Immune-response profiles induced by human immunodeficiency virus type 1 vaccine DNA, protein or mixed-modality immunization: increased protection from pathogenic simian-human immunodeficiency virus viraemia with protein/DNA combination.

Current data suggest that prophylactic human immunodeficiency virus type 1 (HIV) vaccines will be most efficacious if they elicit a combination of adaptive humoral and T-cell responses. Here, we explored the use of different vaccine strategies in heterologous prime-boost regimes and evaluated the breadth and nature of immune responses in rhesus monkeys induced by epidermally delivered plasmid DNA or recombinant HIV proteins formulated in the AS02A adjuvant system. These immunogens were administered alone or as either prime or boost in mixed-modality regimes. DNA immunization alone induced cell-mediated immune (CMI) responses, with a strong bias towards Th1-type cytokines, and no detectable antibodies to the vaccine antigens. Whenever adjuvanted protein was used as a vaccine, either alone or in a regime combined with DNA, high-titre antibody responses to all vaccine antigens were detected in addition to strong Th1- and Th2-type CMI responses. As the vaccine antigens included HIV-1 Env, Nef and Tat, as well as simian immunodeficiency virus (SIV)mac239 Nef, the animals were subsequently exposed to a heterologous, pathogenic simian-human immunodeficiency virus (SHIV)89.6p challenge. Protection against sustained high virus load was observed to some degree in all vaccinated groups. Suppression of virus replication to levels below detection was observed most frequently in the group immunized with protein followed by DNA immunization, and similarly in the group immunized with DNA alone. Interestingly, control of virus replication was associated with increased SIV Nef- and Gag-specific gamma interferon responses observed immediately following challenge.

Induced recruitment of NK cells to lymph nodes provides IFN-gamma for T(H)1 priming.

Naive T cells are stimulated by antigen-presenting dendritic cells (DCs) in secondary lymphoid organs, but whether other types of cell participate in T cell priming is unclear. Here we show in mice that natural killer (NK) cells, which are normally excluded from lymph nodes, are rapidly recruited in a CCR7-independent, CXCR3-dependent manner to lymph nodes on stimulation by the injection of mature DCs. Recruitment of NK cells is also induced by some, but not all, adjuvants and correlates with the induction of T helper cell type 1 (T(H)1) responses. NK cell depletion and reconstitution experiments show that NK cells provide an early source of interferon-gamma (IFN-gamma) that is necessary for T(H)1 polarization. Taken together, our results identify an induced pathway of NK cell migration in antigen-stimulated lymph nodes and a mechanism by which some adjuvants may facilitate T(H)1 responses.

Uric acid promotes tumor immune rejection.

Uric acid released from dying cells has been shown recently to act as a danger signal for the immune system, stimulating dendritic cell maturation and enhancing T-cell responses to foreign antigens. Stimulation of dendritic cell maturation by uric acid has been proposed as a mechanism by which the immune system could generate responses against tumors. We show here that uric acid levels are elevated in tumors undergoing immune rejection and that the inhibition of uric acid production, by systemic administration of allopurinol, or the removal of uric acid, by administration of uricase, delayed tumor immune rejection, whereas subcutaneous administration of crystalline uric acid enhanced the rejection process.

Imiquimod and resiquimod in a mouse model: adjuvants for DNA vaccination by particle-mediated immunotherapeutic delivery.

Imiquimod, an immune response modifier and inducer of cytokines in vitro and in vivo, has been shown to have potent antiviral and antitumour activity and to act as an adjuvant for protein vaccination. We have undertaken studies in mice to investigate the potential of imiquimod and resiquimod to adjuvant DNA vaccination. These imidazoquinolines were administered by subcutaneous injection at the vaccination site immediately after particle-mediated immunotherapeutic delivery of plasmid DNA using a gene gun. Imiquimod was found to increase the number and maturation status of dendritic cells in draining lymph nodes, and to enhance antigen-specific CD4(+) and CD8(+) T cell responses, as assessed by analyses of clonal expansion, and the quantity and kinetics of cytokine production from these cells in lymph nodes and spleens collected after vaccination. A more substantial increase in IFN-gamma-producing, compared with IL-4-producing CD4(+) T cells suggested that imiquimod biased the immune response towards a predominance of Th1 cells. The analogue resiquimod was found to be to produce a similar Th1 biased immune response with a 10-fold reduced dose compared with imiquimod. Collectively, these studies suggest that both imiquimod and resiquimod may be suitable adjuvants for therapeutic DNA vaccines requiring induction of potent cytotoxic T cell responses.

Tumor cell-derived nitric oxide is involved in the immune-rejection of an immunogenic murine lymphoma.

The roles played by host-derived nitric oxide (NO) in the growth and subsequent immune rejection of a immunogenic murine lymphoma were investigated by growing the tumor in mice in which the gene for either inducible NO synthase (iNOS) or endothelial NOS (eNOS) had been ablated. This showed that NO from tumor-infiltrating host cells had no significant effect on either tumor growth or immune rejection, although measurements of tumor nitrite levels and protein nitration showed that there had been significant NO production in the rejected tumors, in both the eNOS and iNOS knockout mice. Inhibition of both tumor and host NOS activities, with an iNOS-selective inhibitor (1400W), a nonselective NOS inhibitor [Nomega-nitro-L-arginine methyl ester (L-NAME)], or scavenging NO with a ruthenium-based scavenger, significantly delayed tumor rejection, while having no appreciable effect on tumor growth. Incubation of tumor cells with medium taken from cultured splenocytes, that had been isolated from immunized animals and activated by incubating them with irradiated tumor cells, resulted in an increase in tumor cell NOS activity and an increase in tumor cell apoptosis, which could be inhibited using L-NAME. We propose that, during the immune rejection of this tumor model, there is induction of tumor NOS activity by cytokines secreted by activated lymphocytes within the tumor and that this results in increased levels of tumor NO that induce tumor cell apoptosis and facilitate immune rejection of the tumor.

