The aim of this procedure is to widen the spinal canal by using minimally invasive techniques to do hemipartial laminectomy and bilateral flavectomy in patients with cervical spinal stenosis due to ligamentum flavum hypertrophy.A 66-year-old man presented with increasing neck and right shoulder pain for one year to Koç University Hospital. He reported a three-month history of numbness in his hands. The Japanese Orthopedic Association (JOA) and Visual Analogue Scale (VAS) scores were 15 and 8, respectively. Preoperative magnetic resonance imaging (MRI) revealed spinal canal stenosis at the C3-4 level secondary to ligamentum flavum hypertrophy. Hemi-partial laminectomy at the C3 level, flavectomy, and bilateral decompression were performed using the right unilateral approach. The patient's complaints of symptoms considerably decreased three months later. The VAS and JOA scores were 2 and 16, respectively.This minimally invasive approach can be an alternative to classic laminectomy in patients who have radiculopathy and myelopathy due to posterior origin spinal stenosis in order to safely resolve pain and neurologic dysfunction. IntroductionFor several years, various techniques have been evaluated to determine the most accurate treatment for cervical spondylosis causing myelopathy and radiculopathy. Even though many studies have been conducted in the last decades, there still is not a consensus on the optimal surgical management. In general, surgical approaches can be divided into anterior, posterior and anterior and posterior cervical canal decompression approaches. Each approach can be supplemented by fusion. The anterior approach typically comprises anterior cervical discectomy with fusion and anterior cervical corpectomy with fusion, whereas laminectomy in the presence and/or absence of instrumentation and/or laminoplasty is included in the posterior approach. Long-term follow-up studies on laminectomy for multilevel cervical stenosis have reported that the procedure causes serious postoperative complications, such as segmental instability, progressive kyphosis, swan-neck deformity, and perineural adhesions; laminoplasty, which is a commonly used procedure for treating cervical stenosis owing to its posterior origin and the hypertrophy or ossification of the ligamentum flavum, was developed to avoid these complications -6. FurthThis study aimed to introduce a minimally invasive alternative approach for the treatment of cervical stenosis resulting from ligamentum flavum hypertrophySurgical techniqueA 66-year-old man presented with increasing neck and right shoulder pain for one year at Koç University Hospital. He reported a three-month history of numbness in his hands. The Japanese Orthopedic Association (JOA) and Visual Analogue Scale (VAS) scores were 15 and 8, respectively. Preoperative magnetic resonance imaging (MRI) revealed spinal canal stenosis at the C3-4 level secondary to ligamentum flavum hypertrophy  ,8. The dKamioka et al.  reportedConventional laminectomy causes cervical instability and kyphosis when facet joints are resected ,20. DynaOur technique has two basic advantages over standard laminectomy: 1. it preserves the biomechanical functions of the posterior muscle complex with minimal skin incisions and 2. it maintains joint integrity via partial laminectomy. The reported technique protects the neural arc, which provides stability to the cervical spine and spinous processes to which some parts of the cervical muscle complex attach. Despite the many advantages, our technique is appropriate for only selected patients with posteriorly originated hypertrophy or ossification of the ligamentum flavum that is not accompanied by anteriorly originated spinal stenosis.ConclusionThis minimally invasive posterior approach can be safely applied as an alternative to classic laminectomy to relieve pain and neurological dysfunctions in myelopathy caused by posteriorly-originated compressive spinal stenosis. The technique has many advantages, such as shorter hospital stay, less postoperative pain, and decreased morbidity compared with the standard laminectomy and laminoplasty techniques. It also has important advantages of preserving the neural arc, partial hemilaminectomy, and limited muscle dissection. These advantages include less postoperative pain medication and quicker return to resuming daily life.


We report a case of branch retinal artery occlusion (BRAO) that occurred after percutaneous coronary intervention (PCI). A 59-year-old man with no other previous diseases presented visual acuity deterioration in the left eye 24 hours after PCI. Fundus examination revealed ischemia at the temporal branch of the retinal artery associated with inner layer edema. Prompt treatment was performed with ocular digital massage and paracentesis of the anterior chamber. However, at discharge, the patient had a persistent visual loss with a central scotoma that persisted at 35-day follow-up without improvement of the visual acuity. The patient did not suffer from any other systemic complications. Retinal infarction should be considered a potential complication of PCI. Patients and health care providers should be aware of any visual signs. Permanent visual disability can be prevented by immediate diagnosis and prompt intervention. Percutaneous coronary intervention (PCI) is currently indicated for the management of patients presenting with coronary artery disease (CAD). Advances in technology, development of more potent and effective antiplatelet therapy, and judicious use of PCI are increasing the safety of the procedure. However, major complications during and after PCI still occur that can be related to the access site, intubation of the coronary artery ostia, or the intervention itself. The most common problems occur in the context of coronary intubation and target vessel or site intervention . HoweverIn November 2020, a 59-year-old man, an ex-smoker with negative previous medical history of heart disease, was admitted to the emergency department of Sondrio Hospital , 48 hours after the onset of episodes of rest angina associated with atypical angina symptoms .Admission ECG showed a heart rate of 95 bpm and a slight ST segment depression in the lateral leads. Measurement of cardiac biomarkers showed a rise in high-sensitivity troponin I . Double antiplatelet therapy (DAPT) with aspirin 100 mg/day and clopidogrel 75 mg/day was started on admission, and the same day, the patient underwent coronary angiography by right transradial access. The angiogram showed 2-vessel CAD (coronary artery disease) with subocclusion of the proximal circumflex coronary artery (LCx) and critical stenosis of the proximal and middle anterior descending coronary artery (LAD) Figures . After aTwenty-four hours after PCI, the patient reported the onset of a left central scotoma and an ophthalmological evaluation was requested. The left visual acuity was reduced to 0.7 logMAR with a central scotoma. There was no alteration in the anterior segment. Intraocular pressure (IOP) was 10 mmHg. Fundus examination revealed a white embolus in the temporal branch of the central retinal artery . A compuBranch retinal artery occlusion (BRAO) is one common cause of retinal vascular disease; it is caused by a reduction in arterial blood flow, resulting in retinal ischemia which can lead to severe vision loss and visual field defects despite therapy. Commonly, it is associated with systemic risk factors including hypertension, diabetes, carotid artery disease, and atherosclerosis. Few studies suggest that different retinal complications could be observed after coronary angiography procedures. Eye involvement includes central retinal artery occlusion (CRAO) , 4, branAn immediate treatment is required if the occlusion occurs within 24 hours from the procedure. Conventional therapy consists in the reduction of intraocular pressure with hypotensive drugs, digital massage of the eyelid, and anterior chamber paracentesis. In some cases, thrombolytics can be infused into the carotid artery, or a laser embolectomy can be performed. The goal of the treatment is reperfusion of the blocked retinal vessels particularly when it is done within 6 h, because retinal ischemic damage after 240 min is normally permanent . HoweverWith the growing occurrence of PCI, all physicians involved in patient care should be worried in keeping special attention towards ocular signs. Visual symptoms and retinal artery occlusions caused by embolization in patients after PCI could occur even 24 hours after catheterization. Prompt diagnosis and early treatment may help improve the outcome and preserve vision deterioration.


Pierre-Robin sequence (PRS) patients frequently exhibit symptoms of airway obstruction due to multiple etiologies, predominantly from glossoptosis and tongue base obstruction. Rarely, these patients can have palatal mass and even rarer is one of neural origin. To date, there are few reports of heterotopic neural tissue causing airway obstruction in literature, and there are only two reports related to PRS. The objective of this report is to detail a PRS patient with obstructive airway symptoms that resolved after removal of a right-sided soft palatal mass containing heterotopic neural tissue. A 5-month-old boy with a past medical history of cleft palate, PRS status-post-mandibular distraction osteogenesis was hospitalized after continuing respiratory distress. Imaging showed a cystic submucosal mass that arose from the right soft palate. Trans-palatal and trans-oral approaches were applied for the removal. The patient tolerated the procedure well and his obstructive events have resolved at follow-up.  Airway obstruction in a pediatrics patient warrants urgent workup as it can lead to clinical disaster , 2. SpecThe present study reports a PRS patient with obstructive airway signs due to a right-sided soft palatal mass containing heterotopic neural tissue, whose symptoms resolved after removal of the mass. The intention of this report is to alert practitioners in the future who come across similar presentations.A 5-month-old boy, who was born at term with a past medical history of PRS, isolated cleft palate, retrognathia status-post mandibular distraction osteogenesis treatment and gastrostomy tube dependence, presented to the emergency department after worsening apneic events throughout the past few weeks. Of note, he had successfully undergone mandibular distraction and was discharged a month earlier after distractor removal. The patient had a normal direct laryngoscopy and bronchoscopy at that time. After the discharge, the patient was followed by an Otolaryngologist in the clinic who performed routine flexible laryngoscopies and noted a right-sided soft palatal mass.The patient was hospitalized for respiratory difficulty. During his hospitalization, the patient had episodic desaturations down to 65%, which were improved minimally with suctioning and repositioning. While asleep, the patient developed intermittent increased work of breathing, nasal flaring, subcostal and suprasternal retractions which necessitated increased respiratory support and ICU placement. Inpatient magnetic resonance imaging (MRI) exhibited a 1.6 × 1.4 × 1.5-cm right-sided cystic submucosal mass arising from soft palate with no aggressive features or invasion .Given the persistent obstructive symptoms, surgical removal of the mass was necessitated. During the operation, an obvious cleft palate was visualized along with two uvulas . The rigAfter removal of the mass, a 16-French nasal trumpet was passed down the right nasopharynx, indicating that the nasopharyngeal airway appeared markedly improved. Given the proximity of surgical intervention near the carotid artery and a potential carotid artery rupture, the patient was sent for inpatient observation for 7 days post-operatively. Final surgical pathology of the mass showed benign neuroglial tissue, consistent with heterotopic tissue. While in observation, the patient stabilized to breathing on room air without concern. He was then discharged, and at follow-up, his obstructive events have resolved and his sleeping has dramatically improved.Airway obstruction in newborn children can be devastating and requires urgent workup. PRS is a well-known clinical sequence that characteristically leads to airway obstruction, which is often managed surgically. Soft palate masses are a rare cause of airway obstruction.The presented data are scarce regarding oropharyngeal masses containing heterotopic neural tissue, which led to airway obstruction in PRS patients. Some cases of heterotopic neural tissue in the pharyngeal area leading to airway obstruction were reported previously but neither patient had PRS . There aThe soft palatal mass containing heterotopic neural tissue could be lethal. It was reported a PRS newborn with a huge left-sided soft palatal mass extending into the infratemporal fossa passed away due to a tracheostomy dislodgement . The masAnother report detailed a case of heterotopic neural tissue involving the PPS in a PRS patient which led to airway obstruction, which had also been successfully removed . The repThe pathogenesis of heterotopic neuroglial tissue is unclear, but several mechanisms have been proposed. One hypothesis is that during early embryogenesis, displacement of totipotent neuroectodermal cells arises and develops into mature neural tissue. Another hypothesis is abnormal migration of glial cells from the olfactory bulbs .The present report has described an unusual source of airway obstruction in a PRS patient who underwent corrective airway surgery. We encourage clinicians to explore other etiologies in PRS patients with continuing symptoms of airway obstruction.Heterotopic neural tissue resembling a mass arising from the soft palate can lead to respiratory compromise in PRS patients. Close follow-up and surgical intervention are warranted for these patients with necessary surgical preparation and planning.


