Boosting BCG-primed mice with a recombinant adenovirus expressing M. tuberculosis antigen 85A by different routes has very different effects on protection against aerosol challenge with M. tuberculosis. Mice boosted intradermally make very strong splenic CD4 and CD8 Th1 cytokine responses to antigen 85A, but show no change in lung mycobacterial burden over BCG primed animals. In contrast intranasally boosted mice show greatly reduced mycobacterial burden and make a much weaker splenic response but a very strong lung CD4 and CD8 response to antigen 85A and an increased response to PPD. This is associated with the presence in the lung of multifunctional T cells, with high median fluorescence intensities and integrated median fluorescence intensities for IFNγ, IL-2 and TNF. In contrast, mice immunized with BCG alone have few antigen-specific cells in the lung and a low proportion of multi-functional cells although individual cells have high median fluorescence intensities. Successful immunization regimes appear to induce antigen-specific cells with abundant intracellular cytokine staining.

The KDM4/JMJD2 family of histone demethylases is amplified in human cancers. However, little is known about their physiologic or tumorigenic roles. We have identified a conserved and unappreciated role for the JMJD2A/KDM4A H3K9/36 tridemethylase in cell cycle progression. We demonstrate that JMJD2A protein levels are regulated in a cell cycle-dependent manner and that JMJD2A overexpression increased chromatin accessibility, S phase progression, and altered replication timing of specific genomic loci. These phenotypes depended on JMJD2A enzymatic activity. Strikingly, depletion of the only C. elegans homolog, JMJD-2, slowed DNA replication and increased ATR/p53-dependent apoptosis. Importantly, overexpression of HP1γ antagonized JMJD2A-dependent progression through S phase, and depletion of HPL-2 rescued the DNA replication-related phenotypes in jmjd-2(-/-) animals. Our findings describe a highly conserved model whereby JMJD2A regulates DNA replication by antagonizing HP1γ and controlling chromatin accessibility.

Identification of correlates of protection for infectious diseases including malaria is a major challenge and has become one of the main obstacles in developing effective vaccines. We investigated protection against liver-stage malaria conferred by vaccination with adenoviral and Modified Vaccinia Ankara (MVA) vectors expressing pre-erythrocytic malaria antigens. By classifying CD8+ T cells into effector (TE), effector/memory (TEM) and central memory (TCM) subsets using CD62L and CD127 markers, we found striking differences in T cell memory generation. While MVA induced accelerated TCM generation, which could be efficiently boosted by subsequent adenoviral administration, it failed to protect against malaria. In contrast, adenoviral (Ad) vectors, which permit persistent antigen delivery, elicit a prolonged TE and TEM response that requires long intervals for an efficient boost. A preferential TEM phenotype was maintained in liver, blood and spleen following Ad/MVA prime-boost regimens and animals were protected against malaria sporozoite challenge. Blood CD8+ TEM cells correlated with protection against malaria liver-stage infection, assessed by estimation of number of parasites emerging from the liver into the blood. The protective ability of antigen-specific TEM cells was confirmed by transfer experiments into naive recipient mice. Thus, we identify persistent CD8 TEM populations as essential for vaccine-induced pre-erythrocytic protection against malaria, a finding that has important implications for logical vaccine design.

Adjuvants are substances that enhance immune responses and thus improve the efficacy of vaccination. Few adjuvants are available for use in humans, and the one that is most commonly used (alum) often induces suboptimal immunity for protection against many pathogens. There is thus an obvious need to develop new and improved adjuvants. We have therefore taken an approach to adjuvant discovery that uses in silico modeling and structure-based drug-design. As proof-of-principle we chose to target the interaction of the chemokines CCL22 and CCL17 with their receptor CCR4.

SummaryProtecting against both liver and blood stages of infection is a long-sought goal of malaria vaccine design. Recently, we described the use of replication-defective viral vaccine vectors expressing the malaria antigen merozoite surface protein-1 (MSP-1) as an antimalarial vaccine strategy that elicits potent and protective antibody responses against blood-stage parasites. Here, we show that vaccine-induced MSP-1-specific CD4+ T cells provide essential help for protective B cell responses, and CD8+ T cells mediate significant antiparasitic activity against liver-stage parasites. Enhanced survival is subsequently seen in immunized mice following challenge with sporozoites, which mimics the natural route of infection more closely than when using infected red blood cells. This effect is evident both in the presence and absence of protective antibodies and is associated with decreased parasite burden in the liver followed by enhanced induction of the cytokine IFN-γ in the serum. Multistage immunity against malaria can thus be achieved by using viral vectors recombinant for MSP-1.

