Search

An ideal therapeutic anti-SARS-CoV-2 antibody would resist viral escape1-3, have activity against diverse sarbecoviruses4-7, and be highly protective through viral neutralization8-11 and effector functions12,13. Understanding how these properties relate to each other and vary across epitopes would aid the development of therapeutic antibodies and guide vaccine design. Here we comprehensively characterize escape, breadth and potency across a panel of SARS-CoV-2 antibodies targeting the receptor-binding domain (RBD). Despite a trade-off between in vitro neutralization potency and breadth of sarbecovirus binding, we identify neutralizing antibodies with exceptional sarbecovirus breadth and a corresponding resistance to SARS-CoV-2 escape. One of these antibodies, S2H97, binds with high affinity across all sarbecovirus clades to a cryptic epitope and prophylactically protects hamsters from viral challenge. Antibodies that target the angiotensin-converting enzyme 2 (ACE2) receptor-binding motif (RBM) typically have poor breadth and are readily escaped by mutations despite high neutralization potency. Nevertheless, we also characterize a potent RBM antibody (S2E128) with breadth across sarbecoviruses related to SARS-CoV-2 and a high barrier to viral escape. These data highlight principles underlying variation in escape, breadth and potency among antibodies that target the RBD, and identify epitopes and features to prioritize for therapeutic development against the current and potential future pandemics.

SARS-CoV-2 can mutate and evade immunity, with consequences for efficacy of emerging vaccines and antibody therapeutics. Here, we demonstrate that the immunodominant SARS-CoV-2 spike (S) receptor binding motif (RBM) is a highly variable region of S and provide epidemiological, clinical, and molecular characterization of a prevalent, sentinel RBM mutation, N439K. We demonstrate N439K S protein has enhanced binding affinity to the hACE2 receptor, and N439K viruses have similar in vitro replication fitness and cause infections with similar clinical outcomes as compared to wild type. We show the N439K mutation confers resistance against several neutralizing monoclonal antibodies, including one authorized for emergency use by the US Food and Drug Administration (FDA), and reduces the activity of some polyclonal sera from persons recovered from infection. Immune evasion mutations that maintain virulence and fitness such as N439K can emerge within SARS-CoV-2 S, highlighting the need for ongoing molecular surveillance to guide development and usage of vaccines and therapeutics.

Purpose It is clinically challenging to integrate genomic-classifier results that report a numeric risk of recurrence into treatment recommendations for localized prostate cancer, which are founded in the framework of risk groups. We aimed to develop a novel clinical-genomic risk grouping system that can readily be incorporated into treatment guidelines for localized prostate cancer. Materials and Methods Two multicenter cohorts (n = 991) were used for training and validation of the clinical-genomic risk groups, and two additional cohorts (n = 5,937) were used for reclassification analyses. Competing risks analysis was used to estimate the risk of distant metastasis. Time-dependent c-indices were constructed to compare clinicopathologic risk models with the clinical-genomic risk groups. Results With a median follow-up of 8 years for patients in the training cohort, 10-year distant metastasis rates for National Comprehensive Cancer Network (NCCN) low, favorable-intermediate, unfavorable-intermediate, and high-risk were 7.3%, 9.2%, 38.0%, and 39.5%, respectively. In contrast, the three-tier clinical-genomic risk groups had 10-year distant metastasis rates of 3.5%, 29.4%, and 54.6%, for low-, intermediate-, and high-risk, respectively, which were consistent in the validation cohort (0%, 25.9%, and 55.2%, respectively). C-indices for the clinical-genomic risk grouping system (0.84; 95% CI, 0.61 to 0.93) were improved over NCCN (0.73; 95% CI, 0.60 to 0.86) and Cancer of the Prostate Risk Assessment (0.74; 95% CI, 0.65 to 0.84), and 30% of patients using NCCN low/intermediate/high would be reclassified by the new three-tier system and 67% of patients would be reclassified from NCCN six-tier (very-low- to very-high-risk) by the new six-tier system. Conclusion A commercially available genomic classifier in combination with standard clinicopathologic variables can generate a simple-to-use clinical-genomic risk grouping that more accurately identifies patients at low, intermediate, and high risk for metastasis and can be easily incorporated into current guidelines to better risk-stratify patients.

