The influenza virus is a global threat to human health causing unpredictable yet recurring pandemics, the last four emerging over the course of a hundred years. As our knowledge of influenza virus evolution, distribution, and transmission has increased, paths to pandemic preparedness have become apparent. In the 1950s, the World Health Organization (WHO) established a global influenza surveillance network that is now composed of institutions in 122 member states. This and other surveillance networks monitor circulating influenza strains in humans and animal reservoirs and are primed to detect influenza strains with pandemic potential. Both the United States Centers for Disease Control and Prevention and the WHO have also developed pandemic risk assessment tools that evaluate specific aspects of emerging influenza strains to develop a systematic process of determining research and funding priorities according to the risk of emergence and potential impact. Here, we review the history of influenza pandemic preparedness and the current state of preparedness, and we propose additional measures for improvement. We also comment on the intersection between the influenza pandemic preparedness network and the current SARS-CoV-2 crisis. We must continually evaluate and revise our risk assessment and pandemic preparedness plans and incorporate new information gathered from research and global crises. The long history of combating and planning for influenza pandemics should inform the fight against novel coronaviruses such as SARS-Cov-2. Richard Webby and co-workers at St. Jude Children’s Research Hospital in Memphis, USA review the history of preparing for influenza pandemics, including the global influenza surveillance network set up by the World Health Organization (WHO) in the 1950s. The 2009 H1N1 pandemic prompted WHO and the US Centers for Disease Control and Prevention to develop more detailed risk assessment tools drawing on laboratory research, genomics, industrial vaccine development, and surveillance of emerging animal strains that might transfer to humans. These tools and experience are proving successful in containing the H7N9 influenza that emerged in 2013, and could serve as models for managing coronaviruses, whose pandemic potential has only become apparent in the past two decades. This adaptation has provided ample hosts from which zoonotic strains may transmit to humans. The influenza virus has eight gene segments, each of which can be substituted through genetic reassortment when two or more viruses infect the same cell to ultimately produce a novel variant2.Influenza has plagued humanity for centuries. Influenza A and B viruses are endemic in humans and responsible for annual epidemics across the globe. In addition to annual epidemics, influenza A viruses were responsible for four pandemics from 1918 to 2009  and United States Centers for Disease Control and Prevention (CDC) created scoring systems to evaluate prepandemic influenza viruses in three primary categories: (1) properties of the virus, (2) attributes of the human population, and (3) viral ecology and epidemiology5.Host diversity and gene segmentation were responsible for the 2009 H1N1 pandemic, in which a series of reassortment events among avian, human H3N2, and swine H1N1 viruses via swine produced a novel influenza strain that was transmitted among humans  was established within the WHO10. This initiative started with the designation of the World Influenza Centre at the Medical Research Council of Great Britain. The impetus for establishing this program was articulated as being driven by memories of the 1918 pandemic, the highly infectious nature of the disease, and the speed of its spread and economic impact on countries. Correspondingly, the stated objectives of the GIP were to plan against possible recurrence of a pandemic, devise control methods, and limit economic impact from which influenza pandemic preparedness was essentially born. A further review of the driving forces behind GIP implementation shows how astute the supporters of these early efforts were and how their statements still hold true. In his announcement of the GIP, Payne from the WHO Division of Communicable Disease Services, although it was unclear at that time how influenza maintained itself between seasonal epidemics, surmised that (1) successful vaccination against influenza depends on knowledge of the virus causing epidemics, (2) continuous vigilance is necessary to detect new and potentially dangerous strains of virus at the earliest possible moment, and (3) epidemiological reports can be correctly interpreted only in terms of laboratory studies of the viruses responsible10. Despite the WHO influenza monitoring system ) currently having been expanded to 144 laboratories in 122 countries11, every one of these tenants rings true today.It is generally acknowledged that influenza pandemics have long been a part of human history. Reports describe bouts of influenza-like respiratory disease as far back as 412 BC, with fifteen likely influenza pandemics occurring after the 1700s when descriptions became more reliable12. Within the last 20 years, multiple influenza zoonotic events have been detected  and neuraminidase (NA) glycoproteins on the surface of the virus are generally considered drivers of influenza infectivity and pathogenesis, studies have shown that internal genes  can confer disease severity characteristics to novel influenza strains27.Laboratory research can reveal crucial insights into the pathogenesis of different influenza strains and guide assessments of risk. The reassortment of genetic material from influenza strain coinfections is a driver of pandemic virus emergence29. In addition, the ferret influenza model is leveraged to predict pathogenicity and transmissibility in humans, even discerning between direct and aerosol-contact modes of transmission31.Pathogenesis studies are facilitated through in vitro and in vivo laboratory models that provide key information on viral traits and host immune response. For example, avian and mammalian culture methods are used as surrogate indicators for human infection and vaccine protectionWith much focus at the time on avian H5N1 viruses, the 2009 H1N1 pandemic illuminated the need for a standardized system to assess the potential risks of emerging and circulating influenza strains and to allocate the limited time and financial resources available to research, prevention, and treatment. After the 2009 pandemic, two major risk assessment tools were developed to meet this need, combining insights from surveillance and laboratory work to develop a general framework for overall potential pandemic risk. These tools are careful to state that they do not predict which pandemic strain will emerge next but rather outline systematic methods for deciding research and funding priorities for emerging strains. Both require input from subject matter experts who score individual elements for a given virus.32. The IRAT evaluates prepandemic influenza viruses not currently circulating in humans based on ten characteristics, weighted according to perceived pandemic risk: (1) genomic variation, (2) receptor binding, (3) transmission in animal models, (4) antiviral susceptibility, (5) immunity in humans, (6) disease severity, (7) antigenic similarity to currently circulating strains in humans, (8) global distribution in animals, (9) animal species infected, and (10) human infections32.In 2010, the CDC created the Influenza Risk Assessment Tool (IRAT) to guide strategic decisions for future influenza pandemic preparedness4. The TIPRA was modeled closely after the IRAT, with 9 risk elements: (1) receptor binding properties, (2) genomic characteristics, (3) transmission in animal models, (4) susceptibility to antiviral treatment, (5) human infection, (6) disease severity, (7) population immunity, (8) geographic distribution in animals, and (9) infections in animals. The TIPRA estimates the risk of sustained human-to-human transmission of emerging viruses.In 2016, the WHO developed the Tool for Influenza Pandemic Risk Assessment (TIPRA) to estimate the pandemic risk of emerging influenza strains33. Below, we discuss the strengths and weaknesses of these elements and offer suggestions for improvement.Both the IRAT and the TIPRA are multielement additive models with weighted categories, with each weight determined through expert consultation and discussion. Figure The risk elements defined by the IRAT and TIPRA are grouped into three general categories Fig. : (1) vir26. For example, if a virus is markedly divergent from endemic strains, especially in the HA or NA genes, it has a higher probability of presenting novel antigens to the population, making pandemic spread more likely. If novel viruses are already circulating in mammals, they may contain genetic signatures associated with human infectivity and/or virulence and would score more highly in this element. Such considerations should be factored into assessing the potential pandemic risk of novel influenza viruses.When assessing the pandemic risk of an influenza virus, genomic characteristics can help predict how viral strains will behave before the behavior is observed in the field. General patterns of virulence and transmission are associated with specific genetic markers in the influenza genome34. Influenza viruses use cell surface glycans with sialic acid to bind and enter cells. Most of these cell surface glycans on human airway epithelial cells contain α2,6 linkages, whereas those on avian cells typically contain α2,3 linkages35. Therefore, influenza strains well adapted to α2,3 linkages must gain mutations that facilitate binding to α2,6 linkages to cross the species barrier into humans. Swine species provide a prime environment for the selection of receptor binding mutations, as cells in the trachea of swine contain a mixture of α2,3 and α2,6 linkages on their surface37. Receptor binding can be assessed by glycan-binding assays, but recent data suggest that primary human cell models are more physiologically relevant indicators of receptor binding properties38. These properties are factored into pandemic risk potential estimates.Receptor binding is a key barrier that avian, and possible mammalian, influenza viruses must overcome to infect and transmit in humans40. In the direct-contact model, infected and noninfected ferrets are housed together, sharing space, food, and water. In the aerosol-contact model, infected and noninfected ferrets are housed near each other to permit airborne transmission of respiratory droplets without direct contact. These two models can provide crucial information about whether prepandemic strains are likely to demonstrate sustained human-to-human transmission. For example, a strain that transmits via direct and aerosol contact in an animal model has a higher risk potential of sustained transmission than one that transmits via direct contact alone.The ferret model is particularly important for the prediction of human transmissibility, measuring both direct- and aerosol-contact–mediated transmission42. However, novel therapeutics against influenza strains resistant to current treatment options typically take years of research and clinical trials to produce. Thus, susceptibility to antiviral treatment should be a major factor for risk assessments of prepandemic strains. This criterion is important to guide the allocation of research and resources towards prepandemic strains that have the highest chance of presenting major treatment challenges should they cross the host-species barrier.Once a novel influenza virus reaches the pandemic level, the determination of appropriate treatment options is vital43. Therefore, emerging influenza viruses that are antigenically similar to seasonal influenza strains endemic in humans are more likely to be hindered by population immunity. Conversely, influenza strains originating from animal reservoirs with novel HA or NA subtypes are more likely to cause pandemics.Influenza pandemic risk assessments must also consider population immunity to emerging strains. Antibody responses to HA and NA surface glycoproteins are primary markers of protective immunity44, are postulated to be of higher risk than an H14 virus that has never been found in humans. However, despite circulating for over 20 years, human-to-human transmission of H5N1 is rare, and infection is typically acquired through close contact with infected birds, lowering its pandemic risk47. Thus, quantities of past human infections do not necessarily correlate directly with the ability for pandemic spread.Unfortunately, our best predictor of human infection potential is provided by evidence of past human infection. As such, viruses such as the A/goose/Guangdong/1/96-lineage H5N1 viruses, which have caused hundreds of human infections with a mortality rate of ~60%48. Although SARS-CoV-2 vaccines are being produced in record time, with candidates moving from preclinical testing to phase III trials in under 1 year49, countless individuals may become infected and succumb to the virus even with expedited vaccine