Aislamiento de anticuerpos neutralizantes anti-SARS-

CoV-2 y evaluación de nuevos fármacos antivirales

Jaeson Calla, PhD

Assistant Project Scientist

School of Medicine
University of California San Diego
 High-content anti-SARS-CoV-2 compound screening

(a) The SARS-CoV-2 (at 25TCID50) infected Vero E6 cells were detected by high-content imaging of the control condition. Fluorescent signals: green, anti- SARS-CoV NP mAb;
blue, Hoechst.
(b) Percentage of the infected Vero E6 of the control conditions.
 High-content anti-SARS-CoV-2 compound screening

(c, d) The high-content images of the infected Vero E6 cells treated with hydroxychloroquine (c) and ivermectin (d) (the left panel). The percentage of virus inhibition (blue) and cell
viability (red) was shown in the right panel (n = 3 biological replicates). (e, f) The production of infectious SARS-CoV-2 in Vero E6 cells was evaluated by plaque reduction assay after 48 h
of hydroxychloroquine (e) and ivermectin (f) treatment (n = 2 biological replicates)
High-content anti-SARS-CoV-2 compound screening
Dose-dependent anti-SARS-CoV-2 effects of six
candidates at the post-infectious phase.
Dose-dependent anti-SARS-CoV-2 effects of six
candidates at the post-infectious phase.
Dose-dependent anti-SARS-CoV-2 effects of six
candidates at the post-infectious phase.
Dose-dependent anti-SARS-CoV-2 effects of B. rotunda extract and
panduratin A at the pre-entry phase.
Dose-dependent anti-SARS-CoV-2 effects of B. rotunda extract and
panduratin A at the pre-entry phase.
Rogers et al., Science 369, 956–963 (2020)
21 August 2020
SARS-CoV-2 neutralizing antibody isolation strategy.
 Location of the major surface protein
circumsporozoite protein (CSP) on Plasmodium falciparum
sporozoites
 CSP and PvTRAP proteins are involved in hepatocyte recognition and
binding in a mammalian host
Circumsporozoite protein
(CSP)

Thrombospondin-related
adhesive protein (PvTRAP)

6MB3
López et al. 2017. What Is Known about the Immune Response Induced by Plasmodium vivax Malaria Vaccine Candidates?.
Front. Immunol., 13.
4/16/21 Winzeler Lab Update 15
 HSP70 DAPI Plasma membrane Merge

Ab 4
A
CSP location on Plasmodium sporozoites

B
Ab 3

C
Ab 2

D
Ab 1

4/16/21 Winzeler Lab Update 16

 CSP protein loca4on in P. berghei SPZ
PfCSP_Abs HSP70 DAPI Merge

A
Ab 4
CSP location on Plasmodium berghei sporozoites

B
Ab 3

C
Ab 2

D
Ab 1
 Role of CSP monoclonal antibodies during exoerythrocytic
infection in human liver cells – Invasion assays
Experimental strategy

CSP Antibodies
5x103 HC04 cells 5x103 HC04 cells 5x103 HC04 cells

2.5x103 P. berghei Anopheles darlingi

GFP SPZ

CSP Antibodies Operetta High content imaging system

PerkinElmer

1h – 4C - Shaking 1h – 4C - Shaking

2.5x103 P. berghei
GFP SPZ

Negative control Infection control

37 C, 5%CO2, 48h IF

4/16/21 Winzeler Lab Update 18

 CSP location on Plasmodium berghei GFP sporozoites

Ab 1 Ab 2 Ab 3 Ab 4

C
A

D
HSP70
DAPI
Plasma membrane
Merge
 Role of S monoclonal antibodies during SARS-Cov-2 infection in
human liver cells – Invasion assays
Experimental strategy

5x103 Vero cells 5x103 Vero cells 5x103 Vero cells S Antibodies

2.5x103 SARS-Cov-2

2.5x103 SARS-Cov-2

CSP Antibodies Operetta High content imaging system

PerkinElmer

1h – 4C - Shaking 1h – 4C - Shaking

Negative control Infection control 2.5x103 SARS-Cov-2

37 C, 5%CO2, 48h IF

4/16/21 Winzeler Lab Update 20

 S monoclonal antibodies and invasion assays

Ab1 Ab1
5x103 Vero cells CSP Antibodies
CSP-1512-C-terminal CSP-1512-C-terminal

** **
*
0.3 0.20

0.15

% Infection
% Infection

0.2

0.10
S Antibodies
0.1
0.05

1h – 4C - Shaking
1h – 4C - Shaking
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Ab [µg/mL] Ab [µg/mL]

High content imaging results

4/16/21 Winzeler Lab Update 21
 Spike monoclonal antibodies and invasion assays

Vero+SARS-Cov-2 Vero+Ab+SARS-Cov-2
5x103 Vero cells

Spike Mn Antibodies

1h – 4C - Shaking

Flow Sorting results

COVID-19 cohort functional screening.
Antibody functional activity by epitope specificities
High Throughput Screening for Novel Antimalarial
Compounds at UCSD
From molecule to medicine: MMV's R&D process
© MMV 2011

Compound library
Find hits
RESEARCH

1– 2 years
SCREENING
(those compounds able to kill
the parasite)

