Cross-Reacting Antibodies Enhance Dengue Virus Infection in Humans

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Science  07 May 2010:
Vol. 328, Issue 5979, pp. 745-748
DOI: 10.1126/science.1185181

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Dengue virus co-circulates as four serotypes, and sequential infections with more than one serotype are common. One hypothesis for the increased severity seen in secondary infections is antibody-dependent enhancement (ADE) leading to increased replication in Fc receptor–bearing cells. In this study, we have generated a panel of human monoclonal antibodies to dengue virus. Antibodies to the structural precursor-membrane protein (prM) form a major component of the response. These antibodies are highly cross-reactive among the dengue virus serotypes and, even at high concentrations, do not neutralize infection but potently promote ADE. We propose that the partial cleavage of prM from the viral surface reduces the density of antigen available for viral neutralization, leaving dengue viruses susceptible to ADE by antibody to prM, a finding that has implications for future vaccine design.

Dengue virus (DENV) is a mosquito-borne virus infection found in tropical and subtropical areas of the world, with an estimated 50 to 100 million infections per year (1). A sequence variation of 30 to 35% allows DENV to be divided into four serotypes, and infection with one serotype does not provide protection from infection with the other serotypes, so that secondary or sequential infections are common (2, 3). Serious complications of dengue haemorrhagic fever (DHF) are more likely during secondary versus primary infections (2, 3).

In 1977, Halstead suggested antibody-dependent enhancement (ADE) to explain severe DENV infections (4). ADE has been widely studied and results from the high sequence divergence between DENV so that antibody to the first infection may not be of sufficient avidity to neutralize a secondary infection (5). The partial cross-reactivity may cause a degree of opsonization that promotes virus uptake into Fc-bearing cells such as monocytes and macrophages—a major site of DENV replication in vivo—leading to increased virus replication.

DENV envelope contains 180 copies of the E glycoprotein, which can be found in either dimeric or trimeric conformation (6). The structural precursor-membrane protein (prM) is a 166-amino-acid protein intimately associated in a 1:1 fashion with domain II of E (7) and is believed to act as a chaperone for the folding of E and to prevent the premature fusion of virus to membranes inside the producing cell. prM contains a furin cleavage site and is cleaved into a C-terminal M portion containing a transmembrane domain that remains associated with the virus particle, and an N-terminal 91-amino-acid precursor fragment that dissociates upon release of the virus from the infected cell.

B cells from seven DENV-infected individuals (table S1) were used to produce human mAb by using the method of Traggiai (8). Culture supernatants were screened against structural antigens by using whole virus and against nonstructural protein 1 (NS1) by means of enzyme-linked immunosorbent assay (ELISA).

Of 3020 cell lines, 301 screened positive, 73% reacted to the whole-virus ELISA for structural antigens, and 27% reacted to NS1. Positive supernatants were tested for reactivity to specific DENV antigens by means of nonreducing Western blot (Fig. 1A). When the supernatants that reacted to whole DENV were tested, 78% gave a positive signal by means of Western blot, and all of these reacted to either E or prM, with no reactivity to capsid. The antibody-to-prM (anti-prM) response was substantial at 60% [95% confidence interval (CI), 67.3 to 52.2%] as compared with the response to E (40%); subgroup analysis of each of the individual cases is shown in table S2.

Fig. 1

Specificity of 301 human antibodies. (A) Western Blot of infected cell lysates (nonreduced, probed individually as single lane strips) showing reactivity of antibodies with dengue NS1, E, and prM proteins. (B to G) Cross-reactivity of human mAb within the DENV serotypes [(B) to (D)] or between the DENV group and JEV [(E) to (G)].

