Intranasal inoculation of SARS-CoV-2 leads to fatal neuroinvasion in K18-hACE2 transgenic mice, limiting the usefulness of this model. Aerosol exposure to SARS-CoV-2 causes robust respiratory infection and anosmia without fatal neuroinvasion, providing a better model for studying disease pathogenesis and drug discovery. Preliminary data also shows the aerosol model induces lung inflammation and reduces immune responses upon antiviral treatment.

1. Welcome!
Valeria Fumagalli, PhD
Dynamics of Immune Responses
San Raffaele University
Postdoctoral Research Fellow
Nancy Mounogou Kouassi, PhD
Viral Zoonoses - One Health
Leibniz Institute of Virology
Postdoctoral Fellow
Novel Animal Models and
Methods for Studying COVID-19:
Golden Hamsters and Aerosolized
SARS-CoV-2 Delivery

2. 4 August 2022
Valeria Fumagalli
Dynamics of Immune Responses
Administration of aerosolized SARS-CoV-2 to
K18-hACE2 mice uncouples respiratory
infection from fatal neuroinvasion
2
Buxco Webinar

3. Mirela
Kuka
Micol
Rava’
Pietro
Di Lucia
Chiara
Perucchini
Eleonora
Sala
Davide
Marotta
Elisa
Bono
Leonardo
Gisutini
Chiara Laura
Federica Moalli
Francesco Andreata
Cristian Beccaria
Xenia Ficht
Caroline Kruger
Keigo Kawashima
San Raffale:
José M. Garcia-Manteiga, Center for Omics Sciences
Patrizia D’Adamo, Animal behavior Facility
Vania Broccoli, Division of Neuroscience
Alessandro Sessa, Division of Neuroscience
Marco Bianchi, Chromatin dynamics Unit
Lorenza Ronfani, CFCM
Alessio Cantore, TIGET
INGM:
De Francesco Raffaele
Lorena Donnici
Lara Manganaro
Università degli Studi di Napoli Federico II:
Vincenzo Summa
Rolando Cannalire
Bianca Partini
Valentina Venzin
Marta Mainetti
Marta Mangione
Giulia Nosetto
Maria Nelli
Rosanna Santella
Matteo Iannacone
Luca Guidotti
Marta
Grillo
Collaborators & Funding
Acknowledgements

4. Results
K18-hACE2
Intranasal Injection (IN)
McCray et al., 2007
SARS-CoV-2 (D614G)
1x105 TCID50 (25 ul)

5. V. Fumagalli et al., Sci. Immunol., 10.1126/sciimmunol.abl9929 (2021)
Results
0 2 4 6
75
80
85
90
95
100
105
110
Days post infection
Percentage
of
initial
weight
(%)
SARS CoV-2 IN (n = 17-29)
PBS (n = 9-11)
***
***
0 2 4 6
0
5
10
15
Days post infection
Clinical
Score
SARS CoV-2 IN (n = 16)
PBS (n = 8)
***
***
0 2 4 6
0
20
40
60
80
100
Days post infection
Survival
(%)
SARS CoV-2 IN (n = 32)
PBS (n = 26)
***
K18-hACE2
Intranasal Injection (IN)
McCray et al., 2007
SARS-CoV-2 (D614G)
1x105 TCID50 (25 ul)

6. V. Fumagalli et al., Sci. Immunol., 10.1126/sciimmunol.abl9929 (2021)
Results
0 2 4 6
75
80
85
90
95
100
105
110
Days post infection
Percentage
of
initial
weight
(%)
SARS CoV-2 IN (n = 17-29)
PBS (n = 9-11)
***
***
0 2 4 6
0
5
10
15
Days post infection
Clinical
Score
SARS CoV-2 IN (n = 16)
PBS (n = 8)
***
***
PBS
SARS-CoV-2 IN
D3 post CoV-2 D6 post CoV-2
0 2 4 6
0
20
40
60
80
100
Days post infection
Survival
(%)
SARS CoV-2 IN (n = 32)
PBS (n = 26)
***
K18-hACE2
Intranasal Injection (IN)
McCray et al., 2007
SARS-CoV-2 (D614G)
1x105 TCID50 (25 ul)

