Oral cancer is one of the life threatening disease which requires an availability of a biomarker for its early detection and also for effective treatment strategies. The current study is done to evaluate the efficacy of one such biomarker i.e. TNF- α as an indicator for oral precancer and oral cancer. To evaluate the efficacy of Tumour necrosis factor - alpha (TNF)-α as a salivary biomarker in histopathologically diagnosed cases of oral leukoplakia and Oral squamous cell carcinoma. To correlate the levels of TNF- α with varying histologic grading in Oral Squamous Cell Carcinoma and dysplasia grading in Oral leukoplakia or Hyperkeratosis. The study group included 90 subjects that were divided into three groups. OSCC (n=30), leukoplakia (n=30) and controls (n=30). Cases were selected based on inclusion and exclusion criteria of the study. Salivary samples were then collected from all three groups. Salivary levels of TNF-α were estimated using Enzyme Linked Immunosorbent Assay (ELISA). The data on concentration gradients obtained were subjected to appropriate statistical analysis. The results of the present study demonstrated higher levels of salivary TNF-α in individuals with OSCC compared to leukoplakia and healthy control subjects with a high level of statistical significance. ROC curve analysis along with diagnostic parameter calculation also revealed that salivary TNF-α to be a better medium for detecting OSCC. There is also an increase in the salivary TNF-α levels with increase in the histological grade of differentiation in OSCC as well as leukoplakia. The present study concludes that salivary TNF – α can be used as a prognostic biomarker of OSCC. In view of the elevated levels of TNF – α in saliva of individuals with severe dysplasia, it can also be used to monitor the malignant transformation to leukoplakia to OSCC.  Oral cancers being the most common of Head and neck cancers, accounts for 354,864 new cases and 177,384 cancer related deaths worldwide in 2018. Cancers of lip and oral cavity are highly frequent in southern Asia and also leading cause of death in men of India and Sri Lanka . Oral cancer comprises about 80–90% of head and neck cancers, with the most common variant being oral squamous cell carcinoma (OSCC) . The combined influence of genetic and environmental exposure to carcinogens resulting in genetic damage. These accumulated genetic damages contribute to the formation of OSCC which may sometimes predispose from a clinically evident oral potentially malignant disorders (OPMD), the most common being the Oral leukoplakia . Most of these oral cancers show a strong association with tobacco and smoking habits, which upon chronic exposure results in changes in the oral mucosa resulting in the development of OPMDs and OSCC. Despite the development of treatment modalities which can bring about the improvement in the survival and prognosis of oral cancer, it still continues to be the life-threatening disease as there is definite absence of signs which would help in the early detection of OSCC. To date, there are various gold standard management and diagnostic options for oral cancer which are based on biopsy and histopathological analysis. Gold standard procedures are not convenient in screening procedures of oral cancer among high-risk population . Hence, it is likely that few biomarkers are needed to predict the evolution of OSCC from an OPMD and also to screen the high-risk patients with tobacco and/or smoking habits. It is of very much importance that the combined early detection and the effective treatment strategies could help in the prevention and the good prognostic outcomes of oral cancer .The development of cytokines as biomarkers for detecting malignancies has helped in realizing the importance of inflammation-mediated carcinogenesis in OPMDs and in oral cancer. The most common of which include tumor necrosis factor alpha (TNF-α), interleukins (IL)-6 and -8, vascular endothelial growth factor (VEGF), IL -4 and -10 . TNF – α, a widely expressed pro-inflammatory cytokine, has been shown diagnostic utility in oral cancer and is found more abundant in saliva compared to that in serum .TNF-α is a pleiotropic, pro- inflammatory cytokine, and is a double-edged sword being the pro and anti- tumorigenic i.e. TNF- α can be cytotoxic to tumor cells, inhibiting the progression of the tumor or causing necrosis and it can also stimulates angiogenesis, proliferation, migration and survival of tumour cells in most cancer cells . The constraint in the number of studies conducted to detect the actual response of TNF- α limits the knowledge of its role in oral precancer and oral cancer.Thus, with the above facts, the objective of the present study was to evaluate the utility and to determine the role of salivary TNF- α in OPMDs and OSCC correlating with the grades of dysplasia and histological differentiation respectively using ELISA technique.Study subjects and the source of the dataThe subjects are patients who presented to Kamineni Institute of Dental Sciences, Narketpally, Nalgonda; MNJ Institute of Oncology, Redhills, Hyderabad and Government Dental College, Afzalgunj Hyderabad. The study included 90 subjects that were divided into three groups. Group A included 30 oral leukoplakia patients who were clinically and histopathologically diagnosed into different grades of oral epithelial dysplasia (OED). WHO in year 2005 classified OED as mild, moderate, severe and carcinoma in situ according to the presence and severity of cellular atypia and the architectural features. Group B included 30 patients of OSCC who were clinically and histopathologically diagnosed with varying grades i.e., well / moderately / poorly differentiated oral squamous cell carcinoma. Group C included healthy controls with age, sex match and periodontal status confirmed by Community periodontal index (CPI) matched to that of group A and B. The group A and B individuals were clinically diagnosed and were not currently undergoing or having undergone any form of definitive therapy in the form of surgery, radiation, chemotherapy or any other adjunctive treatments for OSCC. Histopathological gradingThe histopathological specimens were observed under light microscope (4-40 X magnification) by an Oral and Maxillofacial Pathologist expert(s). The histopathological grading was done based on the following grading systems: Leukoplakia- according to WHO grading of Oral Epithelial Dysplasia, OSCC were graded using Broder’s grading system.The study received Institutional ethical committee approval from Kamineni Institute of Dental Sciences, Hyderabad. Written informed consents were taken from all the participating subjects.Exclusion criteriaIndividuals with any other underlying illnesses that would raise levels of cytokines in saliva were excluded. Individuals with history of local therapeutic medications for any other oral lesions or medications such as anti-histaminics, anti-hypertensives, anti-cholinergics, antidepressants, bronchodilators, or pathological dry mouth syndrome, or inability to collect sufficient saliva samples on a reliable basis.Saliva collectionoC till further analysis. One freeze thaw cycle was done per sample. Salivary samples were collected from subjects during 10:00 AM to 12:00 PM under non-stimulatory condition. Participants were guided to refrain from either eating or drinking at least one hour before collection of salivary samples. Subjects were asked to rinse out their mouth with water at least 5 min prior to saliva collection. Samples were obtained by requesting subjects to swallow first, tilt their head forward, and expectorate all saliva into the sterile disposable tubes. The collected saliva is centrifuged at 3,500 rpm for 10 minutes to remove debris. The supernatant is collected into 1.5ml eppendorf tubes. These tubes with the supernatant sample was stored at -80TNF- α estimationEstimation of salivary TNF- α was done by using DeQuantoTM Human TNF alpha ELISA kit, # QT 4001. The assay was carried out according to the manufacturer’s instructions with some modifications, because the kits were primarily designed for serum samples wherein the concentrations of these constituents are generally high. The technique of ELISA followed here was sandwich method. The method briefly follows: 2SO4) to each well was added. The resultant product is yellow. The changes in colour intensity and the absorbance at 450 nm and 600 nm was read using ELISA microplate reader . The samples/ test specimens were brought to the room temperature. Addition of 100 μl of test/specimen samples to each well of pre - TNF- α antibody coated 96-well microtiter plate, which was s incubated for 1 hour and washed. Followed by addition of 100 μl of detection antibody to each well of the microtiter plate and incubated for 1 hour at ~25°C followed by washing. Then adding of 100 μl of streptavidin - HRP to each well of the microtiter plate and incubate for 30 minutes at ~25°C, followed by adding 100 μl of TMB substrate solution to each well of the microtiter plate and incubated for 15 minutes in dark at ~25°C is done. To stop the reaction, 50 μl of stop solution  values of standards (S1 S6) against their concentrations . The OD Statistical analysisAll statistical procedures were performed with SPSS version SPSS 19.0 (PASW statistics). Data were tested for normality using Ryan- joiner test. Descriptive statistics like mean, median, interquartile range and standard deviation were calculated. The Kruskal-Wallis analysis of variance and the Mann-Whitney U test were used to evaluate differences between multiple group and unpaired observations respectively.Demographic characteristicsThe present study was conducted to evaluate the role of salivary TNF- α in oral leukoplakia and OSCC using ELISA technique. The age and sex matched 30 control subjects, 30 leukoplakia subjects and 30 OSCC subjects were included. All the oral leukoplakia and OSCC subjects either had tobacco/pan chewing/smoking habits. The oral leukoplakia lesions were from buccal mucosa (n=19), palate (n= 1), tongue (n= 2), floor of the mouth (n= 3) and retromolar trigone (n= 5). Similarly, the OSCC lesions were from buccal