We next assessed the serotype specificity of the human antibodies by means of dot blot against the four viral serotypes, which showed a divergence in the cross-reactivity between the antibody-to-NS1 (anti-NS1) and structural [antibody-to-E (anti-E) and anti-prM] groups of antibodies. Half of the anti-NS1 showed limited cross-reactivity among DENV, whereas most of the antibodies directed to structural proteins showed full cross-reactivity against all virus serotypes (Fig. 1, B to D). Because these antibodies were made from secondary cases of DENV infection, we investigated primary anti-prM responses. Western blotting of DENV-infected cell lysates demonstrates that cross-reactive anti-prM responses are made during the primary infection (fig. S1), although as has been reported before, the anti-prM response is amplified after secondary infection (9).

Lastly, we tested cross-reactivity to the related flavivirus Japanese encephalitis virus (JEV), which co-circulates with DENV in some parts of southeast Asia (Fig. 1, E to G). Only 3% of the antibodies to prM cross-reacted with JEV, which is in contrast to the antibodies that recognized envelope, which showed 64% cross-reactivity. The relative specificity of anti-prM to DENV may reflect the lower sequence conservation between prM sequences (35% DENV versus JEV) as compared with that of E (50%); a comparison of sequence conservation among other members of the family flaviviridae can be found in table S3.

Six monoclonal anti-prM mAbs were produced; Western blotting showed that at least five of six react with the cleaved precursor peptide, and reactivity was lost to reduced antigen, implying that they recognize conformational epitopes (fig. S2).

In general, the antibodies to prM were unable to completely neutralize infection (Fig. 2A). Instead, neutralization plateaued between 10 and 60%, and the partial neutralization was largely cross-reactive among the four virus serotypes; the only exceptions were mAb 5F9 and 135.3, both of which showed almost 100% neutralization of DENV4 at high antibody concentrations. This partial neutralization was in contrast to results seen with pooled convalescent dengue serum (PCS) or anti-envelope monoclonal antibodies, in which neutralization approached or reached 100%.

Fig. 2

Partial neutralization but potent enhancement by human monoclonal antibodies to prM. Neutralization assays (A) and enhancement assays (B) were performed with the six human anti-prM mAbs (clones 3-147, 58/5, 2F5, 2G4, 5F9, and 135.3), mouse anti-E mAb (4G2), and purified immunoglobulin (Ig) from pooled dengue convalescent serum (PCS) and pooled non–dengue immune serum (PND) were used as controls (mean ± SE from three independent experiments). Culture supernatants from 20 anti-E and 20 anti-prM cell lines, all of which were specific to DENV2 and cross-reactive with other DENV serotypes, were assayed in neutralization (C) and ADE assays (D) to DENV2 strain 16681. Neutralization was performed by means of focus-forming assay on Vero cells by using a 1:2 dilution of supernatant, whereas ADE was performed by using a 1:100 dilution on U937 cells; infection was read with fluorescence-activated cell sorting (FACS) by using 4G2.

Next, we performed ADE assays using U937 cells as targets in which virus was preincubated with an increasing titer of antibody before addition to the Fc receptor–bearing cells. Enhancement of infection was seen with all six human monoclonal antibodies to prM with a peak of nearly a 105-fold increase (Fig. 2B), which is consistent with a report of ADE with mouse anti-prM mAb (10).

To ascertain whether the results with these six anti-prM mAbs were representative, we tested the enhancing and neutralizing capacity of a further 20 anti-prM and 20 anti-E cell lines as well as two irrelevant human antibodies (Fig. 2, C and D). None of the antibodies to prM showed a high level of neutralization (19 of 19 showed <80%), whereas 12 of 20 antibodies to E showed >90% and 6 of 20 showed 100% neutralization. All of the antibodies to prM showed ADE of 10- to 800-fold, whereas the antibodies to E showed even more variable ADE (2- to 900-fold).

