This 4-plasmid HIV-1 DNA candidate vaccine was assessed as safe and well tolerated in healthy uninfected adults. Importantly, DNA immunization induced both HIV-1–specific antibody and T cell responses to the Env proteins of the 3 major subtypes of HIV-1. We have shown that DNA as a gene-delivery platform for HIV-1 vaccine antigen expression can induce significant immune responses that are sustained for at least 1 year in the majority of antigen-naive subjects. The concept of using bacteria-derived plasmid DNA to deliver vaccine antigens has many attractive features, including ease and flexibility of construction, scalable manufacturing capacity, stability, intracellular production of vaccine antigen, transient expression, no induction of antivector immunity, induction of a balanced CD4+ and CD8+ T cell response as well as of antibody, and lack of significant local or systemic reactogenicity. The broad immunogenicity of the VRC HIV-1 DNA candidate vaccine probably reflects a combination of factors, including optimization of vector design, manufacturing methods, delivery, sample processing, and immunological end-point measurements.
A notable feature of the immunogenicity analysis was the response to Env antigens relative to Gag, Pol, and Nef. The dominant antigenicity of Env was observed for both antibody and T cell responses, with EnvA peptides eliciting the greatest T cell responses and EnvC binding the most vaccine-induced antibody. All the genes encoding the vaccine antigens were codon modified to improve expression. The Env constructs received additional modification to remove the cleavage site, the fusion peptide of gp41, a region of the interspace between the 2 gp41 heptad repeats, and the cytoplasmic domain [3
], but there is no indication that these changes were responsible for liberating additional T cell epitopes. The gag/pol/nef
construct was designed to remove the frame shift, resulting in improved expression of Pol and Nef, at the expense of 66 aa from the carboxy-terminus of Gag [18
]. This construct was immunogenic in preclinical testing and exhibited strong protein expression in vitro [4
]. Here, the Gag, Pol, and Nef antigens elicited only a modest induction of CD4+
T cell responses despite representing 50% of the plasmid mixture by weight. It is not known why the induction of Gag-, Pol-, and Nef-specific responses were lower than Env-specific responses, but there are several possible explanations for increased Env immunogenicity. First, since Env is a membrane glycoprotein, its intracellular trafficking and mode of antigen presentation differs from those of the internal viral proteins. Second, Gag, Pol, and Nef are not glycosylated, are synthesized in the cytoplasm, and localize to a subcellular compartment different from that of Env. Therefore, it is possible that the steps involved in antigen processing and the kinetics of antigen presentation may differ. Finally, our experiments in animal models indicate that Env contains more CD4+
T cell epitopes than does Gag or other proteins [19
]. Thus, the increased immunogenicity of Env is potentially an intrinsic property of the expressed antigen.
Antibody responses induced by this 4-plasmid combination vaccine were detected by IP-Western blotting and research ELISA for vaccine-specific antigens as well as by commercial ELISA and Western blotting for native viral proteins (). Analysis of neutralization did not reveal significant activity. On the basis of animal studies, high levels of binding antibody and neutralizing activity are not anticipated after DNA immunization alone but can be achieved after subsequent boosting with replication-defective recombinant adenovirus vectors [20
]. The induction of measurable antibody to clades A, B, and C Env proteins with DNA alone in humans is encouraging and suggests that DNA vaccination is a viable strategy for priming both cellular and humoral responses. Whether the response to all the Env proteins indicates cross-reactivity between clades or between clade-specific antibodies is not known. As multivalent vaccines become more immunogenic and clinical evaluation expands, the complexity of interpreting routine commercial diagnostic tests will increase. Alternative approaches to serological diagnosis, such as gene-based diagnostic approaches, will be needed in the future.
This DNA vaccine consistently induced T cell responses. Envspecific CD4+
T cell responses were present in nearly all subjects. Detection of this response may be attributed to the development of a highly sensitive flow cytometry–based assay that detects IFN-γ
and/or IL-2 on the same channel. Other studies have shown that most of the CD4+
T cell response can be captured by the combination of these 2 cytokines [21
]; if IFN-γ
alone is used as the end point, a portion of the CD4+
T cell response is missed. CD8+
T cells are key effectors for clearing virus-infected cells [23
] and have been associated with control of lentivirus replication in both nonhuman primates (NHPs) and humans [24
]. Twelve (34%) of the 35 vaccine recipients who received the 4-mg or 8-mg doses had detectable HIV Env–specific CD8+
T cell responses at week 12. The magnitude of positive responses ranged from just above background to 0.65% of total CD8+
T cells. When detected, the magnitude of the CD8+
T cell response is comparable to that seen in NHP studies of similar vaccines, although typically CD8+
T cell responses can be detected in 100% of the animals [33
]. An interesting feature of the T cell response was that, over the course of a year, a higher frequency of EnvA-specific CD4+
T cells made IL-2 only. Reports that show that IFN-γ
– producing CD4+
T cells are not long lived [35
] and that IL-2– producing CD8+
T cells have a greater proliferative capacity in HIV-1–infected patients with nonprogressive disease [36
] suggest that these IL-2–producing T cells may have the capacity for expansion after subsequent antigen exposure.
Demonstrating the safety and immunogenicity of a multigene, multiclade HIV DNA vaccine is an important step in the development of a globally relevant vaccination strategy. The capacity to make precise measurements of vaccine-induced T cell responses will improve the chances of defining correlates of immune protection in future trials.