mucosa (n=7), alveolar mucosa (n=2), palate (n=4), tongue (n=8), floor of the mouth (n= 4), retromolar trigone (n= 2) and oropharynx (n= 3). Based on the histopathological grading of OSCC, 13 (43.33%) patients had well differentiated, 10 (33.33%) had moderately differentiated and 7 (23.33%) had poorly differentiated lesions. Similarly, based on the histopathological grading of the leukoplakia , 10 (33.33%) patients had mild epithelial dysplasia, 10 (33.33%) patients had moderate epithelial dysplasia and 10 (33.33%) patients had severe epithelial dysplasia. The study population in all the groups were predominantly males and the gender wise distribution of different groups is illustrated in . DemograComparison of concentration of salivary TNF - α levels among different groupsThe mean and standard deviation of the salivary levels of TNF-α in OSCC, leukoplakia and control groups are calculated and are 63.94 ± 56.05, 28.96 ± 20.94 and 5.75 ±3.98 respectively. The comparison of these values among the three groups are illustrated in . The desThe test for mean score analysis  procedure is used to compare mean scores of more than two groups. The test showed a highly significant (p = 0.00) difference between the two groups (p<0.01) .Comparison of TNF – α levels in different grades of OED (Leukoplakia)Among 30 patients of leukoplakia, 10 (33.33%) patients had mild epithelial dysplasia, 10 (33.33%) patients had moderate epithelial dysplasia and 10 (33.33%) patients had severe epithelial dysplasia. Comparison of salivary TNF – α levels in these different grades of oral epithelial dysplasia (leukoplakia) is done by using one – way ANOVA test, the results showed that there is no significant difference among different grades dysplasia. The p value of 0.084565 which is non- significant (p>0.01) is obtained . Comparison of TNF – α levels in different grades of OSCCFor 30 patients in OSCC, 13 (43.33%) patients had well differentiated, 10 (33.33%) had moderately differentiated and 7 (23.33%) had poorly differentiated lesion. Comparison of salivary TNF – α levels in these grades of OSCC is done by using one – way ANOVA test, which showed a highly significant increase in PDSCC compared to MDSCC and WDSCC with a p value of 0.000424 (p<0.01) . ROC curve analysisROC curve analyses revealed that salivary TNF-α could serve as valuable biomarker for differentiating leukoplakia  from healthy controls with an area under the curve (AUC) of 0.968 . Significant difference between salivary TNF - α levels of leukoplakia and controls with AUC was found to be 0.00 (p<0.001). At the cut- off value of 10.25 pg/ml, the salivary TNF-α showed a sensitivity and specificity of 90 % and 93.3% respectively . ROC curve analyses revealed that salivary TNF-α could serve as valuable biomarker for differentiating OSCC from healthy controls with an area under the curve (AUC) of 0.997 . Significant difference between salivary TNF - α levels of OSCC and controls with AUC was found to be 0.00 (p<0.001). At the cut- off value of 15.100 pg/ml, the salivary TNF-α showed a sensitivity and specificity of 100 % and 96.7% respectively .Diagnostic utilityThe results of the present study demonstrated higher level of salivary TNF-α production in OSCC while comparing to premalignant disorder and healthy control subjectswith a high level of statistical significance. The comparison of mean levels between different grades of oral epithelial dysplasia showed non- significant increase from mild to severe dysplasia. The mean values between different grades of OSCC showed a highly significant increase in salivary TNF-α levels from WDSCC to PDSCC. ROC curve analysis along with diagnostic parameter calculation reveals salivary TNF-α to be a better medium for detecting OSCC.OSCC comprises of the tumour epithelium and the surrounding connective tissue stroma which constitutes the tumour microenvironment (TME) within which varying populations of mesenchymal cells, extracellular matrix, and inflammatory cells are present . TME provides the cross- talk between the tumour cells and the stromal elements like inflammatory cells & cancer associated fibroblasts contributing to the development, growth, invasion and metastasis of the tumour. This crosstalk is mediated by the generation of several inflammatory cytokines such as TNF – α. These cytokines attract and recruit more inflammatory cells to the tumour microenvironment to further enhance the proliferation and survival of genetically altered tumour cells .Figure 2).TNF-α receptors are expressed on both epithelial and stromal cells. TNF receptor 1 (TNFR-1) and TNFR-2, TNFR-1 is ubiquitously expressed while TNFR-2 is expressed in immune cells. TNF binds to TNF receptor I and/or II (TNFR1 and 2) to activate a receptor signalling complex, which consists of several death-domain-containing proteins, and to transduce two major intracellular signalling pathways: Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) and the caspase cascade . Ligand binding to TNFR1 induces activation of the activator protein -1 (AP1) transcription factors or IκB kinases (IKKs) that, in turn, activate NF-κB. NF-κB activation also importantly induces negative regulators of apoptosis such as FLIPL, BCL-2 and superoxide dismutase. If NF-κβ activation is inadequate, apoptosis is mediated through caspase 8 and and mitochondrial pathways   compared to that of OED (28.96 ± 20.94) and control groups (5.75 ±3.98). These values are statistically significant. These results were in concordance with the studies of Rhodus et al., (2005); Saheb Jamee et al., (2008); Juretic et al., (2012); Krishnan et al., (2014).Rhodus et al., (2005) showed that NF-κB-dependent cytokines i.e TNF-α, IL-1α, IL-6, and IL-8 were elevated in the whole unstimulated saliva of subjects with OSCC compared with PML and controls. ie. TNF- α levels of OSCC are 28.9 ± 14.6, PML with 10.5 ± 7.4 and controls with 3.0 ± 1.9. They have concluded that it is possible that the overexpression of NF-κB correlates with the over-expression of these pro- inflammatory, pro- angiogenic cytokines which may result in the progression of oral cancer from OPMD . SahebJamee et al., (2008) studied the concentrations of salivary TNF-α, IL-1, 6 and IL- 8 using ELISA and concluded that the concentration of salivary cytokine in OSCC was higher than control group with mean values of OSCC with 35.2 ± 51.8 pg/ ml and controls with 4.1 ± 2.1 pg/ml. Juretic et al., (2012) conducted a study to determine the salivary concentrations of TNF-α in patients with premalignant lesions (PML) and malignant lesions using ELISA and observed that the mean values of TNF-α are greater in OSCC (0.739 ± 0.176) than PML (0.601 ± 0.178) and controls (0.013 ± 0.033) . Krishnan et al., (2014) conducted a study to validate the efficacy of TNF- α in diagnosing premalignant oral lesions and OSCC. The salivary TNF- α levels are greater in OSCC with a mean value of 311.9+ 95.3 than PML with mean value of 136.8+59.6 and controls with 4.5+ 2.5. There is also a statistically significant increase in the mean values of salivary TNF – α levels in different grades of OSCC is observed in the present study. The median values of salivary TNF – α levels of WDSCC, MDSCC and PDSCC are as 32.5, 41.75 and 150 pg/ml respectively. These results are in concordance with Krishnan et al., (2014); where evaluation of correlation of the levels of salivary and serum TNF- α with clinic-pathologic factors is done.Though there are various studies correlating the concentration of the TNF- α levels with histological differentiation of OSCC, but there are very limited studies correlating with the degree of dysplasia. So, the present study addresses that issue. There is an increase in the mean values of salivary TNF – α levels in different grades of OED from mild dysplasia to severe dysplasia, but the values are not statistically significant. This altered cytokine responsiveness is tightly associated with the development of oral cancer. In normal cells, stimulation with proinflammatory cytokines leads to growth inhibition, while in oral cancer cells stimulation with proinflammatory cytokines leads to up regulation of posi¬tive cell cycle regulators such as nuclear factor kappa B (NF-κB). Various studies have confirmed that aberrant activation of NF-κβ has been implicated in the development and progression of human cancers, including head and neck squamous cell carcinoma. This results in upregulation of proinflammatory cytokines, proangiogenic factors and anti- apoptotic factors responsible for the tumour growth. Likewise, the constitute production of TNF- α, will be associated with tumour prognosis .The sensitivity and specificity values of TNF – α in OSCC are 100 % and 96.7% respectively and sensitivity and specificity values of TNF – α in OED are 90 % and 93.3% respectively. In reference to these above mentioned values the validity and reliability are very high indicating that salivary TNF – α can be used as a biomarker in OSCC and OED. Though salivary markers could not identify the location from which the tumor originated, but they can identify the individuals at risk. These kind of salivary tests can be managed by non- specialists in remote areas as a screening tool to refer the patients affected for careful evaluation to detect the early stage diseases. Saliva tests can also be used to monitor patients in postoperative management of OSCC and may prove beneficial in chemoprevention trials. But testing for biomarkers in body fluids must be done in a prospective, blinded fashion in clinical settings requiring definite detection of disease. The efficiency of these biomarkers can only be estimated only based on a well-designed, prospective multi-institutional trial with larger sample size.As the present study found that there is an increase in expression of salivary TNF- α levels from controls to leukoplakia to OSCC, this reinforces the hypothesis of TNF- α being a pro- tumorigenic. The present study also supports the utility of saliva in biomarker estimation and in evaluating TNF- α as a prognostic marker, also as an indicator for the neoplastic transformation from OPMD to cancer and in mass screening programmes.