7. V. Fumagalli et al., Sci. Immunol., 10.1126/sciimmunol.abl9929 (2021)
Results
0 2 4 6
75
80
85
90
95
100
105
110
Days post infection
Percentage
of
initial
weight
(%)
SARS CoV-2 IN (n = 17-29)
PBS (n = 9-11)
***
***
0 2 4 6
0
5
10
15
Days post infection
Clinical
Score
SARS CoV-2 IN (n = 16)
PBS (n = 8)
***
***
PBS
SARS-CoV-2 IN
D3 post CoV-2 D6 post CoV-2
by Broccoli V. and Sessa A.
0 2 4 6
0
20
40
60
80
100
Days post infection
Survival
(%)
SARS CoV-2 IN (n = 32)
PBS (n = 26)
***
K18-hACE2
Intranasal Injection (IN)
McCray et al., 2007
SARS-CoV-2 (D614G)
1x105 TCID50 (25 ul)

8. V. Fumagalli et al., Sci. Immunol., 10.1126/sciimmunol.abl9929 (2021)
0 2 4 6
75
80
85
90
95
100
105
110
Days post infection
Percentage
of
initial
weight
(%)
SARS CoV-2 IN (n = 17-29)
PBS (n = 9-11)
***
***
0 2 4 6
0
5
10
15
Days post infection
Clinical
Score
SARS CoV-2 IN (n = 16)
PBS (n = 8)
***
***
PBS
SARS-CoV-2 IN
D3 post CoV-2 D6 post CoV-2
by Broccoli V. and Sessa A.
0 2 4 6
0
20
40
60
80
100
Days post infection
Survival
(%)
SARS CoV-2 IN (n = 32)
PBS (n = 26)
***
D3 post CoV-2 D6 post CoV-2
PBS
SARS-CoV-2 IN
K18-hACE2
Intranasal Injection (IN)
McCray et al., 2007
SARS-CoV-2 (D614G)
1x105 TCID50 (25 ul)
Intranasal inoculation of SARS-CoV-2 leads to fatal
neuroinvasion in K18-hACE2 Tg mice
Results

9. Pitfalls and Solutions
High Central Nervous System infection leads to fatal encephalitis, which
rarely occurs in patients with COVID-19

10. Pitfalls and Solutions
High Central Nervous System infection leads to fatal encephalitis, which
rarely occurs in patients with COVID-19
Limits the usefulness of these mouse models,
hampering studies on disease pathogenesis (i.e., long-term consequences) as well as on drug discovery.

11. High Central Nervous System infection leads to fatal encephalitis, which
rarely occurs in patients with COVID-19
Limits the usefulness of these mouse models,
hampering studies on disease pathogenesis (i.e., long-term consequences) as well as on drug discovery.
Pitfalls and Solutions

12. Buxco Inhalation Exposure System - Data Sciences International
Novel COVID-19 platform: nose-only inhalation tower system
Controller
Mouse port Temperature / Humidity probe
Nebulizer head
2,5 - 4 mm
Tower balance
Pressure -0.5 mmHg
Method

13. V. Fumagalli et al., Sci. Immunol., 10.1126/sciimmunol.abl9929 (2021)
Results
0 2 4 6
0
20
40
60
80
100
Days post infection
Survival
(%)
SARS CoV-2 IN (n = 32)
SARS-CoV-2 AR (n = 26)
***
0 2 4 6
0
5
10
15
Days post infection
Clinical
Score
SARS-CoV-2 IN (n = 16)
SARS CoV-2 AR (n = 16)
***
***
0 2 4 6
75
80
85
90
95
100
105
110
Days post infection
Percentage
of
initial
weight
(%)
SARS CoV-2 IN (n = 17-29)
SARS CoV-2 AR (n = 9-23)
PBS (n = 9-11)
**
**

14. V. Fumagalli et al., Sci. Immunol., 10.1126/sciimmunol.abl9929 (2021)
Results
0 2 4 6
0
20
40
60
80
100
Days post infection
Survival
(%)
SARS CoV-2 IN (n = 32)
SARS-CoV-2 AR (n = 26)
***
0 2 4 6
0
5
10
15
Days post infection
Clinical
Score
SARS-CoV-2 IN (n = 16)
SARS CoV-2 AR (n = 16)
***
***
0 2 4 6
75
80
85
90
95
100
105
110
Days post infection
Percentage
of
initial
weight
(%)
SARS CoV-2 IN (n = 17-29)
SARS CoV-2 AR (n = 9-23)
PBS (n = 9-11)
**
**
D3 post CoV-2 D6 post CoV-2
SARS-CoV-2 IN
SARS-CoV-2 AR

