Dengue is a worsening global health problem. The study of dengue epidemiology and pathogenesis of disease has increased considerably over the past decade. Numerous dengue vaccine candidates are now in pre-clinical and clinical development and two candidates have generated sufficient clinical data to support planning efficacy trials.38
The PRNT assay, or variations of the same, remains at the center of these efforts.
The PRNT has been used to document the development of neutralizing, DENV type-specific antibody to support basic science research and vaccine development efforts for nearly four decades. Understanding neutralizing antibody (NAb) “profiles” at the individual and population levels allow investigators to document the occurrence of a DENV infection, correlate immune status to clinical outcomes (i.e., asymptomatic versus symptomatic DENV infection), and functions as a marker of dengue vaccine immunogenicity. There is general consensus that if defining a surrogate marker of immunity/protection is possible for dengue, it will most likely be accomplished using quantitative NAb titers. Once vaccine efficacy trials are completed and clinical benefit of vaccination established, national regulatory authorities will likely evaluate vaccine candidate immunogenicity based on the ability to reproducibly generate NAbs. As such, many vaccine developers are transitioning to second generation neutralization assays (e.g., microneutralization assay) with greater potential for automation, higher throughput, and validation.
Despite the importance of NAb in understanding immune responses to DENV infection and vaccination and efforts by the WHO and Pediatric Dengue Vaccine Initiative (PDVI), there has been no standardization or harmonization of the PRNT. As a consequence, it has been difficult and potentially misleading to compare and contrast results and data across studies and institutions. The WHO recently offered its guidance and recommendations on PRNT assay performance (http://whqlibdoc.who.int/hq/2007/WHO_IVB_07.07_eng.pdf
Standardization of biologic assays, such as the PRNT, requires consistency among several assay conditions and harmonization between laboratories performing the assay. Standardization and thorough characterization of critical reagents (e.g., cell line, viral strains, use of complement), optimization of assay methods and sample preparation (e.g., incubation time), strict adherence to a single operating procedure (i.e., SOP), and the use of internal and external controls is key. Reproducibility of assay performance (e.g., lower bounds of reliable assay performance) within and between laboratories is also essential. Achieving these ends is typically accomplished using a well-characterized reference panel of natural (i.e., patient) or artificially generated (i.e., spiking serum with known virus and concentration) samples.
In our experiments, we attempted to examine the performance aspects of the PRNT using a well-characterized panel of patient sera representing acute and late convalescent, primary and secondary, and DENV type-specific DENV infections. All assays were performed by the same two technicians who were blinded to the previous serologic and virologic characterization of the samples and used the same assay procedure and method. The objective of the study was to reproduce the assay methods currently in use and identify how the differences in conditions (materials and methods) could impact assay results.
Our study showed significant variability in PRNT results depending on the assay conditions. There was considerable variation in the DENV NAb titers generated by the PRNT depending on the control viral passage and cell line used and the use of complement. The variation in PRNT titers was demonstrated not only to be impacted by manipulating a single condition but also by interaction between two or more conditions.
The effect of viral passage on NAb titers was significant but not uniform across all DENV types. There was varying effect on titer depending on the virus (wild type versus prototype) and passage (prototype versus prototype in tissue culture). The low passage wild type virus represents a different virus strain (genotype) compared with the prototype viruses and genetic diversity could impact NAb titer distinct from passage level. However, the differences in NAb titers between prototype and higher passaged prototype viruses appear to indicate passage level alone can impact assay results. Unfortunately, viral strains from the experiments are no longer available, and it is not possible to quantitate the impact of genetic diversity on NAb titer. A limited exploration of E gene sequences of viruses representative of those used in this study revealed significant divergence between low passage wild type and prototype viruses. There was no significant divergence between prototype viruses and prototype viruses serially passed in tissue culture. Therefore, although genetic variation could account for assay result differences observed between wild type and prototype viruses, it could not account for the differences observed between using prototype and prototype in tissue culture viruses.