Purpose To perform the first meta-analysis of the performance of the genomic classifier test, Decipher, in men with prostate cancer postprostatectomy. Methods MEDLINE, EMBASE, and the Decipher genomic resource information database were searched for published reports between 2011 and 2016 of men treated by prostatectomy that assessed the benefit of the Decipher test. Multivariable Cox proportional hazards models fit to individual patient data were performed; meta-analyses were conducted by pooling the study-specific hazard ratios (HRs) using random-effects modeling. Extent of heterogeneity between studies was determined with the I2 test. Results Five studies (975 total patients, and 855 patients with individual patient-level data) were eligible for analysis, with a median follow-up of 8 years. Of the total cohort, 60.9%, 22.6%, and 16.5% of patients were classified by Decipher as low, intermediate, and high risk, respectively. The 10-year cumulative incidence metastases rates were 5.5%, 15.0%, and 26.7% ( P < .001), respectively, for the three risk classifications. Pooling the study-specific Decipher HRs across the five studies resulted in an HR of 1.52 (95% CI, 1.39 to 1.67; I2 = 0%) per 0.1 unit. In multivariable analysis of individual patient data, adjusting for clinicopathologic variables, Decipher remained a statistically significant predictor of metastasis (HR, 1.30; 95% CI, 1.14 to 1.47; P < .001) per 0.1 unit. The C-index for 10-year distant metastasis of the clinical model alone was 0.76; this increased to 0.81 with inclusion of Decipher. Conclusion The genomic classifier test, Decipher, can independently improve prognostication of patients postprostatectomy, as well as within nearly all clinicopathologic, demographic, and treatment subgroups. Future study of how to best incorporate genomic testing in clinical decision-making and subsequent treatment recommendations is warranted.

Intracerebral infection of mice with mouse hepatitis virus, a member of the Coronaviridae family, reproducibly results in an acute encephalomyelitis that progresses to a chronic demyelinating disease. The ensuing neuropathology during the chronic stage of disease is primarily immune mediated and similar to that of the human demyelinating disease multiple sclerosis. Secretion of chemokines within the CNS signals the infiltration of leukocytes, which results in destruction of white matter and neurological impairment. The CC chemokine ligand (CCL)5 is localized in white matter tracts undergoing demyelination, suggesting that this chemokine participates in the pathogenesis of disease by attracting inflammatory cells into the CNS. In this study, we administer a mAb directed against CCL5 to mice with established mouse hepatitis virus-induced demyelination and impaired motor skills. Anti-CCL5 treatment decreased T cell accumulation within the CNS based, in part, on viral Ag specificity, indicating the ability to differentially target select populations of T cells. In addition, administration of anti-CCL5 improved neurological function and significantly (p < or = 0.005) reduced the severity of demyelination and macrophage accumulation within the CNS. These results demonstrate that the severity of CNS disease can be reduced through the use of a neutralizing mAb directed against CCL5 in a viral model of demyelination.

Intracranial infection of C57BL/6 mice with mouse hepatitis virus (MHV) results in an acute encephalomyelitis followed by a demyelinating disease similar in pathology to the human disease multiple sclerosis (MS). T cells participate in both defense and disease progression following MHV infection. Expression of chemokine receptors on activated T cells is important in allowing these cells to traffic into and accumulate within the central nervous system (CNS) of MHV-infected mice. The present study evaluated the contributions of CCR5 to the activation and trafficking of virus-specific CD8(+) T cells into the MHV-infected CNS mice. Comparable numbers of virus-specific CD8(+) T cells derived from immunized CCR5(+/+) or CCR5(-/-) mice were present within the CNS of MHV-infected RAG1(-/-) mice following adoptive transfer, indicating that CCR5 is not required for trafficking of these cells into the CNS. RAG1(-/-) recipients of CCR5(-/-)-derived CD8(+) T cells exhibited a modest, yet significant (P