Vaccines based on virus-like particles (VLPs) can induce potent B cell responses. Some non-chimeric VLP-based vaccines are highly successful licensed products . Chimeric VLPs are designed to take advantage of the VLP framework by decorating the VLP with a different antigen. Despite decades of effort, there have been few licensed chimeric VLP vaccines. Classic approaches to create chimeric VLPs are either genetic fusion or chemical conjugation, using cross-linkers from lysine on the VLP to cysteine on the antigen. We describe the principles that make these classic approaches challenging, in particular for complex, full-length antigens bearing multiple post-translational modifications. We then review recent advances in conjugation approaches for protein-based non-enveloped VLPs or nanoparticles, to overcome such challenges. This includes the use of strong non-covalent assembly methods (stick), unnatural amino acids for bio-orthogonal chemistry (click), and spontaneous isopeptide bond formation by SpyTag/SpyCatcher (glue). Existing applications of these methods are outlined and we critically consider the key practical issues, with particular insight on Tag/Catcher plug-and-display decoration. Finally, we highlight the potential for modular particle decoration to accelerate vaccine generation and prepare for pandemic threats in human and veterinary realms. The medical and commercial success of virus-like particle (VLP) vaccines against hepatitis B virus  and human papillomavirus  is attributed to their particulate nature , 2. The via major histocompatibility complex (MHC) cross-presentation . Given that many VLPs are icosahedral, the symmetry at the fusion site should match the symmetry of the intended fusion. Other important antigens are heteromultimeric, such as in herpesviruses.Escherichia coli , the Hytönen lab decorated norovirus VLPs Figure  45). tr. tr45). To leverage one of the strongest non-covalent interactions, VLNPs may be biotinylated chemically or using biotin ligase. Biotinylated VLNPs are subsequently recognized by a member of the avidin family Figure . Avidin Flock house virus coat protein was genetically fused to part of the human anthrax toxin receptor (ANTXR2). This fusion allows decoration with anthrax protective antigen, which binds with 170 pM dissociation constant . High tiCovalent reaction between the target antigen and particle brings the obvious advantage of stability. This includes not only stability during storage but also stability after injection, where the lower concentration and altered conditions  favor dissociation of non-covalently linked antigen from the particle. Covalent conjugation also facilitates analysis of decoration. A simple SDS-PAGE can quantify what proportion of particle subunits are coupled to antigen and how much non-covalently associated antigen remain . Covalenvia cell-free protein synthesis  to nanoparticles . SNAP-TaSortase is an enzyme from Gram-positive bacteria that covalently joins proteins with a C-terminal LPXTGX motif to other proteins with an N-terminal oligoglycine motif Figure  65). So. So65). via native chemical ligation  being joined by a peptide bond Figure  68). Sp. Sp68). ligation . Split iligation . Some ofgens see . HoweverElectrostatic interactions have been utilized to align cysteine residues to form preferred disulfide bonds, yielding EILs . Attemptvia side chains, so each partner can react independently of the location within a protein, i.e., either at the N- or C-terminus, or internally within a protein sequence  and a B cell epitope . Linking antigen to scFv via SpyTag/SpyCatcher allowed targeting of antigen to DEC205-positive DCs for cytotoxic T cell stimulation (The first application of Catcher/Tag technology toward immune modulation was the development of SpyAvidins—variants of streptavidin bearing either SpyTag or SpyCatcher on specific subunits of the tetramer. SpyAvidin-MHC eicosamers (bearing 20 streptavidin subunits) surpassed conventional MHC tetramers in stimulating T cell activation . In anotmulation . The sammulation .Plasmodium falciparum blood-stage CIDR antigens or the transmission-blocking ookinete antigen Pfs25 .There is a shortage of side-by-side comparisons of vaccine platforms in the literature. As an initial test of different immunization platforms, mice were immunized with Pfs25 displayed on (i) SpyCatcher-AP205 VLPs using plug-and-display conjugation, (ii) Pfs25-VLPs from chemical decoration of Qβ, or (iii) genetic fusion of Pfs25 to the nanoparticle IMX313. Qβ display generated the highest anti-Pfs25 titer. However, SpyCatcher-VLP display gave the highest antibody quality, assessed by the ability of a given concentration of antibody to block malaria transmission to mosquitoes, using a standard membrane feeding assay Figure , 55. GivThrane and co-workers demonstrated fusion of SpyTag to the N- and C-termini of the AP205 coat protein. Spy-VLPs  were conjugated to a range of antigens from diverse viral and bacterial pathogens (up to 118 kDa) . Spy-VLPThe range of antigens and platforms linked using Tag/Catcher at the time of submission is summarized in Table Decoration of VLNPs with multiple ligands is technically difficult, but may be important for protecting against different strains of a pathogen or even against distinct diseases . For claP. falciparum transmission-blocking antigens, enhancing the response compared with monomeric antigen  coat protein VP1  in vivo . Upon im in vivo .Here, we describe some key issues for modular decoration, moving from proof of principle toward real world application. We illustrate these challenges for SpyTag/SpyCatcher-based assembly, but many issues may be generalized to other decoration technologies.Catcher/Tag decoration appears feasible for a range of non-enveloped VLPs , 91, 97.Yields for multimeric scaffolds of Catcher/Tag-VLPs (AP205 or IMX313) are still at the research scale of 1–20 mg/L using shaking-flask culture , 82, 83.E. coli-expressed Spy-VLPs has been demonstrated, important for well-controlled comparison between immunogens  Figure C 74); t; t74); tChimeric VLPs such as RTS,S  will contain immunologically relevant sequences other than those of the pathogen of primary interest. It is preferable that such sequences are not of human origin, to minimize the risk of autoimmune disease. For RTS,S, one vaccine construct raises antibodies against HBV and malaria. Would pre-existing antibodies against the platform prevent a new vaccine providing protection? For RTS,S in a typical three dose treatment, booster doses do improve the malaria protection even though anti-HBsAg antibodies are already present . SimilarThere is limited information whether SpyCatcher or SpyTag contains MHC class II epitopes, which might contribute to B cell memory. Deletion of the N-terminal region of SpyCatcher to give ΔN1SpyCatcher  reduced antibody titers against SpyCatcher . We showE. coli and Salmonella Typhimurium displaying SpyCatcher-linked pneumococcal antigens  are spherical membranous nanoparticles released from the outer membrane of Gram-negative bacteria. OMVs can be produced at large scale, displaying proteins of interest on their surface or packaging proteins stably inside. SpyTag was first used for OMV display through expression of a SpyTag-OmpA fusion, allowing conjugation of an enzyme for detoxification of nerve agents . SpyTag antigens . In addiantigens . The samantigens .In the future, Tag/Catcher display may facilitate comparison between other non-protein vaccination platforms. Such platforms are beyond our scope to cover in detail, but include emulsions, inorganic NPs, immune-stimulating complexes, and lipid-based NPs  , 111.Virus-like particles are pre-eminent vaccine platforms for inducing antibody responses; a central challenge for the field is to develop platforms giving potent T cell and B cell responses, important for the most intractable pathogens such as tuberculosis or HIV , 113. ThWhere VLNPs may not be able to provide a sufficient T cell response, viral platforms are leading the way at inducing cytotoxic T cell responses. SpyTag fusion has recently enabled modular modification of certain virus models, although immune stimulation has not been tested. The envelope protein GP64 from baculovirus was fused with SpyTag and labeled with SpyCatcher-labeled quantum dots for tracking and visualization in insect cells . SpyTag Beyond infectious diseases, VLP platforms may be adopted for therapeutic use. Allergens on VLPs may suppress allergic immune responses, while avoiding side-effects from direct activation of mast cells . New theIn all fields of science, progress can come faster when one can move from patient crafting of success to a reproducible assembly line –126. VacKB and MH conceived and wrote the manuscript.via spontaneous amide bond formation (EP2534484) and co-founded SpyBiotech Ltd.KB is a consultant and co-founder of Genie Biotech Ltd. MH is an inventor on a patent concerning peptide targeting


Bilophila wadsworthia (BwSarD). Here we report the structural and biochemical characterization of a sulfoacetaldehyde reductase from the human gut fermenting bacterium Bifidobacterium kashiwanohense (BkTauF). BkTauF belongs to the M-ADH family, but is distantly related to BwSarD (28% sequence identity). The crystal structures of BkTauF in the apo form and in a binary complex with NAD+ were determined at 1.9 and 3.0 Å resolution, respectively. Mutagenesis studies were carried out to investigate the involvement of active site residues in binding the sulfonate substrate. Our studies demonstrate the presence of sulfoacetaldehyde reductase in Bifidobacteria, with a possible role in isethionate production as a byproduct of taurine nitrogen assimilation.Hydroxyethylsulfonate (isethionate (Ise)) present in mammalian tissues is thought to be derived from aminoethylsulfonate (taurine), as a byproduct of taurine nitrogen assimilation by certain anaerobic bacteria inhabiting the taurine-rich mammalian gut. In previously studied pathways occurring in environmental bacteria, isethionate is generated by the enzyme sulfoacetaldehyde reductase IsfD, belonging to the short-chain dehydrogenase/reductase (SDR) family. An unrelated sulfoacetaldehyde reductase SarD, belonging to the metal-dependent alcohol dehydrogenase superfamily (M-ADH), was recently discovered in the human gut sulfite-reducing bacterium


