Previous dengue or Zika virus exposure can drive to infection enhancement or neutralisation of other flaviviruses

Diseases caused by mosquito-borne flaviviruses, such as dengue virus (DENV), West Nile virus (WNV), yellow fever virus (YFV) and more recently Zika virus (ZIKV), are responsible for thousands of deaths and millions of hospitalisations worldwide each year. 1 In 2015, ZIKV, associated with congenital Zika syndrome, emerged in Brazil. The outbreak introduced a new “player” into the complex epidemiologic Brazilian scenario where other members of the Flaviviridae family, such as dengue 1-4, were already co-circulating. Therefore, one of the biggest ongoing concerns remains therefore, whether cross-reactivity due to previous flavivirus infection or vaccination can protect from or intensify disease in ZIKV-infected patients.

Sera from DENV exposed human and animals have been demonstrated to cross-react and enhance ZIKV infection in vitro; 2 ^, 3 ^, 4 ^, 5 nevertheless such cross-reactivity could neither be confirmed experimentally in vivo 6 nor in patients with acute ZIKV and a history of previous exposure to DENV. 7

The aim of this study was to investigate whether immune-sera from DENV and ZIKV infected patients would cross-react in vitro with other Flaviviridae family members such as YFV, WNV, Rocio virus (ROCV), Ilheus virus (ILHV) and Saint Louis virus (SLEV), assessing how such cross-reactivity hinders or enhances infection.

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METHODS

Cell and viruses - BHK-21 and K562 (ATCC) cell lines were maintained respectively in minimum essential medium (MEM) and RPMI medium supplemented with 10% foetal bovine serum (FBS), 1% penicillin and 1% streptomycin and incubated at 37ºC with 5% CO₂.

In the plaque reduction neutralisation test (PRNT) and antibody-dependent enhancement (ADE) assays the following strains were used: ZIKV PE243 strain, DENV-1, -2 and -4 (isolated from infected patients in the state of Pernambuco), 8 DENV-3 (derived from an infectious clone), 9 YFV 17D strain, YFV-prM/E-WNV, YFV-prM/E-ILHV, YFV-prM/E-SLEV and YFV-PrM/E-ROCV (viral chimeras built on a YFV 17D strain backbone where prM and E genes of YFV were exchanged for those of each flavivirus) (Gil et al., unpublished observation).

Human serum samples - A total of 19 sera were obtained from two cohort studies performed at the Universidade Federal da Paraíba (UFPB) (four samples) and at the Departamento de Virologia Instituto Aggeu Magalhães (IAM), Fundação Oswaldo Cruz (15 samples). Dengue and Zika fever cases were confirmed via virus isolation and/or viral RNA detection by reverse transcriptase-polymerase chain reaction (RT-PCR) for dengue and ZIKV as previously described 10 ^- 11 and/or via positive serology for anti-DENV or anti-ZIKV IgM ELISA. 11 Seroconversion was determined by the presence of circulating specific IgG antibodies. Primary or secondary infections were determined according to the presence/absence of circulating anti-dengue IgG at the beginning of the ZIKV infection (Table I). Primary anti-DENV positive samples were collected before the 2015 ZIKV outbreak. Samples were named as follows: (i) DENV - samples were IgG positive for DENV and IgG negative for ZIKV; (ii) ZIKV - samples with active ZIKV infection and IgG negative for DENV; and (iii) ZIKV/DENV - samples with active ZIKV infection and IgG positive for DENV. Written informed consent was obtained from all subjects and the study was approved by the ethic committees of the UFPB (protocol #032/2009/CEP/HULW/UFPB) and IAM (CEP: 11/11).

TABLE I

Summary of dengue virus (DENV)- and Zica virus (ZIKV)-infected patients enrolled in the study

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F: female; IgG: immunoglobulin G; M: male; NEG: negative; POS: positive.

Enhancement of virus infection in vitro assay - An ADE assay was performed with DENV and ZIKV immune sera collected from patients to test the augmentation of infection in FcɣRII-expressing K562 cell lines. Briefly, sera were serially diluted (1:10 to 1:10240), added to virus aliquots with 8 × 10⁴ plaque forming units (PFU) for each studied virus and incubated for 20 minutes at room temperature. 1 × 10⁵ K562 cells were added per well of a 96 well plates in order to obtain an multiplicity of infection (MOI) of 0.8 and were incubated for 1 hour at 37ºC. Cells were centrifuged for 5 minutes at 450 g and 25ºC, after which supernatants were aspirated, fresh medium was added to the wells and plates were incubated for 48 hours at 37ºC, 5% CO₂. Cells were stained using a mouse monoclonal antibody anti flavivirus E protein (hybridoma D1-4G2-4-15, ATCC HB-112), prepared as tissue culture supernatant from hybridomas obtained from ATCC. The immune complex was detected with an anti-mouse Ab conjugated to FITC (Sigma, Saint Louis, MO, USA). Fluorescent cells were then quantified by flow cytometry and results were expressed as percentage of positive events. ADE assay was performed in duplicate and repeated once.