Finding hits trends:

Test hits
• HTS allows to screen thousands
1– 2 years
LEAD IDENTIFICATION

in vitro and in vivo

in the laboratory, for drug-like qualities
to produce a lead compound of compounds to identify
potent drug candidates and
Improve lead discover new potential drug
1– 2 years
LEAD OPTIMIZATION

compound's properties
by re-enginnering or optimizing it
to remove any undesirable features until it
targets
can be considered a drug candidate
• Shift to phenotypic screening
TRANSLATIONAL

Test the safety

of the drug candidate
from target-based screening
NON-CLINICAL
DEVELOPMENT
1– 5 years

in the lab
NB: All laboratory work is conducted to ICH* Good Laboratory
Practice standards
• Looking for drugs active at the
Phase I clinical trials
to determine the safety and appropriate dose
exoerythrocytic and
intraerythrocytic stages
PHASE I

1.5 years

of the drug in humans – healthy volunteers

without the disease (~100 people)
NB: All clinical trials (Phase I, II & III) are conducted to
ICH Good Clinical Practice standards

Phase II clinical trials

DEVELOPMENT

to ascertain safety, and ability of the drug

PHASE II

2 years

to cure malaria – also known as 'proof-of-concept'

(~100 – 600 patients)

Phase III clinical trials

to compare the safety and efficacy of the drug,
PHASE III

head-to-head, against the best currently

3 years

available treatment (~3000 patients)

Registration
by a stringent drug regulatory authority
REGISTRATION

6 months–2 years

and/or WHO prequalification that thoroughly

evaluates all aspects of the medicine in order
to decide if it should be deemed a legitimate
pharmaceutical product

Total 10–15 years *ICH: The International Conference on Harmonisation

of Technical Requirements for Registration of Pharmaceuticals for Human Use

For further information see www.mmv.org

 It takes a Village to Run Antimalarial HTS
(Current UCSD Malaria Screening Team as of 03-09-2017)

Pamela Peter Jenya Biniam Jaeson

Colleen Megan Korina McLean Madeline

Former

Stephan
Chris&n Melanie Alan Eddy David
 HTS Screening Equipment, Skaggs School of Pharmacy Building
(4th floor Room ….)
Compounds Transferring robots

Pintool Biomek FX (Beckman Coulter) AcousHc Transfer System

(ATS) (BIOSERO)

Liquid/cell Dispensers Read Out

MultiFlo FX Reagent Dispenser (BioTek) Bo<le Valve (GNF) Envision (PerkinElmer)

 Assay 1: P. falciparum Asexual Blood Stage Assay (ABS)

P. falciparum (Dd2 or 3D7)

3 ul screening for final parasitemia of 0.3% Add 2 ul of 10x SYBR
media Green in lysis buffer Measure fluorescence

0 -24 72 96 hrs
hrs hrs hrs

throughput limited by compound transfer Hme and incubator space

~24x 1536w plates per run

Slide: courtesy of Stephan

Modifications by Jenya
Assay 2: P. berghei-Luciferase (Pb-Luc) Liver Stage Assay

throughput limited by mosquitoes

~12x 1536w plates/week
Slide: courtesy of Stephan
Modifications by Jenya
 Assay 3: HepG2 Cytotoxicity (Tox) Assay

CellTiterGlo

throughput limited by compound transfer rate

(~ 12 1536w plates 18,432 wells per week, about 829,440 wells per year) Slide: courtesy of Stephan
Modifica8ons by Jenya
Envision Reads for Single Point Concentration
PbLuc Assay
Envision Reads for Single Point Concentra:on
HepG2 Cytotoxicity Assay
 Example of Hit Maps and Data Analysis for Single
Point Concentration Screening

Normalized growth = (A compound Signal – ATQ Signal)/(DMSO Signal-ATQ Signal)

(InhibiVon = 1-Growth)
 Data Analysis: IC50 Determination, Prism Output
150
150

100

Growth normalized to DMSO and ATQ

100
Growth normalized to DMSO and ATQ

50
50

10-4 10-3 10-2 10-1 100 101

10-4 10-3 10-2 10-1 100 101

ATQ average
Concentration, uM -50
-50 DMSO averaged
Concentration, uM Blue Juice
MMV690086 MMV690092 MMV690071 MMV690148
PURO (2 mM)
MMV690872 MMV690906 MMV692137 MMV024948

200 200

150 150
Growth normalized to DMSO and ATQ

Growth normalized to DMSO and ATQ

100 100

50 50

10-4 10-3 10-2 10-1 100 101 10-4 10-3 10-2 10-1 100 101

-50 Concentration, uM -50 Concentration, uM

MMV690151 MMV023985 MMV024195 MMV024101

MMV690150 MMV689944 MMV676528 MMV020510
MMV692882 MMV690873 MMV690875 MMV692140
MMV019721 MMV692708 MMV692706 MMV692709
 Acknowledgements

Winzeler Lab

• Elizabeth Winzeler
• Sabine Ottilie
• Nimisha Mittal
• Frances Rocamora
• Tuo Yang
• Karla Godinez
• Madeline Luth
• Krypton Karolino
• All lab members for support for
support and making science
fun! Thank you so much!

Funding: