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Embryotoxic impact of Zika virus in a rhesus macaque in vitro implantation model†.

ABSTRACT: Zika virus (ZIKV) infection is associated with adverse pregnancy outcomes in humans, and infection in the first trimester can lead to miscarriage and stillbirth. Vertical and sexual transmissions of ZIKV have been demonstrated, yet the impact of infection during the initial stages of pregnancy remains unexplored. Here we defined the impact of ZIKV on early embryonic and placental development with a rhesus macaque model. During in vitro fertilization (IVF), macaque gametes were inoculated with a physiologically relevant dose of 5.48log10 plaque-forming units (PFU) of Zika virus/H.sapiens-tc/PUR/2015/PRVABC59_v3c2. Exposure at fertilization did not alter blastocyst formation rates compared to controls. To determine the impact of ZIKV exposure at implantation, hatched blastocysts were incubated with 3.26log10, 4.26log10, or 5.26log10 PFU, or not exposed to ZIKV, followed by extended embryo culture for 10 days. ZIKV exposure negatively impacted attachment, growth, and survival in comparison to controls, with exposure to 5.26log10 PFU ZIKV resulting in embryonic degeneration by day 2. Embryonic secretion of pregnancy hormones was lower in ZIKV-exposed embryos. Increasing levels of infectious virus were detected in the culture media post-exposure, suggesting that the trophectoderm is susceptible to productive ZIKV infection. These results demonstrate that ZIKV exposure severely impacts the zona-free blastocyst, whereas exposure at the time of fertilization does not hinder blastocyst formation. Overall, early stages of pregnancy may be profoundly sensitive to infection and pregnancy loss, and the negative impact of ZIKV infection on pregnancy outcomes may be underestimated.

SUBMITTER: Block LN

PROVIDER: S-EPMC7124964 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundZika 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 findingsWe 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/significanceThese 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.

Project description:We sought to determine the predominant cell type (macrophage, T lymphocyte, B lymphocyte, mast cell) within the area of implantation of the prototypical polypropylene mesh, Gynemesh PS (Ethicon, Somerville, NJ); and to determine the phenotypic profile (M1 proinflammatory, M2 antiinflammatory) of the macrophage response to 3 different polypropylene meshes: Gynemesh PS (Ethicon), and 2 lower-weight, higher-porosity meshes, UltraPro (Ethicon) and Restorelle (Coloplast, Humblebaek, Denmark).Sacrocolpopexy was performed following hysterectomy in rhesus macaques. Sham-operated animals served as controls. At 12 weeks postsurgery, the vagina-mesh complex was excised and the host inflammatory response was evaluated. Hematoxylin and eosin was used to perform routine histomorphologic evaluation. Identification of leukocyte (CD45(+)) subsets was performed by immunolabeling for CD68 (macrophage), CD3 (T lymphocyte), CD20 (B lymphocyte), and CD117 (mast cell). M1 and M2 macrophage subsets were identified using immunolabeling (CD86(+) and CD206(+), respectively), and further evaluation was performed using enzyme-linked immunosorbent assay for 2 M1 (tumor necrosis factor-alpha and interleukin [IL]-12) and 2 M2 (IL-4 and IL-10) cytokines.Histomorphologic evaluation showed a dense cellular response surrounding each mesh fiber. CD45(+) leukocytes accounted for 21.4 ± 5.4% of total cells within the perimesh area captured in a ×20 field, with macrophages as the predominant leukocyte subset (10.5 ± 3.9% of total cells) followed by T lymphocytes (7.3 ± 1.7%), B lymphocytes (3.0 ± 1.2%), and mast cells (0.2 ± 0.2%). The response was observed to be more diffuse with increasing distance from the fiber surface. Few leukocytes of any type were observed in sham-operated animals. Immunolabeling revealed polarization of the macrophage response toward the M1 phenotype in all mesh groups. However, the ratio of M2:M1 macrophages was increased in the fiber area in UltraPro (P = .033) and Restorelle (P = .016) compared to Gynemesh PS. In addition, a shift toward increased expression of the antiinflammatory cytokine IL-10 was observed in Restorelle as compared to Gynemesh PS (P = .011).The host response to mesh consists predominantly of activated, proinflammatory M1 macrophages at 12 weeks postsurgery. However, this response is attenuated with implantation of lighter-weight, higher-porosity mesh. While additional work is required to establish causal relationships, these results suggest a link among the host inflammatory response, mesh textile properties, and clinical outcomes in the repair of pelvic organ prolapse.

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Embryotoxic impact of Zika virus in a rhesus macaque in vitro implantation model†.

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