Induction of autoantibody production but not autoimmune disease in HEL transgenic mice vaccinated with HEL in combination with CpG or control oligodeoxynucleotides

CpG oligodeoxynucleotides (ODN) are synthetic DNA sequences that mimic bacterial DNA, and bind to the TLR9 receptor. The cells that express TLR9, B cells and dendritic cells, are stimulated by CpG ODN and induce innate and acquired immune responses. Because CpG ODN induce antigen-independent immune activation there has been much interest in the possibility that they may break self tolerance. To test this hypothesis we used a tolerance model with hen egg lysozyme (HEL)-transgenic (Tg) mice, anti-HEL Ig-Tg mice and double (Dbl)-Tg mice injected with CpG ODN alone or together with HEL self antigen. When cultured in vitro, tolerant B cells responded to CpG ODN in a similar way as the non-tolerant Ig-Tg B cells in terms of cell proliferation, NFkappaB activation and CD69 expression. Despite these potent in vitro stimulatory effects of CpG ODN alone, HEL-Tg mice injected with CpG ODN alone, or in combination with low dose antigen (4 microg HEL), surprisingly did not produce any detectable anti-HEL Ab. However, HEL-Tg or Dbl-Tg mice immunized with CpG ODN plus higher doses of self antigen showed strong antigen-specific humoral responses. Surprisingly, control non-CpG ODN also had partial activity for breaking tolerance and inducing autoantibody production when administered in combination with self antigen, though not when used alone. Despite the production of high titers of anti-HEL Ab in the immunized HEL-Tg mice, no evidence of autoimmune disease was detected. We conclude that immunization with CpG or control ODN in the presence of a high dose of exogenous self antigen, but not treatment with ODN alone, can break tolerance to self antigen without inducing autoimmune disease in this system.     

Liposomal SLA co-incorporated with PO CpG ODNs or PS CpG ODNs induce the same protection against the murine model of leishmaniasis

First generation Leishmania vaccines consisting of whole killed parasites with or without adjuvants have reached phase 3 trial and failed to show enough efficacy mainly due to the lack of an appropriate adjuvant. In this study, the nuclease-resistant phosphorothioate CpG oligodeoxynucleotides (PS CpG) or nuclease-sensitive phosphodiester CpG ODNs (PO CpG) were used as adjuvants to enhance immunogenicity and rate of protection against leishmaniasis. Due to the susceptibility of PO CpG to nuclease degradation, an efficient liposomal delivery system was developed to protect them from degradation. 1, 2-dioleoyl-3-trimethylammonium-propane (DOTAP) as a cationic lipid was used because of its unique adjuvanticity and electrostatic interaction with negatively charged CpG ODNs. To evaluate the role of liposomal formulation in protection rate and enhanced immune response, BALB/c mice were immunized subcutaneously with liposomal soluble Leishmania antigens (SLA) co-incorporated with PO CpG (Lip-SLA-PO CpG), Lip-SLA-PS CpG, SLA+PO CpG, SLA+PS CpG, SLA or buffer. As criteria for protection, footpad swelling at the site of challenge, parasite loads, the levels of IFN-γ and IL-4, and the IgG subtypes were evaluated. The groups of mice receiving Lip-SLA-PO CpG or Lip-SLA-PS CpG showed a high protection rate compared with the control groups. In addition, there was no significant difference in immune response generation between mice immunized with PS CpG and the group receiving PO CpG when incorporated into the liposomes. The results suggested that liposomal form of PO CpG might be used instead of PS CpG in future vaccine formulations as an efficient adjuvant.     

TLR agonists as vaccine adjuvants: comparison of CpG ODN and Resiquimod (R-848)

TLR ligands that mimic pathogen associated molecular patterns and activate immune cells via Toll-like receptors (TLRs) are being developed for use in humans as therapy against a variety of diseases as well as vaccine adjuvants. These include imidazoquinoline compounds such as Imiquimod and Resiquimod (R-848) that bind to TLR7 and 8, as well as CpG oligodeoxynucleotides (CpG ODN) that bind to TLR9. This study was aimed at comparing CpG ODN and R-848 for their potential use as vaccine adjuvants and to determine whether there are additive or synergistic effects when they are used together. Using HBsAg as a model antigen in mice, we show CpG ODN to be superior to R-848 for augmenting both humoral and cell mediated immune responses.     

