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.

Novel strategies using DNA for the induction of mucosal immunity.

The mucosal surfaces are the primary sites for transmission of most infectious diseases. However, most conventional vaccines are administered parenterally [e.g., by intramuscular (IM) or intradermal (ID) injection] and induce systemic but rarely mucosal immunity. Novel vaccination strategies capable of inducing both systemic and mucosal immune responses could greatly reduce infection and morbidity worldwide. One of the most exciting advances in vaccine technology in recent years has been the development of DNA vaccines, through which the antigen is synthesized in vivo after direct introduction of its encoding sequences. The vast majority of DNA vaccines have been delivered parenterally; however, in recent years a number of studies have reported successful mucosal immunization with DNA vaccines. The induction of strong immune responses following the introduction of DNA appears to be partly due to the potent adjuvant effect of unmethylated immunostimulatory CpG motifs present in the DNA backbone. Synthetic oligodeoxynucleotides (ODN) containing such immunostimulatory CpG motifs are potent adjuvants systemically and mucosally in mice, and have synergistic action with other adjuvants, such as alum and cholera toxin (CT). This article highlights the recent advances in vaccination strategies using DNA delivered to mucosal surfaces either as an antigen-encoding plasmid or as an adjuvant.

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.

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.

Immunization against hepatitis B virus by mucosal administration of antigen-antibody complexes.

Antigen-antibody complexes have been shown to enhance immune responses against several antigens given by parenteral immunization. Herein, we have evaluated the potential of administering such immunostimulatory complexes by a mucosal route. Hepatitis B surface antigen (HBsAg) complexed with antibodies against HBsAg (anti-HBs) (HBsAg/Ab) was administered to BALB/c mice by intranasal inhalation. HBsAg by itself did not induce immune responses, whereas with HBsAg/Ab complexes, both systemic and mucosal immune responses were observed and these could be modulated by adjuvants. With HBsAg/Ab (1 or 10 microg), anti-HBs antibodies induced were predominantly of the IgG1 isotype (Th2-like). In contrast, anti-HBs induced by HBsAg/Ab plus cholera toxin (CT) or oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs (CpG) (1 microg each) were predominantly IgG2a (Th1-like). Results from this study indicate that HBsAg/Ab complexes can induce strong humoral immune responses when delivered by a noninvasive route, whether used alone or in combination with other mucosal adjuvants.

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.

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.

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.

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.

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).

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.

CpG DNA as mucosal adjuvant.

We have previously found synthetic oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs to be a potent adjuvant to protein administered by intramuscular injection or intranasal inhalation to BALB/c mice. Herein we have further evaluated the potential of CpG ODN as a mucosal adjuvant to purified hepatitis B surface antigen (HBsAg) when administered alone or with cholera toxin (CT). CpG ODN and CT both augmented systemic (humoral and cellular) and mucosal immune responses against HBsAg, and these could be further enhanced with higher doses of adjuvant or boosting. Overall, antibody isotypes with CT alone were predominantly IgG1 (Th2-like) whereas they were predominantly IgG2a (Th1-like) with CpG ODN alone or in combination with CT. Results from this study indicate that stimulatory CpG ODN are promising new adjuvants for mucosal vaccination strategies, whether used alone or in combination with other mucosal adjuvants.

CpG DNA is a potent enhancer of systemic and mucosal immune responses against hepatitis B surface antigen with intranasal administration to mice.

Mucosal immunity is difficult to induce with subunit vaccines unless such vaccines are administered with a mucosal adjuvant such as cholera toxin (CT); however, CT is toxic in humans. Synthetic oligodeoxynucleotides containing immunostimulatory CpG motifs (CpG) are potent adjuvants for the induction of Th1-like systemic immune responses against parenterally delivered proteins. Here, we show in mice that intranasal delivery of hepatitis B surface Ag, which alone has no effect, elicits good immune responses when given with CpG oligodeoxynucleotides and/or CT. Overall, CpG is superior to CT for the induction of humoral and cell-mediated systemic immunity as well as mucosal immune responses (IgA) at local (lung) and distant (feces) sites. Furthermore, CpG and CT act synergistically, giving stronger responses than those observed with 10 times more of either adjuvant alone. Ab isotypes were predominantly IgG1 (Th2-like) with CT, mixed IgG1/IgG2a (Th0) with CpG, and predominantly IgG2a (Th1-like) with CpG and CT together.

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.

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.

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.

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.

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.

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.