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 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.     

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.     

In vivo oral administration effects of various oligodeoxynucleotides containing synthetic immunostimulatory motifs in the immune response to pseudorabies attenuated virus vaccine in newborn piglets

Numerous studies have demonstrated that oligonucleotides containing CpG motifs (CpG ODN) are efficient immunoadjuvants to various antigens administered by parenteral routes to mice. Recently, it has been found that CpG ODNs also is a promising mucosal adjuvant in mice. To date, there have been no studies to screen the optimal CpG sequence and modified ODN backbone to piglets in vivo, when delivered by oral route. We have previously demonstrated that human-specific CpG ODN is a potent adjuvant to pseudorabies live attenuated virus (PRV) vaccine when administered subcutaneously (SC) or ocularly in piglets. In this study, we screened and evaluated the optimal CpG sequences (porcine-specific, human-specific, mouse-specific ODN) and optimal backbone (SOS-backbone consisting of a nuclease-resistant phosphorothioate guanosines at the 5' and the 3'-end and with a phosphodiester (O) in the center and phosphorothioate (S) backbone (S-backbone)) to PRV vaccine delivered orally in piglets. The proliferation of peripheral blood mononuclear cells (PBMCs), IFN-gamma and IL-4 in serum, and the titre of IgG, IgG2/IgG1 isotype in serum and IgA in intestinal washings and feces to PRV vaccine were tested at different time-points. The results suggested that, CpG ODNs augmented systemic (IgG in serum, T-cell proliferation) and mucosal (IgA in intestinal washings and feces) immune responses against antigen. CpG ODNs stimulated both T-helper type1 (Th1) (IgG2) and Th2 (IgA) responses when delivered orally. With the same backbone, the porcine-specific ODN-induced responses were comparable with human-specific ODNs, but stronger than mouse-specific CpG ODNs. SOS-backbone induced a stronger IFN-gamma and proliferative responses than S-backbone, while antibody responses induced by SOS-backbones were slightly less or similar with S-backbone. The in vivo data demonstrate for the first time that porcine-specific and human-specific ODNs both are optimal sequences for mucosal system in piglets.     

CpG DNA as a Th1-promoting adjuvant in immunization against Trypanosoma cruzi

Th1-type immune response plays a critical role in resistance to Trypanosoma cruzi infection. We asked whether a synthetic oligodeoxynucleotide that contains immunostimulatory CpG motifs (CpG ODN), known to promote a Th1 response, could act as an adjuvant in immunization with parasite antigens. Mice immunized with a whole homogenate (WH) of T. cruzi antigens co-administered with CpG ODN presented high titers of T. cruzi antibodies (IgG2a isotype), strong delayed type hypersensitivity and a Th1-dominated (IFN-gamma and IL-12) cytokine profile. Furthermore, WH plus CpG ODN protected mice from challenge with an otherwise lethal dose of bloodstream trypomastigotes. As reported for leishmaniasis and malaria, CpG ODN holds considerable promise as an adjuvant for future vaccines against T. cruzi.     

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.     

Enhanced immune response to T-independent antigen by using CpG oligodeoxynucleotides encapsulated in liposomes

Immunostimulatory CpG oligodeoxynucleotides (ODN) have been tested as immunoadjuvants for various vaccines including T-cell independent (TI) antigens. Findings from previous reports suggest that close physical association of CpG ODN to the antigen could enhance its adjuvant effect. As an alternative to chemical conjugation of CpG ODN to the antigen, the current study is aimed at determining the benefit of using liposomes as a carrier for CpG ODN to improve the immune response to biotinylated liposomes (Bx-liposomes), a model of a TI antigen. Liposomes with suboptimal concentration of hapten (1% biotin) were not immunogenic. However, when CpG ODN encapsulated in Bx-liposomes were used to immunize mice, a hapten-specific response was obtained as indicated by antibody-mediated elimination of re-administered Bx-liposomes. CpG ODN co-administered with empty Bx-liposomes could not achieve the same effect, indicating the requirement for encapsulation of the adjuvant. Using both intravenous (i.v.) and subcutaneous (s.c.) immunization methods, it was found that IgM levels, but not IgG levels were elevated. Immunization in nude mice confirmed that the immune response obtained was TI. The use of non-CpG ODN and an ODN with alternatively flanked CpG motifs showed no adjuvant effect. Incorporation of poly(ethylene)glycol (PEG)-modified lipid in liposomes enhanced the immune response even further. In conclusion, our data shows that liposomes are a useful delivery vehicle for CpG ODN as an immune adjuvant for TI antigens.     

