Am. J. Trop. Med. Hyg., 80(1), 2009, pp. 61–65 Copyright © 2009 by The American Society of Tropical Medicine and Hygiene
Short Report: An Automated Dengue Virus Microneutralization Plaque Assay Performed in Human Fcγ Receptor-expressing CV-1 Cells W. W. Shanaka I. Rodrigo, Danielle C. Alcena, Robert C. Rose, Xia Jin, and Jacob J. Schlesinger* Departments of Pathology and Laboratory Medicine, Microbiology and Immunology, and Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York
Abstract. We describe microneutralization assays that used automated 96-well enzyme-linked immunospot (ELISPOT) readout instrumentation to measure human anti-dengue virus (DENV) antibodies in CV-1 cells that were stably transfected to express human FcγRIIA (CD32) using conventional Vero cells as a comparator. Classic plaque reduction neutralization test (PRNT) end-point titers were determined by probit analysis. Neutralization titers against DENV measured in CV-1 transfectants were expressed in terms of both conventional 50% to 90% PRNT end-point titers and differential infectivity of antibody-treated virus in control and CD32-expressing CV-1 cells. Significantly reduced PRNT titers and strikingly heightened infectivity (up to 100-fold) of antibody-treated DENV was observed in CV-1 CD32 transfectants compared with that observed in control CV-1 or Vero cells. Because DENVs may preferentially replicate in CD32-expressing monocytes/macrophages and dendritic cells, in vivo, it is possible that CD32 introduced into a conventional DENV neutralization assay might provide results that better correlate with protection. Dengue virus (DENV) human immune serum volumes are often limited, especially from children and infants, and DENV plaque neutralization assays, conventionally performed in cells of monkey kidney origin (i.e., Vero, LLC-MK2, CV-1), use relatively large formats (e.g., 6-well cluster plates). The DENV microneutralization assays that use such cell types have been described using an enzyme-linked immunosorbent assay (ELISA) format1 or immunofluorescent microscopy.2 In this work, we describe two microneutralization plaque assays that adopted widely used 96-well enzyme-linked immunospot (ELISPOT) readout instrumentation to measure DENV antibodies in African green monkey Vero and CV-1 cells used in DENV plaque assays. The first assay method used Vero cells, which are conventionally used in DENV neutralization tests,3 as a comparator and to calibrate the ELISPOT instrument for DENV plaque counting to determine traditional DENV plaque reduction neutralization test (PRNT) end-point serum titers against each of the four DENV serotypes. The second method used CV-1 cells that were engineered to constitutively express functional human FcγRIIA (CD32), or were stably transfected with an empty vector to serve as a control. This modification was introduced with the notion that neutralization measurements performed in such cells would better reflect the antibody effect on DENV infectivity for CD32-expressing cells of monocyte/macrophage (Mo/MΦ) lineage, the putative in vivo targets of DENV.4–7 Reference strain DENVs representative of each of the four DENV serotypes (DENV-1 16007; DENV-2 16681; DENV-3 16562; DENV-4 1036) were gifts of Dr. Richard Kinney (CDC, Ft. Collins, CO) and were propagated in C6/36 (Aedes albopictus) mosquito cells. For the microassays, Vero or CV-1 cell monolayers (2.5 − 4.0 × 104 cells/well) were established in 96-well plates. All sera were heat-inactivated (56°C, 30 minutes). The method for preparing and delivering DENVantibody mixtures to cell monolayers and monolayer fixation was essentially that described for an ELISA-based neutralization test,1 except for our use of a 0.75% methyl cellulose overlay
that was removed before cell fixation and immunostaining. The DENV plaques were developed by an indirect immunostaining method (Vectastain ABC, Vector Laboratories, Burlingame, CA) employing an anti-NS1 protein monoclonal antibody (9A9)8 that is broadly and equivalently reactive against all DENV serotypes. Plaques formed by DENV-4 and those of the other DENV serotypes were developed two and three days post-infection, respectively. Figure 1A shows immunostained DENV-2 plaques that developed after addition of graded amounts of DENV-2 to Vero cells or control and CD32-expressing CV-1 cell monolayers formed in 96-well plates. Plaques were counted and recorded using automated ELISPOT instrumentation (ImmunoSpot Plate Reader; ImmunoSpot version 3.0, Cellular Technology Ltd., Shaker Heights, OH). Plaque identification criteria were established for each