To determine the ability of Saccadic Vector Optokinetic Perimetry (SVOP) to detect and characterise visual field defects in children with brain tumours using eye-tracking technology, as current techniques for assessment of visual fields in young children can be subjective and lack useful detail.Case-series study of children receiving treatment and follow-up for brain tumours at the Royal Hospital for Sick Children in Edinburgh from April 2008 to August 2013. Patients underwent SVOP testing and the results were compared with clinically expected visual field patterns determined by a consensus panel after review of clinical findings, neuroimaging, and where possible other forms of visual field assessment.Sixteen patients participated in this study . Twelve children (75%) successfully performed SVOP testing. SVOP had a sensitivity of 100% and a specificity of 50% . In the true positive and true negative SVOP results, the characteristics of the SVOP plots showed agreement with the expected visual field. Six patients were able to perform both SVOP and Goldmann perimetry, these demonstrated similar visual fields in every case.SVOP is a highly sensitive test that may prove to be extremely useful for assessing the visual field in young children with brain tumours, as it is able to characterise the central 30° of visual field in greater detail than previously possible with older techniques. Central nervous system (CNS) tumours are the commonest solid tumours of childhood and the most common cause of childhood cancer death. Primary malignant brain tumours have an age-adjusted incidence rate of 3.27/100,000 in children aged 0–14 years at time of diagnosis. The most frequently occurring tumours are pilocytic astrocytomas (26%) and Primitive Neuroectodermal Tumours (PNET) including medulloblastoma (22%). Other gliomas  occur less commonly [Current strategies for children with brain tumours aim to maintain survival rates with minimal treatment related long-term sequelae. Hence their management requires multidisciplinary team (MDT) input in treatment and monitoring . These cDespite the potential benefits, there is a dearth of effective visual field testing methods appropriate for children under 5 years that can provide reliable detail greater than that gained from the confrontation method. Confrontation in children typically requires one examiner to gain a child’s fixation, and a second examiner to introduce dynamic stimuli into various part of the child’s visual field from the rear and side while the first examiner observes the child’s gaze for a reaction. The technique allows only an approximate assessment of visual field defects and in children its usefulness is limited by the attention and concentration of the child. Obtaining reliable and objective field testing in healthy young children is challenging and frequently more difficult in children who are ill as a result of brain tumours. The difficulties associated with performing standard automated perimetry (SAP) in children are well known. The main problems children have with SAP are understanding how to perform the test and maintaining central fixation , 7. ManuSaccadic Vector Optokinetic Perimetry (SVOP) is an objective visual field assessment technique developed specifically for young children unable to perform conventional forms of perimetry , 13. It The aim of this study was to determine the feasibility of SVOP in the detection and characterisation of visual field defects in children with brain tumours, and to authenticate the SVOP results by comparing them with clinically expected visual field patterns following review of neuroimaging, and where possible other forms of visual field assessment.The families of children with known or suspected brain tumours managed at the Royal Hospital for Sick Children in Edinburgh from April 2008 to August 2013 were invited to participate in the study.Each patient underwent binocular SVOP visual field testing. In addition, monocular testing was also performed if the patient was considered of a suitable developmental ability. Many of the participants were tested repeatedly over months or years of follow-up. This study reports the first SVOP testing episode for each child.All patients had undergone MRI neuroimaging as part of their oncological workup and ongoing treatment. A consensus panel comprising an experienced ophthalmologist (BF), neurologist (RM) and neuro-radiologist (GW), reviewed the MR images for the scanning session for each patient that was closest in time to their SVOP test date. Care was taken to ensure that none of the patients had undergone interventions such as radiotherapy or surgery in the interval between the MR imaging session and the SVOP test. The panel was masked to the SVOP results. Based on radiological appearances, and with clinical information including full clinical history, VA, ocular motility, optic disc appearances and results of other VF testing methods, e.g. confrontation or Goldmann (when available), the panel predicted an expected visual field deficit for each patient.Additionally, an experienced ophthalmologist (CS), masked to patient details and imaging, independently interpreted and described the measured SVOP visual field for each child in the study. Abnormal visual fields were those which had two or more contiguous points “unseen” in the same quadrant or vertical hemifield, or three or more non-contiguous “unseen” points in one quadrant or vertical hemifield. If a visual field was classified as abnormal, the defect was categorised using standard neuro-ophthalmology visual field abnormality terms.The panel-predicted visual fields were then compared with the SVOP results and a two-by-two contingency table was utilised to determine the sensitivity and specificity  of the SVOP test in detecting a clinically significant visual field abnormality.When children became capable of performing Goldman visual field testing during follow-up, the pattern of visual field abnormality obtained using Goldman testing was compared with the pattern of visual field abnormality obtained using SVOP. The pair of tests with the shortest time interval between them was used.The SVOP system comprises a personal computer (PC), a patient display  and an eye tracker. During the course of this study two different models of eye tracker (X50 and IS-1 models from Tobii Technology) were used on different occasions. Figure Prior to any SVOP test, a calibration procedure was performed, to produce accurate gaze data for the patient being tested. During a calibration procedure, patients were required to look at the SVOP display screen and follow a visual stimulus with their gaze to five different screen locations. During this procedure, the eye tracker measures characteristics of the subject’s eyes (such as pupil position and shape) and used them together with a mathematical 3D eye model to calculate gaze position. The calibration stimulus was a cartoon character which moved to each of the five screen locations whereupon a simple short animation was played to hold the attention of the child. The calibration procedure lasted ~20–30 s.Each SVOP test began with a central fixation target. For older children a high contrast circular fixation target was used while for younger children the fixation target was a cartoon face to encourage attention and maintain interest. Fixation targets were of an angular diameter of ~1.5°. Real-time gaze data allowed the system to identify when the patient was looking at the fixation target. The instant this was detected, the fixation target disappeared and a test stimulus was presented at a location in their visual field. The on-screen location and size of the test stimulus was calculated in real-time and was based on the visual field angle to be tested and the position of the patient’s eyes relative to the fixation target at that instant. A software algorithm (details previously published ) analyse2, respectively. For reference, this stimulus brightness level is equivalent to 14 dB on the scale used by the Humphrey Field Analyser (HFA). The binocular test pattern used was a pattern consisting of 40 points (with 10 points in each quadrant) arranged with eccentricity out to 25°  and duration 200 ms. Through the use of a calibrated LCD , the lum25° Fig. . The monThis study was approved by the Lothian Regional Ethics Committee. Signed consent was obtained from all participants and/or their parent or guardian.Sixteen  patients already receiving treatment and follow-up for brain tumours participated in this study. The mean age at the time of the reported SVOP test was 7.2 years (range 2.9–15 years). Patients 4, 8 and 9 were tested using an IS-1 eye tracker while the remaining thirteen patients were tested using an X50 eye tracker. The average test time for completed tests was 5 min 4 s (range 1 min 5 s to 9 min 7 s). The large range in test times can be attributed to the different testing strategies used for younger and older children and also the size of visual field defect present. Younger children were presented with small cartoon animations when they successfully detected visual field test stimuli. This animation sequence introduced an additional 2–3 s per “seen” stimulus. In addition, “unseen” stimuli are always retested. Consequently, a patient with a complete hemianopia would have half their visual field points retested, thus lengthening the test time.The average interval between the SVOP test and the closest neuroimaging session, used by the consensus panel, was 52 days (range 5–146 days).Twelve patients . The decision to abort these tests was made by the tester when it became clear that the test was not proceeding correctly. This was known because the system would continually try to regain the child’s fixation and not test any visual field points.A two-by-two contingency table was used to compare the gross correlation of ‘normal’ versus ‘abnormal’ visual field between the consensus panel prediction and SVOP findings for the twelve successfully completed tests. Table  There weEye journal’s website.Table Patients 1, 2, 7, 8 and 15 were effectively monocular due to poor or absent VA in one eye as a result of optic nerve or chiasmal involvement by tumour. Patients 3, 5, 9 and 14 all completed monocular and binocular SVOP tests; the test which best demonstrates the VF abnormality has been included in the table. Only three of the younger patients  who were unable to comply with monocular occlusion, had purely binocular SVOP testing.Six children  became capable of performing Goldmann Visual Field testing during follow-up. One had a right hemianopia on Goldmann testing, two patients had a left hemianopia, one had a right homonomous hemianopia, and two had a full visual field. The pattern of visual field abnormality present on SVOP testing was the same in every case. Figure SVOP was successfully performed in the majority (75%) of this cohort of children with brain tumours, even in those under three years of age. Successful completion of a test was associated with 100% sensitivity in detecting a visual field defect when compared with ophthalmic assessment and/or panel consensus following