Shigella is the leading cause of global diarrheal deaths that currently lacks a licensed vaccine. Shigellosis drives antimicrobial resistance and leads to economic impact through linear growth faltering. Today, there is a robust pipeline of vaccines in clinical development which are broadly divided into parenteral glycoconjugate vaccines, consisting of O-antigen conjugated to carrier proteins, and oral live attenuated vaccines, which incorporate targeted genetic mutations seeking to optimize the balance between reactogenicity, immunogenicity and ultimately protection. Proof of efficacy has previously been shown with both approaches but for various reasons no vaccine has been licensed to date. In this report, we outline the requirements for a Shigella vaccine and describe the current pipeline in the context of the many candidates that have previously failed or been abandoned. The report refers to papers from individual vaccine developers in this special supplement of Vaccines which is focused on Shigella vaccines. Once readouts of safety and immunogenicity from current trials of lead candidate vaccines among the target population of young children in low- and middle-income countries are available, the likely time to licensure of a first Shigella vaccine will become clearer. Shigella. The pathogenesis of shigellosis involves the invasion of the colonic mucosa which results in inflammation. Clinical presentation is either with bloody diarrhea, otherwise known as dysentery, or watery diarrhea. Although dysentery is the classical presentation of shigellosis, watery diarrhea is the more common presentation. While dysentery is highly suggestive of shigellosis, presentation with watery diarrhea is clinically indistinguishable from the many other etiologies of watery diarrhea. Although human-restricted, shigellosis is highly transmissible by the fecal–oral route, contaminated food and water and fomites. It is estimated that 10 s to 100 s of bacteria are sufficient to cause disease [Shigella is responsible for a large global burden of disease and is the leading bacterial cause of diarrheal deaths worldwide. Children under 5 years of age in low- and middle-income countries (LMICs) are most affected, particularly in the second year of life [Shigellosis is caused by an infection of the gastrointestinal tract with Gram-negative bacteria belonging to the genus  of life ,3,4,5. T of life . Shigell of life .Shigella, and three of these have multiple serotypes. This large number of Shigella serotypes presents a challenge for vaccine development, since immunologic protection is largely serotype-specific. Shigella flexneri is the most important species globally, particularly in low-income countries, and consists of 15 serotypes, the most common of which is S. flexneri 2a, followed by 3a and 6. Shigella sonnei has just one serotype and is the dominant species in countries that are undergoing industrial development. Shigella dysenteriae has 15 serotypes, the most significant being Shigella dysenteriae type 1, which was responsible for epidemic outbreaks with high-case fatality rates that seem to have disappeared in the 21st century. Shigella boydii, which has 19 serotypes, is responsible for a small minority of shigellosis cases and is mainly detected in South Asia [There are four species of Shigella is a major cause of linear growth faltering among young children in LMICs [Shigella. Finally, even when initiated, the use of antibiotics for the treatment of Shigellas is becoming increasingly compromised by the rise in antimicrobial resistance among circulating Shigella isolates [In addition to the direct morbidity and mortality due to diarrhea, in LMICs ,9. Currein LMICs . Otherwiisolates ,12,13.Shigella a pressing global public health need. Several approaches have been adopted by vaccine developers for the prevention of shigellosis over the years. These broadly divide between oral whole cell approaches, consisting mostly of live attenuated but also inactivated vaccines, and parenteral subunit approaches, mostly glycoconjugates, which are designed to target the immune response to key Shigella antigens. There is evidence for efficacy with both of these vaccine approaches. In the 1960s, a live attenuated vaccine developed by David Mel of the Yugoslav Army was shown to be efficacious both among military recruits [S. sonnei O-antigen glycoconjugate vaccine developed by John Robbins and colleagues at the National Institutes for Health (NIH), USA [All of these factors make the development of a vaccine against recruits  and chilrecruits  in formeIH), USA . In a suIH), USA .Shigella vaccine development and a large numbers of clinical trials  document [Shigella vaccine must be safe, immunogenic and efficacious against moderate-to-severe diarrhea (MSD) due to Shigella infection among the key target population of infants and young children under 5 years in LMICs. Efficacy should be achieved through a primary immunization schedule of one to two doses delivered during the first 12 months of life with or without an additional booster dose. The PPC document stipulates 60% or more efficacy against Shigella MSD caused by vaccine serotypes and a minimum duration of protection between 24 months and 5 years. A vaccine should be safe and not immunologically interfere when coadministered with other recommended vaccines, and should be stable for two years at 2–8 °C. It should be cost-effective so that price is not a barrier to access in LMICs. The PPC document includes both oral and injectable  routes of administration.Key attributes for document . A ShigeShigella [Shigella isolates from the Global Enterics Multicenter Study (GEMS) [Shigella flexneri 2a, 3a and 6, as well as Shigella sonnei O-antigens could provide direct coverage against 64% of global Shigella isolates [S. flexneri serotypes which has been observed in guinea pigs [Non-human primate studies , controlShigella  all provy (GEMS)  suggestsisolates . Coveragnea pigs .Shigella type 3 secretion system (T3SS), particular IpaB, IpaC and IpaD [Shigella into epithelial cells. A potential advantage of whole-cell approaches to vaccine development is the inclusion of Ipa proteins along with other potentially protective protein antigens.Nonetheless, there is also a body of evidence implicating surface protein antigens common to multiple serotypes in protection against shigellosis. Most prominent among these are the Ipa proteins which form the needle and extracellular complexes of the and IpaD . The T3SShigella vaccines. Both phase 3 studies with NIH S. sonnei glycoconjugate vaccines found a strong association between serum O-antigen IgG and protective efficacy [Shigella vaccines and natural infection with wild-type Shigella, fecal O-antigen IgA has been closely implicated in protection. This difference is likely indicative of parenteral vaccines inducing systemic immunity, in contrast to natural infection and oral vaccines directly inducing mucosal immunity within the gastrointestinal tract.The relevant immunological mediators and mechanisms of protection are likely multifactorial. The most recognized is serum IgG to LPS O-antigen which forms the main immunological response to parenterally administered glycoconjugate efficacy ,17. Subsefficacy . In the Shigella CHIM studies suggest that serum IgG to IpaB could also be a correlate of protection [M), and CD4+ and CD8+ T cells being proposed as having roles in protection against shigellosis [Recent data from otection . Effortsotection . As wellotection ,29. Sevegellosis ,31,32.Shigella is a human-restricted pathogen and even non-human primates require much higher bacterial inocula to induce shigellosis compared to humans, all of these models have limitations. For this reason, there has been a long-standing emphasis on assessing candidate Shigella vaccines in humans. Aligned with this, the establishment of the Shigella CHIM at three centers in the United States, has permitted the early assessment of vaccine efficacy in humans [Several animal models have been used to study shigellosis and assess the potential protective efficacy of candidate vaccines. These include the mouse pulmonary infection , guinea n humans ,37,38. Cn humans , clinican humans  and labon humans  for thesOral whole cell vaccines can be divided between whole cell killed  and liveShigella vaccine by Kiyoshi Shiga, adopted the whole-cell killed approach [The earliest vaccines, beginning with the first attempt at a approach . Key to approach .S. sonnei (SsWc) [S. flexneri 2a (Sf2aWC) [Relatively recently, formalin-inactivated i (SsWc)  and S. f(Sf2aWC)  monovale(Sf2aWC) , it has Shigella strains using media containing the antibiotic streptomycin. This resulted in the strains becoming both streptomycin dependent (SmD) and attenuated, though the exact genetic mutations leading to attenuation were unknown. Although SmD Shigella vaccines were tested in subjects over multiple studies in Yugoslavia and the US, they had a number of issues. First, the primary immunization schedule consisted of four doses. Second, protection was relatively short-lived, lasting for a year. Third, some vaccine lots lost their streptomycin dependence. Finally, there were issues with manufacturing. These vaccines were never licensed and were eventually abandoned.Studies by Mel and colleagues from the 1960s and 1970s ,15 pavedS. flexneri 2a T32 strain by multiple passages on nutrient agar [S. flexneri 2a T32 strain containing an S. sonnei plasmid. This enabled the co-expression of O-antigens of S. flexneri 2a and S. sonnei. In field studies in China, FS was reported to give 61–65% protection against S. flexneri 2a and 57–72% S. sonnei shigellosis [Around the same time, another live attenuated vaccine, the Vadizen live vaccine, was developed in Romania by Istrati from the ent agar . During ent agar . Of notegellosis . The curShigella vaccines with targeted genetic mutations. However, similar challenges are faced by all live attenuated vaccine candidates in balancing acceptable levels of reactogenicity with sufficient immunogenicity to confer protection. Below, we report on a selection of such vaccines that have been tested in clinical trials.More recently, the advent of whole genome sequencing enabled the development of well-defined live attenuated Shigella vaccines based on the well-characterized S. flexneri 2a 2457T strain. CVD 1203 contained mutations in the aroA and virG genes that encode proteins involved in amino acid biosynthesis and intra- and intercellular motility. Although immunogenic, the vaccine was excessively reactogenic, causing dysentery in 72% of recipients in a phase 1 study [guaBA, virG, set and sen genes were deleted. guaBA is required for guanine biosynthesis, while set and sen encode the ShET1 and ShET2 Shigella enterotoxins, respectively. This new approach resulted in tolerability in a phase 1 study but limited immunogenicity [The Center for Vaccine Development at the University of Maryland has developed a number of live attenuated  1 study  and was genicity .guaBA mutation but reverted to using wild-type virG, either