Sterically stabilized cationic liposomes improve the uptake and immunostimulatory activity of CpG oligonucleotides.

Immunostimulatory CpG oligonucleotides (ODN) show promise as immune adjuvants, anti-allergens, and immunoprotective agents. Increasing the bioavailability and duration of action of CpG ODN should improve their therapeutic utility. Encapsulating ODN in sterically stabilized cationic liposomes provides protection from serum nucleases while facilitating uptake by B cells, dendritic cells, and macrophages. In a pathogen challenge model, sterically stabilized cationic liposomes encapsulation doubled the duration of CpG ODN-induced immune protection. In an immunization model, coencapsulation of CpG ODN with protein Ag (OVA) magnified the resultant Ag-specific IFN-gamma and IgG responses by 15- to 40-fold compared with Ag plus CpG ODN alone. These findings support the use of sterically stabilized cationic liposomes to significantly enhance the therapeutic efficacy of CpG ODN.     

Advances in vaccine adjuvants.

Currently, aluminum salts and MF59 are the only vaccine adjuvants approved for human use. With the development of new-generation vaccines (including recombinant subunit and mucosal vaccines) that are less immunogenic, the search for more potent vaccine adjuvants has intensified. Of the novel compounds recently evaluated in human trials, immunostimulatory molecules such as the lipopolysaccharide derived MPL and the saponin derivative QS21 appear most promising, although doubts have been raised as to their safety in humans. Preclinical work with particulate adjuvants, such as the MF59 microemulsion and lipid-particle immune-stimulating complexes (Iscoms), suggest that these molecules are also potent elicitors of humoral and cellular immune responses. In addition, preclinical data on CpG oligonucleotides appear to be encouraging, particularly with respect to their ability to selectively manipulate immune responses. While all these adjuvants show promise, further work is needed to better define the mechanisms of adjuvant action. Ultimately, the development of more potent adjuvants may allow vaccines to be used as therapeutic, rather than prophylactic, agents.     

Parenteral and mucosal prime-boost immunization strategies in mice with hepatitis B surface antigen and CpG DNA

Synthetic oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs (CpG ODN) are potent adjuvants to protein antigens administered by parenteral or mucosal routes to BALB/c mice. To date, there have been no studies using combined parenteral/mucosal approaches with CpG DNA as adjuvant. In this study we evaluated different parenteral prime-mucosal boost and mucosal prime-parenteral boost strategies using hepatitis B surface antigen (HBsAg) alone or with different adjuvants: aluminum hydroxide (alum), cholera toxin (CT), CpG ODN. In addition, since CpG ODN has previously been shown to act synergistically with other adjuvants after parenteral or mucosal delivery, we also evaluated adjuvant combinations: alum+CpG ODN and CT+CpG ODN. The effects of adjuvant and administration strategy on systemic and mucosal humoral responses were measured, as well as cell-mediated immune responses (cytotoxic T lymphocyte activity). These results were compared to parenteral only or mucosal only strategies. Our findings demonstrate that parenteral immunization can prime for mucosal responses even when different lymph nodes were being targeted. HBsAg-specific immune responses (IgG in plasma, cytotoxic T lymphocytes) induced by parenteral prime could all be significantly enhanced by mucosal boosting and despite the fact that intramuscular immunization alone could not induce mucosal IgA, it could prime for a subsequent mucosal boost. In addition, the presence of adjuvant at time of boosting could influence the nature of subsequent immune responses (Th1 vs. Th2). Mice primed intranasally could have their systemic immune responses boosted with a parenteral administration and it was also possible to enhance mucosal responses induced by intranasal prime with an intramuscular boost.     

质粒骨架的CpG修饰对DNA疫苗免疫原性的影响

目的：探讨不同类型的CpG对DNA疫苗免疫应答的影响。方法：将3种不同类型的CpG通过骨架改造的方式引入核酸疫苗的质粒载体骨架中作为内源性佐剂,以LacZ为模式抗原,对其免疫小鼠后特异性抗体水平、细胞免疫水平和细胞因子水平进行比较和分析。结果：3种不同类型的CpG序列在体内能够不同程度地增强免疫小鼠的特异性抗体水平和细胞免疫水平,并且不同类型的CpG序列可能具有不同的免疫调节作用。结论：作为内源性佐剂,CpG免疫刺激DNA序列可不同程度地提高模式抗原特异性的免疫反应,可根据不同抗原的特点加以利用。     

CpG RNA: identification of novel single-stranded RNA that stimulates human CD14+CD11c+ monocytes

