Previous studies have reported increased cross-modal auditory and visual cortical activation in cochlear implant (CI) users, suggesting cross-modal reorganization of both visual and auditory cortices in CI users as a consequence of sensory deprivation and restoration. How these processes affect the functional connectivity of the auditory and visual system in CI users is however unknown. We here investigated task-induced intra-modal functional connectivity between hemispheres for both visual and auditory cortices and cross-modal functional connectivity between visual and auditory cortices using functional near infrared spectroscopy in post-lingually deaf CI users and age-matched normal hearing controls. Compared to controls, CI users exhibited decreased intra-modal functional connectivity between hemispheres and increased cross-modal functional connectivity between visual and left auditory cortices for both visual and auditory stimulus processing. Importantly, the difference between cross-modal functional connectivity for visual and for auditory stimuli correlated with speech recognition outcome in CI users. Higher cross-modal connectivity for auditory than for visual stimuli was associated with better speech recognition abilities, pointing to a new pattern of functional reorganization that is related to successful hearing restoration with a CI. Specifically, the lack of visual or auditory sensory input induces a shrinkage of the corresponding cortical representations, and is usually paralleled by an invasion of the spared sensory modalities7. In the case of deafness, modern cochlear implant (CI) technology allows a partial hearing recovery8. Hearing recovery with CIs also induces cortical reorganization that may be qualitatively different from changes previously induced during the period of deafness. Specifically, CI users have shown higher cross-modal cortical activation in both visual and auditory regions when compared to normal hearing (NH) controls12. It is often suggested that this pattern of reorganization of auditory cortex reflects leftover cortical changes previously induced by deafness. On the other hand, a recruitment of visual cortex for auditory processing presumably reflects cortical changes associated with the adaptation to the new CI input. This distinction is important, as strong cross-modal reorganization of auditory cortex impacts negatively on CI outcome10, whereas cross-modal changes in visual cortex seem to support speech understanding in CI users12.Cortical changes induced by sensory deprivation have been robustly reported in blind and deaf individuals14. Supporting evidence comes from animal studies showing increased anatomical projections from visual to auditory cortices in both early- and late-deaf versus normal-hearing cats17, even though these changes are very specific and the general pattern of cross-modal connectivity seems not significantly altered in deaf animals18. In humans, it has also been found that visual input can modulate auditory responses, which is compatible with the idea of direct cross-modal interactions20. Alternatively, such mediation could be implemented through a separate multi-sensory region, such as in parietal cortex. Indeed enhanced structural connectivity between parietal cortex and low level visual cortices has been observed in deaf individuals22. Importantly, both theories predict increased functional connectivity between visual and auditory areas in both deaf individuals and CI users. Unfortunately, in sharp contrast to numerous structural and task-induced functional connectivity studies in blind and deaf individuals27, little is known about cross-modal connectivity in CI users, despite the significance of such observations in suggesting an increase of information flow between visual and auditory cortices. Reasons for this lack of evidence are mostly related to safety concerns with regards to metal implants in a magnetic resonance imaging scanner, which precludes a proper assessment of structural or functional connectivity in CI users.The mechanisms underlying cross-modal reorganization in CI users remain poorly understood. One theory suggests that the process is mediated by the enhancement of pre-existing connections between visual and auditory cortex28. This work demonstrates that fNIRS can be used to assess potential changes in functional connectivity patterns in CI users. In the current study we therefore re-analyzed a previous fNIRS dataset in which CI users and controls were presented with visual and auditory stimuli9. The aim was to investigate whether task-induced cross-modal functional connectivity between visual and auditory cortices is indeed elevated in CI users as predicted from the possible mechanisms underlying cross-modal reorganization. Additionally, following the recent resting state connectivity results28 we expected decreased task-induced intra-modal functional connectivity between hemispheres in both visual and auditory areas in CI users compared to NH controls, reflecting intra-modal plasticity in CI users. Since cross-modal reorganization may help to explain variability in clinical outcome10, we assessed the relationship between cross-modal functional connectivity and speech recognition abilities in CI users. Specifically, with previous evidence pointing towards a negative role of cross-modal activation in auditory cortex and a positive role of cross-modal activation in visual cortex, we predicted speech recognition to have a positive relationship with cross-modal connectivity for auditory stimuli and a negative relationship with cross-modal connectivity for visual stimuli.Functional near-infrared spectroscopy (fNIRS) provides the unique opportunity to close this gap. A recent fNIRS study reported for the first time decreased resting state functional connectivity between left and right auditory areas in CI users compared to NH controls29. One CI user was excluded due to contamination of noisy signal from movement. The remaining nineteen CI users were all unilaterally implanted, three in the left ear and the other sixteen in the right. All CI users had been continuously using their devices for at least 6 months prior to the experiment  comprising twenty post-lingually deaf CI users and twenty NH individuals participated in the study. All participants had normal or corrected-to-normal vision, and none had a history of neurological or psychiatric illness. Four participants were left-handed and the others were right-handed as assessed by the Edinburgh Handedness Inventory10. Checkerboards (image dimensions are 1024 × 1024 pixels) were radial and consisted of twenty rings, each of which was divided into 18 sectors of interchanging white and black color. This layout compensated for the increase in receptive-field size with eccentricity31. There were four pairs of checkerboard patterns with different proportions of white pixels, corresponding to luminance ratios of 12.5%, 25%, 37.5%, and 50%. The contrast between white and black pixels was identical in all images. Contrast reversals were presented at 2 Hz for 10 seconds. All visual stimuli were presented on a 24-inch monitor at a distance of 150 cm. The visual angle of the checkerboards was 10.5°.The visual stimuli were circular checkerboard patterns adopted from a previous study32. The reversed words were the same German words played backwards. The goal here was to compromise the intelligibility but maintain the overall spectral properties. Word trains of three consecutive words/reversed words were presented with an inter-stimulus interval of 1.3 s and a total duration of 5 s. Within each word train, the words/reversed words were either repeated or not repeated, resulting in four conditions (words/reversed words x repeated/unrepeated) in total. Tones, adopted from Zhang and colleagues (2011), were 1 kHz pure tones of 40 ms duration with 5 ms linear onset and offset ramps. Bursts of five tones were presented with an inter-stimulus interval of 0.7 s and a total duration of 3 s. All auditory stimuli were presented through two loudspeakers located in front of the participants, 21 degrees to the right and to the left at ear height. The loudness level for both tones and words was adjusted individually to a subjectively-reported comfortable loudness level.Auditory stimuli consisted of words, reversed words and tones were all sampled at 44.1 kHz. Disyllabic German words each had a duration of 800 ms and were adopted from a previous study35. A silent nature documentary was presented in the center of the screen during the auditory session, which lasted for around 30 mins. Within both visual and auditory sessions, all the blocks were randomly presented in sequence. The order of the visual and the auditory sessions was counterbalanced across participants.The experiment consisted of a visual and an auditory session Fig. . In the 29 and health state. CI participants additionally answered a questionnaire of CI-related questions such as the duration of deafness or CI implantation. For the visual session, participants were instructed to fixate at the center of the screen continuously, and to press a button at the end of the stimulus to indicate whether the stimulus belonged to a higher (50% or 37.5%) or to a lower (25% or 12.5%) luminance ratio. In the auditory session, participants were instructed to fixate at the center of the screen and to avoid saccades as much as possible (closing eyes was discouraged). The task was to focus on the silent documentary and to ignore the sound presentation. Participants were informed that after the experiment a questionnaire would be given related to the video content and that the answers would be evaluated. Prior to the data recording, the participants received training for the visual task, which was repeated until the hit rate exceeded 75%. After the experiment, CI users performed three speech recognition tests which were the Freiburg monosyllabic words test36, the Oldenburg sentences test (OLSA) in quiet, and the OLSA test in noise37. All speech recognition tests were auditory only without any visual component. The Freiburg monosyllabic word test measures single word recognition and consists of twenty lists of monosyllabic words. One list was randomly selected for each measurement, and the percentage of correctly repeated words by the participant was calculated as the test score. The OLSA test measures word recognition within a sentence structure. The OLSA test in quiet (OLSA_q) measures the percentage of correctly identified words within a sentence at a sound intensity level of 65 dB SPL. The OLSA test in noise (OLSA_n) uses an adaptive procedure to estimate the speech recognition rate, which was defined as the signal-to-noise ratio at which the participants achieved 50% correctly identified words within a sentence. The OLSA_n test was only performed for participants who achieved at least at 50% correct rate in the OLSA_q test to avoid frustration for the participants, since speech recognition in noise is dramatically more difficult and demanding compared to speech recognition in quiet. This resulted in one CI user without an OLSA_n test score . All participants answered a set of questionnaires including handedness38.Functional near-infrared spectroscopy (fNIRS) was recorded using a NIRScout 816 device  with eight LED sources (intensity 5 mW/wavelength) and twelve detectors. The optodes were placed over the left and the right temporal areas, centered around positions T7 and T8 in the 10–20 system, and over the left and the right occipital areas, centered around O1 and O2. Within each area, two sources and three detectors were placed, with each source and its neighboring detector being separated by 3 cm Fig. . Each so41, only HbR concentrations were analyzed in the current study. In four CI users, two channels were blocked by the transmitter of the CI device and had to be excluded from subsequent analyses. In addition, all channels selected in the previous report of this same dataset  were excluded. In this previous study only channels with the highest beta values estimated from a general linear model (GLM) in occipital and temporal areas for visual and auditory stimuli were statistically analyzed. This was done for two reasons. Firstly, most previous animal studies suggest that cross-modal take-over in deafness may be strongest in the secondary visual and auditory areas rather than primary areas42. We reasoned that channels showing highest activation for visual checkerboard and auditory tone stimuli could be functionally identified as capturing responses from more primary visual and auditory cortices. Thus by excluding these channels our analysis focused better on secondary visual and auditory areas. Secondly, since this same data set has been previously reported showing higher cross-modal activation in both visual and auditory areas in CI users compared to NH controls9, this allowed us to explore the unreported data.The fNIRS data were imported into Matlab and transformed to concentration levels (unit: mM) of oxygenated (HbO) and deoxygenated hemoglobin (HbR) using the NILAB toolbox . Since HbR concentrations are thought to be more spatially specific than HbO concentrations9, were correlated to all other channels using Pearson correlations. This was done separately for the 20 minutes visual session time series and for the 30 minutes auditory session time series. A Fisher’s z transformation was used to transform the resulting r values to z values for subsequent analysis. Fisher’s z values were averaged separately to estimate intra-modal connectivity and cross-modal connectivity. Specifically, intra-modal connectivity consisted of correlations between left and right auditory areas (Corr_1) and between left and right visual areas (Corr_2), and cross-modal connectivity consisted of correlations between left visual and left auditory areas (Corr_3), left visual and right auditory areas (Corr_4), right visual and left auditory areas (Corr_5), and right visual and right auditory areas (Corr_6). The z values were averaged separately for visual and auditory stimuli conditions  and modality  as within-subject factors and group  as a