An Attenuated Zika Virus Encoding Non-Glycosylated Envelope (E) and Non-Structural Protein 1 (NS1) Confers Complete Protection against Lethal Challenge in a Mouse Model

doi:10.3390/vaccines7030112

. 2019 Sep 12;7(3):112.

doi: 10.3390/vaccines7030112.

An Attenuated Zika Virus Encoding Non-Glycosylated Envelope (E) and Non-Structural Protein 1 (NS1) Confers Complete Protection against Lethal Challenge in a Mouse Model

Arun S Annamalai^{1

2}, Aryamav Pattnaik^{3

4}, Bikash R Sahoo^{5

6}, Zack P Guinn⁷, Brianna L Bullard^{8

9}, Eric A Weaver^{10

11}, David Steffen¹², Sathish Kumar Natarajan^{13

14}, Thomas M Petro^{15

16}, Asit K Pattnaik^{17

18}

Affiliations

¹ School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, NE 68583, USA. tiger05009@gmail.com.
² Nebraska Center for Virology, University of Nebraska-Lincoln, Lincoln, NE 68583, USA. tiger05009@gmail.com.
³ School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, NE 68583, USA. aryamavpattnaik@ymail.com.
⁴ Nebraska Center for Virology, University of Nebraska-Lincoln, Lincoln, NE 68583, USA. aryamavpattnaik@ymail.com.
⁵ School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, NE 68583, USA. bsahoo@huskers.unl.edu.
⁶ Nebraska Center for Virology, University of Nebraska-Lincoln, Lincoln, NE 68583, USA. bsahoo@huskers.unl.edu.
⁷ Department of Oral Biology, University of Nebraska Medical Center, Lincoln, NE 68583, USA. zack.guinn@huskers.unl.edu.
⁸ Nebraska Center for Virology, University of Nebraska-Lincoln, Lincoln, NE 68583, USA. bbullard@huskers.unl.edu.
⁹ School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, NE 68583, USA. bbullard@huskers.unl.edu.
¹⁰ Nebraska Center for Virology, University of Nebraska-Lincoln, Lincoln, NE 68583, USA. eweaver2@unl.edu.
¹¹ School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, NE 68583, USA. eweaver2@unl.edu.
¹² School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, NE 68583, USA. dsteffen1@unl.edu.
¹³ Nebraska Center for Virology, University of Nebraska-Lincoln, Lincoln, NE 68583, USA. snatarajan2@unl.edu.
¹⁴ Department of Nutrition and Health Sciences, University of Nebraska-Lincoln, Lincoln, NE 68583, USA. snatarajan2@unl.edu.
¹⁵ Nebraska Center for Virology, University of Nebraska-Lincoln, Lincoln, NE 68583, USA. tpetro@unmc.edu.
¹⁶ Department of Oral Biology, University of Nebraska Medical Center, Lincoln, NE 68583, USA. tpetro@unmc.edu.
¹⁷ School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, NE 68583, USA. apattnaik2@unl.edu.
¹⁸ Nebraska Center for Virology, University of Nebraska-Lincoln, Lincoln, NE 68583, USA. apattnaik2@unl.edu.

PMID: 31547297
PMCID: PMC6789518
DOI: 10.3390/vaccines7030112

An Attenuated Zika Virus Encoding Non-Glycosylated Envelope (E) and Non-Structural Protein 1 (NS1) Confers Complete Protection against Lethal Challenge in a Mouse Model

Arun S Annamalai et al. Vaccines (Basel). 2019.

. 2019 Sep 12;7(3):112.

doi: 10.3390/vaccines7030112.

