Tauopathies are a subclass of neurodegenerative diseases characterizedby an accumulation of microtubule binding tau fibrils in brain regions.Diseases such as Alzheimer’s (AD), chronic traumatic encephalopathy(CTE), Pick’s disease (PiD), and corticobasal degeneration(CBD) belong to this subclass. Development of tracers which can visualizeand discriminate between different tauopathies is of clinical importancein the diagnosis of various tauopathies. Currently, several tau tracersare available for in vivo imaging using a positron emission tomography(PET) technique. Among these tracers, PBB3 is reported to bind tovarious types of tau fibrils with comparable binding affinities. In contrast, tau tracer AV-1451 is reported to bind to specific typesof tau fibrils (in particular to AD-associated and CTE) with higherbinding affinity and only show nonspecific or weaker binding towardtau fibrils dominant with 3R isoforms (associated with PiD). The taufibrils associated with different tauopathies can adopt differentmicrostructures with different binding site microenvironments. Byusing detailed studies of the binding profiles of tau tracers fordifferent types of tau fibrils, it may be possible to design tracerswith high selectivity toward a specific tauopathy. The microstructuresfor the tau fibrils from patients with AD, PiD, and CTE have recentlybeen demonstrated by cryogenic electron microscopy (cryo-EM) measurementsallowing structure-based in silico simulations. In the present study,we have performed a multiscale computational study involving moleculardocking, molecular dynamics, free energy calculations, and QM fragmentationcalculations to understand the binding profiles of tau tracer AV-1451and its potential use for diagnosis of AD, CTE, and PiD tauopathies.Our computational study reveals that different affinity binding sitesexist for AV-1451 in the tau fibrils associated with different tauopathies.The binding affinity of this tracer toward different tau fibrils goesin this order: PiD > AD > CTE. The interaction energies for differenttau fibril–tracer complexes using the QM fragmentation schemealso showed the same trend. However, by carrying out molecular dynamicssimulations for the AD-derived tau fibrils in organic solvents, wefound additional high affinity binding sites for AV-1451. The AV-1451binding profile in these cryptic sites correctly explains the preferentialbinding of this tracer toward the AD fibrils when compared with thePiD fibrils. This study clearly demonstrates having a cryo-EM structureis still not sufficient for the structure-based tracer discovery forcertain targets, as they may have “potential but hidden”high affinity binding sites, and we need additional strategies toidentify them. It is proposed that the hyperphosphorylation of tauproteins leads to aggregate formation, and tau fibrils with straight,twisted, or paired helical filaments are usually formed. They makeup for highly insoluble neurofibrillary tangles (NFTs) which leadto a breakdown of the microtubules and to malfunctioning of neurons.It has been a main objective to estimate the population of such NFTsas they can be directly correlated to premorbid cognitive dysfunction6 and neuronal loss and disease progress. Manyradiolabeled tau tracers have so far been developed and tested invivo for imaging of regional accumulation of tau in AD and other tauopathies.9 The first generation tau tracers such as the FDDNP, PBB3, and theTHK series compounds AV-1451 and T808 are tracers binding to differenttauopathies,9 while the second generation tracerssuch as JNJ-311, MK-6240, Ro-948, and PI-2620 seem to exhibit considerablyhigh binding affinity toward tau fibrils in AD.10 While the focus so far has been on AD, onlya few tau tracers have been tested for binding affinity toward taufibrils from different tauopathies like PiD, CTE, CBD, progressivesupranuclear palsy (PSP), and argyrophilic grain disease (AgD).11 These diseases are generally referred to as non-AD tauopathies sincethey are associated with tau fibril accumulation but not with theco-occurrence of amyloid beta fibrils as in AD.Several neurodegenerative diseases are characterized by accumulationof certain biomolecular aggregates in different brain regions at theintraneuronal and extraneuronal compartments.5 The tau protein can exist in six isoforms, andthe number of amino acid residues in each form can range from 352to 441. Depending upon the specific isoform, the tau protein can havethree (3R) or four repeat (4R) units which bind to microtubules andstabilize their microstructure.5 It hasbeen reported that one or other isoforms dominate a specific caseof tauopathy. For example in AD, CTE, and Down’s syndrome,the 3R and 4R isoforms dominate, while in Huntington’s disease,PSP, AgD, and CBD are dominated by the 4R isoform.9 Pick bodies of Pick’s disease are dominated by 3Risoforms. Due to recent developments in cryogenic electron microscopy(cryo-EM) measurements, the structures for many fibrils have now beensolved which were elusive earlier.13 Thus, the structures for taufibrils from AD, CTE, and PiD patients have become available recently.13 The first report of a tau fibrilstructure from an AD brain (will be referred to as AD-tau) was demonstratedby Fitzpatrick et al. in 2017 which showed a double C-type structure(where the outer C filament surrounds the inner filament). The fibrilgrowth occurs in a direction perpendicular to this double C-type structure,and infinitely many fragments are aligned in parallel to make up forthe insoluble neurofibrillary tangles  also had a similar structure except that it had a wideropening in the closed region  had an altogetherdifferent microstructure with a more open region and with additionalfolds at the C-terminal region have demonstrated significant binding to non-AD tauopathies.22 The PBB3 tau tracer was the first tau tracer to detect non-AD tauopathieslike FTD, PSP, and CBD.18 The tracer also wasreported to bind in an AD brain to multiple binding sites with highbinding affinity.18 Autoradiography studieswith brain samples from patients with AD, PiD, and PSP have demonstratedthat PI-2620 binds to these tauopathies, but no specific binding wasreported for the brain slices of nondemented subjects.23 Similar studies with AD- and PSP-derived brainhomogenates suggested that AV-1451 binds to tau fibrils associatedwith AD with high specificity but nonspecifically was binding to PSP.The elevated signals for this tracer when compared to controls inthe brain regions such as pallidum, midbrain, dentate nucleus of thecerebellum, thalamus, caudate nucleus, and frontal regions can beassociated with PSP.19It is of great clinical interest to develop tau tracers which canbind to a specific type of microstructure of tau fibrils and therebydetect different variants of tauopathy. It might be necessary to developtau tracers which can light up multiple tauopathies by binding toboth tau microstructures with 3R and 4R units and that exist bothas straight and paired-helical filaments. In the latter case, theaim is to design a unique tracer to detect all possible tauopathies.Since the tau accumulation in different brain regions has typicaldistribution patterns, the tauopathies can be subclassed into specifictypes based on the spatiotemporal distribution of tau fibrils.20 Football players with a history of repetitivehead injuries tend to develop CTE neurodegenerative disease, and arecent study with AV-1451 in living players could reveal increasedtau deposition in the brain when compared to normal nonplayers.24 Further, it has been reported that AV-1451 alsobinds to CBD-associated tau fibrils, and its retention in motor cortex,corticospinal tract, and basal ganglia could be correlated to CBD.26 Autoradiography studies withbrain tissues from patients with Pick’s disease also showedAV-1451 binding to tau deposits associated with Pick’s disease.27 However, it was found that the binding affinityfor AD-associated tau fibrils was stronger than for the PiD case.27 Another study with post-mortem brain tissuesfrom patients with different tauopathies showed that the tracer bindingto PiD tauopathy is moderate, and that the tracer uptake can be usedto delineate AD and PiD cases from other tauopathies.28 All these experimental studies reveal that AV-1451 bindsto both 3R+4R tau fibrils (as in AD) as well as to either 4R tau (asin CBD) or 3R tau fibrils (as in PiD),27 but the bindingof AV-1451 to non-AD-associated tau fibrils has been reported to beeither nonspecific or weak when compared to AD.27 Overall, AV-1451 appears promising to image a wide range of non-ADtauopathies such as PSP, CTE, and CBD in addition to AD. It is worthrecalling that this tracer is also reported to bind to a number ofoff-targets such as monoamine oxidase-A, monoamine oxidase-B (MAO-B),and neuromelanin-containing cells from the substantia nigra.30 We have recently studied the off-target binding of AV-1451 to theMAO-B target using in vivo and computational studies.31In addition to its potency as a diagnostic tracer for AD and PSPtauopathies, AV-1451 also displayed its ability to image CTE effectively.13 We carried out blind molecular docking to find out various possiblebinding sites for AV-1451 in three different tau fibrils. Moleculardynamics simulations in the isothermal–isobaric ensemble forthe AV-1451 bound to different binding sites of tau fibrils were carriedout to address the stability of fibril–tracer complexes andto establish the equilibrium structures of tracer-bound tau fibrils.Finally free energy calculations based on the MM-GBSA approach32 were carried out to estimate the relative bindingaffinity of AV-1451 in different binding sites of different tau fibrils.To validate the force-field-based binding affinity, we also carriedout calculations using the QM fragmentation scheme35 and computedthe total interaction energy between the fibril and tracer at theM06-2X/6-31+G* level of theory which is known to describe the stabilityof intermolecular complexes having even weaker interactions.36In this work, we intend to address the mechanism behind the bindingaffinity of AV-1451 ,sites with moderate binding affinity , and sites with not sosignificant binding affinity  are seen. In the case of the AD-taufibril, site 1 is the one associated with high binding affinity, andsites 3 and 4 are associated