between-subject factor, were performed to investigate differences in cross-modal and intra-modal functional connectivity between CI users and NH controls. Since previous studies showed evidence of hemisphere-specific cross-modal reorganization of auditory cortex in post-lingually deaf CI usersres Fig. . All staTo investigate the relationship between cross-modal connectivity and speech recognition, cross-modal connectivity (Corr_3–6) was first normalized by subtracting intra-modal connectivity (Corr_1 & 2) to account for individual differences. Additionally, since cross-modal connectivity for visual and for auditory stimuli was predicted to be negatively and positively associated with speech recognition performance respectively, the normalized cross-modal connectivity for auditory stimuli was subtracted from cross-modal connectivity for visual stimuli and correlated with the individual speech recognition ability using Spearman’s Rho correlation to account for non-linearity.Figure 1,36 = 171.487, p < 0.0001, ŋp2 = 0.826) with higher intra-modal connectivity than cross-modal connectivity, a significant main effect of stimulus type  with higher connectivity for visual than for auditory stimuli, a significant interaction between modality and group , and a significant interaction between modality and stimulus type . The interaction between modality and group was the focus of the current study and thus was followed up by averaging between visual and auditory stimuli. The results showed significantly decreased intra-modal connectivity in CI users compared to NH controls , but no significant difference in cross-modal connectivity between the groups . Additionally, intra-modal connectivity was larger than cross-modal connectivity for both CI users  and NH controls . The result therefore shows that the interaction was driven by differences in intra-modal connectivity , modality  and group  for the right auditory area revealed a significant main effect of modality  with higher intra-modal connectivity than cross-modal connectivity, a significant stimulus type effect  with higher connectivity for visual than auditory stimuli, a significant interaction between modality and group , and a significant interaction between modality and stimulus type . Again the interaction between modality and group was followed-up with both stimuli averaged. The results showed significantly decreased intra-modal connectivity , as well as significantly increased cross-modal connectivity in CI users compared to NH controls  and NH controls .The repeated measures three-way ANOVA for the left auditory area revealed a significant modality effect . This association was robust against potential outliers. Outliers were identified via Mahalanobis distance and bootstrappingWe compared intra-modal and cross-modal functional connectivity between a group of post-lingually deaf CI users and a group of age- matched NH controls. Post-lingually deaf CI users exhibited reduced intra-modal connectivity within visual and auditory areas on the one hand and greater cross-modal connectivity between visual and auditory areas in the left hemisphere on the other. In particular, the latter observation was found irrespective of the type of sensory processing  involved. CI users thus recruited both auditory and visual cortices for processing both visual and auditory uni-sensory stimuli. Moreover, cross-modal functional connectivity was correlated with individual speech recognition skills in CI users. The current approach reveals important additional information which is complementary to the previous report. Specifically, the cross-modal activation in both visual and auditory cortices were not simply higher in CI users, they were also more synchronized temporally with the intra-modal activation. This additional temporal information strengthens the hypothesis that auditory cortex is recruited for visual processing and visual cortex is recruited for auditory processing in CI users.24. In particular, animal studies have revealed abnormally increased structural connectivity between visual and auditory sensory cortices not only in early deafness but also in adult-onset deafness17. Thus the current finding of greater cross-modal functional connectivity in post-lingually deaf CI users may reflect left-over changes in connectivity previously induced during the period of deafness. However, previous animal studies focused exclusively on projections from visual to auditory cortex17 and little is known about possible projections from auditory to visual cortex. In contrast, our results indicate increased functional connectivity in CI users for processing both visual and auditory stimuli, which may suggest a bi-directional connection between visual and auditory cortices. Unfortunately, the fNIRS approach adopted here limits the observation to functional connectivity only, and the underlying anatomical pathways remain unclear. While the investigation of structural connectivity in CI users is not possible due to safety concerns, fNIRS provides a first opportunity to explore patterns of functional connectivity in CI users. Our current study was exploratory in nature and therefore does not allow us to draw strong conclusions. However, it provides first evidence suggesting altered cross-modal connectivity patterns in CI users, a finding that should be followed up in future studies. Future studies are encouraged to also explore the possibility of projections from auditory to visual cortex in both deaf and implanted animals. An auditory-to-visual connectivity study may shed light on whether sensory deprivation inflates bidirectional structural connectivity or whether hearing restoration with a CI results in systematic connectivity changes.Increased structural and functional connectivity has been previously observed in deaf animals and, likewise, in deaf humansThe visual and auditory tasks used in the present study were different in nature, since the visual task was an active discrimination task and the auditory task was a passive listening task while watching a silent video. However, one should note that the present study was not designed to compare between visual and auditory stimuli, but instead to compare between CI users and NH controls. Since both CI users and NH controls were presented with the identical stimuli, the imbalance between the visual and the auditory task was unlikely to influence the observed group difference. Another concern may be raised on the presence of visual stimuli in the auditory paradigm. While we cannot rule out a possible influence of visual stimuli in the auditory task on cross-modal connectivity, we argue that such influence would interfere with existing correlation since the auditory stimuli were temporally independent from the video stream. Supporting this argument, we found overall higher z values, which represents correlations, for visual than for auditory stimuli, suggesting that the presence of a video stream introduces potential interference to the correlation in the auditory session. Altogether, we believe that our observation of functional connectivity pattern changes in CI users compared to NH controls cannot be attributed solely to the presence of visual input during the auditory session. Nevertheless, more studies are required to confirm our findings.45. However it remains unclear whether this reorganization is lateralized. Some studies have reported effects primarily in the right hemisphere46, others in the left47, and some have reported no hemispheric differences at all50. Several factors such as type of stimuli47 and experience with sign language51 have been suggested to contribute to this discrepancy. The current study identified increased cross-modal functional connectivity between the left auditory cortex and both right and left visual cortices. The result is consistent with a recent study showing increased functional connectivity between left auditory cortex and right visual cortex in the elderly with hearing loss52. Additionally, the effect in the left hemisphere may be related to the use of speech stimuli. Language processing is left-lateralized in most individuals55. However, whether or how these factors contribute to the cross-modal take-over remains to be investigated. More studies are required to systematically dissociate the potential influence of language processing on deprivation-induced and restoration-related functional connectivity changes in CI users.There is clear evidence of cross-modal reorganization of both visual and auditory cortices in both deaf individuals and CI users28. However, decreased connectivity between hemispheres in visual areas for visual stimuli in CI users was also observed, which may seem rather surprising. Several other studies have also indicated changes in hearing-impaired individuals in visual processing. Specifically, mixed reports exist on whether visual activation in visual cortex may be lower or higher in deaf and CI users compared to NH controls57. Furthermore, altered visual cortex activation may be related to improved visual abilities in deaf individuals. At least one study found an association between visual-evoked potential P1 amplitudes and faster visual detection response times in deaf individuals58. Additionally, another study found increased stimulus-specific adaptation for visual stimuli in CI users, which potentially suggests increased encoding efficiency for visual stimuli38. As a result, it may be that decreased functional connectivity between hemispheres in visual areas contributes to improved visual processing in CI users, such as during lip reading60. Unfortunately visual task performance was not assessed in our study, so it is not feasible to evaluate this hypothesis directly. Future study should follow up on this and determine whether decreased functional connectivity within visual areas serves as the underlying mechanisms for altered visual processing and performance in CI users.On the other hand, a decreased intra-modal connectivity in CI users compared to NH controls was observed. For auditory processing, less temporal consistency was found between hemispheres in CI users. This result is consistent with the degraded auditory input with CIs and has also been previously reported in both animals and humansConsistent with our hypothesis, cross-modal functional connectivity correlated with speech recognition. Specifically, individuals with higher cross-modal functional connectivity for auditory than for visual stimuli performed better than individuals exhibiting the opposite pattern. However, one should note that the current investigation revealed a correlation only with one out of three speech recognition tests performed. Unlike the OLSA tests measuring speech recognition in sentences, the Freiburg monosyllabic words test measures recognition of single words. Our result suggests that the observed changes in functional connectivity may be sensitive to only speech recognition in quiet without sentence structure information. Future studies are required to systematically investigate the relationship between functional connectivity patterns and different aspects of speech recognition. Additionally, the current correlation is consistent and complementary to our previous findings, suggesting that cross-modal activation in the visual cortex may be beneficial for speech recognition whereas cross-modal activation in the auditory cortex may be maladaptive. However, a few differences should be noted. Specifically in the previous report we used only auditory words in the correlation analysis, and in contrast both tones and words were used in the present study. Unfortunately since all the auditory conditions were randomly presented in sequence, the current experimental design did not allow us to investigate functional connectivity patterns for different type of auditory stimuli. Our results show on average increased cross-modal functional connectivity between visual cortices and the left auditory cortex, regardless of the type of auditory stimuli. Future studies should investigate systematically whether different type of auditory stimuli may have different influence on cross-modal functional connectivity.10. Note however that there may still be a functional purpose for increased cross-modal functional connectivity for visual processing. Our group recently reported a positive correlation between cross-modal activation in auditory cortex for face stimuli and lip reading46. Thus increased cross-modal connectivity for visual processing may facilitate lip reading and thereby support daily life communication in CI users. In other words, future studies should jointly consider both aural speech recognition and lip reading ability to better estimate CI benefit and the relationship with cross-modal reorganization in CI users3.Since communication in daily life often involves integration between visual and auditory modalities, the correlation result suggests that speech recognition based on degraded auditory input from a CI can be improved by recruiting visual areas. On the other hand, since visual perception of flickering checkerboards is not necessarily an inherently multisensory experience, it may not be beneficial to recruit auditory cortex for processing simple visual stimuli, hence the negative correlation between functional connectivity for visual stimuli and speech recognitionThe current study provides first evidence of altered cross-modal functional connectivity in CI users. Increased cross-modal functional connectivity between visual and auditory areas and decreased intra-modal connectivity between hemispheres was found when CI users were compared to NH controls. This finding extends the observation of cross-modal reorganization in both visual and auditory cortices, by indicating increased information flow between the sensory cortices. Structural connectivity cannot be assessed in CI users. The current functional connectivity study may have identified a mechanism underlying cross-modal reorganization in post-lingually deaf CI users. Changes in functional connectivity could serve as a new, complementary tool for predicting clinical outcome after cochlear implantation.


