Pre-proenkephalin 1 is Downregulated Under Unloading and is Involved in Osteoblast Biology.

Pre-proenkephalin 1 (Penk1) is a pro-neuropeptide that belongs to the typical opioid peptide's family, having analgesic properties. We previously found Penk1 to be the most downregulated gene in a whole gene profiling analysis performed in osteoblasts subjected to microgravity as a model of mechanical unloading. In this work, Penk1 downregulation was confirmed in the bones of two in vivo models of mechanical unloading: tail-suspended and botulinum toxin A (botox)-injected mice. Consistently, in the sera from healthy volunteers subjected to bed rest, we observed an inverse correlation between PENK1 and bed rest duration. These results prompted us to investigate a role for this factor in bone. Penk1 was highly expressed in mouse bone, but its global deletion failed to impact bone metabolism in vivo. Indeed, Penk1 knock out (Penk1-/-) mice did not show an overt bone phenotype compared to the WT littermates. Conversely, in vitro Penk1 gene expression progressively increased during osteoblast differentiation and its transient silencing in mature osteoblasts by siRNAs upregulated the transcription of the Sost1 gene encoding sclerostin, and decreased Wnt3a and Col1a1 mRNAs, suggesting an altered osteoblast activity due to an impairment of the Wnt pathway. In line with this, osteoblasts treated with the Penk1 encoded peptide, Met-enkephalin, showed an increase of Osx and Col1a1 mRNAs and enhanced nodule mineralization. Interestingly, primary osteoblasts isolated from Penk1-/- mice showed lower metabolic activity, ALP activity, and nodule mineralization, as well as a lower number of CFU-F compared to osteoblasts isolated from WT mice, suggesting that, unlike the transient inhibition, the chronic Penk1 deletion affects both osteoblast differentiation and activity. Taken together, these results highlight a role for Penk1 in the regulation of the response of the bone to mechanical unloading, potentially acting on osteoblast differentiation and activity in a cell-autonomous manner.

24-Hour movement behaviors and executive functions in preschoolers: A compositional and isotemporal reallocation analysis.

This cross-sectional study explored the relationship between 24-hour movement behaviors and executive function (EF) in preschool children. A total of 426 Han Chinese preschoolers (231 males; 3.8 ± 0.6 years old) from Zhuhai, Guangdong Province, China were selected from October 2021 to December 2021. Accelerometers were used to measure physical activity (PA) and sedentary behavior (SB), while sleep duration was obtained via a parent-report questionnaire. Components of EF (cognitive flexibility, inhibitory control, and working memory) were assessed using computerized behavioral tasks. The daily composition was significantly associated with inhibitory control and working memory. Inhibitory control improvements were linked to the addition of moderate-to-vigorous physical activity (MVPA) at the expense of SB and sleep. The reallocation between MVPA, SB, sleep, and light physical activity yielded a significant association with working memory.

Parental role-modeling on 24-hour movement behaviors among preschoolers during the COVID-19 pandemic.

INTRODUCTION: Little is known about the association between parents' and children's 24-h movement behaviors during the pandemic. This cross-sectional study examined the association between the 24-h movement behaviors of parents and their preschoolers and investigated sex differences in this association. METHODS: A total of 1740 preschoolers (4.5 ± 0.8 years old, 50.3% boys) and their parents (35.4 ± 4.9 years old, 24.3% males) in China participated in this study and provided valid and complete data. Parents completed an online survey or a written questionnaire in the period between October and December 2020. Preschoolers' and parents' movement behaviors (physical activity [PA], sedentary behavior [SB], screen time, and sleep) and demographic information were reported by the parents. Generalized linear models and logistic regression models were performed. RESULTS: Positive associations were found between parents' and preschoolers' moderate-to-vigorous intensity PA (ß = 0.28; 95% confidence interval [CI]: 0.20, 0.36), total PA (ß = 0.21; 95% CI: 0.17, 0.24), and sleep (ß = 0.05; 95% CI: 0.03, 0.06) with no apparent sex difference. No significant association was found between parents' and preschoolers' SB or screen time. Girls were more likely to meet all three 24-h movement guidelines when their parents met them (odds ratio = 2.38; 95% CI: 1.42, 4.01), but the relationship was not significant for boys. CONCLUSIONS: Parental role-modeling was positively associated with children's PA and sleep. This finding suggests that supporting parents' movement behaviors has the potential to promote a healthy lifestyle among preschoolers.