production. Existing seasonal or pandemic influenza vaccine stockpiles may minimize the impact of an emerging strain if they are sufficiently antigenically related to the emerging virus. As the antigenic distance between seasonal and pandemic strains increases, the efficacy of existing vaccines decreases50. This particular element is one of the few present in only one of the risk assessment tools (the IRAT).A large determinant of our ability to control the spread of pandemic influenza strains is the availability of vaccines to increase population immunity and limit the number of naïve hosts for infection. However, changes to influenza vaccines typically require at least 6 months from conception to final licensing and distribution51. The ferret is the gold standard to predict and/or study how influenza strains behave in humans because ferrets recapitulate human influenza infections and have a prominence of α2,6 linkages on their airway epithelial cells53. The infectability of ferrets provides some indication of how well a virus is adapted to infect humans.Many different animal models are used in influenza research, including mice, chickens, pigs, hamsters, guinea pigs, and ferretsAs interaction between an infected animal population and humans is a clear requirement for pandemic virus emergence, the host animal reservoir of a virus is an important consideration. For example, a virus circulating widely in backyard poultry is more likely to come into contact with a human host than a virus in a population of wild birds.Geographic distribution in animals considers both the location and the speed of spread of prepandemic strains. Assessing viral locality indicates how easily a virus can be contained. Strains that are only locally distributed exhibit slow to moderate spread that occurs for understandable and predictable reasons  or are contained in single animal populations have a low to moderate risk of causing pandemics. Strains that are distributed widely, show unpredictable spread to other populations, or spread rapidly have a high risk of causing pandemics. A virus with a wider geographic distribution is also more likely to encounter a susceptible human host.56. Therefore, we use the recent H7N9 outbreaks to evaluate our current pandemic risk assessment and assess the strengths and weaknesses of our current level of preparedness.H1, H2, and H3 are the only influenza subtypes known to cause pandemics. However, recent outbreaks of the H5, H7 and H9 subtypes have raised alarms regarding their pandemic potential57. H7N9 is a novel reassortment avian lineage virus that most likely acquired internal genes from an H9N2 avian virus, an HA gene from a duck H7N3 virus, and an NA gene from an H7N9 virus circulating in migratory birds26. Human H7N9 outbreaks occurred in six waves in China, the latest occurring in 2017, with most cases linked to direct exposure to birds58.H7N9 was first isolated in humans in March 2013 and has since caused 1,567 human cases and 615 deaths, a concerning mortality rate26, and candidate prepandemic H7N9 vaccines were identified and recommended by the WHO by the summer of 201359. Subsequently, many researchers began studying H7N9 in vitro and in vivo.Early identification and risk assessments led to a quick response from the research community. The novel strain responsible for the first case in March 2013 was reviewed according to the pandemic risk assessment model by August of that year26. These mutations may contribute to its high pathogenicity and mortality rate in humans.The H7N9 genome contains mutations that increase human receptor binding and replication, a lack of multiple basic amino acids at the HA cleavage site (leading to low pathogenicity and undetected spread in birds), and an internal NS-associated gene constellation from H9N2 that led to high pathogenicity when reassorted into the highly pathogenic H5N1 outbreak in 199732. H7N9 scored highest for risk potential among 14 animal-origin viruses, ranking high for both emergence and impact risk32. H7N9 is concerning for its pandemic risk, and many resources in research and surveillance are consequently dedicated to this strain.H7N9 emergence was a driving factor for developing the TIPRA, and by early 2018, this tool was used to evaluate the risk of this and similar prepandemic strains60. H7N9 is not yet capable of sustained human-to-human transmission, a key determinate in causing pandemics. Since its first infection in humans, many resources have been dedicated to this response to this virus, resulting in a vast knowledge base. With prepandemic vaccines stockpiled, cell line and animal models optimized, and the surveillance system on alert, we are relatively confident that a quick response can be mounted should H7N9 gain the ability of sustained human-to-human transmission. However, we should always be evaluating and updating our current pandemic plans as we gain new information.Since 2013, the H7N9 virus has diverged into two major, antigenically distinct genetic lineages. Fortunately, there is evidence to suggest that stockpiled prepandemic vaccines developed against the early 2013 H7N9 viruses will be effective against each lineage. The vaccination of humans with these stockpiled vaccines elicited antibodies that were able to mediate antibody-dependent cellular cytotoxicity and neuraminidase inhibition of the antigenically drifted A(H7N9) virusesAs we move into the future of influenza pandemic preparedness, we must continue to improve metrics for pandemic risk assessments and our ability to respond to and mitigate such events. Areas for continued investment include surveillance and coordination among research laboratories, risk assessment criteria, vaccines and therapeutics, and clear and cohesive public health strategies and messaging. Fig. Effective influenza pandemic responses rely on rapid detection and identification of emerging strains. Expanded sampling of animal species and human infections increases the likelihood that such strains will be detected early. Continued collaboration among human and animal health influenza surveillance centers will ensure that reagents are appropriately shared, developed, and validated. Furthermore, these centers must be poised to respond when prepandemic influenza strains are identified, including maintaining adequate containment facilities and identification reagents.To ensure the ability of these centers to identify and respond to emerging strains, surveillance efforts should be broadened. Increased funding of pandemic preparedness efforts would allow for expanded sampling of zoonotic viruses, as well as the development of reagents and techniques to more precisely assess the risk posed by these viruses. Furthermore, increased support of such efforts would result in a larger arsenal of countermeasures, such as therapeutics, which may be deployed when high-risk prepandemic influenza strains are identified. Funding for surveillance is, shortsightedly, very difficult to maintain long term in the absence of emerging events.61. Higher mutation rates are linked to greater population diversity and thus have higher potential to evade host immune responses and develop resistance to antivirals63. Tools that can quickly identify strains with high mutation rates or RNA-dependent RNA polymerases with a high propensity for introducing mutations during replication can inform the evaluation of strains for their pandemic potential. Our ability to predict pandemic risk also requires a far larger arsenal of molecular markers of viral phenotypes. The sequencing of viruses is becoming increasingly cheaper and of higher throughput, and the more information that we can glean from sequencing data alone, the stronger our preparedness will be.The study of differential genetic mutation rates among influenza RNA-dependent RNA polymerases may be particularly relevant to pandemic risk assessments. Although the overall mutation rate of influenza has been explored, new methods can compare mutation rates among different influenza strainsAs our understanding of influenza viral dynamics and disease pathology grows, we must use this knowledge to expand and refine the assessment criteria for evaluating prepandemic strains. Although the risk assessment categories comprehensively represent our current understanding of the factors contributing to emerging influenza strains, other viral and host factors may play an underappreciated role in influenza pandemic probability.65. The HA0 precursor is activated via cleavage into the HA1/HA2 complex, a high-energy complex that undergoes conformational changes at low pH to enable membrane fusion66. This pH-dependent conformational change suggests that the stability of HA subtypes varies in differing environments. Accordingly, the pH at which HA0 cleavage occurs differs among subtypes. Influenza subtypes endemic in humans are relatively stable, whereas emerging subtypes in humans have a higher pH of activation. Swine isolates have activation pH values that span those found in human isolates, and avian isolates have wide-ranging activation pH values. Mutations that alter HA stability occur across several subtypes in different species67. Therefore, pH-dependent HA stability may have important implications for influenza virion transmission within mammalian hosts. In support of this, multiple studies in mice and ferrets revealed that the stabilization of H5 contributes to gain-of-function airborne transmission of H5N1 in ferrets and facilitates enhanced upper respiratory tract replication70.For example, we suggest that the acid stability variability between HAs of different subtypes should be considered as a new factor in the assessment criteria. During the viral life cycle, HA binds to the terminal sialic acids of host cell receptors for virion internalization to initiate infections. HA is translated, glycosylated, and acetylated into the trimeric precursor protein HA0, which is functionally inactive and cannot facilitate endocytosis or macropinocytosis for viral entry into cellsAlthough the complete mechanism of pH-dependent HA stabilization is unknown, environmental stability most likely plays a role. A lower activation pH of H5N1 is associated with greater environmental persistence and may prevent premature extracellular activation in the mammalian respiratory tract. To fully understand this mechanism and the association of HA stability with host range, further studies are needed. However, early findings on HA stability in viral adaptation to human hosts suggest that HA activation pH should be included in the influenza risk assessment framework.Our ability to combat future influenza pandemics lies largely in the therapeutics and vaccines at our disposal. When the next influenza pandemic strain emerges, controlling its spread will be determined by our ability to treat infected patients and prevent others from becoming infected. Although no one-size-fits-all treatments are available, either therapeutically or prophylactically, the development of such treatments is being actively investigated.71. Though these therapeutics have been beneficial, an expanded repertoire of therapeutics and therapeutic classes is needed to effectively combat seasonal and pandemic influenza strains. For this reason, new treatment strategies are being explored and developed. For example, baloxavir targets the endonuclease activity of the influenza acidic polymerase protein, thereby preventing the polymerase from performing its essential cap-snatching function and disrupting influenza gene transcription72. However, mutations that confer resistance to baloxavir have already been detected, specifically, a mutation causing an I38T substitution in the acidic polymerase protein. An isolate with this mutation was reported in a patient treated with baloxavir in Japan73. Although baloxavir is still promising, the emergence of resistant influenza strains must continue to be monitored, and additional therapeutics must be developed.Until recently, only three useful antiviral therapeutics have been licensed for use within the United States: oseltamivir, zanamivir, and peramivir. All three are NA inhibitors and are most effective when taken early in infections74. These vaccines are specific to strains with minimal cross-reactivity and confer 40% to 60% protective efficacy when well matched75. However, limited cross-reactivity decreases the likelihood that seasonal vaccines can protect against pandemic strains. Furthermore, the vaccine production process is not ideal during a pandemic. Isolating a pandemic strain, growing it into a seed stock, propagating it into large quantities, inactivating and purifying it, and then mass distributing it is a lengthy and cumbersome process. Further, if the pandemic strain is highly pathogenic, biosafety regulations mandate their handling in high-containment facilities, limiting manufacturing capacity. It is possible to genetically modify these highly pathogenic viruses to where they can be handled at lower containment, but the associated process and required testing add complications and time to the vaccine production timeline. Two approaches