Ennio De Gregorio

Genome-wide analysis of the Drosophila immune response by using oligonucleotide microarrays

To identify new Drosophila genes involved in the immune response, we monitored the gene expression profile of adult flies in response to microbial infection by using high-density oligonucleotide microarrays encompassing nearly the full Drosophila genome. Of 13,197 genes tested, we have characterized 230 induced and 170 repressed by microbial infection, most of which had not previously been associated with the immune response. Many of these genes can be assigned to specific aspects of the immune response, including recognition, phagocytosis, coagulation, melanization, activation of NF-κB transcription factors, synthesis of antimicrobial peptides, production of reactive oxygen species, and regulation of iron metabolism. Additionally, we found a large number of genes with unknown function that may be involved in control and execution of the immune response. Determining the function of these genes represents an important challenge for improving our knowledge of innate immunity. Complete results may be found at http://www.fruitfly.org/expression/immunity/.

The Toll and Imd pathways are the major regulators of the immune response in Drosophila

Microarray studies have shown recently that microbial infection leads to extensive changes in the Drosophila gene expression programme. However, little is known about the control of most of the fly immune‐responsive genes, except for the antimicrobial peptide (AMP)‐encoding genes, which are regulated by the Toll and Imd pathways. Here, we used oligonucleotide microarrays to monitor the effect of mutations affecting the Toll and Imd pathways on the expression programme induced by septic injury in Drosophila adults. We found that the Toll and Imd cascades control the majority of the genes regulated by microbial infection in addition to AMP genes and are involved in nearly all known Drosophila innate immune reactions. However, we identified some genes controlled by septic injury that are not affected in double mutant flies where both Toll and Imd pathways are defective, suggesting that other unidentified signalling cascades are activated by infection. Interestingly, we observed that some Drosophila immune‐responsive genes are located in gene clusters, which often are transcriptionally co‐regulated.

How Drosophila combats microbial infection: a model to study innate immunity and host-pathogen interactions

During the past year, dramatic progress has been achieved in our understanding of Drosophila immune reactions. The completion of the Drosophila genome sequencing project, microarray analysis and the use of genetic screens have led to the identification of several new genes required to combat microbial infection, filling in some important gaps in the understanding of innate immunity. At the same time, this insect was used as a model for the study of host-pathogen interactions. The recent major advances on the mechanisms by which this insect defends itself against intrusion of pathogens are discussed in this review.

New adjuvants for human vaccines.

Despite their obvious benefits, decades of research and hundreds of pre-clinical candidates, only a handful of adjuvants are approved for prophylactic vaccination of humans. The slow pace of development is due to a number of knowledge gaps, the most important of which is the complexity involved in designing adjuvants that are both potent and well tolerated. Recent advances in our understanding of innate immunity have led to the identification of immune pathways and adjuvant formulations more suitable for clinical advancement. One area of particular interest is the discovery of agonists that target the toll-like receptors. This review highlights recent progress of clinically approved vaccine adjuvants and identifies potential novel adjuvants that can broaden the development of new vaccines against infectious diseases.

An Immune-Responsive Serpin Regulates the Melanization Cascade in Drosophila

In arthropods, the melanization reaction is associated with multiple host defense mechanisms leading to the sequestration and killing of invading microorganisms. Arthropod melanization is controlled by a cascade of serine proteases that ultimately activates the enzyme prophenoloxidase (PPO), which, in turn, catalyzes the synthesis of melanin. Here we report the biochemical and genetic characterization of a Drosophila serine protease inhibitor protein, Serpin-27A, which regulates the melanization cascade through the specific inhibition of the terminal protease prophenoloxidase-activating enzyme. Our data demonstrate that Serpin-27A is required to restrict the phenoloxidase activity to the site of injury or infection, preventing the insect from excessive melanization.

Vaccine adjuvants alum and MF59 induce rapid recruitment of neutrophils and monocytes that participate in antigen transport to draining lymph nodes.

