100 Years of BCG Immunization: Past, Present, and Future.

The 100th anniversary of the introduction of Bacille-Calmette-Guérin (BCG) as a tuberculosis (TB) vaccine is an occasion warranting further investigation of the early attempts which culminated in the introduction of BCG as a TB vaccine, as well as of subsequent recognition of failures, new findings that broaden its applications, outstanding questions, and approaches towards the development of novel vaccine candidates [...].

Targeting Inhibition of Accumulation and Function of Myeloid-Derived Suppressor Cells by Artemisinin via PI3K/AKT, mTOR, and MAPK Pathways Enhances Anti-PD-L1 Immunotherapy in Melanoma and Liver Tumors.

Despite the remarkable success and efficacy of immune checkpoint blockade (ICB) therapy such as anti-PD-L1 antibody in treating cancers, myeloid-derived suppressor cells (MDSCs) that lead to the formation of the protumor immunosuppressive microenvironment are one of the major contributors to ICB resistance. Therefore, inhibition of MDSC accumulation and function is critical for further enhancing the therapeutic efficacy of anti-PD-L1 antibody in a majority of cancer patients. Artemisinin (ART), the most effective antimalarial drug with tumoricidal and immunoregulatory activities, is a potential option for cancer treatment. Although ART is reported to reduce MDSC levels in 4T1 breast tumor model and improve the therapeutic efficacy of anti-PD-L1 antibody in T cell lymphoma-bearing mice, how ART influences MDSC accumulation, function, and molecular pathways as well as MDSC-mediated anti-PD-L1 resistance in melanoma or liver tumors remains unknown. Here, we reported that ART blocks the accumulation and function of MDSCs by polarizing M2-like tumor-promoting phenotype towards M1-like antitumor one. This switch is regulated via PI3K/AKT, mTOR, and MAPK signaling pathways. Targeting MDSCs by ART could significantly reduce tumor growth in various mouse models. More importantly, the ART therapy remarkably enhanced the efficacy of anti-PD-L1 immunotherapy in tumor-bearing mice through promoting antitumor T cell infiltration and proliferation. These findings indicate that ART controls the functional polarization of MDSCs and targeting MDSCs by ART provides a novel therapeutic strategy to enhance anti-PD-L1 cancer immunotherapy.

In vitro Evidence of Human Immune Responsiveness Shows the Improved Potential of a Recombinant BCG Strain for Bladder Cancer Treatment.

The live attenuated mycobacterial strain BCG, in use as vaccine against tuberculosis, is considered the gold standard for primary therapy of carcinoma in situ of the bladder. Despite its limitations, to date it has not been surpassed by any other treatment. Our group has developed a recombinant BCG strain expressing the detoxified S1 pertussis toxin (rBCG-S1PT) that proved more effective than wild type BCG (WT-BCG) in increasing survival time in an experimental mouse model of bladder cancer, due to the well-known adjuvant properties of pertussis toxin. Here, we investigated the capacity of rBCG-S1PT to stimulate human immune responses, in comparison to WT-BCG, using an in vitro stimulation assay based on human whole blood cells that allows for a comprehensive evaluation of leukocyte activation. Blood leukocytes stimulated with rBCG-S1PT produced increased levels of IL-6, IL-8, and IL-10 as compared to WT-BCG, but comparable levels of IL-1ß, IL-2, IFN-γ, and TNF-α. Stimulation of blood cells with the recombinant BCG strain also enhanced the expression of CD25 and CD69 on human CD4+ T cells. PBMC stimulated with rBCG-S1PT induced higher cytotoxicity to MB49 bladder cancer cells than WT-BCG-stimulated PBMC. These results suggest that the rBCG-S1PT strain is able to activate an immune response in human leukocytes that is higher than that induced by WT-BCG for parameters linked to better prognosis in bladder cancer (regulation of immune and early inflammatory responses), while fully comparable to WT-BCG for classical inflammatory parameters. This establishes rBCG-S1PT as a new highly effective candidate as immunotherapeutic agent against bladder cancer.

