Plaque reduction neutralization antibody test does not accurately predict protection against dengue infection in Ratchaburi cohort, Thailand

  • Chukiat Sirivichayakul2Email author,

    Affiliated with

    • Arunee Sabchareon2,

      Affiliated with

      • Kriengsak Limkittikul2 and

        Affiliated with

        • Sutee Yoksan2

          Affiliated with

          Virology Journal201411:48

          DOI: 10.1186/1743-422X-11-48

          Received: 22 November 2013

          Accepted: 7 March 2014

          Published: 12 March 2014

          Abstract

          Background

          The plaque reduction neutralization test (PRNT) is currently the best and most widely accepted approach to measuring virus-neutralizing and protective antibodies to dengue virus, and in assessing the immunogenicity of a dengue vaccine. However, the correlation between presence of dengue-neutralizing antibody and protection from infection is not absolute.

          Findings

          In a cohort study in Ratchaburi Province, Thailand, 48 subjects with serologically confirmed symptomatic dengue infection were tested for pre-existing dengue neutralizing antibody using PRNT. Nine subjects had quite high pre-existing PRNT50 titers (titer >90) to subsequent infecting dengue serotypes, but still had symptomatic infections.

          Conclusion

          This report provides evidence that PRNT may not be a good test for predicting protection against subsequent dengue infection.

          Keywords

          Dengue Plaque reduction neutralization test Neutralizing antibody

          Findings

          Background

          The plaque reduction neutralization test (PRNT) is a method for measuring antibodies that neutralize and prevent virions from infecting cultured cells. It is currently the most virus-specific serological test among the flaviviruses, and serotype-specific test among the dengue viruses [1]. PRNT has been widely used in assessing the protective neutralizing antibody response for Japanese-encephalitis vaccines [24].

          For dengue, PRNT is the best and most widely accepted approach to measuring virus-neutralizing and protective antibodies [1], and assessing the immunogenicity of dengue vaccine [58]. However, the correlation between the presence of virus-neutralizing antibody and protection from infection is not absolute. This report aims to provide additional data on the correlation of pre-existing dengue-neutralizing antibody and protection from subsequent dengue infection.

          Methods

          In the cohort study conducted among school children in Ratchaburi Province, Thailand [9], we prospectively collected serum samples annually from all subjects. Acute and convalescent serum samples were also collected from each febrile subject, irrespective of clinical diagnosis. Clinical diagnosis was performed by a pediatrician who was unaware of the dengue diagnostic test results. Clinical diagnoses of dengue fever (DF), dengue hemorrhagic fever (DHF), and DHF severity were made using the WHO criteria (1997) [10]. All blood samples were drawn into serum separator tubes, allowed to clot at room temperature for 1–2 hours, then stored at 4°C. Sera were separated into aliquots within 24 hours and stored at −70°C until laboratory testing. Dengue diagnostic testing was performed at the Center for Vaccine Development, Institute of Molecular Biosciences, Mahidol University, Salaya, Nakhonpathom, Thailand (CVD). Acute and convalescent sera were tested for dengue-virus-specific IgM/IgG by enzyme-linked immunosorbent assay (ELISA) using slightly modified method from that described previously [11]. The sensitivity of this test was 97% in paired sera [11]. An IgM anti-dengue level ≥ 1 unit in acute serum, or seroconversion of either IgM or IgG in paired sera, was considered indicative of acute dengue infection. Primary dengue infection was diagnosed when the IgM:IgG ratio was >1:1.8. Serum samples from acute dengue cases were tested for dengue-virus serotype by inoculation into Toxorhynchites splendens mosquitoes with immunofluorescence detection and serotyping [12].