Technical issues in construction of nucleic acid vaccines.

Delivery of antigen-encoding genes by nucleic acid vaccine vectors offers tremendous advantages in power and flexibility over conventional antigen delivery systems. Of the many forms of nucleic acid vaccine that can be constructed, circular DNA plasmids are the simplest. In this article, we consider the components that make up a generic DNA plasmid vaccine. We discuss some of the issues encountered when optimizing these vectors to exploit their potential for in vivo expression and presentation of antigens and thereby maximizing the immune response.

Characterization of CC-chemokine receptor 7 expression on murine T cells in lymphoid tissues.

Expression of the lymph node homing and CC-chemokine receptor 7 (CCR7), with L-selectin (CD62L), has been shown to divide human memory T cells into two functionally distinct subsets. We generated a polyclonal antibody against murine CCR7 and used this antibody to study CCR7 expression on murine T-cell subsets. Using flow cytometric staining of T cells for visualisation expression of CCR7 in association with CD62L and CD44, a major population of CD4 or CD8 T cells expressing CCR7 were found to be CD62Lhigh CD44low, which would suggest a naïve cell phenotype. By analogy with human studies, memory cells could be subdivided into CCR7high CD62Lhigh CD44high (central memory) and CCR7low CD62Llow CD44high (effector memory). The proportions of these populations were different in lymph node, blood and spleen. Functional, short-term in vitro polyclonal stimulation of blood, spleen and lymph node cells from naive mice demonstrated that CCR7high CD4 T cells produced predominantly interleukin (IL)-2, whereas CCR7low CD4 T cells produced both IL-2 and interferon-gamma (IFN-gamma). However, in contrast to previously published reports, the CCR7high CD8 T-cell subpopulation produced both IFN-gamma and IL-2. Analysis of effector T cells, induced by immunization in vivo, showed that a proportion of activated naïve CD4 T cells down-regulated CCR7 only after multiple cell divisions, and this coincided with the down-regulation of CD62L and production of IL-4 and IFN-gamma. Finally, analysis of effector T cells during the phase of maximal clonal expansion of secondary immune responses in vivo indicated that the vast majority of both IL-2- and IFN-gamma-producing cells are CCR7low, while few cytokine-expressing CCR7high T cells were detected. Our results support the hypothesis, developed from studies with human cells, that CCR7 may separate functionally different murine memory T-cell subpopulations, but indicate additional complexity in that CCR7high CD8 T cells also may produce IFN-gamma.

Local cooperation dominates over competition between CD4+ T cells of different antigen/MHC specificity.

Interactions between CD4(+) T cells in vivo are controlled by a balance between cooperation and competition. In this study the interaction between two populations of CD4(+) T cells of different MHC/peptide specificity was probed at different precursor frequencies, delivering one or both Ags to APC using particle-mediated DNA delivery. Expansion of clonal populations of Ag (OVA and pigeon cytochrome c-specific) CD4(+) T cells was limited at higher precursor frequencies, presumably reflecting intraclonal competition. In contrast, a strong enhancement of the number of cells expressing IFN-gamma, IL-4, and IL-2 was observed in populations of cells at low precursor frequency in the presence of a high frequency of activated cells of a different Ag specificity. The helper effect was most potent when both Ags were delivered to the same dendritic cell (i.e., linked). This reflects the requirement of epicrine or paracrine help for optimal activation of T cell clones at low frequency. A measure of help was also delivered in an endocrine manner (unlinked), especially for Th1 responses, suggesting that there is also limited diffusion of cytokines between dendritic cell clusters. The dominant effects of cooperation over competition between CD4(+) T cells responding to different Ags may have important implications in terms of the efficacy of multivalent vaccines.

Instruction of naive CD4+ T cells by polarized CD4+ T cells within dendritic cell clusters.

Cooperation between CD4(+) T cells can enhance the response and modulate the cytokine profile, and defining these parameters has become a major issue for multivalent-vaccine strategies. We explored cooperation using adoptive transfer of two populations of TCR transgenic T cells of different specificity. One was transferred without prior activation, whereas the second was activated for five days by antigen stimulation under polarizing culture conditions. Both populations were transferred into a single adoptive host and then primed by particle-mediated DNA delivery. Polarized Th1 cells (inducers) raised the frequency of IFN-gamma(+) cells within a naive (target) population, whereas Th2 inducers raised the frequency of IL-4(+) and reduced that of IL-2(+) cells. These effects were obtained when the genes for both antigens were on the same particle, favoring presentation by the same dendritic cell, but not when on different particles delivered to different dendritic cells. Autonomy of DC clusters allows linked sets of antigens (e.g. from a single pathogen) to maintain cytokine bias, but allows other independent responses, each with their own set of autonomous clusters.