In vitro evidence of human immune responsiveness shows the improved potential of a recombinant BCG strain for bladder cancer treatment

The live attenuated mycobacterial strain BCG, in use as vaccine against tuberculosis, is considered the gold standard for primary therapy of carcinoma in situ of the bladder. Despite its limitations, to date it has not been surpassed by any other treatment. Our group has developed a recombinant BCG strain expressing the detoxified S1 pertussis toxin (rBCG-S1PT) that proved more effective than wild type BCG (WT-BCG) in increasing survival time in an experimental mouse model of bladder cancer, due to the well-known adjuvant properties of pertussis toxin. Here, we investigated the capacity of rBCG-S1PT to stimulate human immune responses, in comparison to WT-BCG, using an in vitro stimulation assay based on human whole blood cells that allows for a comprehensive evaluation of leukocyte activation. Blood leukocytes stimulated with rBCG-S1PT produced increased levels of IL-6, IL-8, and IL-10 as compared to WT-BCG, but comparable levels of IL-1ß, IL-2, IFN-?, and TNF-a. Stimulation of blood cells with the recombinant BCG strain also enhanced the expression of CD25 and CD69 on human CD4+ T cells. PBMC stimulated with rBCG-S1PT induced higher cytotoxicity to MB49 bladder cancer cells than WT-BCG-stimulated PBMC. These results suggest that the rBCG-S1PT strain is able to activate an immune response in human leukocytes that is higher than that induced by WT-BCG for parameters linked to better prognosis in bladder cancer (regulation of immune and early inflammatory responses), while fully comparable to WT-BCG for classical inflammatory parameters. This establishes rBCG-S1PT as a new highly effective candidate as immunotherapeutic agent against bladder cancer.

In vitro evidence of human immune responsiveness shows the improved potential of a recombinant BCG strain for bladder cancer treatment

The live attenuated mycobacterial strain BCG, in use as vaccine against tuberculosis, is considered the gold standard for primary therapy of carcinoma in situ of the bladder. Despite its limitations, to date it has not been surpassed by any other treatment. Our group has developed a recombinant BCG strain expressing the detoxified S1 pertussis toxin (rBCG-S1PT) that proved more effective than wild type BCG (WT-BCG) in increasing survival time in an experimental mouse model of bladder cancer, due to the well-known adjuvant properties of pertussis toxin. Here, we investigated the capacity of rBCG-S1PT to stimulate human immune responses, in comparison to WT-BCG, using an in vitro stimulation assay based on human whole blood cells that allows for a comprehensive evaluation of leukocyte activation. Blood leukocytes stimulated with rBCG-S1PT produced increased levels of IL-6, IL-8, and IL-10 as compared to WT-BCG, but comparable levels of IL-1ß, IL-2, IFN-?, and TNF-a. Stimulation of blood cells with the recombinant BCG strain also enhanced the expression of CD25 and CD69 on human CD4+ T cells. PBMC stimulated with rBCG-S1PT induced higher cytotoxicity to MB49 bladder cancer cells than WT-BCG-stimulated PBMC. These results suggest that the rBCG-S1PT strain is able to activate an immune response in human leukocytes that is higher than that induced by WT-BCG for parameters linked to better prognosis in bladder cancer (regulation of immune and early inflammatory responses), while fully comparable to WT-BCG for classical inflammatory parameters. This establishes rBCG-S1PT as a new highly effective candidate as immunotherapeutic agent against bladder cancer.

Changing Priorities in Vaccinology: Antibiotic Resistance Moving to the Top.

Antimicrobial resistance (AMR) is currently the most alarming issue for human health. AMR already causes 700,000 deaths/year. It is estimated that 10 million deaths due to AMR will occur every year after 2050. This equals the number of people dying of cancer every year in present times. International institutions such as G20, World Bank, World Health Organization (WHO), UN General Assembly, European Union, and the UK and USA governments are calling for new antibiotics. To underline this emergency, a list of antibiotic-resistant "priority pathogens" has been published by WHO. It contains 12 families of bacteria that represent the greatest danger for human health. Resistance to multiple antibiotics is particularly relevant for the Gram-negative bacteria present in the list. The ability of these bacteria to develop mechanisms to resist treatment could be transmitted with genetic material, allowing other bacteria to become drug resistant. Although the search for new antimicrobial drugs remains a top priority, the pipeline for new antibiotics is not promising, and alternative solutions are needed. A possible answer to AMR is vaccination. In fact, while antibiotic resistance emerges rapidly, vaccines can lead to a much longer lasting control of infections. New technologies, such as the high-throughput cloning of human B cells from convalescent or vaccinated people, allow for finding new protective antigens (Ags) that could not be identified with conventional technologies. Antibodies produced by convalescent B cell clones can be screened for their ability to bind, block, and kill bacteria, using novel high-throughput microscopy platforms that rapidly capture digital images, or by conventional technologies such as bactericidal, opsono-phagocytosis and FACS assays. Selected antibodies expressed by recombinant DNA techniques can be used for passive immunization in animal models and tested for protection. Antibodies providing the best protection can be employed to identify new Ags and then used for generating highly specific recombinant Fab fragments. Co-crystallization of Ags bound to Fab fragments will allow us to determine the structure and characteristics of new Ags. This structure-based Ag design will bring to a new generation of vaccines able to target previously elusive infections, thereby offering an effective solution to the problem of AMR.