may be used to engineer more effective vaccines: (1) universal influenza vaccines that induce broad cross-reactivity against many influenza subtypes and (2) vaccine platforms that are scalable and can be rapidly produced in response to an emerging novel strain.The best defense against seasonal or pandemic influenza is vaccination. Currently, seasonal vaccines are administered annually; these vaccines contain H1N1, H3N2, and influenza B antigens, with a standardized amount of HA as the primary antigenic target76. Multiple antigenic targets for a more broadly protective vaccine are being explored, all with varying benefits and challenges. One such target is NA. Anti-HA and anti-NA antibodies both correlate with protection from infection and disease, with anti-NA antibodies serving as an independent correlate of protection78.Universal influenza vaccines are currently being investigated79. Anti-HA antibodies typically have limited cross-reactivity because of their specificity to the HA head region, which undergoes regular antigenic drift in its epitopes80. Anti-NA antibodies confer a larger breadth of protection, with polyclonal sera generally having some limited inhibitory activity against NAs of the same subtype to which the sera were generated81. Although NA undergoes antigenic drift at a lower rate than HA, leading to more conserved epitopes across strains and broader protection83, whether this rate is maintained with widespread NA-based vaccinations and thereby focused immune pressure on NA is unclear.Anti-HA antibodies are most strongly associated with preventing infections, whereas anti-NA antibody titers correlate with reduced amount and duration of viral shedding and reduced duration and severity of symptoms during infections84. Anti-HA stalk antibodies may be generated by vaccinating with the HA stalk protein lacking the globular head because the head domain is immunodominant85. However, the removal of the head of the HA protein may result in improper conformation and glycosylation of the stalk and, therefore, the induction of antibodies that do not appropriately recognize wild-type epitopes85. Immunization with chimeric HA proteins composed of the stalk region of the H1 and H3 subtypes matched to novel avian head domains that humans are naïve to may circumvent this limitation, thereby selectively directing the antibody response to the HA stalk while priming anti-avian antibodies86. This strategy has promise, but age-dependent differences in anti-HA stalk immunity, HA stalk escape mutants, and potential autoreactivity of anti-HA stalk antibodies all warrant further exploration before this method of vaccination is widely implemented89.The HA stalk is another antigenic target under investigation. Although the head of HA undergoes antigenic drift at a considerable rate and is highly variable across subtypes, the HA stalk region is more conserved. Broadly neutralizing anti-HA stalk antibodies have been isolated from humans, but these antibodies comprise only a minor fraction of polyclonal sera91.Until a universal influenza vaccine is developed, a more practical approach to pandemic preparedness may entail developing scalable platforms to produce a pandemic vaccine quickly. One such platform is the production of mRNA-based vaccines. Conceptually, mRNA-based vaccines are quite simple; upon the administration of an mRNA encoding the vaccine target, the mRNA is internalized, and the host cell machinery then translates it into a protein that is both displayed on the cell surface for immune recognition and passed through the proteolytic pathway for peptide presentation. These vaccines are advantageous, particularly in the context of pandemic responses, because the production process is rapid and scalable, uses in vitro reactions (rather than embryonated chicken eggs), and can begin as soon as the antigen nucleotide sequence is known. Furthermore, the immunogenicity of mRNA-based vaccines may be adjusted by incorporating synthetic nucleotides and encapsulating them in various delivery vehicles to improve cellular uptake92. The use of mRNA-based vaccines in pandemics is particularly highlighted by the SARS-CoV-2 pandemic. Multiple groups are developing mRNA-based vaccines for SARS-CoV-2. Moderna generated a vaccine with an mRNA encoding the SARS-CoV-2 spike protein mRNA-1273. Only 66 days occurred from the publication of the spike protein sequence to the enrollment of the first phase I clinical trial participant, a record-breaking timeline93. The mRNA-1273 vaccine is currently being tested in a phase III clinical trial, and preliminary results from the phase I trial show robust antibody responses to the vaccine in humans94. Outstanding questions of safety and efficacy remain, but those questions will most likely be answered in the coming months. Because mRNA can be used to produce any antigen, provided the sequence is available, this strategy may be particularly useful for influenza control. This strategy can be leveraged not only for pandemic responses but also for seasonal vaccine production, decreasing the time between strain selection and vaccine administration, thus providing more flexibility for strain selection.Early studies of mRNA-based vaccines for influenza have shown promising protection profiles in mice, ferrets, and swine against a range of influenza strains95. Additionally, Moderna, Johnson & Johnson, and AstraZeneca are investigating vaccines in phase III clinical trials96. All of these treatments were developed and tested preclinically and clinically in collaboration with university and government research institutions, illustrating the value of these collaborations. Maintaining such partnerships will better position us to respond to future pandemics.Although preparation for the next influenza pandemic falls under the purview of government-led surveillance and research, the SARS-CoV-2 pandemic demonstrated the value of industry responses when pandemic-causing viruses emerge. Pharmaceutical companies contribute considerable resources to develop antivirals, including monoclonal antibody therapies from Regeneron and Eli Lilly currently under investigation in phase III clinical trialsA critical component of pandemic management is coherent and cohesive public messaging. The SARS-CoV-2 pandemic highlighted the inadequacy of scientific communication to the public, resulting in mixed messaging and unclear public health guidance. Science is an iterative process; therefore, guidance and conclusions about SARS-CoV-2 pathology and management evolved as information became known. However, scientific conclusions are presented as fact rather than ever-evolving ideas, which leads to public misunderstanding of how the scientific process is conducted and the perception that experts are changing their minds. A critical component of preparing for the next pandemic, influenza or otherwise, will be restoring faith in science. We must be transparent in how we perform science and communicate our findings. Furthermore, we must be united in presenting the conclusions that only sound evidence suggests. As we cope with the effects of the SARS-CoV-2 pandemic and prepare for the next pandemic, we must restore trust in our institutions or risk unmitigated loss of human life and faith in science.97. The SARS-CoV-2 pandemic raises questions about our overall pandemic preparedness and allows some reflection on the overlap of influenza risk assessments and other viruses with pandemic potential. The SARS-CoV-2 pandemic also serves as a model to evaluate our current pandemic workflow for its strengths and weaknesses.Before SARS-CoV-2 emerged in late 2019, most of the influenza research community anticipated that the next pandemic would be caused by a novel influenza strainA vital aspect of pandemic preparedness is surveillance. Influenza surveillance infrastructure and networks continuously monitor circulating strains in animal populations and identify prepandemic strains before they cross the species barrier. The GISRS has identified several prepandemic influenza strains, potentially providing a crucial advantage to prepare before the next influenza pandemic emerges and to contain such prepandemic strains.98. The SARS and MERS outbreaks led to increased research on and surveillance of coronaviruses, but the time and resources to develop these surveillance and response frameworks have not been as extensive as those for influenza. This raises questions about the use of our resources, especially now that we fully understand the pandemic potential of noninfluenza viruses. Can we add to our current influenza risk assessment framework to account for other prepandemic viruses? The expertise and infrastructure developed over the past 70 years for influenza surveillance may serve as a model for coronavirus surveillance, if not directly incorporate it. We should also develop methods to identify other virus families with pandemic potential and consider expanding our surveillance to include these viruses.Such networks were much less developed for SARS-CoV-2 because coronaviruses were identified only in the past two decades as potential pandemic-causing viruses60. Antiviral research has uncovered resistance mechanisms of different influenza strains and optimized therapies100. However, coronavirus-specific antivirals were not developed before the SARS-CoV-2 pandemic101.Another aspect of pandemic preparedness is the ability to respond once a pandemic-causing virus emerges. For influenza, this response has two major categories: vaccines and antivirals. Routine production of seasonal influenza vaccines provides the knowledge and infrastructure needed to quickly develop, test, and produce pandemic vaccines. Prepandemic vaccines are also stockpiled for strains at high risk of causing pandemics49, no coronavirus vaccine platforms were approved before the pandemic. Fortunately, previous and current work on SARS and MERS vaccines accelerated vaccine production, but novel noninfluenza pandemic-causing viruses will require more time for vaccine development. New vaccine platforms, such as mRNA vaccines91, which can be applied to an array of antigens, may decrease the time required for vaccine production in response to pandemics of almost any viral origin48.Although the effort to develop a vaccine for SARS-CoV-2 is unprecedented and will most likely produce multiple vaccines in record time102. Human cases of prepandemic strains are quickly identified and closely monitored to contain any potential widespread outbreaks that may lead to a pandemic. However, when SARS-CoV-2 emerged, diagnostic testing kits required months to develop and widely distribute across the United States, which led to delayed case identification and contact tracing. Such delays are disastrous for containing or mitigating viral transmission. Therefore, the surveillance of all viruses with pandemic potential is crucial for pandemic preparedness. Testing should be developed for all potential classes of pandemic-causing viruses and be easily adapted to identify specific species or strains.Because influenza research has generated many laboratory tests to identify the presence of influenza and its subtypes, we are well prepared to quickly develop testing for novel strains that emerge, especially for prepandemic strains detected in influenza surveillanceThe three key lessons learned from the SARS-CoV-2 pandemic are (1) to continue and enhance the surveillance of prepandemic viruses, (2) to generate a method/platform for rapidly developing and producing pandemic vaccines, and (3) to implement strategies for prompt diagnostic testing responses. Our experience with SARS-CoV-2 will hone our pandemic preparedness workflows, including those for influenza, to better meet the challenges of pandemics in the future.Over the past hundred years, influenza has caused four global pandemics. The risks of influenza viruses are well known, and constant genetic drift and shifts in the influenza genome pose a continual threat to novel emergent strains. The 2009 H1N1 pandemic exposed weaknesses in our pandemic preparedness that have since been improved by increased surveillance, dedicated influenza research, and pandemic risk assessment tools. Determining which strains pose the most risk and laying out systematic workflows for resource allocation and research emphasis are now priorities.Identifying and monitoring prepandemic influenza viruses such as H7N9 suggest that this workflow is functioning well and will give us an advantage should one of these viruses transition from prepandemic to pandemic. Continual routine influenza surveillance in animal populations known to be reservoirs for influenza and understanding the evolution of influenza viruses may reveal important patterns in mutations and recombination events to consider. However, the threat of a completely new strain emerging that is not currently on our radar is always present, highlighted by the SARS-CoV-2 pandemic. We must continually evaluate and update our pandemic workflows to stay ahead of the next deadly strain. With diligence, countless lives may be saved when the next pandemic arrives.