          We randomly selected 48 subjects with acute dengue infection in the year 2006. Pre-infection sera were retrieved from the previous annual serum samples and tested for pre-existing dengue- and Japanese encephalitis-neutralizing antibody using PRNT, as described by Russell et al.[13]. In the tests, conducted at the CVD, monkey kidney-derived LLC-MK2 cells were used for virus production and PRNT. The dengue viruses (D) used in the assay were D1 (16007), D2 (16681), D3 (16562), and D4 (1036). LLC-MK2 cells were seeded in 6-well plates at 1 × 105 cells/well, and incubated for 6–8 days. Neutralizing sera were diluted to 1:5, followed by ten-fold serial dilutions using phosphate buffer solution (PBS) pH 7.5 with 30% fetal bovine serum, mixed with virus (for a final starting dilution of 1:10), and incubated. Following infection, cells were overlaid with 3.0% carboxymethyl cellulose with neutral red added. Plaques were visualized and counted after cultivation for 7 days. Data were interpreted using the Probit model with the SPSS program, and PRNT endpoint titers were expressed as the reciprocal of the last serum dilution. The PRNT titer was calculated based on a 50% reduction in plaque count (PRNT50).

          Results

          Tables 1, 2, 3 show the pre-existing dengue PRNT50 titers in the sera of subjects in February 2006, date of subsequent dengue illness, clinical diagnosis, and the serotype isolated. Among 48 subjects with serologically confirmed dengue infection, dengue viruses could be identified in 31 (64.6%) subjects, comprising 16 D1; 1 D2; 3 D3; and 11 D4. Only 5 (10.4%) subjects had primary infections.
          Table 1

          Pre-existing PRNT50 titer and subsequent dengue infection in subjects with low titer (<90) to subsequent infecting serotype

          Subject code

          PRNT50 titer (Feb 2006)

          Date of illness

          Clinical diagnosis

          ELISA test resulta

          Serotype isolated

          D1

          D2

          D3

          D4

          JE

          03-146

          <10

          <10

          <10

          <10

          155

          11/10/2006

          DF

          Secondary

          D1

          05-119

          <10

          <10

          <10

          <10

          235

          9/6/2006

          Pharyngitis

          Secondary

          D1

          05-181

          <10

          <10

          <10

          <10

          <10

          21/8/2006

          DF

          Primary

          D1

          05-310

          10

          <10

          167

          <10

          250

          21/4/2006

          DF

          Secondary

          D1

          07-479

          <10

          <10

          <10

          <10

          29

          23/7/2006

          DF

          Secondary

          D1

          07-383

          13

          <10

          <10

          <10

          <10

          10/9/2006

          DF

          Primary

          D1

          04-276

          13

          <10

          <10

          <10

          <10

          13/3/2006

          DF

          Primary

          D1

          05-357

          40

          29

          27

          <10

          303

          10/10/2006

          Bronchitis

          Secondary

          D1

          05-002

          50

          <10

          <10

          <10

          396

          25/11/2006

          DF

          Secondary

          D1

          03-097

          75

          1134

          24

          20

          1307

          7/10/2006

          DF

          Secondary

          D1

          05-339

          <10

          <10

          <10

          <10

          503

          28/8/2006

          Pharyngitis

          Secondary

          D2

          04-322

          17

          <10

          <10

          <10

          165

          9/11/2006

          DF

          Secondary

          D3

          04-325

          <10

          <10

          <10

          <10

          685

          23/10/2006

          DHF gr2

          Secondary

          D3

          02-189

          <10

          10

          12

          <10

          <10

          6/7/2006

          DF

          Primary

          D3

          01-227

          49

          <10

          <10

          <10

          590

          19/2/2006

          Pharyngitis

          Secondary

          D4

          01-254

          12450

          3348

          32

          <10

          78

          28/2/2006

          AGE

          Secondary

          D4

          01-384

          <10

          <10

          <10

          <10

          92

          19/12/2006

          Pharyngitis

          Secondary

          D4

          06-164

          210

          540

          12040

          21

          76

          30/8/2006

          DF

          Secondary

          D4

          01-124

          228

          135

          516

          39

          28

          31/3/2006

          DF

          Secondary

          D4

          05-257

          3141

          194

          272

          41

          726

          15/10/2006

          Common cold

          Secondary

          D4

          01-224

          195

          2901

          220

          50

          802

          9/3/2006

          DHF gr1

          Secondary

          D4

          05-378

          6238

          2204

          676

          75

          43

          28/7/2006

          DF

          Secondary

          D4

          aELISA result showed either primary or secondary infection.

