An Innovative AI-based primer design tool for precise and accurate detection of SARS-CoV-2 variants of concern.

As the COVID-19 pandemic winds down, it leaves behind the serious concern that future, even more disruptive pandemics may eventually surface. One of the crucial steps in handling the SARS-CoV-2 pandemic was being able to detect the presence of the virus in an accurate and timely manner, to then develop policies counteracting the spread. Nevertheless, as the pandemic evolved, new variants with potentially dangerous mutations appeared. Faced by these developments, it becomes clear that there is a need for fast and reliable techniques to create highly specific molecular tests, able to uniquely identify VOCs. Using an automated pipeline built around evolutionary algorithms, we designed primer sets for SARS-CoV-2 (main lineage) and for VOC, B.1.1.7 (Alpha) and B.1.1.529 (Omicron). Starting from sequences openly available in the GISAID repository, our pipeline was able to deliver the primer sets for the main lineage and each variant in a matter of hours. Preliminary in-silico validation showed that the sequences in the primer sets featured high accuracy. A pilot test in a laboratory setting confirmed the results: the developed primers were favorably compared against existing commercial versions for the main lineage, and the specific versions for the VOCs B.1.1.7 and B.1.1.529 were clinically tested successfully.

Barriers Influencing Vaccine Development Timelines, Identification, Causal Analysis, and Prioritization of Key Barriers by KOLs in General and Covid-19 Vaccine R&D.

A frequently mentioned factor holding back the introduction of new vaccines on the market are their prohibitively long development timelines. These hamper their potential societal benefit and impairs the ability to quickly respond to emerging new pathogens. This is especially worrisome since new pathogens are emerging at all-time high rates of over one per year, and many age-old pathogens are still not vaccine preventable.Through interviews with 20 key-opinion-leaders (KOLs), this study identified innovation barriers that increase vaccine development timelines. These innovation barriers were visualized, and their underlying causes revealed by means of qualitative root cause analysis. Based on a survey the innovation barriers were quantitatively ranked based on their relative impact on both regular, and Covid-19 vaccine development timelines. KOLs identified 20 key innovation barriers, and mapping these barriers onto the Vaccine Innovation Cycle model revealed that all phases of vaccine development were affected. Affected by most barriers is the area between the preclinical studies and the market entry. Difficult hand-off between academia and industry, lack of funding, and lack of knowledge of pathogen targets were often mentioned as causes. Quantitative survey responses from 93 KOLs showed that general vaccine development and Covid-19 vaccine development are impacted by distinct sets of innovation barriers. For the general vaccine development three barriers were perceived of the highest impact; limited ROI for vaccines addressing disease with limited market size, limited ROI for vaccines compared to non-vaccine projects, and academia not being able to progress beyond proof of principle. Of highest impact on Covid-19 vaccine development, are lack of knowledge concerning pathogen target, high risk of upscaling unlicensed vaccines, and proof of principle not meeting late-stage requirements. In conclusion, the current study demonstrates that barriers hampering timelines in vaccine development are present across the Vaccine Innovation Cycle. Prioritizing the impact of barriers in general, and in Covid-19 vaccine development, shows clear differences that can be used to inform policies to speed up development in both war and peace time.

A survival of the fittest strategy for the selection of genotypes by which drug responders and non-responders can be predicted in small groups.

Phenotype Prediction Scores (PPS) might be powerful tools to predict traits or the efficacy of treatments based on combinations of Single-Nucleotide Polymorphism (SNPs) in large samples. We developed a novel method to produce PPS models for small samples sizes. The set of SNPs is first filtered on those known to be relevant in biological pathways involved in a clinical condition, and then further filtered repeatedly in a survival strategy to select stabile positive/negative risk alleles. This method is applied on Female Sexual Interest/Arousal Disorder (FSIAD), for which two subtypes has been proposed: 1) a relatively insensitive excitatory system in the brain for sexual cues, and 2) a dysfunctional activation of brain mechanisms for sexual inhibition. A double-blind, randomized, placebo-controlled cross-over experiment was conducted on 129 women with FSIAD. The women received three different on-demand drug-combination treatments during 3 two-week periods: testosterone (0.5 mg) + sildenafil (50 mg), testosterone (0.5 mg) + buspirone (10 mg), or matching placebos. The resulted PPS were independently validated on patient-level and group-level. The AUC scores for T+S of the derivation set was 0.867 (95% CI = 0.796-0.939; p<0.001) and was 0.890 (95% CI = 0.778-1.000; p<0.001) on the validation set. For T+B the AUC of the derivation set was 0.957 (95% CI = 0.921-0.992; p<0.001) and 0.869 (95% CI = 0.746-0.992; p<0.001) for the validation set. Both formulas could reliably predict for each drug who benefit from the on-demand drugs and could therefore be useful in clinical practice.