The unprecedented challenges of developing effective vaccines against intracellular pathogens such as HIV, malaria, and tuberculosis have resulted in more rational approaches to vaccine development. Apart from the recent advances in the design and selection of improved epitopes and adjuvants, there are also ongoing efforts to optimize delivery platforms. The unprecedented challenges of developing effective vaccines against intracellular pathogens such as HIV, malaria, and tuberculosis have resulted in more rational approaches to vaccine development. Apart from the recent advances in the design and selection of improved epitopes and adjuvants, there are also ongoing efforts to optimize delivery platforms. Viral vectors are the best-characterized delivery tools because of their intrinsic adjuvant capability, unique cellular tropism, and ability to trigger robust adaptive immune responses. However, a known limitation of viral vectors is preexisting immunity, and ongoing efforts are aimed at developing novel vector platforms with lower seroprevalence. It is also becoming increasingly clear that different vectors, even those derived from phylogenetically similar viruses, can elicit substantially distinct immune responses, in terms of quantity, quality, and location, which can ultimately affect immune protection. This review provides a summary of the status of viral vector development for HIV vaccines, with a particular focus on novel viral vectors and the types of adaptive immune responses that they induce. Since Edward Jenner first demonstrated immunity to smallpox by inoculating a 13-year-old boy with vaccinia virus over 200 years ago, various vaccines effective against numerous microorganisms have been developed. However, the challenges of developing vaccines protective against intracellular pathogens such as human immunodeficiency virus type 1 (HIV-1) has necessitated the adoption of more rational approaches to vaccine design based on the systematic design of epitopes, the use of immunogenic adjuvants, and the selection of appropriate delivery platforms. Viral vectors belong to one such platform and have inherent adjuvant capability in the form of pathogen-associated molecular patterns that can trigger innate immune responses through their engagement of specific pattern recognition receptors. More importantly, different viral vectors may exhibit distinct cellular tropism, with specific innate and adaptive immune phenotypes that render them optimally poised for inducing immunological memory against particular pathogens. Although preexistent immunity is an important factor in vector selection, a better understanding of the immune correlates of protection will ultimately guide vaccine design. In this review, we will summarize the current status of viral vector-based HIV vaccines, highlighting the effects of various vectors on vaccine immunogenicity, safety, and efficacy. This will include a description of various virus vectors that have been used in HIV vaccine development, which include nonreplicating and replicating adenovirus type 5 (Ad5), alternative-serotype adenovirus, poxvirus, lymphocytic choriomeningitis virus (LCMV), cytomegalovirus (CMV), vesicular stomatitis virus (VSV), and attenuated immunodeficiency viruses.Given the complexities of HIV infection, it is increasingly clear that a successful vaccine must elicit multiple arms of adaptive immunity. One way to accomplish this goal is through the selection of distinct vaccine prime-boost platforms.1V2-specific IgG antibodies as inversely associated with infection rates  and antibody responses. The RV144 trial, which is the most successful HIV vaccine trial to date, resulted in 31.2% protection from HIV infection and utilized a heterologous canarypox virus vector (ALVAC strain) priming-protein boosting approach -infected macaques results in increased viral replication –7. Many –8–Furthermore, the important role of CD4 T cells in facilitating both the innate and adaptive immune systems has led to suggestions that vaccine modalities that preferentially induce CD4 T cell responses may be necessary for optimal vaccine-mediated protection. Indeed, it has been shown that CD4 T cells may be necessary for the effective control of certain diseases such as tuberculosis , 15, Wes18–24–However, prior studies have demonstrated that CD4 T cell-biased vaccines may be detrimental in the context of chronic viral infections , 28. In in silico development of mosaic antigens that maximize immune responses to global circulating strains. A demonstration of this novel approach was reported in prior studies that demonstrate that mosaic antigens induce a greater depth and breadth of immune responses relative to consensus antigens  and is colloquially referred to as the “Thai trial,” resulted in 31% protection from HIV acquisition associated with Env Vtibodies . Preventtibodies . To builIn total, three additional poxvirus vectors have garnered significant attention as candidate HIV vaccine vectors. These vectors can be segregated into two groups, (i) orthopoxvirus-derived NYVAC (from the Copenhagen vaccine strain) and MVA and (ii) avipoxvirus-derived ALVAC (canarypox virus) and fowlpox virus. All four vectors are replication incompetent in mammalian cells –93. The –+ T cell and antibody responses in the NYVAC-primed animals  are short-lived but provide immediate cytotoxic function . TherefoOf the various replication-competent vaccine vectors currently under development, recombinant human CMV (rhCMV) and replication-competent recombinant VSV (rVSV) , 107 havAs a classical member of the herpesvirus family, rhCMV persists in the host, providing a constant source of antigen necessary for the maintenance of CD8 T cells with an effector memory phenotype. Similar to novel rLCMV vectors genetically lacking LCMV GP, the immunogenicity of rhCMV vectors is not limited by preexisting antivector immunity and they can therefore be used repeatedly, even in CMV-positive monkeys, to induce effector memory CD8 T cells . This prrVSV-based vectors have garnered renewed interest, given the recent highly successful phase III trials results of an rVSV-based vaccine for Ebola virus , anotherEfforts are ongoing to develop and test other replicating vectors that might also elicit rapid effector memory T cell responses. These efforts include recombinant replication-competent Ad4, Ad5, and Ad26 vectors –121, Sen–In addition, one of the most salient examples of immune protection in the SIV infection model is that induced by SIVΔnef . It has 126–As mentioned earlier, an advantage of replicating vectors is their enhanced antigen expression, which could potentially drive robust adaptive immune responses. High and permanent transgene expression can also be achieved by immunization with HIV-based lentiviral vectors, which integrate into the genome and provide persistent antigen expression, even in nondividing cells –131. One–Until now, we have discussed various vaccine vectors that are used for active immunization, which refers to the induction of the host immune response after exposure to an antigen. However, a main problem with HIV vaccine design is the difficulty of generating broadly neutralizing antibodies (bNAbs). This could be circumvented by cloning bNAb genes into viral vectors to directly induce the expression of bNAbs at the injection site, bypassing all of the steps that are required for the generation of bNAbs, including isotype switching, consecutive germinal center reactions, and somatic hypermutation. Recently, an adeno-associated virus (AAV) vector that constitutively expresses HIV-specific antibodies in a humanized mouse model of HIV infection has shown promise , 134. ThThe successful development of an HIV vaccine remains an unprecedented challenge. We have summarized here various virus vectors that are being developed in the quest to overcome this challenge . One of


Nonocclusive mesenteric ischemia (NOMI) is a mesenteric arterial spasm and intestinal ischemia. This disease is a highly lethal disease because diagnosis and decision of appropriate treatments are often difficult. Operations cannot resolve the spasms and may worsen the situation. However, the safety and effectiveness of catheterization for NOMI with aortic dissection (AD) have not yet been elucidated. Here, we report a successful case of early diagnosis and treatment of NOMI with type B AD involving the superior mesenteric artery (SMA) using the intra-arterial infusion of a vasodilator via the SMA.An 83-year-old man was admitted to our hospital because of abdominal pain after a motor accident. We performed intestinal resection and splenectomy for intestinal perforation and splenic hemorrhage and treated conservatively for acute AD, liver injury, renal hematoma, and pneumothorax. On postoperative day (POD) 2, the patient had localized abdominal pain. Follow-up computed tomography suggested a smaller superior mesenteric vein sign and segmental lack of enhancement in the intestinal wall and ascites without SMA occlusion. Thus, the patient was diagnosed with NOMI. Although the patient had type B AD including the SMA, we performed selective mesenteric arteriography and transcatheter papaverine infusion via the SMA and prostaglandin via the peripheral vein. Seven days post treatment, mesenteric blood flow improved and intestinal wall enhancement was restored.The intra-arterial infusion of a vasodilator is highly efficient and safety treatment option for NOMI with type B AD. Prompt and accurate management can prevent massive small bowel resection, and this procedure is essential in resolving a spasm independent of whether a necrotic bowel has been resected. Nonocclusive mesenteric ischemia (NOMI) refers to a mesenteric arterial spasm and intestinal ischemia and is a highly lethal disease; the mortality rate of NOMI is reported to be 70–90% , 2. TreaAn 83-year-old man was admitted to our hospital because of abdominal pain after a motor accident. Focused assessment with sonography suggested the presence of blood in the abdomen. Contrast-enhanced computed tomography (CT) was performed Fig. , which iWe present a successful case of catheter-directed infusion into the SMA for a patient with NOMI complicated with traumatic type B AD involving the SMA. Vasodilator infusion via arterial catheter has been reported as an effective treatment for NOMI and many reports suggested efficacy of this strategies –7. In 19First, the catheterization of and arterial infusion for the dissected aorta are essential, and more safety procedures were required than expected. We performed catheter-directed infusion of a vasodilator into the SMA because we believe that vasodilator therapies are the most important management option for all patients with NOMI; the resection of necrotic bowels cannot resolve spasms, which can recur by additional invasion, especially in such severe conditions . AlthougSecond, contrast-enhanced CT considering vital signs, physical examinations, and laboratory data is essential for the accurate and prompt evaluation of NOMI . CT imagThe intra-arterial infusion of a vasodilator is highly efficient and can be considered as a feasible treatment option for NOMI with type B AD. Prompt and accurate management can prevent massive small bowel resection. Furthermore, this procedure is essential in resolving a spasm independent of whether a necrotic bowel has been resected.


