Needs of R&D to fight COVID in Japan
Progress and challenges
Takaji Wakita
National Institute of Infectious Diseases,
JapanDaily number of COVID-19 cases, Japan
Cluster surveillanceCases from Europe

Jan 15First confirmedcase

Dr. Motoi Suzuki

Local governors’
responses

State-of-emergency
Stay home request
Total cases (Sep 12)
74,544 (590 / 1M)
Total deaths (Sep 12)
1,423 (11 / 1M)

Adjusted CFR: 7.2%

Adjusted CFR: 0.9%

Concept of “cluster detection and containment”

Activities during 14 days
before onset✖

✖
✖

COVID-19

Identify possible common
source/opportunity ofSARS-CoV-2 infection

Health Care
Worker✖

COVID-19✖
✖ ✖

Need Apps andmore!

Contact tracing to breaktransmission chain

Health Care
WorkerDr. Tomoe Shimada, NIID, IDSC

Molecular Tracing of COVID-19 cases:
A genome epidemiological study of SARS-CoV-2 using
whole genome sequencing (WGS)
PremiseSARS-CoV-2 genome
Mode of molecular evolution on the SARS-CoV-2 genome:
SNV
single mutation every 2 weeks, in a random manner
Step 1. Link cases or clusters with molecular epidemiology✓ To prove suspected epidemiological links andsupport epi investigations (time, place and person)

2 weeks

2 weeks

Step 2. Inform response decision-making for targetedinterventions
Dr. Makoto Kuroda, Pathogen Genomics Center, NIID

Haplotype network analysis of viral gnomes in
Diamond princess Cruise ship outbreakA

Phylogenetic analysis

B

SNV network analysis

Tsuyoshi Sekizuka et al. PNAS 2020;117:33:20198-20201©2020 by National Academy of SciencesPrimary large peakfrom mid-March

Secondary peakfrom mid-JuneCombination of epidemiological characteristics and whole genome sequenceGeographic
distribution

Age
distribution

Clusters

WGS

Imported/domestic• Multi-source surveillance• Detailed transmission
dynamics• Design of strategy

Identification of anti-SARS-CoV-2 drugs from drugrepositioning
Compounds

Biochemistry

Virology

RIKEN, Hokkaido U.
Kyushu U.

Pharmaceutical companies ( > 30)

in silico, Informatics
RIKEN, AIST, NAIST
Nagahama U.
Tokyo U. Sci.
Synergy plot100"#$%= 0.77! &"#' %=1.18! &10- 1synergy

10-210- 310- 410- 515 1050!

fold-change

Hokkaido UniversityChiba Cancer Research Center
National Institute ofTokyo University of Science
Infectious Diseases
University of Tokyo
National Cancer Center
RIKEN
Kyoto University
Nihon University
Osaka University
Chubu University
Okayama University"#$%=0.35! &"#' %=0.91! &421.7
1.5
10.5
0.3!

10010- 1

85!

10- 2
10- 3

Mathematics
Kyushu U., NCGM
U. Tokyo, Indiana U

10- 4
10- 5

15 1050!

Clinics

AMED・BINDS

Nagasaki U.

Approved Drugs
Unique compounds

Anti-COVID-19
agentsMechanisms of virus
infection/replication

Dr. Koichi Watashi(Dept Virol II, NIID)

Higher antiviral activity of NFV and CEP than the current drugcandidates

Nelfinavir

5 compounds< Antiviral activity >IC50 IC90(mM) (mM)

Viral RNA (log-scale)

306 compounds

Cepharanthine

Concentration (mM)

IC50= 0.77 mM
IC90= 1.18 mM

IC50= 0.35 mM
IC90= 0.91 mM

Remdesivir

1.81 3.89

Favipiravir (Avigan®)> 64 > 64

Ciclesonide (Alvesco®)

3.67 6.82

Hydroxychloroquine (Plaquenil®) 2.04 7.05

Lopinavir (Kaletra®)1.73 3.61

Nelfinavir (Viracept®)0.77 1.18

Cepahanthine (Cepahanthin®)0.35 0.91

Target molecules of NFV and CEP
SARS-CoV-2 Spikeentry

Sepharanthine

replicationassembly
ACE2 (receptor)

SARS-CoV-2
Main Protease

NFV (mM):Nelfinavir

Protease activity (RLU)

Time course
6000(-)
0.1
0.2
0.5
124000
2000
0020

4060

incubation time (min)Synergistic antiviral effect of NFV/CEP combination
Improvement of antiviral therapy → Multidrug treatment

NFV (mM)
01.08
1.30
1.56
1.87
2.24

100

10
1
0.1
0.0100

0.78

1010

1.25

2.00

3.20

5.12

: CEP (mM)"#$%= 0.77! &"#' %=1.18! &-1

Synergy plot
Synergistic effect

10synergy

10- 2-3

10- 4
10- 5

15 1050!

fold-change

viral RNA (%)

SARS-CoV-2 RNA

10-1!

