Retinal cell survival requires an equilibrium between oxygen, reactive oxygen species, and antioxidant molecules that counteract oxidative stress damage. Oxidative stress alters cell homeostasis and elicits a protective cell response, which is most relevant in photoreceptors and retinal ganglion cells, neurons with a high metabolic rate that are continuously subject to light/oxidative stress insults. We analyze how the alteration of cellular endogenous pathways for protection against oxidative stress leads to retinal dysfunction in prevalent  as well as in rare genetic visual disorders . We also highlight some of the key molecular actors and discuss potential therapies using antioxidants agents, modulators of gene expression and inducers of cytoprotective signaling pathways to treat damaging oxidative stress effects and ameliorate severe phenotypic symptoms in multifactorial and rare retinal dystrophies. Oxidative stress (OS) alters cellular homeostasis and elicits a cell response that depends on the severity and the type of insult. Essentially, there is a limiting stress threshold: below the threshold, cells elicit protective mechanisms designed to ensure survival. Conversely, if stress surpasses the threshold or the activation of protective mechanisms fail, cells trigger alternative signaling pathways that eventually lead to apoptosis, necrosis, pyroptosis, or autophagic cell death ,2,3,4.2•−), oxygen peroxide (H2O2), hydroxyl radical (OH•), peroxy radical, and the second messenger nitric oxide (NO•) (which interacts with superoxide anion to form peroxynitrate (ONOO−)), are the major sort of ROS present at the cellular level. Usually, there is an equilibrium between oxidative species and antioxidant defense mechanisms, which are mediated by enzymes responsible for metabolizing or neutralizing ROS, e.g., catalase, glutathione peroxidase, or superoxide dismutase (SODs) [Cell survival requires an equilibrium between oxygen and antioxidant molecules. Reactive oxygen species (ROS) such as superoxide anion (Oe (SODs) ,6,7,8.ROS are generated in many enzymatic processes and in redox reactions. Mitochondrial respiratory chain is one of the main ROS sources in cells. In the inner mitochondrial membrane, electrons are transported and oxygen is converted into water. Under hypoxia conditions this process is not completed, resulting in an increase of superoxide anions. The nicotinamide adenine dinucleotide phosphate oxidase (NOX) is the main source of ROS derived from superoxide anions. NOX is an oxidase family of seven members, and among them is NOX4, which generates superoxide anions and hydrogen peroxide .These free radicals are small molecules/ions that are reactive with small activation energies and short lifetimes . There aAir pollution and cigarette smoke are environmental toxic factors that induce the production of ROS. Exposure of the eye to air pollutants increase the risk of suffering glaucoma due to the increase of ROS and the consequent inflammatory response . CigaretHumans are essentially diurnal animals and are constantly exposed to sunlight, which induces lipid and protein oxidation. In fact, mammalian retinas are especially sensitive to light  stress. Photoreceptors and RPE cells generate high levels of ROS due to different factors: (i) the retina is a neuronal tissue characterized by extremely high oxygen consumption related to a highly demanding metabolism; (ii) photoreceptors are constantly exposed to light and contain several photosensitizer molecules; (iii) photoreceptor membranous disks are enriched in polyunsaturated fatty acids, which are particularly sensitive to oxidation damage; and (iv) the daily recycling of photoreceptor disks damaged by oxidative stress is performed via phagocytosis by RPE cells, and consequently, oxidative damage to photoreceptors induces accumulation of ROS in the RPE. Photooxidation causes lipofuscin accumulation, which is the oxidation byproduct of lipids and lipoproteins containing photo-oxidable fluorophores ,16. HighAn inflammatory response is a natural defense mechanism against injuries. Innate immunity is considered the immediate immune response to an insult or pathogen. The retina is equipped with a highly sensitive innate immune system that responds with three key pathways: migration of microglia cells, activation of the complement system to opsonize cellular debris, and inflammasome assembly in the RPE (reviewed in ). To achSeveral chronic diseases are associated with protein oxidation due to high ROS production. Protein oxidation activates the release of inflammation regulatory proteins such as peroxiredoxin 2 (PRDX2). The role of OS as a key trigger of pathogenic inflammation in chronic inflammatory diseases has been well established. Indeed, pro-inflammatory cytokines such as TNF-α, interleukin-1β, or interferon-γ induce ROS production in RPE cells. In fact, these pro-inflammatory cytokines are upregulated in the eyes of patients suffering glaucoma, age-related macular degeneration (AMD), diabetic retinopathy, or retinal vein occlusion. Particularly, endothelial cells affected by inflammation show phenotypic changes due to the increase of the expression of inflammatory mediators and cytokines as well as iNOS activation .As mentioned, one of the first response of the retina to external damaging stimulus, such as oxidative stress, is reactive gliosis, in which astrocytes, microglial, and macroglial cells are activated. Microglia cells are resident retinal macrophages that confer neuroprotection against ROS damage and other injuries. In healthy retinas, microglial cells phagocyte accumulated waste products and cellular debris but under oxidative stress, hypoxia, or inherited mutations, NFκB-mediated inflammatory responses are activated, and microglial cells acquire amoeboid morphology, proliferate, and migrate to the sites of injury, as deteted by Iba-1 positive cells in the photoreceptor layer ,22. OxidOn the other hand, macroglia Müller cells, which constitute the columns of the retinal tissue and with multiple connections with retinal neurons, microglia, astrocytes, and endothelial cells, modulate different responses according to the severity of the stimulus. The activation of these macroglial cells involves hypertrophy, which entails overexpression of vimentin (an intermediate filament) and the glial-acidic fibrillary protein (GFAP), considered a hallmark of retinal stress. As an immediate response to acute non-permanent stimuli, Müller cells promote the secretion of trophic and antioxidant protective factors, but upon chronification, their secretory role may be clearly deleterious for neuronal cells .Endoplasmic reticulum (ER) is the main cellular compartment responsible for protein biosynthesis and folding. This organelle is one of the main cell sensors to stress insults. Alterations of the ER redox status can negatively affect protein folding and result in ER stress . ER streThe retina and RPE of mice exposed to cigarette smoke showed an increase of CHOP, a sensor that triggers cell apoptosis. In cultured human RPE cells, inhibition of ER stress attenuated apoptosis and cell death induced by oxidants including hydroquinone and CSE ,29. Usua2+ overload, and altered protein synthesis rate in the retina  in photoreceptors cells. Chronically activated UPR in degenerating retinas activate pro-apoptotic programs associated with oxidative stress, pro-inflammatory signaling, dysfunctional autophagy, free cytosolic Caiewed in ).isocitrate dehydrogenase-3β gene , are associated with photoreceptor degeneration and result in RP [Photoreceptor outer segments are densely packed with membranes and opsin proteins . In factlt in RP .cis-retinal in the visual cycle can lead to the formation of compounds that are reactive with short wavelength light, generating free radicals [The isomerization of 11-radicals . The higradicals .On the other hand, mTOR levels or activity might be altered in some inherited visual disorders. The activity of mTOR, a key regulator of cellular metabolism , is regulated by phosphorylation. Several studies showed that the phosphorylation of mTOR was reduced in the dorsal cones of retinitis pigmentosa mouse models. In addition, L/M opsin protein levels were also decreased, probably reflecting a reduction in translation, which is under the control of mTOR. Thus, mTOR phosphorylation status in RP might be low due to the cells suffering from some type of nutrient deprivation and/or metabolic dysregulation , furtherAlthough ROS are toxic agents for cell viability, particularly in chronic conditions, they can also play a beneficious role when present at low levels because these molecules activate cell protective mechanisms upon injury.The balance between oxidants/antioxidants is finely regulated to avoid ROS to overcome a damaging threshold. Cells enable different mechanisms to counter the OS with antioxidants, which can be produced endogenously in an endocrine or paracrine pathway, or exogenously, acquired from food or supplements. In either case, antioxidants work as scavengers to neutralize free radicals, preventing and repairing the damage caused by ROS, thus antioxidants enhance immune defense and reduce the risk of suffering some diseases.Antioxidant enzymes play a key role in the first response to reduce ROS levels, hence redox regulation by transcription factors is essential in determining gene expression profiles and coordinating cell response under oxidative stress conditions . Cells aThe nuclear transcription factor κB (NF-κB) belongs to a transcription factor family involved in inflammatory and immunity pathways. NF-κB also plays an important role in developmental processes, cell growth and survival, proliferation and several pathological conditions. NF-κB plays a dual role upon oxidative stress: despite in most cases works as an antioxidant agent promoting cell survival, it may also function as an oxidant agent and causes cell death. NF-κB influence ROS levels increasing antioxidant proteins expression , whose function is to protect cells against oxidative stress by reducing or neutralizing ROS. On the other hand, NF-κB plays an important role in activating inflammatory pathways because it also targets genes encoding enzymes that trigger ROS production , and their increase in activity can eventually lead to cell death ,39.The nuclear factor- erythroid 2-related factor 2  is a transcription factor (TF) that regulates antioxidant defenses. In normal conditions, NRF2 is lowly expressed and localizes at the cytoplasm, where it interacts with KEAP1, which favors its ubiquitination and degradation via proteasome. In contrast, under oxidative stress conditions, NRF2 expression levels increase and it is translocated to the nucleus, where dimerizes with MAF proteins and binds to gene promoters that encode antioxidant enzymes such as SODs, catalase, S-transferase glutathione, which in combination, scavenge ROS ,7,8.NRF2- and NF-κB-pathways regulate the fine redox cellular balance, oxidative stress, and the inflammatory response. Imbalance between NRF2 and NF-κB pathways is associated with a significant number of diseases ranging from neurodegeneration and autoimmune disorders to cancer. The interaction between these two pathways is carried through complex molecular interactions that usually depend on the cell type and the tissue context. These interactions act through transcriptional and post-transcriptional mechanisms, and allow finely tuned dynamic responses to a changing environment ,40. TherIn response to environmental stress  eukaryotic cells shut down protein synthesis in a stereotypic manner to preserve anabolic energy to repair stress induced damage. The translational arrest is selective: while the translation of constitutively expressed genes (housekeeping genes) is hampered, the translation of stress-inducible transcripts encoding heat shock proteins among others is enhanced or favored . To protOn top of the signaling cascades responsible for cell translation reprogramming upon stress are the family of serine/threonine kinases. Some members of this family of environmental stress sensors are: (i) PKR, a double strand RNA-dependent kinase, activated with viral infections, heat, and UV radiation; (ii) PERK/PEK, an ER protein that is active in response to the