with moderate binding affinity referto 1. SimilaIn order to further analyze the individual contributions to thetotal binding free energy, we have also plotted the residue-wise contributions,and this has been done only for the site with maximum binding affinityrefer to 4.Since 35 In this case, the fibrilsare cut along the peptide bonds and capped with hydrogens, and theinteractions are obtained as the sum over the contributions from theindividual amino acids.39 The total interactionenergy with fibrils is referred to as ΔEfibril in ΔEsolvent, and this contribution mimicsthe solvation free energy part of the MM-GBSA scheme. The differenceis that, in the current case, the tracer–solvent interactionenergies are computed through the explicit solvent model, while inMM-GBSA, the solvent is treated implicitly.32 As can be seen, the solvation energies are very significant in thecase of AD-tau and CTE-tau fibrils, while in the case of PiD-tau,the solvent contributions are relatively smaller. Further, it is strikingto notice that the interaction energies between AV-1451 with differenttau fibrils show the same trend as was observed from the MM-GBSA approach for thetau fibrils from AD and CTE are comparable, the latter one had a coresite suitable for the binding of a steroid-like hydrophobic molecule.So, we speculated that there can be certain core binding sites inAD-associated tau fibrils. Since in cyro-EM experiments the measurementsare made in aqueous conditions, these sites may be hidden, but inthe vicinity of small organic molecules, these sites become accessible.Interestingly, in the human genome, as many as >35% proteins are proposedto have such cryptic binding sites.41 Even the populartargets such as β-secretase and sirtuins are crystallized inclosed/holo form, but when cocrystallized in the presence of inhibitors,they crystallize in apo form.41 The identificationof such hidden sites computationally is challenging, and currentlythere are certain methods available: (i) long time scale moleculardynamics simulations,42 (ii) moleculardynamics simulations of the target in an organic solvent or mixedsolvent,44 and (iii) flexible docking. In the secondapproach, the target biomolecule is studied in the presence of smallorganic solvents such as benzene, n-hexane, or methaneor in a mixture of solvents.44 The equilibrated structurefrom such studies will have hidden sites exposed to facilitate thebinding of organic molecules. So, by doing molecular docking, usingsuch a fibril structure with exposed core sites will reveal additionalbinding for tracers which are hydrophobic in nature. In fact, a similarorganic profiling study carried out for AD-derived tau fibrils inthe benzene solvent and subsequent molecular docking study using arepresentative configuration resulted in displaying three additionalcore binding sites, as shown in The MM-GBSA- and QM fragmentation-based freeenergies and interaction energies suggest that the AV-1451 has largerbinding affinity toward PiD-derived tau fibrils than the AD-derivedtau fibrils. However, as we discussed above, the autoradiography studieswith brain homogenates suggested the opposite.320 Here, by using an integrated computational modelingapproach, we investigated in the present study the binding propertiesof AV-1451 to tau fibrils from AD, CTE, and PiD using the structuresfor these tau fibrils currently available from recent cryo-EM measurements.We employed combined molecular docking, molecular dynamics, and implicitsolvent (MM-GBSA)-based free energy calculations to estimate the relativebinding affinity of AV-1451 in different binding sites of the threedifferent tau fibrils. In the cases of AD-tau and PiD-tau, both highaffinity and moderate affinity binding sites were predicted, whilein the case of CTE-associated tau, all the binding sites displayedcomparable binding affinity. The binding affinity of AV-1451 in thecore binding site of PiD-associated tau was the highest; however,when compared to surface sites, their availability for tracer bindingis controlled by kinetic factors.45 Wetherefore speculate that the surface sites might be more easily availabledue to favorable binding kinetics than the high affinity core sites.In order to validate the force-field-based results of binding freeenergies, the QM fragmentation scheme was employed to compute theinteraction energy between AV-1451 and different fibrils. Rewardingly,the results from the two approaches are consistent. The studies usingcryo-EM structures for tau fibrils showed that the binding affinityof AV-1451 toward PiD tau fibrils was larger than that of the AD-taufibrils which was controversial with results from autoradiographystudies using brain homogenates.28 However, thecryptic sites found in AD-tau fibrils are found to be associated withhigh affinity for this tracer, and now the binding specificity forthis tracer toward AD-fibrils is correctly reproduced as observedin experiments. The current modeling study shows that the cryo-EMstructure alone may not be sufficient for certain targets, as theymay have hidden high affinity binding sites. Further, the study showsthat the AV-1451 association with the core site is driven mostly byhydrophobic interaction and partly by intermolecular hydrogen bondinginteraction with certain polar residues. Our findings underline thepossibility to design tracers that are more specific to certain typesof tauopathies by optimizing interactions with local microenvironmentsin the core binding sites of the corresponding tau fibrils.The objective of the present study was to gain a deeper understandingabout the underlying mechanism behind the use of certain tracers tovisualize different types of tauopathies, both AD and non-AD. Fromin vitro experiments, the tracer AV-1451 has been shown to bind totau fibrils from AD, PSP, CBD, CTE, and other non-AD tauopathies.Studies using brain homogenates from patients with different tauopathieshave reported high binding affinity for AV-1451 for the tau fibrilsassociated with AD when compared to fibrils associated with non-ADtauopathies.413 In certain cases, the structures for paired-helicalfilaments are reported, and in order to be consistent in all cases,we have used the structure of a single protofibril structure as ourtarget. Moreover, in our earlier studies, we found that the sitesappearing at the interfacial region of paired helical filaments arenot the ones associated with high binding affinity.39 Followed by molecular docking, we have carried out moleculardynamics calculations for the AV-1451 tracer when bound to variousbinding sites in three different tau fibrils. Since the locationsof binding sites are spatially well separated from each other, wehave carried out a single molecular dynamics study for each fibril–tracercomplex. It would be computationally very demanding to carry out individualMD for each AV-1451 bound to different binding sites of the tau fibrils.The configurations from molecular dynamics simulations were used forthe subsequent binding free energy calculations by employing the molecularmechanics-generalized Born surface area approach. In order to furthervalidate the force-field-based binding free energies, we have alsoemployed the QM fragmentation scheme which provides the total interactionenergy between the fibril–tracer as the sum over interactionenergies with various amino acid fragments. The advantage is thatnow the fibril interaction energies can be easily obtained at thedensity functional level of theory or even the MP2 level of theory.Below we elaborate on the computational details.We have used molecular docking to find various binding sites forthe AV-1451 tracer within tau protofibrils extracted from Alzheimer’s,CTE, and Pick’s disease patients. In particular, the tau fibrilstructures are based on cryo-EM measurements.4.146 The optimized AV-1451 structure has been usedas input for the molecular docking study with three different targetfibrils using the Autodock4.0 software.47 The AD-tau fibril structure was based on the PDB structure withreference number 5O3T,10 while the CTE-tau and PiD-tau structuresare based on the structures with PDB ids 6NWQ and 6GX5, respectively.13 The AD-tau has a pentamer unit, while the remaining two fibrilsonly have trimer units. So, to be consistent, we have built the pentamerprotofibril structure by replicating the units along the fibril growthaxis for these two cases. For the AD-tau fibrils, the number of gridpoints chosen in three directions for the grid box was 220 ×190 × 130. For the CTE-tau and PiD-tau, the number of grid pointswas chosen as 250 × 220 × 115 and 300 × 220 ×135, respectively. Since the binding sites for the tau fibrils arenot known previously, the grid box dimension has been chosen to coverthe whole fibril, and so, the docking software can identify both coreand surface sites. The 500 low energy configurations were stored frommolecular docking for further analysis. The binding poses with highbinding affinity in each of the independent binding sites were chosenfor subsequent molecular dynamics simulations. The high binding affinitybinding sites for AV-1451 in tau fibrils from AD, CTE, and PiD patientsare shown in The molecularstructure for the tracer AV-1451 was built using the Molden software,and the geometry was optimized in the gas phase using density functionaltheory  by employing the Gaussian09 software.4.248 as implementedin Gaussian09.46 In this approach, thecharges are obtained by best fitting to the molecular electrostaticpotential. In particular, the charge calculations are performed usingthe B3LYP/6-31G* level of theory. In the molecular dynamics simulations,the FF99SB force field has been used to describe the fibrils. Thegeneral Amber force field and TIP3P were, respectively, used to describeAV-1451 and water molecules. The fibril–tracer complexes weresolvated in the water solvent, and a sufficient number of counterionswere added to neutralize the whole system. The molecular dynamicssimulations were carried out using Amber 16 software.49 First the minimization run, followed by constant volumesimulation and an equilibration simulation in the isothermal–isobaricensemble, was carried out. The temperature was maintained at 300 Kalong with 1 atmospheric pressure to mimic ambient experimental conditions.The temperature and pressure were regulated by connecting