Understanding the immune composition of a given tumor is critical to assess its potential responsiveness to cancer immunotherapy. This is especially true for tumors that are intrinsically resistant to immunotherapies, such as GBM. Unfortunately, studies on the functional heterogeneity and associated molecular targets of immune-suppressive cells in vivo have been lacking. Here we report an integrated multi-dimensional analysis of the mutational profiles and single-cell transcriptomics of 60,024 glioma and stromal cells from 16 human samples. We identified molecular signatures of seven distinct macrophage subtypes, each with prognostic clinical value. The three inflammatory subtypes showed hallmarks of TNF/NFκB pathway enrichment and are associated with good outcomes; in contrast, four immunosuppressive subtypes with metabolic pathway hallmarks  are associated with poor survival. In addition, we resolved an ongoing controversy in the field regarding the roles of brain resident macrophages, microglia, vs. bone marrow derived macrophages (BMDM) in gliomas. Our data show compelling evidence that microglia are pro-inflammatory and are associated with good survival while BMDMs are mostly immune-suppressive and associated with poor survival. In addition, deciphering immune-suppressive macrophage and Treg molecular signatures enabled us to identify previously unknown immunotherapy targets. In a proof of principle study, we showed that S100A4, a calcium binding protein previously shown to mediate metastasis, was universally upregulated in both innate and adaptive immune suppressor cells, and implantation of gliomas in S100a4-/- host mice significantly extended survival and resulted in pro-inflammatory immune landscape, compared to same glioma cells implanted in B6 control hosts. This functional validation study shows that S100A4 is a highly promising therapeutic target for GBM immunotherapy.


Plasmodium falciparum infections. However, methods to identify and sequence these genomic regions are currently limited.Targeted next-generation sequencing offers the potential for consistent, deep coverage of information-rich genomic regions to characterize polyclonal A bioinformatic pipeline and multiplex methods were developed to identify and simultaneously sequence 100 targets and applied to dried blood spot (DBS) controls and field isolates from Mozambique. For comparison, whole-genome sequencing data were generated for the same controls.P. falciparum heterozygome. For this study, 93 microhaplotypes with high diversity (median expected heterozygosity = 0.7) were selected along with 7 drug resistance loci. The sequencing method achieved very high coverage (median 99%), specificity (99.8%), and sensitivity (90% for haplotypes with 5% within sample frequency in dried blood spots with 100 parasites/µL). In silico analyses revealed that microhaplotypes provided much higher resolution to discriminate related from unrelated polyclonal infections than biallelic single-nucleotide polymorphism barcodes.Using publicly available genomes, 4465 high-diversity genomic regions suited for targeted sequencing were identified, representing the P. falciparum genome, and can be tailored to simultaneously address multiple questions of interest in various epidemiological settings.The bioinformatic and laboratory methods outlined here provide a flexible tool for efficient, low-cost, high-throughput interrogation of the  Plasmodium falciparum genome, followed by a robust polymerase chain reaction–based multiplexing method to simultaneously amplify and sequence these microhaplotypes.A novel bioinformatics pipeline was created to select and characterize the most diverse and tractable short-range sequences (microhaplotypes) in the  Malaria genomics has been applied to generate actionable data to inform control and elimination efforts, for example, tracking the spread of drug resistance and evaluating the response to vaccine candidates . To realP. falciparum diversity [Recent advances in next-generation sequencing (NGS) allow targeted deep sequencing of short, highly variable regions with numerous alleles (microhaplotypes) , 17, preiversity , 27.P. falciparum genome using NGS. However, bioinformatics pipelines to identify informative targets are currently lacking. Furthermore, finding sensitive and high-throughput laboratory methods for targeted sequencing of these markers in a cost-effective manner remains a major challenge. In this study, we describe the initial evaluation of a bioinformatic pipeline to identify high-value P. falciparum microhaplotypes and a robust polymerase chain reaction (PCR)–based laboratory method that allows sequencing of hundreds of these microhaplotypes in a single reaction. This study evaluates the performance of the laboratory method and the information content of a selection of approximately 100 microhaplotypes across a range of in silico analyses, mixture controls, and field samples.With thousands of whole-genome sequencing (WGS) data available , 29, it P. falciparum 3D7 reference genome were determined using tandem-repeat-finder [Tandem repeats longer than 50 bp within the t-finder . Windowst-finder . Windowst-finder . WindowsP. falciparum parasites were mixed at different proportions with uninfected human whole blood to obtain a range of parasite densities  (Mock dried blood spot (DBS) samples were prepared as previously described . In brief blood) . Field DPrimers were designed for 100 selected genomic regions of the microhaplotypes and drug resistance markers using the CleanPlex algorithm (Paragon Genomics). Amplification of the 200-plex oligo pool was performed with some modifications of the CleanPlex protocol (Paragon Genomics) . SamplesP. falciparum 3D7 reference genome (version 3) with BWA-MEM software [Selective whole-genome amplification and WGS were performed following a previously optimized protocol . After dsoftware . Variantsoftware .Population-level allele frequencies were calculated for 91 microhaplotypes and variant loci across Pf3k samples from Ghana, which had the largest number of publicly available WGS. Simulated genomes were created with a single multinomial draw given allele frequencies across all loci. Relatedness between genomes was simulated parameterizing a Bernoulli draw by the expected relatedness over the number of loci, masking the selected loci, and making both genome pairs equivalent at each masked locus. Relatedness was calculated by converting each locus to a boolean vector, concatenating all loci in a sample to a single vector, then calculating Jaccard distance between sample pairs.P. falciparum to identify short genomic regions that exhibit high population-level genetic diversity, here termed “heterozygome” windows  in the global parasite population, and 356 had HE > 0.7  and differentiated (Jost D ≥ 0.21) within Africa were combined with 11 molecular markers of drug resistance for primer design for 2 × 150 Illumina sequencing. From primer design, 7 markers of drug resistance had workable primers and the top 93 microhaplotypes with primers were selected for a total of 100 loci . The selA 2-step multiplex PCR-based assay was optimized using CleanPlex chemistry (Paragon Genomics) for evenness of coverage and detection of minority clones. The assay was evaluated on a range of DBS controls containing known proportions of laboratory strains . A high Compared with targeted sequencing, WGS provides broader evaluation of the genome but at the expense of sequence depth in specific regions of interest. To compare the sensitivity of targeted vs WGS for detection of microhaplotype loci, WGS data were generated from the same DBS mixture controls, using selective whole-genome amplification, at high depth and coverage . From thTo compare the accuracy of complexity of infection (COI) estimation and genetic relatedness between infections by microhaplotype and SNP-based approaches, we extracted molecular SNP barcodes (n = 24 ), SpotMaPolyclonal infections are common in many endemic areas, making it difficult to estimate genetic relatedness between infections. To evaluate the discriminatory power of microhaplotypes vs SNPs, a simple simulation of infection pairs with varying numbers of clones and related parasites was performed. Perfect detection of alleles was assumed, to isolate the information content of the loci from the sensitivity of the laboratory method. For monoclonal infections, all methods performed similarly and were able to easily discriminate unrelated parasites from siblings and clones . HoweverP. falciparum DBS samples from southern Mozambique were genotyped using the microhaplotype panel. Similar to the control samples, a high level of uniformity in the average number of reads per target  between the heterozygosity of the microhaplotypes in the global parasite population and the observed heterozygosity in Mozambique , high-diversity microhaplotypes should provide the required resolution. Based on our analysis, many of these loci are diverse across multiple geographic settings. If the goal is to perform spatial assignment of infections, microhaplotypes with high genetic differentiation between infections from relevant geographic regions should be considered. These may be more specific to particular contexts, as signals of genetic differentiation are more varied geographically and at different spatial scales. Thus, the availability of globally diverse windows across different transmission zones should allow the development of a core panel, with the potential to add, for example, a subset of highly differentiated microhaplotypes for geographic regions of interest or evenly distributed windows across the genome for Identity by decent analysis. It should be noted that many of the microhaplotypes identified herein may be under balancing or directional selection. As such, they may not be appropriate for analyses that mandate neutral markers . If neutP. falciparum