15. V. Fumagalli et al., Sci. Immunol., 10.1126/sciimmunol.abl9929 (2021)
0 2 4 6
0
20
40
60
80
100
Days post infection
Survival
(%)
SARS CoV-2 IN (n = 32)
SARS-CoV-2 AR (n = 26)
***
0 2 4 6
0
5
10
15
Days post infection
Clinical
Score
SARS-CoV-2 IN (n = 16)
SARS CoV-2 AR (n = 16)
***
***
0 2 4 6
75
80
85
90
95
100
105
110
Days post infection
Percentage
of
initial
weight
(%)
SARS CoV-2 IN (n = 17-29)
SARS CoV-2 AR (n = 9-23)
PBS (n = 9-11)
**
**
D3 post CoV-2 D6 post CoV-2
SARS-CoV-2 IN
SARS-CoV-2 AR
D3 post CoV-2 D6 post CoV-2
SARS-CoV-2 IN
SARS-CoV-2 AR
PBS
Aerosolized SARS-CoV-2
uncouples respiratory infection from fatal neuroinvasion
Results

16. V. Fumagalli et al., Sci. Immunol., 10.1126/sciimmunol.abl9929 (2021)
D3 post CoV-2 D6 post CoV-2
PBS
SARS-CoV-2 IN
SARS-CoV-2 AR
Results

17. V. Fumagalli et al., Sci. Immunol., 10.1126/sciimmunol.abl9929 (2021)
D3 post CoV-2 D6 post CoV-2
PBS
SARS-CoV-2 IN
SARS-CoV-2 AR
Patrizia D’Adamo
PBS
Female
bedding
Male
bedding
Male
mouse
0
10
20
30
40
Sniffing
time
(sec)
Day 3 Day 6 Day 3 Day 6
***
n.d.
Results

18. V. Fumagalli et al., Sci. Immunol., 10.1126/sciimmunol.abl9929 (2021)
D3 post CoV-2 D6 post CoV-2
PBS
SARS-CoV-2 IN
SARS-CoV-2 AR
Patrizia D’Adamo
PBS
SARS-CoV-2 IN
SARS-CoV-2 AR
Female
bedding
Male
bedding
Male
mouse
0
10
20
30
40
Sniffing
time
(sec)
Day 3 Day 6 Day 3 Day 6
***
n.d.
Results

19. V. Fumagalli et al., Sci. Immunol., 10.1126/sciimmunol.abl9929 (2021)
D3 post CoV-2 D6 post CoV-2
PBS
SARS-CoV-2 IN
SARS-CoV-2 AR
Patrizia D’Adamo
PBS
SARS-CoV-2 IN
SARS-CoV-2 AR
D3 post CoV-2 D6 post CoV-2
PBS
SARS-CoV-2 IN
SARS-CoV-2 AR
Female
bedding
Male
bedding
Male
mouse
0
10
20
30
40
Sniffing
time
(sec)
Day 3 Day 6 Day 3 Day 6
***
n.d.
Aerosol exposure to SARS-CoV-2 leads to anosmia
in the absence of Central Nervous System infection
Results

20. Unpublished
Mona Khan et al, Cell (184), 2021
OLFACTORY
BULBS
OLFACTORY
EPITHELIUM
Olfactory epithelium is the main target
for aerosolized SARS-CoV-2
Results

21. K18-hACE2
d0
Analyses
(d18)
SARS-CoV-2
2x10^5
d1
Veichle
Antiviral X
d2 d3
X X
X X
X X
X X
X X
X X
Veichle X X X X X X
-
+
+
P.O.
AR
Anlyses
Peripheral Blood
Day 7
Preliminary and Confidential data
Università degli studi di Napoli Federico II
Vincenzo Summa
Rolando Cannalire
Results

22. Preliminary and Confidential data
Università degli studi di Napoli Federico II
Vincenzo Summa
Rolando Cannalire
K18-hACE2
d0
Analyses
(d18)
SARS-CoV-2
2x10^5
d1
Veichle
Antiviral X
d2 d3
X X
X X
X X
X X
X X
X X
Veichle X X X X X X
-
+
+
P.O.
AR
Anlyses
Peripheral Blood
Day 7
IFN-g
TNF-a
Vehicle Antiviral X
IFNg TNF IFNg TNF GzB
0.0
0.5
1.0
2
3
4
5
6
%
CD8
T
cell
/
PB
Vehicle no infected
Vehicle infected
Antiviral X
D7 post CoV-2
Immune response is reduced upon antiviral treatment
Results