the systemto the Langevin thermostat and Berendsen’s barostat, respectively.The time step for the integration of equation of motion was set tobe 2 fs. The time scale for the production runs was around 50 ns.During the simulation, various energetics and density properties weretested for convergence, and the simulation time scale is found tobe sufficient enough. The trajectories corresponding to the last 10ns have been used for computing the free energies of binding by employingthe MM-GBSA approach. In this approach, the fibril–ligand interactionsare computed by adding electrostatic and van der Waals interactionsbetween the two subsystems. However, the polar part of solvation freeenergies is computed by solving the Generalized Born equation, whilethe nonpolar part of the solvation free energies is computed fromthe solvent accessible surface area. The energies are computed forall three subsystems namely AV-1451, fibril, and the fibril–AV-1451complex, and the binding free energies are obtained as the differencebetween the free energy of the complex to individual systems.The moleculardynamics simulations for complexes of AV-1451 with AD-tau, CTE-tau,and PiD-tau fibrils were carried out subsequently. As we mentionedabove, the input orientations of the AV-1451 tracer within the fibrilscorrespond to the binding poses with the least free energy of bindingfrom the molecular docking studies. The charges for AV-1451 were obtainedby employing the CHELPG approach4.350In order to expose the cryptic sitesin AD-associated tau fibrils, this fibril has been studied in thebenzene solvent. The fibril has been embedded in the benzene solvent(approximately 5000 in number), and molecular dynamics calculationsinvolving minimization, in constant volume ensemble, and isothermalisobaric ensemble were carried out as described above in the caseof the water solvent. The benzene solvent has been described usingthe GAFF force field, and the charges were obtained using the sameapproach as used for AV-1451. A short production run for a time scaleof 5 ns has been carried out. The final configuration from this simulationhas been used as the input configuration for molecular docking whichdisplayed additional binding sites for AV-1451 as shown in 4.43 and CO–CH3 groups.Also, it is possible to compute the interaction of dipeptides withthe ligand so that one can also obtain the interaction energies withan account for three-body interactions. Further, the water–ligandinteraction energies can be computed as well with an explicit treatmentof the solvent. So, we can estimate the interactions due to the fibrilalone and due to the water solvent. The interactions between ligandand fibril fragments can be computed using various levels of theorysuch as dispersion corrected density functional theory, MO6-2X andMP2, thus, methods known to be effective in describing the dispersioninteractions. In the present study, we have computed the fibril–tracersinteractions using the MO6-2X/6-31+G** level of theory. Further, theindividual residue-wise contributions from each amino acid in fibrilsare available, and these data have been used to validate the residue-wisedecomposition of binding free energies as obtained using the MM-GBSAapproach.There are many reports showing the successof free energy calculation methods such as the MM-GBSA or molecularmechanics-Poisson–Boltzmann surface area (MM-PBSA) approaches.However, when the experimental binding affinity data are not available,it is recommended to compute the binding free energies with more thanone computational approach to further validate the predicted results.Moreover, the relative binding affinities of tracers in differentfibrils are usually very difficult to predict as the accuracy in bindingfree energy required for reliable prediction should be within a fewkcal/mol. Since the AV-1451 binding affinity to tau fibrils from CTEand PiD is not available from experimental studies, we aimed to validatethe binding free energy data with the more accurate QM fragmentation-basedapproach. We have developed an in-house fragmentation scheme, whichcan fragment the whole fibril into individual amino acids with thetotal fibril–ligand interaction energies computed as the sumover the fragment contributions. The fibrils are cut along the peptidebonds, and then each individual amino acid is capped either with hydrogensor with NH–CH


Despite the vast importance of the fovea for human vision, the molecular mechanisms guiding the development of this region are largely unknown. MicroRNAs (miRNAs) are small post-transcriptional regulators known to orchestrate developmental transitions and cell fate specification in the retina. Here, we have characterized the transcriptional landscape of the developing rhesus monkey retina. Our data indicates that non-human primate fovea development is significantly accelerated compared to the equivalent retinal region at the other side of the optic nerve head, as described previously. Notably, we also identify several miRNAs differentially expressed in the presumptive fovea, including miR-15b-5p, miR-342-5p, miR-30b-5p, miR-103-3p, miR-93-5p as well as the miRNA cluster miR-183/-96/-182. Interestingly, miR-342-5p is enriched in the nasal primate retina and in the peripheral developing mouse retina, while miR-15b is enriched in the temporal primate retina and increases over time in the mouse retina in a central-to-periphery gradient. Together our data constitutes the first characterization of the developing rhesus monkey retinal miRNome and provides novel datasets to attain a more comprehensive understanding of foveal development.Rod and cone photoreceptors differ in their shape, photopigment expression, synaptic connection patterns, light sensitivity, and distribution across the retina. Although rods greatly outnumber cones, human vision is mostly dependent on cone photoreceptors since cones are essential for our sharp visual acuity and color discrimination. In humans and other primates, the  Sight is often considered our most fundamental sense to perceive and navigate the world and, as a result, vision loss has a devastating impact on everyday life. Visual perception begins when photons of light enter the eye and are absorbed by the photoreceptors, the light-sensitive cells of the retina. There are two classes of photoreceptors named rods and cones because of their distinctive morphologies. While both populations contribute to the information transmitted to the visual centers of the brain by the optic nerve, these two cell types serve different purposes: rods are highly sensitive to light and provide relatively coarse, colorless images, while cones require considerably brighter light and are responsible for our sharp chromatic vision and spatial acuity .macula lutea (macula), where the cone concentration is about 200-fold higher than the most eccentric retinal regions  corresponding with the center of the visual field. At the foveal pit, all photoreceptors are densely packed cones and there are virtually no rods , one of the most prevalent types of photoreceptor degeneration, affects millions of people worldwide, and causes irreversible vision loss from the selective degeneration of the photoreceptors of the fovea . It has During retinal development, different classes of retinal populations are consecutively added in a well-known sequence that is conserved in all vertebrates : RetinalmiRbase.org) (in vivo (in vitro (MicroRNAs (miRNAs) are small RNA molecules known to regulate several aspects of development. To date, over 2,000 miRNAs have been recorded in miRbase (ase.org)  and bothase.org) . The ess, an Old World non-human primate. We have generated transcriptomic profiles of rhesus retinas at three developmental time points, spanning the major stages of development, and we have used miRNA-sequencing technologies to identify miRNAs differentially expressed in the presumptive fovea  compared to its equivalent region at the other side of the optic nerve head  at early stages of retinal development. In addition, we have chosen miRNAs with significant differential expression between retinal regions and we have validated their expression using in situ hybridization in mouse and human samples. Together, our data provides invaluable resources for studies aimed at understanding the role of miRNAs in retinal development as well as datasets to broaden our knowledge of foveal development.Given the vast importance of miRNAs as developmental regulators, we have sought to characterize the miRNome of the early developing non-human primate retina, specifically the rhesus monkey  from the prospective fovea  and the equivalent region at the other side of the optic nerve head , from three different time points spanning the three trimesters . Rhesus monkey trimesters are divided by 55-day increments  . We perfWe used the expression of cell type-enriched genes as a read-out of the timing of retinal histogenesis . As expeSimilarly, genes known to be expressed in both mouse and human RGCs exhibit specific expression at different time points . For exaIn addition to the cell-specific markers, other genes also exhibit transcriptional differences between the temporal and nasal regions of the developing rhesus eye , 2. Notan = 3 samples for each anatomical region, six samples total). After NGS profiling, an average of 29.9 million reads were obtained per sample, and the data was mapped to miRBase (release 20) and normalized. Principal Component Analysis (PCA) was performed by including the top 50 microRNAs that varied the most across all samples using normalized reads. As shown in miRNA libraries were obtained from retinal punches from the temporal side of the retina (presumptive fovea) and the nasal side of the optic nerve head as described above, at 50 days gestational age , miR-15b-5p , miR-30b-5p , miR-103-3p , and miR-93-5p . However, the expression levels of miR-369-3p are fairly low .Importantly, our experiments indicate that several miRNAs are differentially expressed in the different regions of the developing primate retina . For exairly low . In contInterestingly, miRNAs belonging to the same families often show similar expression profiles: all members of the miR-17/-20/-93/-106/-519 family are enriched over 2-fold in the temporal samples, miR-15b and miR-16 are enriched 3.85-fold and 