Enhancement of infectious disease vaccines through TLR9-dependent recognition of CpG DNA.

The adaptive immune system-with its remarkable ability to generate antigen-specific antibodies and T lymphocytes against pathogens never before "seen" by an organism-is one of the marvels of evolution. However, to generate these responses, the adaptive immune system requires activation by the innate immune system. Toll-like receptors (TLRs) are perhaps the best-understood family of innate immune receptors for detecting infections and stimulating adaptive immune responses. TLR9 appears to have evolved to recognize infections by a subtle structural difference between eukaryotic and prokaryotic/viral DNA; only the former frequently methylates CpG dinucleotides. Used as vaccine adjuvants, synthetic oligodeoxynucleotide (ODN) ligands for TLR9--CpG ODN--greatly enhance the speed and strength of the immune responses to vaccination.

CpG ODN can re-direct the Th bias of established Th2 immune responses in adult and young mice.

Induction of an appropriate immune response is essential for successful immunization. For example, Th1 type immune responses are necessary for the control of intracellular infections whereas Th2 type responses are more useful for the control of extracellular infections. Immunostimulatory CpG ODN (oligonucleotides containing unmethylated cytosine and guanine dinucleotides in specific base contexts) act as potent adjuvants and have been shown to induce Th1 type immune responses with a number of different antigens. This study investigates the effect of CpG ODN on the Th bias of immune responses generated against the hepatitis B major surface antigen (HBsAg) in adult (6-8 weeks old) and young (<1 week old) BALB/c mice. It also investigates the potential of CpG DNA to reverse a pre-established Th2 response generated as an adult or as a neonate, following re-exposure to HBsAg in adult life. Both adult and young mice immunized with HBsAg/CpG ODN had a Th1 biased immune response (strong cytotoxic T-lymphocyte (CTL) induction, IgG2a>>IgG1). In contrast, mice immunized with HBsAg/alum had a Th2 type immune response (poor CTL, IgG1>>IgG2a). More importantly, when animals were immunized with HBsAg/alum and boosted with HBsAg/CpG ODN, the CpG ODN were able to re-direct the Th2 response pre-established by alum, whereas the animals receiving the primary immunization with HBsAg/CpG ODN and later boosted with HBsAg/alum maintained their Th1 bias, even after the boost with alum. These data suggest that CpG ODN have the ability to augment both humoral and cell mediated immune responses and override the Th2 bias created by alum, even in very young animals, which are known to have a Th2 biased immune system.

The potential of CpG oligodeoxynucleotides as mucosal adjuvants.

The development of mucosal vaccines for humans has been hindered by the lack of safe yet effective mucosal adjuvants. Bacterial toxins are commonly used as adjuvants in animal models, but they are too toxic for use in humans. A novel class of adjuvant is CpG DNA, which contains unmethylated CpG dinucleotides in particular base contexts (CpG motifs). CpG DNA is most often coadministered with antigen in the form of synthetic oligodeoxynucleotides (CpG ODN), which are made with a nuclease-resistant phosphorothioate backbone. The vast majority of studies using CpG DNA as adjuvant have been with parenteral delivery; recently, however, mucosal immunization with CpG DNA as adjuvant has also been shown to induce both systemic (humoral and cellular) and mucosal antigen-specific immune responses. This review will highlight the recent uses of CpG DNA as an adjuvant at mucosal surfaces.

Immune-mediated destruction of transfected myocytes following DNA vaccination occurs via multiple mechanisms.

The delivery of antigenic proteins in the context of a DNA vaccine leads to the intracellular synthesis of antigen and the induction of both humoral and cellular immune responses. Subsequent to immune activation, any transfected cell expressing the immunogenic protein should, by the rules of immunology, become a legitimate target for removal by immune-mediated mechanisms. Herein, we have used an indirect assay of myocyte integrity following intra-muscular (i.m.) delivery of a DNA vaccine, in mice with various immune deficiencies, to determine which immunological mechanisms may be involved in destruction of antigen-expressing cells. We demonstrate that destruction of antigen- expressing myocytes following i.m. injection of a DNA vaccine is dependent on major histocompatibility complex (MHC) class II restricted CD4+ T cell activation, but is not mediated solely by MHC I-restricted or perforin-mediated lysis and appears to have a component that is antibody-mediated. Although we studied myocytes, the results likely represent what happens to any transfected cell expressing a foreign antigen. This study underscores the ability of DNA vaccines at inducing antigen-specific immune responses that include a number of effector mechanisms. From the perspective of gene therapy, this study highlights the significance of immune activation when considering strategies where maintenance of therapeutic gene expression is desired.

