Natural infection by Zika virus but not DNA vaccination consistently elicits antibodies that compete with two potently neutralising monoclonal antibodies targeting distinct epitopes.

BACKGROUND: Autochthonous transmission of Zika virus (ZIKV) has been reported in 87 countries since 2015. Although most infections are mild, there is risk of Guillain-Barré syndrome and adverse pregnancy outcomes. Vaccines are urgently needed to prevent Zika, but sufficient understanding of humoral responses and tools to assess ZIKV-specific immunity are lacking. METHODS: We developed a blockade-of-binding (BOB) ELISA using A9E and G9E, two strongly neutralising ZIKV-specific monoclonal antibodies, which do not react with dengue virus. Receiver operating characteristic curve analysis assessed A9E and G9E BOB serodiagnostic performance. BOB was then applied to samples from a surveillance cohort in Risaralda, Colombia, and phase 1 ZIKV vaccine trial samples, comparing results against traditional serologic tests. FINDINGS: In the validation sample set (n = 120), A9E BOB has a sensitivity of 93.5% (95% CI: 79.3, 98.9) and specificity 97.8 (95% CI: 92.2, 99.6). G9E BOB had a sensitivity of 100% (95% CI: 89.0, 100.0) and specificity 100% (95% CI: 95.9, 100). Serum from natural infections consistently tested positive in these assays for up to one year, and reactivity tracks well with ZIKV infection status among sera from endemic areas with complicated flavivirus exposures. Interestingly, a leading ZIKV vaccine candidate elicited minimal BOB reactivity despite generating neutralising antibody responses. INTERPRETATION: In conclusion, A9E and G9E BOB assays are sensitive and specific assays for detecting antibodies elicited by recent or remote ZIKV infections. Given the additional ability of these BOB assays to detect immune responses that target different epitopes, further development of these assays is well justified for applications including flavivirus surveillance, translational vaccinology research and as potential serologic correlates of protective immunity against Zika. FUNDING: R21 AI129532 (PI: S. Becker-Dreps), CDCBAA 2017-N-18041 (PI: A. M. de Silva), Thrasher Fund (PI: M. H. Collins), K22 AI137306 (PI: M. H. Collins).

Arboviruses as an unappreciated cause of non-malarial acute febrile illness in the Dschang Health District of western Cameroon.

Acute febrile illness is a common problem managed by clinicians and health systems globally, particularly in the Tropics. In many regions, malaria is a leading and potentially deadly cause of fever; however, myriad alternative etiologies exist. Identifying the cause of fever allows optimal management, but this depends on many factors including thorough knowledge of circulating infections. Arboviruses such as dengue (DENV) cause fever and may be underdiagnosed in sub-Saharan Africa where malaria is a major focus. We examined cases of fever in western Cameroon that tested negative for malaria and found 13.5% (13/96) were due to DENV, with 75% (9/12) of these being DENV serotype 2 infections. Two complete DENV2 genomes were obtained and clustered closely to recent isolates from Senegal and Burkina Faso. The seroprevalence of DENV in this region was 24.8% (96/387). Neutralizing antibodies to DENV2 were detected in all (15/15) seropositive samples tested. Chikungunya (CHIKV) is an arthritogenic alphavirus that is transmitted by Aedes mosquitoes, the same principal vector as DENV. The seroprevalence for CHIKV was 15.7% (67/427); however, CHIKV did not cause a single case of fever in the 96 subjects tested. Of note, being seropositive for one arbovirus was associated with being seropositive for the other (Χ2 = 16.8, p<0.001). Taken together, these data indicate that Aedes-transmitted arboviruses are endemic in western Cameroon and are likely a common but underappreciated cause of febrile illness. This work supports the need for additional study of arboviruses in sub-Saharan Africa and efforts to improve diagnostic capacity, surveillance systems, and arbovirus prevention strategies.

Susceptibility to endemic Aedes-borne viruses among pregnant women in Risaralda, Colombia.

OBJECTIVES: Aedes-borne viruses (ABV) affect humans on every inhabited continent and frequently cause epidemics. Recent epidemics of chikungunya and Zika viruses (ZIKV) highlight that preparedness for future epidemics requires assessment of susceptibility, particularly among high-risk groups. We sought to determine immunity against the three major circulating ABV among pregnant women in an ABV-endemic area of Colombia. METHODS: A cross-sectional seroprevalence study was performed, enrolling women presenting to Labor and Delivery. Cord blood and maternal peripheral blood samples were obtained. IgG seroprevalence to flaviviruses and chikungunya was determined by ELISA. An abbreviated neutralization test was used to estimate the frequency and magnitude of immunity to Zika and four dengue serotypes. Cluster analyses explored epidemiologic factors associated with seroprevalence. RESULTS: Most women exhibited high levels of neutralizing antibodies to one or more ABV; however, nearly 20% were seronegative for flaviviruses. Our research took place after the epidemic peak of the ZIKV outbreak in Colombia in 2016. However, only 20% of pregnant women had high levels of Zika-neutralizing antibodies consistent with likely protective immunity to ZIKV. CONCLUSION: Hence, a high proportion of pregnant women in Risaralda remain susceptible to one or more ABV including the teratogenic ZIKV, indicating a risk for future epidemics in this region.

