We show that co-expression of interleukin 15 (IL-15) and IL-15 receptor ␣ (IL-15R␣) in the same cell allows for the intracellular interaction of the two proteins early after translation, resulting in increased stability and secretion of both molecules as a complex. In the absence of co-expressed IL-15R␣, a large portion of the produced IL-15 is rapidly degraded immediately after synthesis. Co-injection into mice of IL-15 and IL-15R␣ expression plasmids led to significantly increased levels of the cytokine in serum as well as increased biological activity of IL-15. Examination of natural killer cells and T lymphocytes in mouse organs showed a great expansion of both cell types in the lung, liver, and spleen. The presence of IL-15R␣ also increased the number of CD44 high memory cells with effector phenotype (CD44 high CD62L؊). Thus, mutual stabilization of IL-15 and IL-15R␣ leads to remarkable increases in production, stability, and tissue availability of bioactive IL-15 in vivo. The in vivo data show that the most potent form of IL-15 is as part of a complex with its receptor ␣ either on the surface of the producing cells or as a soluble extracellular complex. These results explain the reason for coordinate expression of IL-15 and IL-15R␣ in the same cell and suggest that the IL-15R␣ is part of the active IL-15 cytokine rather than part of the receptor. 2 is a pleiotropic cytokine produced in many tissues. It is a member of the four ␣-helix bundle family of cytokines and was initially described as a T cell proliferation factor (1, 2). IL-15 shares with interleukin-2 (IL-2) a common receptor complex, consisting of the IL-2 receptors ␤ and ␥ chains (3). Both IL-2 and IL-15 use an additional private receptor subunit responsible for the specificity of binding, the IL-2 receptor ␣ (IL-2R␣) and IL-15 receptor ␣ (IL-15R␣), respectively. Both molecules have a similar ligand-binding motif (sushi domain) as well as a relatively short intracellular tail (13 amino acids for human IL-2R␣ and 41 amino acids for human IL-15R␣). In contrast to IL-2R␣, which displays a lower affinity for IL-2 (K d ϳ 10 Ϫ8 M) and is expressed mainly on activated T cells, IL-15R␣ has a high affinity for IL-15 (K d ϳ10 Ϫ11 M), and its mRNA has a wide tissue distribution (4). IL-15Ϫ/Ϫ and IL-15R␣ Ϫ/Ϫ mice have profound defects in NK, NK-T, intraepithelial lymphocytes, and memory CD8ϩ T cells, indicating that IL-15 is essential for the homeostatic maintenance and function of these cells (5, 6). In contrast, IL-2 Ϫ/Ϫ and IL-2R␣ Ϫ/Ϫ mice develop autoimmune diseases with increased frequency of activated T and B cells (7,8). Despite the clear results on the positive role of IL-15R␣ for IL-15 function, several investigators have reported inhibitory effects of IL-15R␣ on IL-15 function. Injection in mice of a soluble recombinant form of IL-15R␣ protein (IL-15sR␣) was reported to suppress natural killer (NK) cell proliferation and T-dependent immune responses in vivo (9). Addition of IL-15sR␣ in vitro was reported to block the response of cell lines to IL-1...

We have previously shown that macaques vaccinated with DNA vectors expressing SIVmac239 antigens developed potent immune responses able to reduce viremia upon high-dose SIVmac251 challenge. To further improve vaccine-induced immunity and protection, we combined the SIVmac239 DNA vaccine with protein immunization using inactivated SIVmac239 viral particles as protein source. Twenty-six weeks after the last vaccination, the animals were challenged intrarectally at weekly intervals with a titrated dose of the heterologous SIVsmE660. Two of DNA-protein coimmunized macaques did not become infected after 14 challenges, but all controls were infected by 11 challenges. Vaccinated macaques showed modest protection from SIVsmE660 acquisition compared with naïve controls (P = 0.050; stratified for TRIM5α genotype). Vaccinees had significantly lower peak (1.6 log, P = 0.0048) and chronic phase viremia (P = 0.044), with 73% of the vaccinees suppressing viral replication to levels below assay detection during the 40-wk follow-up. Vaccine-induced immune responses associated significantly with virus control: binding antibody titers and the presence of rectal IgG to SIVsmE660 Env correlated with delayed SIVsmE660 acquisition; SIV-specific cytotoxic T cells, prechallenge CD4 + effector memory, and postchallenge CD8 + transitional memory cells correlated with control of viremia. Thus, SIVmac239 DNA and proteinbased vaccine protocols were able to achieve high, persistent, broad, and effective cellular and humoral immune responses able to delay heterologous SIVsmE660 infection and to provide long-term control of viremia. These studies support a role of DNA and protein-based vaccines for development of an efficacious HIV/AIDS vaccine.T he use of a combination vaccine consisting of the recombinant Canarypox ALVAC-HIV (vCP1521; containing Gag, PR, and Env) together with gp120 Env protein (AIDSVAX B/E) resulted in modest, but statistically significant protection from infection in the RV144 vaccine trial conducted in Thailand (1). The limited efficacy and the fact that the vaccine-induced responses waned over time suggest that improved vaccine designs are needed to achieve long-lasting cross-clade-specific immune responses able to prevent infection. Rhesus macaque simian immunodeficiency virus (SIV) challenge models provide an excellent system to test different vaccine modalities and to compare efficacy using different challenge viruses and infection routes.DNA as priming immunization together with boosting by recombinant viral vectors is a vaccine platform widely used in the HIV/SIV field. DNA as the only vaccine component has been considered poorly immunogenic in humans, although recent results showed that in vivo DNA electroporation (EP) results in more efficient vaccine delivery, a higher frequency of responders, and higher, longer-lasting immunity than needle/syringe delivery (2). Similarly, the inclusion of DNA encoding the cytokine IL-12 as molecular adjuvant has been shown to be advantageous (3). These recent data suggest that DN...