CpG-containing oligodeoxynucleotides augment and switch the immune responses of cattle to bovine herpesvirus-1 glycoprotein D

The adjuvanticity of a synthetic oligodeoxynucleotide containing unmethylated CpG motifs (CpG ODN) was determined in cattle. Calves were immunized with a truncated secreted version of glycoprotein D (tgD) of bovine herpes virus-1 (BHV-1) formulated with alum, CpG ODN, or a combination of both. BHV-1 tgD formulated with CpG ODN or with alum and CpG ODN induced a stronger and more balanced immune response than tgD in alum. This level of immunity was of sufficient magnitude to minimize weight loss and significantly reduce the duration of virus shedding after intranasal viral challenge. Local tissue reactions generated by CpG ODN were very mild and transient, whereas reactions induced by alum or a combination of CpG ODN and alum were moderate in severity and duration. These data demonstrate that CpG ODN causes minimal injection site reactions and yet acts as an effective adjuvant in cattle.     

Cutaneous CpG adjuvant conditioning to enhance vaccine responses

Adjuvant activity of the Toll receptor 9 agonist CpG 1826 was compared when given subcutaneously (s.c.) together with ovalbumin (s.c.[CpG + Ova]), or when given by either s.c. or intradermally (i.d.) routes two days prior to s.c. ovalbumin. Frequencies of CD8 + effector (T_EFF) and central memory (T_CM) T cells along with total IgG, IgG2c, and IgG1 titres were measured to ascertain how timing and location of CpG conditioning influenced vaccination outcome. Prior treatment with CpG enhanced T_EFF, T_CM, as well as total IgG responses. T_EFF and T_CM responses were greatest when CpG was given intradermally and prior to s.c. ovalbumin, conditions that eliminated the fraction of T_CM ‘non-responders’ observed after s.c.[CpG + Ova] vaccination. IgG responses were polarized toward IgG2c after early s.c. CpG but toward IgG1 after early i.d. CpG. Separating CpG adjuvant and antigen application in time and space can improve vaccination outcome.     

Comparison of the protective effects of killed Burkholderia pseudomallei and CpG oligodeoxynucleotide against live challenge

Melioidosis is a fatal disease caused by Burkholderia pseudomallei. Currently there is no vaccine available. Synthetic oligodeoxynucleotides with unmethylated CpG dinucleotide motifs (CpG ODN) can stimulate vertebrate immune cells and clear certain pathogens that are susceptible to a strong Th1 response. In our previous study, pretreatment with CpG ODN alone or CpG-ODN with cationic liposomes for 2–10 or 30 days before B. pseudomallei infection in mice conferred 80–100% protection. In the present study we investigated the protective effect of CpG-ODN together with heat-killed (HK) or paraformaldehyde-killed B. pseudomallei (PP). HK or PP were used to immunize BALB/c mice twice at 15-day intervals before intra-peritoneal challenge with 5LD50 of B. pseudomallei and observed for 30 days. We found that PP could significantly protect mice (60%) with an increased survival time (24.8 ± 11.63 days) while in the HK and PBS groups, all infected mice died within 6 days. Although either CpG ODN or PP conferred significant protection, giving them in combination did not enhance it further. Serum IFN-γ levels on day-5 (before challenge) of the PP and PP + CpG ODN groups were significantly higher than those of the PBS control group. The results further support the importance of IFN-γ in host protection against B. pseudomallei and suggest further study on paraformaldehyde-killed bacteria as a component of a future B. pseudomallei vaccine.     

Feeding pregnant rats a protein-restricted diet persistently alters the methylation of specific cytosines in the hepatic PPAR alpha promoter of the offspring

Induction of an altered phenotype by prenatal under-nutrition involves changes in the epigenetic regulation of specific genes. We investigated the effect of feeding pregnant rats a protein-restricted (PR) diet with different amounts of folic acid on the methylation of individual CpG dinucleotides in the hepatic PPAR alpha promoter in juvenile offspring, and the effect of the maternal PR diet on CpG methylation in adult offspring. Pregnant rats (five per group) were fed 180 g/kg casein (control) or 90 g/kg casein with 1 mg/kg folic acid (PR), or 90 g/kg casein and 5 mg/kg folic acid (PRF). Offspring were killed on postnatal day 34 (five males and females per group) and day 80 (five males per group). Methylation of sixteen CpG dinucleotides in the PPAR alpha promoter was measured by pyrosequencing. Mean PPAR alpha promoter methylation in the PR offspring (4.5 %) was 26 % lower than controls (6.1 %) due to specific reduction at CpG dinucleotides 2 (40 %), 3 (43 %), 4 (33 %) and 16 (48 %) (P < 0.05). There was no significant difference in methylation at these CpG between control and PRF offspring. Methylation of CpG 5 and 8 was higher (47 and 63 %, respectively, P < 0.05) in the PRF offspring than control or PR offspring. The methylation pattern in day 80 PR offspring was comparable to day 34 PR offspring. These data show for the first time that prenatal nutrition induces differential changes to the methylation of individual CpG dinucleotides in juvenile rats which persist in adults.     