Synthetic immunostimulatory nucleic acids such as CpG DNA are being harnessed therapeutically as vaccine adjuvants, anticancer or antiallergic agents. Efforts to identify nucleic acid-based agents capable of more specifically modulating the immune system are being developed. The current study identifies a novel class of single-stranded oligoribonucleotides (ORN) containing unmethylated CpG motifs and a poly(G) run at the 3' end (CpG ORN) that directly stimulate human CD14+CD11c+ monocytes but not dendritic cells or B cells. CpG ORN activate NF-kappaB and p38 MAPK, resulting in IL-6 and IL-12 production and costimulatory molecule up-regulation but not IFNalpha. Methylation of cytosine at the 5' portion in core CpG motif abrogates such activation. TLR3, 7, 8, or 9 alone did not confer response to CpG ORN, in contrast to previously reported respective nucleic acid ligands. These data suggest that CpG ORN represent a novel class of synthetic immunostimulatory nucleic acids with distinct target cells, receptors, and functions from that of previously known immunomodulatory nucleic acids.     

An immunomodulatory GpG oligonucleotide for the treatment of autoimmunity via the innate and adaptive immune systems

Bacterial DNA and immunostimulatory CpG oligodeoxynucleotides (ODNs) activate the innate immune system to produce proinflammatory cytokines. Shown to be potent Th1-like adjuvants, stimulatory CpG motifs are currently used as effective therapeutic vaccines for various animal models of infectious diseases, tumors, allergies, and autoimmune diseases. In this study, we show that the application of an immunomodulatory GpG ODN, with a single base switch from CpG to GpG, can effectively inhibit the activation of Th1 T cells associated with autoimmune disease. Moreover, this immunomodulatory GpG ODN suppresses the severity of experimental autoimmune encephalomyelitis in mice, a prototypic Th1-mediated animal disease model for multiple sclerosis.     

CpG oligodeoxynucleotides up-regulate antibacterial systems and induce protection against bacterial challenge in rainbow trout (Oncorhynchus mykiss)

The effects of unmethylated CpG oligodeoxynucleotides (ODN) on the mammalian immune system are relatively well studied but much less is known of their effects on the immune systems of different fish species. Here we show that CpG ODNs significantly enhance the survival of rainbow trout (Oncorhynchus mykiss) following bacterial challenge when used both as stand-alone prophylactic agents, or as adjuvants to a commercially available vaccine. They are also capable of increasing serum lysozyme activity in vivo and stimulating the production of chemoattractant factors for rainbow trout head kidney (HK) leucocytes in vitro.     

Treatment of infectious diseases with immunostimulatory oligodeoxynucleotides containing CpG motifs

Bacterial DNA with CpG motifs can efficiently stimulate the vertebrate immune system. Thus, synthetic oligodeoxynucleotides that contain such CpG motifs (CpG-ODN) are currently used in preclinical and clinical studies to develop new allergy or cancer therapies and vaccine adjuvants. Recent animal studies indicate that CpG-ODN therapies can also be used for successful treatment of infections caused by bacteria, parasites or viruses. In these experiments, innate and adaptive immune responses against pathogens were augmented by CpG-ODN and subsequently induced resistance against infectious diseases. The stimulation of dendritic cells played a central role for the therapeutic effect of CpG-ODN. However, CpG-ODN can also have negative side effects, which accelerate disease progression in some viral infections. Clinical studies with CpG-ODN will determine their potential for the therapy of infectious diseases in humans.     

Adjuvants--essential components of new generation vaccines

Adjuvants are essential components of vaccines that augment an immunological reaction of organism. New vaccines based on recombinant proteins and DNA, are more save than traditional vaccines but they are less immunogenic. Therefore, there is an urgent need for the development of new, improved vaccine adjuvants. There are two classes of adjuvants: vaccine delivery systems (e.g. emulsions, microparticles, immune-stimulating complexes ISCOMs, liposomes) and immunostimulatory adjuvants (e.g. lipopolysaccharide, monophosphoryl lipid A, CpG DNA, or muramylpeptides). The discovery of more potent and safer adjuvants may allow to development better prophylactic and therapeutic vaccines against chronic infectious (e.g., HSV, HIV, HCV, HBV, HPV, or Helicobacter pylori) and noninfectious diseases as multiple sclerosis, insulin-dependent diabetes, rheumatoid arthritis, allergy and tumors (e.g., melanoma, breast, or colon cancer).     