Studies were performed to investigate the contributions of the CC chemokine receptor CCR5 in host defense and disease development following intracranial infection with mouse hepatitis virus (MHV). T cell recruitment was impaired in MHV-infected CCR5(-/-) mice at day 7 postinfection (pi), which correlated with increased (P < or = 0.03) titers within the brain. However, by day 12 pi, T cell infiltration into the CNS of infected CCR5(-/-) and CCR5(+/+) mice was similar and both strains exhibited comparable viral titers, indicating that CCR5 expression is not essential for host defense. Following MHV infection of CCR5(+/+) mice, greater than 50% of cells expressing CCR5 antigen were activated macrophage/microglia (determined by F4/80 antigen expression). In addition, infected CCR5(-/-) mice exhibited reduced (P < or = 0.02) macrophage (CD45(high)F4/80(+)) infiltration, which correlated with a significant reduction (P < or = 0.001) in the severity of demyelination compared to CCR5(+/+) mice. These data indicate that CCR5 contributes to MHV-induced demyelination by allowing macrophages to traffic into the CNS.

The non-nitrogenous bisphosphonate clodronate disodium (CD) inhibits bone resorption and is approved for horses ≥ 4 yr of age. Extra-label use in juvenile exercising horses is a concern due to potential interference with normal bone growth and development. The objective was to determine the effects of single and repeated doses of CD on bone optical density (radiographic bone aluminum equivalence; RBAE) and lameness score (LS) in juvenile, exercising horses. Quarter Horses [n = 32 (16 geldings, 16 fillies); 500 ± 13 d of age; body weight (BW) = 336 ± 26 kg]) were used to test the hypothesis that horses receiving CD would have greater RBAE and reduced LS, and additional doses of CD would accentuate these effects. In a 168-d trial, horses were housed in individual stalls, and fed 1% BW/d concentrate and ad libitum Coastal bermudagrass hay. Horses were exercised 5 d/wk following a phase-based progressive workload using a free stall exerciser; Phase I (d 0 to 84) mimicked sales preparation whereas Phase II (d 85 to 168) simulated early training. Horses were stratified by age, BW, sex, and initial RBAE into control (CON; n = 8), single dose CD (1X; d 84; n = 8), 2-doses CD (2X; d 0 and d 84; n = 8), and four-doses CD (4X; d 0, d 42, d 84, d 126; n = 8). On injection d (0, 42, 84, and 126), horses were administered either 1.8 mg/kg BW CD (OSPHOS) or isovolumetric saline. On d 0, 84, and 168, dorsalpalmar and lateral-medial radiographs of the left third metacarpus and metatarsus were captured. An aluminum step wedge was included in each image and raw images were analyzed (Quantity One Basic, BioRad) to determine total bone RBAE at 1 cm distal to the nutrient foramen. On d 0 and d 168, horses were evaluated by a veterinarian unaware of treatment group and assigned a LS according to the American Association of Equine Practitioners Lameness Scale. The RBAE and LS data were analyzed via PROC MIXED and PROC GENMOD, respectively, in SAS. For each view, RBAE increased from d 0 to d 84 and remained increased to d 168 (P ≤ 0.02), regardless of treatment, likely due to progressing exercise protocol. On d 0, 29 horses were LS 0, 1 horse was LS 1, and 2 horses were LS 2; LS did not differ among treatment groups (P = 0.61). On d 168, 5 horses were LS 0, 13 horses were LS 2, 11 horses were LS 3, and 1 horse was LS 4; LS did not differ among treatments (P = 0.16). Based on these results, the hypothesis that single or repeated CD administration would increase RBAE and decrease lameness in juvenile, exercised Quarter Horses was rejected. [ABSTRACT FROM AUTHOR]