2.65-fold, respectively, and miR-130a/-130b/-454/-301 are also all expressed at higher levels in the temporal region of the eye . This suPrevious studies have indicated that miRNAs coordinately regulate protein levels and thus, miRNAs that target the same complexes are often co-expressed . We haveMacaca mulatta database but known in other species  could also exhibit higher expression levels in the temporal samples. Finally, the progenitors of the fovea could possess unique properties and miRNA signatures.in situ hybridization (ISH) , E16.5, and postnatal day 3 (P3) by on (ISH) . All theon (ISH) . miR-15bon (ISH) . Intereson (ISH) . By E16.on (ISH) . Intereson (ISH) . Finallyon (ISH) , and thion (ISH)  show neiThe experiments using murine samples indicate that several of the miRNAs identified in our miRNA-seq screening are developmentally regulated and that miR-15b and miR-342-5p also show compartmentalized expression with central-to-peripheral differences. To further assess whether these expression patterns are conserved in primates and relevant to human biology, we used human fetal retina tissue to test miRNA expression of our top candidates . Since tRemarkably, miR-15b is expressed at higher levels in the temporal side of the optic nerve head  and miR-Our most advanced visual abilities such as reading and recognizing faces are dependent on the highly-specialized structure of the fovea. Unfortunately, the current understanding of retinal development is primarily based on mouse studies. Since the mouse retina does not contain a fovea and the rod-to-cone ratio in rodents resembles the most eccentric regions of the human retina, the molecular events that lead to the formation of the macula and the cone-dominated fovea remain largely unknown.Macaca, probably the most extensively used non-human primate model . Comparison between diurnal (foveated) and nocturnal (afoveated) New World primates suggested that alterations in cell cycle kinetics could explain some of the differences between these models, including the higher production of cones in foveated species . HoweverPrior studies have revealed that miRNAs are key regulators of the temporal changes that allow progenitors to produce different cell populations as development proceeds . SimilarMacaca mulatta) were bred and identified as pregnant using established methods  at the University of California at Davis. Normal, healthy adult female rhesus monkeys , we slowly cut the cornea from the puncture toward the corneo-scleral junction. We successively rotated the eye 90° and made three more cuts and we gently removed the lens. Then, using one of the cuts, we carefully inserted the lower blade of the scissors between the sclera/RPE and the retina and we cut all the way to the optic nerve head being careful not to damage the retina. We repeated using the other cuts at the corneo-scleral divide. Next, the sclera, RPE, and choroid were carefully removed with fine forceps  to dissect the retina away from the rest of the tissues. We performed two cuts in the dorsal and ventral part of the retina to open its cup shape and the temporal and nasal samples were obtained using 2.5 mm biopsy punches (World Precision Instruments) at equidistant regions about 0.5 mm from the ONH. As the total size of the retina changes during development, the percentage of retina captured at the different stages varied in the different samples. At 50 days gestational age, the biopsy captured more than half of the retina from the ONH to the ora serrata, thus extending beyond the foveal anlage.Pregnant CD-1 IGS females were obtained from Charles River and housed until embryos or neonates were at the proper developmental stage for dissection and fixation. All animals were used with approval from the University of California Davis IACUC. Dams were euthanized and embryos were dissected and fixed for ISH as described below.n = 6) were obtained from discarded de-identified human fetal tissue with permission of the University of California, Davis Institutional Review Board. The age for the human specimens was estimated by clinic intakes.Eyes  at −80°C. Then, total RNA was obtained from all the samples using the Total RNA Purification plus micro kit , and we used an Agilent Bioanalyzer 2100 to evaluate the quality of the RNA obtained.® Stranded Total RNA (with Ribo-Zero Gold) preparation kit.The sequencing experiments were conducted by Exiqon (Denmark). The library preparation was performed using Illumina TruSeqThe starting material  of total RNA was depleted of rRNAs using ribo-zero gold  magnetic bead-based capture-probe system (Illumina Inc.). The remaining RNA  was subsequently purified (RNAcleanXP) and fragmented using enzymatic fragmentation. Then, first strand synthesis and second strand synthesis were performed, and the double stranded cDNA was purified (AMPure XP). The cDNA was end repaired, 3′ adenylated and Illumina sequencing adaptors ligated onto the fragments ends, and the library was purified (AMPure XP). The stranded libraries were amplified with PCR and purified (AMPure XP). The libraries size distribution was validated and quality inspected on a Bioanalyzer (high sensitivity DNA chip). High quality libraries were quantified using qPCR, the concentration normalized, and the samples pooled. The library pool(s) were re-quantified with qPCR and optimal concentration of the library pool used to generate the clusters on the surface of a flowcell before sequencing on a Nextseq500/High Output sequencing kit (51 cycles according to the manufacturer instructions (Illumina Inc.) using 50-bp single-end reads and 30 million reads.Our data analysis pipeline is based on the Tuxedo software package, including Bowtie2 (v. 2.2.2), Tophat (v2.0.11), and Cufflinks (v2.2.1). CummeRbund was used for post-processing Cufflinks and Cuffdiff results. The heatmap.2 function contained within the ggplot2 R package was used to produce all heat maps. Transcriptomic heat maps were produced by selecting genes that represent specific retinal cell types based on established literature using normalized CPM values.For miRNA-sequencing, we used the same samples that were used for RNA-seq. A total of 500 ng of total RNA was converted into microRNA NGS libraries using NEBNEXT library generation kit (New England Biolabs Inc.) according to the manufacturer’s instructions. Each individual RNA sample had adaptors ligated to its 3′ and 5′ ends and converted into cDNA. Then the cDNA was pre-amplified with specific primers containing sample specific indexes. After 18 PCR cycles the libraries were purified on QiaQuick columns and the insert efficiency evaluated by a Bioanalyzer 2100 instrument on high sensitivity DNA chip (Agilent Inc.). The microRNA cDNA libraries were size fractionated on a LabChip XT  and a band representing adaptors and 15–40 bp insert excised using the manufacturer’s instructions. Samples were then quantified using qPCR and concentration standards. Based on quality of the inserts and the concentration measurements the libraries were pooled in equimolar concentrations (libraries to be pooled are of the same concentration). The library pool(s) were finally quantified again with qPCR and optimal concentration of the library pool used to generate the clusters on the surface of a flowcell before sequencing using v2 sequencing methodology according to the manufacturer instructions (Illumina Inc.). Samples were sequenced on the Illumina NextSeq 500 system.Following sequencing, intensity correction and base calling (into BCL files), FASTQ files were generated using the appropriate bcl2fastq software (Illumina Inc.) which includes quality scoring of each individual base in a read. We found that the vast majority of the data has a Q score greater than 30 (>99.9% correct), indicating that high quality data was obtained for all samples.Principal Component Analysis was performed on miRNA samples using the base R function. To produce the hierarchically clustered heat map, the miRNA-seq data were initially filtered by removing any miRNAs that had a False Discovery Rate (FDR) of greater than 0.001 to improve readability of the heat map. All miRNAs with an FDR of < 0.001 were then hierarchically clustered using the built-in hierarchical clustering algorithm in the heatmap.2 function. The color-key for each heat map was created using predetermined break points to bin the TMM value into colors for each marker. The volcano plot was also obtained using the base R volcano plot function.All samples were collected and quickly fixed in a modified Carnoy’s fixative overnight at 4°C. For the mouse embryonic samples, we fixed whole heads while postnatal day 3 and human fetal samples were fixed as whole eyes. A small hole was made with an 18 gauge needle at the corneal limbus to facilitate the fixation. After fixation, samples were dehydrated and embedded in paraffin as described elsewhere . HorizonSections were prepared as described previously . SectionSamples were prepared as described previously . Next, sIn situ hybridization were imaged using an Axio Imager M2 with ApoTome2 microscope system (Zeiss) using tile scan options (ZEN imaging software), and immunolabeling experiments were documented using a Fluoview FV3000 confocal microscope (Olympus). Images were processed using Fiji (ImageJ software), and figures were prepared in Adobe Photoshop 2000.GSE168475The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: NCBI Gene Expression Omnibus, accession no: The studies involving human participants were reviewed and approved by University of California Davis Institutional Review Board, University of California, Davis. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements. The animal study was reviewed and approved by Institutional Animal Care and Use Committee; University of California, Davis.EF, ML, AMi, SC, JW, and NS conducted experiments and/or analyses. AMo conducted sample collection, contributed to the study design, and revised the manuscript. AFT identified, selected, and monitored the pregnancies sonographically, and collected the specimens for analysis, contributed to the study design, and revised the manuscript. SS and ALT supervised and designed the study, conducted experiments, and wrote the manuscript. All authors contributed to the manuscript 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.