review of neuroimaging. In most of these cases the specific character of the measured visual field defect was compatible with the expected visual field defect when considering the anatomy of the visual pathways. In case 14, the initial field defect detected by the first SVOP differed from the expected defect, despite a repeatable test. However, a nasal defect detected in that eye is plausible given that the gliotic scarring in the left temporal lobe is immediately adjacent to the temporal aspect of the prechiasmal left optic nerve. In this case the panel prediction was based on a predominantly retrochiasmal tumour when in fact the area of contact was in the anterior visual pathway.Subsequent Goldmann and SVOP testing demonstrated recovery of a full visual field in either eye.Overall, six children became capable of performing Goldmann visual fields testing during follow-up. In each case, the pattern of visual field abnormality was the same with Goldman testing and SVOP testing. This finding supports our contention that SVOP assessment at an early age in our cohort was reliable. We have previously published comparison studies of Humphrey Standard Automated Perimetry and SVOP in adults, with favourable results .One of the false positive SVOP tests (case 8) showed poor quality eye tracking with a prolonged test time (9 min 7 s). A potential loss of interest and concentration from the child could have resulted in false positive algorithm decisions and scattered unseen points. However, the bilateral optic tract dysmyelination seen in this patient could have a measurable effect on the visual field. The constricted visual field defect recorded for the remaining false positive case (case 9), whilst not anticipated by the consensus panel, could in fact represent a true constriction due to previous raised intracranial pressure.Test failure occurred in four children and the reasons for this have been elucidated. All four of the failures related to poor eye tracking. In two patients the eye tracker was unable to detect the eyes sufficiently, while the remaining two patient’s eyes were detected but the gaze data showed an offset when compared with the coordinates of the on-screen fixation targets. In each scenario, the SVOP system was not able to determine that the patient was correctly fixating on the displayed fixation targets and hence would not proceed to test visual field points. The decision to abort these tests was made by the tester when it became clear that the test was not proceeding in a satisfactory manner.In patients where eye tracking is poor a complete result is often not possible. Moreover, it is possible that incomplete test results should be considered unreliable because of prolonged test time and reduced interest from the child. In future SVOP studies it will be important to assess the factors that contribute to unreliable tests so that a useful indication can be provided to the tester.In this study, three patients were tested using a different model eye tracker than the remaining patients. Improvements to eye-tracking technology are continuingly being made and it was thought that a newer model of eye tracker would improve the SVOP test. Both of the eye trackers used in this study have since been discontinued by Tobii Technology due to continuing development of their eye trackers and our research group have more recently been performing studies using another eye tracker model .There are few reliable alternatives for assessing visual field loss in young children which can show the same level of detail as SVOP. Manual visual field testing approaches, such as that used by the BEFIE test  or the KAn advantage of SVOP is its ability to characterise the nature and extent of visual field defects, which have important functional implications for the child and it may be particularly useful if changes in tumour size or characteristics seen in neuroimaging are accompanied by functional visual field change detected by SVOP. The automated nature of SVOP means that minimal experience is required to perform the test or operate the system. Children find the test easy, and as it takes ~5 min to perform. With the use of engaging animations to hold and maintain concentration, a meaningful test result was obtained in the majority of cases. In this small cohort SVOP testing has demonstrated the potential for monitoring visual field changes in young patients with brain tumours in greater detail than was previously possible.Future studies using SVOP will focus on longitudinal follow-up of a cohort of children with visual pathway tumours to (i) determine the repeatability and reliability of the test, and (ii) demonstrate changes of visual field defects in relation to progression of tumour size over time, and response to medical or surgical interventions. For anterior visual pathway tumours, the use of OCT may be used to confirm focal deficits in the RNFL corresponding to visual field defects mapped by SVOP.Assessment of visual fields is an important component of the assessment of visual pathway tumours in peadiatric neuro-oncology.Previous commonly used techniques such as confrontational visual field assessment lacked objective detail in young children, whilst standardised kinetic perimetry (Goldmann), or automated perimetry, were difficult for young children to perform reliably.Saccadic Vector Optokinetic Perimetry (SVOP) is a new, objective technique for visual field testing designed for young children.We assessed the ability of SVOP to detect expected or known visual field defects in a cohort of sixteen children with visual pathway tumours.SVOP demonstrated 100% sensitivity and 50% specificity when compared with expected visual field abnormality.The diagnosis and management of young children with visual pathway tumours would be aided by SVOP testing due to its ability to characterise the central visual field in detail.Supplementary Files


A successful HIV-1 vaccine will likely need to elicit broadly neutralizing antibodies (bNAbs) that target the envelope glycoprotein (Env) spike on the virus. Native-like recombinant Env trimers of the SOSIP design now serve as a platform for achieving this challenging goal. However, SOSIP trimers usually do not bind efficiently to the inferred germline precursors of bNAbs (gl-bNAbs). We hypothesized that the inherent flexibilities of the V1 and V2 variable loops in the Env trimer contribute to the poor recognition of gl-bNAb epitopes at the trimer apex that extensively involve V2 residues. To reduce local V2 flexibility and improve the binding of V2-dependent bNAbs and gl-bNAbs, we designed BG505 SOSIP.664 trimer variants containing newly created disulfide bonds intended to stabilize the V2 loop in an optimally antigenic configuration. The first variant, I184C/E190C, contained a new disulfide bond within the V2 loop, whereas the second variant, E153C/R178C, had a new disulfide bond that cross-linked V2 and V1. The resulting engineered native-like trimer variants were both more reactive with and were neutralized by V2 bNAbs and gl-bNAbs, a finding that may be valuable in the design of germline targeting and boosting trimer immunogens to create an antigenic conformation optimal for HIV vaccine development. There remains an urgent need to create an effective vaccine to reduce the further spread of HIV-1. Many vaccine design strategies are now based on the elicitation of broadly neutralizing antibodies (bNAbs)N-linked glycans. Three or four additional glycans are present on ectodomain of gp41  –25. In g–26–26–36–39–As noted above, the BG505 and other genotypes of the SOSIP.664 native-like trimer now serve as platforms for structure-guided immunogen design as part of bNAb-development programs , 42–45. 42–48–52–The availability of high-resolution structural information on the architecture of BG505 and other SOSIP trimers is a critical guide to design improvements, including those to the V2 bNAb epitopes. The V1V2 domain folds into a four stranded anti-parallel β-sheet Greek key motif in which strands A and B are connected by the flexible V1 loop and strands C and D by the flexible V2 loop , 26, 43.43–Here, we sought to stabilize the V1 and V2 domains by introducing disulfide bonds either within the V2 loop or between the V1 and V2 loops. We show that both strategies can improve the presentation of V2 bNAb epitopes. Thus, the resulting SOSIP trimer variants have improved reactivity with V2 bNAbs and their inferred germline (gl) precursors. Moreover, the corresponding virus mutants are more sensitive to neutralization by V2 bNAbs. These trimer-improvement methods may therefore play a role in bNAb-induction strategies.A–C). To stabilize both domains simultaneously, we constructed three mutants, K155C/F176C, I154C/Y177C, and E153C/R178C, in which the C-terminal part of V1 is now linked to the N-terminal part of V2 by an engineered interloop disulfide bond (C). This category of mutant is hereafter referred to by the term V1-V2 disulfide bond. The positions for the Cys substitutions were chosen because of the close proximity of residues Lys155 and Phe176 (5.5 Å distance between the β-carbons (Cβ) atoms; PDB code 5CEZ), Ile154 and Tyr177 (5.4 Å), and Glu153 and Arg178 (4.1 Å); at such distances, it seemed feasible that a disulfide bond could be formed (C).We designed disulfide bond mutants to stabilize the flexible V1 and V2 domains on BG505 SOSIP.664 trimers , A–C. Toide bond C. This ce formed  Fig. 1CCA–C. To A–C). This category of mutant is hereafter referred to by the term V2-internal disulfide bond. The positions for the Cys substitutions were chosen because a twin cysteine motif is present at the corresponding positions in the V2 loops of SIV and HIV-2 Env and has been suggested to lead to the formation of a loop-stabilizing disulfide bond (D) (C).We also elected to stabilize the V2 loop by creating a new disulfide bond within it. Accordingly, we designed two mutants: Q183C/E190C and I184C/E190C , A–C. Thide bond D (18). Abond (D) C.A–C), and the resulting proteins were expressed transiently in 293T cells. We then assessed the yield, trimer formation, and antigenicity of each mutant, using the unpurified culture supernatants. All three V1-V2 disulfide mutants were expressed at acceptable yields , but a BN-PAGE gel showed that trimer formation was severely impaired (E). The implication is that the disulfide bond–linked V1V2 domains are no longer able to form the intersubunit interactions at trimer apex that are important for trimer formation. The conformationally nonselective 2G12 and VRC01 bNAbs bound the various V1-V2 disulfide bond mutant proteins efficiently . However, the conformationally sensitive (trimer-specific or trimer-preferring) bNAbs PG9, PG16, and PGT145 failed to bind to the K155C/F176C