Probiotics as a treatment for prenatal maternal anxiety and depression: a double-blind randomized pilot trial.

Probiotic use may be an efficacious treatment option to effectively manage symptoms of prenatal maternal anxiety and depression. Our primary aim was to test feasibility and acceptability for a probiotic randomized controlled trial (RCT) in pregnant women with pre-existing symptoms. This double-blind pilot RCT included 40 pregnant women with low-risk pregnancies and elevated depressive symptoms and/or anxiety. Once daily, participants orally consumed a probiotic (Ecologic Barrier) or a placebo, from 26 to 30 weeks gestation until delivery. A priori key progression criteria for primary outcomes were determined to decide whether or not a full RCT was feasible and acceptable. Secondary outcomes included depressive symptoms, anxiety, stress, and maternal bonding to offspring. In 19 months, 1573 women were screened; following screening, 155 women (10%) were invited for participation, of whom 135 (87%) received study information, and 40 women (30%) were included. Four out of six a priori determined criteria for success on feasibility and acceptability were met. After 8 weeks of intervention, there was no significant difference between the probiotic and placebo groups for secondary outcomes. The pilot trial was feasible and acceptable, but hampered by recruitment method and study design. Secondary endpoints did not reveal differences between the groups for improving maternal mood.

Lower Respiratory Tract Infection Trends in East and South-East Asia: In the Light of Economic and Health Care Development.

This study explored to what degree economic development and improvement of healthcare are associated with lower respiratory tract infection (LRTI) mortality. A correlation analysis between LRTI mortality and Gross Domestic Product (GDP) per capita, and the Health Access and Quality Index (HAQI), respectively was conducted for 15 countries in East and South-East Asia. The results revealed a dramatic decrease in LRTI mortality in total populations for lower-middle income (LMI) countries but at the same time an increase in upper-middle income (UMI) and high-income (HI) countries. A highly significant (P < .001) growth-dependent relationship between LRTI mortality and economic growth was observed. Improvements in HAQI were significantly associated with a decrease in LRTI mortality in LMI countries, but an increase in UMI and HI countries. The decline of LRTI mortality amongst children in LMI countries is an encouraging trend and efforts against LRTI must be continued, though not at the expense of preparing health systems for the growing burden.

Classification and specific primer design for accurate detection of SARS-CoV-2 using deep learning.

In this paper, deep learning is coupled with explainable artificial intelligence techniques for the discovery of representative genomic sequences in SARS-CoV-2. A convolutional neural network classifier is first trained on 553 sequences from the National Genomics Data Center repository, separating the genome of different virus strains from the Coronavirus family with 98.73% accuracy. The network's behavior is then analyzed, to discover sequences used by the model to identify SARS-CoV-2, ultimately uncovering sequences exclusive to it. The discovered sequences are validated on samples from the National Center for Biotechnology Information and Global Initiative on Sharing All Influenza Data repositories, and are proven to be able to separate SARS-CoV-2 from different virus strains with near-perfect accuracy. Next, one of the sequences is selected to generate a primer set, and tested against other state-of-the-art primer sets, obtaining competitive results. Finally, the primer is synthesized and tested on patient samples (n = 6 previously tested positive), delivering a sensitivity similar to routine diagnostic methods, and 100% specificity. The proposed methodology has a substantial added value over existing methods, as it is able to both automatically identify promising primer sets for a virus from a limited amount of data, and deliver effective results in a minimal amount of time. Considering the possibility of future pandemics, these characteristics are invaluable to promptly create specific detection methods for diagnostics.