          AGE: acute gastroenteritis; D: dengue virus; DF: dengue fever; DHF: dengue hemorrhagic fever; gr: grade; JE: Japanese encephalitis virus; PRNT50: 50% plaque reduction neutralization.

          Table 2

          Pre-existing PRNT50 titer and subsequent dengue infection in subjects with high titer (>90) to subsequent infecting serotype

          Subject code

          PRNT50 titer (Feb 2006)

          Date of illness

          Clinical diagnosis

          ELISA test resulta

          Serotype isolated

          D1

          D2

          D3

          D4

          JE

          06-043

          121

          224

          83

          <10

          11

          14/8/2006

          DF

          Secondary

          D1

          05-021

          133

          <10

          <10

          <10

          72

          26/4/2006

          DF

          Secondary

          D1

          05-074

          173

          1136

          73

          31

          503

          9/5/2006

          DF

          Secondary

          D1

          06-082

          317

          <10

          18

          <10

          760

          14/6/2006

          Viral infection

          Secondary

          D1

          01-286

          581

          <10

          <10

          <10

          <10

          19/4/2006

          DF

          Secondary

          D1

          01-141

          1848

          821

          4521

          328

          738

          6/11/2006

          Pharyngitis

          Secondary

          D1

          07-119

          5291

          84

          332

          98

          207

          28/7/2006

          DF

          Secondary

          D4

          05-244

          4916

          1305

          1006

          145

          393

          21/9/2006

          DF

          Secondary

          D4

          04-378

          11682

          853

          417

          261

          5615

          5/7/2006

          DF

          Secondary

          D4

          aELISA result showed either primary or secondary infection.

          D: dengue virus; DF: dengue fever; JE: Japanese encephalitis virus; PRNT50: 50% plaque reduction neutralization.

          Table 3

          Pre-existing PRNT50 titer and subsequent dengue infection among subjects whose subsequent infecting serotypes could not be identified

          Subject code

          PRNT50 titer (Feb 2006)

          Date of illness

          Clinical diagnosis

          ELISA test resulta

          D1

          D2

          D3

          D4

          JE

          01-177

          537

          218

          619

          74

          14

          1/6/2006

          Pharyngitis

          Secondary

          01-437

          473

          297

          1283

          <10

          75

          9/9/2006

          Influenza

          Secondary

          01-456

          14105

          2851

          7872

          235

          594

          27/6/2006

          DF

          Secondary

          01-559

          393

          1606

          249

          65

          4987

          14/8/2006

          viral infection

          Secondary

          02-246

          <10

          <10

          16

          <10

          2201

          3/5/2006

          DF

          Secondary

          02-434

          <10

          <10

          <10

          <10

          1326

          31/3/2006

          Pharyngitis

          Secondary

          02-453

          172

          2053

          126

          53

          14587

          10/3/2006

          DHF gr3

          Secondary

          03-021

          867

          <10

          1026

          <10

          <10

          29/11/2006

          DHF gr1

          Secondary

          05-072

          351

          36

          <10

          174

          140

          1/12/2006

          Viral infection

          Secondary

          05-0112

          <10

          <10

          <10

          <10

          <10

          2/12/2006

          URI

          Primary

          05-209

          310

          48

          753

          14

          76

          29/7/2006

          DHF gr1

          Secondary

          05-239

          3257

          8199

          111

          40

          919

          24/4/2006

          DHF gr1

          Secondary

          05-358

          249

          247

          3934

          34

          267

          10/6/2006

          Viral infection

          Secondary

          06-070

          236

          2687

          10649

          <10

          94

          16/6/2006

          Pharyngitis

          Secondary

          06-124

          472

          5074

          3943

          29

          164

          27/8/2006

          DHF gr1

          Secondary

          06-192

          10

          <10

          <10

          <10

          42

          26/8/2006

          Pharyngitis

          Secondary

          07-310

          137

          43

          23

          198

          21

          7/8/2006

          Influenza

          Secondary

          aThe ELISA result showed either primary or secondary infection.

          D: dengue virus; DF: dengue fever; DHF: dengue hemorrhagic fever; gr: grade; JE: Japanese encephalitis virus; PRNT50: 50% plaque reduction neutralization.