Human peripheral blood cells differentially recognize and respond to two distinct CPG motifs

Oligodeoxynucleotides (ODN) that contain unmethylated CpG dinucleotides trigger a strong innate immune response in vertebrates. CpG ODN show promise as vaccine adjuvants, anti-allergens, and immunoprotective agents in animal models. Their transition to clinical use requires the identification of motifs that are optimally stimulatory in humans. Analysis of hundreds of novel ODN resulted in the identification and characterization of two structurally distinct "clusters" of immunostimulatory CpG ODN. One cluster ("D") preferentially stimulates IFN-gamma production by NK cells, whereas the other ("K") stimulates cell proliferation and the production of IL-6 and IgM by monocytes and B cells. The distinct immunostimulatory properties of K and D ODN can improve the design of CpG-based products to achieve specific therapeutic goals.     

Recent developments in adjuvants for vaccines against infectious diseases

New generation vaccines, particularly those based on recombinant proteins and DNA, are likely to be less reactogenic than traditional vaccines, but are also less immunogenic. Therefore, there is an urgent need for the development of new and improved vaccine adjuvants. Adjuvants can be broadly separated into two classes, based on their principal mechanisms of action; vaccine delivery systems and 'immunostimulatory adjuvants'. Vaccine delivery systems are generally particulate e.g. emulsions, microparticles, iscoms and liposomes, and mainly function to target associated antigens into antigen presenting cells (APC). In contrast, immunostimulatory adjuvants are predominantly derived from pathogens and often represent pathogen associated molecular patterns (PAMP) e.g. LPS, MPL, CpG DNA, which activate cells of the innate immune system. Once activated, cells of innate immunity drive and focus the acquired immune response. In some studies, delivery systems and immunostimulatory agents have been combined to prepare adjuvant delivery systems, which are designed for more effective delivery of the immunostimulatory adjuvant into APC. Recent progress in innate immunity is beginning to yield insight into the initiation of immune responses and the ways in which immunostimulatory adjuvants may enhance this process. However, a rational approach to the development of new and more effective vaccine adjuvants will require much further work to better define the mechanisms of action of existing adjuvants. The discovery of more potent adjuvants may allow the development of vaccines against infectious agents such as HIV which do not naturally elicit protective immunity. New adjuvants may also allow vaccines to be delivered mucosally.     

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.     

Adjuvant formulations and delivery systems for DNA vaccines

DNA vaccines have become a reliable and major means to elicit immune responses in the past decade. We and others have attempted to obtain stronger, more long lasting, and optimized immune responses, subsequent to the pioneering works demonstrating the ability of plasmid DNA to raise specific immune responses. Advances in molecular biology and biotechnology allow the application of various adjuvants, immunologic agents that increase the antigenic response, in DNA vaccines. Adjuvants can be broadly separated into two classes based on their origin-genetic and conventional. Genetic adjuvants are expression vectors of cytokines or other molecules that can modulate immune responses when administered with a vaccine antigen. Conventional adjuvants are chemical compounds that enhance, prolong, or modulate antigen-specific immune responses. The use of an appropriate adjuvant is pivotal in optimizing the response to DNA vaccines. Moreover, DNA vaccines themselves possess their own adjuvant activity because of the presence of unmethylated CpG motifs in particular base contents. The route of inoculation is also a critical factor in determining the outcome of vaccination. It is well known that intramuscular injection preferentially induces Th1-type immunity, whereas particle bombardment by gene gun predominantly induces Th2-type response. This article focuses on providing the detailed procedure to construct genetic adjuvant plasmids and prepare DNA vaccines formulated with conventional adjuvants. We also offer a practical guide for the procedure of intramuscular DNA injection.     

Plasmid vectors encoding cholera toxin or the heat-labile enterotoxin from Escherichia coli are strong adjuvants for DNA vaccines

Two plasmid vectors encoding the A and B subunits of cholera toxin (CT) and two additional vectors encoding the A and B subunits of the Escherichia coli heat-labile enterotoxin (LT) were evaluated for their ability to serve as genetic adjuvants for particle-mediated DNA vaccines administered to the epidermis of laboratory animals. Both the CT and the LT vectors strongly augmented Th1 cytokine responses (gamma interferon [IFN-gamma]) to multiple viral antigens when codelivered with DNA vaccines. In addition, Th2 cytokine responses (interleukin 4 [IL-4]) were also augmented by both sets of vectors, with the effects of the LT vectors on IL-4 responses being more antigen dependent. The activities of both sets of vectors on antibody responses were antigen dependent and ranged from no effect to sharp reductions in the immunoglobulin G1 (IgG1)-to-IgG2a ratios. Overall, the LT vectors exhibited stronger adjuvant effects in terms of T-cell responses than did the CT vectors, and this was correlated with the induction of greater levels of cyclic AMP by the LT vectors following vector transfection into cultured cells. The adjuvant effects observed in vivo were due to the biological effects of the encoded proteins and not due to CpG motifs in the bacterial genes. Interestingly, the individual LT A and B subunit vectors exhibited partial adjuvant activity that was strongly influenced by the presence or absence of signal peptide coding sequences directing the encoded subunit to either intracellular or extracellular locations. Particle-mediated delivery of either the CT or LT adjuvant vectors in rodents and domestic pigs was well tolerated, suggesting that bacterial toxin-based genetic adjuvants may be a safe and effective strategy to enhance the potency of both prophylactic and therapeutic DNA vaccines for the induction of strong cellular immunity.     

