These data demonstrate, for the first time, that an immunostimulatory cytokine plasmid and the timing of cytokine administration can significantly affect the immune responses elicited to a vaccine in humans. Administration of the plasmid DNA encoding IL-2/Ig given 48 hours after a plasmid DNA vaccine comprising HIV-1 clade A, B, and C envelopes and a clade B/Gag/Pol/Nef polyprotein significantly augmented T-cell responses, compared with IL-2/Ig simultaneous administration. These observations are consistent with findings from studies in murine and nonhuman primate models [13
], and they have important implications for future development of cytokine therapies used to augment immune responses [36
Augmented T-cell immune responses were observed with both IFN-γ and IL-2 ELISPOT assays, testing responses in fresh cells to homologous peptides from EnvA, EnvB, and EnvC. ELISPOT assays performed in frozen cells with potential T-cell epitope peptides showed a similar response pattern, albeit with lower rates of response than in fresh cells, indicating the likely increased sensitivity with the use of fresh cells and/or matched peptides in this setting [38
ICS assays with potential T-cell epitope peptides on frozen cells showed a trend toward a more durable response at 1 year in the IL-2/Ig group that received IL-2/Ig 48 hours after vaccination, compared with the groups that received IL-2/Ig simultaneously with the DNA vaccine or the DNA vaccine alone. These data suggest that IL-2/Ig administration 48 hours after vaccination may affect the duration as well as the magnitude of T-cell responses. CD4+ responses were generally seen more frequently than CD8+ responses. ELISPOT responses were largely to the envelope proteins, and few responses were seen to Gag, Pol, or Nef.
As expected with a DNA-only vaccine, the humoral responses were relatively weak. Low-level but higher antibody responses were measured in the delayed IL-2/Ig compared with the simultaneous IL-2/Ig group suggesting that cytokine timing may influence antibody responses as well. Thus, further studies with more robust vaccine-elicited antibody responses are needed to assess the impact of cytokine administration on the humoral response.
IL-2/Ig plasmid was generally safe and well tolerated given alone or in combination with the DNA HIV vaccine. Unlike experience with IL-2 given parenterally at higher doses, which can be associated with significant toxicity [39
], only 2 episodes of greater than moderate systemic reactogenicity were seen when IL-2/Ig was administered intramuscularly. Local reactions consisting of mild pain or tenderness at the injection site were the most commonly encountered side effects. No differences were seen in rates of local or systemic reactogenicity among the various treatment groups. The excellent safety profile of the IL-2/Ig delivered by DNA plasmid is likely to be a result of the lower dose of IL-2 delivered only to the vaccine site, probably with little systemic absorption [42
The observation that administration of IL-2/Ig given 48 hours after DNA vaccination augments immune responses compared with simultaneous administration of DNA vaccine and IL-2/Ig is consistent with observations in mice and monkeys. The mechanism for the augmentation effect of IL-2/Ig given 48 hours after vaccination is not known. Because the IL-2/Ig plasmid probably has peak expression 1 day after administration, with expression rapidly tapering by the second day [42
], the precise timing of IL-2 stimulation of antigen-specific cells may be a critical factor. Simultaneous administration of IL-2 and antigen may stimulate a broad cellular response, with relatively fewer antigen-specific cells stimulated by IL-2 than when IL-2 is administered 48 hours after these cells have already been stimulated by vaccine antigens. This would presumably mimic the events in natural infection, wherein IL-2 is typically secreted as part of a cytokine cascade after antigen is presented by antigen-presenting cells and triggers T cells. Therefore, it is not surprising that plasmid IL-2/Ig is most effective when delivered after the antigen. It is also possible that simultaneous administration of IL-2/Ig with vaccine antigen impairs the elicitation of the T cell responses. This further indicates the importance of the timing of cytokine administration on immune response, which was also observed in the animal models [24
]. Ultimately, the mechanism is unclear, but we suspect it may be related to improved antigen presentation and T lymphocyte priming. The possibility of the adjuvant affecting T lymphocyte phenotype is of interest and worth exploring in future studies but was not specifically addressed in ours.
Taken together, these data demonstrate, for the first time in humans, that cytokine administration can augment immune responses to vaccination but that the timing of administration can have a significant effect on whether augmentation occurs. Further studies will be required to determine the optimal timing of regimens. These observations may have important implications for the design and interpretation of studies that use cytokines as adjuvants in humans [36
], although it is likely that optimal timing of administration of the cytokine may be different for each cytokine and for the type of immune response sought.