The selection of control virus for any given assay is often dictated by the intended use of the assay. If the assay is being used to characterize regional epidemiology then representative strains (i.e., regional) may be chosen. Assays being used to define vaccine candidate immunogenicity use homologous viruses (i.e., vaccine parent strain). It is currently unknown whether vaccine candidates showing efficacy against DENV strains circulating in, for example, Asia will show similar efficacy against strains circulating in the Americas, Western Pacific, or Africa. Therefore, demonstration of vaccine immunogenicity against non-parental viruses (heterologous strains) may be considered important by vaccine developers seeking a global indication. Until large-scale efficacy trials are conducted the answer to this question will likely remain elusive.
The effect of cell line on NAb titers was significant but not uniform across all DENV types. BHK-21 cells appeared to exert a more uniform effect (lower titers for DENV-1, -2, and -4); a larger sample size may have revealed a significant reduction in NAb titer across all DENV types.
Choice of cell line is also likely impacted by intended assay use. Of the cell lines used in these experiments, a specific derivation of Vero cells are the only cells certified by the WHO for production of live virus dengue vaccine candidates and use in the PRNT.30
Because of this, dengue vaccine developers may be partial to using Vero cells.
The significant lowering effect of DENV-1 and -3 infections on DENV-4 NAb titers is interesting. The observation may be explained by the antigenic similarities between DENV-1 and -3 viruses and a shared reduced ability to cross-neutralize the DENV-4 control virus.39
A second point to consider is that the primary infecting DENV type was not known for the secondary infections. One would assume higher DENV-4 titers would have been observed if the primary infection were caused by a DENV-4 virus (i.e., DENV-4 primary infection followed by DENV-1 or -3 secondary infection).
The addition of complement resulted in a significant rise in titers across all DENV types. Neutralization of DENVs does not require complement and its use in PRNT assays is not recommended.30
Significant interaction effects were observed for virus passage and the use of complement, cell line and the use of complement, and cell line and virus passage. These results were not unexpected considering the significant effects each individual condition had on assay results. The interaction effects contribute to the high variability in titers generated by this assay, suggesting that not only is the choice of assay conditions important but also which conditions are used in combination.
One limitation in our data is the small sample size. Although statistical analyses were applied to the data, the results are considered descriptive and designed to highlight, but not quantify, differences in NAb data generated by PRNT assays under varied testing conditions. Accurately quantifying differences between different assay conditions and attempting to identify “optimal” assay methods would require repeating these experiments with expanded sample panel sizes. A second limitation is the absence of genetic characterizations of the viruses used in the assays. A comparison of virus sequences would have added clarity to understanding assay variation and the varied ability of patient sera to neutralize control viruses. Because of limited sera volume, assays could only be performed once for each condition. Inherent biologic inter-assay variability is a potential issue that would need to be addressed in subsequent experiments. A sera panel composed of more primary infections was desired but these samples are very difficult to acquire in Bangkok using hospital-based studies. An expanded analysis of PRNT assay conditions without the confounding of heterotypic antibody found in secondary infections would have been ideal.
The authors' intent was not to generate a consensus protocol or make recommendations on individual assay conditions. As mentioned, this effort has already been undertaken and the results published by the WHO. The intent of conducting these experiments was to explore how varying individual testing conditions impact assay results. The intra-assay variation experienced when conditions remain constant presupposed variation would be observed when key conditions were modified. The occurrence of intra- and interassay variation is expected with any biologic assay; conducting this series of experiments was an attempt to understand the magnitude of the variation. The data generated during this study supports the contention that significant variation in NAb titers occur when key assay conditions are varied. Furthermore, the data highlight the potential hazards of comparing NAb readouts between laboratories; these concerns are likely most relevant when comparing dengue vaccine candidate immunogenicity. Without the broad implementation of standardized assay reagents and harmonization of assay methods, comparison of NAb data across different vaccine candidates should be approached with caution. If inter-laboratory and manufacturer data comparisons are desired, efforts to standardize and harmonize the PRNT must continue.