ClinicalTrials.gov, the databases of conferences were queried to identify RCTs evaluating the efficacies and toxicities of intensive therapies used for first-line treatment of mCRC patients. The search included articles dated from the inception of these resources until March 31, 2017. We estimated HRs for OS and PFS and RRs for ORR, the R0 resection rate, and toxicities. Ten RCTs comprising 2,506 patients were included in this network meta-analysis. The PFS of patients administered FOLFOXIRI plus target therapy experienced prolonged PFS and OS and improved ORRs compared with FOLFOX/FOLFIRI plus target therapy . There were no significant differences between PFS, OS, ORRs, or R0 resection rates and toxicities of patients administered FOLFOXIRI and FOLFOX/FOLFIRI plus target therapy. Further, FOLFOXIRI plus target therapy did not increase toxicities compared with FOLFOX/FOLFIRI plus target therapy. FOLFOXIRI plus target therapy when administered as first-line treatment of patients with mCRC is the best choice and did not increase toxicities. The patients with RAS/BRAF mutations could benefit from FOLFOXIRI plus Bev. FOLFOXIRI is as effective as FOLFOX/FOLFIRI plus target therapy.Whether the intensive administration of folinic acid, 5-fluorouracil, oxaliplatin and irinotecan (FOLFOXIRI) alone or combined with target therapy as first-line treatment could improve the prognosis of metastatic colorectal cancer (mCRC) patients is controversial. PubMed, the Cochrane Collaboration Central Register of Controlled Clinical Trials, Cochrane Systematic Reviews,  RAS or BRAF mutations [First-line treatment using folinic acid, fluorouracil, and oxaliplatin (FOLFOX) or folinic acid, 5-fluorouracil, and irinotecan (FOLFIRI) combined with target therapy prolongs the overall survival (OS) of patients with metastatic colorectal cancer (mCRC) patients compared with dual chemotherapy, particularly when administered to patients without utations –7. A stuutations . HoweverRandomized controlled trials (RCTs) compared the efficacy and toxicities of triple chemotherapy  plus target therapy with those of triple or double chemotherapy (FOLFOX or FOLFIRI) plus target therapy –11. The The titles and abstracts of 248 studies were reviewed. After the initial screen, we performed a detailed assessment of potentially eligible papers and selected 11 papers and 2 abstracts of 10 randomized controlled trials (RCT) , 9–14  Figure . There wThe pooled RRs of ORR of patients administered individual regimens compared with FOLFOX or FOLFIRI plus target therapy in the network meta-analysis shows that the ORR of patients administered FOLFOXIRI plus target therapy was higher compared with that of FOLFOX or FOLFIRI plus target therapy  Figure . There wThe pooled RRs of toxicities of patients administered individual regimens compared with double chemotherapy plus targeted therapy in the network meta-analysis shows no increase in toxicities between FOLFOXIRI and FOLFOX or FOLFIRI plus target therapy Figure . FurtherRAS/BRAF mutations and the patients without RAS mutations. For the patients with RAS mutations (n = 454), FOLFOXIRI plus Bevacizumab (Bev) significantly prolonged the PFS compared with FOLFOX or FOLFIRI plus Bev . For the patients with BRAF mutations (n = 54), FOLFOXIRI plus Bev prolonged the PFS compared with FOLFOX or FOLFIRI plus Bev, but the difference was not significant . For the patients without RAS mutations (n = 292), FOLFOXIRI plus Bev significantly prolonged the PFS compared with FOLFOX or FOLFIRI plus Bev   .RAS/BRAF mutations could benefit from FOLFOXIRI plus Bev compared with FOLFOX or FOLFIRI plus Bev. Triple chemotherapy used in some studies was as effective as double chemotherapy plus target therapy and was associated with acceptable toxicities. Therefore, patients who cannot afford target therapy may have the option of choosing FOLFOXIRI as their first-line treatment.The results of this network meta-analysis show that first-line treatment using FOLFOXIRI plus target therapy prolonged PFS and OS and improved the ORR and R0 resection rate of patients with mCRC without a significant increase in toxicities. The patients with BRAF mutations, the cytotoxic triplet FOLFOXIRI plus Bev is an option. But the grade of this evidence was IIB [This study is the first to our knowledge to conduct a network meta-analysis that ranks the efficacy and toxicities of intensive therapy strategies. NCCN 2017 version 2 guidelines recommend a choice of FOLFOX, FOLFIRI, CapeOx, infusion 5-FU/LV or capecitabine, or FOLFOXIRI, with or without targeted agents as initial therapy for mCRC patients appropriate for intensive therapy. But which one is the best choice was still controversial. The 2016 ESMO guidelines recommend that the goal for treating patients with potentially resectable or nonresectable mCRC who have the ability to withstand intensive therapy should achieve shrinkage of clinically relevant tumors to the maximum extent possible . Further was IIB .Considering the absence of sufficient evidence, the application of intensive therapeutic strategies for treating patients with mCRC is limited. The results of our network meta-analysis suggest that patients with mCRC may benefit from FOLFOXIRI plus target therapy as first-line treatment. Therefore, FOLFOXIRI or FOLFOXIRI plus Bev may be a more suitable option for patients able to withstand intensive treatment. Moreover, the analysis of toxicities presented here show that FOLFOXIRI did not significantly increase toxicities compared with those associated with FOLFOX or FOLFIRI plus target therapy. And FOLFOXIRI plus target therapy also did not increase toxicities compared with those associated with FOLFOX or FOLFIRI plus target therapy, except for neutropenia.The number of patients with mCRC who can afford target treatment is limited, and the efficacies and toxicities of triple chemotherapy and double chemotherapy plus target chemotherapy were not determined by RCTs. The results of our indirect comparison using network meta-analysis suggest that patients treated with FOLFOXIRI achieved similar PFS, OS, ORR, and R0 resection rates compared with patients treated with double chemotherapy plus target chemotherapy. Therefore, patients with mCRC who cannot afford target therapy may choose FOLFOXIRI as their first-line treatment.This study did not analyze patients’ ages or their performance status because only some certain criteria for the patients administered FOLFOXIRI with or without target therapy were referred in the included studies but not consistent. For example, in the STEAM study , patientRAS and BRAF mutations is not currently required for treatment using Bev, and the HRs of all patients without data for RAS mutational status included in the studies were administered bevacizumab but not an anti-EGFR antibody. Third, the number of eligible studies was small. Therefore, future RCTs must include larger numbers of subjects.Our network meta-analysis has certain limitations. First, target therapy has been used as first-line treatment for mCRC since 2004. Thus, target therapy was not administered to study and control groups in the GONO study , which mRAS/BRAF mutations could benefit from FOLFOXIRI plus Bev. Further, FOLFOXIRI is as effective as double chemotherapy plus target therapy.We conclude that FOLFOXIRI plus target therapy administered as first-line treatment is the best choice for patients with mCRC, because it is not significantly associated with increased toxicities. The patients with ClinicalTrials.gov, and the databases of the European Society for Medical Oncology and American Society of Clinical. The search included articles dated from the inception of these resources until March 31, 2017  guidelines . We searTwo investigators  independently reviewed the entire text of eligible studies. Information was extracted and inserted into an electronic database that included patient characteristics, inclusion and exclusion criteria, treatment protocols, and outcomes. Any disagreement between reviewers was discussed with the other coauthors until a consensus was reached.Outcomes of this study included progression-free survival (PFS), OS, ORR, and the R0 resection rate. Random-effects models were used to account for the heterogeneity among studies. Standard meta-analysis was performed using Stata 12.1 . Network meta-analysis was performed using a netmeta package developed according to the theories of a classical frequentist setting included in the R language framework.