23. Intranasal inoculation
of SARS-CoV-2
Aerosol exposure
to SARS-CoV-2
Clinical picture of encephalitis and
pneumonia, resulting in death after 5-6
days post infection
Robust respiratory infection, anosmia, lung
inflammation, and fibrin deposition without
fatal neuroinvasion.
Conclusions

24. Preliminary and Confidential data
n
o
n
i
n
f
e
c
t
e
d
D
a
y
3
D
a
y
6
102
103
104
105
106
Lung
CoV-2
RNA
/
ng
tot
RNA
n
o
n
i
n
f
e
c
t
e
d
D
a
y
3
D
a
y
6
102
103
104
105
106
NT
CoV-2
RNA
/
ng
tot
RNA
n
o
n
i
n
f
e
c
t
e
d
D
a
y
3
D
a
y
6
102
103
104
105
106
BRAIN
RNA
copy
n
/
ng
tot
RNA
Marco Bianchi, HSR
Raffaele De Francesco, INGM
hy-ACE2
hyACE2 exposed to aerosolized SARS-CoV-2
-4
-3
-2
-1
Lung
Exon
2-3
ACE2
/
Gapdh
ns
ns
C57BL76 WT
K18-hACE2
hyACE2
mouse
ACE2
human
ACE2
New mouse models to study SARS-CoV-2 pathogenesis
Outlook

25. Preliminary and Confidential data
Marco Bianchi, HSR
Raffaele De Francesco, INGM
hy-ACE2 BCR KI S309
Targeted Igk
locus Promoter
Vk
genes Igk
constant genes
VJ S309
Targeted IgH
locus Promoter VDJ S309
VH
and DH
genes IgH
constant genes
Gate on Lin neg
(CD3, CD8, CD11b, Ly6g, F480)
Peripheral Blood
New mouse models to study SARS-CoV-2 pathogenesis
Outlook

26. Thank you

27. Leibniz Institute of Virology (LIV)
Viral Zoonoses - One Health
Nancy Mounogou Kouassi, Ph.D.
SARS-CoV-2 pathogenesis in high-risk groups:
Lessons learned from influenza
BUXCO Webinar
04.08.2022

28. Influenza Epidemics, Pandemics & High-Risk Groups

29. Identification and Translation of Pathomechanisms
BSL 1-3+
Human Cohorts
Preclinical Animal Models

30. Sex differences in influenza pathogenesis

31. Sex hormones are involved in influenza A virus pathogenesis

32. COVID-19 Pandemic
Quelle: Tobias Hoffmann, Robert Koch-Institut (RKI), 2020.

33. Sex hormones act beyond reproduction
Gogos et al., international journal of endocrinology, 2015

34. Sex hormone effects in the lung
Card & Zeldin, Proc Am Thorac Soc, 2009

35. The golden hamster model mimics key features of
SARS-CoV-2 pathogenesis observed in COVID-19 patients
Stanelle-Bertram*, Beck*, Mounogou*, Schaumburg* et al., unpublished

36. Sex differences in SARS-CoV-2 pathogenesis
Stanelle-Bertram*, Beck*, Mounogou*, Schaumburg* et al., unpublished

37. Sex differences in SARS-CoV-2 pathogenesis
Males present delayed recovery compared to females.
Stanelle-Bertram*, Beck*, Mounogou*, Schaumburg* et al., unpublished

38. Sex differences in SARS-CoV-2 pathogenesis
Males present delayed recovery compared to females.
Stanelle-Bertram*, Beck*, Mounogou*, Schaumburg* et al., unpublished

39. Sex differences in SARS-CoV-2 pathogenesis
Stanelle-Bertram*, Beck*, Mounogou*, Schaumburg* et al., unpublished
Buxco® Small Animal Whole Body Plethysmography : indirect respiratory measurements in conscious subjects.

40. Sex differences in SARS-CoV-2 pathogenesis
Stanelle-Bertram*, Beck*, Mounogou*, Schaumburg* et al., unpublished
Males present impaired lung function compared to females.

41. Sex differences in SARS-CoV-2 pathogenesis
Stanelle-Bertram*, Beck*, Mounogou*, Schaumburg* et al., unpublished
Males present impaired lung function compared to females.

