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. 2021 Mar 12;15(3):e0009195.
doi: 10.1371/journal.pntd.0009195. eCollection 2021 Mar.

Zika virus-like particle vaccine protects AG129 mice and rhesus macaques against Zika virus

Lo Vang  1 , Christopher S Morello  1 , Jason Mendy  1 , Danielle Thompson  1 , Darly Manayani  2 , Ben Guenther  1 , Justin Julander  3 , Daniel Sanford  4 , Amit Jain  1 , Amish Patel  1 , Paul Shabram  1 , Jonathan Smith  2 , Jeff Alexander  1   2
Affiliations

Zika virus-like particle vaccine protects AG129 mice and rhesus macaques against Zika virus

Lo Vang et al. PLoS Negl Trop Dis. .

Abstract

Background: Zika virus (ZIKV), a mosquito-borne flavivirus, is a re-emerging virus that constitutes a public health threat due to its recent global spread, recurrent outbreaks, and infections that are associated with neurological abnormalities in developing fetuses and Guillain-Barré syndrome in adults. To date, there are no approved vaccines against ZIKV infection. Various preclinical and clinical development programs are currently ongoing in an effort to bring forward a vaccine for ZIKV.

Methodology/principle findings: We have developed a ZIKV vaccine candidate based on Virus-Like-Particles (VLPs) produced in HEK293 mammalian cells using the prM (a precursor to M protein) and envelope (E) structural protein genes from ZIKV. Transient transfection of cells via plasmid and electroporation produced VLPs which were subsequently purified by column chromatography yielding approximately 2mg/L. Initially, immunogenicity and efficacy were evaluated in AG129 mice using a dose titration of VLP with and without Alhydrogel 2% (alum) adjuvant. We found that VLP with and without alum elicited ZIKV-specific serum neutralizing antibodies (nAbs) and that titers correlated with protection. A follow-up immunogenicity and efficacy study in rhesus macaques was performed using VLP formulated with alum. Multiple neutralization assay methods were performed on immune sera including a plaque reduction neutralization test, a microneutralization assay, and a Zika virus Renilla luciferase neutralization assay. All of these assays indicate that following immunization, VLP induces high titer nAbs which correlate with protection against ZIKV challenge.

Conclusions/significance: These studies confirm that ZIKV VLPs could be efficiently generated and purified. Upon VLP immunization, in both mice and NHPs, nAb was induced that correlate with protection against ZIKV challenge. These studies support translational efforts in developing a ZIKV VLP vaccine for evaluation in human clinical trials.

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Conflict of interest statement

I have read the journal’s policy and the authors of this manuscript have the following competing interests: Jonathan Smith and Darly Manayani have no competing interests. Jeff Alexander is a paid consultant of Emergent. Justin Julander and Daniel Sanford are paid employees of Utah State University and Battelle Biomedical Research Center, respectively. Lo Vang, Chris Morello, Jason Mendy, Danielle Thompson, Ben Guenther, Amit Jain, Amish Patel, and Paul Shabram are paid employees of Emergent BioSolutions Inc.