DENV serotype based on direct visual counting in magnified wells for programming the instrument. Counting parameter settings (sensitivity, background balance, diffuse spot process, and spot separation) were established to quantify individual and overlapping plaques. We manually rejected counts obtained from damaged monolayers or those with staining artifacts. The efficiency of DENV-2 plaque formation was comparable among the three cell lines (Figure 1B), and a linear correlation was observed between DENV-2 plaque counts and a broad range of input DENV-2 plaque-forming units (PFU) ( Figure 1C). The efficiency of plaque formation determined by immunostaining with mAb 9A9 was comparable among all four DENV serotypes (data not shown). Having established optimal immunostaining conditions and verified the accuracy of instrumental plaque counting, we first measured the neutralizing activity of serum from a South Asian adult with no clinical history of dengue fever (DF), against each of the four DENV serotypes in Vero cells. Figure 2A shows representative immunostained DENV plaques developed in a 96-well microneutralization PRNT assay. GraphPad Prism 5 (GraphPad Software Inc, San Diego, CA) was used to analyze instrument readout data, by sigmoidal dose-response curve fit (Figure 2B), and DENV serotype–specific PRNT50 end-points determined by probit analysis.9 The standard error of instrumental plaque counts was generally less than 20% of the mean among quadruplicate wells. The neutralization profile was that
* Address correspondence to Jacob J. Schlesinger, University of Rochester Medical Center, Box 689, 601 Crittenden Blvd, Rochester, NY 14642. E-mail:
[email protected]
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Figure 1. Efficiency of DENV-2 plaque formation in Vero cells and CV-1 transfectants. Graded amounts of DENV-2 (16681) were added to Vero or CV-1 transfectant cell monolayers (2.5 − 4.0 × 104 cells/well) in 96-well plates. Virus plaques were visualized by indirect immuno staining with a DENV serogroup-reactive mouse anti-NS1 monoclonal antibody (9A9) three days post-infection. A, Representative wells from an assay performed in triplicate with Vero cells or CV-1 cells stably transfected with a control “empty” vector or CD32. B, Immunostained plaques (“spots”) were counted and recorded by a C.T.L. ImmunoSpot Series 5 Analyzer. The efficiency of DENV-2 infection was comparable among the Vero and CV-1 cell types. Error bars show the standard deviation of triplicate measurements. C, Comparative efficiency of DENV-2 plaque formation among Vero cells and CV-1 transfectants. Regression analysis verified linearity of the monoclonal antibody immunostain signal over a broad range of input DENV-2 plaque forming units (PFUs).
expected from a multiply DENV–infected adult from a region where all DENV serotypes co-circulate. Neutralizing activity was expressed in two ways using the transfected CV-1 cell lines: 1) by differential infectivity of DENV immune complexes (ICs) for CD32-expressing CV-1 cells versus control CV-1 cells (Figure 3) as we described with COS cell FcγR transient transfectants10; and, 2) in terms of conventional PRNT end-point titers determined simultaneously in Vero cells and the CV-1 transfectants (Figure 4). To explore the capacity of CD32-expressing CV-1 cells to mediate DENV-2 IC infectivity under varying virus and antibody concentrations, we prepared DENV-2 ICs by mixing serial concentrations of the pooled convalescent DENV immune serum with a range of DENV-2 multiplicity of infection (MOIs) in a 4 × 5 checkerboard fashion before addition to CV-1 transfectant monolayers. At relatively small virus input amounts and low serum dilutions DENV-2 was largely neutralized in control CV-1 cells (Figure 3A). Notably, DENV-2 IC infectivity was markedly enhanced (up to ~100-fold) in CD32-expressing CV-1 cells
(Figure 3B) compared with DENV-2 IC infectivity in control CV-1 cells. We next sought to compare the efficiency of DENV neutralization between Vero cell and CV-1 transfectants in parallel PRNT assays. Figure 4 shows PRNT50-90 end-point titers against DENVs tested in a comparative microneutralization assay in Vero cells, and control and CD32-expressing CV-1 cells. Here, three sources of DENV cross-reactive human serum were used: 1) serum from the South Asian individual with no history of DF (Figure 4A); 2) pooled convalescent serum from Asian and Puerto Rican DF and dengue hemorrhagic fever (DHF) patients collectively representing natural infection with all four DENV serotypes (Figure 4B); and, 3) pooled convalescent sera from Puerto Rican patients with secondary DF contracted in 1978, before DENV-4 emerged on the island11 (Figure 4C). For each cell type, the PRNT50-90 end-point titers against DENV-2 were roughly 50 to 100-fold greater with the most broadly reactive DF/DHF convalescent serum pools than with the asymptomatic individual’s serum. PRNT50-90 titers
