposome complex (Lipoplex(O)) induces potent immunomodulatory activity in humans as well as in mice. Additionally, Lipoplex(O) enhances the production of IgG2a specific to antigenic protein in mice. Most importantly, immunization of mice with several peptides co-encapsulated with Lipoplex(O) without carriers significantly induces each peptide-specific IgG2a production in a TLR9-dependent manner. A peptide-specific monoclonal antibody produced against hepatocellular carcinoma-associated antigen has functional effects on the cancer cells. CONCLUSIONS: Our overall results show that Lipoplex(O) is a potent adjuvant and that complexes of peptide and Lipoplex(O) are extremely useful for B cell epitope screening and antibody production without carriers. Therefore, our strategy may be promptly used for the development of therapeutic antibodies by rapid screening of potent B cell epitopes.

Adjuvants and delivery systems in veterinary vaccinology: current state and future developments.

Modern adjuvants should induce strong and balanced immune responses, and it is often desirable to induce specific types of immunity. As an example, efficient Th1-immunity-inducing adjuvants are highly in demand. Such adjuvants promote good cell-mediated immunity against subunit vaccines that have low immunogenicity themselves. The development of such adjuvants may take advantage of the increased knowledge of the molecular mechanisms and factors controlling these responses. However, knowledge of such molecular details of immune mechanisms is relatively scarce for species other than humans and laboratory rodents, and in addition, there are special considerations pertaining to the use of adjuvants in veterinary animals, such as production and companion animals. With a focus on veterinary animals, this review highlights a number of approaches being pursued, including cytokines, CpG oligonucleotides, microparticles and liposomes.

Targeted liposomal delivery of TLR9 ligands activates spontaneous antitumor immunity in an autochthonous cancer model.

Accessibility of tumors for highly effective local treatment represents a major challenge for anticancer therapy. Immunostimulatory oligodeoxynucleotides (ODN) with CpG motifs are ligands of TLR9, which prime spontaneous antitumor immunity, but are less effective when applied systemically. We therefore developed a liposome-based agent for selective delivery of CpG-ODN into the tumor environment. A peptide that specifically targets angiogenic endothelial cells in a transgenic tumor model for islet cell carcinogenesis was engrafted into CpG-ODN containing liposomes. Intravenous injection of these liposomes resulted in specific accumulation around tumor vessels, increased uptake by tumor-resident macrophages, and retention over time. In contrast, nontargeted liposomes did not localize to the tumor vasculature. Consequently, only vascular targeting of CpG-ODN liposomes provoked a marked inflammatory response at vessel walls with enhanced CD8(+) and CD4(+) T cell infiltration and, importantly, activation of spontaneous, tumor-specific cytotoxicity. In a therapeutic setting, 40% of tumor-bearing, transgenic mice survived beyond week 45 after systemic administration of vascular-directed CpG-ODN liposomes. In contrast, control mice survived up to 30 wk. Therapeutic efficacy was further improved by increasing the frequency of tumor-specific effector cells through adoptive transfers. NK cells and CD8(+) T cells were major effectors which induced tumor cell death and acted in conjunction with antivascular effects. Thus, tumor homing with CpG-ODN-loaded liposomes is as potent as direct injection of free CpG-ODN and has the potential to overcome some major limitations of conventional CpG-ODN monotherapy.

Poly(ethylene glycol)-Mediated Assembly of Vaccine Particles to Improve Stability and Immunogenicity.

We report the one-step assembly of vaccine particles by encapsulating ovalbumin (OVA) and cytosine-phosphate-guanine oligodeoxynucleotides (CpG) into poly(ethylene glycol) (PEG)-mediated zeolitic imidazolate framework-8 nanoparticles (OVA-CpG@ZIF-8 NPs), where PEG improves the stability and dispersity of ZIF-8 NPs and the NPs protect the encapsulated OVA and CpG to circumvent the cold chain issue. Compared with free OVA and OVA-encapsulated ZIF-8 (OVA@ZIF-8) NPs, OVA-CpG@ZIF-8 NPs can enhance antigen uptake, cross-presentation, dendritic cell (DC) maturation, production of specific antibody and cytokines, and CD4+ T and CD8+ T cell activation. More importantly, the vaccine particles retain their bioactivity against enzymatic degradation, elevated temperatures, and long-term storage at ambient temperature. The study highlights the importance of PEG-mediated ZIF-8 NPs as a vaccine delivery system for the promising application of effective and cold chain-independent vaccination against diseases.

CpG Oligodeoxynucleotide Developed to Activate Primate Immune Responses Promotes Antitumoral Effects in Combination with a Neoantigen-Based mRNA Cancer Vaccine.

