Antibody discovery efficiency using different pretreatments.

<p>Antibody discovery efficiency using different pretreatments.</p

Comparison of <i>in vitro</i> and <i>in vivo</i> expression profiles of different promoters.

<p>(A) Luciferase expression levels in HaCat keratinocyte cells shows that CAG promoter greatly outperforms SFFV, EF1α, and UbC promoters (n = 4). (B) Luciferase expression levels in HEK293 cells similar expression from CAG and EF1α promoters (n = 4). (C) Luciferase expression levels in Balb/c mice shows that CAG promoter greatly outperforms SFFV, EF1α, and UbC promoters (n = 6; two shots in three mice). (D) Expression profile of each construct reveals a rapid decline in expression each day after delivery and ultimately, reaching background levels by day 3. Overall, the CAG promoter yields the highest expression at day 1 and day 2 (****: p<0.01, independent t-test).</p

Effect of different skin pretreatment strategies on luciferase expression.

<p>(A) 1% SDS treatment leads to an increase in luciferase expression one day after delivery compared to other treatments and mock treated control (**, ***: p<0.01, independent t-test) (n = 6; two shots in three mice).</p

Comparison of luciferase expression profiles in TNFα, IFNγ, and IFNα receptor knockout (KO) mice.

<p>(A-C) A rapid decline in luciferase expression for the CAG promoter is observed in all three KO strains as well as the wild-type C57BL6 strain (n = 5).</p

Effect of different skin pretreatment strategies on anti-huCLDN4 Ab response.

<p>(A) Sandpaper treatment results in a significant increase in MFI against HEK293 expressing huCLDN4 compared to other treatments and mock treated control (n = 5; p < 0.001, independent t-test). Samples were analyzed at a 1:100 dilution. (B) A comparison of FACS- and Mirrorball-based screen of hybridoma supernatants on HEK293 huCLDN4 and parental HEK293 cells demonstrates an 89% (51/57) overlap between the two methods.</p

Luciferase expression profiles after gene gun-based DNA delivery.

<p>Luciferase expression profile in Balb/c (A) and C57BL6 (B) mice for three different gas pressures (150, 300, and 400 psi) at days 1, 2, and 3 post-delivery (n = 5 mice).</p

A Neutralizing Anti-gH/gL Monoclonal Antibody Is Protective in the Guinea Pig Model of Congenital CMV Infection

<div><p>Human cytomegalovirus (HCMV) is the most common cause of congenital virus infection. Congenital HCMV infection occurs in 0.2–1% of all births, and causes birth defects and developmental abnormalities, including sensorineural hearing loss and developmental delay. Several key studies have established the guinea pig as a tractable model for the study of congenital HCMV infection and have shown that polyclonal antibodies can be protective <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004060#ppat.1004060-Bratcher1" target="_blank">[1]</a>–<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004060#ppat.1004060-Chatterjee1" target="_blank">[3]</a>. In this study, we demonstrate that an anti-guinea pig CMV (GPCMV) glycoprotein H/glycoprotein L neutralizing monoclonal antibody protects against fetal infection and loss in the guinea pig. Furthermore, we have delineated the kinetics of GPCMV congenital infection, from maternal infection (salivary glands, seroconversion, placenta) to fetal infection (fetus and amniotic fluid). Our studies support the hypothesis that a neutralizing monoclonal antibody targeting an envelope GPCMV glycoprotein can protect the fetus from infection and may shed light on the therapeutic intervention of HCMV congenital infection in humans.</p></div

Viral kinetics of fetal infection over 21 days plotted from three separate studies.

<p>43 pregnant guinea pigs were inoculated by subcutaneous injection with 4×10³ PFU of pathogenic stock IVP8 at day 21 gestation and sacrificed at 1, 3, 7, 11, 15, and 21 days post-infection. Each glyph (dot) represents the proportion of infected fetuses, with the size of the glyph proportional to the litter size. The solid lines represent the proportion of infected fetuses in a litter from a beta-binomial model fit to all three cohorts. The dashed segment of each line is extrapolated beyond the cohort data and is model based. See statistical section in methods for more details.</p

Effect of 1968/GPFc antibody on maternal mortality and congenital GPCMV infection^A^B.

A<p>Pregnant guinea pigs were infected with 4×10³ PFU of IVP8 at the start of the 2^nd trimester.</p>B<p>Cumulative results at 21 days post-infection.</p>C<p>Virus detected by quantitative PCR.</p>D<p>Virus detected by nested PCR.</p>E<p>Antibody administered I.P. one day prior to infection at 8 mg/kg dose and then twice per week for 3 weeks with a total of six doses (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004060#ppat-1004060-g005" target="_blank">Figure 5</a> for study scheme).</p>F<p>n/a, not applicable, no fetuses were alive at end of study.</p>G<p>Averaged in-house historical data from infected pregnant guinea pigs without antibody treatment.</p

Temporal relationship between placental and fetal infection over 21 days.

<p>43 pregnant guinea pigs were inoculated by subcutaneous injection with 4×10³ PFU of pathogenic stock IVP8 at day 21 gestation and sacrificed at 1, 3, 7, 11, 15, and 21 days post-infection. Each glyph represents an infected mother with the size of the glyph proportional to the number of infected placentas and/or fetuses recovered. The proportion of infected placentas (x-axis) and infected fetuses (y-axis) was determined by qPCR and nested PCR, respectively. Data was compiled from three separate experiments.</p