The use of CpG DNA as a mucosal vaccine adjuvant.

CpG DNA has been shown to be a potent adjuvant in many disease models. Most studies using CpG DNA as adjuvant have used parenteral delivery, but more effective protection against mucosal pathogens could be achieved with effective mucosal immunization. Recently, mucosal immunization with CpG DNA as an adjuvant has been shown to induce both systemic (humoral and cellular) and mucosal antigen-specific immune responses. This review will concentrate on the use of CpG DNA as an adjuvant for the induction of mucosal immunity.

Identification of methylated CpG motifs as inhibitors of the immune stimulatory CpG motifs.

The unmethylated CpG motifs within E. coli DNA (EC) cause immune stimulation. In contrast, mammalian DNA such as calf thymus (CT) DNA had been thought to be immunologically inert. In this article, we demonstrate that CT DNA unexpectedly specifically inhibits the immune activation by EC but not that by endotoxin. This inhibitory effect was mediated in the signaling pathway activated by EC since CT DNA markedly inhibited the CpG-induced nuclear translocation of the transcription factors, NF-kappaB and AP-1. In addition, CT DNA significantly inhibited the synergistic immune activation by EC and endotoxin. The mechanism of the inhibition by CT DNA probably did not involve the inhibition of the cellular uptake of EC. Using a CpG-depleted plasmid, we demonstrated that CpG methylation played an important role in the inhibition by CT DNA. Compared with unmethylated plasmid DNA, CpG-methylated DNA inhibited the immune activation by EC to the same extent as did CT DNA. Importantly, the inhibitory effect of CT DNA was also observed in vivo. Our results suggest that methylated DNA may be applied to alleviate the unwanted immune stimulation and inflammation in systemic inflammatory response syndrome and in gene therapy with plasmid DNA.

Mucosal immunization of mice using CpG DNA and/or mutants of the heat-labile enterotoxin of Escherichia coli as adjuvants.

Cholera toxin (CT) and the Escherichia coli heat-labile enterotoxin (LT) are potent mucosal adjuvants in animals associated, at least in part, with their ability to induce cAMP. While toxicity generally precludes their use in humans, a number of different subunit or genetically detoxified mutants of CT and LT have been developed. Another type of adjuvant that has been shown to be effective at mucosal surfaces comprises synthetic oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs (CpG ODN). We have previously demonstrated a synergy between CpG ODN and native toxins after intranasal (IN) administration to mice, and herein have examined whether this synergy is linked to the cAMP activity. The adjuvanticity of CpG ODN was evaluated with IN and oral delivery of tetanus toxoid or the hepatitis B surface antigen, relative to and in combination with native LT holotoxin (LTh), three active site mutants (LTS61F, LTA69G, LTE112K), a protease site mutant (LTR192G), and the B subunit of LT (LTB). At an equivalent dose, the adjuvants could generally be divided into two groups: one that included CpG ODN, LTh, LTR192G, and LTA69G which acted as strong adjuvants; and the second which comprised LTB, LTS61F, and LTE112K, which produced significantly weaker immune responses. When CpG ODN was co-administered with bacterial toxin-derivatives, in most cases, no synergy between CpG and the LT derivatives was found for strength of the humoral response. Nevertheless, for both routes and antigens, CpG ODN combined with any LT derivative induced a more Type 1-like response than LT derivative alone. These results suggest that while the synergy seen previously with native toxins may have been due in part to inherent cAMP activity, it may have also depended on the particular antigen used and the route of immunization.

Priming of immune responses to hepatitis B surface antigen in young mice immunized in the presence of maternally derived antibodies.