PURPOSE: The purpose of our research was to identify and evaluate synthetic phosphorothioate-modified CPG oligodeoxynucleotides (CPG-ODNs) activating innate and adaptive immune responses. Furthermore, combined treatment with CpG and an mRNA cancer vaccine was evaluated in melanoma models as a therapeutic approach. METHODS: A molecular assay was used to screen new CpG molecules; mouse modeling and pathological analysis were used to confirm the antitumor effect of CpG alone or in combination with an mRNA vaccine. Finally, safety was assessed by monitoring blood biochemistry. RESULTS: We first screened and identified a new CpG-B class ODN (CpG2018B) that effectively stimulated type II interferons in both mouse plasmacytoid dendritic cells (pDCs) and human peripheral blood mononuclear cells (PBMCs). In addition, CpG2018B promoted cytokine production mainly via toll-like receptor 9 (TLR9) pathways. We further demonstrated that intratumoral (IT) injection of CpG2018B inhibited melanoma growth in syngeneic models and could turn "cold" tumors into "hot" tumors. Then, CpG2018B and an mRNA-based neoantigen cancer vaccine were encapsulated in lipid nanoparticles (LNPs) and intratumorally injected into melanoma mouse models. Interestingly, vaccination with CpG or the mRNA vaccine alone could inhibit tumor growth, while combination of CpG with the mRNA vaccine enhanced the antitumor effect. Finally, we described the long-term safety and tolerability of CpG2018B and mRNA therapy in mice model. CONCLUSION: We identified a novel CpG-B class ODN to promote the immune response, and CpG combined with mRNA cancer vaccines is an attractive candidate approach for immunostimulatory sequence (ISS)-based therapeutic strategies.

Respiratory FimA-Specific Secretory IgA Antibodies Upregulated by DC-Targeting Nasal Double DNA Adjuvant Are Essential for Elimination of Porphyromonas gingivalis.

Our previous studies showed that a combination of a DNA plasmid encoding Flt3 ligand (pFL) and CpG oligodeoxynucleotides 1826 (CpG ODN) (FL/CpG) as a nasal adjuvant provoked antigen-specific immune responses. In this study, we investigated the efficacy of a nasal vaccine consisting of FimA as the structural subunit of Porphyromonas gingivalis (P. gingivalis) fimbriae and FL/CpG for the induction of FimA-specific antibody (Ab) responses and their protective roles against nasal and lung infection by P. gingivalis, a keystone pathogen in the etiology of periodontal disease. C57BL/6 mice were nasally immunized with recombinant FimA (rFimA) plus FL/CpG three times at weekly intervals. As a control, mice were given nasal rFimA alone. Nasal washes (NWs) and bronchoalveolar lavage fluid (BALF) of mice given nasal rFimA plus FL/CpG resulted in increased levels of rFimA-specific secretory IgA (SIgA) and IgG Ab responses when compared with those in controls. Significantly increased numbers of CD8- or CD11b-expressing mature-type dendritic cells (DCs) were detected in the respiratory inductive and effector tissues of mice given rFimA plus FL/CpG. Additionally, significantly upregulated Th1/Th2-type cytokine responses by rFimA-stimulated CD4+ T cells were noted in the respiratory effector tissues. When mice were challenged with live P. gingivalis via the nasal route, mice immunized nasally with rFimA plus FL/CpG inhibited P. gingivalis colonization in the nasal cavities and lungs. In contrast, controls failed to show protection. Of interest, when IgA-deficient mice given nasal rFimA plus FL/CpG were challenged with nasal P. gingivalis, the inhibition of bacterial colonization in the respiratory tracts was not seen. Taken together, these results show that nasal FL/CpG effectively enhanced DCs and provided balanced Th1- and Th2-type cytokine response-mediated rFimA-specific IgA protective immunity in the respiratory tract against P. gingivalis. A nasal administration with rFimA and FL/CpG could be a candidate for potent mucosal vaccines for the elimination of inhaled P. gingivalis in periodontal patients.

CpG oligonucleotides induce an immune response of odontoblasts through the TLR9, MyD88 and NF-kappaB pathways.

Odontoblasts are the first-line defense cells against invading microorganisms. Toll-like receptors (TLRs) play a crucial role in innate immunity, and TLR9 is involved in the recognition of microbial DNA. This study aimed to investigate whether odontoblasts can respond to CpG DNA and to determine the intracellular signaling pathways triggered by CpG DNA. We found that the mouse odontoblast-like cell line MDPC-23 constitutively expressed TLR9. Exposure to CpG ODN induced a potent proinflammatory response based on an increase of IL-6 and TNF-alpha expression. Pretreatment with an inhibitory MyD88 peptide or a specific inhibitor for TLR9, NF-kappaB or IkappaBalpha markedly inhibited CpG ODN-induced IL-6 and TNF-alpha expression. Moreover, the CpG ODN-mediated increase of kappaB-luciferase activity in MDPC-23 cells was suppressed by the overexpression of dominant negative mutants of TLR9, MyD88 and IkappaBalpha, but not by the dominant negative mutant of TLR4. This result suggests a possible role for the CpG DNA-mediated immune response in odontoblasts and indicates that TLR9, MyD88 and NF-kappaB are involved in this process.

Mannose-Modified Liposome Co-Delivery of Human Papillomavirus Type 16 E7 Peptide and CpG Oligodeoxynucleotide Adjuvant Enhances Antitumor Activity Against Established Large TC-1 Grafted Tumors in Mice.

