Plasticity of a critical antigenic determinant in the West Nile virus NY99 envelope protein domain III.

Figure 5

Binding of monoclonal and polyclonal antibodies to NY99ic WT and T332 mutants

Binding of WNV MIAF, WNV EIII, 5H10, and 7H2 to NY99ic WT and T332 mutants infected cell lysates in Western blots. Bands shown are from the ~50kD region of the gel, corresponding to the size of intact E protein. Coomassie staining and blotting with WNV MIAF are included to confirm the loading of approximately equivalent amounts of protein.

In general, binding by the monoclonal antibodies 5H10 and 7H2 to NY99ic WT and NY99ic T332 mutant cell lysates in Western blots corresponded well with the strength of neutralization (Figure 5). For 5H10, all NY99ic T332 mutants with neutralization indices of 0.9 or less had very faint or non-visible bands in a Western blots except for NY99ic T332E,. For 7H2, NY99ic T332 mutants with neutralization indices between 1.7 and 1.0 (T332A, T332G, T332I, T332L, T332M, T332N, and T332P+S66R) there is variation between strong and weak/non-visible binding, but neutralization indices ≥1.8 corresponded to strong binding, similar to the NY99ic WT, and neutralization ≤0.7 corresponded to weak or undetectable binding.

Despite having a strong impact on neutralization by polyclonal anti-EIII serum, mutations at T332 had no impact on binding by the polyclonal anti-EIII serum in a Western blot. This is likely because inoculation with recombinant EIII elicits non-functional antibodies against epitopes that are not accessible in the context of an intact, infectious virion but are accessible in the context of an infected cell lysate.

DISCUSSION

The recovery of viable WNV NY99ic mutants encoding every possible amino acid at E-332 is consistent with related observations regarding the ability of E-332 to tolerate substitution. The extreme plasticity of WNV E-332 has been suggested by both its naturally occurring variation and by the previously reported recovery of neutralization escape mutants that retain a wild-type virulence phenotype.(Armstrong et al., 2011; Beasley et al., 2002; Bernardin, 2010; Li et al., 2005; McMullen et al., 2013; Pybus et al., 2012; Sapkal et al., 2011; Zhang et al., 2010) In addition, a panel of packaged replicon reporters (described by Lee et al. as reporter virus particles, or RVPs) was generated that captured the range of possible variation at E-332 and a pool of infectious clone-derived viruses with variation at E-332 was used to examine the selective pressure of neutralizing antibodies.(Lee et al., 2013)

In general, changes to NY99ic T332 had only modest impacts on replication kinetics in mammalian, avian, and mosquito cells, and on virulence in mice. The few significant differences that were observed in cell culture were variable between cell types. For instance, only the NY99ic T332P+S66R mutant appeared significantly impaired for growth in Vero cells, but it was more comparable to NY99ic WT in both C6/36 and DEF cells. Similarly, both NY99ic T332W+S66R and T332Y had titers ≥1log₁₀ PFU/ml lower than NY99ic WT in C6/36 cells on 5 DPI, but replicated similarly to WT in Vero an DEF cells. It has been reported that changes to residues in the surface loop residues of EIII in multiple flaviviruses can have differential impacts on replication in mosquito and mammalian cells.(Erb et al., 2010; Huang et al., 2014; Hung et al., 2004) and this also appears to be the case for WNV E-332. It is worth noting that the substitutions which resulted in cell-type-dependent deficits in peak titer all involved the introduction of either large aromatic rings (T332W+S66R and T332Y) or a cyclic structure (T332P+S66R), raising the possibility that the deficit was not a direct consequence of changing E-332 but of the resulting disruption of neighboring residues. Consistent with the extreme plasticity of E-332 in cell culture, the majority of NY99ic T332 mutants retained a highly virulent phenotype in mice. In fact, all but three mutants caused ≤30% survival following peripheral infection in Swiss Webster mice. Very strong attenuation was only observed with the NY99ic T332P and T332P+S66R mutants. Thus, the detrimental impact of the proline at E-332 observed in Vero cell culture appeared to have a similarly negative effect in vivo. While it cannot be entirely ruled out that some of the observed changes in growth and/or virulence might be influenced by suboptimal codon usage associated with the introduced mutations, the especially strong phenotype of the T332P and T332P+S66R mutants is consistent with the disruptive nature of introducing this cyclic amino acid. Furthermore, although codon usage bias can cause demonstrable impacts on flavivirus replication and virulence, those effects have typically been in the context of changes to large segments of the genome, not single codon substitutions.(de Fabritus et al., 2015; Shen et al., 2015)