The human genome encodes 538 protein kinases that transfer a γ-phosphate group from ATP to serine, threonine, or tyrosine residues. Many of these kinases are associated with human cancer initiation and progression. The recent development of small-molecule kinase inhibitors for the treatment of diverse types of cancer has proven successful in clinical therapy. Significantly, protein kinases are the second most targeted group of drug targets, after the G-protein-coupled receptors. Since the development of the first protein kinase inhibitor, in the early 1980s, 37 kinase inhibitors have received FDA approval for treatment of malignancies such as breast and lung cancer. Furthermore, about 150 kinase-targeted drugs are in clinical phase trials, and many kinase-specific inhibitors are in the preclinical stage of drug development. Nevertheless, many factors confound the clinical efficacy of these molecules. Specific tumor genetics, tumor microenvironment, drug resistance, and pharmacogenomics determine how useful a compound will be in the treatment of a given cancer. This review provides an overview of kinase-targeted drug discovery and development in relation to oncology and highlights the challenges and future potential for kinase-targeted cancer therapies. Kinases are enzymes that transfer a phosphate group to a protein while phosphatases remove a phosphate group from protein. Together, these two enzymatic processes modulate numerous activities of proteins in a cell, often in response to an external stimulus . ApproxiRecent advances in our understanding of the fundamental molecular mechanisms underlying cancer cell signaling have elucidated a crucial role for kinases in the carcinogenesis and metastases of various types of cancer . Since mThe current procedure for developing robust and selective kinase inhibitors has swiftly evolved from synthesizing analogs of staurosporine to sophisticated structure-based design methodologies, facilitated by molecular docking, crystallography and nuclear magnetic resonance spectroscopy , 25. SinTargeting the kinases harboring oncogenic transformational capacity and metastasis has led to a notable change in the clinical management of cancer Fig. . HundredThe development of kinase inhibitors for the treatment of human cancers started in mid 1970s Fig. . In 1978Kinase inhibitors are very efficacious for the treatment of cancer especially targeting specific mutations that chiefly drive tumorigenesis. They are categorized according to their capacity to catalyze the transfer of the terminal phosphate of ATP to the substrates that usually contain a serine, threonine or tyrosine residue Table . Many reType I kinase inhibitors represent ATP-competitors that mimic the purine ring of the adenine moiety of ATP. Functionally, they interact with the conformational phosphorylated active catalytic site of the kinases. These kinase inhibitors bind to the active conformational site and alter the structural conformation otherwise favorable to phosphotransfer , 99. TypN-terminal loop conformation of kinases [Type II kinase inhibitors act by targeting the inactive conformation of kinases and interact with the catalytic site of the unphosphorylated inactive conformation of kinases . Type II kinases , 103. Th kinases , 103. Th kinases , 104. Du kinases . Since c kinases , 103. OvThe third class of kinase inhibitors bind outside the catalytic domain/ATP-binding site and modulates kinase activity in an allosteric manner. Some authors have divided the allosteric inhibitors into two subtypes where type A inhibitors bind to an allosteric site next to the adenine-binding pocket whereas the type B inhibitors bind elsewhere . OverallThese are also called Type IV kinase inhibitors and undergo a reversible interaction outside the ATP pocket, located in the kinase substrate-binding site. These inhibitors don’t compete with ATP and offer a higher degree of selectivity against targeted kinases . SubstraN2 displacement of a leaving group or by reacting with a Michael acceptor incorporated within the kinase inhibitor [The covalent kinase inhibitors form an irreversible covalent bond with the kinase active site and target a catalytic nucleophile cysteine within the active site of the enzyme , 117. Thnhibitor , 121. Conhibitor . Afatininhibitor . Similarnhibitor . Both ofnhibitor . C481 winhibitor . A recennhibitor . Overexpnhibitor . These knhibitor . Other anhibitor . The appnhibitor . The TypBiochemically, kinase inhibitors are classified according to the activation state of the protein kinase target including the nature of DFG-Asp , the C-helix , and the regulatory spine . Apart from type III or allosteric inhibitors, all the FDA-approved kinase inhibitors form hydrogen bonds with one or more hinge residues. Overall, most kinase inhibitors form: (i) hydrophobic contacts with catalytic spine residues; (ii) contact with the RS3 R-spine residue within the C-helix; (iii) interaction with the gatekeeper residue; and (iv) residues that occur just before the DFG-D of the activation segment , 129. ThFrequent mutations in various protein kinases present specific kinase inhibition as a therapeutically relevant approach in oncology. Kinase inhibitors have evolved to target many different regulatory and inhibitory mechanisms. There are various mechanisms by which kinase inhibitors bind to their target kinases broadly classified into kinase inhibitors that bind either covalently or non-covalently to or around the ATP binding site. Primarily, kinases bind with ATP in a cleft between the N- and C-terminal lobes of the kinase domain. In this domain, the adenine group of ATP is bound by two hydrophobic surfaces and interact via hydrogen bonds to the connector of two lobes, called the “hinge region” –132. TheTraditional cancer therapies follow palliative as well as off-targeted approaches in oncology. In contrast, kinase inhibitors symbolize a class of targeted cancer therapeutic agents with limited nonspecific toxicities. So far, 28 inhibitors with activity targeted to one or multiple kinases have been approved for clinical use. With over 500 members, the kinase family has received a high degree of attention from academic researchers as well as pharmaceutical industries . After tRecent kinase developments include precision therapy based on tumor genomic data. The ability to perform genetic studies of tumors and follow-up treatment decisions based on the identification of tumorigenesis drivers has resulted in significant benefits for patients in need of effective systemic therapy. The detailed information regarding all the clinical trials is out of the scope of this mini-review; however, a few important developments are highlighted. A small number of small molecule tyrosine kinase inhibitors have recently received FDA approval for treatment of non-small cell lung cancer (NSCLC) with EGFR mutations or ALK translocations . AfatiniOverexpression of kinases is observed in multiple carcinomas. In recent years, there has been a major paradigm shift in discovery and screening of natural compounds as potential kinase inhibitors. Emerging data has revealed numerous mechanisms by which natural compounds mitigate kinase mutations. Classically, many of the biological actions of small molecule compounds, especially polyphenols, have been credited with their antioxidant properties, either through their reducing capacities or their possible influence on intracellular redox states. These small molecule bioactives can directly bind receptor tyrosine kinases and alter their phosphorylation state to regulate multiple cell signaling pathways Fig. . ElevateFurthermore, myricetin has been reported to target Akt to inhibit cell transformation and proliferation by directly binding to the ATP-binding site of Akt . SimilarDespite numerous advances, scientists are still trying to understand pathophysiology and application of kinase inhibitors for therapeutic benefit in clinical oncology. Kinase inhibition triggers a strong discerning pressure for cells to acquire resistance to chemotherapy through kinase mutations . Thus, tNumerous follow-up strategies are being employed to overcome the challenge of kinase inhibitor resistance. A first approach is to develop inhibitors that can tolerate diverse amino acids at the gatekeeper position , 290. A Even though only a small fraction of the kinome is currently being targeted, kinase inhibitor drug discovery has progressed dramatically in the past decade. Clinical evaluation of kinase inhibitors has shown that therapeutic responses vary widely in individual patients and across patient populations, and seem to depend on many diverse factors. Many new candidate molecules have entered clinical trials, and much more are still at the preclinical stage. Most of the current kinase inhibitor discoveries have developed through rational drug design rather than through random screening and analysis of structure-activity relationships. An important strategy required for future development is to understand the basis of unexpected toxicities related to kinase inhibitors. Improvement in the documentation of toxicities of kinase inhibitor would provide a valuable database for understanding whether there are particular kinases of which inhibition should be avoided or specific substructures that result in problematic metabolites. This strategy will help to develop kinases with better selectivity benefitting the vast patient population. Also, there is a critical need for better ways to monitor target kinase inhibition in humans using minimally invasive techniques. This may include monitoring of cancer biomarkers that may serve as benchmarks for the clinical development of kinase inhibitors. The development of such technologies will help to discover and eradicate tumors using targeted kinase inhibition with minimal toxicities. There is also an urgent need for developing more non-ATP-competitive kinase inhibitors as the current collection of kinase inhibitors is limited to ABL, IKK, AKT, CHK1, MEK, SRC, IGF1R inhibitors , 293–296By transferring the γ-phosphate from the ATP-cofactor onto diverse substrates, kinases regulate key cellular functions. As many human diseases result from mutations and overexpression of kinases, this enzyme class symbolizes an important targeted strategy for drug development. Kinases also play indispensable roles in signaling pathways that regulate tumor cell functions. Deregulation of kinases leads to a variety of pathophysiological changes triggering cancer cell proliferation and metastases. Hyperactivation of kinases also increases anti-apoptotic effects. Currently, about one-third of all protein targets under research in the pharmaceutical industry are kinase-based. Kinase inhibitors represent targeted therapy resultant of the understanding of molecular genetics and molecular signaling pathways. Most of the FDA-approved kinase inhibitors target ATP binding site of kinase enzymes and display therapeutic indications against tumorigenesis. This class of therapeutics represents a transformation from conventional chemotherapy to targeted cancer treatment. Kinase inhibitors have overcome a major drawback of traditional cancer treatment as it effectively discriminates between normal non-malignant cells and rapidly proliferating cancer cells. This leads to fewer off-target effects and low toxicities in the cancer patient population. Kinase inhibitors are also often useful in combination with cytotoxic chemotherapy or radiation therapy. A vital challenge for clinical use of kinase inhibitors in the prevention of drug-resistant cancer stem cells. This phenomenon occurs due to cellular pressure to compensate for the loss of function of an important kinase. Pharmacogenomic factors including gene polymorphisms also contribute to primary kinase drug-resistance. Due to the clinical importance of kinase inhibitors, multiple strategies are required to overcome resistance mechanisms and develop more effective targeted therapies. A key approach is to allosterically induce and stabilize inactive kinase conformations. In the future, scientific advances may eventually allow scientists to combine mutagenesis screens through next generation sequencing and proteomic techniques with the computational modeling of compound interactions with all possible mutant variants of a targeted kinase. This will lead to the development of well-tolerated kinase inhibitors compared to traditional chemotherapeutic treatments. Overall, kinase inhibitors represent a new and promising approach to cancer therapy, one that is already providing beneficial clinical effects.