Krabbe Disease (KD) is an autosomal metabolic disorder that affects both the central and peripheral nervous systems. It is caused by a functional deficiency of the lysosomal enzyme, galactocerebrosidase (GALC), resulting in an accumulation of the toxic metabolite, psychosine. Psychosine accumulation affects many different cellular pathways, leading to severe demyelination. Although there is currently no effective therapy for Krabbe disease, recent gene therapy-based approaches in animal models have indicated a promising outlook for clinical treatment. This review highlights recent findings in the pathogenesis of Krabbe disease, and evaluates AAV-based gene therapy as a promising strategy for treating this devastating pediatric disease. Krabbe Disease (KD) is a devastating pediatric lysosomal storage disorder that affects both the central and peripheral nervous systems. It was first described in detail in 1916 by a Danish neurologist, Knud Krabbe who also lends his name to the disease  Figure .In infants, KD is generally diagnosed by 4 months of age, with death occurring within 2–3 years if left untreated, largely due to organ failure because of severe demyelination .This review describes recent findings that provide insights into pathological mechanisms of KD and evaluates therapeutic avenues thus far explored in treating KD, with a focus on AAV-based gene therapies.In humans, the GALC gene is located on chromosome 14 (14q31.3) and has 17 exons . The GALThe structure of GALC includes an overall fold that comprises three domains: a central triosephosphate isomerase (TIM) barrel, a β-sandwich domain, and a lectin domain that make up the GALC substrate-binding site . While tGALC catalyzes the synthesis of galactose from glycosphingolipids like galactocerebroside and galactosylsphingosine . In the Psychosine accumulation, however, is not unique to deficiencies in GALC. A small glycoprotein, Saposin A (SapA) enhances GALC activity through the formation of a heterodimeric complex with GALC where it helps to orient substrates for effective cleavage . AlthougMultiple epidemiological studies have been conducted on KD but the data has been difficult to reconcile due to the use of different study populations and varying methods of estimation . The accTo date, at least 147 mutations in GALC have been documented, with 80 of them considered to be “severe” due to their impact on GALC activity . MutatioGeographical differences in disease-causing mutations have also been reported. For instance, the missense mutations that are frequently found in KD patients in China  are different than those found in Europe and Japan . In JapaIn all cases and across species, pathogenic mutations in GALC are associated with a dramatic increase in levels of psychosine within the central nervous system (CNS) and peripheral nervous system (PNS). Although psychosine levels are a better biomarker for clinical diagnosis compared with GALC activity, there is some debate concerning its use as a presymptomatic biomarker. Hypoxanthine, a metabolite in the purine nucleotide salvage pathway is elevated in Twitcher mice prior to onset of KD symptoms , and mayKD is unusual among other rare diseases in having a range of animal models available that show reduced GALC activity. These animal models are listed by species and include cats, mice, dogs, sheep, and non-human primates, among which mice have been the most thoroughly characterized in terms of disease progression .Much of the current knowledge on KD was gleaned from characterization of the Twitcher mouse, which arose due to a spontaneous mutation (W339X) at Jackson Labs in 1976. The mouse and human GALC proteins bear 83% identity . The E13A canine model of KD is available on the white terrier (Cairn and West Highland) background. The human and canine versions of GALC share 87% sequence similarity, though the 3' UTR in dogs is 1 kb shorter . Mutant A rhesus macaque model with a two base deletion at positions 387–388 in exon 4 resulting in a frameshift in GALC and subsequent degradation of the resulting protein was first described in 1989 , 57. CliThe common theme in all the animal models and human patients is the psychosine accumulation due to the reduction of functional GALC that results in disease symptoms associated with KD. Preclinical studies, largely centered on the Twitcher mouse, have demonstrated that disease symptoms can be reversed through various therapeutic efforts, indicating the appropriateness of these animal models for developing future therapies.The psychosine hypothesis was proposed nearly 50 years ago and states that a loss in GALC activity leads to psychosine accumulation, which in turn causes the neural pathology seen in KD . The celOligodendrocytes and Schwann cells are particularly sensitive to psychosine-induced apoptosis during the initial stages of myelination and maturation . AlthougPsychosine-mediated loss of oligodendrocytes and Schwann cells leads to widespread demyelination within both the CNS and PNS, and impacts non-neuronal organs as well. For example, demyelination in the spinal cord leads to thymic atrophy, with significant subsequent decreases in the number of CD4 and CD8 T cells in the Twitcher mice , though ex vivo organotypic cerebellar slice culture model , fingolimod, was similarly found to reduce psychosine-induced demyelination in both organotypic cerebellar slice cultures as well as in Twitcher mice—though not to wild-type levels  reduced psychosine-induced demyelination in an re model . This sue levels , 70, 87.Motor dysfunction and muscle weakness are common symptoms among KD patients that are also recapitulated in Twitcher mice, in which psychosine begins accumulating in the muscles soon after birth and increases over time . In the A comparative analysis of the Twitcher and wild-type mice nervous systems by Tandem Mass Tag Spectrometry based approaches indicated differences in more than 400 protein groups . ExtraceThere is a growing appreciation for the role of dysfunctional microglia and astrocytes in demyelination in KD. As opposed to the psychosine hypothesis, the microglial hypothesis suggests a re-evaluation of the order in which the disease cascade occurs, namely that neuroinflammatory processes might precede disease symptoms and demyelination through the activation of astrocytes first, which then activate microglia in an abnormal manner . Post-mohigh/CD206low) assist in myelin degradation in the PNS. It is likely that the recruited GALC−/− macrophages have impaired phagocytosis, accumulate lysosomes and myelin phagosomes and assume an inflammatory phenotype and a globoid appearance. Further analysis showed that treating bone marrow derived macrophages with galactosylceramide  but not psychosine recapitulated the phenotype in vitro. These induced globoid cells had reduced CD206 expression and elevated TNF-α and IL-1β cytokine expression profiles -positive staining, and are often observed early in the progression of the disease—even prior to demyelination or other symptoms. In conditional GALC knockout mice, macrophages (Ly6profiles . A studyprofiles . A similCeramide metabolism in the lysosomes may contribute to increased misfolding and aggregation of proteins . OligodeTherapeutic approaches to treat KD symptoms over the past 20 years are described in the sections below.Hematopoietic stem cell transplantation (HSCT) is currently the gold standard treatment for Krabbe disease. A seminal study in Twitcher mice demonstrated that HSCT administration following lethal irradiation in 10 day old mice could extend their survival from 40 to 80 days. Minor improvements were seen in gait, foraging, grooming, and body weight, but only when HSCT was administered at an early time point . In a suIn human KD patients, current clinical guidelines for applying HSCT differ for early and late onset KD. For early onset KD, HSCT is recommended in infants less than a month old who are asymptomatic. According to a recent study, although infants receiving HSCT prior to 31 days showed no significant difference in 5 and 10 year survival compared to infants receiving HSCT after 31 days, mobility and communication were in fact better in infants receiving HSCT earlier . In latede novo GALC expression in hematopoietic stem cells is toxic . Another challenge is that for HSCT to be successful there needs to be presymptomatic intervention in the early onset KD patients. In this regard, both accurate diagnoses of children with KD as well as pre-symptomatic detection are important obstacles to be surmounted. One potential reason for the limited therapeutic efficacy of bone marrow transplantation seen in KD patients at symptomatic stages of the disease could be due to underlying defects in the BM vascular niche as a result of GALC deficiency . There iis toxic . Howeveris toxic .Propelled by the success of enzyme replacement therapy in other lysosomal storage disorders including Fabry's disease and Gaucher's disease, a research group administered recombinant mouse GALC to Twitcher mice at day P10 intraperitoneally, with subsequent injections every other day . The obsReducing psychosine concentrations in tissues could be another strategy for ameliorating KD symptoms. One attempt used L-cycloserine, which deactivates serine palmitoyltransferase, an enzyme several steps upstream of the biochemical synthesis of psychosine , 112. A ex vivo gene therapy strategies in which long-term transduction of hematopoieitic stem cells is needed, LVs are recognized as better vectors than AAVs, which are primarily non-integrative unless combined with genome integration approaches , these approaches have also included adenoviral and retroviral vectors –118. In proaches . Howeverl models . Of the l models . While tTwo groups led the way in exploring AAV-based treatment for Krabbe disease, highlighting the importance of AAV serotype in ameliorating symptoms , 53. TheAdministration of AAV1 at a total dose of 3.0e13 vgs/kg in the brain parenchyma and ventricles  increased the lifespan of Twitcher mice by 2–3 weeks, showing high GALC expression in the hippocampus and olfactory bulb but undetectable levels in the cerebellum . The micTwo follow-up studies published in 2011 by the Sands group used AAV5 in Twitcher mice, though the route of administration for each was different. AAV5 was administered either IC alone at a dose of approximately 7.20e12 vgs/kg   or it waFrom 2012 to 2020, seven different studies used either AAVrh10 or AAV9 in Twitcher mice as therapeutic preclinical efforts for KD . One of The Wenger laboratory published two studies in Twitcher mice using AAVrh10 