Early vaccination is necessary to protect infants from various infectious diseases. However, this is often unsuccessful largely due to the immaturity of the neonatal immune system. Furthermore, maternally derived antibodies can interfere with active immunization. We have previously shown in young mice that immune responses against several different antigens can be improved by the addition of oligodeoxynucleotides containing immunostimulatory CpG motifs (CpG ODN). In this study we have evaluated immunization of newborn (1-7-day-old) BALB/c mice against hepatitis B surface antigen (HBsAg), with alum and/or CpG ODN, in the presence of high levels of maternal antibody against HBsAg (anti-HBs). Seroconversion rates and anti-HBs titers were compared to those induced by a HBsAg-expressing plasmid, since other studies had suggested DNA vaccines to be superior to protein vaccines in young mice with maternal antibody. HBsAg/alum/CpG ODN was superior to DNA vaccine in inducing HBsAg-specific CTL responses in young mice in the presence of maternally transferred anti-HBs antibodies. However, B cell responses to both HBsAg/alum/CpG ODN and DNA vaccines remained weak in the presence of maternally transferred anti-HBs antibodies.

The potential of oligodeoxynucleotides as mucosal and parenteral adjuvants.

Synthetic oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs (CpG ODN) are potent adjuvants in mice when delivered by parenteral (intramuscular, subcutaneous) and mucosal (intranasal, oral and intrarectal) routes. We have recently shown that with mucosal delivery non-CpG ODN can also have immunostimulatory properties which, in contrast to the Th1-bias characteristic of CpG ODN, are predominantly Th2-like. Herein, using hepatitis B surface antigen (HBsAg) and tetanus toxoid (TT) as model antigens in BALB/c mice, we have examined a number of different ODN (CpG, non-CpG, poly-T, poly-CG) to determine their effects on immune responses after mucosal (oral) and parenteral (IM) immunizations. Our findings demonstrate that with mucosal delivery, there is a Th2-biased immunostimulatory effect that is associated with non-CpG ODN, and that the presence of CpG motifs can shift this towards a Th1 response. The adjuvant effect of non-CpG ODN was much less evident after parenteral immunization.

Intranasal immunization with CpG oligodeoxynucleotides as an adjuvant dramatically increases IgA and protection against herpes simplex virus-2 in the genital tract.

Development of vaccines capable of preventing the transmission or limiting the severity of sexually transmitted viruses, such as HSV and HIV, will likely be dependent on the induction of potent long-lasting mucosal immune responses in the genital tract. Recently, synthetic oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs were shown to serve as potent adjuvants for the induction of mucosal immune responses. Here, we show that intranasal immunization with CpG ODN, plus recombinant glycoprotein B (rgB) of HSV-1, results in significantly elevated levels of specific anti-gB IgA Abs in vaginal washes that remained high throughout the estrous cycle. Additionally, dramatically elevated numbers of specific IgA Ab-secreting cells were present and persisted in the genital tract in response to intravaginal (IVAG) HSV-2 challenge. HSV-2-specific CTL were observed at moderate levels in the spleens of CpG or non-CpG ODN-immunized mice. In contrast, strong CTL responses were observed locally in the genital tissues of both groups following IVAG HSV-2 challenge. Interestingly, mice immunized intranasally with rgB plus CpG ODN, but not non-CpG ODN, were significantly protected following IVAG HSV-2 challenge. Measurement of virus in protected CpG-immunized mice revealed a log lower level of replication within the first few days after infection. In conclusion, these results indicate that intranasal immunization with CpG ODN plus protein mediates immunity in the female genital tract capable of protecting against a sexually transmitted pathogen.

Novel adjuvant systems.

Vaccination remains the single most valuable tool in the prevention of infectious disease. Nevertheless, there exists a need to improve the performance of existing vaccines such that fewer boosts are needed or to develop novel vaccines. For the development of effective vaccines for humans, a great need exists for safe and effective adjuvants. A number of novel adjuvants have been reported in recent years including: i) bacterial toxins such as cholera toxin, CT, and the Escherichia coli heat-labile enterotoxin, LT; ii) less toxic derivatives of CT and LT; iii) endogenous human immunomodulators, such as IL-2, IL-12, GM-CSF; iv) hormones; v) lipopeptides; vi) saponins, such as QS-21; vii) synthetic oligonucleotides containing CpG motifs (CpG ODN); viii) lipid 'A derivatives, such as monophosphoryl lipid A, MPL, and ix) muramyl dipeptide (MDP) derivatives. Herein, we will review recent findings using these novel adjuvant systems.

CpG DNA is an effective oral adjuvant to protein antigens in mice.