In settings where the COVID-19 vaccine supply is constrained, extending the intervals between the first and second doses of the COVID-19 vaccine may allow more people receive their first doses earlier. Our aim is to estimate the health impact of COVID-19 vaccination alongside benefit-risk assessment of different dosing intervals in 13 middle-income countries (MICs) of Europe.We fitted a dynamic transmission model to country-level daily reported COVID-19 mortality in 13 MICs in Europe . A vaccine product with characteristics similar to those of the Oxford/AstraZeneca COVID-19 (AZD1222) vaccine was used in the base case scenario and was complemented by sensitivity analyses around efficacies similar to other COVID-19 vaccines. Both fixed dosing intervals at 4, 8, 12, 16, and 20 weeks and dose-specific intervals that prioritise specific doses for certain age groups were tested. Optimal intervals minimise COVID-19 mortality between March 2021 and December 2022. We incorporated the emergence of variants of concern (VOCs) into the model and conducted a benefit-risk assessment to quantify the tradeoff between health benefits versus adverse events following immunisation.In all countries modelled, optimal strategies are those that prioritise the first doses among older adults (60+ years) or adults (20+ years), which lead to dosing intervals longer than six months. In comparison, a four-week fixed dosing interval may incur 10.1%  more deaths. The rapid waning of the immunity induced by the first dose (i.e. with means ranging 60-120 days as opposed to 360 days in the base case) resulted in shorter optimal dosing intervals of 8-20 weeks. Benefit-risk ratios were the highest for fixed dosing intervals of 8-12 weeks.We infer that longer dosing intervals of over six months could reduce COVID-19 mortality in MICs of Europe. Certain parameters, such as rapid waning of first-dose induced immunity and increased immune escape through the emergence of VOCs, could significantly shorten the optimal dosing intervals.World Health Organization. We searched PubMed with the search terms “(COVID-19 OR coronavirus OR SARS-CoV-2) AND (vaccine) AND (delay second dose OR late second dose)” on 8 November 2021 with no language restrictions. This returned 89 articles, of which 16 articles were relevant to inferring the health impact of different dosing intervals in high-income country settings. Based on the evidence synthesis of the relevant articles, we infer that optimal timing of dosing intervals for two-dose COVID-19 vaccines depends on multiple factors, including pre-existing naturally acquired immunity, serological response after first and second doses, vaccine efficacy and effectiveness after first and second doses, waning dynamics of vaccine-induced immunity, vaccination coverage, vaccine supply rates, variants of concern characteristics and country-specific vaccine prioritisation plans.To the best of our knowledge, this is the first health impact and benefit-risk assessment study of dosing interval strategies for two-dose COVID-19 vaccination outside of high-income settings. We assessed the health impact of COVID-19 vaccination for strategies using different dosing interval approaches after fitting to the COVID-19 mortality data for 13 middle income countries of Europe. We found that the strategy of prioritising the first dose coverage in older adults or adults was the optimal strategy to minimise COVID-19 mortality. Specific variants or vaccine characteristics, such as faster waning duration of the first dose, could significantly modify this conclusion – in which case, fixed dosing intervals between 8 and 20 weeks would be optimal.The optimal dosing intervals for two-dose COVID-19 vaccines are context-specific, depend on the underlying SARS-CoV-2 epidemiology in each country, and are sensitive to variants and vaccine characteristics. We showed that increasing the first dose coverage is key to minimising COVID-19 mortality. However, new evidence on parameters such as variants of concern's transmissibility and severity and (vaccine- or infection-induced) protection waning rate should be incorporated into consideration as they emerge.Alt-text: Unlabelled boxBy October 2021, 22 COVID-19 vaccines were in use globally.Most of the available vaccines involve two doses with recommended between-dose intervals as tested in clinical trials. These dosing intervals are generally 3-4 weeks, although they may differ by vaccine product. However, in practice, countries may use dosing intervals longer than recommended due to a wide range of factors, including but not limited to administrative and logistic constraints (e.g. vaccine clinic capacity), vaccine shortages, and the comparable and potentially higher vaccine efficacy using certain extended dosing intervals.,,,However, existing evidence on these tradeoffs has been limited and almost exclusively based on high-income settings.8Moreover, there are concerns about the benefits of the COVID-19 vaccine versus the harms from adverse events following immunisation (AEFI) depending on the age of the vaccinated individuals, which may change conclusions of the overall health benefits of vaccination.The World Health Organisation commissioned this work to specifically generate country-level evidence outside of high-income settings to address this evidence gap. We used a mathematical modelling approach that incorporated two-dose dynamics to assess the health impacts  of different dosing intervals in middle-income countries (MICs) in Europe between March 2021 and December 2022. We aim to identify the optimal dosing interval strategy that minimises cumulative mortality or maximises the benefit-risk ratio. The evidence generated could inform COVID-19 vaccine policies as vaccines roll out - rapid shifts in policies have been shown feasible.We explored country-level dosing interval policy approaches: (1) by setting fixed dosing intervals of different lengths; (2) by setting coverage targets (e.g. to first cover the entire adult population with the first dose). In the baseline scenario, we based the vaccine characteristics on those of the Oxford/AstraZeneca COVID-19 vaccine AZD1222 .We adapted CovidM, an existing transmission dynamic model of SARS-CoV-2, which has already been extensively described elsewhere,In brief, CovidM is an SEIR-type  compartmental model consisting of 16 age groups (0-4 to 75+ years with five-year increments) to incorporate age-specific susceptibility, clinical fractions, population sizes, and contact patterns. Thirteen compartments were used to capture the population dynamics within and between age group .Figure 11 and V2 in ,v1 and Rv2 in Compared to a classic SEIR model, this framework has several unique features. First, individuals protected by one and two doses are modelled separately , data sparsity issues  or significant changes in ways tallying COVID-19 mortality (n = 2). Country characteristics are captured by population age structures, age-specific contact patterns, changes in the mobility index, epidemic history, and immunity levels before vaccine introduction.The objective of the fitting process is to reproduce daily COVID-19 mortality time-series by country, assuming each observed daily COVID-19 mortality count is sampled from an underlying Poisson distribution. The final model selected maximised the overall likelihood of the entire fitting window. The parameter set associated with the optimal model was identified using differential evolution algorithms for each country.Given this study's relatively short time horizon, we modelled COVID-19 severe cases and mortality as processes, apart from the population dynamics presented in In the base case scenario, we considered a vaccine product with characteristics similar to AZD1222 and incorporated five types of vaccine effects  into the model , a. For Given the substantial uncertainty around the estimates of vaccine efficacies and the possibility that a country may use other (or multiple) vaccine products, we conducted sensitivity analyses over four dimensions around the values for infection- and disease-reducing vaccine efficacies for the first and second doses . This parameter space account for our best understanding of all vaccine products available . There is also limited evidence that suggests higher vaccine efficacy after the second dose when the dosing interval is longer than label recommendations.We implemented a 14-day delay between vaccination (i.e. doses administered) and immunisations (i.e. protection developed).,There is evidence showing the potential waning of the first dose when the second dose has not been administered promptly.n week dosing interval between the first and second doses for all individuals while others may aim to cover all individuals with the first dose before vaccinating anyone with the second dose. We expanded these two general approaches into seven dosing interval strategies  and then moves on to young adults (i.e. 20–59) consistently performs comparably or better than other age-based vaccine prioritisation strategies.We additionally assume the uptake goal for older adults is 90% and for young adults 70%. In other words, when the uptake level has reached 90% among those above 60 years, the vaccination program is considered complete