Occupational risk factors for severe acute respiratory coronavirus virus 2 (SARS-CoV-2) infection among healthcare personnel: A 6-month prospective analysis of the COVID-19 Prevention in Emory Healthcare Personnel (COPE) Study.

OBJECTIVES: To determine the incidence of severe acute respiratory coronavirus virus 2 (SARS-CoV-2) infection among healthcare personnel (HCP) and to assess occupational risks for SARS-CoV-2 infection. DESIGN: Prospective cohort of healthcare personnel (HCP) followed for 6 months from May through December 2020. SETTING: Large academic healthcare system including 4 hospitals and affiliated clinics in Atlanta, Georgia. PARTICIPANTS: HCP, including those with and without direct patient-care activities, working during the coronavirus disease 2019 (COVID-19) pandemic. METHODS: Incident SARS-CoV-2 infections were determined through serologic testing for SARS-CoV-2 IgG at enrollment, at 3 months, and at 6 months. HCP completed monthly surveys regarding occupational activities. Multivariable logistic regression was used to identify occupational factors that increased the risk of SARS-CoV-2 infection. RESULTS: Of the 304 evaluable HCP that were seronegative at enrollment, 26 (9%) seroconverted for SARS-CoV-2 IgG by 6 months. Overall, 219 participants (73%) self-identified as White race, 119 (40%) were nurses, and 121 (40%) worked on inpatient medical-surgical floors. In a multivariable analysis, HCP who identified as Black race were more likely to seroconvert than HCP who identified as White (odds ratio, 4.5; 95% confidence interval, 1.3-14.2). Increased risk for SARS-CoV-2 infection was not identified for any occupational activity, including spending >50% of a typical shift at a patient's bedside, working in a COVID-19 unit, or performing or being present for aerosol-generating procedures (AGPs). CONCLUSIONS: In our study cohort of HCP working in an academic healthcare system, <10% had evidence of SARS-CoV-2 infection over 6 months. No specific occupational activities were identified as increasing risk for SARS-CoV-2 infection.

Occupational risk factors for severe acute respiratory coronavirus virus 2 (SARS-CoV-2) infection among healthcare personnel: A cross-sectional analysis of subjects enrolled in the COVID-19 Prevention in Emory Healthcare Personnel (COPE) study.

Among 353 healthcare personnel in a longitudinal cohort in 4 hospitals in Atlanta, Georgia (May-June 2020), 23 (6.5%) had severe acute respiratory coronavirus virus 2 (SARS-CoV-2) antibodies. Spending >50% of a typical shift at the bedside (OR, 3.4; 95% CI, 1.2-10.5) and black race (OR, 8.4; 95% CI, 2.7-27.4) were associated with SARS-CoV-2 seropositivity.

Application of SARS-CoV-2 Serology to Address Public Health Priorities.

Background: Antibodies against SARS-CoV-2 can be detected by various testing platforms, but a detailed understanding of assay performance is critical. Methods: We developed and validated a simple enzyme-linked immunosorbent assay (ELISA) to detect IgG binding to the receptor-binding domain (RBD) of SARS-CoV-2, which was then applied for surveillance. ELISA results were compared to a set of complimentary serologic assays using a large panel of clinical research samples. Results: The RBD ELISA exhibited robust performance in ROC curve analysis (AUC> 0.99; Se = 89%, Sp = 99.3%). Antibodies were detected in 23/353 (6.5%) healthcare workers, 6/9 RT-PCR-confirmed mild COVID-19 cases, and 0/30 non-COVID-19 cases from an ambulatory site. RBD ELISA showed a positive correlation with neutralizing activity (p = <0.0001, R2 = 0.26). Conclusions: We applied a validated SARS-CoV-2-specific IgG ELISA in multiple contexts and performed orthogonal testing on samples. This study demonstrates the utility of a simple serologic assay for detecting prior SARS-CoV-2 infection, particularly as a tool for efficiently testing large numbers of samples as in population surveillance. Our work also highlights that precise understanding of SARS-CoV-2 infection and immunity at the individual level, particularly with wide availability of vaccination, may be improved by orthogonal testing and/or more complex assays such as multiplex bead assays.

