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Recombinant Envelope-Proteins with Mutations in the Conserved Fusion Loop Allow Specific Serological Diagnosis of Dengue-Infections.

Rockstroh A, Barzon L, Pacenti M, Palù G, Niedrig M, Ulbert S - PLoS Negl Trop Dis (2015)

Bottom Line: We generated insect-cell derived recombinant E-proteins of the four DENV-serotypes which contain point mutations in the FL domain.By using specific mixtures of these mutant antigens, cross-reactivity against heterologous flaviviruses was strongly reduced, enabling sensitive and specific diagnosis of the DENV-infected serum samples in IgG and IgM-measurements.These results have indications for the development of serological DENV-tests with improved specificity.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Fraunhofer Institute for Cell Therapy and Immunology, Leipzig, Germany.

ABSTRACT
Dengue virus (DENV) is a mosquito-borne flavivirus and a major international public health concern in many tropical and sub-tropical areas worldwide. DENV is divided into four major serotypes, and infection with one serotype leads to immunity against the same, but not the other serotypes. The specific diagnosis of DENV-infections via antibody-detection is problematic due to the high degree of cross-reactivity displayed by antibodies against related flaviviruses, such as West Nile virus (WNV), Yellow Fever virus (YFV) or Tick-borne encephalitis virus (TBEV). Especially in areas where several flaviviruses co-circulate or in the context of vaccination e.g. against YFV or TBEV, this severely complicates diagnosis and surveillance. Most flavivirus cross-reactive antibodies are produced against the highly conserved fusion loop (FL) domain in the viral envelope (E) protein. We generated insect-cell derived recombinant E-proteins of the four DENV-serotypes which contain point mutations in the FL domain. By using specific mixtures of these mutant antigens, cross-reactivity against heterologous flaviviruses was strongly reduced, enabling sensitive and specific diagnosis of the DENV-infected serum samples in IgG and IgM-measurements. These results have indications for the development of serological DENV-tests with improved specificity.

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Related in: MedlinePlus

300 ng per well of DENV-2 Ewt (A) and Equad (B) and 160 ng per well of a DENV 1–4 Equad mixture (C) were tested with DENV- WNV- and TBEV- infected and YFV-vaccinated sera compared to negative (NEG) samples in an IgG-ELISA (n = number of individuals).Bottom and top of the boxes are the first and third quartiles. The median signal is depicted as a line inside the box. Whiskers represent the 9th and the 91st percentile. Outliers in B and C are marked with numbers (1–5). Measurements were performed in duplicates in at least two independent experiments.
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pntd.0004218.g004: 300 ng per well of DENV-2 Ewt (A) and Equad (B) and 160 ng per well of a DENV 1–4 Equad mixture (C) were tested with DENV- WNV- and TBEV- infected and YFV-vaccinated sera compared to negative (NEG) samples in an IgG-ELISA (n = number of individuals).Bottom and top of the boxes are the first and third quartiles. The median signal is depicted as a line inside the box. Whiskers represent the 9th and the 91st percentile. Outliers in B and C are marked with numbers (1–5). Measurements were performed in duplicates in at least two independent experiments.

Mentions: First, sera from 38 DENV-, 13 WNV-, 19 TBEV- infected, 8 YFV vaccinated and 7 uninfected individuals were incubated with DENV-2 Ewt protein (Fig 4A). Strong binding for DENV-positive samples was observed (mean absorbance value 2.36), however, several WNV- and TBEV- positive sera (mean values 1.26 and 0.82, respectively) showed cross-reactive signals which were in the range of DENV-infected sera. Samples from YFV-vaccinated individuals showed a reduced cross-reactivity in comparison to WNV- and TBEV-infected serum samples. When using the DENV-2 Equad protein, the cross-reactivities of WNV- and TBEV-infected samples were significantly reduced (mean values 0.106 and 0.160, respectively) (Fig 4B). In addition, the signal intensities of the DENV-positive samples changed. The mean value was reduced to 1.9 and the overall signal range increased from 2.1 to 2.4 in comparison to DENV-2 Ewt. Next, a mixture of the Equad proteins of all four serotypes was prepared (DENV 1–4 Equad Mix). Based on the titration curves (Fig 3) a total amount of 160 ng was found to be optimal in specificity and sensitivity, consisting of 50 ng of DENV 1–3 Equad respectively and 10 ng of DENV-4 Equad. The proportional amount of DENV-4 Equad was reduced compared to the other serotypes because it elicited a higher cross-reactivity with heterologous flaviviral sera (Fig 3D). The mixture was tested with the same serum panel. This resulted in an increase of the 25th percentile from 1.36 to 1.6, demonstrating a higher sensitivity compared to using only DENV-2 Equad. At the same time, cross-reactivity of WNV- and TBEV-positive sera was even further reduced (mean values 0.063 and 0.084, respectively) showing a higher specificity of the test in comparison to using DENV-2 Equad only. The stasticial analysis of the data is shown in S2 Table. The five DENV-positive sera detected as outliers on the lower end of the DENV-panel using the mutant antigens in Fig 4 were numbered. Whereas sera 1, 2 (both unknown DENV serotype infections) and 3 (DENV-3 infection) showed decreased binding to the DENV-2 Equad-protein as compared to the wild type, their signals increased when using the DENV 1–4 Equad mix. Also the signals of samples 4 and 5 (DENV-1 and -2 infections, respectively) decreased with the DENV-2 Equad compared to the wild type but were even lower with the mutant mixture (Fig 4C).