BACKGROUND: Previously, we demonstrated the therapeutic efficacy of a human papillomavirus (HPV) vaccine, including HPV16 E7 peptide and CpG oligodeoxynucleotides (CpG ODN), against small TC-1 grafted tumors. Here, we developed an HPV16 E7 peptide and CpG ODN vaccine delivered using liposomes modified with DC-targeting mannose, Lip E7/CpG, and determined its anti-tumor effects and influence on systemic immune responses and the tumor microenvironment (TME) in a mouse large TC-1 grafted tumor model. METHODS: L-alpha-phosphatidyl choline (SPC), cholesterol (CHOL), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol-2000)] (DSPE-PEG-2000), 1,2-dioleoyl-3-trimethylammonium-propane chloride salt (DOTAP) and Mannose-PEG-DSPE, loaded with HPV16 E7 peptide and CpG ODN, were used to construct the Lip E7/CpG vaccine. The anti-tumor effects and potential mechanism of Lip E7/CpG were assessed by assays of tumor growth inhibition, immune cells, in vivo cytotoxic T lymphocyte (CTL) responses and cytokines, chemokines, CD31, Ki67 and p53 expression in the TME. In addition, toxicity of Lip E7/CpG to major organs was evaluated. RESULTS: Lip E7/CpG had a diameter of 122.21±8.37 nm and remained stable at 4°C for 7 days. Co-delivery of HPV16 E7 peptide and CpG ODN by liposomes exerted potent anti-tumor effects in large (tumor volume ≥200mm3) TC-1 grafted tumor-bearing mice with inhibition rates of 80% and 78% relative to the control and Free E7/CpG groups, respectively. Vaccination significantly increased numbers of CD4+ and CD8+ T cells, and IFN-γ-producing cells in spleens and tumors and enhanced HPV-specific CTL responses, while reducing numbers of inhibitory cells including myeloid-derived suppressor cells and macrophages. Expression of cytokines and chemokines was altered and formation of tumor blood vessels was reduced in the Lip E7/CpG group, indicating possible modulation of the immunosuppressive TME to promote anti-tumor responses. Lip E7/CpG did not cause morphological changes in major organs. CONCLUSION: Lip E7/CpG induced anti-tumor effects by enhancing cellular immunity and improving tumor-associated immunosuppression. Mannose-modified liposomes are the promising vaccine delivery strategy for cancer immunotherapy.

Immune stimulatory antigen loaded particles combined with depletion of regulatory T-cells induce potent tumor specific immunity in a mouse model of melanoma.

Anti-tumor vaccines capable of activating both CD4 and CD8 T cells are preferred for long lasting T cell responses. Induction of a tumor-specific T-cell response can be induced by tumor vaccines that target innate immunity. The ensuing T-cell response depends on efficient antigen presentation from phagocytosed cargo in the antigen presenting cell and is augmented by the presence of Toll-like receptor (TLR) ligands within the cargo. Biodegradable polymers are useful for vaccine delivery in that they are phagocytosed by antigen presenting cells (APCs) and could potentially be loaded with both the antigen and immune stimulatory TLR agents. This study was undertaken to evaluate the effect of poly lactic-co-glycolic acid (PLGA) polymer particles loaded with antigenic tumor lysate and immune stimulatory CpG oligonucleotides on induction of tumor specific immunity in a mouse model of melanoma. We found that after delivery, these immune stimulatory antigen loaded particles (ISAPs) efficiently activated APCs and were incorporated into lysosomal compartments of macrophages and dendritic cells. ISAP vaccination resulted in remarkable T cell proliferation, but only modestly suppressed tumor growth of established melanoma. Due to this discordant effect on tumor immunity we evaluated the role of regulatory T cells (Treg) and found that ISAP vaccination or tumor growth alone induced prolific expansion of tumor specific Treg. When the Treg compartment was suppressed with anti-CD25 antibody, ISAP vaccination induced complete antigen-specific immunity in a prophylactic model. ISAP vaccination is a novel tumor vaccine strategy that is designed to co-load the antigen with a TLR agonist enabling efficient Ag presentation. Targeting of T-reg expansion during vaccination may be necessary for inducing effective tumor-specific immunity.

Strategies to link innate and adaptive immunity when designing vaccine adjuvants.

Adjuvants are important components of vaccine formulations. Their functions include the delivery of antigen, recruitment of specific immune cells to the site of immunization, activation of these cells to create an inflammatory microenvironment, and maturation of antigen-presenting cells for enhancement of antigen-uptake and -presentation in secondary lymphoid tissues. Adjuvants include a large family of molecules and substances, many of which were developed empirically and without knowledge of their specific mechanisms of action. The discovery of pattern recognition receptors including Toll-like-, nucleotide-binding oligomerization domain (NOD)- and mannose-receptors, has significantly advanced the field of adjuvant research. It is now clear that effective adjuvants link innate and adaptive immunity by signaling through a combination of pathogen recognition receptors (PRRs). Research in our lab is focused towards the development of novel adjuvants and immunomodulators that can be used to improve neonatal vaccines for humans and animals. Using a neonatal pig model for pertussis, we are currently analyzing the effectiveness of host defence peptides (HDPs), bacterial DNA and polyphosphazenes as vaccine adjuvants.