We have previously reported that synthetic oligodeoxynucleotides containing immunostimulatory CpG motifs (CpG ODN) are potent adjuvants to protein administered by intramuscular (IM) injection or intranasal (IN) inhalation to BALB/c mice. Herein, we have evaluated oral delivery of CpG ODN with purified hepatitis B surface antigen (HBsAg) or tetanus toxoid (TT) to determine its potential as an adjuvant to oral vaccines. CpG ODN augmented systemic (IgG in plasma, CTL, T-cell proliferation) and mucosal (IgA in lung, vaginal or gut washes, feces and saliva) immune responses against both antigens. CpG stimulated both T-helper type 1 (Th1) (CTL, IgG2a) and Th2 (IgG1, IgA) responses when delivered orally. Results from this study indicate that stimulatory CpG ODN may be effective as an adjuvant with oral vaccines.

Intranasal immunization of mice with CpG DNA induces strong systemic and mucosal responses that are influenced by other mucosal adjuvants and antigen distribution.

BACKGROUND: Synthetic oligodeoxynucleotides (ODN) containing immunostimulatory cytosine-guanine phosphate-linked dinucleotide (CpG) motifs are potent systemic and mucosal adjuvants in mice that have synergistic action with numerous other adjuvants, including alum and cholera toxin (CT). Herein, we evaluate CpG ODN with intranasal (IN) delivery of purified hepatitis B surface antigen (HBsAg), relative to and in combination with CT, Escherichia coli heat labile enterotoxin (LT), the B subunit of CT (CTB), and a nontoxic derivative of LT (LTK63). MATERIALS AND METHODS: BALB/c mice were immunized by IN administration of HBsAg, alone or combined with CT, LT, CTB, or LTK63, and/or CpG ODN, or non-CpG control ODN. In addition, the effect of low-or high-volume administration was assessed, in order to target upper respiratory or entire respiratory tract, respectively. HBsAg-specific systemic (immunoglobulins: IgG, IgG1, IgG2a in plasma) and mucosal (IgA in fecal, lung, vaginal, saliva, and gut samples) humoral responses, as well as cell-mediated immune responses including T-cell proliferation and cytokines (interleukins: IL-4, IL-5; interferon: IFN-gamma) were evaluated. RESULTS: CpG ODN, CT, and LT augmented anti-HBs titers equally, and more so than did CTB or LTK63. CpG ODN acted synergistically with CT and LT, but not CTB or LTK63 to enhance anti-HBs titers. Nevertheless, CpG ODN induced a more Th1-like response for all combinations, compared with the same formulation without CpG. Strength of induced systemic and mucosal immune responses was better with IN delivery of a large volume. A small volume required multiple administrations and higher doses of antigen and adjuvant for equal results. This suggests that delivery of antigen to the lung and/or diges-tive system is superior to delivery to the nasal cavity. CONCLUSIONS: Our results suggest that the synergy between CpG ODN and native toxins (CT, LT) may depend on their enzymatic activity and that the lack of synergy with nontoxic derivatives (LTB, LTK63) arises, since they do not have enzymatic activity. Because both CT and LT are too toxic for use in humans, it is possible that CpG ODN may be combined with bacterial toxin mutants that retain some enzymatic activity to optimize immune augmentation.

Oral, intrarectal and intranasal immunizations using CpG and non-CpG oligodeoxynucleotides as adjuvants.

We have previously demonstrated that synthetic oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs (CpG ODN) are potent adjuvants in mice when delivered by intramuscular, intranasal and subcutaneous routes. Herein, using tetanus toxoid (TT) as a model antigen in BALB/c mice, we compared the ability of CpG ODN to induce mucosal and systemic humoral immune responses when antigen was delivered by three different routes: intrarectal, intranasal and oral. Results showed differences in immune responses with the three routes and also revealed that non-CpG "control" ODN had adjuvant effects when used at mucosal sites. This was unexpected since non-CpG ODN do not have such immunostimulatory effects in vitro or after parenteral immunization. These findings were further investigated after oral delivery of a killed influenza vaccine on its own as well as combined with TT and hepatitis B surface antigen. Our findings demonstrate that with mucosal delivery, there is a Th2 immunostimulatory effect associated with the phosphorothioate ODN backbone, and that the presence of CpG motifs shifts this towards a Th1 response.

The role of CpG in DNA vaccines.