microhaplotypes in translating genomic data into meaningful epidemiologic intelligence. The development of appropriate analytical frameworks will also facilitate the rational selection of informative microhaplotype panels, for example, determining how many and which microhaplotypes provide adequate resolution to answer a set of questions in a range of epidemiologic settings. Availability of the appropriate amplicon panels and calibrated tools will bring us much closer to mapping epidemiologic and genetic data onto operationally relevant measures such as changes in transmission intensity, evaluation of the impact of interventions, accurate classification of local and imported infections, and evaluation of transmission chains [This analysis has identified a set of generally informative markers and has begun to evaluate the utility of using microhaplotypes for high-resolution genotyping of low-density and polyclonal infections. However, recognizing the full potential of genetic data generated from this approach will require additional improvements in downstream analysis. Few analytical tools directly address the challenges of incorporating multiallelic data or evaluation of polyclonal infections; even fewer encompass both. For example, to our knowledge there are no established methods for calculating basic statistics such as population-level allele frequency or formally calculating genetic distance between polyclonal infections using multiallelic markers, forcing the use of ad hoc metrics such as those used here and in prior analyses . Near-ten chains , 50.hrp2/3 deletions), would enable a larger set of use cases to be addressed simultaneously. With such a panel and appropriate analytical tools, an integrated approach to genomic surveillance allowing for routine generation of these valuable data in the areas where they are needed most may be closer to becoming a reality.Incorporating genomics into malaria surveillance in endemic settings will not be trivial, but the availability of efficient and information-rich strategies for generating genomic data from routinely collected field samples, such as that evaluated here, is an important step toward this goal. The ability to simultaneously obtain deep sequence data from targets informing drug resistance and multiple aspects of transmission epidemiology should allow for streamlining and standardization of laboratory and bioinformatic pipelines, allowing these technologies to be transferred more rapidly to endemic settings. Additional components not evaluated here, such as species identification, an expanded number of candidate and validated molecular markers of drug resistance, and markers of diagnostic resistance (eg, The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at jiaa527_suppl_Supplementary_Table_S1Click here for additional data file.jiaa527_suppl_Supplementary_Table_S2Click here for additional data file.jiaa527_suppl_Supplementary_Table_S3Click here for additional data file.jiaa527_suppl_Supplementary_Table_S4Click here for additional data file.jiaa527_suppl_Supplementary_FiguresClick here for additional data file.jiaa527_suppl_Supplementary_MethodsClick here for additional data file.


Although the use of long-read sequencing improves the contiguity of assembled viral genomes compared to short-read methods, assembling complex viral communities remains an open problem. We describe the viralFlye tool for identification and analysis of metagenome-assembled viruses in long-read assemblies. We show it significantly improves viral assemblies and demonstrate that long-reads result in a much larger array of predicted virus-host associations as compared to short-read assemblies. We demonstrate that the identification of novel CRISPR arrays in bacterial genomes from a newly assembled metagenomic sample provides information for predicting novel hosts for novel viruses.The online version contains supplementary material available at (10.1186/s13059-021-02566-x). Since the International Committee on Taxonomy of Viruses has proposed to include MAVs into viral taxonomy studies [Various metagenomic studies have greatly expanded the set of known viral genomes –6 and ha studies , there iSo far, short-read sequencing has been the dominant technology for the discovery of novel MAVs . Such diModern short-read metagenomic assemblies rarely result in a complete assembly of even a single bacterial genome in a bacterial community. Recent progress in long-read sequencing technologies has resulted in many long-read metagenomic projects in the last two years –23 and lWe modified metagenomic long-read assembler metaFlye  to impromulti-edge connected component in the metagenome assembly graph. In contrast to previous approaches that mainly focused on single contigs, the viralFlye pipeline analyzes both single viral contigs and multi-edge viral components. It includes the following steps described in the “Launching the modified metaFlye assembler (that we refer to as metaFlye_v) aimed at viral genome sequencing.Polishing the metaFlye assembly with short reads .Identifying isolated linear and circular contigs in the assembly graph.Identifying multi-edge connected components in the assembly graph (the ComponentTraverser module).Detecting MAVs by verifying the identified linear and circular contigs and connected components as MAVs using the viralVerify tool  and checClassifying viral genomes into linear and circular (the CircularDisconnector module).Predicting the virus-host associations based on CRISPR analysis .A complete viral genome can be represented as an isolated contig (either linear or circular). However, if a viral genome is heterogeneous (represented by multiple closely-related strains or species), it may form a We benchmarked viralFlye using the following four datasets that cover various types of the currently available long read technologies. All datasets were downloaded from SRA in the fastq format.HUMAN_GUT: Long error-prone reads from fecal samples from twelve individuals analyzed in  , and low-coverage, otherwise. Since the single-base accuracy of low-coverage contigs in assemblies of long error-prone is low, we focus on analyzing high-coverage contigs. We note that metaFlye outputs information about the contig coverage but Raven does not—it only provides the read count readCount for each contig. We thus estimate the contig coverage in the Raven assembly using the average read length for each dataset (computed as readCount×readLength/contigLength). This approach may result in a biased estimate of the coverage since the read length for all analyzed datasets has a rather high variance.We classify a contig as minLength  and maxLength . A user can change these default values, e.g., to analyze short circular ssDNA viruses, it would be useful to decrease the minLength parameter to 1.5 kb.viralFlye extracts high-coverage circular and linear isolated contigs from the GFA assembly files with the lengths varying between Table Nine viral genomes assembled in  MK41539 reveals direct terminal repeats (DTRs of length varying from 1905 bp to 2170 bp). Since DTRs often trigger misrepresentations of linear genomes by circular contigs, CircularDisconnector detects and linearizes the misrepresented circular contigs . In , the autTable We further used the assemblies from the original manuscript  to estimCircularDisconnector labeled 28, 4 and 26 circular contigs in ALOHA_VIROME1, ALOHA_VIROME2, and ALOHA_VIROME3 datasets as falsely circularized linear viral contigs. We hypothesize that there are many more linear contigs that were incorrectly classified as circular ones but evaded detection by CircularDisconnector due to highly uneven coverage.Table Although the metaFlye pipeline assembled more circular viral contigs, viralFlye assembled more linear contigs—the result of some circular genomes assembled by meatFlye re-classified as linear by viralFlye. We randomly selected 10 such viral contigs and examined their read alignments profiles. In 7 out of 10 cases, the metaFlye contigs were incorrectly circularized due to the chimeric read connections. These incorrectly circularized linear contigs were detected by metaFlye_v and re-classified as linear viruses. In 1 out of 10 cases, the circular contig constructed by metaFlye was likely correct. In the remaining two cases, the manual analysis was inconclusive due to the complex alignment patters.checkV classified 139 out of 158 linear viral contigs and 150 out of 153 circular viral contigs as complete. The average completeness (estimated with checkV) was 90.7% for linear and 92.8% for circular viral contigs. CircularDisconnector found 16 incorrectly circularized linear contigs in this dataset.Ovis aries) and a sheep parasite Blastocystis spp.. The remaining 26 uncharacterized circular contigs may represent novel and unusual plasmids and circular viruses that have evaded identification by the existing plasmid and virus identification tools .We note that there are 153 high-coverage circular contigs shorter than 1Mb in the metaFlye_v assembly. Since the circular contigs represent isolated loops in the metaFlye_v assembly graph, they likely originate from viral genomes and plasmids. Among 55 non-viral circular contigs, 20 were identified as plasmids using the plasmid identification approach from  and 9 asOn the other hand, metaFlye_v generated 158 non-characterized high-coverage linear contigs in the SHEEP_GUT dataset. These contigs likely originate either from novel and unusual linear viruses or from bacterial genomes with highly non-uniform coverage that resulted in sharp coverage drops. Figuring out which of the uncharacterized circular and linear contigs represent new viruses remains an open problem.Identification of known CRISPR spacers in MAVs is a powerful approach for predicting potential known hosts for novel viruses from metagenomic assemblies , 32. HowIllumina reads from the SHEEP_GUT dataset were assembled using MEGAHIT