Impairments in central reward processing constitute an important aspect of the negative symptoms of schizophrenia. Despite its clinical relevance, the etiology of deficient reward processing in schizophrenia remains largely unknown. Here, we used an epidemiologically informed mouse model of schizophrenia to explore the effects of prenatal immune activation on reward-related functions. The model is based on maternal administration of the viral mimic PolyI:C and has been developed in relation to the epidemiological evidence demonstrating enhanced risk of schizophrenia and related disorders following prenatal maternal infection. We show that prenatal immune activation induces selective deficits in the expression (but not acquisition) of conditioned place preference for a natural reward (sucrose) without changing hedonic or neophobic responses to the reward. On the other hand, prenatal immune activation led to enhanced place preference for the psychostimulant drug cocaine, while it attenuated the locomotor reaction to the drug. The prenatal exposure did not alter negative reinforcement learning as assessed using a contextual fear conditioning paradigm. Our findings suggest that the nature of reward-related abnormalities following prenatal immune challenge depends on the specificity of the reward  as well as on the valence domain (positive vs negative reinforcement learning). Moreover, our data indicate that reward abnormalities emerging in prenatally immune-challenged offspring may, at least in part, stem from an inability to retrieve previously established context–reward associations and to integrate such information for appropriate goal-directed behavior. Negative symptoms represent a class of deficits in the emotional and behavioral domains, which include asociality, poverty of speech, blunted affect and a number of distinct impairments in reward processing.2Schizophrenia is a chronic neuropsychiatric disease characterized by multiple and often co-existing psychopathological symptoms, which are typically referred to as positive, negative and cognitive symptoms.5 They include impairments in positive reinforcement learning, attribution of incentive motivation, effort-value computations and translation of reward-related information into goal-directed behavior.7 On the other hand, the ability to enjoy pleasant stimuli seems to be largely intact in patients with schizophrenia, suggesting that schizophrenia-associated reward abnormalities emerge in the absence of anhedonia.8 Instead, reward-related deficits in this population may primarily stem from difficulties in using internal representations of previous rewarding experiences to drive goal-directed behaviors, which in turn would allow obtaining the desired outcomes.11 These impairments seem to be restricted to positive reinforcement learning, whereas negative reinforcement or aversive learning appears to be largely intact in individuals with schizophrenia.13Abnormalities in central reward processing are a cluster of motivational and behavioral deficits that remain largely unresponsive to currently available antipsychotic drugs and can strongly undermine the daily life quality of affected individuals.9 this class of symptoms has also been documented in unmedicated patients with schizophrenia.14 At least some of the reward abnormalities in schizophrenia may thus have a developmental origin and early onset.15 Yet, the genetic and environmental risk factors of these deficits remain largely unknown. We therefore explored whether schizophrenia-relevant abnormalities in central reward processing may be induced by prenatal exposure to infection, which has been repeatedly implicated in the etiology of schizophrenia and other neurodevelopmental disorders.17In addition to the difficulties in treating reward deficiencies, the underlying etiological mechanisms also remain largely elusive. Although antipsychotic drugs can affect reward-related functions,18 Numerous previous investigations have provided robust evidence for the emergence of long-term functional and structural brain abnormalities following prenatal exposure to PolyI:C, many of which are implicated in schizophrenia and related disorders.19 At least some of the behavioral and cognitive abnormalities can be normalized by antipsychotic drug administration22 and emerge as a function of post-pubertal maturational processes.25 Hence, the prenatal PolyI:C administration model in rodents is characterized by substantial face, construct and predictive validity, and it bares epidemiological relevance to schizophrenia and related disorders. As with many other preclinical models of schizophrenia, however, the PolyI:C model has thus far only received scant attention with respect to modeling deficits relevant to the negative symptoms of schizophrenia.27 Extending the behavioral characterization of the model to reward-related functions is thus expected to provide novel information with regard to etiological processes underlying the emergence of deficits pertinent to the negative symptoms.For this purpose, we used a well-established mouse model of maternal immune activation by the viral mimic polyriboinosinic–polyribocytidilic acid (PolyI:C).29 Because patients with schizophrenia exhibit impairments in other distinct neuropsychological processes that are independent of effort-related behavior, such as for example positive reinforcement learning or reward-based decision making,12 we focused our attention on Pavlovian-based tests that require only minimal levels of effort exertion. We explored the effects of prenatal PolyI:C-induced immune challenge on Pavlovian-based reward behaviors using a well-established contextual reward conditioning task, namely the conditioned place preference (CPP) test.30 Performance in the CPP test is dependent on various hippocampal and cortico-striatal circuits,31 which are known to be altered in schizophrenia.35 Using the CPP test, we evaluated context–reward associations using a natural reward (sucrose) and the psychostimulant drug cocaine. We also assessed the effects of prenatal immune activation on hedonic and neophobic responses to natural sucrose reward in attempts to identify possible relationships between positive reinforcement learning and anhedonic and/or neophobic behavior. Finally, we used a contextual fear conditioning paradigm to explore the effects of prenatal immune activation on negative reinforcement learning that involves contextual processing of aversive events.Recent investigations in animal models of schizophrenia have largely focused on incentive motivation abnormalities that associate with reduced exertion of effort.C57BL6/N pregnant dams on gestation day 17 were randomly assigned to receiving either PolyI:C  or vehicle (NaCl). Adult offspring born to immune-challenged and control mothers were included in the study. All procedures had been previously approved by the Cantonal Veterinarian's Office of Zurich and are fully described in the 36 In addition, we evaluated whether anhedonic behavior and anxiety-related sucrose neophobia could represent confounders influencing the behavioral responses in the sucrose CPP. Finally, to explore the effects of prenatal immune activation on negative contextual conditioning, we measured context–fear-associative learning using a standard contextual fear conditioning paradigm.37 The behavioral paradigms are fully described in the We evaluated positive contextual conditioning for sucrose and cocaine using standard CPP paradigms.t-tests (two tailed), parametric analysis of variance or analysis of covariance, followed by Fisher's least significant difference post hoc group comparisons, or non-parametric Mann–Whitney tests. Statistical analyses are fully described in the All data met the assumptions of normal distribution and equality of variance, or were transformed accordingly. All data were analyzed using Student's F=4.51, P<0.05) and by the subsequent restricted analyses of variance revealing a significant difference between the sucrose-paired and the water-paired chambers in control =5.78, P<0.05), but not in PolyI:C offspring  in the relative time spent in the sucrose- vs water-paired chamber. Representative exploration patterns of PolyI:C and control offspring can be found in In a first series of investigations, we explored the effects of prenatal immune activation on contextual appetitive conditioning for sucrose using a standard sucrose CPP test. Following conditioning, the exploration time between the sucrose-paired and the water-paired (control) chamber was used to assess contextual appetitive conditioning in adult control and PolyI:C offspring. The exploration time was recorded during a 25-min test period and analyzed as a function of 5-min bins. As shown in ffspring . Similarffspring . These affspring . This leper se.Notably, the effects of prenatal immune activation on sucrose CPP emerged in the absence of any difference in general locomotor activity during the test session  or durin38 We used the same 30% sucrose solution as in the preceding CPP test so as to provide a direct comparison between the two experimental paradigms. As shown in Next, we evaluated whether the PolyI:C-induced deficits in CPP for sucrose may be associated with anhedonic behavior toward the primary reinforcer, that is, an inability to experience pleasure from stimuli that others normally find pleasurable. To this end, we compared control and PolyI:C animals in a two-bottle sucrose preference test, which is frequently used to assess anhedonic behavior.We further explored whether PolyI:C offspring might show altered neophobic responses to the same 30% sucrose solution. In this test, sucrose neophobia was indexed by the latency to start consuming sucrose solution when presented the first time in a familiar context. These analyses revealed no significant group differences , indicatGiven that prenatal immune challenge robustly affected the expression of positive contextual conditioning , we nextDuring the conditioning phase of the contextual fear test, the amount of percent time freezing generally increased across the three successive post-shock periods , indicat36 conditioning led to a robust preference for the cocaine-paired chamber in both prenatal treatment groups, whereby the time spent in the cocaine-paired chamber was significantly higher than the time spent in the saline-paired chamber =55.29, P<0.001) =5.48, P<0.05) and by the significant difference in the exploration time of cocaine- and saline-paired chambers between the two prenatal treatment groups =5.48, P<0.05) revealed in subsequent restricted analyses of variance =5.48, P<0.05). These analyses remained significant after inclusion of locomotor activity as covariates in an analysis of covariance =4.63, P<0.01; day 2 included as a covariate: F=4.21, P<0.01). Prenatal treatment did not affect general locomotor activity, as indexed by the total distance moved on the test day  . Interesvariance . Similarvariance : althougtest day . RepreseF=81.38, P<0.001) in square-root-transformed data). Subsequent a priori separation of cocaine and saline data revealed that the cocaine-induced enhancement of locomotor activity was apparent in both experimental groups. This locomotor-stimulating effect of cocaine was, however, blunted in PolyI:C offspring compared with controls, especially within the first 15 min after drug injection =4.33, P<0.05) and by a significant interaction between prenatal treatment and bins =2.50, P<0.05). Subsequent post hoc comparisons revealed significant group differences at several individual time points after cocaine treatment . Significant group differences in locomotor activity were only observed following cocaine, but not saline, administration  into a novel contextual entity (one large chamber made of two subchambers during the test day), a phenomena known to rely on prefrontal–hippocampal functions.50 Accordingly, the