Immunologic mechanisms of seasonal influenza vaccination administered by microneedle patch from a randomized phase I trial.

In a phase 1 randomized, single-center clinical trial, inactivated influenza virus vaccine delivered through dissolvable microneedle patches (MNPs) was found to be safe and immunogenic. Here, we compare the humoral and cellular immunologic responses in a subset of participants receiving influenza vaccination by MNP to the intramuscular (IM) route of administration. We collected serum, plasma, and peripheral blood mononuclear cells in 22 participants up to 180 days post-vaccination. Hemagglutination inhibition (HAI) titers and antibody avidity were similar after MNP and IM vaccination, even though MNP vaccination used a lower antigen dose. MNPs generated higher neuraminidase inhibition (NAI) titers for all three influenza virus vaccine strains tested and triggered a larger percentage of circulating T follicular helper cells (CD4 + CXCR5 + CXCR3 + ICOS + PD-1+) compared to the IM route. Our study indicates that inactivated influenza virus vaccination by MNP produces humoral and cellular immune response that are similar or greater than IM vaccination.

Dengue in Western Uganda: a prospective cohort of children presenting with undifferentiated febrile illness.

BACKGROUND: The spatial distribution and burden of dengue in sub-Saharan Africa remains highly uncertain, despite high levels of ecological suitability. The goal of this study was to describe the epidemiology of dengue among a cohort of febrile children presenting to outpatient facilities located in areas of western Uganda with differing levels of urbanicity and malaria transmission intensity. METHODS: Eligible children were first screened for malaria using rapid diagnostic tests. Children with a negative malaria result were tested for dengue using a combination NS1/IgM/IgG rapid test (SD Bioline Dengue Duo). Confirmatory testing by RT-PCR was performed in a subset of participants. Antigen-capture ELISA was performed to estimate seroprevalence. RESULTS: Only 6 of 1416 (0.42%) children had a positive dengue rapid test, while none of the RT-PCR results were positive. ELISA testing demonstrated reactive IgG antibodies in 28 (2.2%) participants with the highest prevalence seen at the urban site in Mbarara (19 of 392, 4.9%, p < 0.001). CONCLUSIONS: Overall, these findings suggest that dengue, while present, is an uncommon cause of non-malarial, pediatric febrile illness in western Uganda. Further investigation into the eocological factors that sustain low-level transmission in urban settings are urgently needed to reduce the risk of epidemics.

Guidelines for the Detection of NADPH Oxidases by Immunoblot and RT-qPCR.

The identification of NADPH oxidase (NOX) isoforms in tissues is essential for interpreting experiments and for next step decisions regarding cell lines, animal models, and targeted drug design. Two basic methods, immunoblotting and reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR), are important to monitor NOX protein and messenger RNA (mRNA) levels, respectively, for a range of investigations from understanding cell signaling events to judging NOX inhibitor efficacies. For many other genes that are expressed in high abundance, these methods may seem rather simple. However, detecting the low expression levels of endogenous NOX/DUOX is difficult and can be frustrating, so some guidelines would be helpful to those who are facing difficulties. One reason why detection is so difficult is the limited availability of vetted NOX/DUOX antibodies. Many of the commercial antibodies do not perform well in our hands, and dependable antibodies, often generated by academic laboratories, are in limited supply. Another problem is the growing trend in the NOX literature to omit end-user validation of antibodies by not providing appropriate positive and negative controls. With regard to NOX mRNA levels, knockdown of NOX/DUOX has been reported in cell lines with very low endogenous expression (C q values ≥30) or in cell lines devoid of the targeted NOX isoform (e.g., NOX4 expression in NCI-60 cancer cell panel cell line 786-0). These publications propagate misinformation and hinder progress in understanding NOX/DUOX function. This chapter provides overdue guidelines on how to validate a NOX antibody and provides general methodologies to prepare samples for optimal detection. It also includes validated methodology to perform RT-qPCR for the measurement of NOX mRNA levels, and we suggest that RT-qPCR should be performed prior to embarking on NOX protein detection.

Thioxo-dihydroquinazolin-one Compounds as Novel Inhibitors of Myeloperoxidase.