v1.2.9 . To predThe host contigs in the long-read assembly were significantly longer (average/median length 519/135 kb in the long-read assembly as compared to 13/3.5 kb in the short-read assembly). Interestingly, although matching against the CRISPRCasdb library identified more virus-host pairs, these predictions show a very small overlap with the identified intra-sample virus-host pairs. Thus, complementing the traditional matching against the CRISPR database with the intra-sample host predictions significantly increases the number of the predicted virus-host associations.In most cases, the host taxonomy in both datasets and the CRISPRCasdb library was in agreement, at least at the order level  provides information for predicting potential novel hosts for novel viruses and results in a large number of identified virus-host associations. Interestingly, there is a very small overlap between the viral-host pairs identified by the sample-dependent and the sample-independent approaches, suggesting that these approaches are complementary.Although identification of Our study revealed 1036 uncharacterized circular (15967 linear) contigs which can originate from novel and unusual viruses and plasmids. There are multiple reasons that may explain a large number of uncharacterized potentially viral contigs in our assemblies. First, since the existing viral identification tools were trained on known viruses, they are unlikely to classify a contig as viral if it represents a highly diverged virus. Second, since the Stop codon reassignment is common , these tAlthough metagenomic sequencing has greatly expanded our knowledge of the Earth’s virome, extracting complete sequences of viral genomes from metagenomic assemblies remains challenging. Previous studies, aimed at the discovery of novel viruses, often focused on viral contigs in metagenomic assemblies and thus missed an opportunity to sequence complete viral genomes by switching from the contig-based to the assembly graph-based analysis. Emergence of long reads opened a possibility to sequence many complete viral genomes that evaded all attempts to sequence them using short reads.We demonstrated that viralFlye improves identification of complete viruses from long-read metagenomic datasets and has a potential to transform metagenomics-based assembly of novel viruses from a challenging task into a routine procedure. viralFlye recovers up to 2.25 times more complete or nearly complete MAVs, as compared to the previously published pipelines, while reducing the number of misassemblies. We also show that long reads improve the accuracy of virus-host association predictions and reveal many viral quasispecies, populations of multiple viral strains (species) that have structural variations or highly diverged regions.disjointigs and “glues” them into the metagenome assembly graph [metaFlye generates ly graph . This prTo address these complications, we modified the disjointing assembly algorithm in metaFlye by (i) preserving rather than discarding short disjointigs derived from reads covering the entire viral genome and (ii) aggressively filtering possibly chimeric reads that resulted from multiple linear viruses. To enable (i), in the updated metaFlye_v tool, we allow a disjointig to be represented by a single read, provided that such read is supported by other reads. To enable (ii), metaFlye_v detects ends of linear viruses characterized by a sharp drop in coverage by reads; such regions are then subjected to stricter parameters for the chimera detection algorithm . Applyinfreebayes tool [bcftools consensus tool [In the case when both long error-prone reads and short accurate (Illumina) reads are available, viralFlye polishes the metaFlye long-read assembly using short reads. The polishing step reduces the number of base-calling errors and indels in metaFlye assemblies of long error-prone reads (particularly in the case of low-coverage genomes) and thus improves gene prediction, an important step for the success of the downstream virus identification tools , 29. viryes tool  (with thyes tool ) followesus tool  for policompleteness threshold), it often underestimates the completeness in the case of novel viral genomes with limited similarities to known viral genomes. Since metagenomic samples likely contain many viruses without close database references, we have reduced the completeness threshold to 50% and provided an option to change it.There exist multiple viral detection tools that classify each contig into viral and non-viral, such as viralVerify  and checEven though most viral genomes are much shorter than bacterial genomes, environmental viral samples often have high inter- and intra-species heterogeneity . This heviral if it represents a viral genome . Although there exist multiple viral detection tools that classify isolated contigs into viral and non-viral , cl, clvirallength of a connected component Component in an assembly graph (denoted as length(Component)) as the total length of its edges. A connected component is called small if minLength<length(Component)<maxlength, where minLength, and maxLength are thresholds with default values minLength=5 kb and maxLength=1 Mb . The coverage of a connected component Component is defined as the average coverage of its edges normalized by the edge length, i.e, asWe define the numberTraversals such paths .The ComponentTraverser module classifies each small high-coverage connected component of the assembly graph as either viral or non-viral. Given a connected component, it generates random traversals of this component and then launches a viral detection tool on each random traversal. To generate a random traversal, ComponentTraverser launches a random walk that starts from a path formed by a single edge (the longest edge in the component) and gradually extends this path in both directions by randomly adding still untraversed edges until no such edges are found. ComponentTraverser generates viralFlye classifies a multi-edge component as viral if one of the generated traversals is classified as viral by a viral detection tool. It further reports all multi-edge viral components in individual files for further manual examination. Figure direct terminal repeats (DTRs) on both ends that can span 10 kb and more in length [Many linear viruses have n length . Since ln length , it exten length , five vin length  represenCircularDisconnector is based on the observation that the two DTR regions in a linear genome (at the beginning and at the end of the genome) are “glued” into a single region with sharply increased coverage (as compared to the average coverage of this genome) in the assembly graph. The coverage of this region is expected to be nearly twice larger  because it is covered by reads from both the beginning and the end of the genome.jump (coverage drop) if its coverage is larger  than the coverage of the neighboring 100 bp long region by a factor of CoverageJump . To decide whether a circular contig represents a linear genome, CircularDisconnector checks whether there exist coverage jumps/drops in this contig. Figure A position in a contig is classified as a coverage rolling circle replication may span these positions. However, since the number of such reads is typically small, we classify a region between two positions as a low-coverage region if the number of reads spanning both these positions is at least coverageReduction times lower than the average number of reads spanning regions of the same size in the assembled genome .Another indication that a region between two positions in a circular contig actually represents a DTR in a linear virus is the sharp drop in the number of reads spanning these two positions (as compared to the average number of reads spanning segments of the same size). Although we do not expect to see any reads spanning both these positions of a linear genome (and extending beyond them), (i) chimeric reads, (ii) reads arising from a linear genome in the lysogenic stage, and (iii) reads arising from a linear genome concatenation during the sliding window technique [jump and drop separated by distance span=|drop−jump|) satisfy the following two conditions: (i) the region between positions jump and drop is a low-coverage region and (ii) span>minSpan. The default value of minSpan equals the parameter min-overlap  in metaFlye that defines the minimum overlap between disjointigs in the metaFlye gluing procedure .CircularDisconnector examines each circular viral contig in the metaFlye assembly, aligns all reads to this contig, and detects all coverage jumps/drops using the echnique . It furtknown hosts for novel viruses from metagenome assemblies is based on identifying known CRISPR spacers in MAVs [novel hosts for novel viruses.The standard approach for predicting potential  in MAVs , 32. It Given a metagenomic dataset, viralFlye considers all high-coverage circular MAVs, linear MAVs, and multi-edge viral components in its assembly graph (with length varying from 5 kbp to 1 Mb), predicts candidate MAVs using viralVerify, and checks them for completeness using viralComplete (at least 50%). Afterward, it predicts the CRISPR arrays in this assembly using the Minced v. 0.4.2 tool (based on the CRISPR Recognition Tool ) and aliAdditional file 1 AGB vizualization of all viral components from SHEEP_GUT dataset.Additional file 2 Information about all default settings and versions used for each software tool.Additional file 3Table S1. Results of CRISPR-based host prediction results for viralFlye and Megahit assemblies on SHEEP_GUT dataset. Table S2. Results of CRISPR-based host prediction results for viralFlye assemblies on WARWICK_VIROME datasets. Table S3. Results of CRISPR-based host prediction results for viralFlye assemblies on ALOHA_VIROME datasets. Table S4. Results of CRISPR-based host prediction results for viralFlye assemblies on HUMAN_GUT datasets.Additional file 4 Review history.