Vaccine development has the potential to be accelerated by coupling tools such as systems immunology analyses and controlled human infection models to define the protective efficacy of prospective immunogens without expensive and slow phase 2b/3 vaccine studies. Among human challenge models, controlled human malaria infection trials have long been used to evaluate candidate vaccines, and RTS,S/AS01 is the most advanced malaria vaccine candidate, reproducibly demonstrating 40 to 80% protection in human challenge studies in malaria-naïve individuals. Although antibodies are critical for protection after RTS,S/AS01 vaccination, antibody concentrations are inconsistently associated with protection across studies, and the precise mechanism(s) by which vaccine-induced antibodies provide protection remains enigmatic. Using a comprehensive systems serological profiling platform, the humoral correlates of protection against malaria were identified and validated across multiple challenge studies. Rather than antibody concentration, qualitative functional humoral features robustly predicted protection from infection across vaccine regimens. Despite the functional diversity of vaccine-induced immune responses across additional RTS,S/AS01 vaccine studies, the same antibody features, antibody-mediated phagocytosis and engagement of Fc gamma receptor 3A (FCGR3A), were able to predict protection across two additional human challenge studies. Functional validation using monoclonal antibodies confirmed the protective role of Fc-mediated antibody functions in restricting parasite infection both in vitro and in vivo, suggesting that these correlates may mechanistically contribute to parasite restriction and can be used to guide the rational design of an improved vaccine against malaria.

BackgroundNone of the HIV T-cell vaccine candidates that have reached advanced clinical testing have been able to induce protective T cell immunity. A major reason for these failures may have been suboptimal T cell immunogen designs.MethodsTo overcome this problem, we used a novel immunogen design approach that is based on functional T cell response data from more than 1,000 HIV-1 clade B and C infected individuals and which aims to direct the T cell response to the most vulnerable sites of HIV-1.ResultsOur approach identified 16 regions in Gag, Pol, Vif and Nef that were relatively conserved and predominantly targeted by individuals with reduced viral loads. These regions formed the basis of the HIVACAT T-cell Immunogen (HTI) sequence which is 529 amino acids in length, includes more than 50 optimally defined CD4+ and CD8+ T-cell epitopes restricted by a wide range of HLA class I and II molecules and covers viral sites where mutations led to a dramatic reduction in viral replicative fitness. In both, C57BL/6 mice and Indian rhesus macaques immunized with an HTI-expressing DNA plasmid (DNA.HTI) induced broad and balanced T-cell responses to several segments within Gag, Pol, and Vif. DNA.HTI induced robust CD4+ and CD8+ T cell responses that were increased by a booster vaccination using modified virus Ankara (MVA.HTI), expanding the DNA.HTI induced response to up to 3.2% IFN-γ T-cells in macaques. HTI-specific T cells showed a central and effector memory phenotype with a significant fraction of the IFN-γ+ CD8+ T cells being Granzyme B+ and able to degranulate (CD107a+).ConclusionsThese data demonstrate the immunogenicity of a novel HIV-1 T cell vaccine concept that induced broadly balanced responses to vulnerable sites of HIV-1 while avoiding the induction of responses to potential decoy targets that may divert effective T-cell responses towards variable and less protective viral determinants.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0392-5) contains supplementary material, which is available to authorized users.