for that age group and will move on to vaccinating adults. These two uptake thresholds are feasible based on age-specific uptake levels in countries with relatively fast vaccine roll-out in older adults.We assumed the starting date of vaccination programs to be 01 March 2021 in LMICs from WHO press releases. Based on the COVAX vaccine supply forecast to inform vaccine supply,The roll-out timelines specified above are consistent with the slowest adaptors among MICs considered . With these vaccination objectives, we explored the difference between strategies B1 and B2. When vaccine supply is much lower than explored here, B1 and B2 may lead to the same results as their algorithms only diverge after all older adults have been vaccinated with the first doses.To investigate the impacts of vaccine supply delay, we assume the second dose will become available 24 weeks after their initial inoculation dates. As sensitivity analyses, we also explored supply delay levels at 12 and 52 weeks.As vaccines roll out, contact levels within the population may recover. This study assumes contacts gradually recover to near pre-pandemic levels (i.e. 90% recovery) over a year from March 2021 following a sigmoid function. We do not account for reactive public health and social measures in response to surging infections as the action threshold (i.e. the definition of "surging infections") and the action intensity (e.g. a lockdown or a face mask mandate) may vary significantly by country.,,,,The emergence of VOCs may reduce the efficacy of vaccines. We investigated the potential effects of VOC emergence by modifying disease dynamics and vaccine efficacy. To reflect the impact of variants, we applied a 1.5x multiplier to transmissibility.We used the cumulative mortality between 01 March 2021 and 31 December 2022 COVID-19 as the primary decision-making metric. The optimal dosing interval strategy minimises the cumulative mortality. As discussed above, COVID-19 mortality is the product of age-specific infection counts, infection-fatality ratios, and a temporal delay function representing the interval between becoming infected and dying of COVID-19 . We compare dosing interval strategies by calculating the percentage difference in cumulative mortality relative to the reference strategy B1.In line with the previous parameterisation based on AZD1222, we quantified the risk of fatal AEFI based on major thromboembolic events (blood clots) with low platelet count (thrombocytopenia) as reported for the AZD1222. In the UK, up to 01 September 2021, there have been 416 cases in total, of which 45 cases occurred after the second dose.Based on the results of the different dosing intervals in the main analysis, we used these age-specific rates of occurrence after the first and second dose, respectively, times the proportion of fatalities after the first and second dose to estimate the total number of fatal AEFIs based on the number of vaccine doses used in each country.We then traded off the age-specific mortality from COVID-19 versus the age-specific mortality caused by AEFIs. We took the worst-performing dosing strategy in terms of mortality as the baseline to estimate the benefit-risk ratios, which quantify the benefit of additional deaths prevented by the vaccines versus the harm from additional deaths caused by AEFIs. For this analysis, strategy A1 is taken as the baseline to obtain positive results for the benefits (and the benefit-risk ratios) to facilitate interpretation. If one was to consider a no-vaccination strategy , then all of the benefit-risk ratios against no-vaccination are expected to be greater than 1.0 given the estimated much larger rate of outcomes in SARS-CoV-2 positive cases among unvaccinated individuals than vaccinated individuals.https://github.com/yangclaraliu/COVID_Vac_Delay.All the program code and data used in the study are publicly accessible online at The funders were involved in study design, data collection, data analysis, data interpretation, writing of the report, and the decision to submit for publication. We had discussions and received feedback from the members of the WHO Strategic Advisory Group of Experts on Immunization (SAGE) Working group on COVID-19 vaccine impact modelling and Immunisation, and Vaccines related Implementation Research Advisory Committee (IVIR-AC). All authors had full access to all the data in the study and had final responsibility for the decision to submit for publication. This study was approved by the ethics committee (Ref 26318) of the London School of Hygiene & Tropical Medicine.With a 24-week delay in vaccine supply, under strategies A1 and A5, individuals could receive their second doses at the dosing interval prescribed (i.e. 4-20 weeks). Under strategies B1 and B2, the second doses are not provided based on a fixed dosing interval - but on conditions that coverage targets have been met in target populations. The mean dosing intervals under strategy B1 range between 24 and 29 weeks and the median between 22 and 34 weeks. The mean dosing intervals under strategy B2 range between 45 and 57 weeks and the median between 43 and 56 weeks . Under sIn the sensitivity analysis with supply delay of 12 weeks, the dosing intervals for older adults under B1 were significantly shortened; with a supply delay of 52 weeks, the dosing intervals for the entire population were longer than baseline (i.e. 24 weeks) . Due to population age structure, the relationship between vaccine supply and population-level dosing intervals under B1 and B2 are not linear.At the beginning of the simulated vaccine roll-out processes (i.e. 01 March 2021), from model fitting, we estimated immunity levels in 13 MICs in the WHO European region to range from 5.89% to 30.8%.Assuming VOC emergence and a mean first-dose waning duration of 360 days, B1 and B2 were optimal strategies for minimising COVID-19 mortality in all countries investigated and were comparable with each other  a. StratWithout VOC emergence, the advantages of strategy B1 are more evident  b. The nAssuming a mean first-dose waning duration of 120 days, the superiority of strategy B1 is no longer evident. Optimal dosing strategies are shifted towards the range of A2 to A5 . Without VOC emergence, however, the advantage of strategy B1 and B2 over A1-A5 are of smaller magnitude if we consider the positive association between dosing interval and post-second-dose VEs.While varying four dimensions of vaccine efficacy (i.e. first and second doses infection- and disease-reducing effects), we found that strategies B1 and B2 were comparable (within 5% difference) and advantageous for all countries investigated, consistent with the baseline results .The additional deaths prevented by dosing strategies compared to the reference strategy A1 was always greater than the additional deaths due to fatal adverse events caused by these strategies. Most benefit-risk ratios of vaccination strategies in comparison to reference strategy A1 were greater than the additional harms ; in four ratios, the benefits of strategy A2 or B2 were smaller than those of strategy A1 . The additional benefits for strategies A2 and A3 were much greater than the additional harms among all other vaccination strategies, favouring a dosing interval of 8-12 weeks (compared to 4 weeks). The dominant benefit of strategy A3 was even more pronounced in the absence of VOC emergence  B.FigureAlthough we did not include the health and societal benefits of avoiding non-fatal SARS-CoV-2 positive cases, the general trend of additional population health outcomes  follows a similar trend as mortality, which indicates that including additional benefits should result in similar findings as shown in We explored the health implications of different COVID-19 vaccine dosing interval scenarios in 13 middle-income countries in Europe. We explored seven strategies that involved either fixed dosing intervals of between 4 weeks and 20 weeks or a conditional policy that depended on vaccination goals being met. We found that vaccinating all older adults or adults with the first dose (strategies B1 and B2) resulted in the lowest COVID-19 cumulative mortality, while a dosing interval of 4 weeks (strategy A1) resulted in the largest number of cumulative mortality. Small cumulative mortality was associated with an early increase in the proportions of the population vaccinated with the first dose.The optimal dosing strategies we have identified are all significantly longer than label recommendations (i.e. 3-4 weeks), which were based on clinical trial designs. In future vaccine development efforts during a pandemic, besides biological and logistic factors, implementation factors may also be factored in when designing dosing intervals for trials. Additional dosing schedules that allow for vaccine policy flexibility should be explored as soon as possible.The results we have observed are robust to different vaccine characteristics assumptions of vaccine efficacy estimates versus dosing interval relationships and to different vaccine supply delays tested in this study. We conservatively assumed baseline vaccine characteristics based on AZD 1222. Using vaccine candidates with potentially slower first dose waning profile (e.g. mRNA vaccines),,,Under the circulation of