administered either as a combination of IC, ICV, and intravascular (IV) injection or IV alone , 123. InTwo additional studies administered AAV9 to Twitcher mice either IV in combination with ICV and IT , or IV aFinally, most recently, two studies in the canine model of KD , 55 atteThe importance of using the right dose was recently demonstrated in three different studies . In the Twitcher study, 1 day post-BMT treatment, Twitcher mice were injected IV with different doses of AAVrh10 . The 1X dose of AAVrh10 was equivalent to 4e13 vgs/kg, and a dose-dependent increase in lifespan was reported, with the exception of the 4X dose . Interesin vitro cultures, AAV1 and AAV9 both strongly transduce neurons; however, in vivo, following brain injections, AAV1 transduction of neurons in the cortex is low when compared to transduction using AAV9  or P2  , 35. BotTwo studies by Bradbury et al. in the canine model also indicate that the earlier the intervention, the better the outcome , 55. In HSCT has been established as the “standard of care” for pre-symptomatic Krabbe patients because it showed the most effectiveness in slowing the course of the disease and preserving cognitive function . PatientAs one example, results of an AAV/BMT combination therapy in the murine MSP IIIB model indicate that a combined approach does not always result in synergistically improved efficacy. This study surprisingly demonstrated even lower efficacy in the combination therapy treated group than the AAV alone treatment, presumably due to antagonistic effects of the two approaches . In addiGiven that HSCT/BMT is the current gold standard for treating Krabbe disease, nearly half of all the AAV studies investigated the combined efficacy of AAV and BMT in prolonging survival in Twitcher mice. Studies combining AAV with BMT can be difficult to interpret in terms of the observed longevity and overall improvements due to the inherent variability in BMT-mediated engraftment. BMT involves the use of radiation or chemotherapy to eliminate myeloid cells, and depending on the intensity of radiation used (myeloablative or myeloreductive conditioning) or the choice of chemotherapeutic agent (such as busulfan or cyclophosphamide), may cause unintended cellular changes. For instance, Reddy et al.  reported137Cs source; this was followed by an IV injection of unfractionated bone marrow suspension. The combination therapy in this study extended the lifespan in Twitcher mice by an additional 50 days . Additionally, there was decreased astrogliosis, the mice seemed to maintain their weight longer, and showed marked improvement in neuromuscular coordination. Less improvement was observed in neuromuscular strength despite the combined therapy. It is likely that BMT was either providing supplemental GALC for cross-correction  or was reducing overall neuroinflammation. Another study showed that BMT without any immunosuppressive preconditioning had a negligible effect on GALC activity or psychosine levels in the brain or spinal cord (Combining AAV5 (IC) and BMT (myeloreductive) showed a profound effect on multiple aspects of KD in Twitcher mice . The regnal cord . An imponal cord , indicatRafi et al. , 124 comDespite their promise, however, AAVs as vectors for gene therapy are not without their limitations. A key limitation of using AAVs is defining the dose that is both safe and efficacious in human patients, particularly when comparable doses in preclinical models do not show significant side effects. This limitation is particularly relevant for a disease such as Krabbe disease, where high levels of GALC seem to be critical in extending survival as well as neuromuscular function. Furthermore, even if high levels of GALC are achieved at a clinically tolerable dose, it is not known whether AAV-mediated GALC overexpression could result in gain of toxic function. Long term AAV9-mediated overexpression of survival motor neuron (SMN) protein, a protein associated with spinal muscular atrophy, was recently shown to cause toxicity in the sensorimotor circuit in mice .Although less ideal than a single-dose therapeutic, one could envision maintaining adequate levels of GALC across both the CNS and PNS through recurring AAV injections. However, another limitation of using AAVs is that multiple injections of the same virus over time are not possible due to neutralizing antibodies. Nevertheless, the use of substantially different clades of AAVs to evade the immune response could still be a viable strategy. Additional strategies for evading the immune response include using decoy empty capsids to saturate circulating neutralizing antibodies, generating novel viral capsids through directed evolution, and chemically modifying viral capsids , 138. Fu14 vector genomes per kilogram bodyweight (vg/kg) administered to the patients. Assuming the patient body weight to be around 30 lbs, they would have received a dose in excess of 4 × 1015 vgs. Compounding this is the fact that the patients had pre-existing hepatobiliary disease that could have exacerbated toxicity given that most AAVs traffic to the liver irrespective of intended target organ or route of delivery. This data strongly directs setting an upper limit on intrathecal and intravenous AAV dosing though it is still unknown if anti-inflammatory or immune suppressive medications would be helpful in mitigation. In the absence of detailed reports on the clinical trials, it is imperative to proceed with caution when extremely high doses are being considered for therapy.Although rAAVs have traditionally been categorized as relatively non-immunogenic, two recent studies have reported the occurrence of neuroinflammation in the dorsal root ganglia of the spinal cord and an acute syndrome of thrombocytopenia, hepatic and renal toxicity , 140. BoAdditionally, AAV gene therapy in the murine MPS VII model was implicated in hepatocellular carcinoma (HCC) as the malignant cells showed integrated AAV sequences . Logan eex vivo as well as in vivo (using a humanized liver mouse model)  are all variables that require further investigation.Similarly, clonal insertion of wild-type AAV2 genome sequences in human HCC liver biopsies have also raised concerns about the potential genotoxicity of AAV based gene therapy . One impe model) . This stIn conclusion, we would like to highlight the elegant argument that WT AAV insertions in hepatocellular carcinoma do not inform debate over genotoxicity risk of vectorized AAV . InteresGiven the broad expression of GALC in the nervous system, a therapeutic that rescues GALC locally is likely to have limited success. This hypothesis is borne out by multiple studies where localized GALC rescue failed to improve neuromuscular function , 31, 53.In mice, myelination in the spinal cord begins at birth and continues till P60, therefore delaying treatment would allow psychosine to begin impacting early development of cells in the PNS . In suppin vivo without increasing viral dose. For instance, engineering potent transgene cassettes through codon-optimization, selection of promoter, polyA termination sequences, post-regulatory transcriptional elements, and stronger signal secretion sequences are examples of features to consider. In addition, molecular engineering of the GALC enzyme can improve its secretion and uptake efficiency for subsequent cross-correction—a strategy that could be combined with gene therapy approaches. For instance, chimeric GALC with the HIV Tat protein transduction domain was shown to have a 6-fold increase in cross-correction of GALC in vitro  . What waBMT may tone down neuroinflammation based on the status of inflammatory markers such as IL-6, TNFα, MIP-1β, and MCP-1 in the brain . HoweverAAV gene therapies have potentially ushered a new frontier in our ability to tackle dozens of rare diseases, including lysosomal storage disorders such as Krabbe Disease. For KD, two clinical trials using gene therapy are currently underway (both in Phase 1/2). The first of the two is PBKR03 (PassageBio), a GALC transgene in a AAVhu68 vector (AAV9 variant) delivered through the ICM route (NCT04771416). This study will have both a low and high dose in each of two age groups (1–4 and 4–9 months). The second study combines HSCT with AAV; the molecule, FBX-101 (Forge Biologics), is a GALC transgene packaged within an AAVrh10 vector and delivered IV at either a low or high dose following HSCT (NCT04693598). The results from these clinical trials, combined with results from clinical trials of other LSDs such as Metachromatic leukodystrophy (MLD), will be instrumental in helping us refine our approach to these debilitating diseases in the future. For instance, a recently concluded clinical trial using AAVrh10 in 4 children who had pre- or early-symptomatic MLD found that all treated children continued to develop symptoms associated with MLD (NCT01801709). While disappointing, we can speculate on some of the reasons that might be associated with the lack of efficacy observed in the trial. The Arylsulfatase A gene, associated with MLD, is quite similar to GALC in terms of its cellular distribution and function—both enzymes are lysosomal enzymes that process substrates important in myelin homeostasis . Not surIn addition to KD, clinical trials using AAV for treating several others LSDs are currently underway. These diseases include Fabry disease (FLT190 and 4D-310), late onset Pompe disease , Mucopolysaccharidosis Type I (SB-318), Mucopolysaccharidosis Type IIIA (LYS-SAF302), Mucopolysaccharidosis Type IIIB (rAAV2/5-hNAGLU), Mucopolysaccharidosis Type VI (AAV2/8.TBG.hARSB), Gangliosidosis (Tay Sachs and/or Sandhoff disease) , and Gaucher disease Type II (PR001). A more comprehensive overview of AAV-based clinical trials over the past several years was covered recently .In summary, the advent of AAV based gene therapy has significantly expanded the realms of possibility for drug development specifically for autosomal recessive rare diseases like Krabbe disease. Additionally, the Human Genome Project and recent genome-wide association studies have made the near impossible task of annotating the human genome possible and made available the abundant knowledge that helps associate diseases with causal genetic mutations. With increasing exploration of AAVs for gene therapy, the future holds great promise in improving the lives of patients living with debilitating diseases.GN and SA co-wrote and edited the manuscript. RC created GN, RC, FE, and SA were employed by Novartis Institutes for BioMedical Research at the time of research.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.