Protective antiviral immune responses to pseudorabies virus induced by DNA vaccination using dimethyldioctadecylammonium bromide as an adjuvant

To enhance the efficacy of a DNA vaccine against pseudorabies virus (PRV), we evaluated the adjuvant properties of plasmids coding for gamma interferon or interleukin-12, of CpG immunostimulatory motifs, and of the conventional adjuvants dimethyldioctadecylammonium bromide in water (DDA) and sulfolipo-cyclodextrin in squalene in water. We demonstrate that a DNA vaccine combined with DDA, but not with the other adjuvants, induced significantly stronger immune responses than plasmid vaccination alone. Moreover, pigs vaccinated in the presence of DDA were protected against clinical disease and shed significantly less PRV after challenge infection. This is the first study to demonstrate that DDA, a conventional adjuvant, enhances DNA vaccine-induced antiviral immunity.     

Cytosine-phosphate-guanine oligodeoxynucleotides containing GACGTT motifs enhance the immune responses elicited by keyhole limpet hemocyanin antigen in dairy cattle

Adjuvants are important components of vaccine formulations. Effective adjuvants line innate and adaptive immunity by signaling through pathogen recognition receptors. Synthetic cytosine-phosphate-guanine (CpG) oligodeoxynucleotides (ODNs) have been shown to have potentials as adjuvants for vaccines. However, the immunostimulatory effect of CpG is species-specific and depends on the sequence of CpG motifs. A CpG ODN (2135), containing 3 identical copies of GTCGTT motif, was previously reported to have the strongest effects on bovine peripheral blood mononuclear cells (PBMC). Based on the sequence of 2135, we replaced the GTCGTT motif with 11 other sequences containing CG and investigated their effects on bovine lymphocyte proliferation. Results showed that the CpG ODNs containing 3 copies of GACGTT motif had the highest lymphocyte stimulation index (7.91±1.18), which was significantly (P<0.05) higher than that of 2135 (4.25±0.56). The CpG ODNs containing 3 copies of GACGTT motif also significantly increased the mRNA expression of interferon (IFN)-α, interleukin (IL)-12, and IL-21 in bovine PBMC. When dairy cows were immunized with the keyhole limpet hemocyanin (KLH) antigen formulated with CpG ODNs containing 3 copies of GACGTT, production of KLH-specific antibodies in serum and in milk whey was significantly (P<0.05) enhanced. IFN-γ in whole blood stimulated by KLH was also significantly (P<0.05) increased in cows immunized with KLH plus CpG ODNs. Our results indicate that CpG ODNs containing 3 copies of the GACGTT motifs is a potential adjuvant for bovine vaccines.     

Immunostimulatory CpG oligodeoxynucleotides stimulate expression of IL-1beta and interferon-like cytokines in rainbow trout macrophages via a chloroquine-sensitive mechanism.

Synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs are known to stimulate immune responses and are potent adjuvants in higher vertebrates, but so far the effects in fish are poorly described. We here report that CpG ODNs induce IL-1beta expression and production of interferon-like cytokines in rainbow trout head-kidney macrophages, whereas ODNs with an inverted motif (GpC) have a much less stimulatory effect. We further demonstrate that endosomal maturation is essential for CpG signalling, as chloroquine, a compound known to block endosomal acidification, inhibits cytokine expression in the macrophages.     

DNA methyltransferase inhibition may limit cancer cell growth by disrupting ribosome biogenesis

“Mutations” in the pattern of CpG methylation imprinting of the human genome have been correlated with a number of diseases including cancer. In particular, aberrant imprinting of tumor suppressor genes by gain of CpG methylation has been observed in many cancers and thus represents an important alternative pathway to gene “mutation” and tumor progression. Inhibitors of DNA methylation display therapeutic effects in the treatment of certain cancers, and it has been assumed these effects are due to the reversal of “mutant” gene imprinting. However, significant reactivation of imprinted tumor suppressor genes is rarely observed in vivo following treatment with DNA methylation inhibitors. A recent study revealed an unexpected requirement for CpG methylation in the synthesis and assembly of the ribosome, an essential function for cell growth and proliferation. As such, the data provide an unforeseen explanation of the action of DNA methylation inhibitors in restricting cancer cell growth.