attenuation of cocaine-induced hyperactivity may primarily involve reduced dopamine transporter availability in the core region of the nucleus accumbens, which mediates the immediate locomotor effects of acute cocaine treatment.53 Conversely, context–cocaine reward behaviors critically depend on integrated circuits within hippocampal and cortico-striatal loops engaging the nucleus accumbens shell, which together promote context–drug learning, attribution of incentive and predictive properties to contexts, and retrieval of memories.55 These processes emerge after repeated context–cocaine pairings over several days, which likely involves synaptic remodeling and plasticity-related changes within mesocorticolimbic regions.57 For example, an elegant study by Muñoz-Cuevas et al.56 identified rapid spine gain in prefrontal pyramidal neurons following cocaine administration, demonstrating that persistent new spines correlate with the magnitude of cocaine place preference, but not with locomotor sensitization to the drug. Enhanced neuroadaptive changes in mesocorticolimbic structures, together with an overlearning of drug-related cues,55 could thus provide a plausible explanation for the increased cocaine place preference emerging in PolyI:C offspring. Even though indirect evidence for this possibility exists based on previous neurochemical and neuroanatomical studies,48 future investigations will be required to delineate the exact neural mechanisms implicated in these effects.Another main finding of the present study is that prenatal immune activation also altered contextual learning for the psychostimulant drug cocaine. Unlike in the natural reward (sucrose) condition, however, offspring of immune-challenged mothers showed enhanced place preference for the drug reward. At the same time, immune-challenged offspring displayed reduced cocaine-induced locomotor hyperactivity compared with cocaine-treated control offspring. The analyses of locomotor patterning further suggested that these effects represent genuine effects on locomotor activity rather than possible differences in stereotyped behavior. Moreover, the PolyI:C-induced effects on cocaine CPP scores remained significant after controlling for differences in locomotor activity. Our findings thus reveal an intriguing dissociation between the locomotor-enhancing effects of cocaine and its reward-related properties, whereby reduced locomotor activity in prenatally challenged offspring are concomitant to an enhancement of context–cocaine associations. Our data are in line with the notion that the locomotor-stimulating and reward-related effects of cocaine partly rely on distinct neuronal mechanisms and brain subregions.58 According to this theory, sucrose stimuli would fail to elicit a sufficiently large phasic dopamine response to be detected above ‘noise' thresholds in immune-challenged offspring,60 whereas cocaine may elicit stronger phasic signals in immune-challenged offspring compared with controls.60 The latter may occur because of the existence of abnormally large phasic vs tonic dopamine responses to strong dopaminergic stimulants such as cocaine (or cocaine-related cues), similarly to what has been proposed in the context of the aberrant salience hypothesis of schizophrenia.62It also remains elusive whether the diametrically opposite patterns of reward abnormalities  may arise from defects in the same neural circuits, or rather whether they represent independent pathological entities. According to some theoretical accounts, however, seemingly contradictory reward-related phenotypes, such as reduced place preference for sucrose and enhanced place preference for cocaine, could indeed be induced by mutual neural mechanisms. One of these mechanisms may relate to abnormal tonic vs phasic dopamine signaling: it has been hypothesized that abnormalities in tonic vs phasic dopaminergic signals could result in deficient signal-to-noise detectability.64 For example, rat studies have shown that cocaine exposure, but not food exposure, elicits prefrontal-dependent increases in spine size and strength in accumbal medium spiny neurons.65 In addition, a recent study has identified differential expression of the epigenetic regulator MeCP2 and some of its target genes (PP1Cβ or reelin) following passive- or self-administration of cocaine vs food.66 In view of our findings, we speculate that neurodevelopmental disruption by prenatal PolyI:C exposure may differentially alter some of the key neuronal substrates underlying the rewarding effects of natural  and drug  rewards, which in turn may critically determine the direction of reward abnormalities depending on the rewards' nature.An alternative  explanation for the differential effects of prenatal PolyI:C exposure on sucrose- and cocaine-induced CPP relates to a recent observation suggesting that exposure to natural and drug rewards induce partly distinct neuronal changes, in particular within the prefrontal-to-accumbens glutamate pathway and/or downstream medium spiny neurons.42 Furthermore, our results match the large body of evidence suggesting that individuals with schizophrenia show intact negative reinforcement learning, similarly to what we have observed in the contextual fear conditioning test. On the other hand, patients with schizophrenia display clear deficits in positively motivated learning tasks involving rewarding stimuli.68 Besides other abnormalities, affected individuals seem to be incapable of translating value/effort-related information into goal-directed action.6 They also show difficulties in their ability for uncertainty-driven exploration.42 Both of these deficits seem to critically involve prefrontal dysfunctions, which we believe might be implicated in the sucrose place preference deficits that emerged following prenatal immune activation.Whatever precise mechanisms involved, our results are consistent with and corroborate some of the reward-related dysfunctions present in schizophrenia and other neurodevelopmental animal models relevant to this disorder. In line with our observations in the sucrose preference test of anhedonia, patients with schizophrenia typically retain the ability to enjoy pleasurable stimuli.70 The findings from our preclinical model suggests that maternal infection could represent one of the neurodevelopmental factors triggering the appearance of an addiction-prone phenotype in patients with schizophrenia, although epidemiological data supporting such an association are missing thus far. Yet, our findings are in line with the previous work in other schizophrenia-relevant animal models documenting altered responses to natural or drug rewards, including models that are based on neonatal ventral hippocampal lesion,73 prenatal infection75 or mutant expression of DISC1.76 Altogether, these observations provide support for the theory that the predilection for addictive behavior would be ingrained in schizophrenia neuropathology.77 Indeed, it has been proposed that the neuropathological abnormalities that emerge in the brain of individuals with schizophrenia across neurodevelopment create a state of ‘endogenous sensitization', so that functional hyper-responsiveness of dopamine-related circuits, coupled with reduced inhibitory control arising from cortical structures , could result in a hypersensitivity to the rewarding effects of drugs.78 Although current knowledge does not readily allow confirming or refuting this hypothesis, our findings support the notion that neurodevelopmental changes in mesocorticolimbic circuits, which emerge in response to prenatal immune challenge, could potentially also be responsible for drug-related behavioral abnormalities.It is also interesting to note that several studies have reported a high prevalence of substance use disorder in schizophrenia, including abuse of psychostimulant drugs such as cocaine.19 the prenatal PolyI:C administration model in rodents is characterized by substantial face, construct and predictive validity, and it bares epidemiological relevance to schizophrenia and related disorders. As with many other preclinical models of schizophrenia, however, the PolyI:C model has thus far only received scant attention with respect to modeling aspects relevant to the negative symptoms of schizophrenia.27 Given the necessity to establish effective therapeutic interventions against negative symptoms,4 efforts to develop and characterize preclinical models with relevance to this symptom domain are urgently needed.27 The present study may help in achieving this goal by extending the prenatal PolyI:C administration model to reward-related phenotypes that are reminiscent of key features of the negative symptomatology. Together with other models, the PolyI:C model may also help to address the question of whether reward deficits are primary pathological features of schizophrenia, or whether they emerge as a consequence of pharmacotherapy. Although antipsychotic drugs can affect reward-related functions,9 this class of symptoms has also been documented in unmedicated patients with schizophrenia.14 Given the known impact of prenatal PolyI:C exposure on early neurodevelopmental processes and subsequent brain maturation,19 our study provides preclinical evidence for the hypothesis that at least some reward deficits typically seen in individuals with schizophrenia could arise as a pathological consequence of neurodevelopmental insults during pregnancy, such as those elicited by maternal infection.As extensively reviewed elsewhere,79 bipolar disorder80 and schizophrenia.6 According to this scenario, the adverse effects of maternal immune activation on reward-related behaviors could represent a point of entry into a disturbed reward system, whereas the specificity of subsequent disease would be further influenced by additional genetic or environmental factors. This hypothesis would be in line with the emerging evidence suggesting that maternal infection has an etiological role in a variety of neuropsychiatric and neurological disorders with seemingly remote pathologies.82 Against these backgrounds, our findings may encourage future epidemiological studies to determine whether prenatal infection increases the risk of developing discrete neuropsychopathological features  independently of existing diagnostic classifications. Attempts toward this direction are limited to a few studies showing that serologically documented prenatal infection or inflammation is associated with the presence of discrete neuroanatomical or neurocognitive dysfunctions in patients with schizophrenia.84Related to this, our findings may highlight a possible etiological association linking maternal infection to substance abuse disorder and/or dual diagnosis schizophrenia. Moreover, they may have implications for the development of reward-related pathologies independently of existing diagnostic classifications. In fact, immune-mediated reward deficits, such as those described in the present study, could represent an intermediary phenotype with relevance to various neuropsychiatric disorders characterized by reward abnormalities, including autism,In conclusion, our study demonstrates abnormal context–reward associations in an immune-mediated neurodevelopmental mouse model relevant to schizophrenia and related disorders. These deficits likely stem from an inability to retrieve previously established context–reward associations and to integrate such information for appropriate goal-directed behavior, but not from anhedonia, neophobia or primary defects in reinforcement learning and/or incentive salience attribution. As currently available pharmacotherapies have thus far failed to improve reward-related deficits in schizophrenia, our epidemiologically informed mouse model may provide an important preclinical platform for the establishment and characterization of novel therapeutic intervention against these symptoms.