A novel triple adjuvant formulation promotes strong, Th1-biased immune responses and significant antigen retention at the site of injection

Ovalbumin (OVA) was labeled with a near infra-red dye (*OVA) and formulated with the host defense peptide indolicidin (Indol), CpG oligodeoxynucleotide (ODN) 1826 (CpG) and/or poly(p-dicarboxylatophenoxy)-phosphazene (PP4). The immunogenicity of these *OVA formulations was evaluated in mice. All double and triple adjuvant combinations elicited strong antibody responses. *OVA formulated with CpG ODN in combination with indolicidin, PP4 or both induced only IFN-γ, while formulations with indolicidin and/or PP4 promoted predominantly IL-5 production. Overall, both IgG and IFN-γ production was superior when *OVA was combined with CpG/Indol/PP4. Furthermore, mice injected with *OVA formulated with CpG/Indol/PP4 contained detectable *OVA in the injection site two months post immunization. These results indicate that the CpG/Indol/PP4 combination promotes prolonged antigen retention and strong, antigen-specific Th1-biased immune responses.     

Safety and efficacy of CpG-containing oligodeoxynucleotides as immunological adjuvants in rabbits

The efficacy of oligodeoxynucleotides containing unmethylated CpG dinucleotides (CpG ODN) as an adjuvant for rabbits was assessed alone and in combination with aluminum hydroxide (CpG/alum). The CpG/alum combination elicited a greater immune response to several antigens compared to Freund's adjuvant. A non-CpG/alum combination did not have the same effects as CpG/alum suggesting that the adjuvanticity was related to the CpG motifs. In addition, we formulated one of the antigens with combinations of CpG ODN and 30 or 10% Emulsigen (Em) [CpG/Em (30%) and CpG/Em (10%)]. Both CpG/Em (30%) and CpG/Em (10%) were more effective than Em, and equivalent to CpG/alum. The CpG/Em (10%) combination caused minimal tissue damage. Our results demonstrate that the addition of CpG ODN to aluminum hydroxide or to 10% Em significantly improves the efficiency of these adjuvants, without enhancing tissue reaction.     

Induction of protective immunity by vaccination against Chlamydia trachomatis using the major outer membrane protein adjuvanted with CpG oligodeoxynucleotide coupled to the nontoxic B subunit of cholera toxin

The present study was undertaken to test the efficacy of immunization with the native major outer membrane protein (nMOMP) of Chlamydia trachomatis mouse pneumonitis (MoPn) serovar in combination with a novel immunostimulatory adjuvant consisting of CpG oligodeoxynucleotide (ODN) linked to the nontoxic B subunit of cholera toxin (CTB-CpG) to elicit a protective immune response to C. trachomatis. High levels of Chlamydia-specific IgG antibodies were detected in the sera from BALB/c mice immunized intramuscularly and subcutaneously (i.m. + s.c.) with the nMOMP/CTB-CpG vaccine or with nMOMP adjuvanted with a mixture of CT and CpG ODN (CT + CpG). Further, these immunization schemes gave rise to significant T-cell-mediated Chlamydia-specific immune responses. No Chlamydia-specific humoral or cell-mediated immune responses were detected in the control mice vaccinated with ovalbumin together with either CTB-CpG or CT + CpG. Following an intranasal challenge with C. trachomatis the groups of mice immunized with nMOMP plus CTB-CpG, CT + CpG or live C. trachomatis were found to be protected based on their change in body weight and lung weight as well as number of inclusion forming unit recovered from the lungs, as compared with control groups immunized with ovalbumin plus either adjuvants. Interestingly, IFN-γ-producing CD4⁺, but not CD8⁺, T-cells showed a significant correlation with the outcomes of the challenge. In conclusion, nMOMP in combination with the novel adjuvant CTB-CpG elicited a significant antigen-specific antibody and cell-mediated immune responses as well as protection against a pulmonary challenge with C. trachomatis.     