          Of 31 subjects whose infecting dengue serotypes were identified, 14 (45.2%) had pre-existing PRNT50 titers to the infecting serotype < 20. Eight subjects (25.8%) had pre-existing PRNT50 titers to the infecting serotype of between 21 and 75 (Table 1).

          Interestingly, nine (29.0%) subjects had pre-existing PRNT50 titers of > 90 to the subsequent infecting serotypes. Six subjects with D1 infections had pre-existing PRNT50 titers to D1, which ranged from 121 to 1848; geometric mean value 313; median value 245. Two subjects (subjects 01–286 and 05–021) had PRNT50 profiles suggesting previous primary D1 infection, but had secondary symptomatic infections with the same serotype. Three subjects with D4 infections had pre-existing PRNT50 titers to D4, ranging between 98 to 261, geometric mean value 154 (Table 2).

          It is worth noting that many subjects (e.g. subjects 01–456 and 04–378) had pre-existing PRNT50 profiles suggesting secondary dengue infection, but still had symptomatic infections, which were probably tertiary infections (Tables 3 and 2, respectively).

          Discussion

          Dengue viruses comprise 4 serotypes. Infection with one dengue serotype elicits lifelong homotypic immunity, but only short-lived immunity for heterotypic serotypes [14]. Dengue neutralizing antibody has been believed to represent protection against dengue, and the PRNT test has been widely used to measure this neutralizing antibody. Numerous vaccine immunogenicity assessment laboratories consider a seropositive threshold to be 10 [1] and since four of the subjects in this report had PRNT50 titers of 10–13, we arbitrarily divided the subjects into 3 groups, i.e. titer <20, 20–90, and >90. We found that 17 (54.8%) and 9 (29.0%) of 31 subjects had pre-existing PRNT50 titers >20 and >90, respectively, to the subsequent infecting dengue serotype. These data provide partial insight into the correlation between PRNT50 titer and disease protection. This is very important, because PRNT titer is considered an important marker of protection in the development of dengue vaccines. These data are perhaps the most relevant available data, as more valid data on the correlation between pre-existing PRNT50 titer and disease protection in humans requires human challenge with dengue virus, which may not be possible due to ethical issues. This report raises some inconsistencies with our previous understandings. First, the finding in 2 subjects (subjects 01–286 and 05–021 [Table 2]) suggests that previous D1 infection may not induce protection to subsequent symptomatic homotypic dengue infection. Second, a quite high pre-existing PRNT50 titer (>90) may not be able to protect against subsequent symptomatic infection from the respective dengue serotype.

          In a cohort study in Thailand, Endy et al. [15] also found that pre-existing neutralizing antibody directed against infecting dengue serotype (titer >10) was detected in 36%, 67%, and 46% of D3, D2, and D1 infections, respectively. Moreover, only a pre-existing PRNT50 > 100 against the reference D3 strain was associated with milder severity of disease, but not in D2 and D1. This is further confirmed by the finding in a phase-2b dengue-vaccine trial among Thai children that the tetravalent live-attenuated dengue vaccine had a low level of efficacy against D2, despite its high immunogenicity [16].

          There are some possible explanations for the lack of a definite correlation between PRNT50 titer and protection from subsequent dengue infection. One possible explanation is that in our PRNT, we used LLC-MK2 cells, which are not FcγR-expressing cells. In the absence of FcγR, dengue virus-antibody complexes are not able to infect the cells, while these complexes are taken up more efficiently by FcγR-expressing cells, and are still infectious [17]. This is supported by the study of Moi et al.[18], who found that 11 of 18 serum samples from patients with acute secondary dengue infection demonstrated neutralizing activity to the infecting serotype, determined using FcγR-negative BHK cells, but not when determined using FcγR-expressing cells. Another explanation is that the protective PRNT50 titer for dengue may be much higher than the titer of 10, defined for Japanese encephalitis virus, and the protective level of dengue neutralizing antibody should be more accurately defined. This study revealed that subjects with pre-existing PRNT50 titer of up to 1848 against D1, and 261 against D4, still had symptomatic infections due to the respective serotypes, suggesting the protective level should be higher and may differ for different serotypes. Nevertheless, defining the protective-level cut-off point is difficult and challenging. A very large cohort study and long-term follow-up are needed, unless a challenge test in subjects with pre-defined PRNT levels could be conducted. Moreover, as PRNT titers vary significantly depending on testing conditions, such as virus strains, virus passage and cell type [19, 20], optimal testing conditions should be defined.