Vaccine adjuvants such as alum and the oil-in-water emulsion MF59 are used to enhance immune responses towards pure soluble antigens, but their mechanism of action is still largely unclear. Since most adjuvanted vaccines are administered intramuscularly, we studied immune responses in the mouse muscle and found that both adjuvants were potent inducers of chemokine production and promoted rapid recruitment of CD11b(+) cells. The earliest and most abundantly recruited cell type are neutrophils, followed by monocytes, eosinophils and later dendritic cells (DCs) and macrophages. Using fluorescent forms of MF59 and ovalbumin (OVA) antigen, we show that all recruited cell types take up both adjuvant and antigen to transport them to the draining lymph nodes (LNs). There, we found antigen-positive neutrophils and monocytes within hours of injection, later followed by B cells and DCs. Compared to alum, MF59-injection lead to a more prominent neutrophil recruitment and a more efficient antigen re-localization from the injection site to the LN. As antigen-transporting neutrophils were observed in draining LNs, we asked whether these cells play an essential role in MF59-mediated adjuvanticity. However, antibody-mediated neutrophil ablation left MF59-adjuvanticity unaltered. Further studies will reveal whether other single cell types are crucial or whether the different recruited cell populations are redundant with overlapping functions.

The path to a successful vaccine adjuvant--'the long and winding road'.

New generation vaccines will increasingly comprise highly purified recombinant proteins. Unfortunately, these antigens are often poorly immunogenic. Therefore, adjuvants will be required to enable these proteins to become effective vaccines. Although several novel adjuvants have recently emerged, including formulations comprising more than one adjuvant, the approval of vaccines containing novel adjuvants has been slow, particularly in the US. However, despite significant ongoing concerns, the necessary safety data is now emerging to show that new generation adjuvants can be safely used in diverse human populations. In combination with data showing the positive contributions of the adjuvants to the immune response, this safety data should allow several vaccines containing novel adjuvants to obtain licensure within the next few years.

Mechanism of action of licensed vaccine adjuvants

Despite the fact that alum and oil-in-water emulsions have been used for decades as human vaccine adjuvants in a large number of individuals, their mechanism of action is not completely understood. It has been reported that these particulate adjuvants act by increasing antigen availability and uptake by immune cells. However, recent work on alum and on the squalene-based emulsion MF59, has demonstrated that besides antigen delivery functions, these classes of adjuvants can also activate innate immunity pathways in vivo, generating an immunocompetent environment at injection site. Interestingly, it has been demonstrated that alum adjuvanticity depends on the activation of a protein complex called NLPR3/inflammasome, which is required for the correct processing of a number of pro-inflammatory cytokines, including IL1beta. More work needs to be performed to investigate if the inflammasome is also required for the activity of MF59 and of other particulate vaccine adjuvants.

Alum adjuvanticity: Unraveling a century old mystery

The development of vaccine adjuvants for human use has been one of the slowest processes in the history of medicine. For almost one century, aluminium hydroxide (alum) has been the only vaccine adjuvant approved worldwide. Only in the past decade have two oil‐in‐water emulsions and one TLR agonist been approved by the European authorities as new vaccine adjuvants. Despite the fact that alum has been injected into billions of people, its mechanism of action is not fully understood. Recently, several reports have greatly increased our knowledge of the molecular and cellular events triggered by alum; however, the contribution of each of these processes to alum adjuvanticity is still unclear. A study published in this issue of the European Journal of Immunology, together with two recent publications, have demonstrated that the NOD‐like receptor, pyrin domain containing 3 (Nlrp3)‐inflammasome is the molecular target of alum immunostimulatory activity in vitro. Surprisingly, these three studies reported conflicting results on the requirement of the Nlrp3 inflammasome complex for alum adjuvant effects in vivo. This commentary attempts to resolve some of these discrepancies.

Translation driven by an eIF4G core domain in vivo

Most eukaryotic mRNAs possess a 5′ cap structure (m7GpppN) and a 3′ poly(A) tail which promote translation initiation by binding the eukaryotic translation initiation factor (eIF)4E and the poly(A) binding protein (PABP), respectively. eIF4G can bridge between eIF4E and PABP, and—through eIF3—is thought to establish a link to the small ribosomal subunit. We fused the C‐terminal region of human eIF4GI lacking both the eIF4E‐ and PABP‐binding sites, to the IRE binding protein IRP‐1. This chimeric protein suffices to direct the translation of the downstream cistron of bicistronic mRNAs bearing IREs in their intercistronic space in vivo. This function is preserved even when translation via the 5′ end is inhibited. Deletion analysis defined the conserved central domain (amino acids 642–1091) of eIF4G as an autonomous ‘ribosome recruitment core’ and implicated eIF4A as a critical binding partner. Our data reveal the sufficiency of the conserved eIF4G ribosome recruitment core to drive productive mRNA translation in living cells. The C‐terminal third of eIF4G is dispensable, and may serve as a regulatory domain.