Since AIDS was first identified over 25 years ago [1,2], scientific advances have significantly expanded our understanding of the immune system, providing new tools for immune modulation and immunization strategies. The employment of DNA, protein subunits, and recombinant viral vectors in vaccination against HIV have been reviewed elsewhere [3–14]. The current article focuses on the use of new adjuvants as additions to HIV vaccination and immunotherapy regimens. By adjuvant, we refer to an immune potentiator in a vehicle . A wide variety of adjuvants has been tested for their abilities to elicit cellular and humoral responses to HIV antigens in vivo. The goal of studies employing new adjuvants is that their inclusion will promote a stronger and more directed immune response than those generated by current approaches.
Cytokine adjuvants: overview
A number of adjuvants are in use in both animal models as well as in human vaccination [16–18]. These include PAMP (pathogen associated molecular patterns) and co-stimulatory molecules that have been discussed previously and will not be reviewed here [19,20–33]. Currently, the only adjuvant approved for use in human vaccination is alum . While alum does enhance the ability of the immune system to respond to non-self antigens after vaccination, its ability to polarize the immune system is quite poor . More should be expected from the next generations of adjuvants in that they should be able to direct the immune response towards a desired outcome. The employment of Th1-associated cytokines such as interferon (IFN)-γ, interleukin (IL)-2, -12, and -15 [9–14,34–38] in animal models induces type-1, antigen specific cellular immune responses [19,39,40–42]. In contrast, the inclusion of cytokines known to polarize the immune system towards the induction of Th2 responses such as IL-4, IL-10 and granulocyte-macrophage cell stimulating factor [40,43,44] augments antigen-specific humoral immune responses [28,40,45]. Thus cytokine adjuvants may be considered improved to an adjuvant such as alum when the aim of the immunization is to direct the immune response towards a specific Th subtype.
Challenge of an HIV vaccine
Standard methods of vaccination, such as the use of subunit vaccines or whole inactivated virus, have not been able to induce broadly neutralizing immune responses against HIV in their current forms. Thus, the drive to develop a broadly neutralizing approach continues to be an important goal.
The questions posed by this challenge are: what type of immunity needs to be induced for an HIV vaccine to be effective and what types of vaccination can induce these responses? While neutralizing antibodies can be generated in vivo , total antibody responses during infection have limited antiviral effects . Moreover, as infection may theoretically be not only a consequence of viral passage from person to person, but also a result of passage of infected cells from person to person, the use of antibodies as an effective blockade to the establishment of infection may have limitations. Importantly, the STEP Study, the first study of a pure T cell-based approach from a vector platform that induces low level T-cell responses, has been disappointing . Therefore, for an HIV vaccine, the generation of more robust cytotoxic T lymphocyte (CTL) responses that have the potential to be cross reactive against diverse viral HIV strains seems to be needed in addition to the induction of antiviral antibodies. Such a vaccine should be one which induces a massive expansion of HIV-specific CTL which have high cytotoxic potential, are long-lived, and home to mucosal tissues. Accordingly, when infection at mucosal sites occurs, the speed and intensity of the CTL response could feasibly better control or eliminate infection of new target cells. Furthermore other immune parameters should also be expected, including the induction of polyfunctionality, and long lasting T-cell memory.