accumulation of misfolded proteins in the ER; (iii) GCN2, a sensor of intracellular amino acid levels that responds to amino acid deprivation; (iv) HRI, a hemo-regulated kinase that ensures the synthesis of globin chains and hemo during erythrocytes maturation. All of these sensors phosphorylate eIF2α.Met, and GTP), which loads the initiator tRNAiMet on the small ribosomal subunit to start protein synthesis. Translation is usually started when eIF5 hydrolyzes GTP in the pre-initiator complex and its initiation factors are moved out allowing the big ribosomal subunit to couple. The additional ribosomes coupling turns the RNA into a polysome  has been characterized in response to different stress stimulus, for instance, in response to ER stress, oxidative stress induced by a toxic agent like sodium arsenite, or heat shock. These analyses show that there is a dynamic and differential distribution of RNAs in the soluble fraction of the cytoplasm and cytosolic RNPs depending on the stress stimulus . TherefoAgain, neuronal tissues, such as the retina, need a careful balance between the response to oxidative stress and the formation of SGs; between stalling translation and producing the protein levels physiologically required for cell survival.Autophagy is a conserved cell survival pathway that catabolizes damaged proteins and organelles to maintain homeostasis. Nonetheless, in response to different stimuli and cellular contexts, autophagy can also modulate cell viability, either enhancing cell survival or cell death. Autophagy is involved in a number of different processes in the retina participating in development and tissue remodeling and it is also implicated in photoreceptor cell death in response to calcium cytotoxicity, structural damage, and oxidative stress ,47. On tOverall, an impairment of autophagy due to oxidative stress is related with several age-related eye diseases such as dry eye diseases, corneal dystrophy type 2, cataracts and retinal dystrophies like AMD, glaucoma, DR, and retinal artery occlusion ,52.PARK2 mutations are associated with increased ROS and mitochondrial dysfunction in patients with Parkinson disease), and previous work in mouse models suggests that Park2-deficient photoreceptors exhibit ectopic mitochondria localization upon light exposure, thus suggesting that aberrant trafficking of damaged mitochondria might be one of the mechanistic aspects of retinal degeneration [Mitochondria are essential organelles that supply energy to the cell through oxidative phosphorylation (OXPHOS) and are also essential in calcium buffering, cell cycle control and regulation of apoptosis. Mitochondrial activity generates 1–5% ROS in physiological conditions . Severalneration .Under stress, a delicate balance between both mitochondrial fission and fusion is required to maintain functional mitochondria . An exceMitophagy is also relevant for the homeostasis of neuronal long axons subject to ROS and other stress insults. Retinal ganglion cells, whose long axons confluence to form the optic nerve, are particularly dependent on appropriate mitochondrial trafficking and dynamics in excess of ROS production by damaged mitochondria ,58.ER–mitochondria interactions play a key role in the executing and regulating mitochondrial mitochondrial fission and fusion. For instance, in mitochondrial fission, the physical interaction between the membranes of the two organelles is directly demonstrated by the role of the ER tubules in encircling mitochondria and marking the sites of mitochondrial division. Thus, the ER regulates mitochondrial dynamics, and alterations in mitochondrial morphology uniquely reflect cell health. In non-oxidative conditions, the NLRP3 protein, a sensor of OS that triggers the formation of inflammasomes, is located on the ER. However, upon oxidative stress and in response to inflammation, NLRP3 relocates to ER–mitochondria contact sites (MAMs) thus enabling efficient sensing and monitoring of damaged mitochondria, which generate high levels of ROS. The ER and mitochondria reciprocally transmit danger signals through physical contacts, thus reinforcing their communications and triggering multiple and synergistic responses to oxidative stress, among them, promotion of autophagosome formation to degrade damaged mitochondria .To sum up, autophagy plays a protective role against oxidative stress and other cell injuries but the accumulation of autophagosomes due to prolonged insult is also harmful to cells . TherefoELOVL4 cause dominant Stargardt disease. ELOVL4 is an elongase that generates very long chain polyunsaturated fatty acids VLC-PUFA in the retina. Mutations in this gene have pleiotropic effects by directly altering VLC-PUFA signaling, enlarging the membrane rim curvature of photoreceptor disks and impacting on the size of ribbon synapses [The retina presents a high content of lipid composition. For this reason, alterations in lipids composition or balance may affect visual capacity. For instance, mutations in synapses .Lipid peroxidation is a consequence of ROS damage, and polyunsaturated fatty acids (PUFAs) are particularly susceptible to ROS. The high content of lipids in the retina and the constant exposure to light make this organ in special risk for lipid peroxidation. Once initiated by any of several pathways, lipid peroxidation, oxidative damage of membrane lipids, spreads aggressively in a self-propagating chain reaction, amplifying oxidative damage . Lipid pOn the other hand, several studies have shown that accumulation of specific lipids, called lipofuscin, in RPE cells generates reactive oxygen species through phototoxicity. The accumulation of lipofuscin and subsequent generation of ROS upon light damage is a well-known triggering factor of macular degeneration in AMD and Stargardt disease . Recent As aforementioned, oxidative stress induces a variety of structural and functional changes to lipids, proteins, and both nuclear and mitochondrial DNA. For instance, oxidative stress provokes accelerated telomere shortening. Telomeres are specialized structures at the end of chromosomes that contain characteristic repetitive G-rich DNA sequences (TTAGGG) that when damaged or shortened can induce an altered cellular phenotype that promote senescent traits. Some studies show that oxidative stress induces single-stranded breaks in telomeric DNA in RPE cells in vitro and cells with longer replicative life spans, which are more susceptible to oxidative stress and accumulate DNA damage .Moreover, mtDNA is particularly sensitive to oxidative injury because: (i) mtDNA is localized close to the source of ROS production, (ii) it is not covered by histones, (iii) it is a circular intron-less circular DNA with high transcription rate, and (iv) the DNA repair system within the mitochondria appears to be less effective than that in the nucleus. mtDNA damage is particularly detrimental to non-dividing cells such as those in brain, heart skeletal muscles, photoreceptor and other retinal cells, such as and RPE cells . PolymerMef2d renders photoreceptors more susceptible to light-induced damage because they are unable to up-regulate Nrf2 upon oxidative stress [SEMA4A mutations make RPE cells more susceptible to light irradiation, ROS and ER stress [On the other hand, rare genetic mutations can cause specific sensitivity to oxidative and light stress in the retina. At least in mice, haploinsufficiency of e stress . BesidesR stress .Further work is required to identify polymorphic variants in relevant oxidative stress response genes that increase genetic susceptibility to oxidative stress in the retina, and may act as triggering factors in prevalent retinal degeneration pathologies (such as AMD and glaucoma) or as modifier genes in rare mendelian retinal disorders ).Oxidative stress (OS) damages the DNA of the trabecular meshwork (TM), which is responsible for aqueous humor draining of the anterior eye chamber . In glauOptineurin (OPTN) is a glaucoma-causative gene. Perfusion studies show that OPTN expression increases after 2 to 7 days of elevated IOPs (intraocular pressure) [Optineurin, high levels of this protein in the lens and the retina failed to protect against transforming growth factor-β1–induced apoptosis [Optineurin overexpression protects cells from hydrogen peroxide-induced cell death, and the OPTN E50K mutation decreased the neuroprotective effect by compromising mitochondrial membrane integrity, which resulted in cells less fit to survive under stress conditions [Optineurin in glaucoma pathogenesis requires further study, particularly, whether its overexpression can be a protective measure in retina upon oxidative stress.ressure) . OPTN haressure) ; howeverpoptosis . Other inditions . Indeed,AMD is one of the main causes of blindness in western countries, characterized by the loss of RPE cells and photoreceptors in the macula area. AMD retinas display extracellular deposits, known as druses, between Bruch’s membrane and the RPE . RPE celCFH) account for ∼46–71% of the genetic risk for developing AMD. Concerning external triggers, smoking—and consequently, oxidative stress—is the largest modifiable external risk factor for AMD. Genotype and smoking have been independently related to AMD with synergic effects [Usually, RPE damage is observed in early stages of AMD although there is not a direct link between the extent of damage in the RPE caused by OS and AMD progression . RPE-dam effects . Recentl effects .On the other hand, autophagy is a protective homeostatic mechanism designed to remove cellular components including those damaged by oxidative damage. Compromised autophagy contributes in AMD as a result of the RPE dysfunction . Additiomellitus complications. Molecular mechanisms of high glucose levels causing blindness are yet to be completely determined, although chronic hyperglycemia lead to several biochemical changes, among them, the activation of protein kinase C, polyols accumulation through aldose reductase pathway, AGEs production increment and free radical overproduction. These free radicals are generated from glucose auto-oxidation reactions and glycated adducts of proteins. These metabolic changes increase the levels of pro-inflammatory cytokines, chemokines and other mediators of the inflammatory response stimulating an influx of leucocytes and altering vascular permeability, which altogether favors the degeneration of the retina [DR is one of the most common Diabetes e retina ,26,61,94RP, caused by mutations in many different genes, is the most prevalent among rare inherited retinal disorders (IRDs) . The main phenotypic feature of RP is the progressive loss of rods followed by the death of cones. During the last years, OS has been shown to play a relevant role in RP pathogenesis and disease progression. Depending on the RP causative gene, antioxidant treatments might preserve cone function and prolong rod survival. Several studies show that both endogenous OS produced during retinal metabolism, such as lipid peroxidation or DNA damage, and exogenous OS, such as those produced by sunlight exposition, contribute to photoreceptor cell death. Moreover, rod photoreceptor death cause that higher levels of oxygen permeate into the tissue, thus increasing ROS production and inducing cell oxidative damage into the surviving cells (mostly cones), which will eventually result in cone death ,95,96. SIndeed, some RP-causative mutations cause misfolding of highly expressed transmembrane proteins that are involved in photoreception or phototransduction . The accumulation of unfolded or misfolded proteins cause UPR and an increase in oxidative stress in the ER, and elicits a compensative transcriptional program, for instance through PERK and IRE1 pathways, to activate genes encoding antioxidant enzymes. Chronic IRE1 activation causes an increase or mitochondrial ROS in degenerating retinas .Some studies suggest that autophagy could have beneficial effects in RP while others indicate it could negatively affect photoreceptor death. This apparent contradiction may be reconciled if we consider that: (i) RP is a highly heterogenous genetic disease, with many different genes and mutations associated to this disorder, therefore the importance of autophagy in retinal cells might be different depending on the