CpG oligodeoxynucleotides and mobilization of innate mucosal immunity: tasks and tactics

The recent discovery of pathogen-associated molecular patterns (PAMPs) as potential ligands for the evolutionary conserved innate immune receptors termed Toll-like receptors has enabled a modern era of immunotherapy using synthetic mimics of pathogen molecules. Among the PAMPs, bacterial DNA or synthetic oligodeoxynucleotides (ODNs) that contain unmethylated CpG motif shows promising effect on activation of systemic innate immune response. We could document that CpG ODN is capable of mobilizing a potent innate immunity in the mucosal tissues. Thus, intravaginal, intrarrectal, or intragastric delivery of mice with CpG ODN elicits potent innate chemokine responses in the respective mucosal tissues. Interestingly, we could show that the immunostimulatory effect of CpG DNA is much improved when chemically conjugated to the non-toxic B subunit of cholera toxin.     

Immunostimulatory CpG oligonucleotides: Effect on gene expression and utility as vaccine adjuvants

Synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs mimic the immunostimulatory activity of bacterial DNA. CpG ODN directly stimulate B cells and plasmacytoid dendritic cells (pDC), promote the production of Th1 and pro-inflammatory cytokines, and trigger the maturation/activation of professional antigen presenting cells. CpG ODN are finding use as vaccine adjuvants, where they increase the speed, magnitude and duration of vaccine-specific immune responses. For example, CpG ODN significantly prolong the protection induced by AVA (Anthrax Vaccine Adsorbed). Unexpectedly, a majority of animals immunized with CpG-adjuvanted AVA maintain resistance to anthrax infection even after their Ab titers decline to sub-protective levels. This survival is mediated by the de novo production of protective Abs by high affinity long-lived memory B cells. The immunostimulatory activity of CpG ODN was probed at the molecular level by microarray. Results show that a small group of ‘inducers’ rapidly up-regulated a large network genes following CpG treatment of mice. This stimulatory activity is quenched by ‘suppressors’ that down-regulate the expression of targeted genes, including most of the ‘inducers’. These findings shed light on the mechanism underlying CpG-mediated immune activation and therapeutic activity.     

Intravaginal immunization with viral subunit protein plus CpG oligodeoxynucleotides induces protective immunity against HSV-2

Although the genital tract has been considered a poor inductive site for immunization with non-replicating antigens, genital immunization may be important for protection against sexually transmitted infections. Recently, we and others showed that CpG oligodeoxynucleotides (ODNs) serve as potent adjuvants for mucosal immunization. The purpose of this study was to determine whether intravaginal (IVAG) immunization with recombinant glycoprotein B (rgB) of herpes simplex virus type 2 (HSV-2) plus CpG ODN can induce specific immunity and protect against genital HSV-2 challenge. C57BL/6 mice were immunized IVAG with rgB plus CpG ODN, rgB plus non-CpG ODN, or rgB alone and challenged IVAG with HSV-2. Mice immunized with rgB + CpG had higher levels of anti-gB IgA and IgG in the vaginal washes and serum compared to mice immunized with rgB alone. Mice immunized with rgB + CpG also had the highest levels of gB-specific IgG in the nasal washes, however no specific IgA was detected in the nasal washes of any group. Mice immunized IVAG with rgB + CpG showed higher survival and lower pathology scores following genital HSV-2 challenge than mice immunized with rgB + non-CpG ODN or rgB alone. Additionally, vaginal viral titers were lower in the rgB + CpG group after infection. These results clearly show that the genital tract is capable of generating a protective immune response after local intravaginal immunization and that a non-replicating antigen is able to induce such a response when administered with an appropriate adjuvant.     