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Recent studies have revealed the critical role of programmed death-1 (PD-1) in exhaustion of HIV- and SIV-specific CD8+ T cells. In this study, we show that high expression of PD-1 correlates with increased ex vivo spontaneous and CD95/Fas-induced apoptosis, particularly in the “effector-memory” CD8+ T cell population from HIV+ donors. High expression of PD-1 was linked to a proapoptotic phenotype characterized by low expression of Bcl-2 and IL7-Rα, high expression of CD95/Fas and high mitochondrial mass. Expression of PD-1 and CD57 was differentially associated with the maturation status of CD8+ T cells in HIV infection. CD57 was linked to higher apoptosis resistance, with cells expressing a PD-1LCD57H phenotype exhibiting lower levels of cell death. The majority of HIV-specific CD8+ T cells were found to express a PD-1HCD57L or PD-1HCD57H phenotype. No correlation was found between PD-1 expression and ex vivo polyfunctionality of either HIV- or CMV-specific CD8+ T cells. Contrary to CD57, high expression of PD-1 was characterized by translocation of PD-1 into the area of CD95/Fas-capping, an early necessary step of CD95/Fas-induced apoptosis. Thus, our data further support the role of PD-1 as a preapoptotic factor for CD8+ T cells in HIV infection.

HIV sequence diversity and potential decoy epitopes are hurdles in the development of an effective AIDS vaccine. A DNA vaccine candidate comprising of highly conserved p24gag elements (CE) induced robust immunity in all 10 vaccinated macaques, whereas full-length gag DNA vaccination elicited responses to these conserved elements in only 5 of 11 animals, targeting fewer CE per animal. Importantly, boosting CE-primed macaques with DNA expressing full-length p55gag increased both magnitude of CE responses and breadth of Gag immunity, demonstrating alteration of the hierarchy of epitope recognition in the presence of pre-existing CE-specific responses. Inclusion of a conserved element immunogen provides a novel and effective strategy to broaden responses against highly diverse pathogens by avoiding decoy epitopes, while focusing responses to critical viral elements for which few escape pathways exist.

Retroviruses/retroelements provide tools enabling the identification and dissection of basic steps for post-transcriptional regulation of cellular mRNAs. The RNA transport element (RTE) identified in mouse retrotransposons is functionally equivalent to constitutive transport element of Type D retroviruses, yet does not bind directly to the mRNA export receptor NXF1. Here, we report that the RNA-binding motif protein 15 (RBM15) recognizes RTE directly and specifically in vitro and stimulates export and expression of RTE-containing reporter mRNAs in vivo. Tethering of RBM15 to a reporter mRNA showed that RBM15 acts by promoting mRNA export from the nucleus. We also found that RBM15 binds to NXF1 and the two proteins cooperate in stimulating RTE-mediated mRNA export and expression. Thus, RBM15 is a novel mRNA export factor and is part of the NXF1 pathway. We propose that RTE evolved as a high affinity RBM15 ligand to provide a splicing-independent link to NXF1, thereby ensuring efficient nuclear export and expression of retrotransposon transcripts.General mRNA export in eukaryotes is mediated by NXF1 protein orthologues that are conserved from yeast to humans and bind to the export-ready mRNP, targeting them to the nuclear pore complex (NPC) 2 (1-6). NXF1 acts as part of a stable heterodimer with its cofactor p15/NXT1 (7-9). Splicing changes the mRNP protein composition, allowing NXF1-p15 to bind and export to occur, whereas the pre-mRNPs are normally retained in the nucleus until completely spliced (10 -13). In particular, a set of proteins known as exon junction complex (EJC) is deposited onto mRNP as a result of splicing (14), providing critical determinants for the subsequent metabolic steps, including nuclear export, quality control, cytoplasmic trafficking, and translation (15). EJC consists of a stably bound core composed of eIF4AIII, Y14-Magoh, and MLN51/Barentsz that serves as a platform for a multitude of other EJC and EJC-associated factors that are bound more transiently. EJC is thought to commit the spliced mRNPs to nuclear export by providing binding sites for the NXF1-p15 export receptor. In one scenario, the EJC factor UAP56 recruits Aly/REF proteins, which bind directly to NXF1-p15, which in turn tethers the export substrate to the NPC (16 -21). Alternatively, NXF1 may assemble with the spliced mRNP via interactions with non-EJC factors such as SR proteins: SRp20 and 9G8 (22, 23), ASF/SF2 (24), and U2AF (25). Thus, it appears that several pathways lead to the binding of NXF1-p15 with the export-ready mRNP. Upon NXF1-p15-dependent targeting to NPC, such complexes are translocated to the cytoplasm by a yet unknown mechanism. NXF1 is a conserved export receptor for cellular mRNAs (1-6). Proteins of the NXF family can act on nuclear as well as on cytoplasmic mRNA trafficking (26 -28).According to the current model, general mRNA metabolism requires the acquisition of an export signal as a result of splicing, whereas pre-mRNA is generally retained in the nucleus due both to the lack of active export and ...