          Finally, the pre-infection PRNT50 titers are against reference dengue-virus strains. As molecular evolution among dengue viruses has been continuous [21], it may cause antigenic mismatches between the reference dengue virus strains used in the PRNT and infecting viruses, and therefore, mismatch between the pre-existing antibody and the antigen of the infecting homologous serotype. Further studies are needed to clarify these possibilities.

          It is also noted that dengue-naïve but Japanese encephalitis (JE)-immuned subjects shown by PRNT (e.g. subjects 01–384, 04–325, 07–479) showed secondary antibody response to subsequent dengue infection. One subject (subject 04–325) had DHF grade2. These pieces of evidence suggest cross-reactive antibody responses between dengue and JE.

          Declarations

          Acknowledgements

          We thank all subjects and their families for participating in this study and the staff of Ratchaburi Hospital for data collection. We also thank Mr. Paul R Adams for editing this manuscript. This study was funded by the Thai Ministry of Public Health, the Pediatric Dengue Vaccine Initiative, and the Faculty of Tropical Medicine, Mahidol University, Thailand. The funders played no role in study design, or data collection, analysis and interpretation.

          Authors’ Affiliations

          (1)
          Department of Tropical Pediatrics, Faculty of Tropical Medicine, Mahidol University
          (2)
          Center for Vaccine Development, Mahidol University