the wildtype and subsequently the Delta variant as modelled in this study, there is a negative association between dosing intervals and cumulative mortality when mean dosing intervals range between 4 and 26 weeks. This association may not persist with even longer dosing intervals of 43-56 weeks (as in strategy B2). While longer-than-currently recommended dosing intervals may be beneficial in reducing COVID-19 mortality, exceptionally long dosing intervals should be cautioned against. In this study, we tested a set of VOC characteristics approximating the Delta variant (by transmissibility and severity) assumed based on existing literature.When trading off the benefits of the vaccines in preventing deaths versus the harm from deaths caused by adverse events following immunisation, we found that the additional benefits of the strategies compared to strategy A1 exceeded the additional risks for most dosing interval scenarios. The ratio of benefits to risks was highest for strategies A2-A3 , following the age-dependent rates of (prevented) deaths from COVID-19 and adverse events. Including additional outcomes and benefits for society is expected to lead to similar results.,Related studies in high-income country settings of Canada, Netherlands, the United Kingdom, and the United States also found that delaying the second dose of a two-dose COVID-19 vaccine series is beneficial.In our study, we fitted a dynamic transmission model using reported daily COVID-19 mortality in 13 middle-income countries of Europe. We incorporated important epidemic dynamics, including VOC emergence and dosing dynamics . We explored a wide range of dosing interval strategies of prime interest to countries that may face constraints in vaccine supply. We performed extensive sensitivity analyses around key epidemiological parameters like the emergence of variants of concern and vaccine efficacy estimates. We extended the analysis by trading off the vaccines' benefits against the potential harm from adverse events following immunisation with the COVID-19 vaccines.We captured between-country variability through population age structure, contact matrices, mobility and government response stringency index. However, the effect of these variables on SARS-CoV-2 transmission may differ between countries due to additional factors that we were not able to capture. For example, the Google mobility index we used to represent mobility may be less representative in countries with lower smartphone usage. The relationship between the mobility index and interpersonal contacts was established using data from the United Kingdom, which may be partially representative of other countries.We could not extend the fitting window of this study further into 2021 or include spatial diffusion component (as did Cot et al.Our study shows that a dose-specific roll-out strategy that led to an average six-month dosing interval, which is substantially longer than the current label recommendation for most vaccine products available in the European market, may be able to minimise COVID-19 mortality in the MICs in Europe. Countries included in this study have diverse population age structures, contact patterns, and epidemic histories – the overall conclusions are valuable to COVID-19 vaccine dosing policy-making in LMICs elsewhere in the world.YL, FGS, MJ, and KA conceptualised the study. YL, CABP, and FGS developed the model and conducted the health impact assessment and benefit-risk analysis. RCB compiled the evidence on vaccine efficacy estimates. JHK and SF interpreted the comparative analysis of dosing interval strategies. YL wrote the original draft, and all authors contributed to reviewing and editing the manuscript for important intellectual content and have approved the final version.We declare no competing interests.


Vaccine anaphylaxis is rare; however, severe allergic reactions after administration of a coronavirus disease 2019 (COVID-19) vaccines have been reported. Excipients in the vaccine may play a role in severe allergic reactions post-vaccination. Various mechanisms, including IgE-mediated pathways, direct mass cell stimulation via the Mas-related G protein-coupled receptor-X2, and complement pathway activation, have been proposed to cause the anaphylaxis. Skin testing, using the basophil activation test, has been used to clarify the mechanism of the anaphylaxis and provide safety information for the next injection. Here, we review the current evidence and suggested approaches for patients who experienced an immediate severe allergic reaction to the first dose of a COVID-19 vaccine. Owing to the outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), the US Food and Drug Administration (FDA) approved an emergency use authorization or two highly effective coronavirus disease 2019 (COVID-19) vaccines from Pfizer-BioNTech . FollowiSeveral criteria for anaphylaxis diagnosis have been proposed by the National Institutes of Allergy and Infectious Diseases (NIAID)  and the According to NIAID in 2006 , anaphylShimabukuro et al. have reported 21 cases of anaphylaxis according to the Brighton Collaboration case definition criteria for anaphylaxis after administration of 1,893,360 first doses of the Pfizer-BioNTech COVID-19 vaccine from December 14 to 23, 2020, corresponding to an estimated initial rate of 11.1 cases per million doses administered . COVID-1Vaccine hypersensitivity reactions are infrequent, estimated to be 1.31 cases per million vaccine doses from a population-based study . Most reType I hypersensitivity reactions can manifest as severe life-threatening anaphylaxis and typically occur in minutes or within 4 h of exposure. An IgE-mediated systemic reaction to the excipients in the COVID-19 vaccine may play a role in the potential causative mechanism of the anaphylaxis as in Complement activation-related pseudoallergy (CARPA) is another mechanism considered as a cause of the severe allergic reaction. Pre-existing anti-PEG IgM may trigger the classical complement pathway entailing anaphylatoxins  release that causes hypersensitivity reaction –21.Allergic reactions following COVID-19 vaccination can result from vaccine components, including the active immunizing antigen, adjuvants, preservatives, stabilizers, antibiotics, cell culture materials, and leached packaging components . The vacPolyethylene glycol (PEG) has been identified as the cause of severe allergic reactions –25. mRNAPolysorbate 80 is widely used as a stabilizing agent in food and pharmacological products, including vaccines. Previous studies have reported that polysorbate 80 can induce local and systemic allergic reactions, including IgE-mediated and non-immune anaphylaxis –29. CrosAccording to the VAERS data from 55 states and US territories from December 14, 2020 to February 5, 2021, the relative incidence of anaphylaxis following administration of COVID-19 vaccines was two and seven times higher for recipients with a prior history of allergies and recipients who had experienced previous anaphylaxis, respectively . HoweverThe Centers for Disease Control (CDC) has recommended pre-screening using a questionnaire for risk stratification as in Skin tests are recommended for risk evaluation in patients with allergic reactions to COVID-19 vaccines by the WAO, American Academy of Allergy and Immunology (AAAAI), and European Academy of Allergy and Clinical Immunology (EAACI) but there is controversy over the recommendations for revaccination by graded administration –34. The An individual who has a previous history of hypersensitivity to an excipient or has had a severe allergic reaction to the first dose of a COVID-19 vaccine should be evaluated by an allergist . ClinicaMost patients who have had a previous severe allergic reaction to a COVID-19 vaccine can tolerate revaccination. Wolfson et al. have reported that 74% of patients who had an allergic reaction after the first dose of a mRNA vaccine and underwent allergic assessment by a skin test to PEG and polysorbate could be revaccinated safely. The negative predictive value of a negative skin test to PEG has been found to predict a 75% likelihood of tolerating the second dose of the vaccine . This stNon-IgE-mediated allergic reactions may be responsible for COVID-19 allergies. Warren et al. performed allergy assessments by skin testing to PEG and polysorbate 80, basophil activation tests and measurement of PEG IgG levels in 11 patients who were suspected of having an allergic reaction to a mRNA COVID-19 vaccine (The CDC have suggested an interchangeable vaccine platform using a vaccine that contains different excipients for patients who had reactions to excipients in the first dose of the vaccine. The adenoviral vector-based Janssen COVID-19 vaccine can be considered for individuals who had a severe allergic reaction to an mRNA-based COVID-19 vaccine. An mRNA COVID-19 vaccine can be considered for individuals who are contraindicated for the adenoviral vector-based Janssen COVID-19 vaccine. However, vaccination with caution is recommended because of the possibility of cross-reactivity between PEG and polysorbate 80 . AlthougPremedication with an antihistamine is controversial. The WAO , AAAAI , and BASMixing and matching of two different COVID-19 vaccines could be an option