Functional stability of HIV-1 envelope trimer affects accessibility to broadly neutralizing antibodies at its apex

ABSTRACT The trimeric envelope glycoprotein spike (Env) of HIV-1 is the target of vaccine development to elicit broadly neutralizing antibodies (bnAbs). Env trimer instability and heterogeneity in principle make subunit interfaces inconsistent targets for the immune response. Here, we investigate how functional stability of Env relates to neutralization sensitivity to V2 bnAbs and V3 crown antibodies that engage subunit interfaces upon binding to unliganded Env. Env heterogeneity was inferred when antibodies neutralized a mutant Env with a plateau of less than 100% neutralization. A statistically significant correlation was found between the stability of mutant Envs and the MPN of V2 bnAb, PG9, as well as an inverse correlation between stability of Env and neutralization by V3 crown antibody, 447-52D. A number of Env-stabilizing mutations and V2 bnAb-enhancing mutations were identified in Env, but they did not always overlap, indicating distinct requirements of functional stabilization versus antibody recognition. Blocking complex glycosylation of Env affected V2 bnAb recognition, as previously described, but also notably increased functional stability of Env. This study shows how instability and heterogeneity affect antibody sensitivity of HIV-1 Env, which is relevant to vaccine design involving its dynamic apex. IMPORTANCE The Env trimer is the only viral protein on the surface of HIV-1 and is the target of neutralizing antibodies that reduce viral infectivity. Quaternary epitopes at the apex of the spike are recognized by some of the most potent and broadly neutralizing antibodies to date. Being that their glycan-protein hybrid epitopes are at subunit interfaces, the resulting heterogeneity can lead to partial neutralization. Here, we screened for mutations in Env that allowed for complete neutralization by the bnAbs. We found that when mutations outside V2 increased V2 bnAb recognition, they often also increased Env stability-of-function and decreased binding by narrowly neutralizing antibodies to the V3 crown. Three mutations together increased neutralization by V2 bnAb and eliminated binding by V3 crown antibodies. These results may aid the design of immunogens that elicit antibodies to the trimer apex. </jats:p

Correlation Clustering with Low-Rank Matrices

Correlation clustering is a technique for aggregating data based on qualitative information about which pairs of objects are labeled 'similar' or 'dissimilar.' Because the optimization problem is NP-hard, much of the previous literature focuses on finding approximation algorithms. In this paper we explore how to solve the correlation clustering objective exactly when the data to be clustered can be represented by a low-rank matrix. We prove in particular that correlation clustering can be solved in polynomial time when the underlying matrix is positive semidefinite with small constant rank, but that the task remains NP-hard in the presence of even one negative eigenvalue. Based on our theoretical results, we develop an algorithm for efficiently "solving" low-rank positive semidefinite correlation clustering by employing a procedure for zonotope vertex enumeration. We demonstrate the effectiveness and speed of our algorithm by using it to solve several clustering problems on both synthetic and real-world data