The independent acquisition of a S66R mutation by both the NY99ic T332P and T332W mutants was an unexpected finding. Similar acquisitions of a positive charge at a surface exposed E residue has previously been associated with tissue culture adaptation in other encephalitic flaviviruses.(Lee et al., 2004; Lee and Lobigs, 2000) A follow-up comparison of NY99ic WT to the individual NY99ic S66R, T332P, T332P+S66R, T332W, and T332W+S66R mutants revealed that S66R had no detectable impact on amplification and peak titer in Vero cells following a low MOI infection (Supplementary Figure S1). However, although the NY99ic S66R mutant was not attenuated in mice, the presence of S66R did appear to exacerbate the attenuation of NY99ic T332P, and also to increase the LD₅₀ of NY99ic T332W (Table 1) although neither T332W nor T332W+S66R were strongly attenuated. These observed effects of the S66R mutation are consistent with attenuating effects of other tissue culture associated charge changing mutations. More work is required to characterize the role of the S66R mutation and its direct or indirect interaction with mutations at E-332.

In contrast to the limited impact of mutations at E-332 on NY99ic replication and virulence, changes to E-332 had a profound impact on antigenicity. None of the nineteen possible NY99ic T332 mutants retained the very strong neutralization phenotype of NY99ic WT with 7H2, and only the NY99ic T332S mutant retained the very strong neutralization phenotype with 5H10. In fact, nine of the 19 possible NY99ic T332 mutants were weakly neutralized by one or both of the EIII mAbs tested. These results are consistent with those previously reported for the anti-WNV EIII mAbs E33 and E16, which largely failed to neutralize a panel of WNV RVPs with basic, acidic, or aromatic substitutions at E-332.(Lee et al., 2013) All nine mutations that resulted in weak neutralization (NI values <0.4) by 5H10 and/or 7H2 also resulted in moderate (NI values 0.5–1.1) or weak neutralization by the polyclonal anti-EIII rabbit serum, confirming that E-332 is a critical antigenic determinant even in the context of a polyclonal population of antibodies against EIII

The ability of NY99ic to tolerate changes at E-332 that ablate or greatly reduce neutralization by monoclonal and polyclonal antibodies while having little to no impact on replication and virulence phenotypes raises an important consideration for the potential use of EIII-based vaccines or therapeutics against WNV. There are six possible amino acid substitutions that require only a single nucleotide change from the predominant WNV 332 codon of ACG: T332A, T332K, T332M, T332P, T332R, and T332S (Figure 4). Of these six possibilities, our data suggest that T332P is significantly impaired and unlikely to persist in nature but four other mutations (T332A, T332K, T332M, and T332S) are already known to exist in naturally occurring WNV strains. These strains with variation at E-332 were collected over a span of decades (1953–2007) from mosquitoes, a bird, a fruit bat, viremic human blood donors, and febrile human patients.(Armstrong et al., 2011; Beasley et al., 2002; Bernardin, 2010; McMullen et al., 2013; Pybus et al., 2012; Sapkal et al., 2011) Of these mutations, only T332K is associated with large reductions or loss of neutralizing activity by anti-EIII MAbs or polyclonal antiserum. However, alternative amino acids with antigenic and virulence characteristics comparable to T332K, particularly T332D, T332E, and T332H are within a single nucleotide substitution of the other known 332 variants. It is worth noting that single residue EIII mutations in naturally occurring WNV strains that strongly reduce neutralization by polyclonal anti-EIII serum or monoclonal antibodies do not significantly impact neutralization by polyclonal sera raised against whole WNV virions.(Li et al., 2005)

Previous work has shown that the proposed therapeutic mAb E16 exerts sufficient selective pressure on a pool of infectious clone-derived E-332 mutants in vitro to generate a population composed exclusively of T332K and T332R mutants within a single post-transfection passage(Lin et al., 2012b) .Additionally, in vivo experiments have demonstrated that pre-treating mice with the mAb E16 and administering a high but biologically relevant dose of WT WNV can select for neutralization-resistant variants at E-332 which are lethal to the infected animals.(Zhang et al., 2009) Data reported here suggest that WNV could likewise escape from a polyclonal response targeted against EIII. Combined with the previous recovery of 332 variant strains from humans, it seems possible that the administration of an EIII-based subunit vaccine or antibody therapy may potentially select for a resistant population within an infected individual. Such a resistant population would be unlikely to take hold in the wild because humans are dead-end hosts, but could complicate treatment and endanger the individual infected.