with intact (CVD 1204) or deleted (CVD 1208) sen and set genes. The deletion of the Shigella enterotoxins was shown to be required since CVD1204 resulted in unacceptable reactogenicity [Further iterations of CVD 1207 maintained the genicity . CVD 120genicity .Shigella vaccines is the difference in reactogenicity and immunogenicity of the same vaccine among different populations. SC602 is a S. flexneri 2a candidate vaccine developed at the Pasteur Institute, Paris. It is based on the wild-type S. flexneri 2a 494 strain with deletions in virG and iuc which encodes the siderophore, aerobactin. At 104 cfu, SC602 was mildly reactogenic in US adults but immunogenic and protected against fever, dysentery and severe symptoms following challenge with S. flexneri 2a 2457T [Another challenge facing development of live attenuated 2a 2457T . When te2a 2457T .The poor immunogenicity of live attenuated vaccines has also been observed among LMIC populations with licensed vaccines against cholera, rotavirus and polio. Multiple reasons may account for this, a key one being environmental enteric dysfunction (EDD) which commonly occurs in children in LMICs . EDD is Shigella vaccine candidate based on wild-type Shigella dysenteriae type 1 strain SC595 with deleted virG, ent and fep genes, the latter two genes encoding iron chelation proteins. Although well-tolerated in phase 1 trials, the vaccine was poorly immunogenic and has not progressed [The Pasteur Institute also developed SC599, a live attenuated ogressed .Shigella vaccine candidates attenuated through the deletion of aroD which made them auxotrophic for aromatic compounds. SFL124 was based on the moderately virulent parent strain S. flexneri SFL1. The candidate was well tolerated in phase 1 studies in adults in Sweden [In the early 1990s, the National Bacteriology Laboratory in Stockholm developed live n Sweden  and Vietn Sweden , and in n Sweden , but shoaroD deleted from the more virulent S. flexneri 2a 2457T strain. When tested in a phase 1 study in Swedish adults, a narrow safety-immunogenicity profile was demonstrated [Subsequently, SLF1070 was developed with nstrated . NeitherS. sonnei vaccines based on the wild-type Moseley S. sonnei strain with virG deleted. A common problem with S. sonnei strains is loss of the virulence plasmid. However, the plasmid is relatively stable in the Moseley strain. WRSS1, the first-generation vaccine of this series, was immunogenic but resulted in diarrhea and fever among US and Israeli adult volunteers in phase 1 and phase 2 studies [WRAIR developed a series of live attenuated  studies ,62. WRSS studies .senA and senB, WRSS3 has msbB deleted from the virulence plasmid. The removal of msbB, which encodes an acyltransferase, results in the loss of an acyl chain from the lipid A of LPS with a consequent reduction in reactogenicity [Further attenuating mutations were introduced to address the reactogenicity resulting in the WRSS2 and WRSS3 candidate vaccines. While both have deleted enterotoxin genes genicity . Both vagenicity .Shigella vaccine development was the use of the licensed typhoid Ty21a live attenuated vaccine as a vector for the delivery of Shigella sonnei O-antigen. The 5076-1C vaccine was developed by Formal at the University of Maryland in the 1980s [An innovative approach to he 1980s . Though he 1980s , it suffhe 1980s ,68. FurtShigella vaccines, in a similar approach to the Ty21a Shigella candidate 5076-1C, key Shigella antigens have been expressed in E. coli. PGAI 42-1-15 was based on E. coli O8 genetically modified to express S. flexneri 2a O-antigen [S. flexneri 2a CHIM study [E. coli K12 that could express S. flexneri 2a O-antigen and also invade epithelial cells due to the inclusion of the invasion plasmid from S. flexneri 5, with aroD deleted from ECSf2a-2 to reduce reactogenicity. Unacceptable reactogenicity was a problem for ECSf2a-1 [In order to improve the tolerability of live attenuated -antigen . Though IM study . SimilarECSf2a-1 , while EECSf2a-1 . This liE. coli engineered to express Shigella antigens, is the ShigETEC live attenuated vaccine being developed by Eveliqure, Vienna [S. flexneri 2a with Shigella O-antigen and Ipa antigens, the best-characterized targets of protective immunity, removed through the deletion of rfbF and ipaBC, respectively, together with the deletion of setAB to remove enterotoxins. The vaccine was engineered to express a fusion protein of LT and ST, the labile and stable toxins of enterotoxigenic E. coli (ETEC), hence the name ShigETEC. The vaccine was well tolerated and immunogenic in a recent phase 1 trial in Hungary and is set to progress into phase 2 studies [Shigella.An intriguing and almost opposite approach to , Vienna . ShigETE studies . A key oShigella vaccine strategy at the Center for Vaccine Development is to develop a hexavalent Shigella ETEC live attenuated vaccine consisting of six Shigella strains  engineered to express ETEC antigens. This is currently in preclinical development [Similar to the ShigaETEC approach, a new ambitious elopment .Shigella vaccines builds on the successful use of this technology for vaccines against the encapsulated bacteria Haemophilus influenzae b, meningococcus, pneumococcus and, more recently, Salmonella typhi. With the exception of S. sonnei  in protection. This is the key epidemiological observation on which the Shigella glycoconjugate vaccines are based.The glycoconjugate approach to -antigen ), ShigelS. sonnei or S. flexneri 2a chemically linked to recombinant exoprotein A of Pseudomonas aeruginosa (rEPA)  . The vacgellosis . The saf placebo .S. sonnei in the arm receiving S. sonnei O-antigen/rEPA was remarkable considering this was after a single dose of vaccine with participants followed up for a duration of two years. The lack of cases of shigellosis due to S. flexneri 2a in the study prevented any conclusions being made on the efficacy of EcSf2a-2. Given that study findings were published 25 years ago, it is surprising that no glycoconjugate vaccine has yet been licensed. There are perhaps several reasons for this.The finding of 74% efficacy against shigellosis due to Shigella vaccine is young children in LMICs. Thirteen years later, in 2010, findings from a second efficacy study in Israel were published by Paswell and colleagues [S. sonnei O-antigen/rEPA or S. flexneri 2a O-antigen/rEPA. While protection was demonstrated against S. sonnei shigellosis among children aged 3–4 years, protection was not seen in children under three years of age. Loss of protection corresponded to a decrease in serum O-antigen IgG titer further supporting a role for this modality of immunity in protection against shigellosis. Again, insufficient cases of S. flexneri 2a shigellosis precluded any conclusions about the efficacy of S. flexneri 2a O-antigen/rEPA. Neither S. sonnei O-antigen/rEPA nor S. flexneri 2a O-antigen/rEPA were ever commercialized.While protection was demonstrated in military recruits in Israel, the key target population for a global lleagues , this tiE. coli bacteria are genetically engineered to synthesize and chemically couple glycans from exogenous bacteria to protein carriers, exploiting the oligosaccharide transferase PglB [S. dysenteriae type 1 bioconjugate, GVXN SD133-EPA was produced with O-antigen from S. dysenteriae type 1 linked to genetically detoxified exotoxin protein A of Pseudomonas aeruginosa and was found to be safe and immunogenic in a phase 1 study [Limmatech Biologics AG, based in Zurich, developed an innovative glycoconjugate technology known as bioconjugation where ase PglB . As proo 1 study .S. flexneri 2a which, following promising immunogenicity in a phase 1 study in the US, was assessed for efficacy against shigellosis in a CHIM study at Johns Hopkins University. Although the vaccine failed to meet the primary endpoint of protection against all forms of shigellosis, it did protect against more severe shigellosis in a post hoc analysis [Clinical Infectious Diseases (2019 supplement 8). As with the NIH S. sonnei vaccine, protection was closely associated with serum IgG to S. flexneri O-antigen [S. sonnei, flexneri 3a and flexneri 6, as well as S. flexneri 2a. This is currently completing an age-descending dose-finding study in Kenya [Subsequently, the technology was used to develop Flexyn2a-EPA, a vaccine against analysis . This pr-antigen ,79. On tin Kenya .Shigella conjugate vaccine constructs demonstrated that the use of O-antigens of reduced length could result in an enhanced serum IgG response to O-antigen compared with conjugates employing native length O-antigen. This observation provided supporting justification for the synthetic approach to generate Shigella O-antigens for conjugation at the Pasteur Institute. Using an elaborate process involving multiple chemical steps, a series of S. flexneri 2a synthetic O-antigens were generated and conjugated to tetanus toxoid prior to testing in mice for immunogenicity.Separate work by Pozgay and Robbins ,81 with Evaluation in animals led to the selection of a candidate vaccine, SF2a-TT15, with O-antigen consisting of three pentasaccharide repeating units . This vaccine was tested by Cohen in Israeli adults and found to be highly immunogenic, even after a single dose . As withS. sonnei and S. flexneri 2a conjugated to tetanus toxoid with adipic acid dihydrazide as linker. Following a phase 1 descending age study in China to show safety [Using more conventional glycoconjugate technology, Beijing Zhifei Lvzhu Biopharmaceuticals designed and developed a bivalent vaccine, ZF0901, using O-antigen from w safety , ZF0901 w safety .Shigella LPS and Ipa proteins. The initial iteration of Invaplex, native Invaplex or InvaplexNAT, consisted of a complex of Ipa antigens  and LPS extracted from wild-type S. flexneri 2a. Three doses delivered intranasally were well tolerated and immunogenic in phase 1 studies [An interesting alternative subunit vaccine approach to the glycoconjugates is the invasin complex or Invaplex technology developed by WRAIR . Essenti studies ,88 in th studies .AR, was produced using recombinant IpaB and IpaC generated in E. coli, and LPS from S. flexneri 2a which was safe but not consistently immunogenic when delivered intranasally to volunteers in a phase 1 study [AR-Detox) employs an LPS of low reactogenicity purified from S. flexneri 2a with deleted msbB genes. This has allowed the parenteral administration of InvaplexAR-Detox with good safety and immunogenicity results in a recent phase 1 study [Subsequently, a more defined version of Invaplex, artificial Invaplex or Invaplex 1 study . As a fu 1 study . It is sS. flexneri 2a with proteosomes (outer membrane proteins) from meningococcus to give a Proteosome-Shigella flexneri 2a lipopolysaccharide vaccine. This vaccine was only tested in phase 1 and was administered intranasally [In a similar but earlier approach to Invaplex, WRAIR complexed LPS from anasally . It was Shigella, by the deletion of tolR [tolR is a gene involved in maintaining the integrity of the connection between the inner and outer membranes