mutation; (ii) autophagy may play different roles in the death of rods and cones; and finally (iii) moderate levels of autophagy in the photoreceptors may be beneficial whereas excessive autophagy may be deleterious . Again, GLO1 expression have been identified in a Sicilian population. As a result, retinal cells are more sensitive to oxidative stress-damage and eventually die by apoptosis, thus leading to retinal degeneration [To keep oxidation within controllable levels, cells have to activate and deploy endogenous antioxidant defenses. Inactivating mutations or expression deficiency of genes encoding antioxidant enzymes may lead to a faster progression of the disease. Glyoxalase 1 (GLO 1) is a ubiquitous cellular enzyme involved in detoxification of methylglyoxal (MGO), a cytotoxic byproduct of glycolysis. MGO excess can cause oxidative stress through the inactivation of antioxidant detoxification enzymes such as glutathione peroxidase and SOD. Hypomorphic genetic variants associated to diminished neration .CERKL has been implicated as a resilience gene against oxidative stress [CERKL is yet to be determined but several studies show that it is involved in the cellular response to oxidative stress and may play a role in protecting cells against stress injury [CERKL is not only expressed in the retina but also in several adult and fetal tissues in human and mouse. In humans, the highest expression of CERKL is detected in the retina, some brain regions, lung, and kidney [CERKL have been reported to cause RP and cone-rod dystrophy (CRD), thus providing further support to the concept that failure in the endogenous resilience mechanisms to overcome oxidative stress  leads to an accelerated progression of retinal neurodegeneration.In fact, mutations in many different genes involved in a large variety of functions cause mendelian inherited retinal dystrophies (IRDs). Among all the IRD causative genes, only e stress . The pres injury ,102. Bess injury ,101,102.d kidney , while id kidney . MutatioKLHL7, RDH11, CERKL, AIPL1, and USH1G) emerged as validated targets, thus suggesting a tight connection between induced oxidative stress and RP onset and progression [The high genetic heterogeneity of RP not only lies on gene mutation but most probably on gene regulation and ability of photoreceptors and RPE to respond to external toxic stimuli and metabolic challenges as well. In this respect, miRNAS and lncRNAs may regulate relevant IRD-causative genes in response to oxidative stress injuries, as reported for miRNAs , in RPE cells challenged with oxidizing compounds. At least, five RP causative genes  cells and mononuclear phagocytes. Neutralization of TLR2 reduces complement deposition, microglial activation, and protects photoreceptor neurons from oxidative stress-induced degeneration. TLR2 deficiency also promotes RPE resilience .On the other hand, modulation of other cell cytoprotective mechanisms, such as autophagy, including specific organelle-phagy  and mitoThe most interesting conclusions of this type of work is that therapeutic strategies to reduce oxidative stress and inflammation  are promising approaches, which should be concurrently explored as amelioration treatments of symptoms for a wide cohort of patients in addition to gene therapy, which is currently based on the correction of genetic defects caused by mutations and is thus restricted to a handful of patients.Oxidative stress by exogenous or endogenous agents damage the retina and the RPE, triggering apoptosis of photoreceptors and retinal ganglion cells. In prevalent diseases, such as AMD, glaucoma, and diabetic retinopathy, oxidative stress is a trigger of the pathologies, worsening the physiology of retinal cells and accelerating the progression of the disease. In rare genetic disorders, mutations in several causative genes cause metabolic distress and decrease cell resilience to oxidative stress, adding to the progressive attrition of photoreceptors and RGCs, eventually leading to blindness. We hypothesize that other IRD causative genes related to oxidative stress sensing and oxidative stress management, such as genes implicated in the control of autophagy, stress granule formation, or mitochondrial dynamics, will be identified in the near future, widening the landscape of genes and mutations that cause visual disorders. In addition to the more conventional gene therapy tailored to treat specific genetic defects, treatments addressed to increase cytoprotective signaling pathways in front of oxidative stress and to promote retinal cell resilience will provide new avenues to halt the progression of these blinding diseases and restore retinal homeostasis.


Developments of new strategies to restore vision and improving on current strategies by harnessing new advancements in material and electrical sciences, and biological and genetic-based technologies are of upmost health priorities around the world. Federal and private entities are spending billions of dollars on visual prosthetics technologies. This review describes the most current and state-of-the-art bioengineering technologies to restore vision. This includes a thorough description of traditional electrode-based visual prosthetics that have improved substantially since early prototypes. Recent advances in molecular and synthetic biology have transformed vision-assisted technologies; For example, optogenetic technologies that introduce light-responsive proteins offer excellent resolution but cortical applications are restricted by fiber implantation and tissue damage. Other stimulation modalities, such as magnetic fields, have been explored to achieve non-invasive neuromodulation. Miniature magnetic coils are currently being developed to activate select groups of neurons. Magnetically-responsive nanoparticles or exogenous proteins can significantly enhance the coupling between external electromagnetic devices and any neurons affiliated with these modifications. The need to minimize cytotoxic effects for nanoparticle-based therapies will likely restrict the number of usable materials. Nevertheless, advances in identifying and utilizing proteins that respond to magnetic fields may lead to non-invasive, cell-specific stimulation and may overcome many of the limitations that currently exist with other methods. Finally, sensory substitution systems also serve as viable visual prostheses by converting visual input to auditory and somatosensory stimuli. This review also discusses major challenges in the field and offers bioengineering strategies to overcome those. The development of bioelectrical interfaces in the 18th century enthralled scientists looking for strategies to treat brain pathologies and restore vision. Revolutionary experiments by Since then, advancements in neuroimaging, electrophysiology hardware, and surgical equipment have spurred ground-breaking research uncovering intricacies of visual pathways and possible therapeutic targets. Visual prostheses allowing for the restoration of basic abilities promoting object discrimination  and simpThis review describes state-of-the-art electrode-based visual prostheses technologies, and the ongoing development of cutting-edge biological- and genetic-based technologies to restore visual function including nanoparticles, optogenetics, magnetic manipulation and sensory substitution systems. These methods have the capability of artificially encoding sensation i.e. “writing,” into the brain and are gaining considerable interest as next-generation visual therapeutics.In 2015, there were 253 million individuals  blind and moderately-to-severely visually impaired people in the world . By 2050The traditional electrode-based visual prostheses consists of a basic set of components. A video camera is often used to convert light into electrical signals. These analog signals are digitized, and the image is processed by a portable micro-computer. The signals are then wirelessly transmitted to internal componentry with accompanying multi-electrode arrays (MEAs), which directly interface with the neural tissue.The type of the electrode-based prostheses is dictated by the underlying pathophysiology and its location should target a region along the visual pathway that would be the most effective in restoring visual perception. Interfacing too early along the visual pathway could lead to either non-transmitted or significantly corrupted and unintelligible signals. Interfacing at a region later than necessary bypasses functional neuronal circuitry, requiring additional hardware and/or complex image-processing algorithms. There are four major prosthetic design-types, each of which are categorized based on the location of their associated MEAs. Retinal prostheses offer promising rehabilitative potential for a number of retinal-based pathologies, including retinitis pigmentosa and age-related macular degeneration. In 1956, Tassicker developed and implanted the first retinal prostheses, capable of providing the recipient with crude light perception . Nearly 2 . Early research has shown that photodiodes reliant solely on ambient light can induce neurotrophic effects, but are insufficient for phosphene generation . By incoResearch into subretinal implants has proven some initial physiological limitations, though researchers are actively seeking innovative solutions. The uneven photoreceptor density within the retina presents a major challenge. Since the density of cone photoreceptors decreases with increasing retinal eccentricity, uniform phosphene generation throughout the visual field would require MEAs with varying electrode diameters and inter-electrode spacing. Novel MEA designs that promote glial and neuronal migration may allow for lower stimulation levels and more densely packed arrays . Due to Epiretinal prostheses interact with retinal ganglion cells. Owing to their downstream placement, epiretinal prostheses have a wider therapeutic potential than subretinal prostheses. Since extended periods of no photoreceptor input may cause signal corruption and intra-retinal neuronal degeneration, epiretinal prostheses may be the preferred visual prostheses for mid-to-late stage outer retinal pathologies. Epiretinal arrays can even selectively stimulate ganglion cells or bipolar cells based on stimulation parameters, including pulse polarity and duration . Optimiz2 contacts have been implanted around the optic nerve of a human patient  of the thalamus . An LGN Unlike the retina, LGN receptive fields have a consistent spatial density regardless of their visual field eccentricity. Since 60% of the LGN volume is devoted to processing the central 3° of the visual field , lower-d2 columns, which can be further subdivided into smaller columns responsive to orientation axis, color and ocular dominance. Similar to the LGN, these columns maintain a fairly consistent spatial density across the surface of V1 owing to the cortical magnification of central visual fields  allowing them to perform vital functions, such as phototaxis  and enerThe retina has constituted the majority of optogenetic-based visual research because of its accessibility and transparent nature . Since tClinicalTrials.gov NCT02556736, NCT03293524, and NCT03326336). Though biocompatibility concerns are minimal compared to electrode-based prostheses, clinical trials will determine if any long-term immune responses are present  device was demonstrated in 1985 , and quiin vitro (in vivo (Cortical electromagnetic-based prostheses would benefit from superior coil-neuron proximity, permitting numerous small, low-power devices. Recent developments in the construction of micro-scale magnetic coils have the potential to increase the effectiveness and specificity of TMS. A number of micro-coils have demonstrated device efficacy in vitro  and in v(in vivo . While o(in vivo . Advance(in vivo , adding (in vivo .in vitro upon applying an external magnetic field (Nanoparticles are extensively utilized in the fields of drug delivery , biosensic field .Another method is based on magnetic hyperthermia. When exposed to an alternating field, the orientation of a nanoparticle’s magnetic domain oscillates in accordance with the applied frequency. In response to weaker magnetic fields, this causes the nanoparticle to rotate and the particle-medium friction