"#$%=0.35! &"#' %=0.91! &4
2
1.7
1.5
10.5
0.3

010

85!

0.00

0.00
05

0.00

Days aftereffectsymptom onset
Days after symptom onset
Estimated antiviralin clinical settings

1015

2025

05

1015

20

25

05

10

15

2025

2.0

2.5

Days after symptom onset

0.75
Pharmacokinetics
in clinical

50!

0.50
85synergy

0.25

1.00

Antiviral effectfold-change

5 10Synergy plot

0.75
Dose-response
curve"#$%= 0.77! &"#' %=1.18! &

1

1.00

Antiviral effect

Antiviral effect

1.00

42
1.7
1.5
10.5
0.3

0.00

0.50
0.25
0.00

0.0

0.5

1.0

1.5

2.0

2.5

0.50
0.25
0.00

0.0

Days after symptom onset

0.75

0.5

1.0

1.5

2.0

2.5

0.0

Days after symptom onset

0.5

1.0

1.5

Days after symptom onset!
!

"#$%=0.35! &"#' %=0.91! &C5
43
2
10

6
5
45
4
Predicted viral dynamics
43
23ー non-treatment
120ー NFV

65456101525 ー209%

7%

1

NFV+CEP
0510152025

Days after symptom onset

Days after symptom onset

CEP (oral)

NFV (oral) + CEP (intravenous)

3
2
1

30

2

510

15

Cumulative viralload load
Cumulativeviral

54400000
3300000
20000021100000
00
5000005

copies/ml

6

Viral load
(copies/ml, log10)!

CEP (intravenous)

Viral load
(copies/ml, log10)

50

Viral load (copies/ml, log)

5 10Viral load
(copies/ml, log10)

1Viral load(copies/ml, log10)

NFV (oral)copies/ml（x105）

B

05101520257%

6virus elimination

5NFV

4Reduction
0 1 2 3 4 5in transmission

322005101520257%

Without
NFV +
NFV
CEP
no-treat
NFV
CEP
NFV
treatment
CEP+CEP
Reduction time for

Improved
treatment
CEP

1Days after infection

18%

NFV +

The paper is already on line
https://www.biorxiv.org/content/10.1101/2020.04.14.039925v1

Clinical study (jRCT2071200023)
Evaluation of Nelfinavir forasymptomatic and mild COVID-19
A multicenter open-label, blinded outcome,randomized controlled trial(Nagasaki University)

Message
Replication inhibitor
Nelfinavir

oral+Entry Inhibitor

Synergistic antiviral effect

Cepharanthine
injection

Replaceable to anoral drug?

・ We identified other approved drugs from ~3100 non-approved compounds

Concerns for SARS-CoV-2 Vaccine Development
・No vaccine for the similar coronaviruses such as SARSCoV or MERS-CoV.

・SARS-CoV-2 need BSL3 facility to produce inactivatedvaccine.
・Low growth rate of the virus （2x10^7TCID50/ml) wasreported.
・Disease enhancement by the vaccine is reported inSARS and MERS vaccine research.

H Hasegawa, T Suzuki, N Nagata, N Iwata, NIID

Production of SARS-CoV2 Spike protein byBaculovirus Expression Vector System
Baculovirus Expression Vector System: BEVS
Coronavirus

Recombinantbaculovirus

sequenceLarge
scale cell
cultureInsect cells

BEVS System

Examples of vaccine produced by BEVS Human papillomavirus vaccine
 Recombinant Influenza HA vaccinerS vaccine

Eosinophilic infiltration after SARS-CoV
infection to immunized miceControlImmunizedIwata-Yoshikawa et al., J Virol, 2014. 88:8597-614

2020/9/21

Th1Th2
Th1/Th2 balance may affect the severity of eosinophilic
infiltration in immunized and infected mice, which
exacerbate the pneumonia by SARS-CoV.

16

TLR agonist enhance the immunogenicity ofvaccine and reduce vaccine derived side effects

3dpi

10dpi

Iwata-Yoshikawa et al., J Virol, 2014. 88:8597-614
Sekimukai et al., Microbiology and Immunology, 2020
2020/9/21

17

Summary・R&D is most important to fight COVID-19
・ Viral genome epidemiology of SARS-CoV-2 may support

cluster surveillance
・Novel anti-viral therapy is necessary using combination ofcompounds with different mode of actions
・Vaccine development is rapidly ongoing, however, vaccine
efficacy and safety are most important