DECIPHERING THE IMMUNE

RESPONSE TO VIRAL INFECTION

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DECIPHERING THE IMMUNE
RESPONSE TO VIR AL INFECTION

T
he COVID-19 pandemic has revealed an urgent need to advance
our understanding of viral biology, such as mechanisms of infection,
transmission, and interaction with the immune system in order to be
more responsive to outbreaks, expediting the development of new, more
effective therapies and vaccines.

This collection focuses on efforts to unravel this complexity, featuring

groundbreaking studies and analysis platforms that both inform and
expedite the therapeutic discovery and vaccine development process.
Greater understanding of events such as viral entry, replication, and
transmission can provide key information to viral “battle plans” of
invasion. Understanding immune responses to infection can provide
greater insight as to why some individuals are more vulnerable to
infection and complications than others, enabling determination of how
immune responses can be modulated, new therapies identified, providing
inspiration for new approaches for vaccine development.

Sartorius continues to support these early stage investigation by providing

innovative Incucyte® live-cell analysis and iQue® advanced flow cytometry
platforms to provide new critical information, with confidence, as quickly
as possible.

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DECIPHERING THE IMMUNE CONTENTS
RESPONSE TO VIR AL INFECTION

05 08 12
Study Shows How Spying on the Viral Enemy Immunotherapy
Mild Coronavirus with Live-Cell Imaging and Targets Emerging
Infection is Beaten Advanced Flow Cytometry Infectious Diseases
by Immune Response

19 23 26
Blood Clotting Patterns in Dengue Shock Zika Biomarkers
Lungs of COVID-19 Patients Investigation Could Lead to
May Help Explain Apparent Clears CD4 T Cells Prenatal Diagnostic
Differences in Mortality of Wrongdoing

4 | GENengnews.com Cover photo: Jongho Shin / Getty Images

 DECIPHERING THE IMMUNE RESPONSE TO VIRAL INFECTION

Study Shows How Mild Coronavirus Infection

is Beaten by Immune Response

R
esearchers at the Peter Doherty Institute
for Infection and Immunity in Australia
have mapped immune responses in a
patient in response to COVID-19 infection,
demonstrating the body’s ability to fight
the virus and recover from the infection. The
team tested blood samples taken from one
of Australia’s first cases of COVID-19, at four
different time points during the infection. “We
looked at the whole breadth of the immune
response in this patient using the knowledge
we have built over many years of looking at
CDC; photo credit James Gathany

immune responses in patients hospitalized

with influenza,” said Oanh Nguyen, PhD,

ADDITIONAL CONTENT
Blog Article: Real-time visualization
and quantification of neutrophil
activation and function using
Outbreak response in action: Centers for Disease Control and Prevention (CDC) staff support the COVID-19 Live-Cell Analysis
response in the CDC’s Emergency Operations Center (EOC)

