Despite the development of powerful molecular biological techniques and technologies, studies involving research animals remain a key component of discovery biology, and in the discovery and development of new medicines. In 1959, The Principles of Humane Experimental Technique , the 3Rs ( R eplacement, R eduction and R efinement) were developed to provide a framework to ensure animal research was undertaken as humanely as possible. Sixty years since their inception, the extent to which the 3Rs have been adopted and implemented by the global scientific and medical research communities has unfortunately been slow and patchy. However, this situation is changing rapidly as awareness increases, not only of the 3Rs themselves, but of the impact of animal welfare on the reproducibility, reliability and translatability of data from animal studies.

Baculovirus expression is a time-tested technique to produce proteins in insect cells, in high quality and quantity for a range of applications. MultiBac is a baculovirus expression system we developed originally for producing multiprotein complexes comprising many subunits, for structural and mechanistic studies. First introduced in 2004, MultiBac is now in use in many laboratories worldwide, accelerating research programmes in academia and industry. We have continuously optimized our MultiBac system, providing customized reagents and standard operating protocols to facilitate its use also by non-specialists. More recently, we have generated MultiBac genomes tailored for specific purposes, for example, to produce humanized glycoproteins, high-value pharmaceutical targets including kinases, viral polymerases, and virus-like particles (VLPs) as promising vaccine candidates. By altering the host tropism of the baculovirion, we created MultiBacMam, a heterologous DNA delivery toolkit to target mammalian cells, tissues and organisms. Introducing CRISPR/Cas modalities, we set the stage for large-scale genomic engineering applications utilizing this high-capacity DNA delivery tool. Exploiting synthetic biology approaches and bottom-up design, we engage in optimizing the properties of our baculoviral genome, also to improve manufacturing at scale. Here we provide a perspective of our MultiBac system and its developments, past, present and future.

Genome editing technology provides a transformative approach to animal breeding. Otherwise difficult or impossible-to-access genetic variation can now be used in a given target population, with leading examples focussing on animal health and welfare. The race is on for the first food from genome-edited farm animals to reach the shops.

Congenital Chagas disease, caused by Trypanosoma cruzi ( T. cruzi ), is partially responsible for the increasing globalization of Chagas disease despite its low transmission. During congenital transmission, the parasite reaches the fetus by crossing the placental barrier. However, the success or impairment of congenital transmission of the parasite is the product of a complex interaction between the parasite, the maternal and fetus/newborn immune responses and placental factors. There is other evidence apart from the low congenital transmission rates, which suggests the presence of defense mechanisms against T. cruzi . Thus, the typical amastigote nests (intracellular parasites) cannot be observed in placentas from mothers with chronic Chagas disease nor in human placental chorionic villi explants infected in vitro with the parasite. In the latter, only a few parasite antigens and DNA are identified. Accordingly, other infections of the placenta are not commonly observed. All these evidences suggest that the placenta can mount defense mechanisms against T. cruzi .

RTS,S/AS01 is the most advanced vaccine to prevent malaria. It is safe and moderately effective. A large pivotal phase III trial in over 15 000 young children in sub-Saharan Africa completed in 2014 showed that the vaccine could protect around one-third of children (aged 5–17 months) and one-fourth of infants (aged 6–12 weeks) from uncomplicated falciparum malaria. The European Medicines Agency approved licensing and programmatic roll-out of the RTSS vaccine in malaria endemic countries in sub-Saharan Africa. WHO is planning further studies in a large Malaria Vaccine Implementation Programme, in more than 400 000 young African children. With the changing malaria epidemiology in Africa resulting in older children at risk, alternative modes of employment are under evaluation, for example the use of RTS,S/AS01 in older children as part of seasonal malaria prophylaxis. Another strategy is combining mass drug administrations with mass vaccine campaigns for all age groups in regional malaria elimination campaigns. A phase II trial is ongoing to evaluate the safety and immunogenicity of the RTSS in combination with antimalarial drugs in Thailand. Such novel approaches aim to extract the maximum benefit from the well-documented, short-lasting protective efficacy of RTS,S/AS01.