A central goal in vaccinology is the induction of high and sustained antibody responses. Protein-in-adjuvant formulations are commonly used to achieve such responses. However, their clinical development can be limited by the reactogenicity of some of the most potent pre-clinical adjuvants and the cost and complexity of licensing new adjuvants for human use. Also, few adjuvants induce strong cellular immunity which is important for protection against many diseases, such as malaria. We compared classical adjuvants such as alum to new pre-clinical adjuvants and adjuvants in clinical development such as Abisco®100, CoVaccine HT™, Montanide®ISA720 and SE-GLA, for their ability to induce high and sustained antibody responses and T cell responses. These adjuvants induced a broad range of antibody responses when used in a three-shot protein-in-adjuvant regime using the model antigen ovalbumin and leading blood-stage malaria vaccine candidate antigens. Surprisingly, this range of antibody immunogenicity was greatly reduced when a protein-in-adjuvant vaccine was used to boost antibody responses primed by a human adenovirus serotype 5 (AdHu5) vaccine recombinant for the same antigen. This AdHu5-protein regime also induced a more cytophilic antibody response and demonstrated improved efficacy of merozoite surface protein-1 (MSP-1) protein vaccines against a Plasmodium yoelii blood-stage challenge. This indicates that the differential immunogenicity of protein vaccine adjuvants may be largely overcome by prior immunization with recombinant adenovirus, especially for adjuvants that are traditionally considered poorly immunogenic in the context of subunit vaccination, and may circumvent the need for more potent chemical adjuvants.

BackgroundAdjuvants enhance or modify an immune response that is made to an antigen. An antagonist of the chemokine CCR4 receptor can display adjuvant-like properties by diminishing the ability of CD4+CD25+ regulatory T cells (Tregs) to down-regulate immune responses.MethodologyHere, we have used protein modelling to create a plausible chemokine receptor model with the aim of using virtual screening to identify potential small molecule chemokine antagonists. A combination of homology modelling and molecular docking was used to create a model of the CCR4 receptor in order to investigate potential lead compounds that display antagonistic properties. Three-dimensional structure-based virtual screening of the CCR4 receptor identified 116 small molecules that were calculated to have a high affinity for the receptor; these were tested experimentally for CCR4 antagonism. Fifteen of these small molecules were shown to inhibit specifically CCR4-mediated cell migration, including that of CCR4+ Tregs.SignificanceOur CCR4 antagonists act as adjuvants augmenting human T cell proliferation in an in vitro immune response model and compound SP50 increases T cell and antibody responses in vivo when combined with vaccine antigens of Mycobacterium tuberculosis and Plasmodium yoelii in mice.

In the light of the recent emergence of multidrug-resistant and extensively drug-resistant strains of Mycobacterium tuberculosis, the epidemic of tuberculosis (TB) in populations coinfected with human immunodeficiency virus, and the failure of Mycobacterium bovis bacillus Calmette-Guerin (BCG) to protect against disease, new vaccines against TB are urgently needed. Two promising new vaccine candidates are the recombinant ⌬ureC hly ؉ BCG (recBCG), which has been developed to replace the current BCG vaccine strain, and modified vaccinia virus Ankara (MVA) expressing M. tuberculosis antigen 85A (MVA85A), which is a leading candidate vaccine designed to boost the protective efficacy of BCG. In the present study, we examined the effect of MVA85A boosting on the protection afforded at 12 weeks postchallenge by BCG and recBCG by using bacterial CFU as an efficacy readout. recBCG-immunized mice were significantly better protected against aerosol challenge with M. tuberculosis than mice immunized with the parental strain of BCG. Intradermal boosting with MVA85A did not reduce the bacterial burden any further. In order to identify a marker for the development of a protective immune response against M. tuberculosis challenge, we analyzed splenocytes after priming or prime-boosting by using intracytoplasmic cytokine staining and assays for cytokine secretion. Boosting with MVA85A, but not priming with BCG or recBCG, greatly increased the antigen 85A-specific T-cell response, suggesting that the mechanism of protection may differ from that against BCG or recBCG. We show that the numbers of systemic multifunctional cytokine-producing cells did not correlate with protection against aerosol challenge in BALB/c mice. This emphasizes the need for new biomarkers for the evaluation of TB vaccine efficacy.