The interleukin-1 receptor family.

The activity of each member of the IL-1 family of ligands is mediated by its own receptor. Each ligand binds specifically to the extracellular "ligand binding chain" containing three Ig-like regions. With the exception of the IL-1 and IL-36 receptor antagonists, a second chain, termed the "accessory chain", is recruited, forms a heterotrimetic complex with the ligand binding chain and the ligand, and signal transduction is initiated. Each ligand binding or accessory chain shares a common cytosolic segment termed the Toll-IL-1-receptor (TIR) domain. Another family of 13 receptors, termed Toll-like receptors (TLR), have extracellular leucine-rich repeat domains, which bind a broad spectrum of microbial products. All TLR share a nearly identical TIR domain with all members of the IL-1 receptor family. Hence signal transduction and the biological consequences of TLR ligands and IL-1 family ligands are often the same and both receptor families contribute to innate inflammation and host defense. The IL-1 family of receptors is comprised of ten distinct but related gene products. The receptors are indicated by the term IL-1 receptor (IL-1R) followed by a numeral, assigned chronologically by discovery, for example, IL-1R1, IL-1R2, IL-1R3, etc. The ligand binding chain for IL-1α and IL-1ß is IL-1R1 and the accessory chain is IL-1R3. IL-1α, IL-1ß, IL-33 and IL-36 use IL-1R3 as their accessory chain. IL-1R2 is a non-signalling "decoy" receptor that sequesters the IL-1ß and IL-1R3. IL-1R8 exhibits anti-inflammatory properties by reducing IL-1 and TLR signalling. Presently there are two orphan receptors, IL-1R9 and IL-1R10, which have no known function. This review examines the characteristics and functional roles of the IL-1R family in the regulation of innate inflammation, host defense and acquired immunity.

The immunostimulatory effect of IL-1beta in vivo is blocked by antisense peptides complementary to the loop sequence 163-171.

Antisense (AS) peptides complementary to the beta-bulge surface loop VQGEESNDK (Boraschi loop) of the cytokine interleukin-1beta (IL-1beta) have been shown to bind IL-1beta at the Boraschi loop position, and to inhibit some of the IL-1beta-mediated biological effects in vitro. Here we show that primary AS peptide FVITFFSLY inhibits IL-1beta-mediated immunostimulation in vivo in a dose-dependent fashion, while inactive on IL-1beta-induced inflammation, an effect that takes place independently of the Boraschi loop. To the best of our knowledge, this is the first time that an AS peptide has been used successfully in vivo.

Vaccine adjuvants: the dream becomes real.

After about 70 years two new adjuvants have been approved for human vaccines. The first is MF59 developed by the ex-Chiron now Novartis Vaccines and it consists in an oil-in-water emulsion, comprising a low content of biodegradable squalene oil (4.3%) as the dispersed phase, which is stabilized by two non-ionic surfactants (Tween 80 and Span 85), and a low ionic strength citrate buffer as the continuous phase. The second one, defined as AS04, has been developed by GSK Biologics and consists in 3-0-desacyl-4'-monophosphoryl lipid A (MPL) that comes from the cell wall LPS of Gram-negative Salmonella minnesota R595 and is detoxified by mild hydrolytic treatment and purification. It is absorbed on aluminum hydroxide or aluminum phosphate. Thus, new molecules are available to improve the immune response to vaccines also in humans: this is the beginning of a new era in vaccinology.

Immunity against HIV/AIDS, malaria, and tuberculosis during co-infections with neglected infectious diseases: recommendations for the European Union research priorities.