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Figure 2. Dengue virus (DENV) microneutralization in Vero cells. DENV immune serum from a heterologous DENV–infected South Asian adult was titrated against: DENV-1 (16007); DENV-2 (16681); DENV-3 (16562); DENV-4 (1036) in a plaque reduction neutralization test (PRNT) performed in Vero cell monolayers in 96-well plates. Shown are final dilutions of heat-inactivated (56°C, 30 minutes) serum incubated at 37°C with 50 plaque forming unit (PFU) DENV per well for 60 minutes before adsorption to cell monolayers for 90 minutes, followed by phosphate-buffered saline (PBS) washing, and overlayering with 0.75% methylcellulose. A, DENV plaques were immunostained with anti-NS1 monoclonal antibody 9A9 two days or three days post-infection for DENV-4 and DENV-1-3, respectively. Spots were counted in quadruplicate wells and recorded by an ELISPOT instrument. Representative wells corresponding to serially diluted serum are shown for each DENV serotype. B, Spot counts were fitted to a dose-response curve and PRNT50 titers against each DV serotype were calculated by probit analysis,9 both using GraphPad Prism5 software: DENV-1, 1/1843; DENV-2, 1/1115; DENV-3, 1/8873; DENV-4, 1/480. PRNT50 titers with heat-inactivated serum from a flavivirus naïve individual were less than 1/10 against each DENV.
obtained in control CV-1 cells were insignificantly lower than those determined in Vero cells (P = 0.154, Mann-Whitney U test). Notably, DENV-2 PRNT50-90 titers were lower in CD32-expressing CV-1 cells than in control CV-1 cells with both serum sources, the difference being significant with the DF/DHF serum pool (P = 0.016), but not with serum from the asymptomatic individual (P = 0.075). These neutralization patterns were reiterated in PRNT50-90 end-point titers determined with the Puerto Rican DF convalescent serum pool titrated against all four DENV serotypes. Figure 4C shows the PRNT50 and PRNT90 end-point titers. There was no significant difference between neutralization titers obtained in Vero or control CV-1 cells, among DENV serotypes (P > 0.155). PRNT50-90 end-point titers were uniformly lower in CD32-expressing CV-1 cells than in control CV-1 or Vero cells among all four DENV serotypes (P < 0.016). The ELISPOT instrumentation offers the possibility of automated high-throughput DENV plaque assay measurements. Because the PRNT measurements performed in Vero cells were largely done to adapt the instrument to plaque counting, we did not formally compare our results with those obtainable by a conventional large format assay in Vero cells for this proof
of concept study. Nevertheless, the PRNT50-90 profiles were entirely as expected for adult heterotypic DENV immune sera from regions where multiple DENV serotypes have cocirculated.11,12 Furthermore, plaque count variances among replicate wells generally comported with those observed in the classic PRNT assay originally performed with LLC-MK2 rhesus monkey kidney cells in 1 oz. prescription bottles using neutral red staining to visualize DENV plaques.9 Virus neutralization assays generally seek to measure protective antibodies. When performed in conventional cell types, such assays measure antibody blockade of virus attachment or fusion, or both.13 In the case of the mosquitoborne DENVs, conventional assays provide an imperfect correlation between neutralizing antibody titer and protection.14,15 This may not be surprising in view of the susceptibility of Mo/MΦ to infection. Neutralization is conditional: It may be governed by different mechanics in cells that display FcγRs than in cells that do not. Indeed, antibodies may paradoxically enhance DENV replication in Mo/ MΦ by FcγR-mediated antibody-dependent enhancement (ADE), a mechanism widely hypothesized to be central to the pathogenesis of DHF.16
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Figure 3. Heightened dengue virus (DENV)-2 immune complex infectivity in CD32-expressing CV-1 cells. DENV-2 immune complexes were formed with graded virus concentrations and serially diluted pooled DENV convalescent immune serum, in checkerboard fashion, before addition to: A, vector control or B, CD32-R131-expressing CV-1 cell monolayers in quadruplicate.