A combination of Flt3 ligand cDNA and CpG ODN as nasal adjuvant elicits NALT dendritic cells for prolonged mucosal immunity

We explore cellular and molecular mechanisms of nasal adjuvant of a combination of a plasmid encoding the Flt3 ligand cDNA (pFL) and CpG oligodeoxynucleotides (CpG ODN). The double DNA adjuvant given with OVA maintained prolonged OVA-specific secretory IgA (S-IgA) Ab responses in external secretions for more than 25 weeks after the final immunization. Further, both Th1- and Th2-type cytokine responses were induced by this combined adjuvant regimen. The frequencies of plasmacytoid DCs (pDCs) and CD8(+) DCs were significantly increased in nasopharyngeal-associated lymphoreticular tissue (NALT) of mice given the combined adjuvant. Importantly, when we examined adjuvanticity of pFL plus CpG ODN in 2-year-old mice, significant levels of mucosal IgA Ab responses were also induced. These results demonstrate that nasal delivery of a combined DNA adjuvant offers an attractive possibility for the development of an effective mucosal vaccine for the elderly.     

CpG DNA overcomes hyporesponsiveness to hepatitis B vaccine in orangutans

Oligonucleotides containing immunostimulatory CpG motifs (CpG ODN) have been shown to be potent Th1-type adjuvants for augmenting antigen-specific responses in mice against hepatitis B surface antigen (HBsAg). The hepatitis B virus (HBV) infects only humans and great apes and appears to exist among wild chimpanzees and orangutans. An outbreak of HBV among orangutans being rehabilitated for re-introduction to the jungle caused the death of several animals. A prophylactic vaccination program revealed that orangutans are quite hypo-responsive to a current commercial vaccine compared to results obtained previously in humans and chimpanzees. Addition of CpG ODN to hepatitis B vaccine greatly increased the seroconversion rate and the titers of antibody against HBsAg (anti-HBs). This is the first demonstration of CpG DNA in a great ape and the results have important implications for the vaccination of humans against HBV and other diseases.     

Immunostimulatory oligodeoxynucleotides are potent enhancers of protective immunity in mice immunized with recombinant ORFF leishmanial antigen

Unmethylated CpG dinucleotides in bacterial DNA or synthetic oligonucleotides (ODN) have proved as promising adjuvants for promotion of T helper 1 (Th1) type immune response. The potent Th1 like immune activation by CpG-ODNs suggests a possible utility for vaccination against leishmaniasis. We therefore investigated the effect of ODN containing immunostimulatory CG motifs as adjuvant with recombinant ORFF (rORFF) leishmanial antigen. BALB/c mice were vaccinated with the rORFF with or without CpG-ODN as adjuvant and then challenged with Leishmania donovani metacyclic promastigotes. Administration of CpG-ODN alone resulted in partial protection against challenge with L. donovani in BALB/c mice. Combination of rORFF and CpG-ODN showed enhanced reduction in parasite load (84%) when compared to rORFF (56%) vaccinated mice. Immunization with rORFF alone did not induce the typical Th response whereas co-administration of rORFF with CpG-ODN resulted in enhanced production of immunoglobulin G2a and interferon gamma. Our results further demonstrate that CpG-ODN alone or in combination with rORFF resulted in a dose dependent increase of nitric oxide production in activated macrophages. These studies suggest that CpG-ODN are promising immune enhancers for vaccination against visceral leishmaniasis.     

