From the University of Pennsylvania, Philadelphia, PA, USA.
Received: 8 May 2000; accepted: 18 May 2000
Requests for reprints to: David Weiner at above address.
The most powerful and cost-effective way to control infectious disease remains prophylactic vaccination. Traditional vaccination strategies such as live, attenuated or whole, inactivated agents have been very successful in the past. However, for many microorganisms that still lack an effective vaccine, these strategies may not be appropriate, either as a result of safety issues, manufacturing issues or the lack of immune potency. Such issues appear to be central to the question of the development of a vaccine for HIV-1 . Therefore, it is imperative that new approaches to vaccine development continue to be explored. In this regard the article by Boretti et al., in this issue of AIDS, is of particular interest.
Genetic immunization, also known as DNA or polynucleotide immunization, represents a novel approach for achieving specific immune activation. The concept behind genetic immunization is a simple one: genes encoding an antigen (or antigens) specific to a particular pathogen are cloned into a plasmid with an appropriate promoter, and the plasmid DNA is administered to the vaccine recipient. Host cells take up the DNA, and the antigen is expressed. The resultant foreign protein is produced within the host cell and then is processed and presented appropriately to the immune system, inducing a specific immune response. Immunization with DNA thus mimics aspects of live infection, with pathogen protein(s) synthesized endogenously by host cells. This synthesis leads to the induction of a cytotoxic T lymphocyte response via the MHC class I restricted pathway. Local antigen-presenting cells (APC) pick up small amounts of the DNA vaccine and can directly prime CD8 effector cell responses. Alternatively, through cell death or other mechanisms delivering antigen to APC, cross-priming can occur. Concurrently, protein(s) are released into the extracellular fluids. It is believed that this exogenous release of antigen primes the induction of a humoral response, as well as a helper T lymphocyte response via MHC class II restricted antigen presentation by APC, which have taken up the foreign antigen. In theory, genetic immunization confers the same conceptual advantages as immunization with live, attenuated vectors. However, DNA vaccines may suffer in comparison in the immune potency category in that they are not a spreading infection and therefore could pack less of an immune ‘punch’ than live vaccine approaches [reviewed in ref. ].
There have been several simple approaches that have attempted to overcome this issue. Studies have tried combining DNA vaccine priming with live viral vector or subunit boosts to enhance immunogenicity [reviewed in refs. [1–3]]. Some studies  have also focused on targeting the expressed antigen to specific compartments as well as incorporating specific nucleotide tracts that appear to increase immunogenicity of plasmid vectors. Such strategies increase the induced immune responses and are under pre-clinical and clinical examination. However, it would represent an important conceptual advantage if the vector itself could be directed to be a more potent immune driver to orchestrate the resulting immune response.
In this regard, several groups reported that combining IL-12 plasmids along with DNA vaccines for HIV-1 antigens results in enhanced cellular responses [4–6]. Previous work had established that IL-12 protein is a potent driver of T helper 1 type responses [reviewed in ref. ]; however, the recent studies are among the first to test the ability of plasmids to enhance in-vivo plasmid immunity. In challenge studies in rodents , for example, enhanced protection from lethal challenge has been achieved using this approach. Nevertheless, until the present paper by Boretti et al., no data were available on a lentiviral challenge system. In their important study, Boretti et al. used cleverly designed minimalistic, immunogenic defined gene expression vectors encoding the env gene of FIV as well as the first 525 nucleotides of the transmembrane envelope glycoprotein as a DNA vaccine delivered by the gene gun method in an FIV challenge model. Their construct contains no antibiotic resistance markers, only a cytomegalovirus immediate early promoter and SV40 polyadenlyation tract. They are therefore the minimal genetic elements necessary for the induction of DNA vaccine-induced immunity. In these studies, different vectors were simply mixed together to generate the different immunization groups. The authors studied the ability of three different groups of vaccinated or control cats to withstand an intraperitoneal infectious challenge. These groups received a control vaccine, an env vaccine alone or the env vaccine mixed with IL-12 expression vectors. The results are surprisingly clear. No animals seroconverted from the vaccines. This lack of serological response rules out antibody as contributing to the results obtained. Five weeks after challenge four out of four animals in the control group, two out of four in the env DNA group and only one out of four in the env plus IL-12 group seroconverted as an indication of infection after challenge. Quantitative viral load analysis demonstrated that all animals in the control group and all animals in the env alone group were infected. However, only one out of four animals in the env plus IL-12 group was infected. These data provide important confirmation of previous observations made in rodent model systems.
First, neutralizing antibodies were not a factor in the protection as the animals exhibited no serological response to FIV at any time before challenge. This result supports the ability of the cellular immune response to impact on lentiviral challenge in a beneficial manner, thus broadening the weapons against lentiviral challenge at least by analogy. This result is similar to the recent results of Robinson et al.  and our unpublished work, in which challenge protection in a SHIV model did not correlate with neutralizing antibodies. The effect of the IL-12 plasmids in enhancing protection from lentiviral challenge illustrates the power of this simple approach to engineer more effective vaccines. Furthermore, it supports earlier work showing that simply mixing plasmid vectors together allows for increased vaccine potency, an advantage for research investigation. However, further investigation is required to appreciate this work completely. No cellular immune analysis was performed as part of the study, leaving us with many unanswered questions regarding this component of protection. It will be important in further studies to perform lymphocyte subset depletion to delineate the contributions of the individual T cell compartments to the observed protection. Furthermore, it will be useful to transfer these results to a relevant primate model system as the effects of cytokines could vary in different species. However, for the time being it is likely that the study of Boretti et al. appears to bring us a small step closer to a rationally designed HIV vaccine. Vaccines that induce neutralizing antibodies are an important component in protection strategies. It is equally likely, at least in model systems, that protection or viral control can be improved through cytokine plasmid engineering of cellular immunity.
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