Intra-articular corticosteroids (IAC) are administered to horses experiencing joint inflammation, at times with a concurrent articular antibiotic for prophylaxis. However, information regarding the biologic effects of IAC and potential for microbial contamination in IAC-administered joints is limited in young, exercising horses. The objective of this study was to evaluate the impact of IAC on traditional microbial culture, synovial fluid (SF) volume, and the presence of IAC; hypothesizing negative microbial culture, no change of SF volume, and detection of IAC in SF post-administration. To test this, 2-yr-old Quarter Horses {n = 24; body weight (BW) = 409 ± 6 kg; 825 ± 21 d of age; 12 fillies, 12 geldings) were stratified by BW, age, and sex, and randomly assigned to 1 of 3 treatment groups for a 56-d trial. Horses were housed individually in stalls with runs and fed concentrate offered at 0.75% BW/d split evenly every 12 h with ad libitum Coastal bermudagrass hay and water. Every 21 d, BW and body condition score (BCS) were recorded, and concentrate was adjusted accordingly. Horses were exercised 5 d/wk, up to 45 min/d in a progressive workload using a free-stall exerciser. Blood was collected postexercise during wk 1, 4, and 7 via jugular venipuncture and lactate (BL) was measured to monitor increasing workload. Intra-articular treatments consisted of 0 mg (CON; n = 8), 6 mg (CORT6; n = 8), or 12 mg (CORT12; n = 8) of triamcinolone acetonide (TA). On d 0, treatments were administered in one randomly selected radiocarpal joint per horse. Following aseptic preparation, SF from both radiocarpal joints was collected before administration (pre), and on d 7, 14, 28, and 56. Volume of SF was recorded, and standard microbial culture and detection of TA were performed by a commercial laboratory. Data were analyzed using PROC MIXED (BW, BCS, BL, SF volume) and PROC GLM (TA) of SAS. For SF volume, data from the contralateral radiocarpal joint were used as a covariate. Horses increased BW and BCS over time (P ≤ 0.01) across treatments. Post-exercise BL increased from wk 1 to 4 and remained increased through wk 7 (P < 0.01). Microbial culture for all horses and time points were negative. Concentration of TA was not evaluated on d 7 due to limited SF volume, but TA was detected on d 14 and concentration did not differ between CORT6 and CORT12 (P = 0.13). Recorded SF volume did not change from d 0 (pre) to 7 but increased to d 56, regardless of treatment (P < 0.01). These results indicate that, based on standard culture, TA administration did not result in positive microbial culture, or decrease SF volume in young horses undergoing regular exercise, however, TA was detectable to d 14. [ABSTRACT FROM AUTHOR]