Immunoprotective activity of a Salmonid Alphavirus Vaccine: comparison of the immune responses induced by inactivated whole virus antigen formulations based on CpG class B oligonucleotides and poly I:C alone or combined with an oil adjuvant

CpG oligonucleotides and polyinosinic:polycytidylic acid (poly I:C) are toll-like receptor (TLR) agonists that mimic the immunostimulatory properties of bacterial DNA and double-stranded viral RNA respectively, and which have exhibited potential to serve as vaccine adjuvants in previous experiments. Here, a combination of CpGs and poly I:C together with water- or oil-formulated Salmonid Alphavirus (SAV) antigen preparations has been used for a vaccine in Atlantic salmon and tested for protection in SAV challenge trial. The results demonstrate that vaccination with a high dose of the SAV antigen induced protection against challenge with SAV which correlated with production of neutralizing antibodies (NAbs). As the high antigen dose alone induced full protection, no beneficial effect from the addition of CpG and poly I:C could be observed. Nevertheless, these TLR ligands significantly enhanced the levels of NAbs in serum of vaccinated fish. Interestingly, gene expression analysis demonstrated that while addition of oil suppressed the CpG/poly I:C-induced expression of IFN-γ, the upregulation of IFNa1 was substantially enhanced. A low dose of the SAV antigen combined with oil did not induce any detectable levels of NAbs either with or without TLR ligands present, however the addition of CpG and poly I:C to the low SAV antigen dose formulation significantly enhanced the protection against SAV suggesting that CpG/poly I:C may have enhanced a cytotoxic response - a process which is dependent on the up-regulation of type I IFN. These results highlight the immunostimulatory properties of the tested TLR ligands and will serve as a ground for further, more detailed studies aimed to investigate their capacity to serve as adjuvants in vaccine formulations for Atlantic salmon.     

Effects of CpG ODN on CD4+ and CD8+ T subpopulations in the immune response to porcine reproductive and respiratory syndrome killed virus vaccine

CpG ODN is a noval immunostimulatory reagent In this research, the effects of immunostimulatory CpG oligodeoxynucleotides (CpG ODN) on CD4+ and CD8+ T lymphocytes subpopulations in the newborn piglets blood were tested at different time with porcine reproductive and respiratory syndrome killed virus vaccine (PRRS vaccine) with or without CpG ODN. The results suggested that, the CD4+/CD8+ ratio decreased with age in piglets inoculated with vaccine alone or GpC ODN with vaccine or phosphate buffer saline (PBS), however, it was stable in piglets co-inoculated with CpG ODN and PRRS vaccine (p>0.05), the use of CpG ODN can prevent effectively the reduction of the proportion of CD4+ T lymphocytes. High titers of PRRS specific antibody can also be tested in the newborn piglets serum immunized PRRS vaccine and CpG ODN (p<0.05).     

CpG oligodeoxynucleotide enhances immunity against blood-stage malaria infection in mice parenterally immunized with a yeast-expressed 19 kDa carboxyl-terminal fragment of Plasmodium yoelii merozoite surface protein-1 (MSP1(19)) formulated in oil-based Montanides

The 19kDa carboxyl-terminal fragment of Plasmodium yoelii merozoite surface protein-1 (MSP1(19)), an analog of the leading falciparum malaria vaccine candidate, induces protective immunity to challenge infection when formulated with complete/incomplete Freund's adjuvant (CFA/IFA), an adjuvant unsuitable for use in humans. In this study, we investigate Montanide ISA51 and Montanide ISA720 as well as CpG oligodeoxynucleotide (ODN) as adjuvants for induction of immunity to MSP1(19). Mice immunized with MSP1(19) adjuvanted with Montanide ISA51 were protected even though some mice experienced low-grade parasitemia before resolving the infection. Mice immunized with MSP1(19) adjuvanted with Montanide ISA720 showed delayed patent parasitemia with all mice ultimately succumbing to infection. Interestingly, when the synthetic CpG ODN 1826 was included in either Montanide formulation, mice were completely protected with no parasites detected in the blood. MSP1(19)-specific antibodies in MSP1(19)-immunized mice adjuvanted with Montanide ISA51 or Montanide ISA720 showed predominantly IgG1 antibody and low levels of IgG2a. CpG ODN 1826 significantly enhanced both IgG1 and IgG2a antibody responses in Montanide ISA51-adjuvanted mice but significantly enhanced only the IgG2a antibody response in Montanide ISA720-adjuvanted mice. To investigate the relative roles of antibody and CD4(+) T cells in protection, MSP1(19)-immunized mice adjuvanted with Montanide ISA720 and CpG ODN 1826 were depleted of CD4(+) T cells just prior to challenge. Results showed that three of nine immunized/T cell depleted mice died following infection. These results suggest that antibody and CD4(+) T cells are critical for protection following immunization with MSP1(19) adjuvanted with Montanide and CpG ODN and that the formulation of a human malaria vaccine candidate in Montanide ISA720 or ISA51 together with human compatible CpG ODN would be useful for improving efficacy.     

