Improving the potency of immune responses is paramount among issues concerning vaccines against deadly pathogens. IL-28B belongs to the newly described interferon lambda (IFN) family of cytokines, and has not yet been assessed for its potential ability to influence adaptive immune responses or act as a vaccine adjuvant. We compared the ability of plasmid-encoded IL-28B to boost immune responses to a multiclade consensus HIV Gag plasmid during DNA vaccination with that of IL-12. We show here that IL-28B, like IL-12, is capable of robustly enhancing adaptive immunity. Moreover, we describe for the first time how IL-28B reduces regulatory T-cell populations during DNA vaccination, whereas IL-12 increases this cellular subset. We also show that IL-28B, unlike IL-12, is able to increase the percentage of splenic CD8 ؉ T cells in vaccinated animals, and that these cells are more granular and have higher antigen-specific cytolytic degranulation compared with cells taken from animals that received IL-12 as an adjuvant. Lastly, we report that IL-28B can induce 100% protection from mortality after a lethal influenza challenge. These data suggest that IL-28B is a strong candidate for further studies of vaccine or immunotherapy protocols. (Blood. 2009;113:5868-5877)
Protection against infection is the hallmark of immunity and the basis of effective vaccination. For a variety of reasons there is a great demand to develop new, safer and more effective vaccine platforms. In this regard, while ‘first-generation’ DNA vaccines were poorly immunogenic, new genetic ‘optimization’ strategies and the application of in vivo electroporation (EP) have dramatically boosted their potency. We developed a highly optimized plasmid DNA vaccine that expresses the lymphocytic choriomeningitis virus (LCMV) nucleocapsid protein (NP) and evaluated it using the LCMV challenge model, a gold standard for studying infection and immunity. When administered intramuscularly with EP, robust NP-specific cellular and humoral immune responses were elicited, the magnitudes of which approached those following acute LCMV infection. Furthermore, these responses were capable of providing 100% protection against a high-dose, normally lethal virus challenge. This is the first non-infectious vaccine conferring complete protective immunity up to eight weeks after vaccination and demonstrates the potential utility of ‘next-generation’ DNA vaccines.
Plasmid DNA is a promising vaccine platform as it is has been shown to be safe and able to be administered repeatedly without vector interference. Enhancing the potency of DNA vaccination through co-delivery of molecular adjuvants is one strategy currently under investigation. Here we describe the use of the novel chemokine adjuvant CCL27/CTACK to enhance immune responses to an HIV-1 or SIV antigen in mice and rhesus macaques. CCL27 has been shown to play a role in inflammatory responses through Chemotaxis of CCR10+ cells and we hypothesized that CCL27 may modulate adaptive immune responses. Immunizations in mice with HIV-1gag/CCL27 enhanced immune responses both at peripheral and surprisingly mucosal sites. To confirm these findings in a large animal model, we created optimized CCL27 and SIV antigenic plasmid constructs for rhesus macaques. 10 macaques (n=5/group) were immunized intramuscularly with 1mg/construct of antigenic plasmids +/- CCL27 with electroporation. We observed significant IFN-γ secretion and CD8+ T cell proliferation in peripheral blood. Interestingly, CCL27 co-immunized macaques exhibited a trend toward greater effector CD4+T cells in the bronchiolar lavage (BAL). CCL27 co-delivery also elicited greater antigen specific IgA at unique sites including BAL and fecal samples, but not in the periphery. Future studies incorporating CCL27 adjuvant in vaccine or therapy models where eliciting immune responses in the lung are warranted.