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MATERIALS AND METHODS

Generation of WNV T332 Variants

WNV variants encoding amino acid substitutions were generated via targeted mutagenesis of the pWN-AB/pWN-CG NY99ic using the QuikChange Multi Site-Directed Mutagenesis Kit (Stratagene, Cedar Creek, TX) as previously described (Beasley et al., 2005). Initially, four variant plasmid pools were generated using degenerate oligonucleotide primers as described previously (Zhang et al 2010). The four pools collectively included all 19 possible amino acid substitutions at residue T332 (primer sequences available upon request). Following electroporation of in vitro-transcribed RNA into Vero cells, culture supernatants were harvested at 3–5 days and plaque titrated on Vero cell monolayers in 6 well plates. Individual variants were recovered via plaque purification from those plates and amplified in T-25 flasks of Vero cells. When visible CPE was noted (3 days post infection for most variants), multiple aliquots of each viable variant were frozen down and their identities determined via sequencing. Working stocks of recovered variants representing each of the viable amino acid substitutions were then prepared in Vero cells for subsequent experiments. Each variant recovered via this strategy received three additional Vero cell passages following the passage 0 electroporation. In addition, several variants were recovered directly from individual variant pWN-AB plasmids that were generated using the QuikChange Multi kit with specific mutagenesis primers according to previously described methods.(Zhang et al., 2010)

Nucleotide Sequencing of WNV T332 Variants

Viral RNA was purified from tissue culture supernatants using the QIAamp Viral RNA kit (Qiagen, Germantown, MD) according to the manufacturer’s protocol. The prM/E coding region was amplified using the Titan One-Step RT-PCR kit (Roche, Mannheim, Germany) with the WN401 and WN2504A primers.(Beasley et al., 2003) The resulting PCR products were visualized on an agarose gel and the appropriately sized band excised and purified using the QIAquick Gel Extraction kit (Qiagen, Germantown, MD). Traditional Sanger sequencing was performed by the Molecular Genomics Core at the University of Texas Medical Branch using an Applied Biosystems 3130XL instrument using the amplifying primers plus additional internal primers.(Beasley et al., 2003)

Plaque and Temperature Sensitivity Assays

Titers for each virus stock were determined by plaque assays in Vero cells. Briefly, serial 10-fold dilutions of each variant were made in sterile phosphate buffered saline (PBS) and 100μl was used to infect monolayers of Vero cells in 12-well plates. After one hour at room temperature, the plates were washed with PBS and overlaid with a 2ml of a 50:50 mixture of 2XMEM (Gibco, Grand Island, NY) containing 4% bovine growth serum (BGS) (Hyclone, Logan, UT) and 2% agar (Sigma, Portugal). For standard plaque assays, plates were placed at 37°C with 5% CO₂. At 2 days post-infection, 1ml of overlay containing 2% neutral red (Sigma, Irvine, UK) was added to each well, and the plates were wrapped in foil and returned to the incubator. Plaques were read with the aid of a light box on days 3 and 4 post-infection. For the temperature sensitivity assays, infected 6-well plates were overlaid with 4ml of media:agar and placed in either a 37°C or a 41°C incubator with 5% CO₂. On day 3 post-infection the plates were fixed with formalin and stained with crystal violet. Differences in titer and plaque morphology at 37°C and 41°C were recorded. Assays were performed in duplicate.

In vitro Growth Kinetics

Vero, C6/36, and duck embryo fibroblast (DEF) cells were infected in duplicate at an MOI of 0.01 for one hour at room temperature, then washed three times with PBS. Cells were maintained in 6ml MEM supplemented with 2% BGS (Vero) or 2%FBS (C6/36 and DEF). Vero and DEF cells were kept at 37°C with 5% CO₂. C6/36 cells were kept at 28°C in an ungassed incubator. For assays in Vero and DEF cells, two 250μL samples were collected at 0, 0.5, 1, 1.5, 2, 3, and 4 days post-infection and stored at −70°C for subsequent titration. C6/36 cell assays were performed similarly, except that an additional sample was collected at 5 days post-infection. Following sample collection fresh media was added to each sample to maintain a constant volume. All samples were titered via plaque assay on Vero cells

Mouse Neuroinvasiveness and LD₅₀ Determination

For assessment of virulence, cohorts of 10–20 3–4-week-old female Swiss Webster mice (Harlan Laboratories, Houston, TX) were injected with WNV variants in 100µL volumes via the intraperitoneal (i.p.) route. During the initial screening involving all 20 variants, a single 100 pfu dose of virus was used. During subsequent LD₅₀ determination for a subset of viruses, serial 10-fold dilutions ranging from 10³–10⁻¹pfu were used with cohorts of five mice per dose. Following inoculation, mice were observed for 21 days. Mice exhibiting paralysis or signs of severe illness were humanely euthanized and counted as deceased for that day.