Candida. Albeit rare, Candida-associated fungal melanonychia can present as a longitudinal black nail plate streak. The case of a 79-year-old man who developed a solitary linear black streak on his right fourth fingernail after a prior history of recent trauma to the digit’s nail folds is described; the fungal culture grew Candida parapsilosis. Including our patient, Candida-associated longitudinal melanonychia striata has been described in four women and two men ranging in age from 40 to 79 years  at diagnosis. The black streak, present from one month to one year , affected either a hand digit (five patients) or the great toe (one patient). Fungal organisms were visualized on either a potassium hydroxide preparation (one patient), pathologic evaluation of a nail plate specimen (three patients), or both (one patient). Culture grew Candida parapsilosis (two patients), Candidaspecies (two patients), Candida albicans (one patient), and Candida tropicalis (one patient). All of the patients experienced clinical improvement after treatment. Topical treatment (5% amorolfine hydrochloride nail lacquer for two patients or modified Castellani paint and 1% clotrimazole cream for one man) or oral itraconazole (either as monotherapy for two women or combined with 5% amorolfine hydrochloride nail lacquer for one woman) was successfully used. Although the clinical presentation of fungal melanonychia can mimic subungual melanoma when it appears as a solitary black linear vertical nail plate streak, investigative studies--such as a potassium hydroxide preparation, nail plate pathology, nail matrix biopsy, and/or fungal culture--can be used to establish the diagnosis of Candida-associated longitudinal melanonychia striata and exclude the diagnosis of a pigmented melanocytic tumor.Longitudinal melanonychia striata, presenting as a black linear vertical band of the nail plate, can be caused by pigmented lesions and non-pigmented etiologies. A fungal infection of the nail plate, also referred to as onychomycosis or tinea unguim, can result from dermatophytes, non-dermatophyte molds, and  Melanonychia is a black nail. Longitudinal melanonychia striata is a black linear vertical band that extends from the proximal nail fold to the distal end of the nail plate. There are several etiologies for longitudinal melanonychia striata, including pigmented lesions and non-melanocytic causes, such as infections, medications, and systemic diseases ,2.Candida and non-dermatophyte molds can also cause fungal infection of the nail plate; although these usually present as yellow or white discoloration, they can also, albeit less commonly, present as melanonychia [Onychomycosis is a fungal infection of the nail plate and is usually caused by a dermatophyte organism. Common clinical presentations of the nail plate’s dermatophyte tinea infection include distal lateral subungual onychomycosis, proximal subungual onychomycosis, and superficial white onychomycosis. However, nonychia ,4.Candida parapsilosis. Candida species-associated longitudinal melanonychia striata is uncommon; the features of our patient and other individuals with the new onset of a black linear nail plate streak caused by Candida species are reviewed [A man with a solitary linear black streak on a single fingernail presented for evaluation; he had a prior history of recent trauma to the digit’s proximal and lateral nail folds. A fungal culture grew reviewed -8.A 79-year-old man presented for evaluation of a new vertical black streak on the nail of his right fourth fingernail. Four months earlier, he had a manicure of both hands’ thumbs and fingers; the manicurist used a firm instrument to manipulate all of his cuticles. He subsequently developed painful red swelling of the proximal and lateral nail folds on his right fourth digit.Serratia marcescens and Candida albicans, respectively; an anaerobic culture did not isolate any anaerobes.Several clinicians evaluated his acute paronychia. He was empirically treated topically with gentamicin sulfate 0.1% ointment and clotrimazole 1.0% cream; the tender erythema subsequently resolved, and treatment was stopped after two weeks. An aerobic bacterial culture and a fungal culture eventually grew few During the next three months, he developed an asymptomatic pigmented streak on his right fourth fingernail. Cutaneous examination of the nails on his thumbs and fingers showed a 1-mm linear black band on his right fourth finger. It extended from the proximal portion of the lateral nail fold closest to his thumb to the distal tip of his nail  and 1% clotrimazole cream; both medications were applied to the affected nail plate and the adjacent lateral nail fold. An aerobic bacterial culture only demonstrated normal skin flora; however, the fungal culture grew The right fourth fingernail began to show normal-appearing proximal nail plate growth. The pigmented streak completely cleared after three months of daily treatment and the agents were discontinued. Follow-up evaluation of his right fourth fingernail, two months after stopping the medications, showed a normal nail plate without black dyschromia Figure .Longitudinal melanonychia striata usually presents from either melanocytic proliferation or melanocytic activation in the nail matrix. The nail matrix is a melanocyte-containing germinative epithelium that produces the nail plate. Activated melanocytes, within the nail matrix, transfer melanin-rich melanosomes distally into the nail plate cells, which clinically present as a black linear vertical band ,2.Candida albicans) can synthesize melanin in the yeast cells [Melanocytic proliferation is observed with benign and malignant pigmented lesions. Melanocytic activation is associated with many etiologies, including chronic local trauma, dermatologic conditions, endocrine disorders, iatrogenic causes, infection, medications, nutritional deficiencies, pregnancy, and being related racially. In some circumstances of fungal melanonychia, the organisms  at diagnosis. The men ranged in age from 60 to 79 years  when their Candida longitudinal melanonychia striata was diagnosed. Overall, the six patients ranged from 40 to 79 years  [The women with median, 5 years atAll of the women were Korean. In contrast, one of the men was French. Our patient was American -8.A digit of the hand was affected in five of the patients; the great toe was involved in one of the women. Indeed, the thumb or the great toe was the site of fungal melanonychia in four of the patients; both thumbs were affected in one woman. Either the third or the fourth finger was involved in the two men -8. Candida infection to localize to a specific side of the body. The right side was affected in four patients, and the left side was involved in three patients. One woman’s fungal melanonychia occurred on both of her thumbnails [There was no predilection for the umbnails -8.Candida-associated longitudinal melanonychia striata [Trauma to the affected digit had occurred in our patient and the other man . Our pat striata . There w striata ,8. The o striata .Candida longitudinal melanonychia striata ranged from one to 12 months . Until a diagnosis was confirmed, the range of time was one to 12 months for the women . A similar duration of time before documenting the diagnosis was observed in the men: the range was three to 12 months  [The duration of the linear black streak before establishing the diagnosis of  months) -8.A potassium hydroxide preparation of scrapings from the affected nail plate was performed for five of the patients. One woman had a positive initial evaluation and second testing three months later that was negative. A second woman also had a positive examination. The positive potassium hydroxide preparation demonstrated either numerous hyaline pseudohyphae and spores with amorphous brownish pigments surrounding them or scanty round yeasts and no mycelia ,7. The tA piece of the affected nail plate from four patients was sent for pathologic evaluation. The stained specimens with hematoxylin and eosin showed refractile pseudohyphae or ovoid spores or both in the nail bed stratum corneum or round budding yeasts with no mycelial elements ,7. The sCandida parapsilosis (two patients) and Candidaspecies (two patients). The cultures from the other women demonstrated either Candida albicans (one patient) or Candida tropicalis (one patient) [Culture of the affected nail plate or nail fold was performed. The most common fungal organisms were patient) -8.All of the patients showed improvement in their nail plate fungal melanonychia following treatment. The patients received either topical treatment (two men and one woman), systemic treatment (two women), or topical and systemic treatment (one woman). One woman who was successfully treated with systemic itraconazole initially did not show any improvement after a month of oral terbinafine -8.Candida infection. Two women received 200 mg daily of itraconazole for either three or four months [Itraconazole was uniformly efficacious in the management of longitudinal melanonychia striata caused by r months . The thir months .Candida-associated fungal longitudinal melanonychia striata in two of the other patients was also successfully treated topically with 5% amorolfine hydrochloride nail lacquer monotherapy. A 55-year-old woman underwent application of the lacquer twice weekly for three months [e months . The othe months .Our patient was successfully treated with twice-daily topical application of modified Castellani paint and 1% clotrimazole cream. The original formulation of Castellani paint included fuchsin (basic or carbol), alcohol, boric acid, phenol, acetone, resorcinol, and water. The modified version is colorless since the carbolfuchsin is not included. Also, in the current formulation of Castellani paint and modified Castellani paint, the boric acid  is absent ,20.Candida. However, the phenol and resorcinol in Castellani paint and modified Castellani paint have antimicrobial activity. Other modified Castellani paint properties, such as its drying and keratolytic activity, also seem to result in its clinical effectiveness when treating Candida infections [Candida infection.The fuschin in Castellani paint is very active against fections ,20. We aCandida-associated fungal melanonychia can present as a longitudinal black nail plate streak. Candida-associated longitudinal melanonychia striata has been described in four women and two men. Candida parapsilosis (two patients), Candidaspecies (two patients), Candida albicans (one patient), and Candida tropicalis (one patient) grew from the fungal culture of the affected nail plate or nail fold. All of the patients improved clinically after either topical therapy or a systemic antifungal or both. Candida-associated longitudinal melanonychia striata can mimic subungual melanoma when it presents as a solitary black linear vertical nail plate streak; however, investigative studies--such as a potassium hydroxide preparation, nail plate pathology, nail matrix biopsy, and/or fungal culture--can be used to exclude a pigmented melanocytic tumor and establish the diagnosis of a fungal melanonychia.