The failure of antibodies to prM to fully neutralize dengue viruses with a clear plateau in the response was puzzling and suggested that the virus may exist in two populations, one that is susceptible to neutralization and another that is not. Cleavage of prM during viral maturation is believed to be a prerequisite for viral replication, which is exemplified by the very low infectivity of DENV and tick-borne encephalitis viral particles with wholly uncleaved prM (1113). In many virus preparations, prM cleavage is incomplete, and cryogenic electron microscopy (cryo-EM) yields particles that contain both full-length prM and processed M protein, suggesting that a distribution of virus maturation may be present in virus cultures (14, 15). To our knowledge, it has not been formally demonstrated whether only fully processed virus is infectious or whether the virus can contain small numbers of prM molecules at its surface and still remain infectious. The demonstration here that the human antibodies to prM can show partial neutralization implies that some prM-containing particles remain infectious.

The propensity toward incomplete cleavage of prM in DENV leads to two interesting predictions. First, the density of prM at the surface of the virus may not be high enough to allow full neutralization with most antibodies to prM. Instead, viruses with low levels of prM may be susceptible to ADE. Second, viruses that are inherently noninfectious by virtue of displaying a high density of prM may be rendered infectious through ADE.

To investigate the effect of prM cleavage on neutralization and enhancement, virus was produced in cells cultured in the presence of ammonium chloride in order to raise intracellular pH and reduce the efficiency of furin cleavage (12). ELISA assays were performed in order to measure E and prM in the virus preparation; the E assay was calibrated by plotting a standard curve by use of recombinant E protein produced in Sf9 cells. A measure of the number of potential virus particles (virus-equivalent particles), which was derived assuming each particle contained 180 copies of E and the relative density of uncleaved prM, was expressed as the ratio of prM:E. The prM:E ratio was increased by roughly 40 and 80% when virus was cultured in 10 and 20 mM ammonium chloride, respectively (Fig. 3A). As expected, the infectivity of virus produced in the presence of NH4Cl was markedly reduced from 46 to 555 virus-equivalent particles per focus-forming unit (FFU) (Fig. 3B). Although infectivity was reduced, infectious virus produced under each condition remained partially susceptible to neutralization as before, and the titration curves for virus produced in 0, 10, and 20 mM NH4Cl were similar (Fig. 3C).

Fig. 3

Anti-prM can rescue infectivity in virus containing high densities of prM, such as DENV produced in the presence of NH4Cl. (A) The density of uncleaved prM and E were measured with ELISA and expressed as the prM:E ratio. (B) Infectivity was determined in Vero cells, expressed as FFU, and the amount of total virus-equivalent particles was calculated according to the concentration of E protein measured by a sensitive sandwich ELISA. Data are presented as virus-equivalent particles/FFU ratios. (C) Neutralization assays with purified human anti-prM mAb (3-147). (D and E) Enhancement assays of U937 cells read out by FACS based intracellular staining for DENV antigens (4G2) using either (D) a constant amount of infectious virus or (E) constant number of virus particles (mean ± SE from three independent experiments).

We next tested enhancement of these viruses using either a constant amount of infectious virus—fixed FFUs (Fig. 3D)—or a constant amount of virus-equivalent particles (Fig. 3E). These results show that the relatively poorly infectious virus cultured in the presence of NH4Cl can be rendered much more infectious in the presence of enhancing antibodies to prM and indeed can be restored nearly to the level of control virus (Fig. 3E). These results were further exemplified by using virus that was produced in LoVo cells that lack functional furin and therefore produce virus with very low levels of cleaved prM (fig. S3, A to C) (13). Virus produced in LoVo cells as expected had a high prM:E ratio and very low infectivity (< 10 × 10−5 FFU per virus-equivalent particle) but infection could be enhanced in the presence of anti-prM.

Three populations of dengue virus appear to be produced: first, a population containing relatively high levels of prM that are inherently noninfectious but that can be made infectious in the presence of enhancing antibodies to prM; second, a population with an intermediate density of prM at the surface that can infect, but are susceptible to neutralization at high antibody titer; third, a population with low or absent prM at the surface that would not normally be susceptible to neutralization.