Quantity vs. quality: an assessment of the current pipeline for rabies.

OBJECTIVES: To gain insight in the potential of the current pipeline for rabies to decrease the burden of disease by evaluating its relevance to high-risk countries. METHODS: Rabies-related patent documents and clinical trials were retrieved from Espacenet and the WHO ICTRP, respectively. Data were cleaned, modulated and categorised into a pre-defined set of indicators those were used for (statistical) analyses on the number of patent applications, patent quality and type of stakeholders involved for different geographical areas. RESULTS: Analysis of 583 unique patent families applied for in the period 1954-2017 showed a steep growth in the yearly number of patent applications. A significant portion of new patent applications concern Chinese patents with relatively low quality that are filed by a dispersed group of applicants. Excluding these patents, the number of patent applications has been virtually stable over the years. A shift is seen in public stakeholders becoming more prolific as patent applicants. This shift is also reflected in clinical trials; key sponsors of clinical trials include public and private stakeholders originating from high-risk rabies countries. The majority of clinical trials investigate adjustments to existing vaccines that may improve accessibility. CONCLUSION: The results show a discrepancy between the quantity and quality of rabies patent applications that reflects national patent regulations rather than real progress in decreasing the burden of disease. This is in contrast to clinical trials, which focus on incremental innovations that are tested in clinical trials but may nevertheless have a potentially strong impact in high-risk countries.

OBJECTIFS: Comprendre le potentiel du pipeline actuel pour la rage pour réduire la charge de morbidité en évaluant sa pertinence pour les pays à haut risque. MÉTHODES: Les documents de brevet liés à la rage et les essais cliniques ont été récupérés auprès de réseau Espacenet et de l'OMS ICTRP, respectivement. Les données ont été triées, modulées et classées en un ensemble prédéfini d'indicateurs qui a été utilisé pour des analyses (statistiques) sur le nombre de demandes de brevet, la qualité des brevets et le type de parties prenantes impliquées pour différentes zones géographiques. RÉSULTATS: L'analyse de 583 familles de brevets uniques déposées au cours de la période 1954-2017 a montré une forte croissance du nombre annuel de demandes de brevet. Une part importante des nouvelles demandes de brevet concerne des brevets chinois de qualité relativement faible soumises par un groupe dispersé de déposants. A l'exclusion de ces brevets, le nombre de demandes de brevet est resté pratiquement stable au fil des ans. Un changement est observé dans les acteurs publics devenant plus prolifiques en tant que demandeurs de brevet. Ce changement se reflète également dans les essais cliniques; les principaux sponsors des essais cliniques comprennent des parties prenantes publiques et privées originaires de pays à haut risque pour la rage. La majorité des essais cliniques étudient les ajustements aux vaccins existants qui pourraient améliorer l'accessibilité. CONCLUSION: Les résultats montrent un écart entre la quantité et la qualité des demandes de brevet liées à la rage qui reflète les réglementations nationales en matière de brevets plutôt que de réels progrès dans la réduction de la charge de morbidité. Cela contraste avec les essais cliniques, qui se concentrent sur des innovations incrémentales qui sont testées dans des essais cliniques mais qui peuvent néanmoins avoir un impact potentiellement fort dans les pays à haut risque.

Probiotics in pregnancy: protocol of a double-blind randomized controlled pilot trial for pregnant women with depression and anxiety (PIP pilot trial).