A 74-year-old male with a history of metastatic melanoma presents with persistently abnormal small bowel findings on PET-CT scan. The patient had persistent FDG uptake near the ileocolic junction on imaging, concerning for metastatic melanoma. Capsule endoscopy demonstrated ulcerated mucosa in the distal ileum. This area was biopsied and tattooed via retrograde double-balloon enteroscopy to confirm the diagnosis of metastatic melanoma and facilitate subsequent small bowel resection. The case illustrates a unique case of metastatic melanoma to the small bowel and the utility of capsule endoscopy and balloon-assisted enteroscopy to assist in diagnosis and management of metastatic disease. A 74-year-old male with a medical history of metastatic melanoma presents to the outpatient gastroenterology clinic due to persistently abnormal small bowel findings on positron emission tomography-CT (PET-CT) scan. He is otherwise asymptomatic and has no complaints. The following case illustrates a unique and unusual case of metastatic melanoma to the small bowel and the utility of both capsule endoscopy and balloon-assisted deep enteroscopy to assist in diagnosis and preoperative management.A 74-year-old male with a history of previously excised melanoma metastatic to a mesenteric lymph node who had been in remission for 2 years before presenting for additional evaluation of persistently abnormal small bowel findings on a surveillance PET-CT scan. Patient reported feeling well without complaints. He had a medical history of hypothyroidism, GERD, lymphocytic colitis on loperamide therapy, and metastatic melanoma. He was originally diagnosed with melanoma on his back ten years prior to this presentation. His melanoma history includes a left axillary recurrence over 3 years prior to this presentation, initial adjuvant therapy with pembrolizumab that was discontinued after 2 doses due to lymphocytic colitis, and laparoscopic resection of a metastatic mesenteric lymph node that was excised 2 years prior to this presentation. The patient's other surgical, family, and social history are unremarkable.Since his previous lymph node excision two years prior to this presentation, multiple surveillance PET scans had shown intense focal FDG uptake in the distal small bowel, deemed to likely be inflammatory in nature although melanoma recurrence and metastasis remained a possibility. The patient underwent PET-CT scan in the weeks prior to presentation which showed interval increased FDG uptake at the distal small bowel near the ileocolic junction . This fiThis case illustrates the utility of capsule endoscopy to identify small bowel metastasis, an atypical use in the current practice of capsule endoscopy. Furthermore, this case details how retrograde double-balloon enteroscopy, in combination with diagnostic modalities such as PET scans and capsule endoscopy, can assist in the diagnosis of distal small bowel disease including metastasis. This case also illustrates how SPOT tattoo utilization may assist with intraoperative surgical localization of suspicious lesions. A prior small study total of 8 patients) has also shown that capsule endoscopy may reveal metastatic small bowel disease that general endoscopy, push enteroscopy, small bowel follow-through imaging, CT scan, or PET may not detect . Other s patientsA literature review illustrates that little research has been completed regarding retrograde double-balloon enteroscopy in the diagnosis of metastatic melanoma. One study that involved both anterograde and retrograde DBE revealed that preoperative DBE can modify the surgical approach to malignant tumors of the small bowel , althoug


Vaccines are the most effective medical intervention due to their continual success in preventing infections and improving mortality worldwide. Early vaccines were developed empirically however, rational design of vaccines can allow us to optimise their efficacy, by tailoring the immune response. Establishing the immune correlates of protection greatly informs the rational design of vaccines. This facilitates the selection of the best vaccine antigens and the most appropriate vaccine adjuvant to generate optimal memory immune T cell and B cell responses. This review outlines the range of vaccine types that are currently authorised and those under development. We outline the optimal immunological correlates of protection that can be targeted. Finally we review approaches to rational antigen selection and rational vaccine adjuvant design. Harnessing current knowledge on protective immune responses in combination with critical vaccine components is imperative to the prevention of future life-threatening diseases. Vaccines have proven highly successful in reducing mortality and morbidity of infectious diseases on a global scale. The recent COVID-19 viral pandemic and the potential spread and threat of newly emerging infections demands for new efficient vaccines now and in the future . In the Here we describe the principal vaccine concepts ranging from live attenuated to peptide vaccines and their most common advantages and disadvantages. Importantly, depending on the aim of the vaccine its rational design first requires the identification of the immune correlate(s) of vaccine efficacy or the so-called immunological correlates of protection (IMCOP). Next, a suitable immunogenic vaccine antigen has to be identified, and subsequently formulated and presented in order to stimulate the adaptive immune system for the generation of memory immune T and/or B cells. We will address these important decision steps for different types of vaccines, which require a rational approach for the selection of the suitable antigen(s) with low variability, and a critical choice of co-formulated, or built-in (optimized), vaccine adjuvant as antigen delivery system and/or immunomodulator.Live attenuated vaccines (LAV) are widely considered highly effective vaccines, as they closely mimic natural immunity. Indeed, live vaccines are among the most successful vaccines as exemplified by the smallpox vaccine (live vaccinia virus) which led to smallpox eradication in 1980 and the poliomyelitis vaccine which led to polio being almost completely eradicated. Typically, live strains were attenuated by deletion of genes which impeded virulence or conferred auxotrophic phenotypes. LAVs are highly immunogenic, consequently do not require additional adjuvants. A single dose often confers protective immunity, but safety concerns can mean they are not suitable for immunocompromised individuals. For example, the Sabin polio vaccine strains showed issues with safety in a small cohort of recipients due to reversion to a paralytic phenotype resulting in vaccine-associated polio cases, with an incidence of one in 500,000 first time recipients. Attenuation mutations were lost in vaccine-related polio cases and multiple changes in known attenuation mutations were identified in virus excreted from vaccinated children . This po® vaccine is an LAV that incorporates all these characteristics [Additional advantages such as stability on storage, avoiding the need for cold chain and associated costs and challenges can be engineered into LAVs. The Flumisteristics . The trieristics . Recentleristics . This isVibrio cholerae, Burkholderia pseudomallei, Salmonella typhimurium, Francisella tularensis and Pseudomonas aeruginosa.Live attenuated bacterial vaccines have also been developed and licensed, or are at various stages of development, including vaccine candidates for Salmonella typhi strain and incorporates an inactive galE gene encoding an enzyme involved in LPS core oligosaccharide biosynthesis and over 20 other mutations [The typhoid live oral vaccine (Ty21a) is a licensed oral vaccine which was developed by chemical mutagenesis of a utations . It geneutations . Its safutations ,9  vaccines.Neisseria meningitides vaccine MenACWY-CRM (Menveo®) and the MenACWY-TT (Nimenrix®) vaccines comprise purified polysaccharide from four major capsular sub-groups A, C, W and Y conjugated to either diphtheria cross reactive material (CRM) or tetanus toxoid (TT), respectively, are approved for use in infants of 6 weeks and older. The flexibility of the conjugate approach is exemplified by the development of pneumococcal conjugate vaccines (PCV) to protect against Streptococcus pneumoniae. The first PCV was licensed in 2000 and comprised of polysaccharide representing seven serotypes. It was subsequently replaced by versions with broader coverage, namely ten-valent (PCV10) and 13-valent (PCV13) versions.Subunit antigen vaccines contain specific essential antigens to elicit protective responses without any live components and consequently are considered very safe. However, they are generally of lower immunogenicity and thus require additional adjuvant(s) to promote their protective responses. Early subunit vaccines exploited the available knowledge on microbial pathogenesis, for example the diphtheria toxoid and tetanus toxoid antigens, which were prepared by formalin inactivation of the respective bacterial toxins. Other subunit vaccines are based on purified recombinant proteins, such as the hepatitis B surface antigen with an aluminum-based adjuvant in the licensed Hepatitis B vaccine  and the Neisseria meningitides serogroup B [® licensed in 2013 which has had a significant impact on meningococcal disease caused by serogroup B. While this approach has revolutionized vaccine antigen discovery, one key limitation relates to the costs associated with testing the vast array of surface proteins identified ), all of which were tested in mice. Alternative approaches exploit bacterial host interactions including identification of immunoreactive proteins via immunoproteomic approaches or identifying protein-protein interactions. The former approach typically involves probing two dimensional blots prepared from bacterial proteins with serum from infected patients or animals to identify immunoreactive proteins that stimulate serological antibody responses. One limitation of this method is that humoral responses may not be ultimately protective, and identification of potential subunit antigens based on stimulation of a serological response may not identify protective antigens [Several strategies have been devised to identify novel vaccine antigens. Rappuoli and colleagues pioneered the use of reverse vaccinology to identify novel antigens against ogroup B ,14 as adantigens . More reantigens . This inBurkholderia antigens that are protective and stimulate both a serological response and also contain potent T cell epitopes [Identification of bacterial proteins involved in host cell attachment represents another approach to the identification of subunit vaccine antigens capable of stimulating both humoral and cellular responses. Bacterial membrane proteins are probed with human epithelial cells representative of the site of mucosal colonization with subsequent identification of bacterial protein involved in host cell attachment by mass spectroscopy, thereby focusing the field of potential efficacious antigens identified and reducing the burden of testing potentially unnecessary proteins that although present on the microbial surface are not involved in the direct host cell response . This apepitopes .Identification of the most effective epitopes in a subunit protein antigen can result in smaller (20 to 30 amino acid) peptide-based vaccines which can be synthesized in vitro. Consequently, they can be readily purified, are safer and may potentially trigger the desired immunological response. However, such peptide epitopes generally require conjugation to other peptide epitopes or biopolymer carriers in order to overcome their relatively small size and improve immunogenicity. In most cases adjuvant formulations are required. No human peptide prophylactic vaccine has been licensed to date.Vaccines based on in vitro-transcribed mRNA have the potential to transiently express the encoded protein in situ without the adverse effects of viral and DNA-based constructs. Two major types of RNA that are currently studied as vaccines are non-replicating mRNA and virally derived self-amplifying RNA. Both have in common a cap structure, 5′ and 3′ untranslated regions (UTRs), an open-reading frame (ORF), and a 3′ poly(A) tail, while self-amplifying RNA additionally contains replication machinery derived from positive-stranded mRNA viruses, most commonly from alphaviruses such as Sindbis and Semliki-Forest viruses . In ordeIn order to be translated and elicit an antigen-specific immune response, an mRNA-vaccine has to reach the cytosol of its target cells. There are two basic approaches for the delivery of mRNA vaccines: (1) loading of mRNA into dendritic cells (DCs) ex vivo, followed by re-infusion of the transfected DCs, or (2) direct injection of mRNA with or without a carrier into target cells. The most common carriers used for complexing mRNA are protamine (a cationic peptide), lipid or polymer-based nanoparticles. In addition to facilitating efficient cell delivery, the complexation with different carriers may influence immunogenicity and longevity of the mRNA vaccine. As well as inducing specific immune response toward the encoded antigen, once in the endosome, mRNA is recognized by RNA-sensing receptors, namely Toll-like Receptors (TLRs), and other pattern recognition receptors (PRR) contributing to the immunostimulatory features of mRNA vaccine. Alternatively, an early shut-down of antigen expression after the mRNA vaccination due to unwanted PRRs activation might be detrimental underlining the need for fine tuning of mRNA recognition by host immune system.In recent