in Gram-negative bacteria. In addition, htrB, which, like msbB, encodes an acyl transferase, was deleted to reduce reactogenicity, and virG to prevent epithelial invasion. This technology has the advantage of being straightforward to deploy with the potential to produce vaccine at low cost. GVGH considers the vesicles to be a vehicle for the delivery of O-antigen. Another advantage of this approach is that multiple other outer membrane components are presented to the immune system, including many outer membrane proteins [A different subunit approach which has similarities with whole cell killed vaccines is the use of bacterial native outer membrane vesicles (OMVs) as vaccines. The GSK Vaccines Institute for Global Health (GVGH) adopted this approach by increasing the spontaneous release of OMV , which are blebs of outer membrane from the surface of  of tolR . tolR isproteins , though S. sonnei 53G with the above mutations. Though well tolerated, it was poorly immunogenic compared with historic Shigella glycoconjugate vaccines whether administered intramuscularly, intradermally or intranasally in European adults [S. flexneri 1b, 2a and 3a and S. sonnei (with higher O-antigen expression than 1790GAHB) is currently being tested in a phase 1/2 clinical trial [A first OMV candidate vaccine, 1790GAHB, was generated from n adults . This isn adults , it failn adults . A quadral trial .Despite 100 years of vaccine development, there is to date no licensed vaccine to protect against shigellosis, the main cause of childhood bacterial diarrheal death globally. This is despite numerous candidate vaccines having been assessed in clinical trials and clinical efficacy proven with both the early live attenuated approaches of Mel and the Yugoslav Army in the 1960s, and the glycoconjugate approach of Robbins and the NIH in the 1990s. As indicated in this report, the large majority of vaccines tested in humans to date have been live attenuated vaccines. Their lack of success consistently comes down to the difficulty of balancing reactogenicity with sufficient immunogenicity to induce protection.Shigella vaccines [Shigella vaccine should induce protective immunity for at least two years after no more than two doses. The likely need for multivalent Shigella vaccines to provide sufficient breath of coverage against global circulating Shigella strains increases the challenge faced by live attenuated candidates. To date, it has proven exceedingly difficult to achieve an acceptable balance of reactogenicity and immunogenicity with monovalent Shigella vaccines.Where protection was demonstrated with the Mel SmD vaccines, this required multiple doses (a primary series of four doses) and duration of protection was limited to one year. However, according to the recently published WHO Preferred Product Characteristic for vaccines , a ShigeShigella vaccine. Proof of efficacy was demonstrated by Cohen with the NIH S. sonnei O-antigen-rEPA candidate 25 years ago in young Israeli adults [Shigella conjugate vaccines have entered clinical development since 1997. This is perhaps partly a result of the emphasis on pursuing the live attenuated approach by many vaccine developers during the 1990s and 2000s at a time when limited funding was available for Shigella vaccine development.Glycoconjugate technology appears more promising than live attenuated approaches for a licensed i adults  and subsi adults . It is bShigella vaccines with advancing clinical development of several promising glycoconjugate vaccines, as well as live attenuated vaccines advancing. The advent of molecular determination in recent years of pathogen etiology in diarrhea cases in place of exclusive reliance on stool culture [Shigella. Growing worldwide concern about the threat of antimicrobial resistant Shigella [Shigella is a major cause of linear growth faltering [Shigella vaccine development. These factors, together with increased funding for Shigella vaccines and the strong recommendation by the WHO’s Product Development Vaccine Advisory Committee (PDVAC) of the need to develop Shigella vaccines [Following the disappointing results with so many live attenuated candidates, it is reassuring to see a balance across the global portfolio of  culture  has incrShigella  and the Shigella , as wellaltering , are provaccines , are serS. flexneri 2a synthetic O-antigen conjugate vaccine, SF2a-TT15, is completing an age descending, dose-finding study in Kenya [Although glycoconjugate candidates rarely cause issues with reactogenicity compared with live attenuated vaccines, there remains the challenge of whether current candidates will prove to be sufficiently immunogenic among young children in LMICs. The most advanced glycoconjugate vaccine is the quadrivalent bioconjugate candidate, S4V-EPA, from Limmatech. This vaccine is currently completing its phase 2 study in young children in Kenya with the expectation of a readout of interim immunogenicity this year . Meanwhiin Kenya . The GVGin Kenya .Shigella vaccine is a realistic prospect in the next few years. Should these vaccines turn out to be safe but insufficiently immunogenic, there is the possibility of enhancing immune responses using adjuvants. To date, little is known about which adjuvants may potentiate current candidate Shigella vaccines, particularly the glycoconjugates. The use of adjuvants in the clinical trials of these vaccines has so far been limited to alum and effects have been inconsistent. This represents a knowledge gap in Shigella vaccine development. The application of our understanding of new adjuvants from the COVID-19 pandemic may help address this gap but requires formulation, stability and immunogenicity studies with lead candidate vaccines to begin now.The cumulative clinical data from these trials will provide much clarity as to whether a licensed Shigella vaccines for the global pediatric market. Phase 3 studies will be lengthy and costly but ultimately, if able to demonstrate efficacy and confirm the correlate of protection status for O-antigen IgG in children, may permit the licensure of subsequent vaccines on the basis of safety and non-inferior immunogenicity. As a critical enabling activity of this, work is underway at NIBSC to develop a first Shigella International Standard Serum and harmonize Shigella ELISAs [Although there is strong evidence that serum IgG to O-antigen is a correlate of protection in adults , this isa ELISAs .Shigella vaccine for the global pediatric market is unlikely to entice one of the big five multinational vaccine companies to manufacture a Shigella vaccine. It is more likely that companies from the Developing Country Vaccine Manufacturers Network [Shigella vaccines.Studies are currently underway to facilitate and expedite the initiation of phase 3 trials for the most promising candidate vaccines when ready . Each of Network  will parShigella vaccine development has become a graveyard filled with numerous candidates that entered clinical trials and proved to be either too reactogenic, insufficiently immunogenic or both. Nevertheless, proof of efficacy has been attained on more than one occasion and there are currently multiple promising candidates in clinical development, with lead candidates due to read out shortly from studies in the target populations of young children in LMICs.Over the past 100 years,


X-ray photoelectron spectroscopy and electrochemical measurementsreveal the sp3 carbon character and advantageous electrochemicalproperties of a BDD electrode are retained during the milling process.TEM diffraction studies show a dominant (110) crystallographic orientation.Compared with conventional carbon TEM films on metal supports, theBDD-TEM electrodes offer superior thermal, mechanical and electrochemicalstability properties. For the latter, no carbon loss is observed overa wide electrochemical potential range (up to 1.80 V vs RHE) under prolonged testing times (5 h) in acid (comparable withaccelerated stress testing protocols). This result also highlightsthe use of BDD as a corrosion free electrocatalyst TEM support forfundamental studies, and in practical energy conversion applications.High magnification TEM imaging demonstrates resolution of isolated,single atoms on the BDD-TEM electrode during electrodeposition, dueto the low background electron scattering of the BDD surface. Giventhe high thermal conductivity and stability of the BDD-TEM electrodes, in situ monitoring of thermally induced morphological changesis also possible, shown here for the thermally induced crystallizationof amorphous electrodeposited manganese oxide to the electrochemicallyactive γ-phase.The majority of carbon based transmission electron microscopy(TEM)platforms (grids) have a significant sp Thin films (3–30 nm) of carbon floatedover a support substrate are particularly popular due to carbon beinglow electron scattering and electronically conductive. Here the carbonis typically an amorphous carbon film, although graphene oxide5 and graphene6 layershave also been used.Transmission electron microscopy (TEM)is a powerful analyticaltool for characterizing nanomaterials at the atomic level.2 to sp3 bonded carbon and the levelof graphitization.7 Such carbons are useddue to their low cost, electrical conductivity and reduced electrocatalyticactivity towards the energy conversion processes of interest.8 To assess NP structure pre- and post-electrocatalysis,either the NPs or the NPs plus carbon black support, can be placedon the carbon TEM grid.10 Outside of energy applications,carbon film TEM grids have also found use in the elucidation of electrochemicallydriven NP deposition mechanisms.12In electrocatalytic energy conversion applicationscarbon is alsofrequently used as the nanostructured electrocatalyst support forelectrocatalytically active nanoparticles (NPs). The carbon, typicallya carbon black, can take a variety of structures, which range in theratio of spex situ studies, measurements are typicallyundertaken by dipping the TEM grid into an electrolyte solution, performingthe electrochemical process of interest, removing from solution andthen imaging the surface.13 This process typicallyrequires the carbon film to be supported on a metal grid so an electricalcontact can be made. Use of TEM “finder grids”  makes monitoringof the same location, pre- and post-electrochemical treatment mucheasier; this process is termed identical-location (IL-)TEM.16 However, interpretation of the current passed is challenging dueto both the metal support and carbon film acting as an electrode.A few studies have overcome this issue by using bespoke holders whichprevent electrolyte accessing the metal grid.18For 20 Here the SECCM tip was used to both electrodeposit NPs locally19 and measure the local electrocatalytic activityof pre-formed NPs.20 SECCM has also beenused as a method for placing pre-formed NPs onto the working electrodefor in situ electrochemical TEM observation.21More recently, scanning electrochemicalcell microscopy (SECCM),where an electrolyte filled nanopipette is used to create a miniatureelectrochemical cell, has