Infectious diseases remain a major health and socioeconomic problem in many low-income countries, particularly in sub-Saharan Africa. For many years, the three most devastating diseases, HIV/AIDS, malaria, and tuberculosis (TB) have received most of the world's attention. However, in rural and impoverished urban areas, a number of infectious diseases remain neglected and cause massive suffering. It has been calculated that a group of 13 neglected infectious diseases affects over one billion people, corresponding to a sixth of the world's population. These diseases include infections with different types of worms and parasites, cholera, and sleeping sickness, and can cause significant mortality and severe disabilities in low-income countries. For most of these diseases, vaccines are either not available, poorly effective, or too expensive. Moreover, these neglected diseases often occur in individuals who are also affected by HIV/AIDS, malaria, or TB, making the problem even more serious and indicating that co-infections are the rule rather than the exception in many geographical areas. To address the importance of combating co-infections, scientists from 14 different countries in Africa and Europe met in Addis Ababa, Ethiopia, on September 9-11, 2007. The message coming from these scientists is that the only possibility for winning the fight against infections in low-income countries is by studying, in the most global way possible, the complex interaction between different infections and conditions of malnourishment. The new scientific and technical tools of the post-genomic era can allow us to reach this goal. However, a concomitant effort in improving education and social conditions will be needed to make the scientific findings effective.

The interleukin-1 receptor family.

The cytokines IL-1 and IL-18 are key molecules both in the innate and in the adaptive immune response. Their activity is mediated by specific receptors present on the membrane of target cells. It has become apparent that these receptors are members of a larger family of related receptors, most of which are apparently involved in the mechanisms of host defense. Thus, the large Toll/IL-1R (TIR) superfamily encompasses the Ig domain family (IL-1 receptors, IL-18 receptors, and IL-1R-like receptors), the leucine-rich domain family [the Toll-like receptors (TLR) and similar receptors], and a series of TIR domain-containing intracellular adapter molecules. The TIR superfamily is defined by a common intracellular TIR domain, involved in the initiation of signaling. A group of TIR domain-containing adapters (MyD88, TIRAP, TRIF, and TRAM) are differentially recruited to the Toll/IL-1 receptors, contributing to the specificity of signaling. Recent studies have also begun to unravel the mechanisms of negative regulation of the Toll/IL-1 receptors. The orphan receptor TIR8/SIGIRR, a member of TIR superfamily, while unable to initiate signaling, can negatively modulate the TIR-mediated responses. Other negative regulators of the Toll/IL-1R family include T1/ST2, some soluble forms of TLR, and MyD88s. The coordinated positive and negative regulation of the TIR activation ensures the appropriate modulation of the innate and inflammatory responses and avoids the risk of pathological derangement. This chapter will consider in detail the characteristics and functional role of the Ig domain receptor subfamily in the regulation of host defense and their possible role in pathology.

Mucosal delivery of anti-inflammatory IL-1Ra by sporulating recombinant bacteria.

BACKGROUND: Mucosal delivery of therapeutic protein drugs or vaccines is actively investigated, in order to improve bioavailability and avoid side effects associated with systemic administration. Orally administered bacteria, engineered to produce anti-inflammatory cytokines (IL-10, IL-1Ra), have shown localised ameliorating effects in inflammatory gastro-intestinal conditions. However, the possible systemic effects of mucosally delivered recombinant bacteria have not been investigated. RESULTS: B. subtilis was engineered to produce the mature human IL-1 receptor antagonist (IL-1Ra). When recombinant B. subtilis was instilled in the distal colon of rats or rabbits, human IL-1Ra was found both in the intestinal lavage and in the serum of treated animals. The IL-1Ra protein in serum was intact and biologically active. IL-1-induced fever, neutrophilia, hypoglycemia and hypoferremia were inhibited in a dose-dependent fashion by intra-colon administration of IL-1Ra-producing B. subtilis. In the mouse, intra-peritoneal treatment with recombinant B. subtilis could inhibit endotoxin-induced shock and death. Instillation in the rabbit colon of another recombinant B. subtilis strain, which releases bioactive human recombinant IL-1beta upon autolysis, could induce fever and eventually death, similarly to parenteral administration of high doses of IL-1beta. CONCLUSIONS: A novel system of controlled release of pharmacologically active proteins is described, which exploits bacterial autolysis in a non-permissive environment. Mucosal administration of recombinant B. subtilis causes the release of cytoplasmic recombinant proteins, which can then be found in serum and exert their biological activity in vivo systemically.