dissipates heat. With sufficient frequency, the localized heating can induce neuronal activation or inhibition via TRPV1  or TMEM1Voltage-gated ion channels expressed in V1 neurons can also be targeted by magnetoelectric composite nanoparticles. These nanoparticles, consisting of a magnetostrictive core and a piezoelectric shell, exhibit significant elastic coupling and magnetoelectric output . The magNanoparticle materials are only suitable candidates for neurostimulation if they exhibit minimal cytotoxicity; little is known about the long-term health effects of nanoparticle delivery to the brain. Additionally, the method of effectively delivering nanoparticles to the central nervous system is a significant concern. Only a fraction of intravenously-injected nanoparticles successfully reach the brain and only do so by long-term endothelial cell endocytosis or by destroying their cellular membranes . HoweverOngoing efforts have been dedicated to developing genetic-based neuromodulation technologies relying on magnetic changes. Magnetogenetics is a technology that allows cell, temporal, and location specific activation via magnetic fields and could evolve into a powerful non-invasive and effective technique for neurorehabilitation and vision restoration. These technologies mitigate concerns over cytotoxicity and tissue damage because no introduction of synthetic materials is required. A recent work showed the development of a construct encoding for ferritin protein subunits fused to TRPV4 receptors and its effectivity in inducing neural changes . HoweverKryptopterus bicirrhis (In vitro and in vivo animal studies demonstrated that EPG is capable of eliciting neural responses (Several organisms including birds , fish Q and bacticirrhis . The magesponses . A numbeStimulation devices that convert optical information into tactile and auditory sensory inputs may offer alternative sight to individuals who are visually impaired due to stroke or brain injury . TactileThe tongue is an excellent location for a sensory substitution device because it is densely innervated and coated in electrolytic saliva. With a tongue array consisting of over 100 electrode contacts, subjects significantly increased their visual acuity . One draOther sensory substitution research has investigated auditory stimulation as a replacement for visual input. These devices use a number of translational parameters, including substituting pitch or frequency for vertical location, binaural intensity or time-scanning for horizontal position, loudness for brightness, and timbre for color . While tThe total number of visual ailments is increasing rapidly among the general population in both developed and developing nations. Preliminary electrode-based visual prostheses demonstrated that they could induce visual percepts by interfacing with various regions of the visual system but lacked the efficiency to be adopted by blind and visually-impaired individuals in their everyday lives. Contemporary electrode-based visual prostheses have improved substantially since early prototypes. These devices can restore a number of abilities, such as crude object recognition and spatial navigation, and are now becoming a viable therapeutic consideration for blind and visually-impaired individuals. However, even with advanced micromachining and surgical procedures, limited spatial resolution and unavoidable tissue damage may render future electrode-based devices wanting. Nevertheless, non-electrode-based means for neurostimulation are being pursued. Optogenetic technologies introduces light-responsive proteins that can be used to excite or inhibit neural activity. For example, optogenetic technologies that introduce light-responsive proteins offer excellent resolution but cortical applications are restricted by fiber implantation and tissue damage. Other stimulation modalities, such as magnetic fields, have been explored to achieve non-invasive neuromodulation. Miniature magnetic coils are currently being developed to activate select groups of neurons. The poor coupling efficiency between magnetic fields and biological tissue leads to increasing power requirements and reducing achievable resolution. Magnetically-responsive nanoparticles or exogenous proteins can significantly enhance the coupling between external electromagnetic devices and any neurons affiliated with these modifications. The need to minimize cytotoxic effects for nanoparticle-based therapies will likely restrict the number of usable materials. Nevertheless, advances in identifying and utilizing proteins that respond to magnetic fields may lead to non-invasive, cell-specific stimulation and may overcome many of the limitations that currently exist with other methods. Finally, sensory substitution systems also serve as viable visual prostheses by converting visual input to auditory and tactile stimuli.AF and GP conceptualized the manuscript, drafted the manuscript, revised and finalized it, and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In recent years inhaled systems have shown momentum as patient-personalized therapies emerge. A significant improvement in terms of therapeutic efficacy and/or reduction adverse systemic effects is anticipated from their use owing these systems regional accumulation. Nevertheless, whatever safety and efficacy evidence required for inhaled formulations regulatory approval, it still poses an additional hurdle to gaining market access. In contrast with the formal intravenous medicines approval, the narrower adoption of pulmonary administration might rely on discrepancies in pre-clinical and clinical data provided by the marketing authorization holder to the regulatory authorities. Evidences of a diverse and inconsistent regulatory framework led to concerns over toxicity issues and respiratory safety. However, an overall trend to support general concepts of good practices exists. Current regulatory guidelines Uncertainty over the conceivable safety issues over pulmonary administration has dramatically deemed its potential. Inhaled therapeutics or vaccines have the advantage of providing its accumulation in the target tissue avoiding its systemic spread and loss of pharmacological activity. Local drug accumulation with subsequent high local efficacy as it was demonstrated by the available data gathered from respiratory diseases, such as asthma, cystic fibrosis and chronic obstructive pulmonary disease (COPD) . NanoparDifferent indications of approved medicines reflect the need to fight multiple targets over cancer tumorigenesis, e.g., the complex microenvironment, growth rate, angiogenesis, dissemination and immunosuppressive properties. Concerning the clinical implications of innovative tools, in which drug “cocktails” and “sandwich-based” treatments have become more used, one challenge for the pharmaceutical technology is to fulfill unmet medical needs using more precise medicines.nivolumab , and pembrolizumab  highlighted that pharmaceutical companies are still pursuing truly innovative solutions for lung cancer management. This trend can been also seen in the recent approved personalized therapies, such as osimertinib to be used in Epidermal Growth Factor Receptor (EGFR) exon 20 mutated lung carcinomas after failure of first line Tyrosine kinase inhibitors (TKI), or crizotinib  for first line metastatic ROS1 positive lung carcinomas, or even alectinib for anaplastic lymphoma kinase (ALK) positive carcinomas progressing after crizotinib approved for ROS1-positive metastatic non-small cell lung cancer (NSCLC). Up until today, limitations based on systemic toxicity, efficacy and safety of the available therapies along with the lukewarm effectiveness in including more selective, targeted delivery strategies, such as nanomedicines: broadly defined as drug delivery systems (nanoDDS)  .Nanomedicines, were thought as a first-of-a-kind strategy designed to improve patient outcomes trough, (1) promoting selective deliver of their cargo in the lesion; (2) avoidance of adverse effects in healthy cells, and (3) protecting the pharmacological molecule from earlier metabolization . TypicalEver-increasing attention in nanomedicines, either marketed or under clinical trials, have already stressed that it is advisable to encourage a better understanding of this systems features by the regulatory authorities. Efforts already underway include task forces to find a consensus on the requirements for registration and market approval accelerating patient access to those products. Regardless the existing favorable “regulatory environment”Patients and physicians are looking forward to have the augured advanced inhaled nanoDDS pharmaceuticals and its consequent add value. However, despite their promising capabilities, differences in distribution, pharmacology, immunology and toxicology raise numerous issues needing to be addressed to hardness pre-clinical validation of new targets supported by original data collections.Obstacles in the drug development process toward a broad acceptance of inhaled systems opportunities prompted several initiatives , such asEmbracing innovative solutions supported by biomedical data gathered from clinical/translational research studies could support clinical decisions that might contribute to improve lung cancer modest outcomes: primary cause of cancer-related death, with more than 1.6 million yearly deaths worldwide , 11. NowIn 2011, the International Association for the Study of Lung Cancer (IASLC), the American Thoracic Society (ATS), and the European Respiratory Society (ERS) established significant changes to the prior pathologic classification of lung cancer .omics”Conventional histologic-based classification for diagnosis and treatment decision in lung cancer have shown a limited scenario based in the dichotomist classification of small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC) –17. It lMolecular-driven understanding of tumor growth and progression might positively impact areas, such as diagnosis and patient sub-populations outcomes. As metastatic competencies increase, several intracellular events drive the primary tumor communication with the surrounding microenvironment as well as with distant organs. At that point, it is possible to observe the emergence of complex molecular cascades , 19.Systematic disease mutations in oncogenes crucial for sustaining cells tumorigenic potential, are perfect examples of conditions that could dictate patients stratification making possible to achieve outcomes beyond our current therapeutic expectations –22. Key EGFR mutations or ALK rearrangements drove recent progresses in precision medicine field  as first-line treatment for patients with advanced NSCLC harboring somatic mutations in EGFR exons 19 and 21. Higher efficacy  and safety , was reported in a phase III randomized clinical trials  Non-specific immune stimulation ; (2) AdoLung cancers are characterized by the constitutive activation of oncogenes that regulate the abrogation of the normal T-cell's activation, driving tumor escape from the immune surveillance . These tIpilimumab, a fully humanized anti-CTLA-4 mAb that targets protein receptors at the DC surface , pembrolizumab (Keytruda®), atezolizumab (Tecentriq®), avelumab (Bavencio®), and durvalumab (Imfinzi®) are in clinical use for the treatment or co-treatment of a broad range of malignant stages. FDA-approval of nivolumab (2014) has indications for patients with BRAF V600 wild type or mutation-positive unresectable metastatic melanoma. Lung cancer and specifically NSCLC patients already benefits from this therapy, as it becomes the first immunotherapy to be approved as second-line treatment (2015). Indications have now been extended to advanced renal, hepatocellular, and urothelial carcinomas, metastatic squamous cell carcinoma of the head and neck (SCCHN), classical Hodgkin's lymphoma (cHL) and microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer , up-regulated by cytokines, such as INF-γ and IL-4, which are produced after T-cell activation, establishing a feedback loop that attenuates tumors immune responses , 46–48. l cancer . Expectal cancer , 51.ipilimumab (anti-CTLA-4) and nivolumab (anti-PD-1) evaluated in a phase II trial against advanced melanoma, provided evidence of an improvement