Neutralization by MAbs and polyclonal anti-EIII serum

Neutralization of parental and 332 mutant viruses by commercially-available mAbs and polyclonal anti-EIII sera was assessed by determination of neutralization indices, as described previously.(Beasley and Barrett, 2002; Li et al., 2005) The two MAbs used - anti-WNV 5H10 and 7H2 (Bioreliance Corporation, Rockville, MD) - were previously shown to recognize distinct but overlapping EIII epitopes that include E-332.(Beasley and Barrett, 2002) The rabbit anti-WNV antiserum has been described elsewhere.(Beasley et al., 2004) Briefly, serial 10-fold dilutions of all 20 variants were combined with a constant concentration of monoclonal antibody (2.5ng/μL) or polyclonal antiserum (final concentration of 1:50) diluted in MEM/2% BGS and incubated at room temperature (approximately 22°C) for one hour. A “medium only” control was also included for each virus. The virus:antibody mixtures were then used to infect monolayers of Vero cells in 12-well plates for one hour at room temperature. Cells were overlaid with 2ml/well of 2XMEM/agar and placed in a 37°C incubator with 5% CO₂. After two days, 1ml of a second overlay containing 2% neutral red was added. Plaques were visualized with a light box on days 3 and 4 post-infection. NI values were calculated as the log₁₀ reduction in apparent titer in the presence of each antibody/antiserum compared to the no antibody control. Assays were performed in duplicate.

Western blotting

Individual T-25 flasks of Vero cells were infected with viable 332 variants. When early CPE was visible (typically 2 days post-infection), the media was removed and the flask was stored at −70°C overnight. The monolayer was then thawed and lysed in 1ml of 10% SDS for 10 minutes at room temperature. The lysate was then incubated for 30 minutes at 56°C prior to transfer to BSL2. Equal volumes of the resulting cell lysates were then subjected to electrophoresis on 12% SDS polyacrylamide gels under non-reducing conditions. Gels were stained with Coomassie blue for assessment of total protein or transferred to a nitrocellulose membrane for Western blotting. Nitrocellulose membranes were blocked with 3% BSA in TBS, washed, then incubated with primary antibody for one hour at room temperature. Primary antibodies included anti-whole WNV mouse immune ascitic fluid (MIAF) at 1:500, rabbit anti-WNV EIII serum at 1:500, 5H10 at 0.5ng/μL, and 7H2 at 0.25ng/μL. After the primary antibody, the membrane was washed and incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (anti-mouse IgG or anti-rabbit Ig; Sigma, St. Louis, MO) for one hour at room temperature. After additional washing, the membrane was developed with the ECL Western blotting detection reagents (GE Healthcare, Buckinghamshire, UK) and exposed to ECL Hyperfilm (GE Healthcare, Buckinghamshire, UK) for visualization.

Statistical Analysis

Differences in apparent titer at 37°C vs 41°C were compared for NY99ic wild-type (wt) and each T332 mutant using unpaired, two-tailed Student’s T-tests. Variation of log₁₀ pfu/ml titer values for each measured time point in the replication curves, survival times, and neutralization indices were assessed by one-way ANOVA. Individual strains that varied from the NY99ic wt control were identified by post-hoc Dunnett’s multiple comparisons test. Survival rates were compared using the Kaplan Meier log-rank test. LD₅₀ values were calculated using the Spearman Karber method. To assign neutralization indices to a particular group, the appropriate number of clusters was determined using k-means analysis of a pooled dataset of all virus:antibody NI values and NI values were then assigned to a particular cluster using the average method of hierarchical clustering. The significance threshold for all tests was 0.05. Statistical analysis was performed using GraphPad Prism version 6.05 for Windows (GraphPad Software, La Jolla, CA) and R 3.0.2 (R Foundation for Statistical Computing, Vienna, Austria).

​

Highlights

• Residue 332 in domain III of West Nile virus NY99 strain envelope protein is highly mutable.
• Mutations at residue 332 had significant effects on antigenicity of WNV domain III but relatively minor effects on growth in cell cultures or mouse virulence.
• Use of monoclonal antibody therapies or subunit vaccines targeted against domain III could potentially select for variants that retain virulence equivalent to the wild-type virus.

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Supplementary Material

Supplementary Figure S1: Impact of S66R on NY99ic replication in Vero cells:

Replication kinetics of WNV NY99ic WT and selected T332 mutants with or without the S66R mutation in Vero cells with an initial multiplicity of infection of 0.01.

Click here to view.^{(177K, tif)}

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Acknowledgments

This work was supported by funding from UTMB’s Institute for Human Infections and Immunity to DWCB. JAP was supported by pre-doctoral fellowships from the Biodefense Training Program (NIH grant T32-AI060549) and the Sealy Center for Vaccine Development.

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Footnotes

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