been employed with carbon film TEM grids.22 The mostnotable one is the fact carbon can undergo oxidation and corrosionat potentials which are important for energy conversion reactions.These include, for example, during the start–stop cycle ofa proton exchange oxygen reduction reaction (ORR) membrane fuel cell24 and at the potentials required for the oxygen evolution reaction.25 Corrosion of the carbon TEM film also complicatesstudies aimed at investigating electrocatalytically induced changes in NPs. Furthermore, forthermal studies, interactions between the TEM grid metal support andthe nanostructure/carbon film should also be considered. For example,at elevated temperatures, certain metals e.g. nickel,can graphitize the carbon film,27 and metal atoms fromthe underlying support, can evaporate and redeposit, causing contamination.28Whilst carbon is useful as an electrocatalystsupport and TEM filmit does have some drawbacks.2 carbon, as the sp3 carbon bonding results in anincreased mechanical strength and corrosion resistance, both chemicaland electrochemical. BDD is also an excellent conductor of heat.29 When used as an electrode material, in the morecommon oxygen terminated form, the surface properties are such thatthe double layer capacitance is low (<10 μF cm–2) and electrocatalytic processes such as ORR and water electrolysisare significantly kinetically retarded.29 Many of these properties make BDD an ideal NP electrocatalyst support.31 However, BDD has yet to be used routinely as a TEM electrode, dueto the lack of methodologies available to fabricate BDD which is notonly thin enough to be electron beam transparent (∼10 to 100nm thick) but is also handleable. In this paper we describe a procedureto produce free standing (unsupported) BDD-TEM electrodes, and highlighttheir useful properties as corrosion free, temperature stable, TEMsupports for applications of importance in the electrodeposition andenergy conversion fields.Given the above, it is interesting toconsider the suitabilityof boron doped diamond (BDD) for correlative TEM-electrochemical  measurements. BDD is an interesting alternative to sp2SO4 95–97%, Scientific and Chemical Supplies Ltd.) andpotassium nitrate  were used. (b) For metal electrodeposition, manganeseoxide was deposited from a solution containing manganese chloride and potassium chloride  acidified with hydrochloric acid . (c) For electrochemical characterization,hexaamineruthenium(III) chloride  was employed as a redox couple andpotassium nitrate  as supportingelectrolyte. (d) Sulfuric acid  was used for long term electrochemicalstability testing. Where commercial TEM grids were used for comparison,carbon films on a 300 mesh copper or gold support  were employed.All solutions were preparedusing Milli-Q ultrapure water with a resistivity of 18 MΩ cm(Millipore). (a) For acid cleaning, sulfuric acid  such that the material was above the metallic threshold.32 Both surfaces were mechanically (resin bonded) polished, to thinthe material to ∼50–80 μm and produce surfacesof ∼nm surface roughness.33 Lasermicromachining of the BDD was carried out using a 355 nm Nd:YAG 34ns laser (Oxford Lasers) to cut out disks of 3 mm diameter. To removemachining debris the electrodes were acid cleaned by immersing inconcentrated H2SO4 (saturated with KNO3) at ∼200 °C, for 30 min, followed by rinsing with ultrapurewater before cleaning for 30 min in concentrated H2SO4 only, at ∼200 °C.34All BDD was provided by Element SixLtd., Oxford, UK, and was grown using microwave chemical vapor deposition(CVD) to a suitable thickness so that it could be removed from thenon-diamond growth support.i.e. ahole had formed. To reduce surface roughness the disk was mountedin a clamp support for a final low energy polish of both sides ofthe disk simultaneously. This was achieved using a modulated ion beamat a lower accelerating voltage and angle of incidence, for 30 min.To increase the robustness of the Ohmic contact to BDD, a small segmentof the upper portion of the disc was laser roughened (532 nm Nd:YAG15 ns laser).34 The disk was again acidcleaned (vide supra) and electrical contact madeto this lasered region by either sputtering a Ti (10 nm)/Au (400 nm)contact (Moorfield MiniLab 060 Platform), followed by annealing for5 h at 400 °C,32 or by applicationof a conductive carbon ink . Note, as a resultof laser roughening and acid cleaning, the small region of the BDDonto which the contacts are placed, contains a very thin surface layerof non-diamond carbon.34 This is especiallyuseful when making an Ohmic contact using the carbon ink.Argon ion milling and polishing of the BDD disk was carriedoutusing a GATAN precision ion polishing system (PIPS). The BDD was mountedon a post support using glycol-phthalate bonding wax (Agar Scientific),allowing continuous milling as the sample rotated. First one sidewas milled for 2.5 h, then the disk was turned over and the otherside milled for 2 h and then in 15 min intervals until light transmissionthrough the center of the grid was visible 33 After any electrochemical process and before imaging, theBDD-TEM electrode was rinsed by gently dipping in ultrapure waterand then left to dry in a desiccator, held under vacuum.Cyclic voltammetry (CV)was carried out using a three-electrode setup controlled by a potentiostat with a saturated calomel electrode  or an Ag|AgCl electrode  used as the reference and a Pt coil as the counter. Electricalcontact to the BDD-TEM electrode was made using a metal clamp. Forelectrochemical measurements the BDD-TEM electrode was dipped intothe electrolyte, ensuring that the central hole was fully immersedin solution and the electrical contact remained dry.35 with a 1 mm diameter disk onthe BDD-TEM electrode exposed using Kapton tape. A 200 μL dropletof electrolyte solution was placed on the electrode surface for eachmeasurement. Solvent window and capacitance measurements were runin 0.1 M KNO3 at a scan rate of 0.1 V s–1. The electrode response for the redox couple Ru(NH3)63+/2+ was investigated by recording CVs of 1 mMRu(NH3)6Cl3 in 0.1 M KNO3 at a scan rate of 0.1 V s–1 with a step size of1 mV. Solvent windows were defined for a geometric current densityof ±0.4 mA cm–2.32Forelectrochemical characterization, a three-electrode droplet cell set-upwas used36 Contactangle measurements were recorded using a drop shape analyzer  with a water droplet of volume 50μL. Measurements were recorded in triplicate, with the surfacedried carefully in between using a lint free tissue.Surface roughness measurements were made using both an atomicforcemicroscope  and a white light interferometer. Image analysis wasperformed using Gwyddion 2.5.2.6) TEM at 200 kV. In situ TEM heating wasalso achieved in this TEM with a double tilt heating holder . Atom resolution annular dark field (ADF) imageswere recorded in a double aberration-corrected JEOL JEM-ARM200F operatedat 200 kV.Field emissionscanning EM (FE-SEM) was used to image the BDD-TEMelectrode. Images were recorded using the in-lens, secondary electron(SE2), and scanning TEM (STEM) detectors on a Zeiss Gemini FE-SEM500 operating at 20 kV. TEM imaging and electron diffraction on theBDD-TEM electrodes were carried out using a JEOL JEM 2100 . Data from the BDD-TEM electrodes were collectedusing an analysis area of 55 μm diameter, to probe as closeto the hole edge as possible. For the control sample  the data were acquired using a spot size of 110 μmin order to increase the overall count rate. C 1s spectra were obtainedusing take-off angles of 90 and 30° with respect to the surfaceplane. To investigate the different carbon chemical environments atthe electrode surface, all data collected were fitted in CasaXPS usingLorentzian–Gaussian lineshapes and Shirley backgrounds, withasymmetry included for the sp2 bonded carbon C–Cpeak.X-ray photoelectron spectroscopy (XPS) was conducted using a KratosAnalytical Axis Ultra DLD spectrometer with a monochromated Al KαX-ray source (1486.69 eV) in a chamber with a base pressure below1 × 10Compared to non-diamondcarbons, achieving electron beam transparent diamond electrodes ischallenging, due to the limited number of growth methods. Here, atop down approach was employed where the starting point was the productionof freestanding and double-sided polished polycrystalline BDD electrodes,as thin as possible, but still able to be handled easily. For thisreason 50–80 μm thick BDD electrodes were produced byCVD growth with subsequent mechanical polishing, and then cut intodisks of 3 mm diameter to make them suitable for insertion into theTEM holder. To achieve electron beam transparency the electrode wasfirst argon ion milled, on each face, and then the energy of millinglowered to ion “polish” both surfaces simultaneouslyas depicted schematically in ca. 30–150μm in diameter) formed in the center of the BDD disk. The observationof a hole indicates the presence of a region of BDD that is thin enoughto be electron beam transparent around the hole edge. Typically, ifthe hole is increased in size smaller areas of electron beam transparencyresult. For nine BDD-TEM electrodes examined we found an approximatelylinear relationship (R2 = 0.98) betweenthe diameter of the hole and the width of the electron beam transparentarea. As milling results in ripples (vide infra)on the BDD surface, a final lower energy ion polish (1–2 keV)was required .Initially the highest accelerating voltage wasused for the fastestmilling rate to remove the bulk of the BDD. An angle of incidencein the middle of the accessible range was employed. If the angle wasset too high, material was removed more quickly, and a smaller regionof electron beam transparent material resulted. A middle value wasfound to be a time efficient compromise. The exact milling time wasdependent on the starting material thickness. As required 1 at a sl37 anda conductive carbon ink contact and the distance between the BDD-TEM electrode and reference electrodewas kept constant.38 For both electrodes,similar Ru values of 229 ± 1 Ω(Ti/Au contact) and 346 ± 4 Ω (carbon ink contact) wereobtained (see Supporting Information 2),indicating both approaches were valid. Whilst Ti/Au is commonly usedto make an Ohmic electrical contact to BDD,32 it does require access to a sputter system and care is requiredduring sputtering to prevent Au spill over onto the BDD-TEM electrodedue to its concave profile. In contrast, the carbon ink can be paintedonto the laser roughened (and non-diamond carbon containing) areaof the BDD surface by hand. This method thus represents a cheaper,quicker, metal contamination free option, particularly useful forlong-term electrochemical experiments.To apply an electrical contact to the BDD-TEM electrode,two approacheswere investigated: a Ti/Au contact,tact see . To assevia the methodoutlined are shown in ca. 50 μm in width (at 20 kV). 