and I154C/Y177C mutants and reacted only poorly with the E153C/R178C mutant. These data patterns are consistent with the poor formation of trimers seen in the gel-based analysis .The five disulfide bond mutants described above were introduced into a D7324-tagged BG505 SOSIP.664 construct , A–C, animpaired E. The imAFig. S1). The Q183C/E190C mutant failed to form trimers, but a moderate level of trimer formation was observed for the I184C/E190C mutant (E). In the antigenicity studies, the Q183C/E190C mutant bound 2G12 and VRC01 somewhat less well than the SOSIP.664 comparator and did not bind PG9, PG16, and PGT145 at all . In contrast, the I184C/E190C mutant bound comparably to 2G12 and VRC01 and was also reactive with PG9, PG16, and PGT145, albeit to a lesser extent than SOSIP.664 . We judged that the extent of binding of these three V2 bNAbs was sufficient to pursue further studies of this V2-internal disulfide bond mutant (see below).The V2-internal disulfide bond mutants were both slightly less efficiently expressed than the parental BG505 SOSIP.664 construct . Each mutant virus was therefore passaged for a prolonged period in two independent SupT1 T cell cultures to see whether compensatory mutations arose that increased replication efficiency. The K155C/F176C and I154C/Y177C mutants failed to replicate detectably during the 3-month experiment, indicating that their defects were beyond repair. However, the E153C/K178C mutant acquired replication competence in both cultures after 3 weeks (note that position 178 in the LAI clone is a lysine and not the arginine present at the corresponding position in BG505 Env). Proviral DNA was therefore sequenced after 1, 2, and 3 months of culture. In both cultures, a point substitution at position 152 was found to have emerged within 1 month (B). Specifically, glycine 152 had changed to either glutamic acid  or valine . The significance of these observations is that residue 152 is directly adjacent to the cysteine substitution at position 153. The changes at position 152 persisted to the end of the 3-month culture period, but we also found that a second change, S128R, arose in the virus in culture 1 (B).As expected from the studies on the BG505 trimers, none of the three V1-V2 disulfide bond mutant LAI viruses was infectious, as measured in a single round infection assay in TZM-bl cells A. Each m 1 month B. Specifulture 1 B.C). To confirm that the G152E, G152V, and S128R compensatory mutations identified by sequencing the env genes were in fact responsible for the restored infectivity, we introduced these changes individually or in combination into an LAI clone that also contained the Cys153–Cys178 V1-V2 disulfide bond. In all three cases, the resulting virus mutants and the quasispecies present in the evolution cultures at month 3 had comparable infectivities for TZM-bl cells (C). The substitutions at position 152 had the strongest effect, whereas the S128R change modestly increased the infectivity of the G152E mutant. Taken together, the evolutionary repair experiments show that a compensatory substitution at position 152, either G152E or G152V, partially restores the infectivity of an otherwise replication-defective E153C/K178C V1-V2 disulfide bond mutant LAI virus.We next assessed the ability of the E153C/K178C virus quasispecies from the SupT1 cultures to infect TZM-bl cells and found that there was a gradual gain of infectivity during the 3 months of evolutionary rescue. By the end of the culture period, the E153C/K178C mutant virus in SupT1 cultures 1 and 2 showed infectivity levels of ∼50 and ∼100% compared with the parental LAI-PG9 virus, respectively C. To conbl cells C. The suA and BFig. S2). However, the G152V mutation was not beneficial .To assess whether the LAI compensatory mutations at residue 152 would translate to the BG505 SOSIP.664 trimer context, we introduced either the G152E or G152V change into the SOSIP.664 E153C/R178C mutant and expressed the proteins by transiently transfecting 293T cells. The G152E compensatory mutation clearly improved both trimer formation, judged using a BN-PAGE gel and the binding of the conformationally sensitive V2 bNAbs , confirming the results of the HEK 293T transfection (A).We then expressed the BG505 SOSIP.664 WT, E153C/R178C, and E153C/R178C/G152E constructs in 293F cells, purified the resulting Env proteins via 2G12 affinity chromatography, and analyzed them with size-exclusion chromatography (SEC). 2G12 binds to all possible forms of Env , and SEC allowed us to determine the trimer formation efficiency for each Env protein. Compared with SOSIP.664, the SEC profile of the E153C/R178C mutant contained many more monomers and dimers and proportionately far fewer trimers, but trimer formation was clearly increased when the G152E change was also present B, confirsfection A.C). Whereas the trimer yield for the BG505 SOSIP.664 construct was ∼2.0 mg/liter, the corresponding values for the E153C/R178C and E153C/R178C/G152E variants were ∼0.2 and ∼0.6 mg/liter, respectively .The improvement described above was not sufficient to allow the BG505 SOSIP.664 E153C/R178C/G152E trimers to be purified by the 2G12/SEC method. However, PGT145-affinity chromatography is an alternative approach to isolate native-like trimers , 63. We Fig. 3ectively . The yieectively . As expeAFig. S3). Alternative (i.e. aberrantly formed) disulfide bonds were also detected in low abundance, but they were quantitatively and qualitatively similar to ones previously identified in the WT BG505 SOSIP.664 trimer .To confirm that the engineered disulfide bonds had actually formed, the loop-stabilized BG505 SOSIP.664 trimers were trypsin-digested into fragments that were analyzed by MS to identify peptides linked by disulfide bonds . The E154 trimer . We concE) (E) (E). The I184C/E190C V2-internal disulfide bond mutant had a very similar appearance to the SOSIP.664 parental trimer, being predominantly (86%) in the closed native-like conformation (E).Negative-stain (NS) EM showed that the PGT145-purified SOSIP.664 E153C/R178C, E153C/R178C/G152E, and I184C/E190C disulfide bond mutant trimers were all in a native-like conformation. The SOSIP.664 trimer was in a closed, fully native-like conformation (>95%), as reported previously E 42). I. IE) (42E) E (64). TE) (E) , 63. We E) (E) , 63. TheE) (E) E. The I1ormation E.Tm = 65.3 °C versus 66.6 °C), whereas the additional presence of the G152E compensatory mutation reverted the Tm to 66.7 °C (F). The corresponding Tm value of 66.3 °C for the I184C/E190C V2-internal disulfide bond mutant trimer was also almost identical to SOSIP.664 (G). Thus, in contrast to when disulfide bonds are introduced into the core regions of a gp120 monomer (I201C/A433C)  to quantify the thermostability of the modified SOSIP.664 trimers. The E153C/R178C V1-V2 disulfide bond mutant was slightly less thermostable than SOSIP.664   or SOSIPC/A433C) , 65, engThe goal of our studies was to investigate whether we could improve the antigenicity of SOSIP.664 trimers for V2 bNAbs. We therefore tested an extensive panel of bNAbs and non-NAbs for their abilities to bind the disulfide mutant trimers in an ELISA. The various trimers were purified by PGT145 affinity chromatography as described above.A). Taken together, these results imply that the more open appearance of these trimers observed in NS-EM studies is associated with an antigenically less preferred conformation. The introduction of the G152E compensatory mutation improved VRC01, PGT151, and 2G12 binding and was also extremely beneficial to the epitopes for the V3-glycan bNAb PGT121 and the V2 bNAbs PG9, PG16, and CH01; indeed, the latter four bNAbs bound better to the E153C/R178C/G152E mutant than to SOSIP.664 (A and B). An increased binding to CD4bs bNAb CH103 was also observed for the E153C/R178C/G152E mutant (B). However, several non-NAbs also bound more strongly to this mutant trimer, compared with SOSIP.664 (A and B).Compared with BG505 SOSIP.664, the E153C/R178C V1-V2 disulfide mutant bound the VRC01, PGT151, and 2G12 slightly less well, whereas the 19b, 14e, and 17b non-NAbs targeting V3 and CD4i epitopes bound more strongly A. Taken OSIP.664 , A and BE mutant B. HoweveOSIP.664 , A and BB). Although binding to VRC01 was similar between mutant and SOSIP.664 trimer, binding to CH103 was improved for the V2-internal disulfide bond mutant trimer (B). There were no, or only very minor, differences in how the 2G12, PGT121, and PGT151 bNAbs or the F105 and 17b non-NAbs bound to the mutant and parental SOSIP.664 trimers (B).The I184C/E190C V2-internal disulfide bond mutant trimer bound all the V2 bNAbs  better than SOSIP.664. The titration curves showed that the maximum binding levels were substantially higher for the mutant trimers compared with SOSIP.664 B. Althout trimer B. There  trimers B.B) (B). Based on half-maximal binding (EC50) values, gl-PG9 binding to the mutant was increased by 5-fold, and the maximum binding level was also higher (C). A similar pattern was seen for the gl-PG16 and gl-CH01 bNAbs, although EC50 values could not be reliably determined because the titration curves did not reach a plateau (B and C).We also assessed whether the gl-PG9, gl-PG16, and gl-CH01 V2 bNAbs were more reactive with the mutant trimers. As reported previously, all three gl-bNAbs bound detectably to the BG505 SOSIP.664 trimer, albeit with lower affinities than the corresponding mature bNAbs B (66). HB) B. Based o higher C. A simi plateau , B and CB). None of the trimer variants bound gl-PGT145 or gl-VRC26.09 detectably (C).The V1-V2 disulfide bond mutant trimer was slightly more reactive with gl-PG9, gl-PG16, and gl-CH01 compared with SOSIP.664, although the beneficial effects of this engineered disulfide were markedly less than those conferred by the V2-internal disulfide B. None otectably C.Fig. S4). The binding site for gl-PG9 also remained stable over time on the V1-V2 disulfide mutant trimers, although incubation at 37 °C reduced the binding strength by 40% .Binding of V2 bNAbs to the V1-V2 disulfide mutant and SOSIP.664 trimer was not affected by preincubation of up to 10 days at 37 °C, indicating the exceptional robustness of the disulfide mutant BG505 trimers . The data pattern implies that the V2 bNAb and gl-bNAb epitopes are not stabilized in the same way by the two disulfide mutational strategies tested here. Superposition of the Env trimer structure with a V1V2 scaffold bound by PG9 shows that the V2 loop can interfere with V2 bNAb binding (A). Whether the V1-V2 disulfide bond mutant improves binding of V2 bNAbs to the BG505 Env trimer obviously depends on the position of the stabilized V2 loop and might be different for the V1-V2 disulfide and V2-internal disulfide mutant trimers.To