in patient response rate of 61%, compared to 11% for ipilimumab monotherapy, suggesting an exciting potential complementary role in regulating adaptive immunity  can be design to improve lung distribution and deposition providing higher local pharmacological doses with attenuated systemic adverse effects , 58.Recent breakthroughs in the field of molecular biology and developments in RNA interference (RNAi) technology have promoted the therapeutic use of RNAi-mediated strategies in the oncologic field. Particularly, the use of short interfering RNA (siRNA) arose as a reliable therapeutic approach by which harmful genes can be “silenced” by delivering complementary and rationally designed sequences –62. UnliOne of the major challenges in the siRNA field is the dependence of therapeutic efficacy on effective cell targeting. It is accepted that both cell uptake and intracellular trafficking are critical to promote intracellular accumulation of these molecular-target agents. Furthermore, limitations are also found in systemic administration, owing its quick degradation by body nucleases and clearance from the bloodstream. Unlike other molecules, siRNA biological fate is totally determined by the existence and properties of a carrier-mediated delivery system and its ability to overcome both extracellular and intracellular barriers .Nanomedicines might promptly change the limitations already faced with intracellular delivery of naked oligonucleotides. The protection and selective targeting can undoubtedly generate opportunities for molecular-target agent's translation from bench to clinical use can be anticipated.siRNA-nanocarriers engineered to tune selectivity of advanced nanoDDS liposomes, polymeric nanoparticles, micelles, solid lipid nanoparticles and inorganic nanoparticles could be designed in order to promote higher bioavailability, stability and residency times at the lung –67. For in vitro (in B16F10 cell lines), but variations in vivo (mouse). The nanoliposomes prepared with cationic dioleoyl-sn-glycero-3-ethylphosphocholine and cholesterol (ECL) presented the highest pulmonary delivery in vivo (26.2-fold higher than naked siRNA) and also the lowest cytotoxicity in vitro among the formulations tested. According to this study, the intra-tracheal administration of siMcl1 in ECL lipoplexes was able to significantly silence Mcl1 mRNA and its protein levels in the lungs, while reducing the formation of melanoma tumor nodules. These findings support the use of ECL nanoliposomes for pulmonary delivery of therapeutic siRNA to address lung cancer and other respiratory pathologies , an inhaled siRNA-based treatment of asthma are the most used inhaled systems undergoing clinical trials .Whichever the transported pharmacological agent, innovation, drug discovery and subsequent clinical trials have been mostly focused on intravenous administration, a route closely related to drug's adverse effects .Exploring alternative administration routes, e.g., lung administration, are urgently required to offer a selective and more effective therapeutic delivery while preventing drug accumulation at non-targeted tissues. This innovative strategy also aims to reduce clinical adverse effects avoiding dose-limiting toxicities. Advances emerging from the use of nanomedicines are a major source of improvements that can be applied to explore this regional route of administration to handle lung diseases with emphasis in lung carcinomas , 73.in vivo by inhalation   . The entXT C1.1) 6.Tomoda et al.  reportedThe effective transition of novel nanomedicines from experimental into the clinical arena demands a proper assessment of the properties forming the basis of their use (proof of concept plus kinetics plus toxicity). The relationship between the administered dose and the amount of drug deposited in the lung, the regional distribution, the residence time and/or clearance to the lung region or circulatory system need to be assessed for proper therapy planning .For conventional drugs, this information is initially obtained in pre-clinical studies in animals and other test systems, using classical approaches, e.g., by analysis of biological samples  using a combination of techniques, such as high-performance liquid chromatography (HPLC) and mass spectrometry (MS) . In the One strategy complementary to classical approaches for the tracking and quantification of nanoparticle-based complexes consists of using labeled particles, which implies the creation and use of dedicated technology for, (1) the labeling, (2) the visualization at organ, tissue, cellular and/or subcellular level of particles and drug-nanoparticle complexes, and (3) the quantification of nanoparticle-drug complexes, empty nanoparticles, non-complexed therapeutic molecule and degradation products , 80.In vivo, ex vivo, and in vitro test systems are available or under development for the purposes above described, such as Positron Emission Tomography (PET) and Single Photon Emission Computerized Tomography (SPECT) ( (SPECT) . An exce (SPECT) .in vivo owing the breathing-related motion of the subject during image acquisition. Of note, gated imaging can be applied to correct for this; alternatively, in the preclinical setting excision of the organs of interest and ex vivo imaging can solve this problem, at the cost of animal sacrifice. Additionally, resolution of nuclear imaging is usually poor . If higher resolution images are required, tissue dissection, slicing and evaluation using autoradiography can provide accurate information about regional distribution of the administered drug with a resolution close to 50 μm ..in vivo in vivo might be achieved. This strategy, to the best of our knowledge, has so far not been applied to the investigation of drug loaded nanocarriers. However, recent works demonstrate the feasibility of the approach and hence energy-discriminant SPECT might become a powerful tool to gain information about the in vivo behavior of nanomedicines in the near future  and ICH topic M3 (2) lack aspects related to the lung specificity. This underlines the question: if the pre-clinical existing package seems to be insufficient for fostering the approval of the advanced inhaled pharmaceuticals what should be done to avoid this gap? This means a review on many aspects related to the value of non-clinical conventional safety evaluation (ICH Topic M3 (R2), biological issues associated with the active pharmaceutical ingredient bioavailability upon lung deposition but also considering critical the role of excipients, known and new, that are being used for preparing pulmonary delivery formulations.Massive information has been gathered from research over the pulmonary administration route mechanisms and toxicological aspects. Not considering this fragment of evidence on emerging inhaled nanomedicines and their particularities represents a disadvantage for both industry and police makers.Different situations may emerge in this exercise, for the development of medicinal products to administer by the inhalation route, (1) the repurposing of an existing nanomedicine, (2) the repurposing of an approved molecule using an innovative nanomedicine, (3) innovative molecule and formulation with well-characterized nanoparticles, and (4) innovative molecule and formulation with newly developed nanoparticles.Given the relatively large spectrum of proven or potentially useful molecular entities currently available, which show activity in lung cancer, or in non-lung tumors, but with plausible mode of action justifying their study in lung cancer, the hypothesis (1) and (2) might be the most straightforward for improving the tumor access of drugs through their local administration. For both concepts to be applied into nanomedicines development, multiple requests need to be fulfilled in what concerns the characterization of the biological effects and of the methodological tools used, e.g., the inhaled route and the delivery systems .The clinical testing of such medicines demands a previous non-clinical estimation of the benefits (through proof of concepts) and of the risks posed by the new  route of administration. Relevant methodologies are available, as described above, to address the distribution of the nanoDDS across the respiratory tract, their potential accumulation in the tumor vs. healthy cells, the intracellular distribution in relevant T-cells, and the biodistribution from the sites of administration and accumulation into the systemic circulation, with subsequent clearance. The comparison of these aspects within the delivery routes, e.g., the inhaled vs. the approved non-inhalation route  will inform on the type and extension of studies that will be needed to support the entry into human trials.Existing modeling and simulation approaches, together with tests in artificial respiratory tract/lung equipment's are becoming important tools for planning the particle characteristics vs. the intended deposition across the respiratory tract. If the systemic exposure, including tissue distribution of the inhaled formulation will be, as expected, lower than the observed with the approved molecule and formulation, the preclinical requirements will be mostly circumscribed to the local effects in the respiratory tract, meaning that a short-term repeated dose studies in one or two species might suffice to support the entry into human use.On the contrary, depending on the comparative exposures reached systemically with the approved molecule vs. its innovative inhaled nanomedicines, a more extensive program will be needed, fully characterizing the (nano) particles, pre-clinical profile  as well as the local toxicity.This opens up countless opportunities for using the accumulated knowledge on existing molecules developed for non-lung tumors to support and abbreviate their development under inhaled formulations for lung cancer. The simplification of the development programs will allow faster patient access to innovation, but also will allow a faster success or failure together with increased knowledge on the relevance of the targeted cascades on the newly tested lung tumor(s), which are the remit of this manuscript. For now, it is important to focus our attention in toxicological matters rather than pharmacology and pharmacokinetics.The pulmonary route of administration embodies local/regional drug delivery against multiple diseases of the lung and the respiratory tract, e.g., asthma or cystic fibrosis. The systemic access of drugs through the lung has been attempted with limited success, as has been the case on inhaled insulin or calcitonin. Though this exploratory systemic administration shows great potential, information regarding possible advantages and challenges are out of this work scope.The enormously potential value of the development of inhaled nanomedicines has been illustrated in this manuscript specifically for regional management of lung cancer or other cancers invasion in the lung compartment. This incurable disease may embody situations where a cost-benefit could be achieved by the local delivery of anticancer products. Furthermore, pulmonary administration followed by regional nanoDDS deposition would be expected to promote faster and more direct access of drugs to the tumor microenvironment, higher pharmacological concentrations even though with decreasing administered dosing and at the same time, reducing the systemic exposure and associated toxicities.The increasing knowledge on the mechanisms behind tumors environment and the associated cellular/functional cascades, provides evidences on shared mechanisms not exclusive of one tumor type, making possible to approve therapeutic strategies with indications for multiple tumors in different organs. In another words, tumor characterization based on identified mutations rather than lesions in specific tissues/organs open up the rationale for shared therapies among multiple cancer situations independent of their location.Numerous challenges need to be overcome for a successful and efficient pulmonary delivery of antitumor products, including the “construction” of appropriate formulations based on nanoparticles having appropriated quality attributes for reaching and targeting the intended cell, within the “geography” of the respiratory system, taking into