Performance assessment and validation of a plaque reduction neutralization test (PRNT) in support to yellow fever diagnostic and vaccine clinical trials

Dia, Moussa; Bob, Ndeye Sakha; Talla, Cheikh; Dupressoír, Anne; Escadafal, Camille; Thiam, Mareme Seye; Diallo, Amadou; Ndiaye, Oumar; Heraud, Jean Michel; Faye, Oumar; Sall, Amadou Alpha; Faye, Ousmane; Fall, Gamou

doi:10.1002/jmv.28700

Cited by 6 publications

(4 citation statements)

References 29 publications

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“…No positive correlation was detected for the other three viral serotypes DENV2 ( r = 0.36, p = 0.29), DENV3 ( r = −0.42, p = 0.41) and DENV4 ( r = −0.17, p = 0.62). A similar analysis but using PRNT90, which is considered of higher specificity than PRNT50 in the serodiagnostic of Flaviviruses, 26 Spearman coefficients for the correlation analysis between serotype‐dependent neutralization capacity and anti‐DIII titers are statistically significant for sera from early convalescence (File : Figure ). However, for the sera from late convalescence there is a significant decreased in the correlation of these two determinations, and Spearman correlation coefficients are not statistically significant (File : Figure ).…”

Section: Resultsmentioning

confidence: 99%

“…The 24 sera were tittered by ELISA to determine the neutralizing capacity of each serum to compare it with the anti-DIII IgG titer (File S1: Figure S2; Table S2). A similar analysis but using PRNT90, which is considered of higher specificity than PRNT50 in the serodiagnostic of Flaviviruses, 26 Spearman coefficients for the correlation analysis between serotype-dependent neutralization capacity and anti-DIII titers are statistically significant for sera from early convalescence (File S1:…”

Section: Calculations and Statistics Proceduresmentioning

confidence: 99%

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Dominant epitopes of cross‐reactive anti‐domain III human antibody response change from early to late convalescence of infection with dengue virus

González‐Lodeiro,

Martín Dunn,

Martín Prieto

et al. 2024

Journal of Medical Virology

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Cross‐neutralizing activity of human antibody response against Dengue virus complex (DENV) changes importantly over time. Domain III (DIII) of the envelope protein of DENV elicits a potently neutralizing and mostly type‐specific IgG response. We used sera from 24 individuals from early‐ or late convalescence of DENV1 infection to investigate the evolution of anti‐DIII human IgG with the time lapse since the infection. We evaluated the correlation between the serotype‐specific reactivity against recombinant DIII proteins and the neutralization capacity against the four serotypes, and examined its behavior with the time of convalescence. Also, we use a library of 71 alanine mutants of surface‐exposed amino acid residues to investigate the dominant epitopes. In early convalescence anti‐DIII titers and potency of virus neutralization were positively associated with correlation coefficients from 0.82 to 1.0 for the four serotypes. For late convalescence, a positive correlation (r = 0.69) was found only for DENV1. The dominant epitope of the type‐specific response is centered in the FG‐loop (G383, E384, and K385) and includes most of the lateral ridge. The dominant epitope of the anti‐DIII cross‐reactive IgG in secondary infections shifts from the A‐strand during early convalescence to a site centered in residues E314‐H317 of the AB‐loop and I352‐E368 of the DI/DIII interface, in late convalescence. An immunoassay based on the detection of IgG anti‐DIII response can be implemented for detection of infecting serotype in diagnosis of DENV infection, either primary or secondary. Human dominant epitopes of the cross‐reactive circulating antibodies change with time of convalescence.

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Section: Resultsmentioning

confidence: 99%

Section: Calculations and Statistics Proceduresmentioning

confidence: 99%

Dominant epitopes of cross‐reactive anti‐domain III human antibody response change from early to late convalescence of infection with dengue virus

González‐Lodeiro,

Martín Dunn,

Martín Prieto

et al. 2024

Journal of Medical Virology

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“…A recent study assessed the intra- and inter-assay precisions, dilutability, specificity, and lower limit of quantification (LLOQ) using international standard YF serum and sera from vaccinees and human specimens collected through YF surveillance. The results showed that the PRNT assay showed 100% of intra-assay precision, 95.6% of inter-assay precision, 100% of specificity, 100% of LLOQ, and 95.3% of dilutability [ 52 ]. The main limitation in PRNT is that it cannot differentiate the isotype of neutralizing antibodies.…”