39FE-SEM and STEM images ofa typical BDD-TEM electrode fabricated Supporting Information 3, Figure S3). The AFM measurements  shows a WLI imageof the central region of the BDD-TEM electrode covering a 1.2 mm diameterregion. Figure S3a(ii) shows the corresponding x, y topography profile from the hole edgeto 0.6 mm away [red line in Figure S3a(i)] highlighting the increasing non-linear thickness of the BDD movingaway from the hole. The ripples in the surface topography are evidentin the AFM images of topography ]. Such ripples have alsobeen seen when diamond surfaces have undergone ion bombardment.40 The mechanism of ripple formation is still disputeddespite being first observed over twenty years ago.41 The RMS roughness was calculated to be 48.3 nm across the wholearea in Supporting Information 3, Figure S4 for FE-SEM and AFM characterization.The topography of the BDD-TEM electrode was investigatedusingAFM, 3 C–C/C–Hcontribution of approximately 72%, with a sp2 C–Ccharacter of 21% . The C–O contribution is approximately 7%.39 Higher order oxides (e.g. O=C–O)which occur at >288 eV, make a minimal contribution (<1% ofthefitted peak). In order to provide greater surface sensitivity, theXPS take-off angle was decreased to 30°, to reduce the samplingdepth by a factor of two.43 Using the inelasticmean free path calculator (IMFP-TPP2M)44 the penetration depth (for C 1s) reduces from 9.9 to 4.5 nm. Underthe more surface-sensitive conditions at 30°, the main differenceis a reduction in the sp2 carbon component to 9% of thefitted envelope. This result indicates that a significant componentof the sp2 carbon signal in 45 There is also an increase inthe C–O contribution from ∼7% at 90° to ∼13%at 30°, as a result of the lower collection angle being moresurface sensitive; the O termination is only found at the surfaceof the BDD. Adventitious carbon signals are minimal.To assess theimpact of ion milling on the surface chemistry ofthe BDD-TEM electrodes, XPS was employed, 2 carbon character from 21 to 10% for the BDD-TEMelectrode. The value is now close to that seen for the 30° take-offangle BDD control data. This relative reduction in the sp2 carbon content in the more surface-specific geometry confirms thepresence of sub-surface damage from the initial mechanical polish,which is removed during the ion milling and ion polishing process.The data also indicates no subsequent ion milling induced sub-surfacesp2 carbon creation. Table S3, Supporting Information 4, gives fittings of the C 1s data for both electrodesexpressed as percentages of the total fitted envelope. There is ashift of approximately 0.9 eV in the absolute binding energy of theC 1s peak, and thus of the binding energies of each assigned peakin the fitting, for the BDD TEM electrode when compared to the BDDcontrol electrode. The reason for this shift is unknown. To accountfor this, binding energies have been considered relative to the assignedsp3 carbon peak for each electrode .When the 90° take-off angle BDD control data3a is comSupporting Information 4, Figure S5 and Table S4) at room temperatureand at a take-off angle of 90°. The sp2 and sp3 carbon content was found to be 62 and 27% respectively, insimilar proportions to fittings reported in the literature.20 As expected with an increased sp2 carbon content, more significant contributions from C=O,O=C–O and π–π* (2% of the envelopeeach) were observed. Contact angle measurements were recorded to comparethe hydrophobicity and wetting of a BDD-TEM electrode versus a commercial amorphous carbon coated TEM grid . For the BDD-TEM electrode,the disk was only ion milled on one side to prevent formation of thehole which would adversely affect the observed wetting. Contact anglesof 62.2 ± 0.5° (BDD-TEM) and 83.6 ± 1.1° (amorphousC film) were measured showing that the BDD-TEM electrode is more hydrophilicthan the carbon film. This is also advantageous for TEM electrodeaqueous based applications, where uniform wetting of the TEM electrodeis preferred. The value recorded for the BDD-TEM electrode is at theupper end of those recorded on other oxygenated BDD surfaces.29Comparison XPSmeasurements were also obtained on a commercialthin film carbon Au backed (C/Au) TEM grid (Ep) forthe redox couple Ru(NH3)63+/2+ areshown in Supporting Information 6, Figure S7. Wide and featureless solvent windows with values of 3.2 and 3.5V, capacitance values of 5.3 and 4.3 μF cm–2, and ΔEp values of 70 and 68 mVwere obtained for the ion milled/polished BDD-TEM and BDD control electrodes, respectively. The responses forall three parameters for the two differently prepared electrode surfacesare similar with the ion milled/polished surface showing only a smalldecrease in solvent window, and a very slight increase in both capacitanceand ΔEp compared to the controlmaterial.To further investigatethe properties of the ion milled/polished surface compared with amechanically polished control electrode, electrochemical characterizationof the solvent window and electrical double layer capacitance wascarried out. Solvent window, double layer capacitance and CV peakto peak separation data (Δ242SO4) for 1000 CV cycles at 0.1 V s–1  over the potential range 0.80 to 1.60 V versus Ag|AgCl .24 These conditions reflect the extreme positivepotentials a proton exchange membrane fuel cell experiences24 and the typical operational voltages of acidbased electrolyzers.46 Here the experimentwas carried out with both a BDD-TEM electrode and C/Au TEM grid dippedinto solution. A similar immersion depth was used for both (∼2mm). For the former, a carbon conducting ink was used to create theelectrical contact. This avoids Au dissolution from a Ti/Au contactand potential re-deposition (contamination) on other areas of theelectrode, as the sulfuric acid will evaporate during the long timescaleof this experiment.To assess thesusceptibility of the BDD-TEM electrode to electrochemical corrosionthat is carbon dissolution, the electrode was subjected to acceleratedstress testing (AST),Supporting Information 7). EELSspectra (pixel size = 3 nm) were acquired across all areas of theBDD electrode BDD area shown in Supporting Information 7, Figure S8.For each pixel within the EELS spectrum image the BDD thickness, t, was measured using the absolute log ratio method,47 (48), I0 is the area under thezero loss peak and It is the total area under the whole spectrum. Across the lines shownin t of 20.4 nm beforecycling and 21.7 nm after . The difference in thesevalues is within the experimental error of the calculation, whichis estimated to be ca. 5%,47 and indicates the BDD is not electrochemically corroding duringAST. This is also in agreement with no observed change in the shapeof the coastline.To verify the absence of dissolution(corrosion) of the BDD electrodeIL-STEM EELS measurements were carried out around the hole edge. IL-EELScan quantify thickness changes in the same area of the electron beamtransparent BDD in response to the AST cycling treatment. ethod,47 11λ is thversus Ag|AgCl,24 although in practice it is kinetically limited, occurringat much higher potentials.25 With increasingscan number, growth of the reductive peak at 0.90 V versus Ag|AgCl is also observed. This peak is attributed to cathodic strippingof Au surface oxides (AuOx),49 formed during the oxidative part of the scan.The Au support, also exposed to solution, is converted to AuOx at electrode potentials >∼1.2V versus Ag|AgCl. The increasing AuOx response with increasing number of cycles also indicatescorrosionof the thin C film coating the top surface of the Au grid, which resultsin more Au being exposed to solution with time. Unlike BDD, the C/AuTEM grid CV response is still changing after 100 cycles albeit lesssignificantly than during the initial 10 cycles, and by the 1000thcycle a response close to bulk gold is observed.49 This indicates that a significant amount of carbon hasbeen removed or damaged. The extent of corrosion damage to the carbonfilm was confirmed by TEM imaging. 4]− in 0.1 M HClO4. Ahigh driving potential of −0.5 V versus SCEwas employed for a very short period of time, 10 ms, to minimize thesize of the nanostructures electrodeposited. As can be seen underthese conditions observation of both crystalline Au NPs and isolatedsingle Au atoms is possible, 50 Given the mechanical and chemical robustness of the BDD-TEM supports,repeated imaging in the same location is also possible (IL-TEM),33 as shown in To highlight the capability of the BDD-TEM electrode as a combinedelectrodeposition and TEM imaging platform, Au nanostructures wereelectrodeposited on the electrode from a solution containing 1 mM[AuClca. 1500 °C in vacuum and 950 °Cin air51 and is an excellent conductorof heat52 (∼700 W m–1 K–1 at 300K). These properties not only enable BDD-TEM substrates to be usedin high temperature electrochemical applications,53 they also ensure that the temperature the BDD-TEM electrodeexperiences during in-situ TEM heating, matchesthat of the TEM heater. Here, BDD-TEM electrodes were employed toinvestigate the temperature-induced crystallization of electrodepositedamorphous manganese oxide (MnO2). MnO2 is usedcommercially in alkaline batteries due to the low cost and naturalabundance of Mn.54 The electrochemicallyactive γ-phase is favored due to its ability to facilitate protonintercalation.56Diamond also has the advantage it is thermallystable up to 2 on the BDD electrode.The interface between light and dark regions in the image indicatesthe BDD-TEM hole edge. Deposition was achieved by applying a potentialof 1.50 V versus SCE for 50 s in 0.1 M MnCl2 with 0.1 M KCl as a supporting electrolyte, acidified with HCl (0.01M).57 The electrode was then dipped indistilled water to remove any salt residues and left to air dry. Atthis potential and pH, Mn2+ is first oxidized to Mn3+, followed by acid-catalyzed hydrolysis to MnO2. Note, electrodeposition on the BDD-TEM electrode was carried out ex situ prior to placement in the TEM for the subsequentimaging/heating experiments. SAED data of the MnO2, recordedin the region of the red circle in 2 is amorphous due to the diffuse ring in 2-BDD-TEM electrode was heated in situ in the TEMand under vacuum. The electrode was heated first to 50 °C, thenallowed to cool to ambient temperature (in the TEM) and an image/diffractionpattern taken. This process was repeated, with the heating temperatureincreased in 50 °C increments , until crystallization was observeddue to the emergence of a diffraction pattern. Upon reaching 400 °C,TEM [Supporting Information 8, Figure S10). Latticespacingd* values were measured from the SAED pattern [2 ,56 the preferred phase of MnO2 for battery applications.Supporting Information 9, Figure S11. In particular, holes were seen forming due tothermal oxidation of the carbon film,58 which increased in severity with time, in addition to bending ofthe grid. This further emphasizes the wider range of operating/experimentalconditions accessible when using a BDD-TEM electrode.For some studies, it may be of interestfor the nanostructuredmaterial to undergo heating in air, with TEM imaging taking placebefore and after. To assess the suitability of BDD-TEM electrodesunder these conditions, experiments were conducted where both BDD-TEMand commercial C/Cu grids were heated to 200 °C and then 400°C in air for 4 h