Associations of school physical activity environment with comorbid obesity and myopia in children and adolescents: Findings from a Chinese national follow-up study.

BACKGROUND: Overweight and obesity (OWOB) and myopia have become two of the most important issues affecting the health of children and adolescents worldwide. Despite the recognition that the school physical activity (PA) environment is a critical factor for preventing and controlling overweight, obesity (OWOB), and myopia in children and adolescents, research on OWOB and myopia as a comorbidity remains unexplored, with evidence for effective strategies still being inconclusive. Hence, this study aimed to assess the prevalence and progression of comorbid OWOB/myopia and each condition alone, and to explore the association with school PA environment. METHODS: A total of 9814 children and adolescents aged 6-18 years were included from the Chinese National Survey on Students' Constitution and Health follow-up survey conducted from November 2019 to November 2020 in China. Anthropometric measurements, unaided distance vision acuity and non-cycloplegic refraction data were collected to assess OWOB and myopia, while eight indicators from questionnaires for children and adolescents aged 9-18 years were investigated to assess school PA environment. We calculated the one-year incidence and progression rates of comorbid OWOB/myopia, OWOB alone, and myopia alone. Mixed effect logistic regression was evaluated the association between school PA environment and incidence and progression of comorbid OWOB/myopia, OWOB, and myopia. RESULTS: The prevalence of comorbid OWOB/myopia increased from 11.1% in 2019 to 17.9% in 2020, and the incidence of comorbid OWOB/myopia was 10.9%. Children and adolescents experiencing an unfavorable school PA environment had a higher risk of the incidence of comorbid OWOB/myopia compared to a favorable school environment (OR = 1.85, 95% CI: 1.42-2.42). Similar findings were seen in the incidence of obesity (OR = 1.86, 95% CI: 1.26-2.75). Children and adolescents in an unfavorable school PA environment had a higher risk of myopia progression (OR = 1.29, 95% CI: 1.01-1.65). CONCLUSIONS: Obesity and myopia and their comorbidity have been serious among children and adolescents in China. A favorable school PA environment might mitigate the risk of comorbid OWOB/myopia, OWOB, and myopia progression.

24-H movement behaviors and physical fitness in preschoolers: A compositional and isotemporal reallocation analysis.

Background/Objectives: This study examined the relationships between 24-h movement behaviors and physical fitness (PF) in preschool children. Methods: The study was conducted on 474 children aged 3-6 years in Zhuhai. Physical activity (PA) and sedentary behavior (SB) were collected by the accelerometer, and sleep time was assessed through the parent-report questionnaire. Balance, cardiorespiratory fitness (CRF), flexibility, muscle strength, muscular endurance, and speed-agility were measured using a balance beam test, 20 m shuttle run test, sit and reach test, handgrip test, sit-ups, and 4 × 10 m shuttle run test respectively. The compositional data analysis was used to examine the association between 24-h movement behaviors and PF, and the compositional isotemporal substitution analysis was used for the time reallocation. Results: The daily composition, adjusted for age, gender, and body mass index (BMI), was significantly associated with CRF (p < 0.001, r2 = 0.20), flexibility (p < 0.001, r2 = 0.07), muscular strength (p < 0.001, r2 = 0.37), muscular endurance (p < 0.001, r2 = 0.26), and speed-agility (p < 0.001, r2 = 0.26). The addition of moderate-to-vigorous PA (MVPA) at the expense of SB and sleep, MVPA at the cost of sleep, was associated with significant muscular strength and speed-agility improvements respectively. The impact of SB and sleep replacing MVPA is stronger than MVPA replacing SB and sleep on muscular strength. Conclusion: These findings offer useful insight for the replacement of movement behaviors within the recommended range to facilitate PF development in early childhood.

Hepatitis E virus seropositivity in an ethnically diverse community blood donor population.