Myeloperoxidase (MPO) is a key antimicrobial enzyme, playing a normal role in host defense, but also contributing to inflammatory conditions including neuroinflammatory diseases such as Parkinson's and Alzheimer's. We synthesized and characterized more than 50 quinazolin-4(1H)-one derivatives and showed that this class of compounds inhibits MPO with IC50 values as low as 100 nM. Representative compounds showed partially reversible inhibition that was competitive with respect to Amplex Red substrate and did not result in the accumulation of MPO Compound II. Members of this group show promise for therapeutic development for the treatment of diseases in which inflammation plays a pathogenic role.

Ebselen and congeners inhibit NADPH oxidase 2-dependent superoxide generation by interrupting the binding of regulatory subunits.

NADPH oxidases (Nox) are a primary source of reactive oxygen species (ROS), which function in normal physiology and, when overproduced, in pathophysiology. Recent studies using mice deficient in Nox2 identify this isoform as a novel target against Nox2-implicated inflammatory diseases. Nox2 activation depends on the binding of the proline-rich domain of its heterodimeric partner p22phox to p47phox. A high-throughput screen that monitored this interaction via fluorescence polarization identified ebselen and several of its analogs as inhibitors. Medicinal chemistry was performed to explore structure-activity relationships and to optimize potency. Ebselen and analogs potently inhibited Nox1 and Nox2 activity but were less effective against other isoforms. Ebselen also blocked translocation of p47phox to neutrophil membranes. Thus, ebselen and its analogs represent a class of compounds that inhibit ROS generation by interrupting the assembly of Nox2-activating regulatory subunits.

A comparison of three cell types as potential candidates for intervertebral disc therapy: annulus fibrosus cells, chondrocytes, and bone marrow derived cells.

OBJECTIVES: Candidate cell types for disc cell transplantation therapy include anulus fibrosus (AF) cells, chondrocytes, and bone marrow derived cells (BMDCs). We compared the disc matrix production in these three types of cells, before and after stimulation with rhBMP-2. There is no study extant that compares these three cell types to determine the best candidate for the disc cell therapy. METHODS: AF cells, chondrocytes, and BMDCs (iliac crest and femur) were isolated and grown in monolayer. They were treated for 3 days with rhBMP-2. After 3 days, proteoglycan (sGAG) content in the media was quantified. The results were normalized by cell numbers. The mRNA expression of aggrecan, type I collagen, and type II collagen was measured using real-time PCR. Each cell type was also cultured in chamber slides and immunostained for aggrecan, type I collagen, and type II collagen after 3 days of treatment with rhBMP-2. RESULTS: (1) Without rhBMP-2 the chondrocytes produced more proteoglycan (sGAG) as compared to the other two cell types (AF cells and BMDCs). After stimulation with rhBMP-2 the chondrocytes produce even more proteoglycan than the other two cell types. (2) As compared to the other two cell types, in terms of mRNA expression, the chondrocytes expressed more aggrecan, type I collagen, and type II collagen before stimulation with rhBMP-2. After rhBMP-2 stimulation, the chondrocytes expressed even more aggrecan, type I collagen, and type II collagen in proportion to the concentration of rhBMP-2. For the BMDCs there were no changes in type I and II collagen. (3) rhBMP-2 stimulation produced increases in the protein levels of aggrecan, type I and II collagen in all three types of cells. CONCLUSIONS: On balance, according to these results, it would seem that chondrocytes are the best candidate for the disc cell therapy.

Can TGF-beta1 and rhBMP-2 act in synergy to transform bone marrow stem cells to discogenic-type cells?