Nrf2-knockout mice revealed that NRF2 dysfunction is intimately involved in the pathogenesis of various human diseases. Multiple single nucleotide polymorphisms (SNPs) have been detected in human NRF2 locus. An NRF2 gene SNP , located in the upstream promoter region, affects the transcriptional level of NRF2 and thereby the protein level and downstream gene expression. It has been reported that the SNP-617 is associated with various diseases. The onset and exacerbation of the diseases are regulated by genetic predisposition and environmental factors; some people live in the air-polluted environment but are not affected and remain healthy, suggesting the presence of individual differences in the susceptibility to air pollutants. NRF2 polymorphisms may also be associated with the fetal effects of fine particles exposure. Screening high-risk pregnant women genetically susceptible to oxidative stress and prevention by antioxidant interventions to protect fetal development in air-polluted areas should be considered. This article reviews the recent advances in our understanding of the fetal health effects of fine particles and describes potential chemoprevention via the NRF2 pathway to prevent the developmental and reproductive toxicity of fine particles.Air pollution is associated with significant adverse health effects. Recent studies support the idea that inhalation of fine particles can instigate extrapulmonary effects on the cardiovascular system through several pathways. The systemic transfer of ultrafine particles (UFPs) or soluble particle components  is of particular concern. An integral role of reactive oxygen species (ROS)-dependent pathways has been suggested in systemic inflammatory responses and vascular dysfunction at the molecular level. Accumulating lines of evidence suggest that fine particles affect fetal development, giving rise to low birth weight and a reduction in fetal growth, and also affect the immune, cardiovascular, and central nervous systems. Oxidative stress plays an important role in fine particles toxicity; pre-treatment with antioxidants partially suppresses the developmental toxicity of fine particles. On the other hand, Nuclear factor erythroid-derived 2-like 2 (Nfe2l2), also known as NRF2, is a transcription factor essential for inducible and/or constitutive expression of phase II and antioxidant enzymes. Studies using  An integral role of reactive oxygen species (ROS)-dependent pathways has been suggested in systemic pro-inflammatory responses and vascular dysfunction at the molecular level  . Recent diseases , arteriodiseases , neurolodiseases , diabetediseases , fetal ddiseases  , and Nrf2 knockout mice are highly sensitive to oxidative stress caused by DE , located in the upstream promoter region, affects the transcriptional level of NRF2 and thereby the protein level and downstream gene expression , also known as NRF2, is a transcription factor essential for the inducible and/or constitutive expression of phase II and antioxidant enzymes . Recent ed by DE . Most DEed by DE . In thispression . It has pression . It is svia the NRF2 pathway to prevent the developmental and reproductive toxicity of fine particulates.Many studies have suggested that fine particulates air pollution is related to developmental and reproductive . The imp2.5 (particulate matter of diameter <2.5 μm) and UFPs (particulate matter of diameter <0.1 μm). These particles are derived from various sources and by various mechanisms as shown in Particulate matter is a complex mixture of organic and inorganic chemicals, including metals and particulates , and is Several studies have shown that UFPs are more toxic than larger particles . UFPs arDE emissions, are a major source of UFPs in urban environments, and it is these particles that have the capacity to induce the most significant health effects . Previou2.5 affects development and reproduction as have been documented in epidemiological reports  release of proinflammatory mediators from lung cells, 2) affect autonomic nervous system balance by particle interactions with lung receptors or nerves, and 3) translocation of UFPs and soluble particle components into the systemic circulation. Therefore, inhaled UFPs can reach the alveolar region and extrapulmonary organs -1. If these enzymes fail to neutralize the effects of ROS, proinflammatory effects constitute a second tier or superimposed level of oxidative stress. The final tier or superimposed level of oxidative stress is cytotoxicity, including the initiation of programmed cell death.  located in the upstream promoter region affects the transcriptional level of NRF2 and thus the protein level and downstream gene expression. NRF2 rSNP-617 exhibit significantly decreased NRF2 gene expression. SNP-617 was found to be associated with a higher risk of oxidant-induced acute lung injury in humans  have been identified in human NRF2 . The NRFn humans . Individg injury , Parkinsg injury , diabeteg injury , chronicg injury , breast g injury , cerebrog injury , and vasg injury . The pre smokers , and is  smokers   is a useful biomarker for clinical diagnosis  biomarker; personalized prevention of UFPs-induced oxidative stress at the developmental and reproductive stages, by antioxidant interventions based on NRF2 polymorphisms, may be feasible. Activation of NRF2 represses pro-inflammatory reactions induced by oxidative stress and ameliorates various inflammatory diseases. Many NRF2-activating compounds have been developed or are currently being refined (The onset and exacerbation of diseases are regulated by genetic predisposition and environmental factors; some people living in an air-polluted environment are not affected by it and remain healthy. This suggests that there are individual differences in the susceptibility to air pollutants. Many reports mentioned above suggest that iagnosis . In air- refined . Figure This review is a literature review and has not been statistically analyzed. Regarding the health effects of fine particulates, the effects of component analysis have not been analyzed. Although there are these limits, the information summarized in this report highlights the importance of the NRF2-antioxidant pathway and proposes a hypothesis for preventing developmental and reproductive processes implicated in oxidative stress caused by exposure to fine particles in the atmosphere.


NFE2L2 gene and is a member of the cap ‘n’ collar subfamily of basic region leucine zipper transcription factors. Under normal physiological conditions, Nrf2 remains bound to Kelch-like ECH-associated protein 1 in the cytoplasm that ultimately leads to proteasomal degradation. During peripheral neuropathy, Nrf2 can translocate to the nucleus, where it heterodimerizes with muscle aponeurosis fibromatosis proteins and binds to antioxidant response elements (AREs). It is becoming increasingly clear that the Nrf2 interaction with ARE leads to the transcription of several antioxidative enzymes that can ameliorate neuropathy and neuropathic pain in rodent models. Current evidence indicates that the antinociceptive effects of Nrf2 occur via reducing oxidative stress, neuroinflammation, and mitochondrial dysfunction. Here, we will summarize the preclinical evidence supporting the role of Nrf2 signaling pathways and Nrf2 inducers in alleviating peripheral neuropathic pain.Oxidative stress, resulting from an imbalance between the formation of damaging free radicals and availability of protective antioxidants, can contribute to peripheral neuropathic pain conditions. Reactive oxygen and nitrogen species, as well as products of the mitochondrial metabolism such as superoxide anions, hydrogen peroxide, and hydroxyl radicals, are common free radicals. Nuclear factor erythroid 2 (NFE2)-related factor 2 (Nrf2) is a transcription factor encoded by the  In 1994, the International Association for the Study of Pain defined neuropathic pain as “pain initiated or caused by a primary lesion or dysfunction in the nervous system”. This definition was widely criticized due to the ambiguity of the term “dysfunction”. Some non-neurological diseases, such as fibromyalgia or irritable bowel syndrome that involve the central dysfunctions of the nociceptive system, could also fall under the definition of neuropathic pain. Therefore, a new definition was proposed in 2008 defining neuropathic pain as “initiated or caused by a primary lesion or dysfunction in the nervous system” , while iDamage or injury to the peripheral nerves is referred to as peripheral neuropathic pain . PeripheOne mechanism underlying the development and maintenance of peripheral neuropathic pain is oxidative stress, which is an imbalance between the production of damaging free radicals and protective antioxidants, and unchecked free radicals begin damaging cells membranes, proteins, and DNA . The mosOnce neuropathic pain has developed, it is a significant challenge to therapeutically manage. First-line treatments are often anticonvulsants and antidepressants; however, these are not effective for all neuropathic pain patients and come with unwanted negative side effects that make their use suboptimal. Thus, preclinical research is engaged in the discovery of novel neuropathic pain targets to inform the development of next-generation therapeutics to treat peripheral neuropathic pain with better efficacy and lesser side effects . TargetiNFE2L2 gene and a member of the cap ‘n’ collar subfamily of basic region leucine zipper (bZip) transcription factors. Nrf2 contains a bZip domain at the C-terminus that is responsible for the formation of heterodimers with other bZip proteins, such as small muscle aponeurosis fibromatosis (MAF) K, G, and F . T. Tcis-cr+-dependent histone deacetylases that are also involved in protein acetylation and deacetylation besides histone deacetylation ..145].Kim and Chung developed the classical spinal nerve libation model (SNL) model in which the unilateral L5 and/or L6 spinal nerves of rodents at a distal location from the dorsal root ganglion were tightly ligated . This moThe preclinical studies presented in this review highlight the therapeutic potential of Nrf2 inducers for alleviating neuropathic pain in several neuropathy conditions modeled in rodents. Collectively, the conclusion of these studies is that Nrf2 signaling activation leads to alleviation of ROS-activated oxidative stress, neuroinflammation, and mitochondrial dysfunction , osteoarthritis (NCT04638387), and cancer . Data coming out of these clinical trials indicate that Nrf2 inducers possess good efficacy, safety, and tolerability, and some of the Nrf2 inducers and/or signaling pathway are used and/or studied in clinical trials ,168,169.