Increased potency of BioThrax anthrax vaccine with the addition of the C-class CpG oligonucleotide adjuvant CPG 10109

The inclusion of an adjuvant, in addition to the existing aluminum hydroxide, in the formulation of the licensed anthrax vaccine BioThrax may have the potential to positively modify immune responses. Some potential desirable outcomes from the inclusion of an additional adjuvant include increased immune response kinetics, increased response rates, more prolonged antibody decay rates, and the ability to use less antigen per dose or fewer doses to achieve immunity. One promising group of adjuvants that is being investigated with a variety of vaccines and which has been shown to cause many of these effects are oligonucleotides which contain unmethylated CpG motifs. The C-class oligonucleotide CPG 10109, constructed of a mixed phosphorothioate/phosphodiester backbone and containing 3 CpG motifs, was added to various dilutions of BioThrax and used in mouse and guinea pig immunogenicity studies. Anti-protective antigen (PA) IgG ELISAs and the anthrax toxin neutralization assay (TNA) were performed on serum samples from both species. Anti-PA IgG and TNA responses were approximately 10-fold higher after a single dose of undiluted or diluted BioThrax upon addition of 100 microg CPG 10109 in the mouse regardless of the route of immunization. Responses were also significantly greater in the guinea pig after receiving CpG-adjuvanted undiluted BioThrax or CpG-adjuvanted BioThrax diluted 1:5, 1:10 or 1:30 compared to those achieved with BioThrax alone. A guinea pig spore challenge study showed that a single injection of BioThrax vaccine diluted 1:10 in the presence of 25 microg CPG 10109 was as protective as undiluted BioThrax, whereas a single injection of BioThrax diluted 1:10 was not protective. Taken together with the results from the immunogenicity studies, these results suggest that a CpG adjuvant could be used to reduce the dose of active ingredient required to elicit a protective response, and could lead to improved immune response kinetics.     

CpG containing oligodeoxynucleotides are potent adjuvants for parenteral vaccination with the fusion (F) protein of respiratory syncytial virus (RSV)

The feasibility of using oligodeoxynucleotides (ODN) containing unmethylated CpG motifs as parenteral adjuvants for subunit vaccines against RSV was tested in BALB/c mice. Compared with immunization with natural F protein adsorbed to aluminum hydroxide (F/AlOH) adjuvant alone, coadministration of F/AlOH with CpG ODN resulted in statistically significant increases in serum neutralization titers, an enhanced generation of splenic antigen-dependent killer cell precursors, and accelerated clearance of infectious virus from lungs 4 days after challenge. The statistically significant increases in serum IFNγ and anti-F protein IgG2a titers, and significantly diminished pulmonary IL-5 and eosinophilia after challenge indicated that CpG ODN enhanced the ability of F/AlOH to elicit type 1 immune responses. F protein-specific serum IgE titers were also reduced. Further analysis of pulmonary inflammatory cells demonstrated an expansion of CD8⁺ T cells, relative to the CD4⁺ T cell compartment. The potency of CpG ODN was not adversely affected in gene knockout mice devoid of the p35 chain of the IL-12 heterodimer. Taken together, the results suggest a novel formulation for naïve recipients of F protein-based subunit vaccines that does not result in a type 2 phenotype.     

Systemic immune responses to an inactivated,whole H9N2 avian influenza virus vaccine using class B CpG oligonucleotides in chickens