5 | GENengnews.com
 DECIPHERING THE IMMUNE RESPONSE TO VIRAL INFECTION
Chanin_non / Getty Images
co-author of the researchers report, which is published in Nature
Medicine.
“We showed that even though COVID-19 is caused by a
new virus, in an otherwise healthy person, a robust immune
response across different cell types was associated with clinical
recovery, similar to what we see in influenza,” added University
of Melbourne Professor Katherine Kedzierska, PhD, a laboratory
head at the Doherty Institute. Nguyen said the report is the
first to describe broad immune responses to COVID-19. The
researchers’s findings are reported in a paper titled, “Breadth of
concomitant immune responses prior to patient recovery: a case
report of non-severe COVID-19.”
6 | GENengnews.com
The patient was an otherwise healthy, non-smoking woman,
aged 47 years, from Wuhan, Hubei province, in China, who had
travelled to Australia 11 days previously, and presented to an
emergency department in Melbourne, Australia. Her symptoms,
which had started four days before she went to hospital,
included lethargy, sore throat, dry cough, pleuritic chest pain,
mild dyspnea and subjective fevers.
“Three days after the patient was admitted, we saw large
populations of several immune cells, which are often a tell-tale
sign of recovery during seasonal influenza infection, so we
predicted that the patient would recover in three days, which
is what happened,” Nguyen said. The SARS-CoV-2 virus was
detected in nasopharyngeal, sputum and fecal samples from
the patient on presentation at the hospital on day four, and
again on days 5–6, but was undetectable from day 7. She was
discharged to home isolation on day 11. Her symptoms resolved
completely by day 13, and she remained well at day 20, “with
progressive increases in plasma SARS-CoV-2-binding IgM and
IgG antibodies from day 7 until day 20.”
“We have provided evidence on the recruitment of immune
cell populations (ASCs, TFH cells and activated CD4+ and
CD8+ T cells), together with IgM and IgG SARS-CoV-2-binding
antibodies, in the patient’s blood before the resolution of
symptoms,” the authors wrote. “Collectively, our study provides
novel contributions to the understanding of the breadth and
 DECIPHERING THE IMMUNE RESPONSE TO VIRAL INFECTION
Jongho Shin / Getty Images
kinetics of immune responses during a non-severe case of
COVID-19...We propose that these immune parameters
should be characterized in larger cohorts of people with
COVID-19 with different disease severities to determine
whether they could be used to predict disease outcome and
evaluate new interventions that might minimize severity and/
or to inform protective vaccine candidates.”
7 | GENengnews.com
The patient was enrolled for testing through a coronavirus
substudy that is part of the Sentinel Travelers and Research
Preparedness for Emerging Infectious Disease (SETREP-ID)
initiative, which is led by Royal Melbourne Hospital Infectious
Diseases physician Irani Thevarajan, MD, at the Doherty
Institute. SETREP-ID is a platform that enables a broad range
of biological sampling to take place in returned travelers in the
event of a new and unexpected infectious disease outbreak,
which is exactly how COVID-19 started in Australia. “When
COVID-19 emerged, we already had ethics and protocols in
place so we could rapidly start looking at the virus and immune
system in great detail,” Thevarajan said. “Already established
at a number of Melbourne hospitals, we now plan to roll out
SETREP-ID as a national study.”
Thevarajan commented that according to current estimates
more than 80% of COVID-19 cases are mild-to-moderate,
and understanding the immune response in these mild cases
represents key research. “We hope to now expand our work
nationally and internationally to understand why some people
die from COVID-19, and build further knowledge to assist in
the rapid response of COVID-19 and future emerging viruses,”
she stated. n
 DECIPHERING THE IMMUNE RESPONSE TO VIRAL INFECTION

Spying on the Viral Enemy

with Live-Cell Imaging and
Advanced Flow Cytometry
Jill Granger, M.S., Technical Writer, Essen Bioscience/Sartorius

T
he rapid emergence of the COVID-19 pandemic has
scientists working tirelessly with groundbreaking speed
to unravel disease pathology, immune responses, identify
therapeutics, and develop vaccines. Driving quickly toward
understanding these critical elements requires automated,
integrated analysis systems that capture key biological events to
generate new insights that guide experimental decision-making.
Any good battle plan begins with intelligence gathering

to learn the enemy’s vulnerabilities, and viral infection is no
exception. Live-cell analysis and advanced high-throughput
flow cytometry are well-suited for this type of reconnaissance
mission and can be strategically incorporated into research
workflows to capture and analyze important biological events
in real time. This “spying on the enemy” approach can reveal
secrets of viral binding and entry into host cells, the “hi-jacking”
of host transcriptional machinery for replication, the interactions
between the host and immune system, and viral spreading.
NIAD
Colorized scanning electron micrograph of an apoptotic cell (red) heavily infected
with SARS-COV-2 virus particles (yellow), isolated from a patient sample.
Image captured at the NIAID Integrated Research
Facility (IRF) in Fort Detrick, Maryland.
ADDITIONAL CONTENT
Whitepaper: Live-cell Immunocytochemistry
Correlating surface marker expression with
morphological and functional changes in
living cells over time

