Combination Attenuation Offers Strategy for Live Attenuated Coronavirus Vaccines - PubMed (original) (raw)

Combination Attenuation Offers Strategy for Live Attenuated Coronavirus Vaccines

Vineet D Menachery et al. J Virol. 2018.

Abstract

With an ongoing threat posed by circulating zoonotic strains, new strategies are required to prepare for the next emergent coronavirus (CoV). Previously, groups had targeted conserved coronavirus proteins as a strategy to generate live attenuated vaccine strains against current and future CoVs. With this in mind, we explored whether manipulation of CoV NSP16, a conserved 2'O methyltransferase (MTase), could provide a broad attenuation platform against future emergent strains. Using the severe acute respiratory syndrome-CoV mouse model, an NSP16 mutant vaccine was evaluated for protection from heterologous challenge, efficacy in the aging host, and potential for reversion to pathogenesis. Despite some success, concerns for virulence in the aged and potential for reversion makes targeting NSP16 alone an untenable approach. However, combining a 2'O MTase mutation with a previously described CoV fidelity mutant produced a vaccine strain capable of protection from heterologous virus challenge, efficacy in aged mice, and no evidence for reversion. Together, the results indicate that targeting the CoV 2'O MTase in parallel with other conserved attenuating mutations may provide a platform strategy for rapidly generating live attenuated coronavirus vaccines.IMPORTANCE Emergent coronaviruses remain a significant threat to global public health and rapid response vaccine platforms are needed to stem future outbreaks. However, failure of many previous CoV vaccine formulations has clearly highlighted the need to test efficacy under different conditions and especially in vulnerable populations such as the aged and immunocompromised. This study illustrates that despite success in young models, the 2'O methyltransferase mutant carries too much risk for pathogenesis and reversion in vulnerable models to be used as a stand-alone vaccine strategy. Importantly, the 2'O methyltransferase mutation can be paired with other attenuating approaches to provide robust protection from heterologous challenge and in vulnerable populations. Coupled with increased safety and reduced pathogenesis, the study highlights the potential for 2'O methyltransferase attenuation as a major component of future live attenuated coronavirus vaccines.

Keywords: MERS-CoV; SARS-CoV; aged; coronavirus; live attenuated; vaccine.

Copyright © 2018 American Society for Microbiology.

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Figures

FIG 1

FIG 1

Host response to SARS-CoV dNSP16 CoV mirrors response to wild-type virus at early times. (A) NSP14 exonuclease (ExoN), NSP16 (2′O MTase), envelope (E), and S1 portion of the spike (S) protein sequences of the indicated viruses were aligned, and the sequence identities were extracted from the alignments. A heat map of the sequence identity using SARS-CoV Urbani or MERS-CoV EMC as the reference sequence was constructed using Evolview (

www.evolgenius.info/evolview

). The heat map was further rendered and edited in Adobe Illustrator CC 2017. (B and C) Twenty-week-old C57BL/6 mice mock challenged (PBS, gray) or challenged with SARS-CoV MA15 (WT, black) or SARS-CoV dNSP16 (green) at 105 PFU were examined for weight loss (n > 5 for WT and dNSP16 groups) (B) and lung viral titer (C) (n = 5 per group). (D) PCA analysis of total RNA expression at individual times for WT (blue), dNSP16 (green), and mock (black) groups. Numbers (1, 2, 4, and 7) indicate the time point of the sample. (E) Differentially expressed genes relative to the mock group were used to generate clustered expression heat maps comparing expression in WT and dNSP16 following in vivo infection across the time course. P values were determined using a Student t test *, P < 0.05; ***, P < 0.001.

FIG 2

FIG 2

NSP16 vaccine protects from heterologous challenge. (A to C) Ten-week-old BALB/c mice were vaccinated with 105 PFU of SARS-CoV dNSP16 (blue) or mock vaccinated (PBS), monitored for 28 days, and subsequently challenged with 105 PFU of heterologous SARS-CoV expressing SHC014 spike (SHC014-MA15). Mice were examined over a 4-day time course for weight loss (A), day 4 lung virus titer (B), and day 4 lung hemorrhage (C). (D) Plaque reduction neutralization titers of WT SARS-CoV (solid line) or heterologous SHC014-MA15 (hashed line) in sera of dNSP16-vaccinated mice. P values were determined using a Student t test (***, P < 0.001).