consideration the physiopathological changes inherent to the disease itself.Furthermore, any innovative formulation aiming to deliver new or existing therapeutic molecules will need to undergo a pre-clinical and clinical development program for supporting the marketing authorization stage and further human administration. Pre-clinical programs will include proof of concept, PK/PD and toxicology studies, being the extension of the studies determined by the innovative components of the developing medicines.Medicines repurposing will be a consequence of these new approaches, which mat allow a faster application of innovation to multiple situations. The preclinical programs may therefore be reduced and adjusted when the repurposing of a drug is attempted for changing the target organ of efficacy. A faster and better patient access to innovative treatments will be a direct beneficial consequence. These considerations apply to pulmonary delivery of existing medicines approved for use through other administration routes or intended for different organ targeting.Tracking nanoDDS across the pulmonary tract, different lung cells and eventually subcellular systems, demands technical solutions that are emerging, such as imaging technologies. Nevertheless, it is expectable that one of the higher advantages of local pulmonary delivery of antitumor drugs, particularly for lung cancer will be the reduction of the doses needed and reduction of systemic toxicity.The early and continuous dialogue between the scientists involved in the development process and regulators will be fundamental for the design of well-structured, potentially more simplified development programs, pre-clinical and/or clinical, to allow an earlier access and benefit of patients to those innovations.MV, JL, and CS managed the writing/manuscript preparation and data presentation. MV and JL were also responsible for the funding acquisition. BK, IV, BF, and BS-L conducted and prepared the manuscript section about toxicity and regulatory issues. NG provided a critical review on the clinical status and UC conducted the experimental work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This study aimed to assess associations between serum cartilage oligomeric matrix protein (sCOMP) and phenotypic characteristics in late-stage hip and knee Osteoarthritis (OA) as well as its correlation with further serum markers of possible comorbidities in the Ulm Osteoarthritis Study. Moreover, the prognostic relevance of preoperative sCOMP concentrations for short-term functionality and pain outcomes after hip or knee joint replacement was explored. Preoperative serum samples and detailed information about the health status ) of 754 OA patients undergoing total joint replacement were included. Spearman rank-correlation coefficients and multiple linear regression models were used to evaluate the relationships between sCOMP, other serum markers, and health outcomes. There was a significant positive association between sCOMP and markers of renal  and cardiac  impairment. Since renal failure might cause accumulation of sCOMP, additional adjustment with eGFR was performed. Preoperative sCOMP levels in knee OA but not hip OA patients were positively associated with FFbH, WOMAC function sub-scale and total WOMAC scale as well as the post-operative WOMAC stiffness sub-scale six months after surgery. Our data clearly demonstrate an association between sCOMP and renal function as well as other confounding factors, which should be considered in future biomarker studies. Osteoarthritis (OA) of hip and knee joints represents one of the most frequent causes of disability and pain worldwide . BesidesSince metabolic alterations of chondrocytes usually precede the structural damage of cartilage, associated biomarkers are highly relevant for early diagnosis and might also allow a prediction of rapid disease progression. Biomarkers studied in OA are mostly associated with extracellular matrix turnover of cartilage, joint inflammation, or general metabolic parameters ,9. AlthoCOMP, also referred to as thrombospondin 5, is a pentameric glycoprotein, which is expressed in cartilage and numerous other tissues/cells including ligaments, synovium, fibroblasts/myofibroblasts, vascular smooth muscle cells, cardiomyocytes, and various tumor cells . It is kIn the late stages of OA, the elevated synovial fluid and serum levels of COMP (sCOMP) are declining probably due to its depletion in highly degenerated tissue ,18,19. HTherefore, the present analysis focuses on the possible association of sCOMP concentrations with epidemiologic and phenotypic characteristics in late-stage hip and knee OA as well as its correlation with further serum markers associated with possible comorbidities in the well-characterized cohort of the Ulm Osteoarthritis Study . This exAll patients included within the longitudinal Ulm Osteoarthritis Study had advanced OA of the hip or knee and underwent the first unilateral total joint replacement between January 1995 and December 1996 in one of four large hospitals in the southwest of Germany  was obtained by standardized interview and examination performed by trained physicians before surgery (=baseline) as well as six months after surgery (=follow-up). Radiologic evaluation of contralateral joints and both hands was performed to classify patients as having unilateral, bilateral, or generalized OA . Secondary OA was assumed in case of infection, avascular necrosis, osteochondritis dissecans, hemorrhagic diathesis, slipped femoral capital epiphysis, or acetabular dysplasia of the hip and preceding trauma with radiologically and/or surgically confirmed structural joint lesion . MissingNon-fasting serum samples were taken preoperatively by standard venipuncture and stored at −80 °C. Analysis of uric acid, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (AP), calcium, phosphate, and creatinine was performed in a central laboratory at the time of baseline recruitment according to routine methods. The glomerular filtration rate (eGFR) was estimated using the creatinine-based formula of the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) . High-seBaseline sCOMP (sCOMP) was analyzed by means of a commercial sandwich ELISA against human COMP . The assay was run according to the manufacturer’s instructions, using a sample dilution of 1:50. Both inter (CV 15.6%) and intra assay variations (CV 2.8%) were assessed as quality control. The absorbance was detected at 450 nm and a reference wavelength at 630 nm by using the multimode microplate reader Infinite M200 Pro . Participants were categorized into four groups based on the quartiles of serum COMP levels at baseline . The characteristics of the patients were described using descriptive statistics. Distributions of continuous variables were presented as means  or medians  and those of categorical variables as the frequency with the corresponding percentage of the total cohort and within each sCOMP quartile.Based on the skewed distribution observed in serum COMP concentrations, medians were calculated across the strata of the various covariates. Generalized linear models were used to test associations of selected socio-demographic and clinical variables with serum COMP concentration when they were controlled for eGFR or age, gender, BMI, and eGFR. For further analysis, serum COMP concentrations were natural log-transformed, which led to a nearly normal distribution. Partial Spearman rank correlation coefficients, adjusted for age, sex, BMI, and in addition also for eGFR, were calculated to evaluate the relationships between sCOMP and other serum marker concentrations at baseline. Associations between sCOMP and health status scores at six months follow-up  were evaluated using multiple linear regression models adjusted for age, sex, BMI, and eGFR.Statistical analysis was performed with SAS version 9.4  and R version 3.5.1 .Overall, sCOMP quantification could be performed for 754 patients representing 93.2% of the total cohort. Parallel preoperative sCOMP values and FFbH assessment at baseline and six months after the joint replacement were available for 77.1% and 61.7% of the overall cohort, respectively . All WOMAfter adjustment for age, sex, and BMI, the sCOMP concentration of OA patients was positively correlated to parameters commonly associated with cardiac (NT-proBNP) and renal  impairment and negatively correlated with eGFR . MoreoveWith respect to the statistically significant associations between sCOMP and all tested parameters of renal function , an additional adjustment concerning eGFR was included. Due to this consideration, we found a negative association of sCOMP and hsCRP, while the trend for a positive association with AP and GDF-15 was diminished. However, the significant association of cystatin C with sCOMP was preserved after eGFR adjustment.Increased levels of sCOMP were positively associated with age, male gender, and higher BMI  at baselSince patients with knee OA had higher median sCOMP values as compared to those with hip OA  and withOverall, joint replacement led to an improvement in general physical function assessed by the FFbH in both hip  and kneeUsing adjustment for age/sex/BMI and eGFR in hip OA patients, the sCOMP values were not associated with FFbH score, which is an established tool for assessing more general physical function neither at baseline nor six months after joint replacement . In patiIn general, relevant improvement in all WOMAC sub-scales  as well as in the total WOMAC score could be shown in patients with hip  and kneeIn line with the results based on FFbH, with adjustment for age/sex/BMI and eGFR no statistically significant association of sCOMP was found for WOMAC pain, stiffness, and function sub-scales as well as the total score in case of hip OA, neither at baseline nor six months after joint replacement .p = 0.071). Six months after joint replacement there was no association with pain or function sub-scales as well as the total WOMAC score. Interestingly, however, a significant association was observed between baseline sCOMP and the WOMAC stiffness score at this follow-up time-point , implying the consideration of appropriate adjustment of these confounders. Independent of renal function there was a positive association of sCOMP with a distinct subset of biomarkers related to cardiovascular disease including NT-ProBNP and cystatin C, although the self-reported manifest cardiovascular disease was not associated. Moreover, we described that high sCOMP levels were positively associated with the preoperative FFbH and WOMAC sub-scale function as well as the total WOMAC score in knee OA patients, but not in hip OA patients.Fernandes et al. reported that COMP might suit as a diagnostic marker in early asymptomatic OA that did not exhibit any radiological abnormalities . SimilarIn line with previous studies, we also found a positive association with sex, age, and BMI  but noneInterestingly, we observed several associations between sCOMP and other disease-related serum parameters. The strong association with markers of renal function—cystatin C, creatinine, and eGFR—suggests that the COMP serum level is not only dependent on the release from different tissues but might also be modulated by renal function and the capacity of its excretion. In agreement with these results, increased COMP levels have been previously identified in the urine of horses with aseptic joint diseases . TherefoWhile hyperuricemia was previously found to be associated with generalized OA in hip OA patients within the Ulm Osteoarthritis Study , no signAlthough enhanced AST levels might suggest an association between sCOMP and hepatic disease, no association was found with respect to ALT . AST canHowever, high levels of COMP were found to be associated with a lower risk of incident myocardial infarction in a case–control study . This obAlthough the present cohort study could not consider data of healthy controls, our analysis revealed useful information to further characterize the association of sCOMP with clinical parameters in late-stage OA. In fact, for knee OA patients we identified an association between high preoperative sCOMP levels and functional impairment based on the more general FFbH score as well as the joint-specific WOMAC