The level of protection against rotavirus shedding in mice following immunization with a chimeric VP6 protein is dependent on the route and the coadministered adjuvant

Intranasal (i.n.) immunization of BALB/c mice with chimeric murine rotavirus EDIM (epizootic diarrhea of infant mice) VP6 and attenuated E. coli heat-labile toxin (LT), LT(R192G), stimulated >99% protection against rotavirus shedding after EDIM challenge. Here, we evaluated other potential adjuvants with chimeric VP6 administered by two mucosal routes: i.n. and oral. Besides LT(R192G), the adjuvants examined included Adjumer, CpG oligodeoxynucleotides (CpG ODN), chimeric A1 subunit of cholera toxin (CTA1)-DD, and QS-21. All except QS-21 significantly (P<0.05) increased VP6-specific serum IgG responses after i.n. immunization, but none significantly increased these responses when administered orally. The i.n. delivery of chimeric VP6 alone induced both rotavirus IgG1 and IgG2a whose relative titers suggested a skewed Th2-like response. Inclusion of Adjumer greatly increased Th2-like responses, while CpG ODN shifted the response to a less Th2-like response. The adjuvants CTA1-DD, LT(R192G), QS-21 had no significant effect on ratios of IgG1/IgG2a titers. Following EDIM challenge of mice immunized i.n. with chimeric VP6 and either LT(R192G), CTA1-DD, Adjumer or CpG ODN, shedding was reduced >99, 95, 80, 74, respectively, relative to that found in unimmunized mice (P<0.05). QS-21 induced less protection (43%, not significant (N.S.)) while immunization with chimeric VP6 alone reduced shedding by only 16% (N.S.). Oral immunization with chimeric VP6 and all selected adjuvants except QS-21 was less effective than after i.n. immunization, with protection levels of 94 (P<0.05), 71 (P<0.05), 55, 35 and 28% for LT(R192G), QS-21, CpG ODN, CTA1-DD, and Adjumer, respectively, while immunization with chimeric VP6 alone gave no protection. Thus, different adjuvants induced different degrees of protection and oral immunization was generally less effective then the i.n. route.     

Generation of Th1 immune responses to inactivated,gp120-depleted HIV-1 in mice with a dominant Th2 biased immune profile via immunostimulatory [correction of imunostimulatory] oligonucleotides--relevance to AIDS vaccines in developing countries

Vaccination against HIV-1 of hosts with a dominant Th2 immune profile may fail to induce essential protective Th1 immune responses. By using Schistosoma-infected mice, with a pre-existent Th2 immune background, we demonstrate that oligodeoxynucleotides (ODN) containing unmethylated cytosine-phosphate-guanosine (CpG) immunostimulatory sequences co-administered with inactivated, gp120-depleted HIV-1 viral particles (HIV-1 immunogen) lead to potent Th1 anti-HIV-1 immune responses overcoming the Th2 bias. In contrast, Schistosoma-infected mice immunized with HIV-1 immunogen in incomplete Freund's adjuvant only, induced Th2 anti-HIV-1 immune responses. These findings strongly support the advisability of using CpG ODN as a Th1 inducing adjuvant when immunizing human populations with a strong pre-existent Th2 immune profile.     

Immunostimulatory DNA as adjuvant: efficacy of phosphodiester CpG oligonucleotides is enhanced by 3' sequence modifications

The immunostimulatory activity of oligonucleotides containing CpG motifs is well established and represents the basis for an effective vaccine adjuvant. For use in vivo CpG ODN have to be protected from the attack of nucleases to ensure sustained effectiveness. This is usually accomplished by phosphothioate (PTO) modifications of the ODN's backbone. However, PTO modification may induce undesired effects. We, therefore, have attempted to enhance the immunostimulatory activity of CpG phosphodiester ODN by supplementation of the ODN's sequence. We report here, that addition of poly-guanosine runs to the 3'-end of a CpG-PO ODN conveys immunostimulatory activity to CpG-PO ODN in vivo and in vitro. Guanosine-rich PO ODN thus might be an alternative approach to develop effective and safe vaccine adjuvants.     