Recombinant Envelope-Proteins with Mutations in the Conserved Fusion Loop Allow Specific Serological Diagnosis of Dengue-Infections.

Rockstroh A, Barzon L, Pacenti M, Palù G, Niedrig M, Ulbert S - PLoS Negl Trop Dis (2015)

300 ng per well of DENV-2 Ewt (A) and Equad (B) and 160 ng per well of a DENV 1–4 Equad mixture (C) were tested with DENV- WNV- and TBEV- infected and YFV-vaccinated sera compared to negative (NEG) samples in an IgG-ELISA (n = number of individuals).Bottom and top of the boxes are the first and third quartiles. The median signal is depicted as a line inside the box. Whiskers represent the 9th and the 91st percentile. Outliers in B and C are marked with numbers (1–5). Measurements were performed in duplicates in at least two independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4643925&req=5

pntd.0004218.g004: 300 ng per well of DENV-2 Ewt (A) and Equad (B) and 160 ng per well of a DENV 1–4 Equad mixture (C) were tested with DENV- WNV- and TBEV- infected and YFV-vaccinated sera compared to negative (NEG) samples in an IgG-ELISA (n = number of individuals).Bottom and top of the boxes are the first and third quartiles. The median signal is depicted as a line inside the box. Whiskers represent the 9th and the 91st percentile. Outliers in B and C are marked with numbers (1–5). Measurements were performed in duplicates in at least two independent experiments.
Mentions: First, sera from 38 DENV-, 13 WNV-, 19 TBEV- infected, 8 YFV vaccinated and 7 uninfected individuals were incubated with DENV-2 Ewt protein (Fig 4A). Strong binding for DENV-positive samples was observed (mean absorbance value 2.36), however, several WNV- and TBEV- positive sera (mean values 1.26 and 0.82, respectively) showed cross-reactive signals which were in the range of DENV-infected sera. Samples from YFV-vaccinated individuals showed a reduced cross-reactivity in comparison to WNV- and TBEV-infected serum samples. When using the DENV-2 Equad protein, the cross-reactivities of WNV- and TBEV-infected samples were significantly reduced (mean values 0.106 and 0.160, respectively) (Fig 4B). In addition, the signal intensities of the DENV-positive samples changed. The mean value was reduced to 1.9 and the overall signal range increased from 2.1 to 2.4 in comparison to DENV-2 Ewt. Next, a mixture of the Equad proteins of all four serotypes was prepared (DENV 1–4 Equad Mix). Based on the titration curves (Fig 3) a total amount of 160 ng was found to be optimal in specificity and sensitivity, consisting of 50 ng of DENV 1–3 Equad respectively and 10 ng of DENV-4 Equad. The proportional amount of DENV-4 Equad was reduced compared to the other serotypes because it elicited a higher cross-reactivity with heterologous flaviviral sera (Fig 3D). The mixture was tested with the same serum panel. This resulted in an increase of the 25th percentile from 1.36 to 1.6, demonstrating a higher sensitivity compared to using only DENV-2 Equad. At the same time, cross-reactivity of WNV- and TBEV-positive sera was even further reduced (mean values 0.063 and 0.084, respectively) showing a higher specificity of the test in comparison to using DENV-2 Equad only. The stasticial analysis of the data is shown in S2 Table. The five DENV-positive sera detected as outliers on the lower end of the DENV-panel using the mutant antigens in Fig 4 were numbered. Whereas sera 1, 2 (both unknown DENV serotype infections) and 3 (DENV-3 infection) showed decreased binding to the DENV-2 Equad-protein as compared to the wild type, their signals increased when using the DENV 1–4 Equad mix. Also the signals of samples 4 and 5 (DENV-1 and -2 infections, respectively) decreased with the DENV-2 Equad compared to the wild type but were even lower with the mutant mixture (Fig 4C).

Bottom Line: We generated insect-cell derived recombinant E-proteins of the four DENV-serotypes which contain point mutations in the FL domain.By using specific mixtures of these mutant antigens, cross-reactivity against heterologous flaviviruses was strongly reduced, enabling sensitive and specific diagnosis of the DENV-infected serum samples in IgG and IgM-measurements.These results have indications for the development of serological DENV-tests with improved specificity.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Fraunhofer Institute for Cell Therapy and Immunology, Leipzig, Germany.

ABSTRACT
Dengue virus (DENV) is a mosquito-borne flavivirus and a major international public health concern in many tropical and sub-tropical areas worldwide. DENV is divided into four major serotypes, and infection with one serotype leads to immunity against the same, but not the other serotypes. The specific diagnosis of DENV-infections via antibody-detection is problematic due to the high degree of cross-reactivity displayed by antibodies against related flaviviruses, such as West Nile virus (WNV), Yellow Fever virus (YFV) or Tick-borne encephalitis virus (TBEV). Especially in areas where several flaviviruses co-circulate or in the context of vaccination e.g. against YFV or TBEV, this severely complicates diagnosis and surveillance. Most flavivirus cross-reactive antibodies are produced against the highly conserved fusion loop (FL) domain in the viral envelope (E) protein. We generated insect-cell derived recombinant E-proteins of the four DENV-serotypes which contain point mutations in the FL domain. By using specific mixtures of these mutant antigens, cross-reactivity against heterologous flaviviruses was strongly reduced, enabling sensitive and specific diagnosis of the DENV-infected serum samples in IgG and IgM-measurements. These results have indications for the development of serological DENV-tests with improved specificity.

Show MeSH
Related in: MedlinePlus