Authors

Affiliations

¹ School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, NE 68583, USA. tiger05009@gmail.com.
² Nebraska Center for Virology, University of Nebraska-Lincoln, Lincoln, NE 68583, USA. tiger05009@gmail.com.
³ School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, NE 68583, USA. aryamavpattnaik@ymail.com.
⁴ Nebraska Center for Virology, University of Nebraska-Lincoln, Lincoln, NE 68583, USA. aryamavpattnaik@ymail.com.
⁵ School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, NE 68583, USA. bsahoo@huskers.unl.edu.
⁶ Nebraska Center for Virology, University of Nebraska-Lincoln, Lincoln, NE 68583, USA. bsahoo@huskers.unl.edu.
⁷ Department of Oral Biology, University of Nebraska Medical Center, Lincoln, NE 68583, USA. zack.guinn@huskers.unl.edu.
⁸ Nebraska Center for Virology, University of Nebraska-Lincoln, Lincoln, NE 68583, USA. bbullard@huskers.unl.edu.
⁹ School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, NE 68583, USA. bbullard@huskers.unl.edu.
¹⁰ Nebraska Center for Virology, University of Nebraska-Lincoln, Lincoln, NE 68583, USA. eweaver2@unl.edu.
¹¹ School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, NE 68583, USA. eweaver2@unl.edu.
¹² School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, NE 68583, USA. dsteffen1@unl.edu.
¹³ Nebraska Center for Virology, University of Nebraska-Lincoln, Lincoln, NE 68583, USA. snatarajan2@unl.edu.
¹⁴ Department of Nutrition and Health Sciences, University of Nebraska-Lincoln, Lincoln, NE 68583, USA. snatarajan2@unl.edu.
¹⁵ Nebraska Center for Virology, University of Nebraska-Lincoln, Lincoln, NE 68583, USA. tpetro@unmc.edu.
¹⁶ Department of Oral Biology, University of Nebraska Medical Center, Lincoln, NE 68583, USA. tpetro@unmc.edu.
¹⁷ School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, NE 68583, USA. apattnaik2@unl.edu.
¹⁸ Nebraska Center for Virology, University of Nebraska-Lincoln, Lincoln, NE 68583, USA. apattnaik2@unl.edu.

PMID: 31547297
PMCID: PMC6789518
DOI: 10.3390/vaccines7030112

Abstract

Zika virus (ZIKV), a mosquito-transmitted flavivirus, emerged in the last decade causing serious human diseases, including congenital microcephaly in newborns and Guillain-Barré syndrome in adults. Although many vaccine platforms are at various stages of development, no licensed vaccines are currently available. Previously, we described a mutant MR766 ZIKV (m2MR) bearing an E protein mutation (N154A) that prevented its glycosylation, resulting in attenuation and defective neuroinvasion. To further attenuate m2MR for its potential use as a live viral vaccine, we incorporated additional mutations into m2MR by substituting the asparagine residues in the glycosylation sites (N130 and N207) of NS1 with alanine residues. Examination of pathogenic properties revealed that the virus (m5MR) carrying mutations in E (N154A) and NS1 (N130A and N207A) was fully attenuated with no disease signs in infected mice, inducing high levels of humoral and cell-mediated immune responses, and protecting mice from subsequent lethal virus challenge. Furthermore, passive transfer of sera from m5MR-infected mice into naïve animals resulted in complete protection from lethal challenge. The immune sera from m5MR-infected animals neutralized both African and Asian lineage viruses equally well, suggesting that m5MR virus could be developed as a potentially broad live virus vaccine candidate.

Keywords: NS1 protein; Zika virus; attenuation; glycosylation; vaccine candidate.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

**Figure 1**
Characterization of the envelope (E)/non-structural protein 1 (NS1) glycosylation mutant viruses. (A) The ZIKV (Zika virus) genome and the encoded polyprotein, showing glycosylation sites in E (N154) and NS1 (N130 and N207). The asparagine residue at each of these sites was mutated to an alanine residue. (B) Two micrograms of in vitro transcribed RNA, from each of the mutant clones or the wt(rMR) clone, were transfected into Vero cells in 6-well plates. After 5 days, the transfected cells were processed for immunofluorescence staining using 4G2 monoclonal antibody to detect the E protein. Scale bar: 400 μm. Multistep growth of rMR, m2MR, m3MR, m4MR, and m5MR viruses in Vero (C) and C6/36 (D) cells. The data show mean values with error bars representing standard deviation from three independent experiments. Mann–Whitney test and Kruskal–Wallis test were used to determine significance between m2MR and other mutant viruses at different times post-infection. **, p < 0.01; *, p < 0.05; ns, non-significant.