The recent successes of cancer immunotherapy, first and foremost checkpoint blockade therapy, illustrate the power of the immune system to control cancer. As the number of patients receiving this therapy is increasing, the number of patients being resistant or establishing resistance toward immunotherapy is also increasing. We, therefore, need to further understand the mechanisms mediate resistance in order to prevent or overcome those mechanisms. Increasing evidence is being reported that alterations in tumor cell-intrinsic signaling pathways, including the activation of the WNT/β-catenin pathway, are associated with blunted T-cell infiltration. Infiltration of tumor by CD8 T cells is one of the most predictive biomarkers for the response toward immunotherapy and therefore the notion that alterations of certain tumor cell-intrinsic signaling pathways might mediate resistance should be considered. Understanding the molecular and immunological mechanisms mediating resistance will ultimately facilitate the development of effective treatment strategies counteracting immune evasion.

The success of immune checkpoint inhibitor therapies (ICTs) to bring about durable clinical responses in a subset of patients with different cancer histologies is transforming cancer care. However, many patients do not benefit from single-agent ICT, including patients with melanoma and non-small cell lung cancer, which are often considered to be immunogenic tumor types. In addition, several other common solid tumors, such as breast, colon, and prostate cancers, have reported very low response rates. A growing body of evidence suggests that the majority of tumors may be categorized as being primary immune-ignorant tumors, hence precluding response to single-agent ICTs. The molecular mechanisms that govern the immune-ignorant phenotype are under intense investigation. This review focuses on how oncogenic pathways can promote the development of a primary immune-ignorant tumor.

The rapid emergence of antibiotic-resistant bacterial strains has prompted efforts to find new and more efficacious treatment strategies. Targeting virulence factors produced by pathogenic bacteria has gained particular attention in the last few years. One of the inherent advantages of this approach is that it provides less selective pressure for the development of resistance mechanisms. In addition, antivirulence drugs could potentially be the answer for diseases in which the use of conventional antibiotics is counterproductive. That is the case for bacterial toxin-mediated diseases, in which the severity of the symptoms is a consequence of the exotoxins produced by the pathogen. Examples of these are haemolytic-uraemic syndrome produced by Shiga toxins, the profuse and dangerous dehydration caused by Cholera toxin or the life-threatening colitis occasioned by clostridial toxins. This review focuses on the recent advances on the development of small molecules with antitoxin activity against Enterohaemorrhagic Escherichia coli , Vibrio cholerae and Clostridium difficile given their epidemiological importance. The present work includes studies of small molecules with antitoxin properties that act directly on the toxin (direct inhibitors) or that act by preventing expression of the toxin (indirect inhibitors).

The growing incidence of antibiotic-resistant Gram-negative bacterial infections poses a serious threat to public health. Molecules that have yet to be exploited as antibiotics are potent protein toxins called bacteriocins that are produced by Gram-negative bacteria during competition for ecological niches. This review discusses the state of the art regarding the use for therapeutic purposes of two types of Gram-negative bacteriocins: colicin-like bacteriocins (CLBs) and tailocins. In addition to in vitro data, the potency of eight identified CLBs or tailocins has been demonstrated in diverse animal models of infection with no adverse effects for the host. Although the characteristics of bacteriocins will need further study, results obtained thus far regarding their in vivo potency, immunogenicity and low levels of resistance are encouraging. This leads the way for the development of novel treatments using bacteriocins as protein antibiotics.

Antibiotic resistance is a serious public health concern at the global level. Available antibiotics have saved millions of lives, but are progressively losing their efficacy against many bacterial pathogens, and very few new antibiotics are being developed by the pharmaceutical industry. Over the last few decades, progress in understanding the pathogenic process of bacterial infections has led researchers to focus on bacterial virulence factors as potential targets for ‘antivirulence' drugs, i.e. compounds which inhibit the ability of bacteria to cause damage to the host, as opposed to inhibition of bacterial growth which is typical of antibiotics. Hundreds of virulence inhibitors have been examined to date in vitro and/or in animal models, but only a few were entered into clinical trials and none were approved, thus hindering the clinical validation of antivirulence therapy. To breathe new life into antivirulence research and speed-up its transfer to the clinic, antivirulence activities have also been sought in drugs already approved for different therapeutic purposes in humans. If effective, these drugs could be repositioned for antivirulence therapy and have an easier and faster transfer to the clinic. In this work we summarize the approaches which have led to the identification of repurposing candidates with antivirulence activities, and discuss the challenges and opportunities related to antivirulence therapy and drug repurposing. While this approach undoubtedly holds promise for boosting antivirulence drug research, some important issues remain to be addressed in order to make antivirulence drugs viable alternatives to traditional antibacterials.