Cytokine adjuvants and vaccination
Vaccines that have benefited from the use of adjuvants span multiple classes, such as subunit vaccines, vaccines employing viral vectors and DNA vaccines. For example, the addition of adjuvants to administered peptide antigens such as gp120 can augment immune activity in vivo, resulting in durable responses postvaccination . The use of viral vectors as a means of delivering an antigen of interest for vaccination continues to have wide popularity both in academia [50–53] as well as industry [54–56]. Employing viruses such as Modified Vaccinia Virus Ankara (MVA) allows for delivery of antigens into the cytoplasm of target cells, thus providing expression through MHC Class I and subsequent generation of CTL responses [57,58]. In addition to MVA, recombinant adenoviruses are being employed more often as viral vectors in vaccination [51,52,54–57]. While there are a number of adenovirus serotypes, recombinant replication-defective adenovirus serotype 5 (rAd5) is the most commonly employed variant for vaccination against HIV and is able to induce antigen specific CTL responses [51,52,54,59]. The type of immune responses generated by rAd5 vectors has prompted clinical trials by the NIH-VRC as well as pharmaceutical companies such as Merck (see Table 2). These trials have shown this system to be safe and well tolerated in healthy adults in addition to inducing T-cell mediated immune responses . However, data from the STEP study suggests that levels of CTL activity induced by these vectors are not protective in humans . In fact questions of the relationship of the vector to acquisition of infection in these trials is under investigation. Thus, improving the potency of this approach and understanding its limitations are critical. Overall, adjuvants have not been employed as widely in combination with this rAd5 vaccination as they have with other vaccination methods. Data from the STEP trial and other studies may indicate that an analysis of adjuvants in conjunction with recombinant adenovirus-based vectors is warranted.
Out of all currently studied routes of vaccination, DNA vaccines have likely benefited the most from the continuing study and expansion of cytokine adjuvants. DNA vaccination employs the introduction, intramuscularly or intradermally, of a DNA vector encoding a gene of interest . Studies suggest that plasmid DNA vectors can transfect and express in skeletal muscle, macrophages and dendritic cells in vivo . Vaccination with SIV antigens encoded by DNA plasmids leads to reduced viremia in macaques after challenge with SIVmac251 . Responses to DNA vaccination are further increased when it is used in conjunction with in vivo electroporation in animal models [63,64,83]. IFN-γ, IL-2, -12, -15 and -21 have all been employed in multiple studies of DNA vaccines targeted against HIV or SIV, and are generally administered in plasmid form as well [39,65–69]. These adjuvants do, indeed augment CD8 cell responses to antigens delivered via DNA vaccination, with IL-2/Ig and IL-12 in particular creating robust anti-HIV CTL responses [65,66]. Mouse studies using IL-12 in conjunction with a plasmid encoding an HIV Gag/Pol construct show a 4.5-fold increase in specific CD8 cell lysis of target cells over inoculation with the Gag/Pol construct alone . IL-12, both on its own and in co-administration with IL-15 also induces potent type-1 responses in rhesus macaques, leading to substantial control of viremia, lower viral set point, and greatly improved clinical outcomes after challenge with SHIV 89.6P . Additionally, a recent study shows that use of IL-15 in conjunction with DNA encoding SHIV antigens leads to significant protection against SHIV 89.6P challenge in macaques . These studies suggest that the use of IL-12 and IL-15 in DNA vaccination against HIV or SIV leads to the generation of CTL responses that are improved with respect to HIV vaccine development.
Previous studies have suggested that IL-15, in particular, can prime CTL to better degranulate in response to TCR ligation . It may be of some benefit, then, to study further this cytokine in an HIV infection model. Additionally, IFN-γ has shown positive results in inducing CTL activity in mouse models . However, our own studies show that the use of IFN-γ as an adjuvant in DNA vaccination of non-human primates may not yield similar results (Table 1). This result may suggest that adjuvants that work in mouse models may not necessarily show the same potency in primates. To that end, further studies need to be performed with IL-15 in primates, as the employment of this cytokine in mouse vaccination studies has resulted in the generation of HIV-specific CD8 cell responses that are able to function partially independent of CD4 cells. This CTL characteristic can be of crucial importance for an HIV vaccine . Positive results observed in vaccination studies of non-human primates using challenge with highly pathogenic viruses closely related to HIV suggest that DNA vaccination with cytokine adjuvants has potential for vaccines in humans. A number of clinical trials are gearing up or currently underway, including vaccination with HIV-DNA constructs augmented with plasmid IL-12 or IL-15 (Table 2).