Figure 4. Comparative dengue virus (DENV) neutralization in Vero cells and CD32-expressing CV-1 cells. A, DENV immune serum from a heterologous DENV–infected South Asian adult subject without a history of clinically apparent DENV infection, or B, pooled convalescent dengue immune serum from dengue fever (DF) and dengue hemorrhagic fever (DHF) patients who were collectively likely to represent infection with all DENV serotypes were titrated against DENV-2 in 50–90% plaque reduction neutralization tests (PRNT50-90). Microneutralization assays were performed in Vero or CV-1 transfectant cell monolayers, in parallel. PRNT50-90 end-point titers were calculated by probit analysis of triplicate determinations. C, Pooled serum from Puerto Rican patients with secondary DF in 1978 (before DENV-4 was introduced into the island) was titrated in Vero cells and CV-1 transfectants against DENV serotypes 1–4 and PRNT50-90 end-point titers were calculated by probit analysis of quadruplicate determinations.
DENGUE VIRUS MICRONEUTRALIZATION PLAQUE ASSAY
The second assay method described herein, introduction of FcγR display, may therefore offer a number of advantages over conventional DENV neutralization assays. First, and foremost, neutralizing antibody measurements performed in CV-1 cells that display FcγRs and exhibit properties of professional phagocytes (e.g., binding and phagocytosis of IgG opsonized particles and interferon α/β production) may present more stringent criteria for neutralization. Results with such cell types are also more likely to reflect infectivity of DENV ICs for Mo/MΦ, in vivo, and therefore might prove to be more predictive of protection. We have obtained concordant PRNT/ADE results in CD32-expressing CV-1 cells and in normal human CD14+ monocytes (Rodrigo I and others, unpublished data). Second, the methodology is fundamentally that of a conventional PRNT assay and is amenable to further modification in choice of candidate cell type for FcγR-transfection. The automated optical readout instrumentation that we adopted is widely used for ELISPOT determinations, but other micro-readout formats, (e.g., ELISA,1 flow cytometry, or direct visual counting by light or immunofluorescent microscopy2) are possible with these cells. It should be considered, however, that genotypic heterogeneity in plaque size in mixed DENV populations might not be appreciated by an ELISA or flow cytometry-based assay nor would the relative contribution of such variants to the color signal be discernable. Third, the cloned CD32-expressing CV-1 cell lines exhibited remarkable stability and uniformity of receptor expression in continuous cell culture (at least 6 months) and were readily propagated after cryopreservation making them attractive for routine reference laboratory use. Whether use of such FcγR transfectants improves detection of truly protective neutralizing DENV antibodies or warns of ADE, awaits testing with well-characterized DENV strains and sera from vaccinated or naturally infected individuals, who were and were not, protected from subsequent DENV infection. Received June 25, 2008. Accepted for publication September 19, 2008. Acknowledgments: We thank Dr. Eric Henchal (AFRIMS, Bangkok, Thailand) and Ms. Gladys Sather (CDC, Puerto Rico) for human dengue immune sera. Financial support: This work was funded by the Pediatric Dengue Vaccine Initiative of the International Vaccine Institute, Awards TR 03/04 (J.J.S) and TR16 (X.J.). Authors’ addresses: W. W. Shanaka I. Rodrigo, Danielle C. Alcena, Robert C. Rose, Xia Jin, and Jacob J. Schlesinger, University of Rochester Medical Center, Box 689, 601 Crittenden Blvd., Rochester, NY 14642, E-mail:
[email protected].
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