Open Access

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

  • Chukiat Sirivichayakul1Email author,
  • Arunee Sabchareon1,
  • Kriengsak Limkittikul1 and
  • Sutee Yoksan2
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. 10.1089/vim.2008.0007View 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. 10.1016/j.vaccine.2009.12.080PubMedView 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. 10.1016/j.vaccine.2011.01.058PubMedView 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. 10.4161/hv.20071PubMedView 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. 10.1016/S0264-410X(00)00105-5PubMedView 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. 10.1097/01.inf.0000109289.55856.27PubMedView Article
  7. WHO: Dengue: guidelines for diagnosis, treatment, prevention and control. 2009.http://whqlibdoc.who.int/publications/2009/9789241547871_eng.pdf
  8. Guy B, Saville M, Lang J: Development of Sanofi Pasteur tetravalent dengue vaccine. Hum Vaccin 2010,6(9):696-705. 10.4161/hv.6.9.12739View 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. 10.1371/journal.pntd.0001732PubMedPubMed CentralView 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. 10.1086/382280PubMedView 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. 10.1016/S0140-6736(12)61428-7PubMedView 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. 10.1128/JVI.00792-06PubMedPubMed CentralView 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. 10.1371/journal.pntd.0001536PubMedPubMed CentralView 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. 10.4269/ajtmh.2009.08-0625PubMedPubMed CentralView 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. 10.1186/1471-2334-12-233PubMedPubMed CentralView 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

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