BACKGROUND: Maternal prenatal depressive or anxiety symptoms are associated with adverse maternal and infant health outcomes. With prevalence rates of maternal prenatal depression and anxiety ranging between 10 and 20%, attempts to identify effective interventions to reduce symptoms are priority. There are indications that probiotics can reduce symptoms of maternal depression or anxiety. Probiotics ingested by the mother may thus offer a promising and accessible intervention to complement existing treatments. METHODS: The Probiotics in Pregnancy (PIP) pilot trial is a double-blind, placebo-controlled, randomized pilot trial. While one group orally consumes a probiotic mixture (Ecologic® Barrier; 2,5 × 109 colony forming units/g; 2 g; daily), the other group consumes a placebo, from between 26 and 30 weeks gestation until delivery. Subjects are randomly allocated (1:1) to the intervention or placebo group. Forty healthy pregnant women with symptoms of depression or anxiety and uncomplicated pregnancies at randomization will be included. The primary aim is to determine the feasibility and acceptability of a probiotic trial to reduce symptoms of maternal depression or anxiety in pregnancy. The secondary aim is to exploratorily compare the potential effect of probiotics, compared to placebo, on depressive and/or anxiety symptoms, maternal stress (i.e. reported/hair cortisol), maternal vaginal and intestinal microbiota, and by possibly affecting maternal mood and microbiota, maternal bonding to offspring, infant microbiota and infant crying. DISCUSSION: Results of this pilot trial will help determine whether or not to proceed with a full trial after the pilot trial, and if so, whether revisions should be made to the study protocol and procedures before conducting a full randomized controlled trial. Additionally, they are expected to provide insights into whether changes in psychological, behavioral and biological parameters can be attributed to the probiotic intervention. TRIAL REGISTRATION: Netherlands Trial Register, NTR6219 . Registered on 28 February 2017.

Towards a rational design of faecal transplant analogues.

Faecal transplants (microbiota transfer) have shown to be promising therapies having a wide range of therapeutic applications. However, current safety considerations hamper further valorisation. As such, well designed faecal transplant analogues provide an interesting alternative to minimize possible safety aspects. However, to date little knowledge on how to rationally design such analogues exists. Here, we show by applying first order basic graph theory that such analogues dedicated to restoring a specific physiological functionality (a microbial guild) should consist of 5-6 species to maximize stability, efficiency, and minimize safety issues and production costs.

A research agenda to reinforce rabies control: A qualitative and quantitative prioritization.

BACKGROUND: Despite the existence of safe and effective vaccines, rabies disease still causes an estimated 59,000 human deaths a year in the endemic areas in Asia and Africa. These numbers reflect severe drawbacks regarding the implementation of PrEP and PEP in endemic settings, such as lack of political will and low priority given to rabies. Since these contextual factors have proven to be persistent, there is an urgency to improve current strategies or develop novel approaches in order to control rabies disease in the future. METHODS/FINDINGS: This study aimed to identify and systematically prioritize the research needs, through interviews and questionnaires with key-opinion-leaders (KOLs). A total of 46 research needs were identified and prioritized. The top research needs are considered very high priority based on both importance for rabies control and need for improvement. KOLs agree that animal rabies control remains most important for rabies control, while research on human host, agent (rabies virus) and the environment should be prioritized in terms of need for improvement. A wide variety in perceptions is observed between and within the disciplines of virology, public health and veterinary health and between KOLs with more versus those with less experience in the field. CONCLUSION/SIGNIFICANCE: The results of this study give well-defined, prioritized issues that stress the drawbacks that are experienced by KOLs in daily practice. The most important research domains are: 1) cheap and scalable production system for RIG 2) efficacy of dog mass vaccination programs and 3) cheap human vaccines. Addressing these research needs should exist next to and may reinforce current awareness and mass vaccination campaigns. The differences in perspectives between actors revealed in this study are informative for effective execution of the One Health research agenda.

How ownership rights over microorganisms affect infectious disease control and innovation: A root-cause analysis of barriers to data sharing as experienced by key stakeholders.