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 DECIPHERING THE IMMUNE RESPONSE TO VIRAL INFECTION
This can inform research activities such as epitope mapping
of viral antigens, the development of neutralizing antibodies,
identification of biomarkers and therapeutic targets, serology
testing, and ultimately, the development of new vaccines.
Live-cell imaging and analysis provides a unique, dynamic
view into the inner-workings of the viral life cycle and the
interaction of the virus with host’s immune system, providing
key information of cellular health, function, morphology and
activities such as proliferation, cell death, and cell killing and
NETosis. Automated image collection can be performed in vitro
using co-cultures grown on microplates within a lab incubator.
This combination of non-invasive automated image analysis
9 | GENengnews.com
within a stable environment comes with the added benefit of
reduced operator exposure to dangerous pathogens.
Live cell imaging and analysis has provided new insights into
viral infection that have therapeutic implications. Kelly et al.
(2019) studied the cell-to-cell spread of Measles Virus (MeV)
using a recombinant MeV that expressed a fluorescent protein
as a transcription unit. Incucyte® live-cell imaging and analysis
was used to quantitate viral replication, syncytia formation,
and expansion. The study revealed that protein induced by
interferon, bone marrow stromal antigen 2 (BST2), inhibited
cell fusion and cell-cell spread. In an Oxford study by Xiao et
al. (2018), this platform captured viral plaque morphology of
a “toned-down” version of Ebola virus, crafted by combining
influenza virus pseudotyped with a glycoprotein of Ebola.
This Ebola surrogate could be safely handled in BSL1/BSL2
laboratories, enabling the screening of 1280 small molecules for
their ability to inhibit viral entry. Rothan et al. (2019) studied the
use of host cell components as a therapeutic target in dengue
virus (DENV) and Zika virus (ZIKV) infection and replication.
Live-cell imaging was used to quantitate proliferation of ZIKA-
infected cells treated with Bardoxolne methyl (CDDO-me); an
inhibitor of the Hrd1 ubiquitin ligase-mediated ERAD pathway.
Herod et al. (2019) used Incucyte to detect GFP reporter gene
expression to study the effects of the anti-influenza drug Arbidol
(ARB) against the foot-and-mouth disease (FMDV) and equine
 DECIPHERING THE IMMUNE RESPONSE TO VIRAL INFECTION

rhinitis virus (ERAV). For the first time, this drug was shown to
inhibit FMDV replication ERAV infection. Smith et al. (2019) used
live cell imaging to study to study macrophage regulation in a
mouse model of influenza. Incucyte live cell cytotoxicity assays
and apoptosis assays were used to assess IAV viral replication in
GFP-labeled bronchoalveolar lavage cells from infected mice.
The study revealed that Macrophage Migration Inhibitory Factor
(MIF) can promote viral spread, suggesting that it may be a
potential risk marker as well as a therapeutic target in the battle
against viral pneumonia.
In addition to live-cell imaging and analysis, advanced
high-throughput flow cytometry is another powerful tool
for examining viral interactions with the host, the biology
of immune responses, and therapeutic screening. The
iQue3 platform provides a panoramic view of the immune surface proteins of ARPE-19 cells in immunized mouse and
response, giving comprehensive insight into the extent of part of a high-throughput inhibition assay, which identified,
responses, which may shed light on individual vulnerabilities. for the first time, that CD46 was an important factor for CMV
This platform requires small sample volumes for greater entry into cells. Marcandalli et al. (2019) explored the use
efficiency, and can be used to quantify levels of inflammatory of a nanopartical immunogen (DS-Cav1-153-Cav1) in the
mediators (cytokines), analysis of T-cell subsets and activity, development of a vaccine for respiratory syncytial virs (RSV).
NK cell killing, epitope mapping, antibody binding and They tested the neutralization capabilities of mouse and
neutralization non-human primate (NHP) sera in a RSV micro-neutralization
Advanced flow cytometry has already produced some flow cytometry assay using Intellicyt instrumentation and GFP-
valuable insights for viral research. Stein et al. (2019) used labeled RSV and Hep-2 cells. The nanoparticle immunogen
this to assess the presence of antibodies against the cell elicited a neutralizing antibody response that was tenfold