BACKGROUND AND OBJECTIVES: Hepatitis E virus (HEV) is an underrecognized and emerging infectious disease that may threaten the safety of donor blood supply in many parts of the world. We sought to elucidate whether our local community blood supply is at increased susceptibility for transmission of transfusion-associated HEV infections. MATERIALS AND METHODS: We screened 10,002 randomly selected donations over an 8-month period between 2017 and 2018 at the Stanford Blood Center for markers of HEV infection using commercial IgM/IgG serological tests and reverse transcriptase quantitative polymerase chain reaction assays (RT-qPCR). Donor demographic information, including gender, age, self-identified ethnicity, location of residence and recent travel, were obtained from the donor database and used to generate multivariate binary logistic regressions for risk factors of IgG seropositivity. RESULTS: A total of 10,002 blood donations from 7507 unique donors were screened, and there was no detectable HEV RNA by RT-qPCR. The overall seropositivity rate was 12.1% for IgG and 0.56% for IgM. Multivariate analysis of unique donors revealed a significantly higher risk of IgG seropositivity with increasing age, White/Asian ethnicities and residence in certain local counties. CONCLUSION: Although HEV IgG seroprevalence in the San Francisco Bay Area is consistent with ongoing infection, the screening of a large donor population did not identify any viraemic blood donors. While HEV is an underrecognized and emerging infection in other regions, there is no evidence to support routine blood screening for HEV in our local blood supply currently; however, periodic monitoring may still be required to assess the ongoing risk.

Development and evaluation of a blocking ELISA for serological diagnosis of equine infectious anemia.

Equine infectious anemia (EIA) is an important viral disease characterized by persistent infection in equids worldwide. Most EIA cases are life-long virus carriers with low antibody reactions and without the appearance of clinical symptoms. A serological test with high sensitivity and specificity is required to detect inapparent infection. In this study, a B-cell common epitope-based blocking ELISA (bELISA) was developed using a monoclonal antibody together with the EIAV p26 protein labelled with HRP. The test has been evaluated against the standard and with field serum samples globally. This bELISA test can be completed within 75 min, and the sensitivity is higher than those of either the AGID or one commercial cELISA kit. This bELISA assay was 8-16 times more analytically sensitive than AGID, and 2 to 4 times more analytically sensitive than one cELISA kit by testing three sera from the USA, Argentina, and China, respectively. The 353 serum samples from Argentina were tested, in comparison with AGID, the diagnostic sensitivity and specificity of our bELISA assay were 100% (154/154) and 97.0% (193/199), respectively, and the accuracy of the bELISA test was 98.3%. The bELISA test developed in this study is a rapid, sensitive, specific method for the detection of EIAV infection, and could be a promising candidate for use in the monitoring of the EIA epidemic worldwide. KEY POINTS: • A universal epitope-based blocking enzyme-linked immunosorbent assay (bELISA) was developed for detection of antibodies to EIAV. • The bELISA assay can be used to test EIAV serum samples from different regions of the world including North America, South America, Europe, and Asia. • The bELISA assay was evaluated in three different international labs and showed a better performance than other commercial kits.

Secular trend and urban-rural disparity for age at spermarche among Chinese Han boys from 1995 to 2019.

AIM: To examine the secular trend and the urban-rural disparity of spermarche among Chinese Han boys from 1995 to 2019. METHODS: A total of 392 775 boys of Han ethnicity aged 11-18 years were extracted from the 1995, 2000, 2005, 2010, 2014 and 2019 Chinese National Surveys on Students' Constitution and Health. The median age at spermarche was estimated using the status quo data and probit analysis. The chi-square, ANOVA and LSD tests were used to compare the differences between the year-subgroups. U-test was used to compare the difference between urban and rural areas at each year. RESULTS: The median age at spermarche in Chinese Han boys decreased from 14.6 years in 1995 to 13.9 years in 2019 (p < 0.001). The rural boys showed a faster decreasing pace with a 1-year advance of age at spermarche while the urban boys had only a 0.5-year decrement, and there was still a statistically significant difference between urban-rural areas in 2019 (p < 0.001). Similar to urban-rural disparity, the age gap at spermarche between areas with different urbanisation rates became smaller over time. CONCLUSIONS: In general, spermarche in Chinese Han boys showed a decreasing trend, but the pace was slowing down. The urban-rural disparity in puberty development still existed but was gradually narrowing.