FIG 3

FIG 3

SARS-CoV dNSP16 infection risks virulence in aged mice. (A to C) Twelve-month-old BALB/c mice were challenged with 105 PFU of WT SARS-CoV MA15 (black) or dNSP16 (green) or mock challenged (PBS, gray) and then examined for weight loss (A), survival (B), and lung viral titer (C) over a time course. (D to F) Twelve-month-old BALB/c mice were challenged with 100 PFU of WT SARS-CoV MA15 (black) or dNSP16 (green) or mock challenged (PBS, gray) and then examined for weight loss (D), survival (E), lung viral titer (F), airway resistance (PenH) (G), and midtidal expiratory flow (EF50) (H) as measured by whole-body plethysmography (Buxco, DSI). P values were determined using a Student t test *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 4

FIG 4

SARS-CoV dNSP16 vaccination protects aged mice. (A to C) Twelve-month-old BALB/c mice were vaccinated with 100 PFU of SARS-CoV dNSP16 or mock vaccinated (PBS), monitored for 28 days, and subsequently challenged with 105 PFU of WT SARS-CoV MA15. Mice were examined over a 4-day time course for weight loss (A), survival (B), and viral titer (C). P values were determined using a Student t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

FIG 5

FIG 5

SARS-CoV dNSP16 can revert to virulence. (A to C) Eight RAG−/− mice were infected with 105 PFU of SARS-CoV dNSP16, monitored, and euthanized, and lung tissues were harvested after 30 days. Clarified homogenates were then inoculated into 10-week-old BALB/c mice and individually monitored for weight loss (A), survival (B), and input titer (C) prior to infection. Five of eight RAG−/− homogenates (green) produced significant weight loss and lethality and had detectable input titers. Three RAG−/− homogenates (black) had no disease and no detectable virus.

FIG 6

FIG 6

SARS-CoV dNSP16/ExoN is a viable vaccine and protects from homologous and heterologous challenge. (A) Vero cells were infected at an MOI of 0.01 with WT SARS-CoV MA15 (black), dNSP16 (green), dExoN (gray), or dNSP16/ExoN (red). (B to D) Ten-week-old BALB/c mice were challenged with 104 PFU of SARS-CoV MA15 (black) or dNSP16/ExoN (red) and examined for weight loss (A), lung viral titer (B), and lung hemorrhage (C). (E to G) Ten-month-old BALB/c mice were vaccinated with 104 PFU of SARS-CoV dNSP16/ExoN (red) or mock vaccinated (PBS, black), monitored for 28 days, and subsequently challenged with 105 PFU of WT SARS-CoV MA15 (solid line) or SHC014-MA15 (dotted line). Mice were examined over a 4-day time course for weight loss (E), viral titer (F), and lung hemorrhage (G). (H) Plaque reduction neutralization titers of WT SARS-CoV MA15 (solid) or heterologous SHC014-MA15 (dotted) in sera from dNSP16/ExoN-vaccinated mice. P values were determined using a Student t test (***, P < 0.001).

FIG 7

FIG 7

SARS-CoV dNSP16/ExoN protects aged mice and is cleared from immunocompromised mice. (A to D) Twelve-month-old BALB/c mice were challenged with 100 PFU (solid line/closed circles) or 104 PFU (dotted lines/open circles) of WT SARS-CoV MA15 (black) or dNSP16/ExoN (red) and examined for weight loss (A), survival (B), lung viral titer (C), and lung hemorrhage (D) over the time course. (E and F) Twelve-month-old BALB/c mice were vaccinated with 100 PFU of SARS-CoV dNSP16/ExoN (red) or mock vaccinated (PBS, black), monitored for 28 days, and subsequently challenged with 105 PFU of WT SARS-CoV MA15. Mice were examined over a 4-day time course for weight loss (E) and lung viral titer (F). (G and H) Five RAG−/− mice were infected with 104 PFU of SARS-CoV dNSP16/ExoN, monitored, and euthanized, and lung tissues were harvested after 30 days. Clarified homogenates were then inoculated into 10-week-old BALB/c mice and monitored for weight loss (G) and input titer (H). P values were determined using a Student t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

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