19 research outputs found
An Evolved Mxe GyrA Intein for Enhanced Production of Fusion Proteins
Expressing
antibodies as fusions to the non-self-cleaving Mxe GyrA
intein enables site-specific, carboxy-terminal chemical modification
of the antibodies by expressed protein ligation (EPL). Bacterial antibody-intein
fusion protein expression platforms typically yield insoluble inclusion
bodies that require refolding to obtain active antibody-intein fusion
proteins. Previously, we demonstrated that it was possible to employ
yeast surface display to express properly folded single-chain antibody
(scFv)-intein fusions, therefore permitting the direct small-scale
chemical functionalization of scFvs. Here, directed evolution of the
Mxe GyrA intein was performed to improve both the display and secretion
levels of scFv-intein fusion proteins from yeast. The engineered intein
was shown to increase the yeast display levels of eight different
scFvs by up to 3-fold. Additionally, scFv- and green fluorescent protein
(GFP)-intein fusion proteins can be secreted from yeast, and while
fusion of the scFvs to the wild-type intein resulted in low expression
levels, the engineered intein increased scFv-intein production levels
by up to 30-fold. The secreted scFv- and GFP-intein fusion proteins
retained their respective binding and fluorescent activities, and
upon intein release, EPL resulted in carboxy-terminal azide functionalization
of the target proteins. The azide-functionalized scFvs and GFP were
subsequently employed in a copper-free, strain-promoted click reaction
to site-specifically immobilize the proteins on surfaces, and it was
demonstrated that the functionalized, immobilized scFvs retained their
antigen binding specificity. Taken together, the evolved yeast intein
platform provides a robust alternative to bacterial intein expression
systems
Ocular and uteroplacental pathology in a macaque pregnancy with congenital Zika virus infection
<div><p>Congenital Zika virus (ZIKV) infection impacts fetal development and pregnancy outcomes. We infected a pregnant rhesus macaque with a Puerto Rican ZIKV isolate in the first trimester. The pregnancy was complicated by preterm premature rupture of membranes (PPROM), intraamniotic bacterial infection and fetal demise 49 days post infection (gestational day 95). Significant pathology at the maternal-fetal interface included acute chorioamnionitis, placental infarcts, and leukocytoclastic vasculitis of the myometrial radial arteries. ZIKV RNA was disseminated throughout fetal tissues and maternal immune system tissues at necropsy, as assessed by quantitative RT-PCR for viral RNA. Replicating ZIKV was identified in fetal tissues, maternal uterus, and maternal spleen by fluorescent in situ hybridization for viral replication intermediates. Fetal ocular pathology included a choroidal coloboma, suspected anterior segment dysgenesis, and a dysplastic retina. This is the first report of ocular pathology and prolonged viral replication in both maternal and fetal tissues following congenital ZIKV infection in a rhesus macaque. PPROM followed by fetal demise and severe pathology of the visual system have not been described in macaque congenital ZIKV infection previously. While this case of ZIKV infection during pregnancy was complicated by bacterial infection with PPROM, the role of ZIKV on this outcome cannot be precisely defined, and further nonhuman primate studies will determine if increased risk for PPROM or other adverse pregnancy outcomes are associated with congenital ZIKV infection.</p></div
Uteroplacental histopathology from the primary placental disc.
<p>(A) Maternal neutrophils invading chorionic plate (arrow) is diagnostic of acute chorioamnionitis. (B) Villi show increased perivillous fibrin deposition (arrow) and there are multiple remote infarctions (arrow, C). (D) Radial arteries in the myometrium show a pronounced leukocytoclastic vasculitis (arrow) defined as an infiltrative mixture of lymphocytes, eosinophils, and plasma cells into the smooth muscle wall of these vessels.</p
Amniotic fluid (AF) markers confirm rupture of membranes and contamination of pan-collected urine.
<p>(A) An AmniSure<sup>®</sup> test, which measures PAMG-1 protein, was performed on pan urine collection (28 dpi, 45 dpi, 49 dpi) and AF (28 dpi) from the pregnant animal. Nonpregnant control animal urine and pregnant animal AF are included as controls. (B) Relative pixel density of the Amnisure<sup>®</sup> test strip test band and control band. (C) Amniotic fluid protein IGFBP-1 ELISA. Body fluids from the pregnant animal (pan urine collection 28, 42, 45, 49 dpi and AF 28 dpi), nonpregnant negative control male and female urine samples, amniotic fluid from a control pregnancy were evaluated for the presence of IGFBP-1. Dashed lines indicate the upper and lower limits of quantitative accuracy of the assay. In Panels B and C, white bars denote body fluids from the experimental animal and grey bars denote control fluids from other animals in the colony.</p
Maternal and fetal necropsy images.
<p>(A) The uterus was removed in entirety from the abdominal cavity of the dam using sterile instruments and a syringe was used to aspirate the purulent fluid from inside the uterine cavity. (B) The fetus was removed from the uterus and was covered in thick fibrinous material. Placental discs 1 (C) and 2 (D) were covered in the same thick fibrinous maternal that covered the fetus.</p
ZIKV RNA levels in maternal body fluids.
<p>vRNA was measured by quantitative RT-PCR in plasma, urine, amniotic fluid and CSF. The limit of detection is 33 copies/mL and the assay limit of quantification is 100 copies/mL, which are indicated by dashed lines.</p
Tissue histology and viral localization of maternal spleen, maternal uterus, amniotic/chorionic membrane, and fetal lung.
<p>Each tissue was stained with H&E, ISH, and mFISH. ISH shows localization of ZIKV vRNA. mFISH shows replicative intermediates by staining the negative sense RNA strands green and positive sense RNA strands red. Co-localization (yellow) demonstrates dsRNA intermediates. Black arrows denote a germinal center. Asterisks indicate neutrophils. Blue arrows highlight green, red, or yellow fluorescence.</p
Tissues with detectable ZIKV RNA from mother and fetus.
<p>Tissues with detectable ZIKV RNA from mother and fetus.</p
Fetal growth measured by ultrasonography.
<p>(A) Head circumference (HC), biparietal diameter (BPD), and femur length (FL) were measured in weekly ultrasounds. All measurements are depicted as millimeters (mm). The solid lines were derived from reference ranges from Tarantal et al. 2005 to show the mean (black lines) and one, two, and three standard deviations from the mean (grey lines). The HC, BPD, and FL were then plotted along these reference ranges to observe any deviations from the mean. (B) The pGA is plotted against the aGA (based on gestational age estimated from breeding and menstrual history). The pGA is shown separately for each measurement: BPD (triangle), HC (square), and FL (circle).</p
Neutralizing antibody titers following ZIKV infection.
<p>PRNT titers were measured pre and post infection. The x-axis represents the reciprocal serum dilution (log<sub>10</sub>) and the y-axis represents the percent reduction. The dashed lines indicate 90% and 50% reduction.</p