Commercial vaccines against avian influenza viruses (AIV) in chickens consist mainly of inactivated AIV, requiring parenteral administration and co-delivery of an adjuvant. Limitations in T helper 1 or T helper 2 biased responses generated by these vaccines emphasize the need for alternative, more efficacious adjuvants. The Toll-like receptor (TLR) 21 ligand, CpG oligodeoxynucleotides (ODN), has been established as immunomodulatory in chickens. Therefore, the objective of this study was to investigate the adjuvant potential of high (20 μg) and low (2 μg) doses of CpG ODN 2007 (CpG 2007) and CpG ODN 1826 (CpG 1826) when administered to chickens with a formalin-inactivated H9N2 AIV. Antibody responses in sera were evaluated in 90 specific pathogen free (SPF) chickens after intramuscular administration of vaccine formulations at 7 and 21 days post-hatch. Antibody responses were assessed based on haemagglutination inhibition (HI) and virus neutralization (VN) assays; virus-specific IgM and IgY antibody responses were evaluated by ELISA. The results suggest that the vaccine formulation containing low dose CpG 2007 was significantly more effective at generating neutralizing (both HI and VN) responses than formulations with high or low doses of CpG 1826 or high dose CpG 2007. Neutralizing responses elicited by low dose CpG 2007 significantly exceeded those generated by a squalene-based adjuvanted vaccine formulation during peak responses. A significantly higher IgM response was elicited by the formulation containing low dose CpG 2007 compared to high and low doses of 1826. Although the low dose of CpG 2007 elicited a higher IgY response than CpG 1826, the difference was not statistically significant. In conclusion, 2 μg of CpG 2007 is potentially promising as a vaccine adjuvant when delivered intramuscularly with inactivated H9N2 virus to chickens. Future studies may be directed at determining the mucosal antibody responses to the same vaccine formulations.     

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.     

Role of interferon-gamma during CpG oligodeoxynucleotide-adjuvanted immunization with recombinant proteins

Synthetic oligonucleotides (ODNs) containing immunostimulatory CpG motifs (CpG) are a new class of adjuvants suitable for the development of recombinant vaccines. Here we describe that endogenous interferon (IFN) was critical for the adjuvant activity of CpG ODN as genetically deficient mice developed significantly lower IgG antibody titers following immunization with recombinant proteins. In contrast, the absence of endogenous IL-12/IL-23 or IL-4 had little impact on the magnitude of the antibody response but instead caused a dramatic change in the pattern of IgG isotypes. The dependence on IFN-gamma was specific for CpG ODN and it was not observed with other adjuvants tested. IFN-gamma was produced by NK, dendritic cells, CD4+ and CD8+ T cells stimulated in vitro with CpG ODN. Adoptive transfer experiments confirmed that CD4+ or CD8+ T cells were in fact relevant sources of IFN-gamma in vivo. Following CpG ODN injection, splenic dendritic cells from IFN-gamma deficient mice did not up-regulate CD86 or CD40 expression, suggesting a role for these molecules. The importance of CD28 (CD86 ligand) was confirmed using CD28 deficient mice which presented severely impaired immune responses following CpG ODN-assisted immunization.     

Effective induction of CD8+ T-cell response using CpG oligodeoxynucleotides and HER-2/neu-derived peptide co-encapsulated in liposomes

CpG oligodeoxynucleotides (CpG ODN) have been shown to have potent adjuvant activity for a wide range of antigens. Of particular interest is their improved activity when closely associated with the antigen. The purpose of this study is to determine the potential benefit of liposomes as a co-delivery vehicle to enhance the adjuvant activity of CpG ODN for a HER-2/neu-derived peptide to induce CD8+ T-cell response. Immunization studies were performed to evaluate the effectiveness of the liposomal vaccine in BALB/c mice. Mice were immunized with p63-71 encapsulated in liposomes alone or in combination with CpG ODN, as well as p63-71 alone in saline or with peptide-pulsed dendritic cells (DC) as controls. Enzyme-linked immunospot assay (ELISPOT) assay was performed to measure the frequency of splenocytes secreting IFN-gamma as a means to determine the antigen-specific response. It was found that immunization using p63-71 co-encapsulated with CpG ODN within the same liposomes enhanced the antigen-specific IFN-gamma response by more than 100-fold when compared with mice immunized with p63-71 alone. Immunization using free CpG ODN plus p63-71 encapsulated in liposomes or p63-71 and CpG ODN encapsulated in separate liposomes could not achieve the same effect. Using CD8 as a second marker and intracellular flow cytometric analysis, it was found that the IFN-gamma response was contributed by CD8+ T-cells, confirming the induction of cytotoxic T-lymphocytes (CTL) by this vaccination method. This indicates that a close association of HER-2/neu peptide and CpG ODN inside liposomes enhances the CTL epitope delivery and induces CD8+ mediated immune response. These results suggest that a vaccinal approach using liposome delivery system carrying in self-tumoral epitope and CpG ODN as adjuvant may have important implications for cancer therapy.