Section: Discussionmentioning

confidence: 99%

Oropouche Virus Exposure in Febrile Patients during Chikungunya Virus Introduction in the State of Amapá, Amazon Region, Brazil

de Lima,

Dias,

de Souza

et al. 2024

Pathogens

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Oropouche orthobunyavirus (OROV) is an arbovirus transmitted by midges that has been involved in outbreaks throughout Central and South America. In Brazil, human cases have been historically concentrated in the northern region of the country. Oropouche fever in humans range from mild clinical signs to rare neurological events, and is considered a neglected tropical disease in Brazil. Due to the clinical similarities to other arboviruses, such as chikungunya and dengue viruses, OROV infections are likely to be underreported. Chikungunya virus (CHIKV) cases in Brazil were first recognized in 2014 in the states of Amapá and Bahia in the north and northeast regions, respectively. Both OROV and CHIKV cause nonspecific symptoms, making clinical diagnosis difficult in a scenario of arbovirus cocirculation. Aiming to investigate OROV transmission during the CHIKV introduction in the state of Amapá located in the Brazilian Amazon, we conducted a retrospective molecular (RT-qPCR) and serological investigation in febrile cases (N = 166) collected between August 2014 and May 2015. All acute serum samples were negative for OROV RNA using RT-qPCR. However, neutralizing antibodies for OROV were detected using a plaque reduction neutralization test (PRNT90) in 10.24% (17/166) of the patients, with neutralizing antibody titers ranging from 20 to ≥640, suggesting the previous exposure of patients to OROV. Regarding CHIKV, recent exposure was confirmed by the detection of CHIKV RNA in 20.25% (33/163) of the patients and by the detection of anti-CHIKV IgM in 28.57% (44/154) of the patients. The additional detection of anti-CHIKV IgG in 12.58% (19/151) of the febrile patients suggests that some individuals had been previously exposed to CHIKV. Whether the OROV exposure reported here occurred prior or during the CHIKV circulation in Amapá, is unknown, but because those arboviral infections share similar clinical signs and symptoms, a silent circulation of enzootic arboviruses during the introduction of exotic arboviruses may occur, and highlights the importance of syndromic cases’ surveillance to arboviruses in Brazil.

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“…Assay validity criteria included (i) the absence of PFU in the cell control well; (ii) neutralization by positive control sera, and no neutralization by negative control sera; (iii) the observed number of PFU for each triplicate (per each dilution of each mock or infected sample tested) in an experiment were within a 3-fold difference of the median PFU count (Dia et al, 2023; Timiryasova et al, 2013) and; (iv) the observation of 70 to 180 PFU in virus control wells. The median percentage of viral neutralization was estimated for each experiment comparing the median number of PFUs in virus wells to those in wells where the virus was incubated with liver homogenates, positive and negative controls.…”

Section: Methodsmentioning

confidence: 99%

Detection of neutralizing antibodies against arboviruses from liver homogenates

Costa,

Arruda,

Oliveira

et al. 2024

Preprint

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Yellow fever virus (YFV) circulates in a sylvatic cycle between non-human primates (NHPs) and arboreal mosquitoes in Brazil. Passive monitoring of ill or deceased NHPs is a key component of the Brazilian yellow fever (YF) surveillance program. Samples from NHPs carcasses are usually suitable for molecular tests but not for serological assays. As an alternative to the conventional plaque reduction neutralization test (PRNT) based on sera, we tested the utility of liver homogenates from experimentally infected (YFV, Mayaro virus [MAYV], chikungunya virus [CHIKV], or mock) mice to quantify PRNTs. Although homogenates from mock-infected mice showed a low level of nonspecific virus neutralization against YFV, MAYV or CHIKV, homogenates from YFV-, MAYV- and CHIKV-infected mice demonstrated significantly higher levels of virus neutralization compared to controls. Receiver operating characteristic (ROC) curves analyses were performed using the median neutralization values of three technical replicates for each infected group separately or collectively. Results showed scores ≥0.97 (95% CI ≥ 0.89-1.0) for the area under the curve at dilutions 1:20 to 1:80, suggesting that median virus neutralization values effectively differentiated YFV-, MAYV-, or CHIKV-infected groups from controls. Liver homogenates obtained from 25 NHPs carcasses (collected during the 2017 YF outbreak in Brazil) were also tested using the adapted PRNT as well as rapid anti-YFV IgM immunochromatographic tests. Neutralization activity was detected in six NHPs samples that were also positive by PCR and anti-YFV IgM tests and one sample that tested negative by PCR and IgM test. Our results demonstrate the feasibility of using liver homogenates as an alternative approach for serological investigation in viral epidemiologic surveillance.

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Performance assessment and validation of a plaque reduction neutralization test (PRNT) in support to yellow fever diagnostic and vaccine clinical trials

Cited by 6 publications

References 29 publications

Dominant epitopes of cross‐reactive anti‐domain III human antibody response change from early to late convalescence of infection with dengue virus

Dominant epitopes of cross‐reactive anti‐domain III human antibody response change from early to late convalescence of infection with dengue virus

Oropouche Virus Exposure in Febrile Patients during Chikungunya Virus Introduction in the State of Amapá, Amazon Region, Brazil

Detection of neutralizing antibodies against arboviruses from liver homogenates

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