BACKGROUND: Genetic information of pathogens is an essential input for infectious disease control, public health and for research. Efficiency in preventing and responding to global outbreaks relies on timely access to such information. Still, ownership barriers stand in the way of timely sharing of genetic data from pathogens, frustrating efficient public health responses and ultimately the potential use of such resources in innovations. Under a One Health approach, stakeholders, their interests and ownership issues are manifold and need to be investigated. We interviewed key actors from governmental and non-governmental bodies to identify overlapping and conflicting interests, and the overall challenges for sharing pathogen data, to provide essential inputs to the further development of political and practical strategies for improved data sharing practices. METHODS & FINDINGS: To identify and prioritize barriers, 52 Key Opinion Leaders were interviewed. A root-cause analysis was performed to identify causal relations between barriers. Finally, barriers were mapped to the innovation cycle reflecting how they affect the range of surveillance, innovation, and sharing activities. Four main barrier categories were found: compliance to regulations, negative consequences, self-interest, and insufficient incentives for compliance. When grouped in sectors (research institutes, public health organizations, supra-national organizations and industry) stakeholders appear to have similar interests, more than when grouped in domains (human, veterinary and food). Considering the innovation process, most of barriers could be mapped to the initial stages of the innovation cycle as sampling and sequencing phases. These are stages of primary importance to outbreak control and public health response. A minority of barriers applied to later stages in the innovation cycle, which are of more importance to product development. CONCLUSION: Overall, barriers are complex and entangled, due to the diversity of causal factors and their crosscutting features. Therefore, barriers must be addressed in a comprehensive and integrated manner. Stakeholders have different interests highlighting the diversity in motivations for sharing pathogen data: prioritization of public health, basic research, economic welfare and/or innovative capacity. Broad inter-sectorial discussions should start with the alignment of these interests within sectors. The improved sharing of pathogen data, especially in upstream phases of the innovation process, will generate substantial public health benefits through increased availability of data to inform surveillance systems, as well as to allow the (re-)use of data for the development of medical countermeasures to control infectious diseases.

The mechanistic link between health and gut microbiota diversity.

Although numerous reports link a decreased diversity of the gut microbiota to a declined health status, to date no mechanistic motivation for this exists. Here, we show by applying first principles basic graph theory on small networks that higher diversity within such a network indeed leads to more efficient systems and redundancy. Our results quantitatively support earlier hypothetical considerations on gut microbiota richness with respect to these parameters. Our simulations show that higher species diversity leads to higher resilience within small microbiological ecosystems, like being present in the gut microbiota. This notion should provide an ingredient when developing new interventional strategies within the domain of microbiota management.

The underexposed role of food matrices in probiotic products: Reviewing the relationship between carrier matrices and product parameters.

Probiotic microorganisms are increasingly incorporated into food matrices in order to confer proposed health benefits on the consumer. It is important that the health benefits, sensory properties, shelf-life and probiotic gastrointestinal tract (GIT) survival of these products are carefully balanced as they determine functionality and drive consumer acceptance. The strain-specific effects of probiotic species are imperative in this process but carrier matrices may play a pivotal role as well. This study therefore recapitulates the wealth of knowledge on carrier matrices and their interaction with probiotic strains. The most substantiated carrier matrices, factors that influence probiotic functionality and matrix effects on shelf-life, GIT survival and clinical efficacy are reviewed. Results indicate that carrier matrices have a significant impact on the quality of probiotic products. Matrix components, such as proteins, carbohydrates and flavoring agents are shown to alter probiotic efficacy and viability. In vivo studies furthermore revealed strain-dependent matrix effects on the GIT survival of probiotic bacteria. However, only a limited number of studies have specifically addressed the effects of carrier matrices on the aforementioned product-parameters; most studies seem to focus solely on the strain-specific effects of probiotic microorganisms. This hampers the innovation of probiotic products. More human studies, comparing not only different probiotic strains but different carrier matrices as well, are needed to drive the innovation cycle.

Functions of CD40 and Its Ligand, gp39 (CD40L).

Initially, a role for the interaction between CD40, expressed on B cells, and gp39 (CD40L), expressed on activated T cells, has been defined in humoral immunity. CD40-CD40L interaction is an essential signal for B cell proliferation, expression of activation markers, immunoglobulin production, and isotype switching. CD40-CD40L interaction is also required for formation of B memory cells and germinal centers, and signaling through CD40 prevents apoptosis of germinal center B cells. Defective expression of CD40L in humans leads to an inability to produce isotypes other than IgM (hyper IgM syndrome), and to an absence of germinal centers. More recent evidence indicates an expansion of the role of the CD40-CD40L axis in cellular interactions beyond antibody formation. Induced expression of CD40 on monocytes can lead to CD40L-activated monocyte effector mechanisms. In addition, CD40-CD40L interactions are crucially involved in development of autoimmune disease in a number of animal models. CD40-CD40L interactions also impact on growth regulation of certain carcinomas. Manipulation of CD40L has also been used to develop novel strategies for long-term antigen-specific tolerization of peripheral T cells. Finally, the CD40-CD40L axis is involved in thymic selection. Following is a comprehensive overview of CD40L-CD40 interactions in physiological and pathogenic cellular responses and a discussion of the therapeutic ramifications of these interactions.