A vaccine candidate that elicits humoral and cellular responses to multiple sporozoite and liver-stage antigens may be able to confer protection against Plasmodium falciparum malaria; however, a technology for formulating and delivering such a vaccine has remained elusive. Here, we report the preclinical assessment of an optimized DNA vaccine approach that targets four P. falciparum antigens: circumsporozoite protein (CSP), liver stage antigen 1 (LSA1), thrombospondin-related anonymous protein (TRAP), and cell-traversal protein for ookinetes and sporozoites (CelTOS). Synthetic DNA sequences were designed for each antigen with modifications to improve expression and were delivered using in vivo electroporation (EP). Immunogenicity was evaluated in mice and nonhuman primates (NHPs) and assessed by enzyme-linked immunosorbent assay (ELISA), gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) assay, and flow cytometry. In mice, DNA with EP delivery induced antigen-specific IFN-γ production, as measured by ELISpot assay and IgG seroconversion against all antigens. Sustained production of IFN-γ, interleukin-2, and tumor necrosis factor alpha was elicited in both the CD4(+) and CD8(+) T cell compartments. Furthermore, hepatic CD8(+) lymphocytes produced LSA1-specific IFN-γ. The immune responses conferred to mice by this approach translated to the NHP model, which showed cellular responses by ELISpot assay and intracellular cytokine staining. Notably, antigen-specific CD8(+) granzyme B(+) T cells were observed in NHPs. Collectively, the data demonstrate that delivery of gene sequences by DNA/EP encoding malaria parasite antigens is immunogenic in animal models and can harness both the humoral and cellular arms of the immune system.
Plasmid-encoded DNA vaccines appear to be a safe and effective method for delivering antigen; however, the immunogenicity of such vaccines is often suboptimal. Cytokine adjuvants including interleukin (IL)-12, RANTES, granulocyte-macrophage colony-stimulating factor, IL-15, and others have been used to augment the immune response against DNA vaccines. In particular, IL-15 binds to a unique high-affinity receptor, IL-15Ra; is trans-presented to CD8 þ T cells expressing the common bg chain; and has been shown to play a role in the generation, maintenance, and proliferation of antigen-specific CD8 þ T cells. In this study, we took the unique approach of using both a cytokine and its receptor as an adjuvant in an HIV-1 vaccine strategy. To study IL-15Ra expression, a unique monoclonal antibody (KK1.23) was generated to confirm receptor expression in vitro. Coimmunization of IL-15 and IL-15Ra plasmids with HIV-1 antigenic plasmids in mice enhanced the antigenspecific immune response 2-fold over IL-15 immunoadjuvant alone. Furthermore, plasmid-encoded IL-15Ra augments immune responses in the absence of IL-15, suggesting its role as a novel adjuvant. Moreover, pIL-15Ra enhanced the cellular, but not the humoral, immune response as measured by antigen-specific IgG antibody. This is the first report describing that IL-15Ra itself can act as an adjuvant by enhancing an antigen-specific T cell response. Uniquely, pIL-15 and pIL-15Ra adjuvants combined, but not the receptor a chain alone, may be useful as a strategy for generating and maintaining memory CD8 þ T cells in a DNA vaccine.
As the primary site of HIV-1 replication is the mucosa, a desirable feature of a HIV-1 vaccine would be the generation of immune responses at mucosal sites. We created an optimized CCL27 DNA construct as well as optimized SIV gag, pol, and env plasmids for immunizing rhesus macaques. Macaques (n=5/group) were immunized with SIV plasmids +/- CCL27. Both immunization groups had significant IFN-g responses against the encoded SIV antigens (~12,000 SFU/106 PBMCs). CD8+ T cell proliferation as measured by CFSE staining was similar between the groups at ~22%. While systemic immune responses were robust, co-delivery of CCL27 did not adjuvant peripheral responses similar to what was observed in mice. Interestingly, macaques that received CCL27 had greater antigen specific IgA at mucosal sites including bronchial lavage and fecal samples. In addition, CD4+ T cells secreting effector cytokines were enhanced in the CCL27 group at mucosal, but not peripheral sites when compared to the antigenic group alone. The CCL27 group also had more polyfunctional CD8+ T cells in the bronchial lavage than the DNA group post-challenge. These data show that a novel chemokine adjuvant may be capable of targeting immune responses to mucosal sites. This strategy may also be applicable to other diseases than HIV-1 where mucosal immune responses are desirable.
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