determine whether the engineered disulfide bonds increased the presentation of V2 bNAb and gl-bNAb epitopes on the native, virion-associated trimer, we made variants of the BG505.T332N Env-pseudotyped virus containing the E153C/R178C/G152E or I184C/E190C changes. The V2-internal disulfide mutant virus was more sensitive to neutralization by V2 bNAbs compared with BG505 , although there was no difference in the sensitivity of the two viruses to PGT145 and VRC26.09, and gl-PG9 neutralized neither virus . Whether the V2 loop interferes with how different bNAbs see their CD4bs epitopes is likely to be influenced by their angle of approach to the trimer. Because CH103 approaches at a 45° angle relative to the trimer axis, it comes into close proximity with the V2 region; hence any alteration in the orientation of V2 caused by an engineered disulfide could alter access of CH103 to its epitope (B). In contrast, VRC01 approaches the CD4bs at an angle parallel to the virus membrane and at 90° to the trimer axis, which means that its neutralization potency is less likely to be affected by the precise orientation of the V2 loop (B).We noted that both disulfide bond mutant viruses were ∼10-fold more sensitive to neutralization by CH103, whereas there was no difference in sensitivity to VRC01, which also recognizes a CD4bs epitope . However, there were also examples, such as gl-PG9, where stronger trimer binding did not translate into more potent neutralization. The correlation was not statistically significant for the V1-V2 disulfide bond mutant trimer and virus . Thus, stabilization of the V2 domain either by a disulfide bond within the V2 loop or by one that links the V1 and V2 domains can improve the presentation of V2 bNAb and/or gl-bNAb epitopes at the trimer apex. However, the beneficial effects of the engineered disulfide bond(s) are not universal for all the V2-targeted antibodies, and the two strategies do not lead to identical outcomes.Overall, the virus neutralization data and SOSIP.664 trimer-binding data were generally concordant. Compared with their unmodified counterparts, the V1-V2 and V2-internal disulfide bond mutant trimers generally interact more strongly with V2 bNAbs and gl-bNAbs compared with SOSIP.664 trimers, and this is also true for neutralization of the corresponding Env-pseudotyped viruses. Thus, increases in Ab binding to the V2-internal disulfide bond mutant trimer and neutralization of the corresponding mutant virus by the same Ab were correlated , but only two of them (E153C/R178C and I184C/E190C) yielded trimers with reasonable efficiency. Even for these mutants, there was a marked reduction in trimer yield compared with the SOSIP.664 trimer. We assume that the introduced cysteine residues interfere with the intricate process of gp120 folding and trimer assembly, perhaps by forming aberrant disulfide bonds leading to dead-end folding pathways or by distorting the gp120 subunit enough to impede trimer formation  arose in an evolutionary rescue culture of an HxB2 virus clone that was crippled by the deletion of a key glycan at position 262. In that case, the S128N change compensated for a substantial folding defect in the gp120 subunit –70. We h–The third step that was required to isolate native-like disulfide mutant trimers was affinity purification using the trimer-specific PGT145 bNAb. This positive-selection procedure allowed the isolation of stable, native-like V1-V2 and V2-internal disulfide mutant trimers. Nevertheless, the eventual yields of the V1-V2 and V2-internal disulfide trimers described here were still substantially  lower than that of the parental BG505 SOSIP.664 trimer. If these mutant trimers prove to be useful components of a bNAb-induction strategy, additional steps will need to be taken to address this limitation. For example, we have found that expressing a mutant trimer in a stable CHO cell line can sometimes improve the yield by severalfold, and modifying key residues at the gp120-gp41 interface can also be beneficial.The V2-internal disulfide bond did improve the trimer binding of V2 bNAbs. One explanation may be that the flexible V2 loop assumes a conformation in which V2 bNAb epitopes are transiently or permanently occluded on a subpopulation of BG505 SOSIP.664 trimers. Such masking could be mediated by the protein or the glycan components of the V2 loop itself or of the nearby V1 loop. Whatever the explanation, several V2 and one CD4bs bNAb epitopes were better presented on the V2-internal disulfide mutant trimer, perhaps because the additional cross-link stabilizes the loop and reduces its propensity to adopt a conformation in which certain epitopes are at least partially occluded. The disulfide bond connecting the V1 and V2 loops also improved the presentation of V2 bNAb epitopes, although less so than the V2-internal disulfide. We also intend to determine whether the V2-internal and V1-V2 disulfides can be incorporated into SOSIP trimers of other genotypes. The length of the V2 loop may be a variable that affects, in particular, the stabilizing and/or other effects of the V2-internal disulfide. Indeed, removal of 7 amino acids from the BG505 V2 contributed to the engagement of V2 gl-bNAbs .For an HIV-1 Env immunogen to induce bNAbs, it is likely to be necessary that it can first activate naïve B-cells that express appropriate gl-bNAb epitopes as their BCRs. A complex evolutionary process may then drive the emergence of a true bNAb. In this context, we found that the affinity of three inferred V2 gl-bNAb precursors for the V2-internal disulfide mutant was increased compared with SOSIP.664. Such an affinity increase may enhance the probability that naïve B-cells expressing similar gl-NAb epitopes will be found and activated when the modified trimers are used as immunogens. Whether improved antigenicity of the V2-internal disulfide mutant trimer correlates with an improved immunogenicity has to be addressed in future immunization studies. Such studies remain to be initiated, for example using knockin mice that express V2 gl-bNAbs as BCRs , 73. The183–Cys191) of the clade C ZM109F Env-pseudotyped virus has recently been described  motif in V2. However, in contrast to the effect of the disulfide that we introduced into the BG505.T332N virus, the ZM109F mutant virus was not more sensitive to neutralization by bNAb PG9 bNAb epitopes was improved when the V1 and V2 loops were stabilized by the V1-V2 disulfide and more so by the V2-internal disulfide.ECTO residue 664 (improvement of homogeneity and solubility) (ECTO and were constructed by adding the amino acid sequence GSAPTKAKRRVVQREKR after residue 664 in gp41ECTO (The BG505 SOSIP.664 construct has been described elsewhere . It was ubility) . SOSIP.6gp41ECTO . Point mThe various SOSIP Env constructs were expressed by polyethyleneimine-mediated transient transfection (together with the furin gene) of adherent HEK 293T cells or of HEK 293F cells adapted for serum-free suspension cultures, as previously described , 47. ForB, which included 2G12-purified trimers, all other experiments were performed with PGT145-purified trimers.For screening purposes, unpurified 293T-expressed trimers were analyzed directly using the culture supernatants obtained 6 days post-transfection. For extensive analysis of V2 bNAb reactivity, Env trimers were purified from 293F transfection supernatants by affinity chromatography using a PGT145 column or a 2G12 column followed by SEC, as described , 47, 63.Fig. 3Env proteins were analyzed using SDS-PAGE and BN-PAGE, followed by Western blotting or Coomassie Blue dye staining as previously described , 78.m NaHCO3 (pH 8.6) overnight. Serially diluted Abs were added for 2 h, washed, and then detected using horseradish peroxidase–labeled goat anti-human immunoglobulin (Jackson Immunoresearch). We also used a modified D7324-capture ELISA protocol that was optimized for detecting gl-bNAb binding , followed by blocking of the plates with casein blocking buffer. Then purified trimers were transferred to the plates in a concentration of 2.5 μg/ml and incubated at room temperature for 2 h. Next, binding of several antibodies to the BG505 trimers was tested using the following Ab concentrations: 2G12, 0.75 μg/ml; PGT145, 0.75 μg/ml; glPG9, 5.0 μg/ml; glPG16, 5.0 μg/ml; glCH01, 5.0 μg/ml; PG9, 0.75 μg/ml; PG16, 0.75 μg/ml; and CH01, 0.75 μg/ml. After incubating the antibodies for 1 h, similar steps were followed as for the D7324 ELISA to detect antibody binding.Purified BG505 trimers were incubated in PBS at room temperature or at 37 °C for 0, 60, 120, 180, or 240 h, and subsequently antigenicity was assessed with a lectin ELISA. Greiner half-well ELISA plates were coated with m, and association and dissociation were measured for 300 s. Trimer binding to a protein A sensor not loaded by antibody was set as background.Antibody binding to purified BG505 trimers was studied using a ForteBio Octet K2. All assays were performed at 30 °C and with agitation set at 1000 rpm. Trimer and antibody dilutions were made in running buffer  in a final volume of 200 μl/well. Antibody was loaded on protein A sensors (ForteBio) at 1.0 μg/ml in running buffer until a binding threshold of 0.5 nm was reached. Trimers were diluted in running buffer at 80 nPGT145-purified BG505 trimers were analyzed by negative stain EM as previously described , 63, 83.Thermal denaturation of purified Env proteins was studied using a MicroCal VP-Capillary DSC calorimeter  or a nano-DSC calorimeter (TA Instruments) as described previously . All EnvN-glycosidase F solution  in 100 mm ammonium citrate buffer (pH 6.5) for 1 week at 37 °C. The fully deglycosylated, and alkylated samples were digested overnight with trypsin (protein to enzyme ratio of 30:1) at 37 °C and were then analyzed by LC-MS using an Orbitrap Velos ProTM hybrid  mass spectrometer equipped with electron transfer dissociation module and coupled to an Acquity ultra performance liquid chromatography system . Approximately 5 μl  of the tryptic digest was injected onto a C18 PepMapTM 300 column , and the peptides were separated using a linear gradient starting from 3% B to 40% B gradient in 50 min and then 90% B for 10 min followed by re-equilibration at 97% A for 10 min. LC-MS runs were performed with a flow rate of 5 μl/min using mobile phases consisting of solvent A: 99.9% HPLC-grade H2O + 0.1% formic acid and solvent B: 99.9% HPLC-grade CH3CN + 0.1% formic acid. Data were collected using the data-dependent mode. To determine the disulfide connectivity profiles, the five most intense ions in a high-resolution scan in the Orbitrap were subjected to alternating collision-induced dissociation and electron transfer dissociation in the linear ion trap. The resulting data were analyzed