Structural and Functional Insights into Alphavirus-Host Interactions

Alphaviruses are arthropod-transmitted positive sense, single-stranded RNA viruses of the Togaviridae family that cause explosive, global outbreaks with significant morbidity. These viruses are classified into two groups based on genetic relatedness and clinical manifestations. Arthritogenic alphaviruses include chikungunya (CHIKV), Mayaro (MAYV), Ross River (RRV), O’nyong’nyong (ONNV) and generally cause acute and chronic musculoskeletal disease. To date, CHIKV has caused \u3e1.8 million cases in over 40 countries. Encephalitic alphaviruses, including Eastern (EEEV), Venezuelan (VEEV), and Western (WEEV) equine encephalitis viruses, infect the central nervous system and result in encephalitic sequelae. In 2019, outbreaks of EEEV in the United States have resulted in 38 confirmed cases, including 15 deaths, with many more infections suspected but not diagnosed. Despite their epidemic potential, there exist no approved vaccines or treatments for these alphaviruses. Further understanding of alphavirus-host interactions can provide a strategy to mitigate alphavirus infection and disease. These studies will investigate the molecular basis of alphavirus host-engagement, and upon infection, the features required for antibody-mediated neutralization in vitro and in vivo. A recent CRISPR-Cas9 genome-wide screen identified the matrix remodeling-associated protein 8 (Mxra8), a poorly characterized cell adhesion molecule, as a host receptor required for infection by multiple arthritogenic alphaviruses. Deletion of Mxra8 resulted in markedly reduced levels of infection by arthritogenic alphaviruses. Infection was restored upon re-introduction of mouse or human Mxra8 gene in cells. In vivo genetic-editing of Mxra8 or Mxra8 blockade using antibodies reduced CHIKV infection and clinical disease. Mxra8 directly interacts with CHIKV, but not with the encephalitic alphavirus EEEV. Competition binding studies with previously mapped anti-CHIKV antibodies and alanine-scanning mutagenesis revealed that Mxra8 recognizes domains A and B on the CHIKV E2 envelope protein. To determine the structural basis of Mxra8 engagement with CHIKV, we obtained a 2.2 Å crystal structure of Mxra8 and a 4 to 5 Å cryo-electron microscopy reconstruction of Mxra8 bound to CHIKV. Mxra8 contains an unusual Ig-like fold topology and recognizes a quaternary binding site that spans between the CHIKV E2 and E1 proteins on the virion. Evolutionary analysis of the Mxra8 receptor revealed a unique mechanism of evasion of virus-host receptor interactions. Mxra8 of several lineage within the Bovinae family (e.g., cattle, bison, buffalo, and antelope) contained a unique three quasi-identical 5-residue repeats in the virus binding interface, which conferred resistance to alphavirus binding and infection. Removal of this 15-residue insertion from cattle Mxra8 restored alphavirus infection and reciprocally, addition of the 15-residue insertion into mouse Mxra8 conferred resistance to alphavirus infection. A 2.3 Å crystal structure of cattle Mxra8 and cryo-EM docking revealed that the Mxra8 insertion hinders CHIKV engagement and binding. Analysis of Mxra8 gene sequences from animals in the Saint Louis Zoo dated the insertion to at least 5 million years ago. To determine significance of the 15-residue Mxra8 insertion in vivo, we genetically engineered mice with the 15-residue insertion in Mxra8 gene using CRISPR-Cas9. These gene-edited mice exhibited decreased clinical disease and exhibited reduced viral replication and pathogenesis, essentially behaving like Mxra8 knockout mice with respect to CHIKV infection. Understanding the molecular determinants of in vitro neutralization and in vivo protection by antibodies upon alphavirus infection can be informative towards therapeutic and vaccine strategies. We identified a panel of potently neutralizing murine monoclonal antibodies against EEEV. Mapping of these antibodies through alanine-scanning mutagenesis, virus escape, and cryo-EM revealed that the antibodies recognize epitopes in distinct regions of domains A or B of the E2 envelope protein. In vivo administration of EEEV antibodies protected mice from a lethal subcutaneous or aerosol challenge and significantly decreased viral replication in the brain. In addition, we identified human monoclonal antibodies that are cross-reactive and protective against both arthritogenic and encephalitic alphaviruses. These antibodies are non-neutralizing yet bind avidly to cells infected by arthritogenic and encephalitic alphaviruses. In vivo administration of these antibodies protected mice in a CHIKV-induced arthritis model, MAYV-induced arthritis model, and a lethal VEEV challenge model. Competition binding studies revealed that these antibodies recognize an epitope proximal to the E1 fusion loop, a highly conserved region amongst all alphaviruses. Altogether, these studies reveal important determinants of alphavirus-host interactions and humoral immune responses which can inform therapeutic and immunogen design strategies to combat this global epidemic

Genetic Contributions to Age-Related Decline in Executive Function: A 10-Year Longitudinal Study of COMT and BDNF Polymorphisms

Genetic variability in the dopaminergic and neurotrophic systems could contribute to age-related impairments in executive control and memory function. In this study we examined whether genetic polymorphisms for catechol-O-methyltransferase (COMT) and brain-derived neurotrophic factor (BDNF) were related to the trajectory of cognitive decline occurring over a 10-year period in older adults. A single nucleotide polymorphism in the COMT (Val158/108Met) gene affects the concentration of dopamine in the prefrontal cortex. In addition, a Val/Met substitution in the pro-domain for BDNF (Val66Met) affects the regulated secretion and trafficking of BDNF with Met carriers showing reduced secretion and poorer cognitive function. We found that impairments over the 10-year span on a task-switching paradigm did not vary as a function of the COMT polymorphism. However, for the BDNF polymorphism the Met carriers performed worse than Val homozygotes at the first testing session but only the Val homozygotes demonstrated a significant reduction in performance over the 10-year span. Our results argue that the COMT polymorphism does not affect the trajectory of age-related executive control decline, whereas the Val/Val polymorphism for BDNF may promote faster rates of cognitive decay in old age. These results are discussed in relation to the role of BDNF in senescence and the transforming impact of the Met allele on cognitive function in old age

Growth, detection, quantification, and inactivation of SARS-CoV-2

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 is the agent responsible for the coronavirus disease 2019 (COVID-19) global pandemic. SARS-CoV-2 is closely related to SARS-CoV, which caused the 2003 SARS outbreak. Although numerous reagents were developed to study SARS-CoV infections, few have been applicable to evaluating SARS-CoV-2 infection and immunity. Current limitations in studying SARS-CoV-2 include few validated assays with fully replication-competent wild-type virus. We have developed protocols to propagate, quantify, and work with infectious SARS-CoV-2. Here, we describe: (1) virus stock generation, (2) RT-qPCR quantification of SARS-CoV-2 RNA; (3) detection of SARS-CoV-2 antigen by flow cytometry, (4) quantification of infectious SARS-CoV-2 by focus-forming and plaque assays; and (5) validated protocols for virus inactivation. Collectively, these methods can be adapted to a variety of experimental designs, which should accelerate our understanding of SARS-CoV-2 biology and the development of effective countermeasures against COVID-19