**Figure 2**
Glycosylation status and secretion of NS1 protein from the mutant virus-infected cells. Cells in 6-well plates were mock-infected (M) or infected with the mutant viruses (shown on top) at an MOI (multiplicity of infection) of 1. Cell culture supernatants were collected at 48 dpi, and proteins from an equal volume of supernatants were concentrated by acetone precipitation, digested with PNGase F (+), or left undigested (−) and analyzed by Western blotting using anti-NS1 antibody. Relative migration of molecular mass markers (in kDa) is shown on the left.

**Figure 3**
m5MR virus is attenuated in mice. (A) Scheme of virus inoculation and challenge study in three to four-week-old *Ifnar1*^−/− A129 mice. Mice were injected subcutaneously (s.c.) with 1000 PFU (plaque-forming unit) of rMR (n = 6), m2MR (n = 12), and m5MR (n = 12) viruses on day 0. PBS was injected in the control group (n = 6). Clinical scores at 6 dpi for rMR, m2MR, and m5MR virus-infected groups (B) and 10 dpi for m2MR and m5MR virus-infected groups (C). (D) Weight loss and (E) Percent (%) survival of mice in infected or PBS control groups. Viremia in serum at 3 dpi and 6 dpi as measured by genome copy numbers (F) and infectious virus titers (G). Data presented are combined data from two independent experiments. Unpaired Student’s t-test (two-tailed) (for panel (B,C)) and Mann–Whitney test (for panel (F,G)) were used to determine significance between groups. ****, p < 0.0001; ***, p < 0.001; **, p < 0.01; ns, non-significant.

**Figure 4**
The mutant viruses replicate less efficiently in the brain of infected animals. Viral genome copy number (A) and infectious virus titer (B) in the brain of animals infected with rMR (n = 6), m2MR (n = 6), and m5MR (n = 6) viruses at 6 dpi. Infectious virus titers from the spleen (C) and liver (D) of the animals. Unpaired Student’s t-test (two-tailed) was used to determine significance between the groups. ****, p < 0.0001; **, p < 0.01. (E) Hematoxylin and eosin staining of sections of the cerebrum of animals inoculated with PBS, or infected with rMR, m2MR, or m5MR viruses. Representative images of the cerebrum (40x) from animals in each group are shown. Prominent perivascular cuffing in rMR- and m2MR-infected samples are shown with white arrows. Scale bar (at bottom left), 60 µm.

**Figure 5**
Mutant virus-infected animals are protected from lethal challenge. (A) Clinical scores, (B) weight loss, and (C) survival of the animals injected with PBS (n = 6), or infected with m2MR (n = 5), m5MR (n = 6), and after 28 days, challenged s.c. with 10,000 PFU rMR virus. (D) rMR viremia in the plasma following challenge. The horizontal dashed line shows the limit of detection. Unpaired Student’s t-test (two-tailed) for panel A was used to determine significance. ****, p < 0.0001; **, p < 0.01.

**Figure 6**
Humoral and cell-mediated immune responses in mutant virus-infected animals. (A) The viral neutralization titers in serum [expressed as reciprocal of 50% plaque reduction neutralization test (PRNT₅₀) values] from PBS-treated or m2MR- or m5MR-infected animals at 28 dpi. (B) Antibody isotyping in sera by ELISA for PBS-injected or m5MR virus-infected mice. (C) The spleen from m5MR virus-infected (n = 5) or PBS-injected (n = 4) A129 animals were collected at 28 dpi, and the cells were stimulated with ZIKV E specific peptides for CD4+ and CD8+ T cells. ELISPOT was performed to determine spot-forming cells (SFC) per million splenocytes. Mann–Whitney test (for panel (A, B)) and Unpaired Student’s t-test (two-tailed) (for panel (C)) were used to determining significance between groups. ****, p < 0.0001; ***, p < 0.001; **, p < 0.01; ns, non-significant.