INTRODUCTION: The recombinant human bone morphogenic protein-2 (rhBMP-2) is known to increase the proteoglycan production and chondrogenic gene expression in the disc cells. The transforming growth factor-beta 1 (TGF-beta(1)) can transform the bone marrow stem cells (BMDCs) into the disc-like cells. MATERIALS AND METHODS: We carried out an experiment to determine if TGF-beta(1) and rhBMP-2 can act in synergy on BMDCs by increasing the production of sulfated-glycosaminoglycan (sGAG) and affecting the mRNA expression of aggrecan, type I collagen, and type II collagen. The BMDCs were isolated from the iliac crest and femur of a New Zealand white rabbit (1 year). The BMDCs were culured in monolayer and treated for 6 days with TGF-beta(1) 10 ng/ml (group 1), rhBMP-2 200 ng/ml (group 2), and both TGF-beta(1) 10 ng/ml and rhBMP-2 200 ng/ml (group 3: the combined group) in Dulbecco's modified Eagle medium/F-12 with 1% fetal bovine serum. After 6 days, the sGAG content in the media was quantified using 1,9-dimethylmethylene blue staining and the mRNA expression of aggrecan, type I collagen, type II collagen, Sox-9, BMP-2, and BMP-7 were measured with the real-time PCR. The same BMDCs were also cultured in the chamber slide at 3 x 10(4) cells/chamber. After 6 days treatment, the treated cells were immunofluorescence stained with aggrecan, type I collagen, type II collagen, anti-BMP-2, anti-BMP-7 antibodies. After that, we compared the number of positive immunofluorescence stained cells with fluorescence microscope. The sGAG production and mRNA expression for each group were normalized against the same parameters for a non-treatment group. RESULTS AND DISCUSSION: The sGAG production was increased 1.15*, 1.34*, and 1.45* times in the TGF-beta(1) 10 ng/ml group, the rhBMP-2 200 ng/ml group, and the combined group respectively. The mRNA expression of aggrecan was increased 1.28, 3.42*, and 5.34* times, the mRNA expression of type I collagen was increased 0.86, 1.09, 1.17 times, the mRNA expression of type II collagen was increased 3.58*, 3.77*, and 10.78* times, the mRNA expression of Sox-9 was increased 1.29, 2.45, 2.75* times, the mRNA expression of BMP-2 was increased 1.14, 2.07, 4.43* times, and the mRNA expression of BMP-7 was increased 1.16, 1.49, 1.97* times, respectively for each group (* indicates p < 0.05). On the immunofluorescence staining of antibodies, the average positively immunofluorescence stained cells number for aggrecan were 4.2, 15.8*, 10*, and 22* according to the non-treatment group, TGF-beta(1) 10 ng/ml group, rhBMP-2 200 ng/ml group, and the combined group respectively. The average positively immunofluorescence stained cells number for type I collagen were 7, 14.2*, 9.2*, 17.4* and the average positively immunofluorescence stained cells number for type II collagen were 8.5, 28.25*, 20.25*, 42.25* and the average positively immunofluorescence stained cells number for anti-BMP-2 were 5, 16.75*, 8.75*, 27.25* and the average positively immunofluorescence stained cells number for anti-BMP-7 were 3.25, 7.5*, 8.75*, 15.25* (* indicates p < 0.05). CONCLUSIONS: Both TGF-beta(1) and rhBMP-2 alone, can increase proteoglycan production in the BMDCs. However, if they were used in combination, there is a synergistic effect. Similarly, the mRNA expressions of both aggrecan, type II collagen, Sox-9, BMP-2, and BMP-7 except for type I collagen were increased significantly when TGF-beta(1) and rhBMP-2 were combined. The positive immunofluorescence stained cell numbers for aggrecan, type I, II collagen, BMP-2 and BMP-7 were also increased after each TGF-beta(1) and rhBMP-2 treatment, and also more increased significantly in the aggrecan, type I, II collagen, BMP-2, and 7 when they were used jointly.

DNA damage induces a novel p53-survivin signaling pathway regulating cell cycle and apoptosis in acute lymphoblastic leukemia cells.

Survivin is a novel member of the inhibitor of apoptosis protein (IAP) family. Here we report that the chemotherapeutic drug doxorubicin, a DNA-damaging agent, activates a p53-survivin signaling pathway inducing cell cycle arrest and apoptosis in childhood acute lymphoblastic leukemia (ALL). Treatment of wild-type (wt) p53 ALL cells (EU-3 cell line) with doxorubicin caused accumulation of p53, resulting in dramatic down-regulation of survivin, depletion of cells in G(2)/M, and apoptosis (increased sub-G(1) compartment). In contrast, doxorubicin treatment of mutant (mut) p53 cells (EU-6/ALL line) up-regulated survivin and induced G(2)/M arrest without inducing apoptosis. However, treating EU-6 with anti-survivin antisense resensitized these cells to doxorubicin, resulting in apoptosis. With a p53-null cell line (EU-4), although doxorubicin treatment arrested cells in G(2)/M, survivin expression was unchanged, and cells underwent only limited apoptosis. However, re-expression of wt-p53 in EU-4 cells could restore the doxorubicin-p53-survivin pathway, resulting in significantly decreased survivin expression and increased apoptosis in these cells after doxorubicin treatment. Following cotransfection of p53-null EU-4 cells with survivin promoter-luciferase constructs and either wt-p53 or different mut-p53 expression vectors, wt-p53 inhibited survivin promoter activity; p53-mediated inhibition could be abrogated by overexpression of murine double minute2 (MDM2) protein. Together, these studies define a novel p53-survivin signaling pathway activated by DNA damage that results in down-regulation of survivin, cell cycle arrest, and apoptosis. Furthermore, our data indicate that loss of wt-p53 function in tumor cells may contribute to up-regulation of survivin and resistance to DNA-damaging agents.