Older, Less Regulated Medical Marijuana Programs Have Much Greater Enrollment Rates Than Newer ‘Medicalized’ Programs

Twenty-three states and the District of Columbia have passed laws implementing medical marijuana programs. The nineteen programs that were in operation as of October 2014 collectively had over one million participants. All states (including D.C.) with medical marijuana laws require physicians directly or indirectly to authorize the use of marijuana at their discretion, yet little is known about how medical marijuana programs vary regarding adherence to basic principles of medical practice and associated rates of enrollment. To explore this, we analyzed marijuana programs according to seven components of traditional medical care and pharmaceutical regulation. We then examined enrollment rates, while controlling for potentially confounding state characteristics. We found that fourteen of the twenty-four programs were nonmedical and collectively enrolled 99.4 percent of participants nationwide, with enrollment rates twenty times greater than programs deemed to be “medicalized.” Policy makers implementing or amending medical marijuana programs should consider the powerful relationship between less regulation and greater enrollment. Researchers should consider variations across programs when assessing programs’ population-level effects

ELVIS: Entertainment-led video summaries

© ACM, 2010. This is the author's version of the work. It is posted here by permission of ACM for your personal use. Not for redistribution. The definitive version was published in ACM Transactions on Multimedia Computing, Communications, and Applications, 6(3): Article no. 17 (2010) http://doi.acm.org/10.1145/1823746.1823751Video summaries present the user with a condensed and succinct representation of the content of a video stream. Usually this is achieved by attaching degrees of importance to low-level image, audio and text features. However, video content elicits strong and measurable physiological responses in the user, which are potentially rich indicators of what video content is memorable to or emotionally engaging for an individual user. This article proposes a technique that exploits such physiological responses to a given video stream by a given user to produce Entertainment-Led VIdeo Summaries (ELVIS). ELVIS is made up of five analysis phases which correspond to the analyses of five physiological response measures: electro-dermal response (EDR), heart rate (HR), blood volume pulse (BVP), respiration rate (RR), and respiration amplitude (RA). Through these analyses, the temporal locations of the most entertaining video subsegments, as they occur within the video stream as a whole, are automatically identified. The effectiveness of the ELVIS technique is verified through a statistical analysis of data collected during a set of user trials. Our results show that ELVIS is more consistent than RANDOM, EDR, HR, BVP, RR and RA selections in identifying the most entertaining video subsegments for content in the comedy, horror/comedy, and horror genres. Subjective user reports also reveal that ELVIS video summaries are comparatively easy to understand, enjoyable, and informative

The Deuterium to Hydrogen Abundance Ratio Towards a Fourth QSO: HS0105+1619

We report the measurement of the primordial D/H abundance ratio towards QSO \object. The column density of the hydrogen in the $z \simeq 2.536$ Lyman limit system is high, \lnhi $= 19.422 \pm 0.009$ \cmm, allowing for the deuterium to be seen in 5 Lyman series transitions. The measured value of the D/H ratio towards QSO \object is found to be D/H$= 2.54 \pm 0.23 \times 10^{-5}$. The metallicity of the system showing D/H is found to be $\simeq 0.01$ solar, indicating that the measured D/H is the primordial D/H within the measurement errors. The gas which shows D/H is neutral, unlike previous D/H systems which were more highly ionized. Thus, the determination of the D/H ratio becomes more secure since we are measuring it in different astrophysical environments, but the error is larger because we now see more dispersion between measurements. Combined with prior measurements of D/H, the best D/H ratio is now D/H$= 3.0 \pm 0.4 \times 10^{-5}$, which is 10% lower than the previous value. The new values for the baryon to photon ratio, and baryonic matter density derived from D/H are $\eta = 5.6 \pm 0.5 \times 10^{-10}$ and \ob $=0.0205 \pm 0.0018$ respectively.Comment: Minor text and reference changes. To appear in the May 10, 2001 issue of the Astrophysical Journa

Asymptotics and local constancy of characters of p-adic groups

In this paper we study quantitative aspects of trace characters $\Theta_\pi$ of reductive $p$-adic groups when the representation $\pi$ varies. Our approach is based on the local constancy of characters and we survey some other related results. We formulate a conjecture on the behavior of $\Theta_\pi$ relative to the formal degree of $\pi$, which we are able to prove in the case where $\pi$ is a tame supercuspidal. The proof builds on J.-K.~Yu's construction and the structure of Moy-Prasad subgroups.Comment: Proceedings of Simons symposium on the trace formul