Although various epithelia are uniquely structured with specific function, prevalent allergic-type epithelial diseases in children potentially have common or parallel disease processes. These include inflammation and immune response dysregulation stemming from atypical epithelial barrier function and integrity. Two diseases where aetiology and pathogenesis are potentially linked to epithelial vulnerabilities include Paediatric Asthma and Eosinophilic Oesophagitis (EoE). For example, rhinovirus C (RV-C) is a known risk factor for paediatric asthma development and is known to disrupt respiratory epithelial barrier function causing acute inflammation. In addition, EoE, a prevalent atopic condition of the oesophageal epithelium, is characterised by similar innate immune and epithelial responses to viral injury. This review examines the current literature and identifies the gaps in the field defining viral-induced effects on a vulnerable respiratory epithelium and resulting chronic inflammation, drawing from knowledge generated in acute wheezing illness, paediatric asthma and EoE. Besides highlighting the importance of epithelial structure and barrier function in allergic disease pathogenesis regardless of specific epithelial sub-types, this review focuses on the importance of examining other parallel allergic-type disease processes that may uncover commonalities driving disease pathogenesis. This in turn may be beneficial in the development of common therapeutics for current clinical management and disease prevention in the future.The epithelium is integral to the protection of many different biological systems and for the maintenance of biochemical homeostasis. Emerging evidence suggests that particular children have epithelial vulnerabilities leading to dysregulated barrier function and integrity, that resultantly contributes to disease pathogenesis. These epithelial vulnerabilities likely develop  Asthma is a heterogeneous, multifaceted respiratory disorder often emerging in early childhood –4. It isChildren exhibiting severe symptoms or respiratory distress caused by several of these pathologies may be hospitalised as their illness requires intervention –20 and iTo address this further, Oksel et al.  used latCollectively, numerous heterogeneous risk factors contribute towards asthma susceptibility as well as wheezing illness in children. With regards to asthma development, parental history of asthma, host genetics, wheezing illnesses and wheeze phenotype all play a pivotal role as well as exposure to allergens, pathogens and exogenous particulates , 29–31. The airway epithelium is a pseudostratified structure whose complex functions provides protection through structural, mucociliary and innate-immunological barriers. These barriers work synergistically in maintaining epithelial homeostasis and providing a dynamic response to pathogens, allergens and particulate matter. As reviewed by Knight and Holgate  and alsovia a myriad of junctional complexes  have been associated with increased MUC5AC expression during respiratory viral illnesses  and the low density lipoprotein receptor (LDLR) respectively (rs6967330 A/G (Cys529Tyr) is located in the cadherin related family member 3 (CDHR3), the viral receptor for RV-C, the species most prevalent in paediatric wheezing illness  acute wheezing cohort . They foSMAD3, that encodes for the cell signalling protein SMAD3, as well as DDO and METTL24, genes that encode for peroxisomal flavoprotein and methyltransferase 24 respectively  and its associated medical stresses such as oxygenation, steroids use and mechanical ventilation . Hillas 60 hours . In cont60 hours . Moreoverm birth , 122, harm birth  with furin vitro cell cultures with exogenous fibronectin only partially restored wound repair  is the most common virus found in children, particularly in those admitted to hospital with wheezing , 139. AsThe inter-relationships between RV infections, epithelial vulnerability and why some children have persistent wheeze, leading to the development of asthma, is still largely unclear , 151. RhThe effect of RV on the paediatric airway epithelium - and its contribution to wheezing illness and eventual asthma development - is not yet fully understood. One study suggests that receptor specificity determined by the major or minor genotype of RV- A and RV-B can influence host response to the virus . Other sSTAT1) signalling pathway. STAT1 is typically activated in response to RV in epithelial cells and activates a signalling cascade that promotes the expression of anti-viral genes  and a consequent increase in permeability when infected with RV-A1 , leading to fibroblastic repair rather than normal epithelial repair processes , exhibits similar disease characteristics to paediatric asthma including atypical epithelial barrier integrity and dysregulated innate and adaptive immunity . AlthougEosinophilic Oesophagitis (EoE) is an increasingly prevalent atopic condition defined by eosinophilia of at least 15 eosinophils per high power field (eos/hpf) and symptoms of oesophageal dysfunction , 195. AsFibrosis is responsible for EoE symptoms such as dysphagia and food impaction. However, children tend to present with less specific symptoms related with oesophageal inflammation and oesophageal dysmotility such as feeding difficulties, gagging and vomiting , 197. CoCCL26) on 7q11.23, thymic stromal lymphopoietin (TSLP) on 5q22, and Toll-like receptor 3 (TLR3) on 4q35.1, have been connected to EoE by several independent groups (TLR3 SNP (CC or CG at rs3775292), are found more frequently in allergic EoE patients  of epithelial genes promoting Th2 inflammation such as eotaxin-3  on 19q13 has been connected with EoE , 227. EoCAPN14) on chr2p23.1, Filaggrin (FLG) on 1q21 and epithelial serine protease inhibitor SPINK5 on 5q32. Like other atopic diseases such as asthma or eczema, genetic polymorphisms may predispose the epithelium to be more permeable and more vulnerable to damage by the Th2 inflammation. This is thought to increase the amount of contact between antigens and the immune system, and predispose to inappropriate loss of tolerance for these antigens (CAPN14) is a protease in the calpain family that is expressed at the highest level in the esophageal epithelium and upper GI tract. IL-13 induces CAPN14 expression in the esophageal epithelium with consequent loss of barrier function (Inflammation is also known to lead to epithelial barrier dysfunction and genes that build the epithelial barrier have been implicated in EoE pathogenesis. These include calpain 14 (antigens . Calpainfunction . Nethertfunction , 233.CAPN14 is highly expressed in the oesophagus and pharyngeal cells (Although at the moment there is no data that RV or any other virus may induce the epithelium changes that eventually lead to food sensitisation and EoE, it is possible that viruses such as herpes virus or others could act similarly to RV in initiating EoE after infection directly or indirectly through upper airways infection. Indeed, the common cold can involve one or all the sinuses, nasopharynx, oropharynx and larynx. EoE is commonly associated with Eosinophilic Laryngitis and Aerodigestive Dysfunction in children . At the al cells . In asthal cells , or creaal cells , 237. ThIt is inherently clear that RV infection is of critical importance in wheezing illnesses leading to asthma development and diagnosis. RV has been shown to have a dysregulatory effect on the barrier function and integrity of the paediatric airway epithelium, particularly in asthmatic children who may already have intrinsic epithelial vulnerabilities. Many of the effects of RV are related to the child’s epithelial host response which is unable to effectively fight the virus . MoreoveThis review highlights the growing significance and clinical relevance of an innately vulnerable epithelium in different prevalent allergic-type epithelial diseases, and the effect of viral infection, particularly RV-C infection on airway epithelial barrier function and integrity in paediatric asthma, and viral and injury-induced effects on the oesophageal epithelium in EoE. In asthma specifically, it is postulated that innate epithelial vulnerability further potentiates RV-C infection, due to dysregulated host response to the virus. In addition, RV-C may further potentiate this vulnerability and barrier dysregulation compared to other RV species, leading to a persistent cycle of infection. Furthermore, it is suggested that consecutive or repeated insults to the airway of a child with a vulnerable epithelium culminates in persistent wheezing illnesses and eventually asthma development and diagnosis. This is one current research focus. Subsequently, once this has been identified, therapeutic development pipelines can be developed and potentially be extrapolated to and used in other allergic diseases such as EoE. It is important to compare diseases such as paediatric asthma and EoE as they have parallel disease pathologies and there are lessons that can be learnt from identifying commonalities such as epithelial vulnerabilities in children.RW is the Primary Author of this manuscript. KL, IL, AC, and AK all contributed in equal proportions and have joint Senior Authorship over this manuscript. All authors contributed to the article and approved the submitted version.AK is a Rothwell Family Fellow. Dr Ingrid Laing is funded by NHMRC App#1147630.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.