function sub-scale and total score. Moreover, we observed a trend for a positive association with the WOMAC pain sub-scale. Although comparable associations between the sCOMP level and specific WOMAC sub-scales as well as the WOMAC in total have been described previously in patients suffering from knee OA, the numbers of included cases were relatively small and/or the appropriate adjustment of confounders was missing ,46,47,48For hip OA patients, however, no respective associations could be identified in our study, which supports the conclusion of a recent meta-analysis that COMP may have better performance as a biomarker in knee than hip OA . This diOverall, it should be mentioned that the present findings, in particular concerning cardiac impairment, might be influenced by the pre-selected cohort for joint replacement surgery, which excludes patients at high risk for complications. Moreover, the assessment of self-reported comorbidities, although performed by physicians, might represent an additional bias to some extent. Another limitation results from the lack of an adequate control group represented by healthy individuals, which only allowed comparisons between subgroups of OA. Also, the cohorts did not comprise of patients with early stage OA. Strengths of the Ulm Osteoarthritis Study include the well-characterized study population with more than 800 patients and the availability of basal laboratory data as well as serum samples for biomarker analysis from the vast majority of participants, allowing respective multiple adjustments. Due to the German data protection rules we were not able to assess long-term cardiovascular mortality in relation to the identified cardiovascular marker profile in OA patients so far. It is an important future aim to identify OA patients with a higher risk for cardiovascular disease and improve respective prevention strategies.Our findings provide profound evidence of an association between sCOMP and renal function. Therefore, appropriate adjustment might be recommendable for future epidemiological studies focusing on sCOMP in OA and other diseases. Besides age, gender, and BMI we found a distinct profile of markers for cardiovascular risk including NTproBNP, cystatin C, and AST associated with sCOMP values, indicating the need for more detailed investigation in correlation with the clinical manifestation of cardiovascular disease. The data further support the notion that sCOMP is more strongly associated with clinical features of advanced knee OA compared to hip OA. Moreover, sCOMP levels were rather associated with clinical parameters of OA, as assessed by the WOMAC and FFbH scores, than with the radiographic classification by Kellgren and Lawrence. Overall, this study confirmed that sCOMP is a more complex biomarker in the context of OA than previously thought.


History illustrates the remarkable public health impact of mass vaccination, by dramatically improving life expectancy and reducing the burden of infectious diseases and co-morbidities worldwide. It has been perceived that if an individual adhered to the MMR vaccine schedule that immunity to mumps virus (MuV) would be lifelong. Recent mumps outbreaks in individuals who had received two doses of the Measles Mumps Rubella (MMR) vaccine has challenged the efficacy of the MMR vaccine. However, clinical symptoms, complications, viral shedding and transmission associated with mumps infection has been shown to be reduced in vaccinated individuals, demonstrating a benefit of this vaccine. Therefore, the question of what constitutes a good mumps vaccine and how its impact is assessed in this modern era remains to be addressed. Epidemiology of the individuals most affected by the outbreaks (predominantly young adults) and variance in the circulating MuV genotype have been well-described alluding to a collection of influences such as vaccine hesitancy, heterogeneous vaccine uptake, primary, and/or secondary vaccine failures. This review aims to discuss in detail the interplay of factors thought to be contributing to the current mumps outbreaks seen in highly vaccinated populations. In addition, how mumps diagnoses has progressed and impacted the understanding of mumps infection since a mumps vaccine was first developed, the limitations of current laboratory tests in confirming protection in vaccinated individuals and how vaccine effectiveness is quantified are also considered. By highlighting knowledge gaps within this area, this state-of-the-art review proposes a change of perspective regarding the impact of a vaccine in a highly vaccinated population from a clinical, diagnostic and public perspective, highlighting a need for a paradigm shift on what is considered vaccine immunity. MuV is an enveloped, non-segmented, negative-sense, single stranded RNA virus that varies between a spherical and pleiomorphic shape of ~200 nm 85–300 nm) , 2. MuV 00 nm , 2The MuV genome of seven genes consists of 15,384 nucleotides, and encodes six structural proteins and at least two non-structural proteins; the nucleocapsid protein (NP), V protein (V), phosphoprotein (P), matrix (M) protein, fusion (F) protein, small hydrophobic (SH) protein, hemagglutinin-neuraminidase (HN) protein, and large (L) protein. The role of the I protein is not known , 8. The Since 1946 numerous mumps vaccines have been developed worldwide, varying in efficacy and safety profiles but primarily consisting of an attenuated live MuV without an adjuvant –89. CurrThe Jeryl Lynn (JL) vaccine, derived from the genotype A MuV strain was first developed in the USA and has been used extensively in the United Kingdom (UK), Ireland and USA since it was licensed in 1967 . DerivedThe RIT 4385 mumps vaccine, developed from the dominant viral component (JL5) in the JL vaccine strain appears to have comparative safety and efficacy (seroconversion) profiles to the JL vaccine strain , 96–98. Despite the integration of the MMR vaccine into childhood immunization programs, cyclical outbreaks [defined as two or more cases linked by place and time ] of MuV Several reviews, both observational and systematic have demonstrated the clinical benefit of a mumps vaccine , 108, thHistory depicts the remarkable public health impact of mass vaccination. Previously inevitable childhood diseases with potentially debilitating or deadly outcomes have seen their rates plummet worldwide or become successfully eradicated. Immunizations of vaccine preventable diseases are estimated to prevent ~2–3 million deaths per annum and increase life expectancy by ~29 years . More reWith one in eight children in the USA under the age of 2 currently thought to be unvaccinated due to parental choice, the WHO now considers vaccine hesitancy as one of the ten threats to global health in 2019 . VaccineHeterogeneity of immunization coverage in specific populations or geographic locations of susceptibility is also becoming an important epidemiological issue in maintaining proficient population immunity for mumps , 122. ThThe clinical presentation of mumps is pathognomic ; therefore supporting laboratory diagnosis was rarely employed in the past. As the classical symptoms of mumps are not always typical, there may have been a significant number of individuals in the past who may have been infected but were not identified as such. When mumps vaccination was introduced in 1967, the criteria the vaccine had to meet was the proof that it was clinically effective, i.e., that it reduced the risk of disease in vaccinated individuals in real-world conditions over a set period. Such an example was seen the USA; the reported cases  of mumps declined from >100 cases per 100,000 population before 1967 (pre-vaccine era) to 10 cases per 100,000 population in 1977, a reduction of 99% , 131. ToCurrently the laboratory diagnosis of mumps infection in Ireland is based upon two approaches: detection of mumps RNA by reverse transcriptase PCR (RT-PCR) in a buccal swab containing saliva, throat swab or urine specimen, and serological detection of immunoglobulin M (IgM) using a capture assay , 133. BoThere are challenges in using standard serological laboratory diagnostic methods to reliably confirm mumps re-infection of individuals who had been previously naturally infected or vaccinated , 136. BrTherefore, Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) is recommended , 140, anThe case numbers of more recent mumps outbreaks should always be assessed with this question in mind; are the number of mumps cases increasing, or/and are we better at diagnosing an acute infection? The latter seems to be the most probable, as many individuals who are being tested do not present with classical symptoms. In addition to enhanced surveillance of mumps cases, further optimizations of technologies are also occurring; the utilization of next-generation sequencing demonstrated that by editing one 2-fold degenerate nucleotide in the forward primer and three 2-fold degenerate nucleotides in the probe sequence optimized the fluorescence intensity and clinical sensitivity of the real-time RT-PCR when compared to the CDC-developed and WHO-recommended RT-PCR target [(NP) gene] leading to ~11% increase in clinical sensitivity  .Much is not known about the immunological response to the mumps vaccine strain. However, a number of young adults who were vaccinated as children over the last two decades have demonstrated an increased risk of MuV infection with time, which is assumed to be related to a decline of antibodies to sub-protective levels of immunity , 143–146Primary vaccine failure is defined as the lack of a sufficient initial antibody response to a vaccine in a recipient resulting in a lack of protective immune responses , 147. AlHow is a response to a vaccine determined? In pre-licensure studies of the JL and Urabe mumps vaccines, high seroconversion and low failure rates were observed in children after the first vaccine dose , demonstrating that the vaccine induced an antibody response –153. A mn = 616)  . Poor imn = 616) . This isn = 616) . FurtherThe current methods used to determine immunity against mumps cannot discriminate between primary and secondary vaccine failure; only the timing of these tests can assess whether an individual ever mounted an immune response post-vaccination or whether the response is detectable years post-vaccination. Primary vaccine failure encompasses the failure to mount an immune response to a dose of a vaccine, secondary vaccine failure refers to a more gradual loss of immunity after a successful initial response that occurs over a number of years post-vaccination . SeveralWaning immunity is defined as a decline in immunological protection proportional to time since vaccination. Potential waning immunity has been documented in the current mumps outbreaks seen in Europe and the USA, mostly affecting young adults within highly vaccinated populations attending tertiary education who have received two doses of the MMR vaccine in early childhood , 175–181A number of studies from the USA, where a JL vaccine has been used since 1971 have demonstrated waning immunity within the population. The risk of developing clinical mumps was shown to increase by 10–27% for every year post-MMR vaccination , with thIn contrast, other studies appear to contradict, these findings, showing no link between mumps protection and time elapsed following administration of mumps vaccine , 185. LeImplementation of a third dose of the MMR vaccine has been shown to be effective as a stop gap measure in limiting disease spread in outbreak settings situations . IndividTherefore, as waning immunity is thought to be an important factor facilitating mumps outbreaks, the emphasis placed on the quantity/quality of mumps-specific antibodies may need to be re-assessed. It is yet undetermined if the total loss of detectable antibodies correlates to a loss of clinical protection, as the minimal level of neutralizing antibody required for protection against mumps has not yet been defined .Antigenic variation and thus reduced cross-neutralization between the vaccine and circulating strains of different MuV genotypes have been cited as possible explanations for mumps outbreaks in highly vaccinated populations , 189–191In vitro studies of the genotypic distribution and temporal shift