Using a Bayesian machine learning algorithm, bNAb-specific glycan footprints were identified and used to design antigens that selectively alter bNAb antigenicity as a proof-of concept. Our approach provides a new design strategy to predictively modulate antigenicity via the alteration of glycan topography, thereby focusing the humoral immune response on sites of viral vulnerability for HIV.Mounting evidence suggests that glycans, rather than merely serving as a “shield”, contribute critically to antigenicity of the HIV envelope (Env) glycoprotein, representing critical antigenic determinants for many broadly neutralizing antibodies (bNAbs). While many studies have focused on defining the role of individual glycans or groups of proximal glycans in bNAb binding, little is known about the effects of changes in the overall glycan landscape in modulating antibody access and Env antigenicity. Here we developed a systems glycobiology approach to reverse engineer the complexity of HIV glycan heterogeneity to guide antigenicity-based  Carbohydrates on the HIV Env glycoprotein, previously often considered as a “shield” permitting immune evasion, can themselves represent targets for broadly neutralizing antibody (bNAb) recognition. Efforts to define the impact of individual glycans on bNAb recognition have clearly illustrated the critical nature of individual or groups of glycans on bNAb binding. However, glycans represent half the mass of the HIV envelope glycoprotein, representing a lattice of interacting sugars that shape the topographical landscape that alters antibody accessiblity to the underlying protein. However, whether alterations in individual glycans alter the broader interactions among glycans, proximal and distal, has not been heretofore rigorously examined, nor how this lattice may be actively exploited to improve antigenicity. To address this challenge, we describe here a systems glycobiology approach to reverse engineer the complex relationship between bNAb binding and glycan landscape effects on Env proteins spanning across various clades and tiers. Glycan occupancy was interrogated across every potential N-glycan site in 94 recombinant gp120 recombinant antigens. Sequences, glycan occupancy, as well as bNAb binding profiles were integrated across each of the 94-atngeins to generate a machine learning computational model enabling the identification of the glycan site determinants involved in binding to any given bNAb. Moreover, this model was used to generate a panel of novel gp120 variants with augmented selective bNAb binding profiles, further validating the contributions of glycans in Env antigen design. Whether glycan-optimization will additionally influence immunogenicity, particularly on emerging stabilized trimers, is unknown, but this study provides a proof of concept for selectively and agnostically exploiting both proximal and distal viral protein glycosylation in a principled manner to improve target Ab binding profiles. Env glycoproteins on the surface of enveloped viruses, such as HIV –4, DenguGlycans represent more than half the mass of the HIV Env glycoprotein, obscuring nearly the entire surface of the Env trimer. While these glycans were originally believed to shield against an Ab response, over the past decade a number of bNAbs have been identified that actively recognize these glycan themselves. Interestingly, these antibodies usually emerge following extensive evolutionary selection enabling them to generate unusual antigen-recognition domains (Fabs) that are able to reach through, and even utilize glycans, to access the underlying protein surface –36. MoreAntigenic opportunities for manipulating glycans on Env were first elucidated in studies focused on either altering the overall glycans or certain glycans specifically –43. ThesWhile causal relationships between individual glycosylation sites, or groups thereof, and bNAb binding have been defined , 49–56, de novo, in silico protein antigen design model using an iterative antigenicity-guided sequence evolution framework, seeking to predict alterations in the antigen that could selectively improve or impair target antibody binding. These predictions were then used to synthesize novel antigens that successfully bound to target bNAbs with enhanced and selective antigenicity. All together, our work provides proof-of-concept, in an initial HIV Env application, for the advance of multi-variate glycosite-based engineering of immunogens that can focus the humoral immune response on sites of neutralizing vulnerability.In this work we describe a multi-variate, combined experimental/computational glyco-engineering strategy to rationally alter HIV envelope glycosylation with a goal of inducing desirable topographical changes in epitope accessibility. First, a deconvolution model was constructed to reverse-engineer the data in a panel of 94 recombinant HIV Env antigenic profiles screened against a battery of broadly neutralizing and non-neutralizing Abs. Glycan occupancy at each n-linked glycan site (‘sequon’) was assessed by mass spectrometry, and both protein sequence and glycan occupancy were used as variables in the model to elucidate how Ab binding is dependent on them. Next, based on this model, forward-engineering principles aimed at changing the overall antigenic profile of any given HIV Env were ascertained. Using a Bayesian machine learning algorithm permitted determination of key sequon sites that positively or negatively influence Ab binding. Finally, these results were then used in a Over the past decade, significant effort has been invested in the generation of stabilized, native, trimeric HIV Env proteins or minimal scaffolds of sites targeted by bNAbs, aimed at focusing the immune response to sites vulnerable to antibody mediated neutralization. While exciting results continue to accrue, these proteins alone have not proven sufficient to drive neutralizing Ab breadth. Strategies to further optimize the HIV Env glycoprotein for induction of bNAb breadth thus continue to be urgently needed. Given our emerging appreciation for the antigenic nature of the large array of glycans that decorate the HIV Env glycoprotein, here we hypothesized that the glycan shield itself might be strategically modified and exploited in a rational manner, offering a potentially generalizable glyco-engineering approach that could be applied to either trimeric or monomeric Env proteins. For proof-of-concept purposes, we focused our study on a diverse panel of Env monomers.18O isotopic labeling . The complete probability distribution containing 2110 states is overwhelming to exhaustive sampling. Therefore, we used an approximation to provide a statistical estimate of the glycan determinant distribution P(X) given a set of experimental observations. According to Bayes’ theorem, the conditional probabilities of the glycan determinants given an observed data set are:Xopt denotes the vector of the glycan determinants in the model and p(Xopt | data) represents the posterior of Xopt. p(data | Xopt) and p(Xopt) are likelihood and prior of Xopt, respectively. Since the ratio of posterior is only required following MCMC sampling, the equation simplifies:–ln(likelihood(Xopt)) was calculated by mean squared errors (MSE) between the prediction from SVR and the observed data. SVR was trained on a sub-set of glycan site occupancy profiles (Xopt), and examined by means of 10-fold cross-validation randomly dividing the data into training and test sets with fitness estimated by calculating MSE. -ln(prior(Xopt)) was calculated by the sum of squared deviation between the selected and empirical glycan determinants, assuming that the distribution of the site identified between two states  follows a Gaussian distribution:σ2j and <xj> are variance and mean of glycan occupancy at the site xj. To sample the desired probability distribution P(X) we implemented a Metropolis–Hastings MCMC walk, in which the algorithm iteratively computed the posterior at current and next position. With this posterior assumed proportional to P(X), the decision of jumping to the next position was then obtained:Xs indicates the current position and X’ the candidate for the next position; Xs+1 the next position. The distribution α of the next position depends only on the current position value, by definition of the Markov chain. The algorithm accordingly generated a sequence of sample values in which the distribution of values closely approximated the original probability distribution P(X).q-value after multiple-testing adjustment showed statistical significance (q<0.01)  denotes the regression function of SVR and y represents the actual Ab binding fingerprint. A learning curve was used to evaluate the model fitting given the predictors  was introduced and calculated:p(xi) denotes the probability of a sequon being a “determinant” which was obtained from the MCMC-SVR model and d represents a direction  determined by the covariance matrix between individual glycan occupancy pattern versus the Ab fingerprint , where every single residue of the 303 positions in each sequence was converted from a categorized amino acid into a binary vector. Ultimately, three models—glycan alone, sequence alone, and both glycan and sequence—were constructed for each Ab to assess the model prediction performance as well as identify the critical glycan site and protein residues that may tune Ab binding.-ln(posterior(Xopt)) is modified). Given a set of previously identified Ab-specific glycan determinant sites denoted as X, the model in this section attempted to determine the likelihood of the presence or absence of each critical site associated with the optimized Ab binding:xi contained two states . Thus, the model aimed to approximate the joint probability distribution P(X) by implementing the same Bayesian MCMC sampling as earlier (ln(posterior(Xopt)) represented the predicted antigenicity from the trained SVR model given a set of combinatorial sites Xopt.Using the Bayesian MCMC-SVR algorithm that identified the glycan determinants positively or negatively impacting Ab binding, the data were further analyzed to rationally design antigens with selective Ab antigenic-profiles. The same training set  and model algorithm were utilized, but now modified to search for global optimum solutions for best or worst binding (the objective function  earlier .–ln(postP(X), we performed 200,000 iterations to reach convergence from an initial gp120 sequence, and estimated the global sequon occupancy profile for an antigen that yields optimized Ab binding. Each iteration simulated the evolutionary process of mutagenesis, in which the state of a selected site xi was altered between 0 and 1 analogous to random knockout/insertion of the sequon; an evolutionary driving force is assumed to mainly depend on predicted antigenicity. Accordingly, in each iteration determination was made of whether the sequon should be added or removed by computing the ratio of posteriors for the current and previous iteration according to For sampling within the multi-dimensional probability distribution de novo peptide sequence from a gp120 sequence template to accommodate an ideal glycan determinant profile, addition and deletion of sequons was accomplished according to these rules: (1) if a sequon needed to be eliminated, a Asn-to-Gln substitution at the sequon was introduced; (2) if a new sequon needed to be introduced, a N-L-T sequence was substituted into the original sequence .When generating a p-value was corrected to account for multiple hypothesis testing . Glycan pairs with significant mutual exclusion were then identified  were considered to be larger than acceptable. Glycan sites that showed the least effect on bNAb binding from steric hindrance were removed one at a time until an acceptable level of steric hindrance was achieved. The process of removing steric glycan sites was repeated until total degree of steric hindrance was lower than mean+1 s.d.To incorporate the steric hindrance effects of proximal glycans, pairs that negatively impacted one another were identified. Our hypothesis was that if two glycans sterically constrained each other, the glycan occupancy patterns should be mutually exclusive; i.e., they should have strong negative correlation. Therefore, a covariance matrix was constructed containing all pairs of glycan-glycan auto-correlations. To determine statistical significance, a nominal P-value of each pair was calculated, assuming that the distribution of all pairs of correlation coefficients followed a Gaussian process, and an adjusted +/-/PGT128+/- optimized glycoproteins, including a signal peptide-‘MPMGSLQPLATLYLLGMLVASVLA’ at N-terminus and an avitag-‘GLNDIFEAQKIEWHE’ followed by a histag-'HHHHHH’ at the C-terminus, were synthesized and inserted into pcDNA3.1 (Thermo Fisher). Plasmid DNA was purified and verified by sequencing. Plasmids encoding these proteins were transfected into 293-F cells  and proteins were isolated from expression supernatants 6 days after transfection. Briefly, gp120 proteins were purified by metal affinity chromatography using Ni-NTA resin (Qiagen). Fractions containing gp120 were combined and oligomers, trimers and monomers were separated by gel filtration chromatography using a Hi-Load 16/60 Superdex 200pg column . Protein purity was confirmed by SDS-PAGE gel electrophoresis and Western blotting using a mouse monoclonal Ab Chessie 13–39.1  Click here for additional data file.S2 File(PDF)Click here for additional data file.S3 File(ZIP)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.