Movement behaviors and mental health of caregivers of preschoolers in China during the COVID-19 pandemic.

This study aimed to examine the associations between physical activity (PA), sedentary behavior (SB), sleep, and the mental health of caregivers of preschool children following the COVID-19 outbreak. From 5 October to 16 December 2020, responses from 2476 respondents in China were collated through an online survey or a written questionnaire. Movement behaviors (PA, SB, screen time, and sleep), mental health (depression, anxiety, and stress), and demographic information were self-reported by the respondents. Linear mixed models were used for data analysis. Valid responses were received from 2002 caregivers (35.5 ± 4.9 years old, 76.3% females) of children between 3 and 6 years old in China (Hong Kong 3.2%, Shanghai 20.6%, Guangzhou 34.1%, Guiyang 26.7%, Xuzhou 11.3%, Xi'an 4.1%). A higher level of PA was associated with a lower score of depression, while lower SB and longer sleep duration were associated with lower scores of depression, anxiety, and stress. Meeting the Canadian 24-h movement guidelines was associated with less symptoms of depression, anxiety, and stress. Higher PA was associated with lower levels of depression, while longer sleep and lower SB were associated with better scores of depression, anxiety, and stress. Meeting the Canadian 24-h movement guidelines has been associated with better mental health during the COVID-19 pandemic. Interventions to improve mental health among caregivers should involve enhancing their overall movement behaviors.

Movement behaviors and posttraumatic stress disorder during the COVID-19 pandemic: A retrospective study of Chinese university students.

Background/objective: This study aimed to examine the associations between physical activity (PA), sedentary behavior, sleep and posttraumatic stress disorder (PTSD) among undergraduate students during the coronavirus disease 2019 (COVID-19) pandemic in China. Methods: A total of 3178 university students responded to an online questionnaire between December 2020 and January 2021. Participants self-reported the time they spent on PA, screen time and sleep after (over the past seven days) and during the outbreak peak (from January to March 2020). Their sleep quality was measured using the Chinese version of the Pittsburgh Sleep Quality Index. The Chinese version of the Posttraumatic Stress Disorder Checklist - Civilian Version was used to measure PTSD. Logistic regressions and generalized linear mixed models were conducted. Results: The final analysis included data from 2070 university students (20.2 ±â€¯1.3 years old, 37.0% males). The prevalence of PTSD was 7.1%. Better sleep quality both during and after the outbreak peak, and longer sleep duration after the outbreak peak were associated with a lower odds ratio of having PTSD and lower re-experiencing, avoidance and hyperarousal scores. Higher total PA levels during the outbreak peak were associated with a higher odds ratio of having PTSD and higher levels of re-experiencing and avoidance. Conclusion: Sleep quality and duration were negatively associated with PTSD among university students during the COVID-19 pandemic. The associations between PA, screen time and PTSD require further examination. Future interventions to enhance mental health could consider targeting university students' sleep hygiene.

Structure-Based Design of Hepatitis C Virus E2 Glycoprotein Improves Serum Binding and Cross-Neutralization.