**Figure 7**
Passive transfer of sera from m5MR-infected animals protects naïve animals from lethal challenge. (A) The antibody neutralization titer following 2 hpt (0d) and 7-day pt (7d) of pooled sera in A129 mice. (B) Clinical scores, (C) weight loss, and (D) survival of animals challenged with a lethal dose of rMR virus following passive transfer of sera. PRNT₅₀ titer of serum samples from individual animals infected with m5MR virus against PRVABC59 and MEX1–7 strains of ZIKV. The antibody titers are expressed as reciprocal of PRNT₅₀ values. Unpaired Student’s t-test (two-tailed) for panel (B) and Mann–Whitney test for panel (E) were used to determine significance between the groups. ns, non-significant. hpt: hour post transfer of serum. ****, p < 0.0001; ns, non-significant.

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References

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[1] Hills S.L., Russell K., Hennessey M., Williams C., Oster A.M., Fischer M., Mead P. Transmission of Zika Virus Through Sexual Contact with Travelers to Areas of Ongoing Transmission—Continental United States, 2016. MMWR Morb. Mortal. Wkly. Rep. 2016;65:215–216. doi: 10.15585/mmwr.mm6508e2. - DOI - PubMed

[2] Hills S.L., Russell K., Hennessey M., Williams C., Oster A.M., Fischer M., Mead P. Transmission of Zika Virus Through Sexual Contact with Travelers to Areas of Ongoing Transmission—Continental United States, 2016. MMWR Morb. Mortal. Wkly. Rep. 2016;65:215–216. doi: 10.15585/mmwr.mm6508e2. - DOI - PubMed

[3] Musso D., Gubler D.J. Zika Virus. Clin. Microbiol. Rev. 2016;29:487–524. doi: 10.1128/CMR.00072-15. - DOI - PMC - PubMed

[4] Musso D., Gubler D.J. Zika Virus. Clin. Microbiol. Rev. 2016;29:487–524. doi: 10.1128/CMR.00072-15. - DOI - PMC - PubMed

[5] Sharma A., Lal S.K. Zika Virus: Transmission, Detection, Control, and Prevention. Front. Microbiol. 2017;8:110. doi: 10.3389/fmicb.2017.00110. - DOI - PMC - PubMed

[6] Sharma A., Lal S.K. Zika Virus: Transmission, Detection, Control, and Prevention. Front. Microbiol. 2017;8:110. doi: 10.3389/fmicb.2017.00110. - DOI - PMC - PubMed

[7] Bailey M.J., Duehr J., Dulin H., Broecker F., Brown J.A., Arumemi F.O., Bermudez Gonzalez M.C., Leyva-Grado V.H., Evans M.J., Simon V., et al. Human antibodies targeting Zika virus NS1 provide protection against disease in a mouse model. Nat. Commun. 2018;9:4560. doi: 10.1038/s41467-018-07008-0. - DOI - PMC - PubMed

[8] Bailey M.J., Duehr J., Dulin H., Broecker F., Brown J.A., Arumemi F.O., Bermudez Gonzalez M.C., Leyva-Grado V.H., Evans M.J., Simon V., et al. Human antibodies targeting Zika virus NS1 provide protection against disease in a mouse model. Nat. Commun. 2018;9:4560. doi: 10.1038/s41467-018-07008-0. - DOI - PMC - PubMed

[9] Bogoch I.I., Brady O.J., Kraemer M.U.G., German M., Creatore M.I., Kulkarni M.A., Brownstein J.S., Mekaru S.R., Hay S.I., Groot E., et al. Anticipating the international spread of Zika virus from Brazil. Lancet. 2016;387:335–336. doi: 10.1016/S0140-6736(16)00080-5. - DOI - PMC - PubMed

[10] Bogoch I.I., Brady O.J., Kraemer M.U.G., German M., Creatore M.I., Kulkarni M.A., Brownstein J.S., Mekaru S.R., Hay S.I., Groot E., et al. Anticipating the international spread of Zika virus from Brazil. Lancet. 2016;387:335–336. doi: 10.1016/S0140-6736(16)00080-5. - DOI - PMC - PubMed

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An Attenuated Zika Virus Encoding Non-Glycosylated Envelope (E) and Non-Structural Protein 1 (NS1) Confers Complete Protection against Lethal Challenge in a Mouse Model

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An Attenuated Zika Virus Encoding Non-Glycosylated Envelope (E) and Non-Structural Protein 1 (NS1) Confers Complete Protection against Lethal Challenge in a Mouse Model

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Abstract

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