DNA vaccination offers the potential to induce immunity to viral, bacterial, and tumor antigens as well as allergens (9
). This study investigated methods that could possibly enhance this mode of immunization by coinjection of cytokine genes that modulate the magnitude or profile of HIV antigen-specific immune responses. Here, immune analyses were performed using an inbred mouse strain, with the same expression vector for vaccination, the same vector for coadministration of immunomodulators, and comparable levels of protein synthesis. Despite this consistency, different immune responses to Nef and Env were detected, attributable only to the immunogen, i.e., the cDNA insert was the only difference between the alternative vectors. Expression of cytokines and hematopoietic factors from coinjected plasmid vectors can significantly increase antigen-specific cellular or humoral immune responses, and a synergism was observed between selected immunomodulators, but the predominance of a cellular or humoral response was not reversed by the genetic adjuvants tested in this study. Despite similar levels of gene expression, it is unsurprising that the immune responses to these antigens differ. Other factors, such as folding, oligomerization, secretion or localization in different subcellular organelles (endoplasmic reticulum, Golgi apparatus, or cytoplasm), or affinity for major histocompatibility complex-encoded molecules may also affect the character and potency of the response. This finding highlights a significant difference between DNA immunization with genetic adjuvants and traditional subunit or live vector vaccine strategies.
The induction of a strong anti-Nef antibody response and CD4+
Th cell responses indicates that the antigen was not fully sequestered in the cytosol and was capable of priming CD4+
Th and B cells to differentiate and produce cytokines and antibodies. This result suggests that protein may have been secreted from the transfected cell or released from dying cells, as with influenza nucleoprotein (30
), or from immune-mediated lysed cells (8
). Furthermore, an anti-Nef cytotoxic response can be induced when mice are immunized with recombinant vaccinia virus expressing Nef (21
), suggesting that induction of immune responses to an antigen expressed from plasmid DNA can be different from that in response to viral vectors. It is known from previous studies using DNA priming and adenovirus boosting in an Ebola virus model (28
), for example, that 10- to 100-fold increases in antibody titer can be seen when using an adenoviral vector boost compared to a boosting immunization with plasmid DNA alone encoding the same antigen in primates. In contrast to the anti-Nef responses, immunization with a plasmid encoding the full-length membrane-bound Env induced a strong CTL response, suggesting that the number of effective antigen-presenting cells was sufficient to induce a persistently strong anamnestic CTL response through presentation of Env epitopes in class I molecules. Recently, mutations have been identified in this codon-modified Env plasmid that elicit substantial antibody responses by DNA immunization, with comparable levels of protein synthesis (1
; Chakrabarti et al., submitted). This result demonstrates that regions of the Env protein determine its ability to elicit an antibody response by DNA vaccination.
The lower antibody response to Env suggests that the expressed gp160 antigen may not present conformational epitopes that induce the activation and differentiation of B cells. For immunization with DNA, the induction of a predominantly humoral or cellular immune response, therefore, was also an inherent property of the antigen, in contrast to live vector or subunit vaccine strategies. In the last two cases, the effects of the formulation or the inflammatory properties of the virus can dominate adjuvant determinants of the antigen. Although bacterial DNA can induce innate immune responses (10
), pNGVL-Nef and pNGVL-gp160 contain similar numbers and types of known immunostimulatory sequences, suggesting that the DNA sequence of the plasmid is unlikely to account for the differences in Nef and Env immune responses.
It has been observed that primate immune responses induced by DNA vaccination may be less potent than those observed in murine models. However, early results suggest that DNA immunization followed by a heterologous boost may be an extremely effective vaccine regimen in primates (28
). A better understanding of interspecies discrepancies will be required to extrapolate the results of rodent studies to humans. On the other hand, if differences in immune responses are related to inherent properties of an antigen, they could be maintained, possibly with quantitative differences, across species.
Repeated immunization with pNGVL-Nef enhanced the induction of humoral responses, with a significant increase in the IgG1 titers, and did not result in the induction of a CTL response or a significant increase in cell-mediated immunity, suggesting that some genetic vaccines may optimize humoral immunity without increasing cellular immunity after multiple injections. The requirement for multiple immunizations to boost initial antigen-specific cellular immunity may be reduced by coinjection of cytokine-expressing plasmids. This method of cytokine delivery may also represent a safer and more effective alternative to repeated systemic administration of the recombinant protein, which can be toxic (16
) or short-lived due to a low serum half-life (3
). For example, IL-12 exerts side effects when delivered as protein, but coinjection of IL-12 plasmid with the Nef expression vector enhanced Th1 and antibody responses but did not lead to obvious splenomegaly or markedly increased numbers of white blood cells.
We observed an antigen-specific effect on the capacity of individual genetic adjuvants to modulate immune responses. The effects of coinjection differed when pNGVL-gp160 was used instead of pNGVL-Nef; in the former case, GM-CSF, not IL-12, induced the most significant increases in Env-specific CD4+
Th1 and CD8+
CTL responses. These differences in the abilities of the genetic adjuvants to modulate the immune response were dependent on the antigen. The different effects of cytokines on these responses may reflect their underlying mechanisms of action. For example, in the predominantly antibody-driven response to Nef, IL-12 was most effective, likely due to its fundamental role in driving Th1 cell differentiation and in the stimulation of non-B-cell responses (29
). In contrast, the strong cell-mediated immune response to gp160 may not require IL-12 to increase this already polarized response. Instead, the actions of GM-CSF, by boosting the antigen-presenting population that activates T cells (6
), may exert more complementary effects on this response. It is interesting that the FL plasmid did not induce such a potent effect as the GM-CSF plasmid in pNGVL-gp160-immunized mice. The capacity for these two growth factors to stimulate expansion of distinct subsets of dendritic cells may explain this effect (24
). These findings suggest that genetic adjuvants must be chosen with consideration of the antigen-driven profile of the immune response, and an individual genetic adjuvant may not act universally for all DNA vaccines.
A cooperative effect in Th1 cytokine induction between FL and other cytokines was observed at lower stimulatory concentrations of Nef or Env peptide. These results demonstrate that FL alone can increase immune responses after DNA immunization; however, the FL-enhanced cell populations, predominantly dendritic cells, may be more effective in the presentation of antigen to T cells modulated by cytokines, such as IL-12, IL-2, or IL-15. Furthermore, in contrast to the effects of individual cytokines, this cooperative effect between FL and other cytokines was independent of the immunizing antigen; however, the expansion of progenitor populations must be simultaneous with modulation of the effector immune response. Cooperativity was reduced when FL was administered only on the first immunization and cytokines were coinjected on subsequent immunizations. This approach to generate a potent immune response by concurrent stimulation of hematopoiesis and the adaptive immune response may prove more useful at lower doses of antigen or viral challenge. This vaccination strategy is also less dependent on the immunizing antigen and may be more convenient than injection of individual cytokines or growth factors. Such genetic strategies may contribute to vaccine development for HIV and other infectious pathogens.