of MuV suggest that cross neutralization between wild type and vaccine genotypes may be approximately half the concentration measured against the vaccine strain (The JL vaccine strain (genotype A), differs phylogenetically to the circulating MuV (genotype G) . In vitre strain . Pre-infe strain . Santak e strain , 196 alsin vitro plaque reduction neutralizations , or individuals previously exposed to MuV . TherefoDespite its use, the correlation to mumps-specific IgG concentrations and neutralization titers against the JL virus is poor, suggesting that IgG concentrations do not adequately represent a sufficient surrogate correlate of protection . This isAntibody levels of other components of the MMR vaccine have seen similar trends. Waning rubella antibody titers have been observed, despite the number of acute rubella and congenital rubella syndrome cases not increasing. It has also been shown that college students who received rubella vaccination during childhood and had low/no antibody response were able to mount a secondary response when challenged with rubella indicating that an individual's low antibody levels are not always indicative of susceptibility to infection . MeaslesSimilarly, three doses of the Hepatitis B (HBV) vaccine in a cohort of Alaskan natives showed >95% seroconversion in children and young adult post-vaccination and provided long term and durable protection against chronic HBV infection. Although no increase of HBV prevalence were observed 51% individuals had low to undetectable antibody levels after 30 years.These observations suggest that an individual's antibody levels do not indicate susceptibility to infection, that either an antibody titer lower than recommended guidelines is still protective, or/and is an ineffective surrogate of protection. This is emphasized in a study by Amanna et al.;  responseIt is also important to stress that seroconversion rates due to immunization/natural infection only reflects a change of antibody status from negative to positive, but not necessarily the intensity of antibody response. In addition, there is no consistency in the timing of sample collected post-vaccination to test vaccine efficacy, and between the serological tests utilized for detecting mumps antibodies. As a result, documented seroconversion rates of the mumps vaccines used vary widely .This highlights that the assays used to detect immunity to MuV may not always detect an adequate post-vaccination response. Only a small number of serological commercial assays such as the detection of Hepatitis B surface antibody (anti-HBs)  and rubeThough labor-intensive, neutralizing antibodies are considered to be a better correlate of mumps immunity. Antibodies against the haemagglutinin-neuraminidase protein (HN) and nucleoprotein (NP) have been shown to neutralize MuV, however, repeated attempts to define a titer that provides a protective threshold titer have been inconclusive , 228. InIndividuals who lack measurable mumps-specific antibody levels may be susceptible to infection but protected from significant illness as they may be protected by cell-mediated immune memory. Prolonged T-cell responses are reported after other vaccinations; 14–16 years after a single dose of the rubella vaccine RA27/3, a T-cell proliferative response to neutralizing antibody-inducing peptides suggest T helper and B-cell interactions. This indicates that full vaccine effectiveness could be dependent on mounting both an antibody and cell-mediated immune response .Although cell mediated immunity has not been as well-assessed in mumps infection, a lymphoproliferative response was induced in infants vaccinated at 6, 9, or 12 months of age was induced  with antLow levels of mumps-specific memory B-cells have also been documented suggesting that mumps infection or vaccination may not generate a robust B-cell memory , 233. TwAlthough the role and efficacy of T-cell immunity to mumps infection is unclear, there is a possibility that certain MuV strains may be capable of escaping vaccine induced T-cell responses, which may not be considered of significance until B-cell waning immunity comes into play . In indi+ T-cells recognizing a MuV epitope in a HLR-DR restricted manner. In addition, the response of IFN-γ and TNF producing CD8+ T-cells specific to MuV epitopes are lower in vaccinated individuals when compared to individuals who were naturally infected , B-cells , or cytotoxic T-cells. While most plasma cells, produce and secrete large amounts of antibodies, some differentiate into memory cells .Vaccination aims to stimulate the host immunological process and formation of cell-mediated immunological memory via the use of live-attenuated or of inactivated/subunit vaccine components to promote a cell-mediated immune response. Extensive knowledge gaps significantly hinder improvements to the mumps vaccine and prospects for mumps eradication and maintaining proficient population immunity , 187. FeIt has been suggested that wild-type infection could confer a “better quality,” broader and prolonged immuno-activation than vaccine-induced immunity. This is reflected in mean neutralizing antibody titers detected post-mumps vaccination, which were over five times lower than those detected following wild type infection. Similarly, hemagglutination-inhibiting titers after natural disease were 1:9 compared to 1:5 post-vaccination , 219.The use of a live-attenuated virus vaccine is intended to mimic immunological reactions and responses between the host and wild type virus . The curMucosal vaccines such as intra-nasal vaccination have advantages over traditional injectable vaccines as they can induce an effective, more robust immune response without any physical discomfort and more closely replicate the natural route of infection for mumps , 259. B-Such an example is the intranasal immunization of inactivated influenza. With a 70–90% similar efficacy between the injectable and intranasal influenza in healthy individuals this intranasal vaccine can elicit the secretion of haemagglutinin and neuraminidase specific IgA antibodies in the upper respiratory tract, and corresponding IgG antibodies . Live, cInactivated vaccines consisting of heat/chemical or live-attenuating monovalent or multivalent pathogens in animals/cell lines were developed to protect against disease causing microorganisms . Less emCurrently, the least expensive and time effective method to licensure is the comparison of serologic responses of the new vaccine to an existing licensed vaccine, which can lead to a bias on the development of novel vaccines . This meRaymond et al.  has suggVaccine RIT 4385 strain derived from one of the two distinct virus subtypes of the JL vaccine (JL5) showed comparable seroconversion rates despite inducing a significantly lower geometric mean antibody titer when compared to recipients of the JL vaccine, but does not have any longitudinal trials investigating its efficacy, even though there are populations who are currently receiving it , 270.The significant time gap between pathogen emergence and vaccine licensure, could potentially lead to antigenic drift. There is potential that modern biotechnologies could be utilized to design novel vaccine platforms , 272. ClIn addition, despite being extremely pleomorphic, utilizing MHC epitopes as potential B-cell and T-cell vaccine candidates are also being investigated , 275. VaSince the 1990's, significant progress has also been made in developing flexible, amplifiable, scalable, inexpensive, and cold-chain free RNA vaccines, such as synthetic mRNA molecules encoding only the antigen of interest and self-amplifying RNA (sa-RNA) . Such exExponential developments in the “OMIC” area has enabled further vaccine development and understanding of the immunological response and challenges surrounding this area . SystemsAdjuvants, a group of biological and chemical compounds could also be considered to enhance and improve the longevity of the immune response of a vaccine such as the MMR. Adjuvants have been successful in significantly reducing overall antigen dose in vaccine formulations as well as alter and broaden the host response through epitope spreading and qualitatively shaping the effector function of antibodies through subclass selection , 286.The re-purposing of live-attenuated vaccines as TIbV are also being investigated. Trained Immunity based Vaccines (TIbV) elicit heterologous protective effects by inducing a broader, lasting priming of innate immune cells, in addition to the intended specific immunological response and memory of conventional vaccines [reviewed in ]. MMR anThe mumps component of a vaccine is an unpurified product whose potency is measured through a biological assay for the substance rather than through evaluation of integrity of physical form (quantitative PCR after cell culture) . A monovComparing alternative culturing technologies and defining a viral potency range for vaccines could help reduce variability within the MMR vaccine . EnsurinThe efficacy of a vaccine is defined by disease prevention , or complications associated with infection  . DespiteTherefore, does “immunity” refer to sterile immunity or solely to protection from symptomatic infection? What defines an effective vaccine, or what constitutes vaccine failure? Does the medical profession and the “pro-vaccine” message contribute to the public skepticism regarding immunization? Is it time to shift the medical and public perception paradigm from “protection of infection following vaccination” to “protection from serious clinical mumps manifestation”?The lack of definition leads to misinterpretation by health professionals and media of what is truly occurring. Such an example is currently observed with influenza; individuals who have recently being vaccinated against influenza and subsequently become infected with influenza, assume that the vaccine has “failed” even though there is a reduction in symptoms.The current assertion that vaccines “protect against” or “eliminate” the risk of infection may contribute to the misperception about what level of protection a vaccine actually provides (vaccination efficacy) perpetuated by the witnessing of visible clinical disease and outbreaks despite vaccination , 298. ThA paradigm shift in what is considered to be a good vaccine i.e. one that provides protection against serious clinical sequalae, in addition to identifying a reliable laboratory marker for this protection is required . By focuIf we can decide whether the end point of a vaccine is either the prevention of infection or protection against serious sequalae of infection, its efficacy and impact can be determined and will have enormous implications on how vaccine failure can be studied, quantified and interpreted. This teasing out of the immunological response to MuV will ultimately provide potential correlates with robust predictive power, suggest directions for further vaccine improvement, and enable the discovery of potential biomarkers to help create a more efficient diagnostic assay that can discern between different infectious diseases and vaccination vs. disease status. The identification and incorporation of a correlate into diagnostic protocols which can be widely accessible may potentially allow global harmonization of criteria defining immunological protection against mumps.The medical and scientific field needs to inform the public more accurately about what a good vaccine consists of, which may result in a more positive attitude toward vaccines. In the majority of individuals, a vaccine can prevent serious clinical sequalae and associated complications following wild type infections, but also significantly reduce onwards transmission in particular to the cohorts who are not vaccinated due to a contraindication to vaccination. This is the positive and realistic view of vaccination which should be presented rather than the current flawed message of “get the vaccine and be protected from infection.” The public deserves, and will appreciate, a more accurate and informed message.AC, JC, and JH contributed to the conception and design of the review. AC wrote the first draft of the manuscript. JC, TL, and JH contributed to manuscript revision. All authors have read and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.