The limit of detection of the PCR/LDR/Universal Array assay using <i>in vitro</i> transcribed RNA or whole virus.

<p>ND = Not determined</p><p>Pfu/ml = plaque forming units/ml</p><p>ffu/ml = focus forming units/ml.</p><p>^a PCR/LDR was performed on cloned RNA fragments for all viruses except EBOV and DENV while</p><p>^bdilutions of culture supernatants were used for the latter two viruses. <i>Zaire ebolavirus</i>’95 was used for determination of LOD.</p><p>The limit of detection of the PCR/LDR/Universal Array assay using <i>in vitro</i> transcribed RNA or whole virus.</p

Comparison of universal array profile of viral RNA/DNA tested for the corresponding zip-codes.

<p>Normalized average signal intensity for the zip-codes assigned to each virus are presented. The color bars are the signals obtained with the indicated virus (positives). The black bars are the signals produced by the other ten viruses. A signal was considered positive if the intensity of the zip-code spot was at least 10-fold higher than the uniform background level of fluorescence of the array slide. Although a few other viruses produced low-level positive signals for zip18, this did not result in any false positive results since positive signals from at least two addresses was required for a positive identification. In the future, this issue would be rectified by switching to a different zip-code. The average signal intensity for the positives ranged from 31.2 to 123.4, depending on the virus. The average signal intensity for the negatives ranged from 0.3 to 6.2, thus they were not considered positive signals.</p

Schematic of the PCR/LDR assay for detection of VHF viruses.

<p>For each virus (ebolavirus is shown as a representative virus), 1–2 different regions are amplified by RT-PCR using forward and reverse primers, each with minimal degeneracy and all containing universal tails to prevent the formation of primer dimers. Cy-3 labeled downstream LDR primers and single base-discriminating upstream primers with unique zip-code complements (20-30-mers) are targeted to specific sequences/SNPs within the PCR amplicons. Ligation of two adjacent oligonucleotides annealed to a complementary DNA target occurs in the presence of thermostable ligase only if the nucleotides are perfectly matched at the junction [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138484#pone.0138484.ref054" target="_blank">54</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138484#pone.0138484.ref055" target="_blank">55</a>]. The zip-code complements on the 5’ end of fluorescently labeled LDR products anneal to specific complementary zip-code addresses on a universal array [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138484#pone.0138484.ref056" target="_blank">56</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138484#pone.0138484.ref057" target="_blank">57</a>]. A positive signal on the universal array is detected as a fluorescent spot. Primers for the ligation reaction were designed targeting 2 or 3 areas within each PCR amplicon. Each virus could produce a maximum of six ligation products, except for VAR and VACC, for which there were a maximum of 5 each. The detection of 2 or more ligation products was required for the detection and identification of a virus. Representative arrays that detect and identify <i>Ebola Zaire</i>, Lassa and Yellow fever viruses are shown.</p

Details of viral cultures and nucleic acids used in the study: RNA viruses.

<p>* The exact sequence of the stock used is not known. The GenBank sequence of the parent strain is indicated.</p><p>Details of viral cultures and nucleic acids used in the study: RNA viruses.</p

The majority of the CCHFV-M vaccinated mice developed both IgG1 and IgG2c responses following three vaccinations.

<p>Prior to challenge, CCHFV-specific antibody isotypes and the antibody avidity were examined by ELISA against the CCHF_VLP. A) The CCHFV-specific IgG1 and IgG2c response in individual mice following three vaccinations. Pooled sera from IFNAR^-/- and WT C57BL/6 mice vaccinated with empty vector were tested concurrently and had no detectable signal. B) The avidity of the CCHFV-specific antibody response in vaccinated mice following three vaccinations was measured. For (A) and (B) mice that died after CCHFV challenge are shown in red. *Two-way ANOVA, confidence intervals were set to 95%.</p

CCHFV-M vaccinated mice developed CCHFV-specific neutralizing antibodies following three vaccinations, and the response was enhanced with the addition of complement.

<p>Prior to challenge, neutralization titers were measured against the CCHF_VLP, with and without complement. Mice that died following challenge are highlighted in red. *Two-way ANOVA confidence intervals were set to 95%.</p

<i>In vitro</i> expression of the glycoprotein genes from the CCHFV-M DNA vaccine plasmid.

<p>A) The total (permeabilized cells) and surface presence (non-permeabilized cells) of G_C was examined 44 h after transfection of COS-7 cells with wild-type CCHFV-M, optimized CCHFV-M, or empty vector, the maximum expression was seen at 250 ng of each plasmid (shown). B) <i>In vitro</i> expression by Western blot of G_N (37 kDa) and G_C (75 kDa) in COS-7 cells 44 h after transfection of CCHFV-M or empty vector, 250 ng of each plasmid.</p

N-specific antibodies in post-challenge sera of CCHFV-M vaccinated mice.

<p>Sera from vaccinated mice that survived CCHFV challenge (28 days post-challenge) were examined by ELISA for antibodies against N.</p

CCHFV-specific IgG ELISA titers increase following each vaccination.

<p>The CCHFV-specific ELISA titers following three vaccinations are similar in IFNAR^-/- and WT C578BL/6 mice before challenge. A) Mouse sera were pooled in each vaccination group and the CCHFV-specific IgG antibodies were measured by CCHF_VLP ELISA following each vaccination with the optimized CCHFV-M vaccine. Vaccinations were performed at weeks 0, 3, and 6. B) The CCHFV IgG ELISA titers for individual mice 1 week prior to challenge; mice that died after CCHFV challenge are shown in red. *One-way ANOVA, confidence intervals were set to 95%.</p

A DNA vaccine for Crimean-Congo hemorrhagic fever protects against disease and death in two lethal mouse models

<div><p>Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus capable of causing a severe hemorrhagic fever disease in humans. There are currently no licensed vaccines to prevent CCHFV-associated disease. We developed a DNA vaccine expressing the M-segment glycoprotein precursor gene of CCHFV and assessed its immunogenicity and protective efficacy in two lethal mouse models of disease: type I interferon receptor knockout (IFNAR^-/-) mice; and a novel transiently immune suppressed (IS) mouse model. Vaccination of mice by muscle electroporation of the M-segment DNA vaccine elicited strong antigen-specific humoral immune responses with neutralizing titers after three vaccinations in both IFNAR^-/- and IS mouse models. To compare the protective efficacy of the vaccine in the two models, groups of vaccinated mice (7–10 per group) were intraperitoneally (IP) challenged with a lethal dose of CCHFV strain IbAr 10200. Weight loss was markedly reduced in CCHFV DNA-vaccinated mice as compared to controls. Furthermore, whereas all vector-control vaccinated mice succumbed to disease by day 5, the DNA vaccine protected >60% of the animals from lethal disease. Mice from both models developed comparable levels of antibodies, but the IS mice had a more balanced Th1/Th2 response to vaccination. There were no statistical differences in the protective efficacies of the vaccine in the two models. Our results provide the first comparison of these two mouse models for assessing a vaccine against CCHFV and offer supportive data indicating that a DNA vaccine expressing the glycoprotein genes of CCHFV elicits protective immunity against CCHFV.</p></div