years an increasing number of preclinical studies have shown promising results with both self-amplifying and non-replicating mRNA vaccines conferring protection against various pathogens. However, data reporting outcomes of clinical trials are still modest. The first-ever demonstration in humans shows that a prophylactic mRNA-based candidate vaccine against rabies virus induced boostable functional antibodies against a viral antigen when administered intradermally with a needle-free device, although not when injected intramuscularly by a needle-syringe. The vaccine was generally safe with a reasonable tolerability profile . By contA DNA vaccine comprises the DNA that encodes vaccine antigens. After administration of the DNA to the host, antigens are produced in vivo and intended to stimulate an immune response. DNA vaccines contain nucleotides encoding an antigenic portion of a tumor-associated antigen or a target pathogen, such as the viral core region or viral envelope region. Any antigen encoded by a DNA vaccine is expressed intracellularly and consequently can be processed via the endogenous Major histocompatibility complex (MHC) class I pathway. Contrary to an RNA vaccine, protein expression by a DNA vaccine requires the nuclei acid to cross two cellular membranes, namely the plasma, as well as the nuclear membrane. Thus, successful transfection is crucial for DNA vaccine efficacy. Hence, various delivery methods have been developed, including gene gun, jet injection, in vivo electroporation, but also different formulations of DNA, for example encapsulation in lipid nanoparticles, adsorption to polymers, and adsorption or encapsulation in biodegradable nanoparticles. Safety concerns of DNA vaccines mostly relate to potential DNA integration into the host genome and generation of antibodies against the injected DNA. Indeed, DNA integration events were detected in a few studies, while no anti-DNA antibodies were found in animal models. In general, the potential risks of DNA vaccines are considered relatively low, although safety issues may differ from one application to another, and require strict monitoring . To dateDespite the initial studies on DNA vaccines commencing in the 1990s, there are no approved DNA vaccines for use in humans to date. There are, however, a selection of DNA-based vaccines approved by the USDA for veterinary use, including an equine vaccine against West Nile Virus  and caniViruses have proven to be highly efficient vehicles for introducing foreign nucleic acid into target cells. Additionally, viruses are sensed by several intra- and extra-cellular Toll like receptors and intrinsically induce host immune responses upon cell infection. Both of these features, accompanied by extensive knowledge of molecular biology and methods for manipulating the viral genome that are now available, make viruses attractive candidates for vaccine vector development. Hence, recombinant viral vectors have been and are being investigated as vaccines targeting a broad range of viral, bacterial, and protozoan pathogens. They are particularly used in disease areas where classical vaccination strategies have proven ineffective, difficult, or technically impossible.Viral genomes can be manipulated to express any antigen of choice encoded either in the genome  or preseViral vectors present a versatile and modular platform for vaccine development that can be especially valuable in outbreak situation when prompt reaction is needed. The SARS-CoV-2 vector vaccine candidates, which we are currently witnessing with unprecedented development, exemplifies this. Viral vector-based vaccines against SARS-CoV-2 have been tested in the context of non-replicative vectors such as human and chimpanzee adenoviruses and replication-competent vesicular stomatitis virus The chimpanzee adenovirus-vectored vaccine ChAdOx1 nCoV-19, encoding the spike protein of SARS-CoV-2, was immunogenic in pigs and mice, eliciting a robust humoral and cell-mediated response . VaccinaN meningitidis requires treatment with deoxycholate detergent to in-crease the yield and to reduce endotoxin levels. Endotoxins are lipopolysaccharides (LPS) that are potent TLR-4 agonists inducing activation of innate immune responses and inflammatory cytokines secretion. Removal of LPS by detergent treatment is essential to prevent serious adverse effects, however this treatment leads to the removal of potential surface exposed antigens with consequent impact on the long-term stability of the OMV vaccine. Genetic modification of N. meningitidis by deletion of specific genes overcomes both these limitations. Deletion of the rmpM gene results in higher yields of OMV. The RmpM protein anchors the outer membrane to the peptidoglycan layer, and its absence consequently leads to increased OMV release [The most commonly used approaches to bacterial vaccines are based on inactivated whole cells, purified recombinant proteins and protein-polysaccharide conjugates. Genetically engineered live attenuated bacteria are currently not widely used as human vaccines, although this approach has been investigated especially in the case of the BCG vaccine against tuberculosis . Outer M release . The tox release . The comN. meningitidis are available: (i) fusion to the N-terminus of the lipoprotein factor H binding protein followed by internal expression by the OMV-producing strain [Vaccine applications of OMVs can be greatly expanded by the expression of heterologous antigens, where the OMV’s serve as both adjuvant and delivery vehicle. Surface exposure of the antigens might improve the immune response, so targeting to the OMV surface is an important consideration. Two different methods for surface display in g strain , and  in immune hosts and the subsequent monitoring of a drop in protective immune function after challenge. The contribution of antibody-producing B cells can be studied by using B cell-neutralizing antibodies or by specific genetic deletion of the B cell population . Alternatively, the contribution of T cells to immunity in immune hosts can be examined using T cell-depleting antibodies, or mice with genetically deleted T cells (or their subpopulations). Any reduction in immunity relative to wild-type or depleted immune mouse models indicate their contribution.Despite these experimental strategies, the specific contributions of distinct immune effector mechanisms are known for just a few pathogens. This may be due in part to the lack of suitable animal models reflecting human or veterinary disease, the complex changes in life cycle of certain pathogens and their evasion from immune surveillance, as well as the complex nature of immune reactions and synergistic activities of immune cell populations. For example, antibodies may be involved in protection during the acute phase of infection while cell-mediated responses may become critical during the chronic phase of infection. Although animal models may not fully reflect the disease or may not be available, in many cases there are clues to the signature of immune clearance mechanisms that are associated with recovery from, or immunity to, specific infectious diseases. Extrapolations to correlates of immunity can be made based on knowledge from related categories of intracellular versus extracellular pathogens, or related types of micro-organisms with similar life cycles. Actual correlates have been defined or suggested for Hib, pneumococcal and meningococcal vaccines. Moreover, for most tumors we know that in general type-1 immune and STING pathway interferon responses are related to protection . Hence, The systems biology approach can facilitate a better picture of the immune responses in general and to vaccination in humans ; in fact+ T cells and therefore is also dependent on successful cell-mediated immunity. Activated B cells interact with antigen-specific CD4+ helper T cells in the outer cortex of the lymph nodes and undergo proliferation in the presence of cytokines such as IL-4 and IL-5 produced by CD4+ T cells. The antibodies produced bind the organisms and/or their toxins, directly interfering with microbial proliferation via neutralization, opsonization, and complement activation, or will direct other immune cells to phagocytose and destroy the bound microbe. Following a successful induction of humoral immune response, B cells producing affinity matured and isotype-switched antibodies differentiate into quiescent memory B cells , such as dendritic cells and macrophages. Activated APCs provide the critical co-stimulatory signals, which stimulate the T cell receptor (TCR) resulting in T cell proliferation. Following TCR activation by APCs, naïve CD4+ T cells differentiate into either T helper (Th)1, Th2, Th9, Th17, Th22, T follicular helper, and T regulatory cells under unique cytokine-polarized milieus .Should immunological data indicate that a disease can be prevented by an antibody response, it is important to select an appropriate antibody-inducing antigen. Induction of robust antibody responses by vaccines requires inclusion of relevant antigenic epitopes for B cells as well as T cell peptide epitopes to elicit appropriate T helper (Th) cell activation and assistance to B cells. Hence, selection of the optimal antigen is imperative in vaccine design.Streptococcus pneumoniae, Haemophilus influenzae or Salmonella typhi Vi. Saccharide-based vaccines have also been developed in multivalent forms to provide protection against numerous serotypes and serogroups [A successful strategy in the selection of the antigenic determinants as “subunit” vaccines is the isolation and inactivation of essential components of the pathogenic organism . Chemicarogroups .Current strategies utilize reverse vaccinology approaches to select promising glyco- or protein-derived antigens based on structural studies of the target epitopes of potent antibodies ,72. RatiStructural antigen vaccinology is a structural biology approach to design immunogenic antigens. It rationally aims to generate an effective antibody-inducing vaccine antigen, combining experimental methods such as X-ray crystallography, nuclear magnetic resonance (NMR), molecular biology, electron microscopy and mass spectrometry, with computational methods including molecular modeling, virtual screening and epitope prediction . The ideThe aim of this re-engineering process is to obtain an antigen that is more effective in eliciting an optimal B cell response. Based on the analysis of antigen-antibody interaction network, the candidate vaccine antigen may contain only those residues essential to reproduce the protective epitopes. Molecular modeling is extremely helpful in designing optimal re-engineered antigens, through the identification of mutations that stabilize immunogenic conformations of epitopes. Moreover, residues outside the epitope can be mutated to improve antigen production yields. Another important issue in antigen re-engineering is the improvement of antigen thermal stability, through the identification of stability-enhancing mutations or through the insertion of the designed antigen in stable protein scaffolds. Eventually, the immunogenicity and efficacy of the candidate vaccine must be tested in animal models .While glyco- or peptide-based subunit vaccines offer improved safety and more precise targeting, they usually require conjugation to immunogenic molecules , or presentation in multimeric format (virus-like particles or nanoparticles) to achieve optimal immune responses , which cAnother key element for rational vaccine design is the presentation of the antigen in its relevant native-like conformation, enabling optimal antibody recognition of the antigenic epitope within the quaternary protein structure. Several approaches have been developed to modulate these epitopes to favor conformationally relevant states of the antigen , includi®). However, these key epitopes are well exposed only in the pre-fusion trimeric conformation of the RSV-F protein. Structural vaccinology was fundamental to generate stabilized pre-F trimers that preserved key epitopes in the proper conformation to elicit the most neutralizing activity in human sera [N. meningitidis serogroup B (MenB) composed of three recombinant proteins and a bacterial membrane vesicle selected using a reverse vaccinology approach . In this case, specific epitopes belonging to the F glycoprotein  present potent neutralizing activity, 