Vector-based genetically modified vaccines: Exploiting Jenner's legacy.

The global vaccine market is diverse while facing a plethora of novel developments. Genetic modification (GM) techniques facilitate the design of 'smarter' vaccines. For many of the major infectious diseases of humans, like AIDS and malaria, but also for most human neoplastic disorders, still no vaccines are available. It may be speculated that novel GM technologies will significantly contribute to their development. While a promising number of studies is conducted on GM vaccines and GM vaccine technologies, the contribution of GM technology to newly introduced vaccines on the market is disappointingly limited. In this study, the field of vector-based GM vaccines is explored. Data on currently available, actually applied, and newly developed vectors is retrieved from various sources, synthesised and analysed, in order to provide an overview on the use of vector-based technology in the field of GM vaccine development. While still there are only two vector-based vaccines on the human vaccine market, there is ample activity in the fields of patenting, preclinical research, and different stages of clinical research. Results of this study revealed that vector-based vaccines comprise a significant part of all GM vaccines in the pipeline. This study further highlights that poxviruses and adenoviruses are among the most prominent vectors in GM vaccine development. After the approval of the first vectored human vaccine, based on a flavivirus vector, vaccine vector technology, especially based on poxviruses and adenoviruses, holds great promise for future vaccine development. It may lead to cheaper methods for the production of safe vaccines against diseases for which no or less perfect vaccines exist today, thus catering for an unmet medical need. After the introduction of Jenner's vaccinia virus as the first vaccine more than two centuries ago, which eventually led to the recent eradication of smallpox, this and other viruses may now be the basis for constructing vectors that may help us control other major scourges of mankind.

Self-Centric and Altruistic Unmet Needs for Ebola: Barriers to International Preparedness.

OBJECTIVE: Barriers to international Ebola preparedness may be elucidated by identifying heterogeneities in arguments to invest in countermeasures during "peace time." METHODS: For each patent family (related patent documents that differed only by limited alterations to the same invention) concerning Ebola and published until the end of 2014 the oldest patent document was analyzed. Grounded theory coding identified 5 unmet needs for (1) vaccines and therapies, (2) control of outbreaks in endemic areas, (3) detection and control of outbreaks in nonendemic areas, (4) better understanding of filoviruses, and (5) protection against bioterrorism. Odds ratios for unmet needs by geographic regions and institution types were compared by using Pearson's chi-square test. RESULTS: Statistically significant heterogeneities in unmet need profiles were found. US applicants combined self-centric and altruistic arguments, focusing on medical unmet needs and bioterrorism protection. Russian and Asian applicants emphasized self-centric motives, specifically, detection and control of nonendemic outbreaks. A clear, statistically significant mismatch between industry and academia was found: whereas industrial applicants focused on bioterrorism and neglected detection and control of nonendemic outbreaks, academic applicants did the opposite. CONCLUSIONS: This research identified heterogeneities in articulated needs between geographic regions and stakeholder types. Structural articulation of unmet needs may form the basis for attuning stakeholder engagement strategies while progression across the demand-driven value chain might necessitate international concordance. (Disaster Med Public Health Preparedness. 2016;10:644-648).

Lactobacillus plantarum WCFS1 and its host interaction: a dozen years after the genome.

Lactobacillus plantarum WCFS1 is one of the best studied Lactobacilli, notably as its genome was unravelled over 12 years ago. L. plantarum WCFS1 can be grown to high densities, is amenable to genetic transformation and highly robust with a relatively high survival rate during the gastrointestinal passage. In this review, we present and discuss the main insights provided by the functional genomics research on L. plantarum WCFS1 with specific attention for the molecular mechanisms related to its interaction with the human host and its potential to modify the immune system, and induce other health-related benefits. Whereas most insight has been gained in mouse and other model studies, only five human studies have been reported with L. plantarum WCFS1. Hence NCIMB 8826 (the parental strain of L. plantarum WCFS1) in human trials as to capitalize on the wealth of knowledge that is summarized here.