In vivo mucosal delivery of bioactive human interleukin 1 receptor antagonist produced by Streptococcus gordonii.

BACKGROUND: Interleukin-1 (IL-1) is a cytokine involved in the initiation and amplification of the defence response in infectious and inflammatory diseases. IL-1 receptor antagonist (IL-1ra) is an inactive member of the IL-1 family and represents one of the most potent mechanisms for controlling IL-1-dependent inflammation. IL-1ra has proven effective in the therapy of acute and chronic inflammatory diseases in experimental animal models and also in preliminary clinical trials. However, optimisation of therapeutic schedules is still needed. For instance, the use of drug delivery systems targeting specific mucosal sites may be useful to improve topical bioavailability and avoid side effects associated with systemic administration. RESULTS: In order to develop systems for the delivery of IL-1ra to mucosal target sites, a Streptococcus gordonii strain secreting human IL-1ra was constructed. The recombinant IL-1ra produced by S. gordonii was composed of the four amino acid residues RVFP of the fusion partner at the N-terminus, followed by the mature human IL-1ra protein. RFVP/IL-1ra displayed full biological activity in vitro in assays of inhibition of IL-1beta-induced lymphocyte proliferation and was released by recombinant S. gordonii in vivo both at the vaginal and the gastrointestinal mucosa of mice. RFVP/IL-1ra appeared beneficial in the model of ulcerative colitis represented by IL-2-/- mice (knock-out for the interleukin-2 gene), as shown by the body weight increase of IL-2-/- mice locally treated with S. gordonii producing RFVP/IL-1ra. CONCLUSIONS: These results indicate that recombinant S. gordonii can be successfully used as a delivery system for the selective targeting of mucosal surfaces with therapeutic proteins.

Novel human vaccine strategies and the 5th Framework Programme: pushing the envelope.

Mucosal vaccines could result in a great scientific and practical achievement. More than three decades of research in experimental models have shown promising results in stimulating mucosal immune responses, thus, it was expected that within a short time mucosal vaccines for human use could be achieved. Indeed this is not being the case. In the last few years, the most important oral vaccine, the anti-polio developed by Sabin in the fifties, has been progressively abandoned in developed countries to avoid the few cases of disease caused by the vaccine. Furthermore, two recently developed mucosal vaccines for human use against rotavirus diarrhoea and influenza were withdrawn after a short period in the market because of adverse reactions among the vaccinees. This controversial situation has created a difficult future for research on mucosal vaccine at the industrial level. A great help and encouragement for believers in mucosal vaccines has been given by the EU Commission through the 5th Framework Programme (5FP). At the end of the first projects of the 5FP, it is quite clear that mucosal vaccines are experiencing a real renaissance. The Euroconference/Workshop "Novel Strategies of Mucosal Immunisation through Exploitation of Mechanisms of Innate Immunity in Pathogen-Host Interaction", organised under the sponsorship of the EU Commission and reported in this special issue of Vaccine, witnesses a very creative moment of European groups involved in mucosal immunology. This conclusive paper of the issue is intended to describe a positive experience of some European scientists that have been working together in organised fashion within two EU projects. The first, defined by the acronym MUCIMM, was aimed to pave the way to tackle mucosal vaccines with different approaches, mainly that of new delivery systems and adjuvants, that of dissecting the fine mechanisms of basic mucosal responses and that of obtaining meaningful assays to measure human immune responses to mucosal vaccines. The second, the MUCADJ project, was aimed to prove that an intranasally delivered influenza vaccine induces protective levels of immunity in human adult volunteers. The results obtained demonstrate that mucosal vaccines for humans are feasible. It is interesting to note how this model of making biomedical research is flourishing, becoming an example for work organisation. At the same time it is important to underline some of the limits of this cooperative approach and it is also mandatory to spend a word of consciousness regarding the impact that the new European regulations could exert on mucosal vaccines, particularly for those based on live delivery systems. In a world where the damages caused by syringe needles are still largely visible, mucosal vaccines could represent an extremely important tool to fight infections, particularly in the large population of the most impoverished. The EU Commission will still encourage and support mucosal vaccines in the 6FP. The scientific community must decide in which direction has to proceed to avoid regulatory problems that could halt a promising tool to improve human health. The envelope has been pushed, the trip begins.