if an individual is suspected to have had an allergic reaction to the first dose, or if there is a shortage of certain vaccine. Previous studies found priming with the ChadOx1 nCoV-19 vaccine followed by boosting with BNT162b2 resulted in significantly greater humoral and cellular immune responses –43. In sNordstrom et al. reported that individuals who used a heterologous ChadOx1 nCoV-19 and mRNA prime-boost vaccination strategy were 68% less likely to develop a symptomatic COVID-19 infection compared with those who were unvaccinated whereas those who used a homologous ChadOx1 nCoV-19 were 50% less likely to develop a symptomatic COVID-19 infection compared with those who were unvaccinated . AdditioHeterologous priming with the ChadOx1 nCoV-19 vaccine, followed by boosting with BNT162b2, is an effective alternative to induce strong humoral and cellular immune responses and acceptable reactogenicity , 46. ThiAllergic reactions to the COVID-19 vaccines are rare, and various components of the vaccines could be responsible for causing the reaction. To date, PEG and polysorbate 80 have been identified as possible factors for the etiology of reactions to the mRNA vaccine. However, the precise mechanism of action is not yet known. Therefore, the CDC has recommended that individuals with a history of anaphylaxis to PEG should avoid both COVID-19 mRNA vaccines. Furthermore, clinical phenotyping, allergic assessment, and shared decision making before the next injection should be performed in conjunction with an allergist. The future recommendations may change as additional data are obtained.The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The novel COVID-19 virus has resulted in an immense burden in healthcare throughout the world. In addition to respiratory complications, COVID-19 has been associated with hypercoagulability and ischemic changes. We report a case of a patient with COVID-19 who presented with a rapidly progressing necrotizing fasciitis treated in our institution. The novel Coronavirus disease 2019 (COVID-19) is caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Although it is known for its respiratory complications, medical literature has shown that individuals with COVID-19 tend to have a spectrum of clinical manifestations, which includes coagulation abnormalities. Hyper-coagulability can worsen prognosis secondary to increased risk of pulmonary embolism, venous thrombo-embolism, limb ischemia, and digital ischemia. The underlying pathophysiology may be related to a severe inflammatory response leading to cytokine induced endothelial damage, micro-vascular thrombosis, and the development of pro-thrombotic anti-phospholipid antibodies . ElevateThe COVID-19 pandemic has caused substantial disruptions to health care delivery. Initial reports from China have helped us understand the approaches to the management of the infectious complications of COVID-19. An early case series from intensive care units in Wuhan, China described seven patients with peripheral ischemia and elevated D-dimer levels suggesting hypercoagulability A more reNecrotizing fasciitis is a rare condition characterized by widespread necrosis of subcutaneous tissue and fascia. The mortality rate with necrotizing fasciitis has been reported to be as high as 32% . There aWe present a case of a patient who was seen in our hospital with finger ischaemia, gangrene and necrotizing fasciitis of his hand and forearm with confirmed COVID-19. To our knowledge, this is the first case of necrotizing fasciitis of the upper extremity in a patient with confirmed COVID-19 being reported in literature.A 72-year-old male with past medical history of uncontrolled insulin dependent diabetes mellitus (Admission HbA1C of 15.5%), chronic Stage 4 kidney disease, congestive heart failure, peripheral vascular disease and previous foot amputation presented to the emergency department (ED) with complaints of severe progressive pain, swelling and discoloration of the ring and small fingers that started ten days ago. The patient denied a history of trauma to his hand. Upon arrival to the ED, patient’s vital signs included a temperature of 98.7 F, pulse rate of 66/min, respiratory rate of 16/min, and blood pressure of 87/47.While in the ED, physical examination of the patient’s hand revealed a wet gangrene of the right ring and small fingers. In addition, there were hemorrhagic blisters present over the hand as well as over the dorsal forearm . His labA chest Xray obtained at the time of admission demonstrated patchy bilateral infiltrates as well as airspace decrease more on the left side than the right, a pattern that is seen with COVID-19 . At the The patient was taken to surgery by the hand surgery service within a few hours after presenting to the ED. The surgery was performed in a negative pressure room. Adequate personal protective equipment was utilized by the surgical team .The ring and small fingers were found to be black, necrotic and non-viable. Ray amputations of the ring and small fingers were performed. Extensive debridement and removal of all necrotic tissues of the hand and the dorsal compartment of the forearm was performed. Foul smelling dish washer fluid suggestive of necrotizing fasciitis was evident. Biopsy and intra-operative cultures were obtained. Cultures demonstrated infection with Staph aureus and moderate group A Streptococcus pyogenes. Biopsy revealed extensive necrosis and inflammation of tissues.The patient was admitted in the MICU for COVID-19, septic shock and his underlying uncontrolled multiple medical problems. Patient was treated with Azithromycin (Zithromax) and Hydroxychloroquine (Plaquenil) for COVID-19. He was treated with Piperacillin-Tazobactam for his necrotizing fasciitis.On day two of admission, a repeat debridement was performed. Extensive necrosis of the middle finger was identified and a ray amputation of the middle finger was performed.In the following days, the patient underwent repeat debridements of his wound, wound VAC and Integra pre-meshed bilayer wound matrix placement . He demonstrated continued clinical improvement. His WBC, ESR and CRP down trended. Vasopressors were discontinued. He was transferred from the MICU to the medical floors.Throughout the course of his admission, the patient did not require intubation for COVID-19 and he was treated with nasal oxygen and medications for COVID-19.On week 3 of his admission, the patient’s COVID-19 PCR was negative. His wound showed early granulation and no evidence of infection. Week 3 following his admission, the patient was discharged to a sub-acute rehabilitation facility.Split-thickness skin graft (12/1000th of an inch) from the thigh was performed in the following days. At final follow up at 3 months , the wouThe patient subsequently did not follow up with hand surgery. Eight months after his initial admission, the patient was hospitalized at a different institution for his medical problems and passed away from post COVID-19 related lung changes and renal failure .Necrotizing fasciitis can be a life-threatening condition affecting the upper extremity associated with high rates of morbidity and mortality ,8. We weIschemic complications of the upper extremity have been reported in literature in patients with COVID-19 ,3,5. ThiAt the time of his presentation, the patient had several abnormal blood parameters. Recent studies have shown that COVID-19 can result in elevated WBC, D dimer values, ESR and CRP [In a recent review of 42 patients with COVID-19 and Acute Limb Ischaemia (ALI), Wohlauer et al.  found thIn the setting of the patient’s several uncontrolled medical conditions, it might be difficult to attribute the patient’s infection entirely to COVID-19. However, we hypothesize that the patient’s COVID-19 and its associated hyper-coagulopathic manifestations likely contributed to either the onset and/or evolution of the patient’s disease process.The novelty of the case report lies in that there is no current literature on the onset and disease progression of necrotizing fasciitis in the setting of COVID-19. This is the first case report being reported on this combination of life- threatening infections.Further research is needed to understand the ischaemic and secondary infectious complications of COVID-19. We believe that this case report will add to existing knowledge of the patho-mechanism and disease progression of hand ischaemia and infections in the setting of a patient with concomitant COVID-19.There are no current reports in existing literature about necrotizing fasciitis in a patient with COVID-19. In an elderly patient, both COVID 19 and necrotizing fasciitis are extremely life-threatening and our patient presented to us with both conditions. In addition, our patient also presented with several uncontrolled medical co-morbidities. This case illustrates that expeditious multi- disciplinary management in such patients may result in good outcomes.There is currently a paucity of literature on COVID-19 and its various manifestations. Our understanding of its various presentations, specifically in relation to hypercoagulopathy and limb ischaemia is still evolving. We believe that this case report will add to the currently evolving knowledge of COVID-19 and its manifestations.