An effective vaccine for hepatitis C virus (HCV) is a major unmet need, and it requires an antigen that elicits immune responses to key conserved epitopes. Based on structures of antibodies targeting HCV envelope glycoprotein E2, we designed immunogens to modulate the structure and dynamics of E2 and favor induction of broadly neutralizing antibodies (bNAbs) in the context of a vaccine. These designs include a point mutation in a key conserved antigenic site to stabilize its conformation, as well as redesigns of an immunogenic region to add a new N-glycosylation site and mask it from antibody binding. Designs were experimentally characterized for binding to a panel of human monoclonal antibodies (HMAbs) and the coreceptor CD81 to confirm preservation of epitope structure and preferred antigenicity profile. Selected E2 designs were tested for immunogenicity in mice, with and without hypervariable region 1, which is an immunogenic region associated with viral escape. One of these designs showed improvement in polyclonal immune serum binding to HCV pseudoparticles and neutralization of isolates associated with antibody resistance. These results indicate that antigen optimization through structure-based design of the envelope glycoproteins is a promising route to an effective vaccine for HCV.IMPORTANCE Hepatitis C virus infects approximately 1% of the world's population, and no vaccine is currently available. Due to the high variability of HCV and its ability to actively escape the immune response, a goal of HCV vaccine design is to induce neutralizing antibodies that target conserved epitopes. Here, we performed structure-based design of several epitopes of the HCV E2 envelope glycoprotein to engineer its antigenic properties. Designs were tested in vitro and in vivo, demonstrating alteration of the E2 antigenic profile in several cases, and one design led to improvement of cross-neutralization of heterologous viruses. This represents a proof of concept that rational engineering of HCV envelope glycoproteins can be used to modulate E2 antigenicity and optimize a vaccine for this challenging viral target.

Broadly neutralizing antibodies from an individual that naturally cleared multiple hepatitis C virus infections uncover molecular determinants for E2 targeting and vaccine design.

Cumulative evidence supports a role for neutralizing antibodies contributing to spontaneous viral clearance during acute hepatitis C virus (HCV) infection. Information on the timing and specificity of the B cell response associated with clearance is crucial to inform vaccine design. From an individual who cleared three sequential HCV infections with genotypes 1b, 1a and 3a strains, respectively, we employed peripheral B cells to isolate and characterize neutralizing human monoclonal antibodies (HMAbs) to HCV after the genotype 1 infections. The majority of isolated antibodies, designated as HMAbs 212, target conformational epitopes on the envelope glycoprotein E2 and bound broadly to genotype 1-6 E1E2 proteins. Further, some of these antibodies showed neutralization potential against cultured genotype 1-6 viruses. Competition studies with defined broadly neutralizing HCV HMAbs to epitopes in distinct clusters, designated antigenic domains B, C, D and E, revealed that the selected HMAbs compete with B, C and D HMAbs, previously isolated from subjects with chronic HCV infections. Epitope mapping studies revealed domain B and C specificity of these HMAbs 212. Sequential serum samples from the studied subject inhibited the binding of HMAbs 212 to autologous E2 and blocked a representative domain D HMAb. The specificity of this antibody response appears similar to that observed during chronic infection, suggesting that the timing and affinity maturation of the antibody response are the critical determinants in successful and repeated viral clearance. While additional studies should be performed for individuals with clearance or persistence of HCV, our results define epitope determinants for antibody E2 targeting with important implications for the development of a B cell vaccine.

The mean age of menarche among Chinese schoolgirls declined by 6 months from 2005 to 2014.

AIM: This study assessed the trends in the age at menarche in Chinese schoolgirls from the majority Han group and 21 ethnic minorities from 2005 to 2014. We also compared the group differences during the study period. METHODS: A total of 344 230 girls aged 9-18 years were extracted from the 2005, 2010 and 2014 Chinese National Survey on Students' Constitution and Health. The age at menarche for each subgroup was estimated by probit analysis. The chi-square test and Z-test were used to compare the differences between the groups. RESULTS: The overall average age at menarche in Chinese schoolgirls decreased from 12.8 years in 2005 to 12.3 years in 2014. The Han girls and girls from 17 ethnic minorities showed decreasing trends in the age at menarche, while four ethnic minorities demonstrated fluctuating increasing trends over time. The gaps in age at menarche between the Han group and 14 of the ethnic minorities became smaller over the study period and were similar by 2014. CONCLUSION: The overall findings were a decrease in the age at menarche in China and smaller gaps between the majority Han group and ethnic minority groups. Ethnic-specific public health policies are urgently needed on issues such as contraception.

Antigenicity and Immunogenicity of Differentially Glycosylated Hepatitis C Virus E2 Envelope Proteins Expressed in Mammalian and Insect Cells.