To test the relative roles of the antibodies to prM in neutralization and enhancement of primary cells, we looked at human monocytes, which are thought to be a major site of virus replication in vivo. Monocytes can be infected in the absence of antibody and, because they express Fc receptors, infection can be increased through ADE. To our surprise, human anti-prM mAbs failed to show any neutralization activity on primary monocytes and instead, even at concentrations of antibody as high as 30 μg/ml, enhanced infection from 20 to 70% (Fig. 4A) over a large range of antibody concentration.

Fig. 4

The roles of anti-prM on neutralization and enhancement of DENV infection of peripheral blood mononuclear cells (PBMCs). (A) PBMCs were infected with DENV2 in the presence of human anti-prM mAbs; at 24 hours, DENV Ag was stained intracellularly (4G2) and detected with flow cytometry in gated monocytes. PCS, PND, and irrelevant human mAb were used as control. (B) The density of prM on DENV from C6/36 cells and DC were detected with ELISA and presented as prM:E ratio. (C) Neutralization and (D) ADE of infection performed on Vero and U937 cells, respectively, of DENV generated from either C6/36 cells or DC in the presence of PCS or anti-prM mAb (3-147) (mean ± SE from three independent experiments).

Virus was generated in the insect cell line C6/36, which is known to cleave prM inefficiently, and the results we have obtained are therefore analogous to the first encounter with DENV, which was an insect-produced virus after a bite from an infected mosquito. Lastly, we set out to determine whether virus produced in primary mammalian cells contained noncleaved prM and whether antibody to prM had any enhancing capacity on such virus. Virus was produced in immature dendritic cells (DCs) in which cleavage of prM was more efficient than in the insect cell culture but still not complete (Fig. 4B). As with insect-produced virus, the antibodies to prM were unable to fully neutralize DC-produced virus with a clear plateau in efficacy (Fig. 4C) but were still able to enhance infection, although to a lesser degree (Fig. 4D).

This is the first description, using human monoclonal antibodies, of the serological response in DENV infection. Anti-prM is a major component of the response, and most of these antibodies display limited virus neutralization capacity. The combination of partial cleavage of prM, together with substantial cross-reaction between serotypes, makes the anti-prM response particularly susceptible to enhancement. Promotion of such an antibody response toward anti-prM could thus be interpreted as immune evasion or even as an immune enhancement strategy of the virus.

Most current DENV vaccine candidates—whether naturally attenuated, recombinantly attenuated, or chemically inactivated virus or DENV–yellow fever chimeras—contain native dengue prM sequences (16). It may be advisable to design DENV vaccines that minimize the anti-prM response. There is relatively low sequence conservation between DENV prM and sequences from other flaviviruses, and the majority of antibodies to dengue prM do not cross-react with JEV. Chimeric attenuated viruses containing heterologous flaviviral prM sequences may therefore not lead to such cross-reactive anti-prM responses as seen on infection with viruses that contain native dengue prM sequences, although the feasibility of making such chimeras has to our knowledge not yet been tested.

Supporting Online Material

Materials and Methods

Figs. S1 to S3

Tables S1 to S4

References and Notes

  1. We thank W. Supanchaimat, V. Jarupoonphol, S. Jinathongthai, K. Sriruksa, P. Wongsilarat, T. Suphachaiyakit, K. Ratarpa, Y. Sutvigit, and the staff of Khon Kaen and Songkhla hospitals for sample collection; L. Damrikarnlerd, P. Suriyapol, C. Komoltri, S. Udompunturak, N. Tangthawornchaikul, A. Jairangsri, K. Sae-Jang, and S. Supajitkasem for data and clinical database management and statistical analysis; N. Sittisombut and P. Keelapang from Chiang Mai University for sharing knowledge; and C. P. Simmons and B. Wills from the Oxford University Clinical Research Unit, Ho Chi Minh City for samples. This work was supported by the Medical Research Council, UK; the Wellcome Trust, UK; the National Institute for Health Research Biomedical Research Centre funding scheme; the Thailand Tropical Disease Research Program T2; and the Thailand National Centre for Genetic Engineering and Biotechnology.
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