Maternal treatment with a high dose of CpG ODN during gestation alters fetal craniofacial and distal limb development in C57BL/6 mice

Synthetic oligodeoxynucleotides (ODN) containing CpG motifs, characteristic of bacterial DNA, are currently being evaluated as vaccine adjuvants for inducing protective immunity. Recently, there is increasing pressure to vaccinate pregnant women against maternally transmitted diseases including AIDS and tetanus, as well as against potential bio-weapons such as anthrax. CpG vaccines are effective because they trigger transient increases in T(H)1 cytokine production. Recent literature suggests, however, that a shift toward a T(H)1 cytokine profile during pregnancy may increase the risk of fetal morphologic defects. On this basis, we hypothesized that exposure to CpG motifs during pregnancy could result in T(H)1 inflammation leading to adverse effects on fetal development. To address this hypothesis, pregnant C57BL/6 mice were injected with CpG ODN (0-300 microg/dam) and maternal and fetal outcomes were determined. Injection of dams with the highest dose of CpG ODN resulted in markedly increased fetal resorptions and craniofacial/limb defects, while lower doses had little, if any effects. Histological examination of placentas revealed cellular necrosis with mixed inflammation and calcification in the spongiotrophoblast layer and dysregulation of labyrinthine vascular development. Concomitant elevations in maternal serum cytokine levels were observed including interleukin (IL)-2, IL-10 and IL-12. Treatment with 300 microg of non-CpG ODN did not cause any adverse effects. The 300 microg dose of CpG ODN used in the present study is 30-fold higher than the highest dose that has been administered to humans during clinical trials. These results suggest that the induction of T(H)1 cytokines during pregnancy by CpG motifs may potentially increase the risk of fetal loss and morphologic defects in mice, at least at high doses, and support the need for further investigation of teratogenesis that may result from exposure to vaccine adjuvants designed to produce T(H)1 cytokine profile shifts.     

Oral administration of second-generation immunomodulatory oligonucleotides induces mucosal Th1 immune responses and adjuvant activity

CpG DNA induces potent Th1 immune responses through Toll-like receptor 9. In the present study, we used oligonucleotides consisting of a novel 3'-3'-linked structure and synthetic stimulatory motifs, referred as second-generation immunomodulatory oligonucleotides (IMOs). The stimulatory motifs included: CpR, YpG, or R'pG (R = 2'-deoxy-7-deazaguanosine, Y = 2'-deoxy-5-hydroxy-cytidine, and R' = 1-[2'-deoxy-beta-d-ribofuranosyl]-2-oxo-7-deaza-8-methyl-purine). We evaluated the stability of orally administered IMOs in the gastrointestinal (GI) environment and their ability to induce mucosal immune responses in mice, and compared these characteristics with those of a conventional CpG DNA. The IMOs were significantly more stable than CpG DNA following oral administration, and IMOs induced stronger local and systemic immune responses as determined by MIP-1beta, MCP-1, IP-10, and IL-12 production. Mice orally immunized with ovalbumin (OVA) and IMO had higher levels of IgG2a antibodies in serum and IgA antibodies in intestinal mucosa than did mice immunized with OVA and CpG DNA. These studies demonstrate that IMOs are more stable than CpG DNA in the GI tract and can induce more potent mucosal Th1 adjuvant responses. IMOs may prove to be effective oral adjuvants, able to promote strong systemic and mucosal immune responses to oral vaccines and antigens for therapeutic and prophylactic applications.     

Transcutaneous vaccination with virus-like particles

Virus-like particles (VLP) are inert, empty capsids of viruses, which contain no DNA/RNA from the virus itself. However they retain the structure of a virus and they can be engineered to have antigens attached. We have constructed VLP, derived from Rabbit hemorrhagic disease virus, and shown they are highly immunogenic. We tested the capacity of these engineered VLP to induce immune responses when they are administered to mice via the transcutaneous route. This route of vaccination is important, in order to generate mucosal protection. Our data showed that VLP are taken up by dendritic cells (DC), antigen-presenting cells that are essential to initiate acquired immune responses. The VLP induced an increase in expression of CD40, CD80 and CD86 but required an adjuvant, CpG DNA oligo-deoxy nucleotides (ODN) motifs, to enhance these responses. In vivo testing has also shown that the VLP, when wiped on to the skin in conjunction with immunostimulatory CpG, induce Ag-specific immune responses, typified by high levels of IFN-gamma and IgG1.