using the Mascot search engine to identify peptides containing free cysteine residues, and the disulfide bond patterns were then determined manually, as described previously  and cloned into pLAI as SalI–BamHI fragments. The integrity of all plasmids was verified by sequencing. To create a PG9-sensitive LAI variant, four substitutions  were introduced into β-strand C of the V1V2 domain; the substitutions were based on the corresponding residues in the BG505 isolate, which is sensitive to PG9 . The resulting LAI-PG9 virus was then used as the basis for the disulfide mutant viruses described in the results.The pRS1 plasmid expressing the full-length LAI molecular clone (pLAI) was the basis for the WT and mutant viruses used in these studies . Env gene to PG9 . The out6 SupT1 cells were transfected with 5 or 20 μg of LAI-PG9. Virus replication in the cultures was monitored twice a week by visual inspection for the appearance of syncytia and by using a p24-antigen ELISA. The cultures were maintained for 3 months, during which period cell-free supernatants were passaged onto uninfected cells when virus replication was apparent. Smaller quantities of supernatant were used for passaging when the extent of virus replication was sufficient to cause significant levels of cell destruction in the cultures. At regular intervals, cells and filtered supernatant were collected for storage at −80 °C until needed for genotypic and phenotypic analyses. When a substantial increase in virus replication was identified, DNA was extracted from the infected cells using the QIAamp DNA mini kit (Qiagen). The complete proviral env genes were PCR-amplified using primers 1 (5′-ATAAGCTTAGCAGAAGACAGTGGCAATG-3′) and 2 (5′-GCAAAATCCTTTCCAAGCCC-3′) and then sequenced.Evolution experiments were performed as described before , 59, 61.50) was calculated using a sigmoid function in Prism software version 5.0.LAI viruses and BG505 Env-pseudotyped viruses were generated by transfection of 293T cells as described previously , 47, 59.S. W. d. T., I. A. W., J. P. M., and R. W. S. conceptualization; S. W. d. T., M. M.-R., and R. W. S. resources; S. W. d. T., E. P. G., K. S., A. T. d. l. P., and K. B. data curation; S. W. d. T., E. P. G., K. S., A. T. d. l. P., K. B., M. M.-R., W.-H. L., H. D., and R. W. S. formal analysis; S. W. d. T. and R. W. S. investigation; S. W. d. T. visualization; S. W. d. T. methodology; S. W. d. T. and R. W. S. writing-original draft; S. W. d. T. and R. W. S. project administration; S. W. d. T., K. S., H. D., I. A. W., J. P. M., A.B.W., and R. W. S. writing-review and editing; H. D., J. P. M., A.B.W., and R. W. S. supervision; J. P. M., A.B.W., and R. W. S. funding acquisition; A.B.W. software; R. W. S. validation.


Sex chromosomes originate as a pair of homologus autosomes that then follow a general pattern of divergence. This is evident in mammalian sex chromosomes, which have undergone stepwise recombination suppression events that left footprints of evolutionary strata on the X chromosome. The loss of genes on the Y chromosome led to Ohno’s hypothesis of dosage equivalence between XY males and XX females, which is achieved through X-chromosome inactivation (XCI). This process transcriptionally silences all but one X chromosome in each female cell, although 15–30% of human X-linked genes still escape inactivation. There are multiple evolutionary pathways that may lead to a gene escaping XCI, including remaining Y chromosome homology, or female advantage to escape. The conservation of some escape genes across multiple species and the ability of the mouse inactive X to recapitulate human escape status both suggest that escape from XCI is controlled by conserved processes. Evolutionary pressures to minimize dosage imbalances have led to the accumulation of genetic elements that favor either silencing or escape; lack of dosage sensitivity might also allow for the escape of flanking genes near another escapee, if a boundary element is not present between them. Delineation of the elements involved in escape is progressing, but mechanistic understanding of how they interact to allow escape from XCI is still lacking. Although increasingly well-studied in humans and mice, non-trivial challenges to studying escape have impeded progress in other species. Mouse models that can dissect the role of the sex chromosomes distinct from sex of the organism reveal an important contribution for escape genes to multiple diseases. In humans, with their elevated number of escape genes, the phenotypic consequences of sex chromosome aneuplodies and sexual dimorphism in disease both highlight the importance of escape genes. Sex can be determined through various strategies, either environmentally or genetically. The evolution of chromosomal sex determination begins as the result of a sex-determining mutation on an autosome, favoring the loss of recombination at that location (reviewed in 1–5) and five Y chromosomes (Y1–5), while the female has two copies of each X1–5 (DMRT1) present on X5 (5 (Prototherians (monotremes) and therians  appear to have developed their modern sex chromosome systems in tandem, following the divergence between the two lineages about 200 million years ago (MYA) . Monotreach X1–5 . Four ofach X1–5 , with thnt on X5 . The beson X5 , creating de facto sex chromosomes  and a smaller long arm PAR (PAR2) . As initOutside of the PARs, ongoing Y homology still strongly correlates with escape from XCI, most strikingly for those genes that have retained functional Y gametologs . In an aAs discussed above, the step-wise loss of recombination left footprints of evolutionary strata on the X chromosome. There is a clear correlation between these strata and the likelihood of escape from inactivation, with the more recently diverged genes being more likely to escape . It has Selection could also favor escape from XCI if there is a female-specific benefit to ongoing expression from the Xi. Notably, the acquisition of an invasive placenta coincides evolutionarily with the addition of the XAR, which harbors an excess of escape genes. This change in reproductive biology required female eutherian immune systems to both be able to tolerate fetal antigens and defend against parasites and pathogens during pregnancy. The “pregnancy compensation hypothesis” recently proposed that sex-specific differences in both autoimmune diseases  and most cancers  in industrialized environments are due to adaptions that occured to faciliate successive pregnancies in ancestral conditions .The progressive sequestering of recombination between the sex chromosomes likely coincided with different rates of acquisition for genetic elements that favor the spread of silencing. Escape genes may be more prevalent in the XAR due to a paucity of silencing elements in this region. Notably, escape genes are still expressed from older areas of the X chromosome and so must have also acquired genetic elements that still allow expression from the Xi. Domains of silencing and escape require boundaries between the different regions of closed versus open chromatin. Here we discuss the evidence for silencing, escape, and boundary elements, and the potential effects of the physical structure of the Xi. To date, these features have only been studied in humans and mice.The autosomal portion of an unbalanced X;autosome translocation is able to establish stable silencing, although less efficiently than the X chromosome itself, which led to the proposal of “waystations” that propagate the silencing signal along the X chromosome . These wKdm5c) in four random locations on the mouse Xi  that was observed upon integrating a human BAC transgene onto the mouse Xi demonstrated that such elements could be recognized across species  allows the examination of expression from the Xa or Xi within individual nuclei. Biallelic signals (indicative of escape from XCI) were observed in elephant, human, and mouse for several candidate escape genes, although a substantial proportion of cells showed only a single focus of expression  results from the absence of a second sex chromosome, although mosaicism and rearranged X chromosomes (such as an isochromosome) are observed in a significant proportion of cases. Strikingly, the vast majority of 45,X conceptuses are spontaneously aborted, which has been argued to reflect a need for some level of mosaicism . In spitKlinefelter syndrome, usually 47,XXY but sometimes 48,XXXY or 49,XXXXY, occurs in ∼1/650 male births and is accompanied by the inactivation of all but a single X. While infertility and tall stature are common, additional phenotypic consequences vary widely, contributing to an overall increase in morbidity and mortality . In miceThere are reports of sex chromosome aneuploidy in other mammals, and by reviewing this literature in comparison to genome assemblies, Raudsepp et al. suggest the viability of XO individuals is strongly correlated with a smaller PAR . They faDue to the complexity of comparing XX females and XY males, the X chromosome is often excluded from genome-wide association studies, limiting our understanding of its relationship to complex diseases. Conversely, X-linked mutations in hemizygous males are readily detected, and thus a high density of monogenic traits have been assigned despite the relatively low gene density. The X chromosome is subject to unique evolutionary pressures: it spends more time in females, yet the hemizygosity in males allows immediate expression (and therefore positive or negative selection) of new mutations. Thus, the X chromosome has a unique repertoire of genes, and has been described as a “smart and sexy” chromosome due to genes involved in brain and gametogenesis . There aThe X chromosome is home to a variety of immune-related genes. Auto-immunity is often more common in females  and silencing of one X chromosome in females , studies in the last 50 years have revealed unexpected alternatives. In humans over 25% of genes escape from XCI, and over half of these do not have Y gametologs. Although multiple controlling elements have been suggested, it is currently unknown what DNA sequences, chromatin features, or ultrastructural contributions allow genes to escape from XCI. Identification of these elements will provide insights into heterochromatin, and may inform therapeutics aimed at reactivating X-linked genes, silencing autosomes, or avoiding host silencing of gene therapies. In mice, the use of sex-reversed mice to assess the “four core genotypes” allows researchers to distinguish the impact of the sex chromosomes from sex of the animal, and has revealed chromosomal contributions to many diseases . Since hBP and CB each contributed to writing of the review.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.