The development of a prophylactic vaccine for hepatitis C virus (HCV) remains a global health challenge. Cumulative evidence supports the importance of antibodies targeting the HCV E2 envelope glycoprotein to facilitate viral clearance. However, a significant challenge for a B cell-based vaccine is focusing the immune response on conserved E2 epitopes capable of eliciting neutralizing antibodies not associated with viral escape. We hypothesized that glycosylation might influence the antigenicity and immunogenicity of E2. Accordingly, we performed head-to-head molecular, antigenic, and immunogenic comparisons of soluble E2 (sE2) produced in (i) mammalian (HEK293) cells, which confer mostly complex- and high-mannose-type glycans; and (ii) insect (Sf9) cells, which impart mainly paucimannose-type glycans. Mass spectrometry demonstrated that all 11 predicted N-glycosylation sites were utilized in both HEK293- and Sf9-derived sE2, but that N-glycans in insect sE2 were on average smaller and less complex. Both proteins bound CD81 and were recognized by conformation-dependent antibodies. Mouse immunogenicity studies revealed that similar polyclonal antibody responses were generated against antigenic domains A to E of E2. Although neutralizing antibody titers showed that Sf9-derived sE2 induced moderately stronger responses than did HEK293-derived sE2 against the homologous HCV H77c isolate, the two proteins elicited comparable neutralization titers against heterologous isolates. Given that global alteration of HCV E2 glycosylation by expression in different hosts did not appreciably affect antigenicity or overall immunogenicity, a more productive approach to increasing the antibody response to neutralizing epitopes may be complete deletion, rather than just modification, of specific N-glycans proximal to these epitopes.IMPORTANCE The development of a vaccine for hepatitis C virus (HCV) remains a global health challenge. A major challenge for vaccine development is focusing the immune response on conserved regions of the HCV envelope protein, E2, capable of eliciting neutralizing antibodies. Modification of E2 by glycosylation might influence the immunogenicity of E2. Accordingly, we performed molecular and immunogenic comparisons of E2 produced in mammalian and insect cells. Mass spectrometry demonstrated that the predicted glycosylation sites were utilized in both mammalian and insect cell E2, although the glycan types in insect cell E2 were smaller and less complex. Mouse immunogenicity studies revealed similar polyclonal antibody responses. However, insect cell E2 induced stronger neutralizing antibody responses against the homologous isolate used in the vaccine, albeit the two proteins elicited comparable neutralization titers against heterologous isolates. A more productive approach for vaccine development may be complete deletion of specific glycans in the E2 protein.

Inactivation of Escherichia coli O157:H7 in apple cider by resveratrol and naringenin.

The study investigated the efficacy of two GRAS-status phytochemicals, mega-resveratrol (RV) and naringenin (NG) to inactivate Escherichia coli O157:H7 (EHEC) in apple cider. A five-strain mixture of EHEC (∼7 log CFU/ml) was inoculated into cider, followed by the addition of RV (8.7 mM and 13.0 mM) or NG (7.3 mM and 11.0 mM). The cider samples were stored at 4 °C for 14 days and EHEC was enumerated on days 0,1,5,7 and 14. The deleterious effects of RV and NG on EHEC cells were visualized by scanning electron microscopy (SEM), and RT-qPCR was done to determine the effect of phytochemicals on three known acid resistance (AR) systems of EHEC. NG was more effective than RV and reduced EHEC counts by ∼4.5 log CFU/ml by day 14, whereas RV reduced counts by ∼2.5 log CFU/ml compared to controls (P < 0.05). SEM showed that RV and NG resulted in the destruction of EHEC cells, and surviving bacteria appeared 'lemon shaped'. RT-qPCR results revealed that RV and NG downregulated the transcription of AR associated genes in EHEC (P < 0.05). Results suggest the potential use of RV and NG as natural antimicrobial additives to enhance the microbiological safety of apple cider. However, sensory analysis studies are warranted.

Effectiveness of active video game usage on body composition, physical activity level and motor proficiency in children with intellectual disability.