Therapeutic post-infection immunization
In contrast to vaccination, whose aim is to prevent the establishment of productive infection by HIV, immunotherapy regimens using viral antigens are aimed at promoting or augmenting immune responses to HIV in patients who are already infected. The hypothesis for such approaches is that re-exposing the patient's immune system to antigen in a structured fashion may lead to the development of a more potent and directed immune response, which may reduce or eliminate the need for HAART in the infected individual. Design of therapeutic immunizations should likely have the same endpoint as HIV vaccines: expanding HIV-specific CTL. However, the methods used are likely to be different from those utilized in a vaccine. Patients receiving therapeutic immunizations are already HIV positive and thus are immunocompromised to some extent as a result of infection. Therefore, therapeutic immunizations will need to be designed in such a way as to account for the fact that CD4 cell help will be limited at best. The inclusion of adjuvants that promote CD8 cell responses that are more independent of CD4 T-cell help will be of great importance, as will the inclusion of adjuvants that promote better antigen-specific priming of CTL.
Cytokine adjuvants and post-infection immunization
In the same vein as vaccines, immunotherapies have been studied using DNA, whole inactivated gp120-deleted virus and viral vectors [73–78]. Such therapies have shown promise both in animal models  as well as in human clinical trials [77,78]. Specifically, a study employing IL-2 in conjunction with a viral vector delivering SIV antigens showed that this type of immunization of SIVmac251 infected macaques substantially augmented SIV-specific CD8 T-cell responses . Additionally, therapeutic immunization of rhesus macaques with DNA encoding SIV antigens after infection with SIVmac251 resulted in considerably increased cellular immune responses along with long lasting reduction in viral loads after the animals were released from HAART . In regards to augmenting the responses generated by therapeutic immunization, specific cytokines seem to have great potential, especially IL-15 and IL-21. Both cytokines have been shown to induce increases in perforin and granzyme B levels in CD8 cells cultured ex vivo, giving CTL greater cytotoxic potential [71,81]. Moreover, peripheral blood mononuclear cells isolated from HIV positive patients have shown higher increases in granularity in response to IL-15 and IL-21 treatment than those taken from healthy individuals . Additionally, including IL-15 as an adjuvant in therapeutic immunization may help in the induction of antiviral CD8 cell activity by antigen presenting cells. CD8 T cells from HIV infected subjects co-cultured with mature dendritic cells restored the ability of the CD8 cells to inhibit viral replication by a non-cytotoxic mechanism . This effect was associated with the production of IL-15 by the dendritic cells . Such a result suggests that therapeutic immunization of HIV positive patients with antigen co-administered with IL-15 could also promote the stimulation of better CD8 cell anti-HIV responses. This effect is on top of the afore mentioned fact that IL-15 can induce the generation of CD8 cells that function somewhat independently of CD4 cell help .
Increased use of adjuvants in both vaccines and therapeutic immunizations for HIV infection should bring renewed vigor in attempts to induce lasting, robust immune responses against the virus. These adjuvants would boost HIV-specific immunity, modulate immune phenotype, and their use gives the scientific community a greater measure of control over what types of immune responses are driven in response to HIV antigen. Specific adjuvants including IL-12, IL-15 as well specific chemokines can strongly polarize the adaptive immune response towards a desired Th subtype. These look particularly important for clinical evaluation. Some adjuvants elicit strong responses from the innate immune system as well, such as IL-8 , and should be further studied in primate systems. This is an additional benefit of specific adjuvants, the induction of both the adaptive and innate arms of the immune system in response to HIV. While at this time there appears to be no single cytokine that is superior to others for use in vaccination and immune therapy, adjuvants such as IL-12 and IL-15 and specific chemokines such as Mip1α show promise in prophylactic vaccination approaches while IL-15 and IL-21 may prove beneficial for use in immune therapy for HIV infection.
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