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 24th edition of the annual NAT conference  and the 4th edition of the biennial LabEx IGO meeting (Immunotherapy Graft Oncology) were held jointly around a common theme: “New horizons in immunotherapy”, on May 31st and June 1st 2021 to highlight new findings in the fields of transplantation, autoimmunity and cancer. The 24th edition of the annual NAT conference  and the 4th edition of the biennial LabEx IGO meeting (Immunotherapy Graft Oncology) were held jointly and entirely online, on May 31st and June 1st 2021. For this edition, the two events decided to join their forces around a common theme: “New horizons in immunotherapy”, in order to offer an overview of recent advances in immunology, transplantation, autoimmunity and cancer. This NAT and IGO joint meeting have thus revealed the most advanced concepts and work of researchers and clinicians in the field of immunotherapies.The conference program has featured 4 sessions with the 2 first sessions devoted to bioreagents and advanced cell therapy to control immune responses in the context of immunotherapy in transplantation, autoimmunity and cancer. The 3rd session was devoted to understanding new horizons in immunotherapy and the 4th session focused on tissue microenvironment and its role in the development of innovative immunotherapies.The first session was chaired by Dr Paolo Dellabona  and Dr Carole Guillonneau (Centre de Recherche en Transplantation et Immunologie (CRTI), Nantes, France) and presented an overview of different strategies to regulate the immune system in cancer and transplantation through the use of bioreagents.Dr Eliane Piaggio, research director of the translational team at Institut Curie in Paris, works since many years on IL-2 based immunotherapies for disengaging Tregs. IL-2 based immunotherapy is a well-known strategy, used for decades, especially for cancer treatment but with complex issues. In fact, this cytokine is hard to use due to its systemic toxicity at high doses (e.g. cytokine storm) and its lack of specificity between T cell subtypes (effector T cells vs regulatory T cells) at low doses. Dr Piaggo’s team goal was to selectively target the regulatory component of the immune response in the tumoral microenvironment by using IL-2/anti-IL-2 mAb complexes (IL-2Cx). Depending on the epitope recognized by the anti-IL-2 mAb, this immune complex can preferentially act on Tregs, to control autoimmunity, or effector T cells, to induce anti-tumor response  in France and Scientific Director of the company Innate Pharma. For many years, immunomodulatory approaches have focused on immune checkpoint inhibitors or activating bispecific antibodies. This has led to unprecedented successes. However, many patients are not eligible for such strategies and resistance frequently appears. Many strategies target T cells but innate immunity plays an important role in cancer immunity. More specifically, NK cells, capable of direct cytotoxicity and secretion of immunomodulatory cytokines represent an attractive target  presented an antitumor virotherapy strategy based on oncolytic activity of attenuated Measles virus and defect in the type I interferon response in cancer. Oncolytic viruses represent interesting tools because they infect dividing cells, such as tumor cells, and their replication leads to immune cell death of infected cells which stimulates anti-tumor responses. From an immunotherapeutic perspective, non-pathogenic attenuated Measles Virus (MV) can be used thanks to their spontaneous oncolytic activity and use of CD46 as entry receptor which is often overexpressed by tumor cells, especially in malignant pleural mesothelioma (MPM). After establishing a primary cell line bank of MPM, they showed that tumor cells were not all sensible to oncolytic activity of MV. Susceptibility to infection is due to a defect of intracellular type I interferon pathway in infected cells, mainly involved in antiviral response  and Dr Nathalie Labarrière  and focused on advanced cell therapies in auto-immune diseases and cancer., research director at the San Raffaele Telethon Institute for Gene Therapy in Milan, Italy, focused her talk on dendritic cells (DCs) and their impact in autoimmune diseases and transplantation. They identified a subset of tolerogenic DCs producing IL-10 and named DC-10. Since they highlighted a diminution of DC-10 in patients’ blood exhibiting autoimmunity (in Type 1 diabetes and Multiple sclerosis models) or severe inflammation (in Celiac disease model), DC-10 could be defined as biomarker of tolerance and used in clinical applications  and DC-10-based therapy would restore tolerance in these diseases.Dr Silvia Gregoriications . To suppzed mice . They op  was one of the first to perform proof of concept studies showing that antigen specific T cells could be used to boost T cell immunity against viruses and to tackle cancer. He is also a pioneer in using engineered and modified CAR or TCR transduced T cells in human. He gave a much-appreciated talk about overcoming barriers to efficacy for engineered T cells in solid tumors during which he presented results describing a CD4 T cell population directed against a neoantigen derived from the oncogenic mutated BRAFV600E gene expressed in melanoma cell. This population was identified within the Tumor Infiltrating Lymphocytes (TIL) population adoptively transferred to a stage IV acral melanoma patient  who is an associate professor at UCSF (USA). He presented a strategy that he developed to efficiently reprogramming the genome of human immune cell populations using the CRISPR/Cas9 technology. He used this global strategy with a guide library in order to identify relevant targets for modulating T cell responses. In particular, Dr Marson found that RASA2, which is a GTPase-activating Protein (GAP) functioning as a negative regulator for Ras, was a promising target for novel immunotherapeutic strategies  (The next speaker was Dr Alexander Marsonlidated) . Knockin chair of the department of Systems Pharmacology and Translational Therapeutics at University of Pennsylvania . One of his area of interest is molecular understanding of human T cell exhaustion and their role in disease. He tries to understand which T cell population best correlates with clinical outcome in anti-tumor immunotherapy and which is critical to predict and optimize therapeutic responses. He presented results defining a precise human T cell molecular reference atlas. By combining two high throughput approaches (ATAC seq and RNA seq) he provided very detailed analysis of specific gene expression. The work was primarily performed by Josephina Giles and is about to be published.The keynote lecture was given by Prof. John WherrySession III was chaired by Prof Megan Sykes  and Dr Catherine Rabu  and devoted to new horizons in immunotherapy., postdoctoral researcher at the University of Zurich, a specialist of tissue ecosystem profiling by Imaging Mass Spectrometry (IMC). He has a long-standing interest in the study of type 1 diabetes with the objective of understanding the progression of this auto-immune disease by reconstructing evolution of β cells and islet-infiltrating immune cells. To deeply profile this tissue ecosystems, he used IMC, a high-resolution technology coupling a laser ablation system with a time-of-flight mass spectrometry, allowing simultaneous analysis of spatial mapping and cell phenotyping through the detection of over 40 markers. Thanks to this technique, he realized an immune cell profiling on pancreas section from donors at different stages of disease progression: non-diabetic, pre-diabetic with autoantibodies, recent onset and long duration. This longitudinal study revealed that T cells infiltration was dependent and correlated to the islet profile, originated by alteration of β cell phenotype at pre-diabetic stage  heterogeneity and its implication for their clinical application. MSC have clinical potential based on their immunosuppressive and anti-inflammatory properties but their large heterogeneity could impact clinical responses. She draw our attention to the different levels of heterogeneity, starting with the variability due to the donor which requires strong phenotypic characterization . In a first step, she realized a multiomic analysis of fetal bone marrow, combining RNAseq and CITEseq, to identify and annotate each cell types  and Dr Jérôme Martin  and focused on tissue microenvironment in cancer or in inflammatory situations.immunoregulation capacity of inflammation. FcRs are mainly activating receptors, thanks to immunoreceptor tyrosine-based activation motif (ITAM), and participate in many functions in both innate and adaptative immunity. Nevertheless, FcRs bearing inhibitor ITAMs (ITAMi) have been highlighted and might play a role in homeostasis. As opposed to classical ITAM signaling, ITAMi signaling involves the recruitment of diverse inhibitory effectors, like SHP-1, by inducing an intracellular co-aggregation forming clusters termed “inhibisomes”  in Paris, France, began with Fc receptors (FcRs) and their bisomes” . This reMyeloid cells represent a diverse group of cells with multiple roles that can act in promoting tumor growth. Dr Antonio Sica  gave an overview of his recent work in this field. He defined the concept of emergency myelopoiesis and showed in mice models that the RORC1 (retinoic-acid-related orphan receptor)-expressing myeloid cells are enriched in advanced tumors. MDSC (myeloid derived suppressor cells) and TAM (Tumor associated macrophages) expressing RORC1 promote cancer development (high) which overexpress HO-1 (heme-oxygenase-1) that plays a critical role in promoting tumor metastasis and immunosuppression. Interestingly, in metastatic melanoma patients, HO-1 expression on various monocyte populations discriminates survival as a good prognostic marker in case of low expression  and T cells and it interacts with Candida fungal pathogens. Since the 1980s, it has been shown that NK cells have abilities to recognize and kill transformed and virus-infected cells and also extracellular pathogens, such as Candida, responsible of various diseases (from Candida vulvovaginitis to invasive candidiasis). On that context, he presented data describing the first fungal ligands that impair NK cell functions through TIGIT and establishing the role of NK cells in the control of C.albicans infection.The next speaker, Prof Ofer Mandelboim, professor of Molecular Immunology at The Hebrew University, Jerusalem, Israel, presented his work on immune escape mechanisms of tumors and their impact for immunotherapies. She mostly described defects in the HLA class I antigen processing and presentation machinery (APM), occurring by mutations or (post) transcriptional regulations observed in many cancers at variable frequencies (from 30 to 80%). An altered APM in tumors was associated with a worst prognosis, like in oral squamous cell carcinoma, mainly due to a reduced intratumoral density of CD8 T cells , miR-26b-5p and miR-21-3p, involved in downregulating TAP1 protein expression thereby inducing a reduced HLA class I expression in melanoma cells  were only active in subsets of patients (The last talk of the session was given by Dr Shannon Turley, Staff Scientist in the Department of Cancer Immunology at Genentech in San Francisco. Her area of interest is about the patients . Using apatients . In a sepatients , was higpatients . In concThis 24th edition of the NAT conference and 4th edition of the LabEx IGO meeting took place online with more than 200 attendees and allowed interesting exchanges between international specialists in their field around the common theme of new horizons in immunotherapy. The exhaustive program included presentations of latest technical advances used to better understand physiopathology of transplantation, cancer, autoimmune diseases and highlighted the limitations still faced in these diseases. The congress also gave an overview of new active/passive immunotherapies developed based on molecular/cellular compounds to overcome failure of current treatments and to successfully treat patients in the future thanks to shelf cell therapy, genetic engineering, bioreagents and a better understanding of tissue ecosystem interactions in diseases.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.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.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.