PRNT - PRNT 90 % (PRNT₉₀) assays were performed in BHK-21 cells, as previously described. 12 Serial dilutions of previously positive DENV, ZIKV and DENV/ZIKV serum (1:10 to 1:5120) were mixed with 100 PFU of selected flaviviruses (ZIKV-PE243, YFV-17D, YFV-prM/E-WNV, YFV-prM/E-ILHV, YFV-prM/E-SLEV and YFV-PrM/E-ROCV), incubated for 1 hour at 37ºC, then added to the cells and incubated at 37ºC for an additional hour. After 1 hour of incubation, MEM containing 1.5 % carboxymethyl cellulose (CMC), 5% FBS and 1% penicillin-streptomycin were added to each well and plates were incubated at 37ºC, 5 % CO₂ for 96 hours. After this incubation, plates were fixed with 10 % formaldehyde for 1 hour and subsequently stained with 1 % crystal violet for 15 minutes. Plaques were counted and the neutralisation capacity was estimated as the sera concentration causing a 90 % reduction in PFU. This assay was performed in duplicate and repeated once.

Statistical analysis - A nonparametric unpaired Mann-Whitney U test was used to compare the enhancing activity between tests and control. Results with p ≤ 0.05 were considered as statistically significant.

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RESULTS

In vitro flavivirus neutralisation by convalescent-phase DENV, ZIKV and DENV/ZIKV immune sera - Four DENV samples (P322, P122, P156, P234) presented high cross neutralisation titres for ZIKV (1:320, 1:320, 1:5120, 1:5120, respectively) (Table II). Besides, DENV positive sera also neutralised YFV [P132 (1:151), P491(1:261), P306 (1:1006)], WNV [P491 (1:252)], ILHV [P132 (1:320), P234 (1:1280)], SLEV [P156 (1:1007), P234 (1:1280)]. On the other hand, ZIKV samples neutralised ILHV [P05, P15, P67, P71 (1:20 for each)], YFV [P05, P67 (1:20 for each)], WNV [P05 (1:138), P13 (1:20), P67 (1:49), P69 (1:20)], SLEV [P71 (1:20)], ROCV [P15, P67 (1:20 each)] (Table II).

TABLE II

Summary of plaque reduction neutralisation test (PRNT) titres and selected flavivirus infection enhancement by convalescent dengue virus (DENV)- and Zica virus (ZIKV)-infected samples

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ADE: antibody dependent enhancement; IgG: immunoglobulin G; ILHV: Ilheus virus; ROCV: Rocio virus; SLEV: Saint Louis encephalitis virus; WNV: West Nile virus; YFV: yellow fever virus.

DENV positive sera presented neutralising activity for YFV and SLEV than ZIKV samples, the frequencies of positivity for YFV and SLEV were 55.6 and 33.4 percent, respectively (Table III). While the ZIKV samples neutralised WNV, ILHV and ROCV with frequencies of positivity of 40, 40 and 20 %, respectively (Table III).

TABLE III

Frequency of positivity

DENV: dengue virus; IgG: immunoglobulin G; ILHV: Ilheus virus; PRNT: plaque reduction neutralisation test; ROCV: Rocio virus; SLEV: Saint Louis encephalitis virus; WNV: West Nile virus; YFV: yellow fever virus; ZIKV: Zika virus.

DENV or ZIKV immune sera deliver flavivirus virions to FcRFcɣRII-expressing cells - We tested the ability of DENV or ZIKV convalescent sera to promote ADE in the FcRFcɣRII-expressing cell line K562. ZIKV was preincubated with titrered convalescent anti-dengue serum and selected flaviviruses (ZIKV-PE243, YFV-17D, YFV-prM/E-WNV, YFV-prM/E-ILHV, YFV-prM/E-SLEV and YFV-prM/E-ROCV) were preincubated with convalescent DENV, ZIKV and ZIKV/DENV sera and then used to infect K562 cells. In all but three cases DENV sera increased ZIKV infection with a median 1.75-fold increase of infection by ZIKV-PE243 (Fig. A).