Concerns over the extra-label use of bisphosphonates in skeletally immature horses has extended to include its likely re-release from bone into circulation in response to stressors. Therefore, the objective of this study was to be first to quantify the re-release of clodronate in response to controlled stressors. Yearling Quarter horses (n = 32) were stratified by age, body weight (BW), sex, and initial bone optical density into four treatment groups for a 168-d trial. Treatments consisted of control (CON; n = 8), single-dose (1X; d 84; n = 8), 2-dose (2X; d 0 and 84; n = 8), and four-dose groups (4X; d 0, 42, 84, and 126; n = 8). All horses received iso-volumetric intramuscular injections of either 1.8 mg/kg BW clodronate disodium (OSPHOSÒ) or saline (placebo) on d 0, 42, 84, and 126. Horses were housed in individual stalls (3.6 m × 7.2 m). Diets were formulated to meet nutrient requirements including concentrate offered every 12-h and ad libitum coastal bermudagrass (Cynodon dactylon) hay and water. Horses were exercised 5 d/ wk with a progressive workload, and maximum exercise intensity was reached on d 120, verified via heart rate and blood lactate. On d 120, blood samples were collected prior to the start of the exercise bout (pre), and 0-, 0.5-, 1-, 3-, 12-, and 24-h post exercise. Both tuber coxae of each horse were biopsied on d 168 with blood samples collected prior to (pre), and 0, 12, 24, 48, 72, 168, and 336 h post biopsy. Clodronate was quantified in plasma from exercise and biopsy samples using liquid chromatography tandem mass spectrometry. Exercise and biopsy serum were analyzed for cortisol, a marker of stress, while biopsy serum was analyzed for substance P, a pain marker. Data were analyzed using SAS PROC MIXED. Exercise did not result in clodronate re-release as there was no treatment x time interaction or time effect (P ≥ 0.44), but clodronate was dose-dependently greater in treated groups throughout the exercise collection period (P < 0.01). A treatment x time interaction (P ≤ 0.01) was observed for clodronate concentrations surrounding bone biopsy in which concentrations decreased at 168 h post biopsy before increasing at 336 h in 4X horses. Substance P increased over time (P < 0.01) following biopsy, regardless of treatment. Despite decreasing over time (P ≤ 0.01), there were similarly no treatment differences (P ≥ 0.46) nor treatment ´ time interactions (P ≥ 0.78) for cortisol following either exercise or bone insult. In conclusion, submaximal exercise did not result in re-release of clodronate; however, direct bone insult disrupted clodronate concentrations in horses receiving 4 administrations of bisphosphonate despite no treatment impact on stress or pain indicators. [ABSTRACT FROM AUTHOR]

Intra-articular lipopolysaccharide (LPS) is an established model for inducing robust, transient inflammation and increasing cartilage metabolism for up to 24 h following administration. Currently, there is limited information evaluating the long-term effects of this gram-negative endotoxin on the intra-articular environment of young horses. Therefore, the objective of this study was to determine the lasting effects of a single administration of intra-articular LPS on synovial biomarkers of inflammation and cartilage metabolism over 35 wk. Quarter Horse yearlings [n = 10; body weight (BW) = 409 ± 24 kg; 619 ± 16 d of age; fillies, (n = 7) and geldings (n = 3), originating from a single herd were used to test the hypothesis that intraarticular inflammation and cartilage metabolism would not be elevated at 35 wk post-injection. Each horse had one randomly selected radiocarpal joint injected with 0.8 mL of a 0.5 ng LPS solution derived from Escherichia coli O55:B5 (INFL; Sigma-Aldrich, St. Louis, MO) and the contralateral joint received sterile lactated Ringer’s solution (CON) as a control. Synovial fluid was obtained on 0 (before injection of LPS), 2, and 35 wk following arthrocentesis. Synovial fluid samples were analyzed in duplicate for carboxypropeptide of type II collagen (CPII), collagenase cleavage neopeptide (C2C), chondroitin sulfate 846 epitope (CS846), and prostaglandin E2 (PGE2 ) by commercial ELISA (IBEX Pharm, Montreal, Quebec, CA and Arbor Assays, Ann Arbor, MI). Biomarkers were analyzed using PROC MIXED of SAS v9.4 with repeated measures (time). The model included treatment (CON, INFL), time (week), and treatment × time interaction as fixed effects. Synovial biomarkers PGE2, CPII, and CS846 changed over time (P < 0.01) regardless of LPS administration, increasing from 0 to 2 wk and declining to below baseline concentrations at wk 35. There was a treatment × time interaction (P ≤ 0.01) in which INFL joints had decreased catabolic C2C concentrations compared with CON joints at wk 35. Therefore, LPS administration did not increase cartilage metabolism and inflammation levels at 2 or 35 wk post-LPS induction, indicating no negative effects compared with the contralateral controls. This study supports using intra-articular LPS in young horses without negative long-term effects. [ABSTRACT FROM AUTHOR]