Co-administration of polyphosphazenes with CpG oligodeoxynucleotides strongly enhances immune responses in mice immunized with Hepatitis B virus surface antigen.

An emerging paradigm in vaccinology is that multiple adjuvant combinations may be more effective than individual adjuvants in enhancing immune responses to vaccine antigens. We investigated whether the polyphosphazenes used in combination with CpG oligodeoxynucleotides (ODN) were potent adjuvant formulations. BALB/c mice were immunized subcutaneously with Hepatitis B surface antigen (HBsAg) alone, or in various combinations with poly[di(sodium carboxylatophenoxy)phosphazene] (PCPP), poly[di(sodium carboxylatoethylphenoxy)phosphazene] (PCEP) or CpG ODN. All three adjuvants enhanced HBsAg-specific IgG1 antibody responses with PCEP inducing the highest responses. PCEP and CpG ODN significantly enhanced the Th1-associated antibody isotype IgG2a. As expected CpG ODN induced predominantly Th1-type immune responses while PCEP was associated with mixed Th1/Th2 immune responses. Interestingly, PCEP and PCPP synergized with CpG ODN to further enhance antibody responses. Since the mechanisms which mediate the adjuvant activity of polyphosphazenes are not fully understood, we investigated whether PCEP and PCPP could stimulate innate immune responses. Incubation of mouse splenocytes with PCEP or PCPP (in the absence of antigen) stimulated production of IL-4 and IL-12, but only PCEP induced significant IFNgamma production. Additionally, IL-12 was not required for PCEP induced IFNgamma response. We conclude that the polyphosphazene-CpG ODN combination is a potent adjuvant formulation that is more effective in enhancing immune responses than either of the individual adjuvants. In addition, we provide evidence that PCEP and PCPP can stimulate innate cytokine production, suggesting a potential mechanism by which polyphosphazenes achieve their potent adjuvant effects.

Poly[di(sodium carboxylatoethylphenoxy)phosphazene] (PCEP) is a potent enhancer of mixed Th1/Th2 immune responses in mice immunized with influenza virus antigens.

We investigated the ability of a novel polyphosphazene polyelectrolyte, poly[di(sodium carboxylatoethylphenoxy)phosphazene] (PCEP) to enhance antigen-specific immune responses. BALB/c mice were immunized once subcutaneously with either bovine serum albumin (BSA) or influenza virus X:31 antigen alone, or in combination with PCEP, or either of the adjuvants poly[di(sodium carboxylatophenoxy)phosphazene] (PCPP) and alum. Both PCEP and PCPP significantly enhanced serum antigen-specific total IgG, IgG1 and IgG2a antibody titers, and these responses were highest in PCEP-immunized mice. Alum induced only a modest enhancement of antibody responses. Reducing the dose of X:31 antigen by 25-fold had no effect on antibody responses in mice immunized with PCPP and PCEP, but resulted in reduced titers in those immunized with alum. Analysis of X:31 antigen-specific cytokines revealed that alum and PCPP were associated with a predominantly IL-4 response. In contrast, PCEP was associated with production of both IFNgamma and IL-4. We conclude that PCEP is a potent enhancer of antigen-specific Th1 and Th2 immune responses and is a promising adjuvant for vaccine applications.