Figures

Fig 1
Fig 1. Design, production, purification, and characterization of ZIKV VLP vaccine candidate.
(1A) Schematic of ZIKV genome and plasmid DNA encoding ZIKV structural genes: 1) prM derived from the MR766 African strain (orange) with native signal sequence replaced with the human IL2 sequence (yellow) (MYRMQLLSCIALSLALVTNS); 2) E ectodomain derived from the SPH2015 Brazilian strain (grey), and 3) E stem-anchor (S-A) from MR766 (orange). (1B) SDS PAGE Coomassie gel, lane 1, 1μg purified VLP, and lane 2, 2μg purified VLP, approximately 55kDa. (1C) Western blot, lane 1, 0.25μg purified VLP, and lane 2, 0.5μg purified VLP, approximately 55kDa.
Fig 2
Fig 2. Zika VLP mouse study timeline and serum nAb responses.
(2A) Schematic of experiment design. AG129 mice immunized either with Zika VLP or alum only (100μg) as control (n = 10). (2B) Serum RlucNT50 titers post prime on Study Day 14 and (2C) post boost on Study Day 42 were determined. GMTs for each group are shown by lines and values are given above each group. NT50 titers of individual mice are shown by symbols. The assay LOD was 10 (dashed lines) and titers below LOD are shown as 5. Kruskal-Wallis analysis followed by Dunn’s comparison tests on all pairwise comparisons within each time point were performed and statistical significance levels relative to each alum only group are denoted above the GMT value as: ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, or ns (not significant). Additional significant differences between pairwise VLP groups are shown by bars and significance levels.
Fig 3
Fig 3. Survival and weight loss following lethal ZIKV challenge.
On day 44 of the study, mice were given an SC challenge of ~100 CCID50 of ZIKV strain PRVABC59. (3A) Survival was monitored for 28 days and survival rates for each VLP group were compared to the alum only (100μg) group by Fisher’s exact test with significance levels denoted as: ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, or ns (not significant). (3B) Weight loss was monitored for 16 days starting at day 5 post-challenge. Group mean weight loss is shown as a percentage relative to day 5 post-challenge with error bars denoting group standard error of the mean for ease in viewing. An uninfected control group (n = 5) is shown for comparison and the dotted line indicates zero change. Weight changes were compared on days 7 through 15 by one-way ANOVA, and comparisons to the alum only group were by Dunnett’s multiple comparison tests with significance levels denoted as for Fig 3A.
Fig 4
Fig 4. Protection against ZIKV RNAemia following challenge.
(4A) Serum was collected on day 5 post-challenge (Study Day 49) for quantification of ZIKV genomes by RT-qPCR. Each sample was analyzed against ZIKV RNA and GAPDH RNA standard curves and the ZIKV genome equivalents (GE) relative to GAPDH were determined and Log10 transformed. Serum samples with no detectable ZIKV RNA were assigned a Log10 Relative GE value of -1 for graphing and statistical analysis. Group mean Relative GE levels are shown by line, individual mice shown by symbol, and ‘†’ denotes the individual mice that succumbed to infection. All mice in the alum only (100μg) control group died. The dashed line indicates the highest Log relative GE from the sera of 5 naïve mice (-0.77) and is shown for comparison. Statistical significance relative to the alum only group by Kruskal-Wallis test followed by Dunn’s pairwise comparison tests are denoted as: ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, or ns (not significant). All possible pairwise comparisons were performed and there were no statistically significant differences among VLP groups. (4B) Log10 relative ZIKV GE on Study Day 49 (day 5 post-challenge) were plotted compared to mice surviving (protected) and mice succumbing to infection (Not Protected). Group mean Log10 Relative GE are denoted by line, individual titers by symbols, the maximum naïve mouse value by a dashed line as in Fig 4A, and P value was calculated by Mann-Whitney U test.
Fig 5
Fig 5. Neutralizing antibody levels induced by ZIKV VLP vaccination correlates with protection.
(5A) Comparison of Day 42 RlucNT50 titers in mice that survived (Protected) or died (Not Protected) after lethal ZIKV challenge. GMTs is shown by bars, individual titers by symbols, and assay LOD by dashed line. Significance value shown is from Mann-Whitney U test. (5B) Correlation of Day 49 (day 5 post-challenge) relative ZIKV GE compared to Day 42 RlucNT50 titers for all groups. (5C) Correlation of Day 49 (day 5 post-challenge) relative ZIKV GE compared to Day 42 RlucNT50 titers for the VLP 0.1μg dose group only. Log-transformed values for each serum RlucNT50 titer and Relative GE RNAemia load (symbols based on immunization group), with dashed lines representing the assay LOD for RlucNT50 assay (x-axis) or reference level for maximum naïve mouse Relative ZIKV GE as in Fig 4 (y-axis) are provided. R and P values as well as regression lines shown were determined by least squares regression analyses.
Fig 6
Fig 6. Experimental design and serum neutralizing antibody responses to Zika VLP vaccination in NHPs.
(6A) Rhesus macaques (5 per group) were immunized on the indicated days with a dose titration of VLPs formulated with alum (20μg VLP/300μg alum; 5μg VLP/75μg alum; and 1.25μg VLP/18.8μg alum) or alum only (300μg) (black syringes), test bled (red drops), and challenged (gray syringe) on the days shown. (6B) Serum nAbs measured by PRNT50, (6C) microneutralization (MN50) assay, and (6D) Renilla luciferase (Rluc)-based ZIKV neutralization assay (RlucNT50). Test bleed time points are shown at top for each assay. GMTs are shown by bars, individual titers by symbols, and assay detection limits by dotted lines. Levels of significance between groups are shown by lines and were obtained by performing Kruskal-Wallis and Dunn’s pairwise comparison tests denoted by: ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, or ns (not significant).
Fig 7
Fig 7. Correlation between serum neutralization titers determined by MN50 or RlucNT50 and PRNT50.
(7A) Correlations were evaluated for MN50 compared to PRNT50 and for (7B) RlucNT50 compared to PRNT50 day 56 serum samples, with resultant R and P values shown and non-Log transformed values plotted. Closed circles depict individual monkey serum samples that had detectable titers for both assays and that were included in the regression analysis, while open circles depict sera that were negative for at least one assay and excluded from analysis. R and P values and regression lines shown by least squares regression. Dashed lines show assay detection limits. Any titers below the limit of detection were assigned a value of 5 for graphing and visualization purposes.
Fig 8
Fig 8. Protection against ZIKV viremia following challenge.
Following ZIKV challenge on day 56, blood samples were tested for infectious virus by (8A) plaque assay and (8B) RNAemia by RT-qPCR. GMT for each titer is shown by a line, individual titers by symbols, and plaque assay LLOQ (195 PFU/mL) and LOD (61 PFU/mL) by dashed lines. Note that the RT-qPCR assay does not have an established LLOQ or LOD, so all data are presented and samples with undetectable ZIKV RNA shown at 100 GE/mL. Statistical differences between groups (by Kruskal-Wallis analysis plus Dunn’s multiple comparison tests for all pairwise comparisons) are indicated in the text.
Fig 9
Fig 9. Correlate of protection by ZIKV VLP vaccine.
Day 3 post-challenge ZIKV GE/mL (RNAemia) compared to day 56 nAb titers obtained by (9A) PRNT50 and (9B) RlucNT50 assays are plotted for each animal with immunization groups denoted by symbol. R and P values and regression lines are shown and non-Log transformed values plotted. The dashed line shows the LOD for the RlucNT50 assay, and RNA GE/mL levels that were undetectable were assigned a value of 100.
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