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 DECIPHERING THE IMMUNE RESPONSE TO VIRAL INFECTION
greater than the RSV vaccine canidate DS-Cav1. Behzadi
et al. (2019) repurposed the influenza A vaccine platform,
via reverse genetic engineering, to express a neutralizing
antibody against the CMV viral envelope protein (gH). In this
study, advanced flow cytometry was used to perfrom binding
analysis of viral envelope protein for the design of a new human
cytomegalovirus (CMV) vaccine. A study by Niu et al. (2019)
examined the antibody responses of convalescent patients that
had been infected with Zika virus (ZIKV), isolating three mAbs
which bound to ZIKV envolope protein that were most potent
in neutralizing the virus. Intellicyt flow cytometry was used to
perform epitope mapping of the antibodies. The antibodies
were administered to SCID mice that had been administered a
lethal dose of ZIKV and they provided protection.
These examples illustrate the value of using live-cell imaging
and analysis as well as advanced high-throughput flow
cytometry to observe viral infection, replication, and the immune
responses for the development of new therapies and vaccines.
New insights can expose viral vulnerabilities that we can use to
our advantage — and in this case, a picture is worth not just a
thousand words, but perhaps, thousands of lives. n
11 | GENengnews.com
REFERENCES
1 Kelly JT, Human S, Alderman J, Jobe F, Logan L, Rix T, Gonçalves-Carneiro D, Leung C, Thakur
N, Birch J, Bailey D. BST2/Tetherin Overexpression Modulates Morbillivirus Glycoprotein
Production to Inhibit Cell-Cell Fusion. Viruses. 2019 Jul 30;11(8). pii: E692. doi: 10.3390/
v11080692.
2 Xiao JH, Rijal P, Schimanski L, Tharkeshwar AK, Wright E, Annaert W, Townsend A.
Characterization of Influenza Virus Pseudotyped with Ebolavirus Glycoprotein. J Virol.
2018 Jan 30;92(4). pii: e00941-17. doi: 10.1128/JVI.00941-17. Print 2018 Feb 15.
3 Rothan HA, Zhong Y, Sanborn MA, Teoh TC, Ruan J, Yusof R, Hang J, Henderson MJ, Fang S.
Small molecule grp94 inhibitors block dengue and Zika virus replication. Antiviral Res. 2019
Nov;171:104590. doi: 10.1016/j.antiviral.2019.104590. Epub 2019 Aug 14.
4 Herod MR, Adeyemi OO, Ward J, Bentley K, Harris M, Stonehouse N, Polyak S. The
broad-spectrum antiviral drug arbidol inhibits foot-and-mouth disease virus genome
replication. J Gen Virol. 2019 Sep;100(9):1293-1302. doi: 10.1099/jgv.0.001283. Epub
2019 Jun 4.
5 Smith CA, Tyrell DJ, Kulkarni UA, Wood S, Leng L, Zemans RL, Bucala R, Goldstein DR.
Macrophage migration inhibitory factor enhances influenza-associated mortality in mice.
JCI Insight. 2019 Jul 11;4(13). pii: 128034. doi: 10.1172/jci.insight.128034. eCollection 2019
Jul 11.
6 Stein KR, Gardner TJ, Hernandez RE, Kraus TA, Duty JA, Ubarretxena-Belandia I, Moran TM,
Tortorella D. CD46 facilitates entry and dissemination of human cytomegalovirus. Nat
Commun. 2019 Jun 20;10(1):2699. doi: 10.1038/s41467-019-10587-1.
7 Marcandalli J, Fiala B, Ols S, Perotti M, de van der Schueren W, Snijder J, Hodge E, Benhaim
M, Ravichandran R, Carter L, Sheffler W, Brunner L, Lawrenz M, Dubois P, Lanzavecchia A,
Sallusto F, Lee KK, Veesler D, Correnti CE, Stewart LJ, Baker D1, Loré K, Perez L, King NP,
Induction of Potent Neutralizing Antibody Responses by a Designed Protein Nanoparticle
Vaccine for Respiratory Syncytial Virus. Cell. 2019.Mar7;176(6):1420-1431.e17.
doi:10.1016/j.cell.2019.01.046.
8 Niu X, Zhao L, Qu L, Yao Z, et al. Convalescent patient-derived monoclonal
antibodies targeting different epitopes of E protein confer protection against Zika
virus in a neonatal mouse model. Emerg Microbes Infect. 2019;8(1):749-759. doi:
10.1080/22221751.2019.1614885.
9 Behzadi MA, Stein KR, Bermúdez-González MC, Simon V, Nachbagauer R, Tortorella D.
An Influenza Virus Hemagglutinin-Based Vaccine Platform Enables the Generation of
Epitope Specific Human Cytomegalovirus Antibodies. Vaccines (Basel). 2019 Jun 14;7(2).
pii: E51. doi: 10.3390/vaccines7020051.
 DECIPHERING THE IMMUNE RESPONSE TO VIRAL INFECTION

Immunotherapy Targets Emerging Infectious Diseases

BIOLOGICS AND VACCINES GAINST EBOLA, ZIKA, AND CHIKUNGUNYA AT FOREFRONT OF DEVELOPMENT

By Kathy Liszewski

F
ear of deadly contagion drives the action in The Hot
Zone, a television drama inspired by the true story of
Ebola’s first arrival in the United States in 1989. Although
the Ebola strain that breached our borders back then
was eventually found to be incapable of causing illness in
humans, The Hot Zone recreates flashback scenes depicting
the terrifying 1976 Ebola outbreak in Africa before cutting
back to the spectacle of hazmat-suited U.S. Army personnel
euthanizing infected animals and decontaminating lab
facilities. At the Baylor College of Medicine, Jeroen Pollett, PhD, and colleagues are developing
mRNA-encoded antibody therapies. The scientists have leveraged improvements
More recent African outbreaks attest to the deadly nature of
in mRNA vaccine formulations to enhance the transfection of antigen-presenting
this hemorrhagic fever and other emerging infectious diseases cells and subsequent translation to therapeutic proteins. For example, they have
and highlight the diligent efforts to create new therapies. As developed a multivalent antiparasitic mRNA vaccine against Chagas disease.