BACKGROUND: Interventions with active video games (AVGs) can promote physical activity (PA) and health and are compatible with a school setting. The needs of children with intellectual disability (ID) in this area have been neglected. METHODS: A two-arm trial was conducted among 203 students with intellectual disability. The intervention group was prescribed a 12-week intervention with AVG. The control group continued with usual PA. RESULTS: Children's BOT-2 short-form score increased in both the intervention and control groups. However, the AVG intervention had no statistically significant effect on children's body composition, PA and motor proficiency overall, or in analyses of subgroups based on age, body weight and comorbid autism. CONCLUSION: Active video game intervention had no marked effect on body composition, PA and motor proficiency in children with intellectual disability. The reasons for the lack of effectivity of the intervention are discussed; these may provide better guidelines for future AVG intervention in children with intellectual disability.

Global mapping of antibody recognition of the hepatitis C virus E2 glycoprotein: Implications for vaccine design.

The E2 envelope glycoprotein is the primary target of human neutralizing antibody response against hepatitis C virus (HCV), and is thus a major focus of vaccine and immunotherapeutics efforts. There is emerging evidence that E2 is a highly complex, dynamic protein with residues across the protein that are modulating antibody recognition, local and global E2 stability, and viral escape. To comprehensively map these determinants, we performed global E2 alanine scanning with a panel of 16 human monoclonal antibodies (hmAbs), resulting in an unprecedented dataset of the effects of individual alanine substitutions across the E2 protein (355 positions) on antibody recognition. Analysis of shared energetic effects across the antibody panel identified networks of E2 residues involved in antibody recognition and local and global E2 stability, as well as predicted contacts between residues across the entire E2 protein. Further analysis of antibody binding hotspot residues defined groups of residues essential for E2 conformation and recognition for all 14 conformationally dependent E2 antibodies and subsets thereof, as well as residues that enhance antibody recognition when mutated to alanine, providing a potential route to engineer E2 vaccine immunogens. By incorporating E2 sequence variability, we found a number of E2 polymorphic sites that are responsible for loss of neutralizing antibody binding. These data and analyses provide fundamental insights into antibody recognition of E2, highlighting the dynamic and complex nature of this viral envelope glycoprotein, and can serve as a reference for development and rational design of E2-targeting vaccines and immunotherapeutics.

Antibody Response to Hypervariable Region 1 Interferes with Broadly Neutralizing Antibodies to Hepatitis C Virus.

UNLABELLED: Hypervariable region 1 (HVR1) (amino acids [aa] 384 to 410) on the E2 glycoprotein of hepatitis C virus contributes to persistent infection by evolving escape mutations that attenuate binding of inhibitory antibodies and by blocking access of broadly neutralizing antibodies to their epitopes. A third proposed mechanism of immune antagonism is that poorly neutralizing antibodies binding to HVR1 interfere with binding of other superior neutralizing antibodies. Epitope mapping of human monoclonal antibodies (HMAbs) that bind to an adjacent, conserved domain on E2 encompassing aa 412 to 423 revealed two subsets, designated HC33 HMAbs. While both subsets have contact residues within aa 412 to 423, alanine-scanning mutagenesis suggested that one subset, which includes HC33.8, has an additional contact residue within HVR1. To test for interference of anti-HVR1 antibodies with binding of antibodies to aa 412 to 423 and other E2 determinants recognized by broadly neutralizing HMAbs, two murine MAbs against HVR1 (H77.16) and aa 412 to 423 (H77.39) were studied. As expected, H77.39 inhibited the binding of all HC33 HMAbs. Unexpectedly, H77.16 also inhibited the binding of both subsets of HC33 HMAbs. This inhibition also was observed against other broadly neutralizing HMAbs to epitopes outside aa 412 to 423. Combination antibody neutralization studies by the median-effect analysis method with H77.16 and broadly reactive HMAbs revealed antagonism between these antibodies. Structural studies demonstrated conformational flexibility in this antigenic region, which supports the possibility of anti-HVR1 antibodies hindering the binding of broadly neutralizing MAbs. These findings support the hypothesis that anti-HVR1 antibodies can interfere with a protective humoral response against HCV infection. IMPORTANCE: HVR1 contributes to persistent infection by evolving mutations that escape from neutralizing antibodies to HVR1 and by shielding broadly neutralizing antibodies from their epitopes. This study provides insight into a new immune antagonism mechanism by which the binding of antibodies to HVR1 blocks the binding and activity of broadly neutralizing antibodies to HCV. Immunization strategies that avoid the induction of HVR1 antibodies should increase the inhibitory activity of broadly neutralizing anti-HCV antibodies elicited by candidate vaccines.