          References

          1. Roehrig JT, Hombach J, Barrett AD: Guidelines for plaque-reduction neutralization testing of human antibodies to dengue viruses. Virol Immunol 2008, 21:123–132.View Article
          2. Reisler RB, Danner DK, Gibbs PH: Immunogenicity of an inactivated Japanese encephalitis vaccine (JE-VAX) in humans over 20 years at USAMRIID: using PRNT50 as an endpoint for immunogenicity. Vaccine 2010, 28:2436–2441.PubMedView Article
          3. Eder S, Dubischar-Kastner K, Firbas C, Jelinek T, Jilma B, Kaltenboeck A, Knappik M, Kollaritsch H, Kundi M, Paulke-Korinek M, Schuller E, Klade CS: Long term immunity following a booster dose of the inactivated Japanese Encephalitis vaccine IXIARO®, IC51. Vaccine 2011, 29:2607–2612.PubMedView Article
          4. Feroldi E, Pancharoen C, Kosalaraksa P, Watanaveeradej V, Phirangkul K, Capeding MR, Boaz M, Gailhardou S, Bouckenooghe A: Single-dose, live-attenuated Japanese encephalitis vaccine in children aged 12–18 months: randomized, controlled phase 3 immunogenicity and safety trial. Hum Vaccin Immunother 2012, 8:929–937.PubMedView Article
          5. Kochel TJ, Raviprakash K, Hayes CG, Watts DM, Russell KL, Gozalo AS, Phillips IA, Ewing DF, Murphy GS, Porter KR: A dengue virus serotype-1 DNA vaccine induces virus neutralizing antibodies and provides protection from viral challenge in Aotus monkeys. Vaccine 2000, 18:3166–3173.PubMedView Article
          6. Sabchareon A, Lang J, Chanthavanich P, Yoksan S, Forrat R, Attanath P, Sirivichayakul C, Pengsaa K, Pojjaroen-Anant C, Chambonneau L, Saluzzo JF, Bhamarapravati N: Safety and immunogenicity of a three dose regimen of two tetravalent live-attenuated dengue vaccines in five- to twelve-year-old Thai children. Pediatr Infect Dis J 2004, 23:99–109.PubMedView Article
          7. WHO: Dengue: guidelines for diagnosis, treatment, prevention and control. 2009.
          8. Guy B, Saville M, Lang J: Development of Sanofi Pasteur tetravalent dengue vaccine. Hum Vaccin 2010,6(9):696–705.View Article
          9. Sabchareon A, Sirivichayakul C, Limkittikul K, Chanthavanich P, Suvannadabba S, Jiwariyavej V, Dulyachai W, Pengsaa K, Margolis HS, Letson GW: Dengue infection in children in Ratchaburi, Thailand: a cohort study. I. Epidemiology of symptomatic acute dengue infection in children, 2006–2009. PLoS Negl Trop Dis 2012, 6:e1732.PubMed CentralPubMedView Article
          10. World Health Organization: Dengue hemorrhagic fever: diagnosis, treatment, prevention and control. Geneva: WHO; 1997.
          11. Innis BL, Nisalak A, Nimmannitya S, Kusalerdchariya S, Chongswasdi V, Suntayakorn S, Puttisri P, Hoke CH: An enzyme-linked immunosorbent assay to characterize dengue infections when dengue and Japanese encephalitis co-circulate. Am J Trop Med Hyg 1989, 40:418–427.PubMed
          12. Rosen L: The use of Toxorhynchites mosquitoes to detect and propagate dengue and other arboviruses. Am J Trop Med Hyg 1981, 30:177–183.PubMed
          13. Russell PK, Nisalak A, Sukhavachana P, Vivona S: A plaque reduction test for dengue virus neutralizing antibodies. J Immunol 1967, 99:285–290.PubMed
          14. Sabin AB: Research on dengue during World War II. Am J Trop Med Hyg 1952, 1:30–50.PubMed
          15. Endy TP, Nisalak A, Chunsuttitwat S, Vaughn DW, Green S, Ennis FA, Rothman AL, Libraty DH: Relationship of preexisting dengue virus (DV) neutralizing antibody levels to viremia and severity of disease in a prospective cohort study of DV infection in Thailand. J Infect Dis 2004, 189:990–1000.PubMedView Article
          16. Sabchareon A, Wallace D, Sirivichayakul C, Limkittikul K, Chanthavanich P, Suvannadabba S, Jiwariyavej V, Dulyachai W, Pengsaa K, Wartel TA, Moureau A, Saville M, Bouckenooghe A, Viviani S, Tornieporth NG, Lang J: Protective efficacy of the recombinant, live-attenuated, CYD tetravalent dengue vaccine in Thai schoolchildren: a randomised, controlled phase 2b trial. Lancet 2012, 380:1559–1567.PubMedView Article
          17. Rodrigo WW, Jin X, Blackley SD, Rose RC, Schlesinger JJ: Differential enhancement of dengue virus immune complex infectivity mediated by signaling-competent and signaling-incompetent human Fcgamma RIA (CD64) or FcgammaRIIA (CD32). J Virol 2006, 80:10128–10138.PubMed CentralPubMedView Article
          18. Moi ML, Lim CK, Chua KB, Takasaki T, Kurane I: Dengue virus infection-enhancing activity in serum samples with neutralizing activity as determined by using FcγR-expressing cells. PLoS Negl Trop Dis 2012, 6:e1536.PubMed CentralPubMedView Article
          19. Thomas SJ, Nisalak A, Anderson KB, Libraty DH, Kalayanarooj S, Vaughn DW, Putnak R, Gibbons RV, Jarman R, Endy TP: Dengue plaque reduction neutralization test (PRNT) in primary and secondary dengue virus infection: how alteration in assay conditions impact performance. Am J Trop Med Hyg 2009, 81:825–833.PubMed CentralPubMedView Article
          20. Rainwater-Lovett K, Rodriguez-Barraquer I, Cummings DA, Lessler J: Variation in dengue virus plaque reduction neutralization testing: systematic review and pool analysis. BMC Infect Dis 2012, 12:233.PubMed CentralPubMedView Article
          21. Chen SP: Molecular evolution and epidemiology of four serotypes of dengue virus in Thailand from 1973 to 2007. Epidemiol Infect 2012, 14:1–6.

          Copyright

          © Sirivichayakul et al.; licensee BioMed Central Ltd. 2014

          This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://​creativecommons.​org/​publicdomain/​zero/​1.​0/​) applies to the data made available in this article, unless otherwise stated.

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