One of the most exciting developments in the field of vaccine research in recent years has been DNA vaccines, with which immune responses are induced subsequent to the in vivo expression of antigen from directly introduced plasmid DNA. Strong immune responses have been demonstrated in a number of animal models against many viral, bacterial and parasitic pathogens, and several human clinical trials have been undertaken. The strong and long-lasting antigen-specific humoral (antibodies) and cell-mediated (T help, other cytokine functions and cytotoxic T cells) immune responses induced by DNA vaccines appear to be due to the sustained in vivo expression of antigen, efficient antigen presentation and the presence of stimulatory CpG motifs. These features are desirable for the development of prophylactic vaccines against numerous infectious agents. Furthermore, the strong cellular responses are also very desirable for the development of therapeutic DNA vaccines to treat chronic viral infections or cancer. Efforts are now focusing on understanding the mechanisms for the induction of these immune responses, which in turn should aid in the optimization of DNA vaccines. This review will focus on the role of CpG motifs in DNA vaccines.

CpG DNA induces stronger immune responses with less toxicity than other adjuvants.

The ability to augment protective immune responses with minimal side effects is quintessential for a good adjuvant. This study has compared various adjuvants that are used in animal research (Freund's complete and incomplete adjuvants, Titermax Gold), are licensed for human use (alum), or are in clinical testing for humans (monophosphoryl lipid, CpG DNA), for their ability to augment humoral responses to a model antigen (hepatitis B surface antigen) and for the degree of damage they caused in the injected muscle. According to the data, the adjuvant combination CpG DNA+alum had the greatest potential to augment immune responses with minimal side effects at the injection site. Evaluation of antibody isotypes indicated Th2 responses (no IgG2a) with all adjuvants except monophosphoryl lipid and CpG DNA, which gave mixed Th1/Th2 responses (IgG1 and IgG2a). Strong Th1 responses (predominantly IgG2a) were obtained with combinations of CpG DNA with other adjuvants.

Optimization strategies for DNA vaccines.

DNA immunization is a relatively new vaccination strategy that involves the direct introduction into the host of plasmid DNA encoding the desired antigen. The DNA enters host cells and results in immune responses following in vivo expression of the antigen. Although DNA-based immunization works well in animal models for the induction of both humoral and cell-mediated immune responses, its success in humans has been limited. This paper discusses different approaches that have attempted to optimize DNA vaccines, and presents results evaluating some of these approaches in mice.

Mucosal immunization with DNA vaccines.

The mucosal surface area of the gastrointestinal, genitourinary and respiratory tracts is more than 200 times greater than that of the skin and is the primary site of transmission of numerous diseases. The entry of pathogenic organisms at mucosal surfaces can be prevented by mucosal, but not systemic immunity. Vaccines which are delivered by intramuscular (IM) or subcutaneous (SC) injection induce strong systemic responses but generally no mucosal immunity. In contrast, vaccines delivered at mucosal surfaces trigger both mucosal (at local and distant sites) and systemic responses (1,2). Other advantages of mucosal immunization include a broader age range of recipients, the vaccines are easy and non-invasive to administer and there is no risk of needle stick injury and cross contamination (3).

Mucosal immunization with DNA vaccines.

DNA vaccines can induce potent humoral and cellular immune responses in numerous animal models. Most DNA vaccines have been administered parenterally; however, more effective protection against mucosal pathogens could be achieved with mucosal immunization. This review concentrates on the use of DNA vaccines for the induction of mucosal immunity.

DNA vaccines for viral diseases.

DNA vaccines, with which the antigen is synthesized in vivo after direct introduction of its encoding sequences, offer a unique method of immunization that may overcome many of the deficits of traditional antigen-based vaccines. By virtue of the sustained in vivo antigen synthesis and the comprised stimulatory CpG motifs, plasmid DNA vaccines appear to induce strong and long-lasting humoral (antibodies) and cell-mediated (T-help, other cytokine functions and cytotoxic T cells) immune responses without the risk of infection and without boost. Other advantages over traditional antigen-containing vaccines are their low cost, the relative ease with which they are manufactured, their heat stability, the possibility of obtaining multivalent vaccines and the rapid development of new vaccines in response to new strains of pathogens. The antigen-encoding DNA may be in different forms and formulations, and may be introduced into cells of the body by numerous methods. To date, animal models have shown the possibility of producing effective prophylactic DNA vaccines against numerous viruses as well as other infectious pathogens. The strong cellular responses also open up the possibility of effective therapeutic DNA vaccines to treat chronic viral infections.