each. Visual inspection of the grids usingan optical microscope was carried out. For the BDD-TEM electrode novisual changes were observed. In contrast, for C/Cu the same gridcould not be used throughout due to visible damage to the grid afterthe first heating experiment, A top down facile fabrication method forthe production of re-useable,electron beam transparent and electrically conductive BDD-TEM electrodes,using argon ion milling and polishing of thin (<100 μm) BDDhas been demonstrated. In contrast with conventional C TEM grids,which typically require a metal support for handling, the resultingBDD-TEM electrodes self-support. This was due to the non-uniform natureof the milling process where the central region was thinned the most,resulting in a concave profile to both sides of the BDD-TEM grid anda very small hole  in the center.For combined electrochemical-TEM measurements this also meant theelectrochemical response was only due to the BDD and not a combinationof BDD and metal support. The electron beam transparent regions ofthe BDD-TEM electrode were shown to be thin enough to facilitate resolutionof electrochemically deposited and isolated single atoms (of Au).The central hole was also extremely helpful for IL-(S)TEM experiments,where the distinct shape of the hole edge was used to locate specificareas for repeat imaging.3 carbon character with minimal surface damage after ion milling.TEM diffraction studies showed a dominant (110) crystallography ofthe surface. Useful electrochemical properties in terms of large aqueoussolvent window and low double layer capacitance, were also retained.Moreover, the BDD-TEM grid was shown to be chemically and electrochemicallyresistant to carbon corrosion when subject to AST in acid, unlikecommercial C/metal TEM grids. Such properties are extremely usefulwhen developing NP catalyst supports, and are particularly suitablefor correlative electrochemical-TEM experiments, under corrosion freesupport conditions. Thermal stability in air was evaluated  by heating up to 400 °C. TheBDD-TEM electrodes remained intact unlike the C/metal grids. The advantageof having a thermally stable and electrically conductive TEM gridwas highlighted by using the BDD-TEM electrodes in combination with in situ TEM heating to investigate the temperature of crystallizationfor electrodeposited amorphous MnO2. Transition to theelectrochemically-active γ-MnO2 phase was shown at400 °C.A combination of techniques: SEM,TEM (including EELS), WLI, AFMand XPS, contact angle and electrochemical were employed to characterizethe surface and investigate the impact of ion milling on the BDD surfaceand electrochemistry. XPS determined the BDD kept its spFinally, with the current design of BDD-TEM electrode,the centerof the disk contains a very small hole. The mass transport profileat the very edge of the hole will be different to that further away.Future work is focused on the development of BDD-TEM electrodes whichare hole free and where the whole surface is electron beam transparent.Such electrodes should also find promise for combined optical detection-electrochemicalmeasurements.


We will place a particular emphasis on changing ethical considerations over time and the evolution of the model of care from gatekeeping to informed consent. To this end, we did an extensive review of the literature. We identified a trend across successive iterations of the guidelines in both reducing stigma against TGD individuals and shift in ethical considerations from “do no harm” to the core principle of patient autonomy. This has helped reduce barriers to care and connect more people who desire it to gender affirming care (GAC), but in these authors’ opinions does not go far enough in reducing barriers.Transgender and gender diverse (TGD) are terms that refer to individuals whose gender identity differs from sex assigned at birth. TGD individuals may choose any variety of modifications to their gender expression including, but not limited to changing their name, clothing, or hairstyle, starting hormones, or undergoing surgery. Starting in the 1950s, surgeons and endocrinologists began treating what was then known as transsexualism with cross sex hormones and a variety of surgical procedures collectively known as sex reassignment surgery (SRS). Soon after, Harry Benjamin began work to develop standards of care that could be applied to these patients with some uniformity. These guidelines, published by the World Professional Association for Transgender Health (WPATH), are in their 8th iteration. Through each iteration there has been a requirement that patients requesting gender-affirming hormones (GAH) or gender-affirming surgery (GAS) undergo one or more detailed evaluations by a mental health provider through which they must obtain a “letter of readiness,” placing mental health providers in the role of gatekeeper. WPATH specifies eligibility criteria for gender-affirming treatments and general guidelines for the content of letters, but does not include specific details about what must be included, leading to a lack of uniformity in how mental health providers approach performing evaluations and writing letters. This manuscript aims to review practices related to evaluations and letters of readiness for GAS in adults over time as the standards of care have evolved  Gender identity can be expressed through any combination of name, pronouns, hairstyle, clothing, and social role. Some TGD individuals wish to transition medically by taking gender-affirming hormones (GAH) and/or pursuing gender-affirming surgery (GAS) .1 The meHistorically physicians have placed significant barriers in the way of TGD people accessing the care that we now know to be lifesaving. Even today, patients wishing to receive GAC must navigate a system that sometimes requires multiple mental health evaluations for procedures, that is not required of cisgender individuals.The medical and psychiatric communities have used a variety of terms over time to refer to TGD individuals. The first and second editions of DSM described TGD individuals using terms such as transvestism (TV) and transsexualism (TS), and often conflated gender identity with sexuality, by including them alongside diagnoses such as homosexuality and paraphilias. Both the DSM and the International Classification of Diseases (ICD) have continuously changed diagnostic terminology and criteria involving TGD individuals over time, from Gender Identity Disorder in DSM-IV to Gender Dysphoria in DSM-5 to Gender Incongruence in ICD-11.3, renamed the World Profession Association for Transgender Health (WPATH) in 2006, was the first to publish international guidelines for providing GAC to TGD individuals. The WPATH Standards of Care (SOC) are used by many insurance companies and surgeons to determine an individual’s eligibility for GAC. Throughout each iteration, mental health providers are placed in the role of gatekeeper and tasked with conducting mental health evaluations and providing required letters of readiness for TGD individuals who request GAC  according to PRISMA guidelines. The search was comprised of database-specific controlled vocabulary and keyword terms for (1) mental health and (2) TGD-related surgeries. Searches were conducted on December 2, 2020 in MEDLINE (PubMed), the Cochrane Library Databases (Wiley), PsychINFO (EBSCOhost), CINAHL (EBSCOhost), Scopus (Elsevier), and Dissertations and Theses Global (ProQuest). All databases were searched from inception to present without the use of limits or filters. In total, 8,197 results underwent multi-pass deduplication in a citation management system (EndNote), and 4,411 unique entries were uploaded to an online screening software (Rayyan) for title/abstract screening by two independent reviewers. In total, 303 articles were included for full text screening , howeverIn total, 86 articles were included for review. Eleven articles were focused on ethical considerations while the remaining 75 articles focused on the mental health evaluation and process of writing letters of readiness for GAS. Version 8 of the SOC was published in September of 2022 during the review process of this manuscript and is also included as a reference and point of discussion.Fourteen articles were identified in the literature search as published prior to the development of the WPATH SOC version 1 in 1979. Prominent themes included classification, categorization, and diagnosis of TS. Few publications described the components of a mental health evaluation, and inclusion and exclusion criteria, for GAS. Many publications focused exclusively on transgender females, with a paucity of literature examining the experiences of transgender males during this timeframe.Authors emphasized accurate diagnosis of TS, highlighting elements of the psychosocial history including early life cross-dressing, preference for play with the opposite gender toys and friends, and social estrangement around puberty . One autMoney argued that the selection criteria for patients requesting GAS include a psychiatric evaluation to obtain collateral information to confirm the accuracy of the interview, work with the family to foster support of the individual, and proper management of any psychiatric comorbidities . AuthorsOthers focused the role of the psychiatric evaluation on the social lives and roles of the patient. They believed the evaluation should include exploring the patient’s motivation for change for at least 6–12 months , facilitEll recommended evaluation to ensure the patient has “adequate intelligence” to understand realistic expectations of surgery and attempted to highlight the patient’s autonomy in the decision to undergo GAS. He wrote, “That is your decision [to undergo surgery]. It’s up to you to prove that you are a suitable candidate for surgery. It’s not for me to offer it to you. If you decide to go ahead with your plans to pass in the opposite gender role, you do it on your own responsibility” . NotablyIn these earliest publications, one can start to see the beginning framework of modern-day requirements for accessing GAS, including ensuring an accurate diagnosis of gender incongruence; ruling out other possible causes of presentation such as psychosis; ensuring general mental stability; making sure that the patient has undergone at least some time of living in their affirmed gender; and that they are able to understand the consequences of the procedure.The first two versions of the WPATH SOC were written in 1979 and 1980, respectively and are substantially similar to one another. SOC version three was the first to be published in an academic journal in 1985 and changes from the first two versions were documented within this publication. The first two versions required that all recommendations for GAC be completed by licensed psychologists or psychiatrists. The first version recommended that patients requesting GAH and non-genital GAS, spend 3 and 6 months, respectively, living full time in their affirmed gender. These recommendations were rescinded in subsequent versions . Figure Five articles