Macaque Monoclonal Antibodies Targeting Novel Conserved Epitopes within Filovirus Glycoprotein.

UNLABELLED: Filoviruses cause highly lethal viral hemorrhagic fever in humans and nonhuman primates. Current immunotherapeutic options for filoviruses are mostly specific to Ebola virus (EBOV), although other members of Filoviridae such as Sudan virus (SUDV), Bundibugyo virus (BDBV), and Marburg virus (MARV) have also caused sizeable human outbreaks. Here we report a set of pan-ebolavirus and pan-filovirus monoclonal antibodies (MAbs) derived from cynomolgus macaques immunized repeatedly with a mixture of engineered glycoproteins (GPs) and virus-like particles (VLPs) for three different filovirus species. The antibodies recognize novel neutralizing and nonneutralizing epitopes on the filovirus glycoprotein, including conserved conformational epitopes within the core regions of the GP1 subunit and a novel linear epitope within the glycan cap. We further report the first filovirus antibody binding to a highly conserved epitope within the fusion loop of ebolavirus and marburgvirus species. One of the antibodies binding to the core GP1 region of all ebolavirus species and with lower affinity to MARV GP cross neutralized both SUDV and EBOV, the most divergent ebolavirus species. In a mouse model of EBOV infection, this antibody provided 100% protection when administered in two doses and partial, but significant, protection when given once at the peak of viremia 3 days postinfection. Furthermore, we describe novel cocktails of antibodies with enhanced protective efficacy compared to individual MAbs. In summary, the present work describes multiple novel, cross-reactive filovirus epitopes and innovative combination concepts that challenge the current therapeutic models. IMPORTANCE: Filoviruses are among the most deadly human pathogens. The 2014-2015 outbreak of Ebola virus disease (EVD) led to more than 27,000 cases and 11,000 fatalities. While there are five species of Ebolavirus and several strains of marburgvirus, the current immunotherapeutics primarily target Ebola virus. Since the nature of future outbreaks cannot be predicted, there is an urgent need for therapeutics with broad protective efficacy against multiple filoviruses. Here we describe a set of monoclonal antibodies cross-reactive with multiple filovirus species. These antibodies target novel conserved epitopes within the envelope glycoprotein and exhibit protective efficacy in mice. We further present novel concepts for combination of cross-reactive antibodies against multiple epitopes that show enhanced efficacy compared to monotherapy and provide complete protection in mice. These findings set the stage for further evaluation of these antibodies in nonhuman primates and development of effective pan-filovirus immunotherapeutics for use in future outbreaks.

Affinity maturation of a broadly neutralizing human monoclonal antibody that prevents acute hepatitis C virus infection in mice.

Direct-acting antivirals (DAAs) have led to a high cure rate in treated patients with chronic hepatitis C virus (HCV) infection, but this still leaves a large number of treatment failures secondary to the emergence of resistance-associated variants (RAVs). To increase the barrier to resistance, a complementary strategy is to use neutralizing human monoclonal antibodies (HMAbs) to prevent acute infection. However, earlier efforts with the selected antibodies led to RAVs in animal and clinical studies. Therefore, we identified an HMAb that is less likely to elicit RAVs for affinity maturation to increase potency and, more important, breadth of protection. Selected matured antibodies show improved affinity and neutralization against a panel of diverse HCV isolates. Structural and modeling studies reveal that the affinity-matured HMAb mediates virus neutralization, in part, by inducing conformational change to the targeted epitope, and that the maturated light chain is responsible for the improved affinity and breadth of protection. A matured HMAb protected humanized mice when challenged with an infectious HCV human serum inoculum for a prolonged period. However, a single mouse experienced breakthrough infection after 63 days when the serum HMAb concentration dropped by several logs; sequence analysis revealed no viral escape mutation. CONCLUSION: The findings suggest that a single broadly neutralizing antibody can prevent acute HCV infection without inducing RAVs and may complement DAAs to reduce the emergence of RAVs. (Hepatology 2016;64:1922-1933).