42. SARS-CoV-2 impact on sex hormone metabolism
Stanelle-Bertram*, Beck*, Mounogou*, Schaumburg* et al., unpublished
In males, impaired lung function correlates with low testosterone and high estradiol levels.

43. SARS-CoV-2 impact on sex hormone metabolism
Stanelle-Bertram*, Beck*, Mounogou*, Schaumburg* et al., unpublished
In females, impaired lung function correlates with low estradiol levels.

44. CYP19A1
Stanelle-Bertram*, Beck*, Mounogou*, Schaumburg* et al., unpublished
CYP19A1

45. CYP19A1 is more abundantly expressed in the male lung.
SARS-CoV-2 infection increases CYP19A1 transcription particularly in males.
CYP19A1
CYP19A1
Stanelle-Bertram*, Beck*, Mounogou*, Schaumburg* et al., unpublished

46. Stanelle-Bertram*, Beck*, Mounogou*, Schaumburg* et al., unpublished
CYP19A1
SARS-CoV-2 specifically induces CYP19A1 transcription in lung cells in vitro.

47. Letrozole treatment of SARS-CoV-2 infected hamsters
Stanelle-Bertram*, Beck*, Mounogou*, Schaumburg* et al., unpublished

48. Letrozole treatment of SARS-CoV-2 infected hamsters
Stanelle-Bertram*, Beck*, Mounogou*, Schaumburg* et al., unpublished
Males treated with letrozole show improved lung function at 21d p.i.

49. Letrozole treatment of SARS-CoV-2 infected hamsters
Stanelle-Bertram*, Beck*, Mounogou*, Schaumburg* et al., unpublished
Males treated with letrozole show improved lung function at 21d p.i.

50. Letrozole treatment of SARS-CoV-2 infected hamsters
Stanelle-Bertram*, Beck*, Mounogou*, Schaumburg* et al., unpublished

51. Letrozole treatment of SARS-CoV-2 infected hamsters
Stanelle-Bertram*, Beck*, Mounogou*, Schaumburg* et al., unpublished
Males treated with letrozole show improved lung function at 21d p.i. accompanied by
reduced fibrosis of the lung.
Females do not substantially benefit from letrozole treatment.

52. COVID-19 Autopsies:
Hamburg, Tübingen, Rotterdam (n=57)
Stanelle-Bertram*, Beck*, Mounogou*, Schaumburg* et al., unpublished

53. COVID-19 Autopsies:
Hamburg, Tübingen, Rotterdam (n=57)
Stanelle-Bertram*, Beck*, Mounogou*, Schaumburg* et al., unpublished
Men with COVID-19 present elevated CYP19A1 expression
in their lungs at the time point of death.

54. SARS-CoV-2 induced metabolic hits: The HPLG axis
Stanelle-Bertram*, Beck*, Mounogou*, Schaumburg* et al., unpublished

55. 1st hit 1st hit
Stanelle-Bertram*, Beck*, Mounogou*, Schaumburg* et al., unpublished
SARS-CoV-2 induced metabolic hits: The HPLG axis

56. 1st hit 1st hit
2nd hit
Lung (L) axis:
CYP19A1
Stanelle-Bertram*, Beck*, Mounogou*, Schaumburg* et al., unpublished
SARS-CoV-2 induced metabolic hits: The HPLG axis

57. Acknowledgment
Prof. Karin Klingel
Dr. Stephanie Stanelle-Bertram
Dr. Sebastian Beck
Dr. Nancy Kouassi Mounogou
Dr. Berfin Schaumburg
Dr. Zoe Schmal
Martin Zickler
Tian Bai
Fabian Stoll
Annette Gries
Anna Lüttjohann
Hanna Jania
Jenny Ruschinski
Britta Weseloh
Prof. Dr. Wolfgang Baumgärtner
Dr. Georg Beythien
Dr. Kathrin Becker
Prof. Maren von Köckritz-Blickwede
Dr. Claudia Schulz
Prof. Lothar Kreienbrock
Dr. Bettina Schneider
Prof. Dr. Stefan Kluge
Dr. Maria Schröder
Dr. Dominik Jarzcak
Dr. Axel Nierhaus
Prof. Dr. Benjamin Ondruschka
Dr. Fabian Heinrich
Dr. Susanne Krasemann
Prof. Dr. Jörg Heeren
Sandra Ehret
Prof. Jan von der Thüsen
Dr. Debby van Riel

58. Thank you for
participating!