published between 1979 and 1980 were included in this review. Again, emphasis was placed on proper diagnosis, classification and consistency of gender identity over time , 18.Wise and Meyer explored the concept of a continuum between TV and TS, describing that those who experienced gender dysphoria often requested GAS, displayed evidence of strong cross-dressing desires with arousal, history of cross-gender roles, and absence of manic-depressive or psychotic illnesses . RequireVersion 3 broadened the definition of the clinician thereby broadening the scope of providers who could write recommendation letters for GAC. Whereas prior SOC required letters from licensed psychologists or psychiatrists, version 3 allowed initial evaluations from providers with at least a Master’s degree in behavioral science, and when required, a second evaluation from any licensed provider with at least a doctoral degree. Version 3 recommended that all evaluators demonstrate competence in “gender identity matters” and must know the patient, “in a psychotherapeutic relationship,” for at least 6 months . VersionNine articles were published during the timeframe that the SOC version 3 were active (1981–1990). Themes in these publications included increasing focus on selection criteria for GAS and emphasis on the RLT, which was used to ensure proper diagnosis of gender dysphoria. Recommendations for the duration of the RLT ranged anywhere between 1 and 3 years , 23.Proposed components of the mental health evaluation for GAS included a detailed assessment of the duration, intensity, and stability of the gender dysphoria, identification of underlying psychiatric diagnoses and suicidal ideation, a mental status examination to rule out psychosis, and an assessment of intelligence  to comment on the individual’s “capacity and competence” to consent to GAC. The Minnesota Multiphasic Personality Inventory (MMPI), Weschler Adult Intelligence Scale (WAIS), and Lindgren-Pauly Body Image Scale were also used during assessments .Authors developed more specific inclusion and exclusion criteria for undergoing GAS with inclusion criteria including age 21 or older, not legally married, no pending litigation, evidence of gender dysphoria, completion of 1 year of psychotherapy, between 1 and 2 years RLT with ability to “pass convincingly” and “perform successfully” in the opposite gender role, at least 6 months on GAH , reasonably stable mental health , good financial standing with psychotherapy fees , and a pWorld Professional Association for Transgender Health SOC version four was published in 1990. Between version three and version four, DSM-III-R was published in 1987. Version four relied on the DSM-III-R diagnostic criteria for TS as opposed to the DSM-III criteria in version three. The DSM-III-R criteria for TS included a “persistent discomfort and sense of inappropriateness about one’s assigned sex,” “persistent preoccupation for at least 2 years with getting rid of one’s primary and secondary sex characteristics and acquiring the sex characteristics of the other sex,” and that the individual had reached puberty . NotableSix articles were published between 1990 and 1998 while version four was active. Earlier trends continued including emphasizing proper diagnosis of gender dysphoria , 32, howBockting and Coleman, in a move representative of other publications of this era, advocated for a more comprehensive approach to the mental health evaluation and treatment of gender dysphoria. Their treatment model was comprised of five main components: a mental health assessment consisting of psychological testing and clinical interviews with the individual, couple, and/or family; a physical examination; management of comorbid disorders with pharmacotherapy and/or psychotherapy; facilitation of identity formation and sexual identity management through individual and group therapy; and aftercare consisting of individual, couple, and/or family therapy with the option of a gender identity consolidation support group. Psychoeducation was a main thread throughout the treatment model and a variety of treatment “subtasks” such as understanding decision making, sexual functioning and sexual identity exploration, social support, and family of origin intimacy were identified as important. The authors advocated for “a clear separation of gender identity, social sex role, and sexual orientation which allows a wide spectrum of sexual identities and prevents limiting access to GAS to those who conform to a heterosexist paradigm of mental health” .This process can be compared with the Italian SOC for GAS which recommend a multidisciplinary assessment consisting of a psychosocial evaluation and informed consent discussion around treatment options, procedures, and risks. Requirements included 6 months of psychotherapy prior to initiating GAH, 1 year of a RLT prior to GAS, and provision of a court order approving GAS, which could not be granted any sooner than 2 years after starting the process of gender transition. Follow-up was recommended at 6, 12, and 24 months post-GAS to ensure psychosocial adjustment to the affirmed gender role .Other authors continued to refine inclusion and exclusion criteria for GAS by surveying the actual practices of health centers. Inclusion criteria included those who had life-long cross gender identification with inability to live in their sex assigned at birth; a 1–2 years RLT ; and ability to pass “effortlessly and convincingly in society”; completed 1 year of GAH; maintained a stable job; were unmarried or divorced; demonstrated good coping skills and social-emotional stability; had a good support system; and were able to maintain a relationship with a psychotherapist. Exclusion criteria included age under 21 years old, recent death of a parent , unstablThe survey indicated some programs were more lenient around considering individuals with bipolar affective disorder, the ability to pass successfully, and issues around family support. Only three clinics used sexual orientation as a factor in decision for GAS, marking a significant change in the literature from prior decades. Overall, the authors found that 74% of the clinics surveyed did not adhere to WPATH SOC, instead adopting more conservative policies .Published in 1998, version five defined the responsibilities of the mental health professional which included diagnosing the gender disorder, diagnosing and treating co-morbid psychiatric conditions, counseling around GAC, providing psychotherapy, evaluating eligibility and readiness criteria for GAC, and collaborating with medical and surgical colleagues by writing letters of recommendation for GAC . EligibiFive articles were published between 1998 and 2001 while version five was active. Two of these articles were summaries of the SOC , 38. TheMa reviewed the role of the social worker in a multidisciplinary gender clinic in Hong Kong. Psychosocial assessment for GAS included evaluation of performance in affirmed social roles, adaptation to the affirmed gender role during the 1-year RLT and understanding the patient’s identified gender role and the response to the new gender role culturally and interpersonally within the individual’s support network and family unit. She noted five contraindications to GAS: a history of psychosis, sociopathy, severe depression, organic brain dysfunction or “defective intelligence,” success in parental or marital roles, “successful functioning in heterosexual intercourse,” ability to function in the pretransition gender role, and homosexual or TV history with genital pleasure. She proposed a social work practice model for patients who apply for GAS with categorization of TGD individuals into “better-adjusted” and “poorly-adjusted” with different intervention goals and methods for each. For those who were “better-adjusted,” treatment focused on psychoeducation, building coping tools, and mobilization into a peer counselor role, while treatment goals for those who were “poorly-adjusted” focused on building support and resources .Damodaran and Kennedy reviewed the assessment and treatment model used by the Monash gender dysphoria clinic in Melbourne, Australia for patients requesting GAS. All referrals for GAS were assessed independently by two psychiatrists to determine proper diagnosis of gender dysphoria, followed by endocrinology and psychology consultation to develop a comprehensive treatment plan. Requirements included RLT of minimum 18 months and GAH .Miach reviewed the utility of using the Minnesota Multiphasic Personality Inventory-2 (MMPI-2), a revision of the MMPI which was standardized using a more heterogeneous population, in a gender clinic to assess stability of psychopathology prior to GAS, which was only performed on patients aged 21–55 years old. The authors concluded that while the TGD group had a significantly lower level of psychopathology than the control group, they believed that the MMPI-2 was a useful test in assessing readiness for GAC .Published in 2001, version six of the WPATH SOC did not include significant changes to the 10 tasks of the mental health professional  or in thThirteen articles were published between 2001 and 2012. One is a systematic review of evidence for factors that are associated with regret and suicide, and predictive factors of a good psychological and social functioning outcome after GAC. De Cuypere and Vercruysse note that less than one percent of patients regret having GAS or commit suicide, making detection of negative predictive factors in a study nearly impossible. They identified a wide array of positive predictive factors including age at time of request, sex of partner, premorbid social or psychiatric functioning, adequacy of social support system, level of satisfaction with secondary sexual characteristics, and surgical outcomes. Many of these predictive factors were later disproved. They also noted that there were not enough studies to determine whether following the WPATH guidelines was a positive predictive factor. In the end they noted that the evidence for all established evaluation regimens  was at best indeterminate. They recommended that changes to WPATH criteria should redirect focus from gender identity to psychopathology, differential diagnosis, and psychotherapy for severe personality disorders .The literature at this time supports two opposing approaches to requests for GAC, those advocating for a set of strictly enforced eligibility and readiness criteria associated with very thorough evaluations and those who advocate for a more flexible approach. Common approaches to the evaluation for GAC include: taking a detailed social history including current relationships, support systems, income, and social functioning; a sexual development history meant to understand when and how the patient began to identify as TGD and how their transition has affected their life; an evaluation of their coping skills, “psychic functions” and general mental well-being; and a focus on assessing the “correct diagnosis” of gender identity disorder –56. The Those that advocated for a stricter interpretation of the eligibility and readiness criteria emphasized the importance of the RLT , 55, 56.Among groups supporting a flexible interpretation of the SOC, there was a much stronger emphasis on the supportive role of the mental health provider in the gender transition process , 52, 53.The lack of emphasis on informed consent by both groups of authors mirrors the discussion of informed consent within the SOC, which up through version six, had a relatively narrow definition and role specifically related to risks and benefits of surgery. As far back as version one, the SOC states “hormonal and surgical sex reassignment are procedures which must be requested by, and performed only with the agreement of, the patient having informed consent…[these procedures] may be conducted or administered only after the patient applicant has received full and complete explanations, preferably in writing, in words understood by the patient applicant, of all risks inherent in the requested procedures . “This rguidelines meant to be flexible to account for different practices in different places. Compared to version six, a significantly expanded section on the “Tasks of the Mental Health Provider” was added, offering some instructions on what to include in the assessment of the patient for GAS. For the first time the SOC expand on what it means to obtain informed consent and describe a process where the mental health provider is expected to guide a conversation around gender identity and how different treatments and procedures might affect TGD individuals psychologically, socially, and physically. Other recommendations include “at a minimum, assessment of gender identity and gender dysphoria, history and development of gender dysphoric feelings, the impact of stigma attached to gender non-conformity on mental health, and the availability of support from family, friends, and peers.” There is also a change to the recommended content of the letters: switching from “The initial and evolving gender, sexual, and other psychiatric diagnoses” to “Results of the client’s psychosocial assessment, including any diagnoses”, indicating a shift in the focus away from diagnosis toward the psychosocial assessment. Version 7 also adds two new tasks for the mental health provider including “Educate and advocate on behalf of clients within their community  and assist clients with making changes in identity documents” and “Provide information and referral for peer support” vs. external . For internal surgeries, criteria are the same as for top surgery with the addition of a required 12 months of GAH. For external surgeries the criteria are the same as for internal, with the addition of required 12 months of living in the patient’s affirmed gender identity .Twenty-three articles were published while version 7 of the SOC have been active. Themes include identifying the role of psychometric testing in GAC evaluations, expanding the discussion around informed consent for GAC, and revising the requirements for letter writers.A systematic review evaluated the accuracy of psychometric tests in those requesting GAC, identifying only two published manuscripts that met their inclusion criteria, both of which were of poor quality; this led them to question the utility of psychometric tests in in TGD patients . Keo-MeiMany authors describe the process of informed consent for GAC , 62–76. Practice patterns and opinions on who should write letters of readiness and how many letters should be required vary widely. Many letters that surgeons receive are cursory, and short and non-personal letters correlate with poor surgical outcomes . SeveralThe Mount Sinai Gender Clinic describes an integrated multidisciplinary model where a patient will see a primary care doctor, endocrinologist, social worker, psychiatrist, and obtain any necessary lab work in a single visit, significantly reducing barriers to care. The criteria in this model focus on informed consent, the social determinants of health, being physically ready for surgery, and putting measurable goals on psychiatric stability, while deemphasizing the gender dysphoria diagnosis. Their study showed that people who received their evaluation over a 2-year period were more likely to meet their in-house criteria than they were to meet criteria as set forth in WPATH SOC. The Mount Sinai criteria allowed for significantly decreased barriers to care, allowing more people to progress through desired GAC in a timely fashion .Standards of care version 8, published in September 2022, includes major updates to the guidelines around GAS. This version explicitly highlights the importance of informed decision making, patient autonomy, and harm reduction models of care, as well as emphasizing the flexibility of the guidelines which the authors note can be modified by the healthcare provider in consultation with the TGD individual.Version 8 lays out the roles of the assessor which are to identify the presence of gender incongruence and any co-existing mental health concerns, provide information on GAC, support the TGD individual in their decision-making, and to assess for capacity to consent to GAC. The authors emphasize the collaborative nature of this decision-making process between the assessor and the TGD individual, as well as recommending TGD care occur in a multidisciplinary team model when possible.Version 8 recommends that providers who assess TGD individuals for GAC hold at least a Master’s level degree and have sufficient knowledge in diagnosing gender incongruence and distinguishing it from other diagnoses which may present similarly. These changes allow for non-mental health providers to be the main assessors for GAC.Version 8 recommends reducing the number of evaluations prior to GAS to a single evaluation in an effort to reduce barriers to care for the TGD population. Notably, the authors have removed the recommendations around content of the letter of readiness for GAC. The guidelines note that the complexity of the assessment process may differ from patient to patient, based on the type of GAC requested and the specific characteristics of the patient. Version eight directly states that psychometric testing and psychotherapy are not requirements to pursue GAC. While evaluations should continue to identify co-existing mental health diagnoses, version 8 highlights that the presence of a mental health diagnosis should not prevent access to GAC unless the mental health symptoms directly interfere with capacity to provide informed consent for treatment or interfere with receiving treatment. Version 8 recommends that perioperative matters, such as travel requirements, presence of stable, safe housing, hygiene/healthy living, any activity restrictions, and aftercare optimization, be discussed by the surgeon prior to GAS. In terms of eligibility criteria, the authors recommend a reduced duration of GAH from 12 months (from version 7) to 6 months (in version 8) prior to pursuing GAS involving reproductive organs .A total of eleven articles explored ethical considerations of conducting mental health evaluations and writing letters of readiness for GAS, including a comparison of the ethical principles prioritized within the “gatekeeping” model vs. the informed consent model for GAC and the differential treatment of TGD individuals compared to cisgender individuals seeking similar surgical procedures.Many authors compare the informed consent model of care for TGD individuals to the WPATH SOC model. In the informed consent model, the role of the health practitioner is to provide TGD patients with information about risks, side effects, benefits, and possible consequences of undergoing GAC, and to obtain informed consent from the patient . Cavanauvia such systems like the WPATH SOC  to write letters for surgery . In pracThere are few published guides for writing letters of readiness for GAC. The WPATH SOC provide vague guidelines as to the information to include within the letter itself, which, in addition to a lack of consistency in implementation of the SOC, lead to a huge variety in current practices around letter writing and limit their usefulness to surgical providers . There iThe reviewed articles included opinion manuscripts, published SOC, and proposed models for how to design and operate GAC clinics, however, this narrative review is limited by a lack of peer reviewed clinical trials that assess the evidence for the GAC practices described here. As a result, it is challenging to comment on the effectiveness of various interventions over time.The WPATH SOC have evolved significantly over time with regards to their treatment of TGD individuals. Review of the literature shows a clear progression of practices from paternalistic gatekeeping toward increasing emphasis on patient autonomy and informed consent. Mental health evaluations, still required by SOC version eight are almost entirely unique as a requirement for GAS, apart from some bariatric and transplant surgeries. Individuals who wish to pursue GAC are required to get approval for treatments that their cisgender peers may pursue without such evaluations. While there may be some benefits from these evaluations in helping to optimize a patient socially, emotionally, and psychologically for GAC, the increased stigma and burden placed on patients by having a blanket requirement for such evaluations leads us to seriously question the readiness evaluation requirements in SOC version 8, despite a reduction in the requirements compared to previous SOC. This burden is made worse by limited access to providers knowledgeable and competent in conducting GAC evaluations, writing letters of readiness, and a lack of consistency in the application and interpretations of the SOC by both providers and insurance companies. Other barriers to care created by multiple letter requirements include the often-prohibitive cost of getting multiple evaluations and the delay in receiving their medical or surgical treatments due to extensive wait times to see a mental health provider. This barrier will in theory be ameliorated by updates to SOC in version 8, but multiple letters are likely to at least be required by insurance companies for some time. Overall, the shift from gate keeping to informed consent has been a net positive for patients by reducing barriers to care and improving patient autonomy, but the mental health evaluation is still an unnecessary barrier for many people. Further research is necessary to develop a standardized evaluation and letter template for providers to access, as well as further study into who can most benefit from an evaluation in the first place.The original contributions presented in this study are included in the article/TA and KK contributed to the conception and design of the study under the guidance of RL and AJ, reviewed and analyzed the literature, and wrote the manuscript. AW organized the literature search and wrote the “Methods” section. RL and AJ assisted in review and revision of the completed manuscript. All authors approved of the submitted version.