10 to 100 times greater than that observed for clinically used monoclonal antibody palivizumab . HThe potential improvement in the identification of antigenic determinants expressed on cancer cells enabled the design of therapeutic subunit vaccine to treat cancer ,85,86. A+ and CD8+ T cell responses are worthy methods to de-sign vaccines using reverse vaccinology approaches [Listeria monocytogenes as a model pathogen Kono and co-workers and Calderon-Gonzalez and co-workers and DCs loaded with peptides of two virulence factors of this pathogen, listeriolysin O (LLO) and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) prepared vaccines that confer listeriosis protection [L. monocytogenes challenge. This approach helps to predict other epitopes for vaccines against listeriosis and other pathogens as mycobacteria and streptococci [A considerable challenge in vaccinology is the design of vaccines against pathogens for which antibody immune responses are not protective . To deveproaches . Using Lotection ,91. Lateotection   and new ways to present antigen. Optimization of the immune response with the use of different types of adjuvant has received less attention. Typically, when preliminary studies with a “standard” adjuvant does not show the desired (protective) response, the researchers will seek another type of antigen, instead of searching for an appropriate adjuvant that may provide the suitable or preferred immune response. Often the desired adjuvant or formulation is unknown or not available. Nevertheless, there have been noteworthy achievements in improving existing vaccines by introducing specific adjuvants as well as new vaccine delivery methods ,94.Live attenuated vaccines, however, do not usually require adjuvants since the resulting immune response is a result from the attenuated microbe and its attempt to multiply. However, adjuvants come in many forms  and may Molecularly defined subunit vaccines are generally safer but often lack immunogenicity, due to the absence of key additional structural components needed for efficient activation of innate immunity. Such vaccines require the coadministration of an adjuvant . Few adjIt is not straightforward to translate the newly discovered mechanisms of adjuvant activity to generally applicable approaches for rationally designed vaccines. Thus, a thorough knowledge of adjuvants and their immunological effects is needed to realize the full potential of rational vaccine design. . These cThe most common and long-time used immunoadjuvant alum has been extensively reviewed before ,101, butE. coli type) is the most toxic and its recognition and binding by the TLR4/MD-2 binary complex can lead to sepsis. Changing the number and length of the acyl chains or the phosphate decorations decreases its recognition by the immune system receptor [E. coli type lipid A makes the molecule only moderately active. The phosphate groups can be absent or replaced by monosaccharides [l-arabinose to neutralize their negative charge altering immune recognition [d-Glc disaccharide [The lipid A molecule is the immunostimulant component of Gram-negative bacterial lipopolysaccharide (LPS). It consists of a β-(1→6)-linked diglucosamine backbone with different patterns of acylation and phosphorylation . The lipreceptor ,108. A vreceptor  or more receptor . The remcharides ,113,114 ognition ,116. In ccharide ,118. Theccharide . Lipid Accharide , and casccharide ,123,124.ccharide .Brucella abortus LPS showed promising results in various formulations [Bacteroides thetaiotaomicron and Prevotella intermedia [O-deacylated lipooligosaccharide from E. coli J5 [S. minnesota R595, which reduces significantly the TLR4-driven activity and toxicity. Decreased TLR4 activation induced by MPL derives from a less efficient dimerization of TLR4/MD-2/MPL complexes due to the absence of the phosphate group which the weakens proteins’ interaction at the dimerization interface [For the structural reasons explained above, some natural LPS could be used as vaccine adjuvants. For example, ulations ,127, as termedia . There a coli J5  or the b coli J5 . MPL is nterface ,133,134.nterface ,136.To improve specific adjuvant properties and to modify or enhance immune stimulating activity, different synthetic lipid A analogues and lipid A mimetics have been prepared as vaccine adjuvant candidates. Chemical synthesis provides access to structurally defined molecules free of any biological contaminations and allows tremendous improvements in structure-activity relationships studies. Basically, all synthetic lipid A mimetics mirrored the basic architecture of their parent lipid A and were composed of a hydrophilic polar/charged head group and a hydrophobic lipid region. Simplification of the lipid A structure by replacing one or both glucosamine (GlcN) rings for linear or branched aglycons. Thus, GSK Biologicals developed aminoalkyl glucosaminide phosphates (AGPs) where the proximal GlcN ring of lipid A was omitted furnishing in this way a polar head group with rationalized structure . VariablCalliphora vicina. A striking feature of larval hemolymph is that the hemocytes possess a cytotoxic activity functionally analogous to human NK cells. When stimulated by bacteria, these larvae produce high levels of potent defensive molecules typical of the insect immune system which accumulate in the hemolymph [Alloferons are a group of natural peptides isolated from insects that can stimulate human natural killer (NK)cell cytotoxicity towards cancer cells. These peptides are originally isolated from the hemolymph of maggots from the blowfly emolymph . These iemolymph  belonginemolymph . Allostaemolymph . Hence, Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is a cytoplasmic pattern recognition receptor involved in both innate as well as adaptive immune responses and therefore constitutes an excellent target for rationally designed vaccine adjuvant ligands . Muramyl1)) is predominantly responsible for the efficacy of Freund’s complete adjuvant, it suffers from pyrogenicity and rapid elimination as a single administrated molecule [2) and temurtide (3) emerged as the most interesting candidates for further development as vaccine adjuvants, while muramyl tripeptide phosphatidylethanolamine  (4), B30-MDP (5) and romurtide (MDP-Lys (L18) (6) were the most prominent lipophilic derivatives, which tend to augment the Th1-type immune reaction [Although MDP  as exemplified by murabutide or muradimetide [Substitution of the molecule . The d-idimetide . Murabutdimetide , therefodimetide . Its adjdimetide . Recentldimetide . Temurtidimetide .4) was evaluated in Phase I clinical trials as a vaccine adjuvant in human immunodeficiency virus type I vaccines, but failed to improve their immunogenicity, while causing increased reactogenicity [N-stearoyl-l-Lys, led to the discovery of MDP-Lys(L18) . A combination of MDP-Lys(L18) and B30-MDP has shown promising results in mice, which produced higher antibody titres against rHBsAg after intraperitoneal injection [7), which proved effective as an adjuvant for Borrelia burgdorferi antigen rOspA, also features a B30-acylated Lys-extension [8) carries a N-cinnamoyl-l-Lys moiety and showed promising results in a mouse model by increasing the levels of anti-HBs antibodies [N-acetylmuramyl moiety (MurNAc) is replaced by a hydrophobic group. The Trans-feruloyl moiety has recently been identified as an excellent MurNAc mimetic, resulting in a low nanomolar NOD2 agonist (9), which induced ovalbumin-specific IgG titers in a mouse model of adjuvancy [The introduction of lipophilic groups into the structures of NOD2 agonists has been shown to strongly enhance the cellular immune response and overall increased the immunostimulatory adjuvant activity of the compounds. MTP-PE (genicity . Decoratgenicity . Furthernjection  and incrnjection . A closextension . Similartibodies . Desmuradjuvancy .Quillaja saponin (QS) family [A number of adjuvant analogues have been developed based on natural product adjuvants leveraging detailed structure–activity relationship studies, such as synthetic saponin variants derived from QS-21 a and the) family .In particular, QS-21 is a saponin natural product with a long history and great potential as an adjuvant. It elicits both antibody and cellular immune responses, including cytotoxic T lymphocytes, and has been recently approved in combination with MPLA as part of the AS01 adjuvant system in vaccines against malaria and shingles . HoweverAdditional studies on the detailed immunological profile of these saponins are warranted and in progress, with the aim to elucidate the precise functional and molecular roles of these adjuvants, also in the context of adjuvant systems and anti-cancer vaccines.The spread of COVID-19 highlighted the need for swift vaccine development in a global pandemic and over 320 vaccine trials were conducted worldwide at time of writing ,172. HisVLPs are macromolecular self-assembling structures that closely resemble the native forms of viruses. One of the superior features of these “stunt viruses” is that they are non-infectious since they lack viral genetic content. Therefore, VLPs are safer than whole-pathogen-based vaccines such as those containing attenuated viruses. VLPs can be developed through expression of individual viral structural proteins following transfection which can self-assemble into the VLPs before being released into the extracellular environment from the producer cells. The success and licensing of the multivalent VLP-based vaccines for human papilloma virus and Hepatitis B validated the safety and efficacy of the vaccine concept for VLPs ,174, whiIn the case of SARS-CoV2 for example, four structural proteins of SARS-CoV-2, namely, spike, envelope, membrane and nucleocapsid could be cloned within proper expression vector(s) and expressed in a suitable producer cell line that could range from mammalian to insect, to even yeast or plant cells. The immunogenicity and strength of protective capacity of these vaccine candidates could be further amplified by the use of a proper Th1 immunity-supporting biological adjuvants such as CpG oligodeoxynucleotides (CpG ODN hereafter) . DevelopSpecific sequences of single stranded synthetic oligodeoxynucleotides containing unmethylated cytosine-phosphate-guanine oligodeoxynucleotide motifs (CpG ODN) stimulate type I interferon production via TLR9. Based on their differential activation of immune cells, four major classes of synthetic CpG ODNs have been defined. The K class ODNs are potent B cell activators that stimulate TNF-α secretion not interferon-α (IFNα), while D, C-, and P-class ODNs induce variable amounts of IFNα secretion ,178. TheThe complexity of the natural immune response and the potential immune escape mechanism(s) of particular pathogens complicate the design and its predictive value of a successful vaccine and the selection of critical vaccine elements. The vaccine components described above are very useful tools to design considerably different vaccine concepts that are necessary to evoke the desired immunological correlates of protection and, if necessary, to outsmart the pathogen by artificially induced immune responses. In times of emerging infections, the speed of efficient vaccine development becomes a critical factor to prevent health threatening disease. Hence, having a detailed knowledge and the availability of critical vaccine components is imperative to the prevention of future life-threatening diseases.Specifically, versatile vaccines, which consist of elements that are readily available, to rapidly produce the antigens expected to provide successful immune targets are highly needed in emergency situations. Therefore, vaccines based on genetic approaches, such as RNA- and DNA vaccines, or recombinant vector vaccines, or synthetic peptide vaccines are very useful tools which can be engaged rapidly as soon as the required genetic sequence of the presumed target antigen becomes available. Mathematical modeling and deep learning artificial intelligence (AI) applications involving the prediction of viral mutation(s) may undoubtedly help to anticipate the next generation of viral disease. For example, with RNA sequences of one generation of a virus acting as inputs, and the RNA sequences of the next generation acting as outputs, an algorithm was developed to predict RNA sequences of successive generations . In addi