in The Hot Zone, the challenges faced by everyday scientists

sometimes prompt them to rise to the occasion and become
real-life microbe-fighting heroes. They are already making
headway developing novel immunotherapeutic strategies to
attack and neutralize menacing and highly infectious viruses.
(Immunotherapy is also targeting other areas from cancer to
antibiotic resistance.)
ADDITIONAL CONTENT
Whitepaper: Evolution of immune cell killing assays
This whitepaper demonstrates how the technique
of real-time live-cell analysis has evolved to meet
the changing requirements of immuno-oncology
research.

12 | GENengnews.com
 DECIPHERING THE IMMUNE RESPONSE TO VIRAL INFECTION

The inaugural “Immunotherapy for Infectious Diseases”

conference was recently held “to provide a forum for exchange
of results and ideas and to establish a community of interested
parties in the interphase between academia and industry,”
commented meeting organizer Magnus Hook, PhD, professor,
director, Center for Infectious and Inflammatory Diseases,
Texas A&M Health Center. A second conference is planned for
2020 in Italy.
Conference topics included coaxing lung cells to combat
infections, leveraging new technological innovations to create
mRNA vaccines, assessing unique therapeutic monoclonal
antibodies (mAbs) against Ebola, Zika, and other infectious
diseases, and utilizing electroporation-assisted delivery of naked
DNA to create “designer DNA vaccines.”
LURING LUNGS TO ATTACK PATHOGENS
The epithelium of the lung, akin to a military perimeter, defends
against infection both as a passive barrier and as a battery of
active killing components, noted Burton F. Dickey, MD, professor
and chair of the department of pulmonary medicine at the
University of Texas MD Anderson Cancer Center. “Rather than
outsourcing all active antimicrobial defenses to leukocytes,
barrier epithelial cells, which face the onslaught of pathogen
attack, have developed their own potent innate defenses,”
Dickey explained.
Researchers at MD Anderson Cancer Center led by Burton F. Dickey, MD (right), have
shown that airway epithelial cells sense the presence of pathogens through innate
immune receptors such as Toll-like receptors (TLRs). Pathogen sensing and response
steps unfold as follows: (1) TLRs bind molecules shed by microbes. (2) Cells are
prompted to kill pathogens by releasing reactive oxygen species and antimicrobial
peptides. (3) Cells also recruit leukocytes to assist in pathogen defense. The response
steps can be induced by aerosolized drugs that mimic pathogen-associated
molecules.
Dickey along with Scott E. Evans, MD, associate professor in
the same department, and colleagues, developed a strategy to
induce lung cells to ramp up their defenses. The team identified
a synergistic combination of two Toll-like receptor (TLR) agonists
that, when inhaled, could induce rapid lung resistance to
infection from more than 15 bacteria, viruses, and fungi.
According to Dickey, the discovery was exciting, but also
puzzling. “There was,” Dickey pointed out, “no obvious link
between the two agonists that consisted of a diacylated

13 | GENengnews.com
 DECIPHERING THE IMMUNE RESPONSE TO VIRAL INFECTION
lipopeptide ligand for TLR2/6 (that is, Pam2CSK4) and a class
C unmethylated 2'-deoxyribocytidine-phosphate-guanosine
(CpG) ligand for TLR9 (that is, ODN M362).
The investigators next pursued the biology of the effect.
“The combo (Pam2-ODN) induced production of reactive
oxygen species without reliance on type I interferon signaling.
Essentially, the lung epithelial cells were producing ‘Clorox’ to kill
pathogens,” quipped Dickey.
The scientists also recently discovered the mechanism of
action, which presented another surprise. Dickey reported, “The
ODN in the mix binds a cytoplasmic DNA sensor that is required
for the rather magical effect in which all three players are
engaged to induce pathogen killing.”
The PAM2- ODN therapeutic is currently in a Phase IIa
trial. “ We are testing its ability to block bronchitis caused
by rhinovirus in a challenge study,” Dickey said. However,
other uses include treating cancer patients undergoing
myeloablative chemotherapy, who are very susceptible
to pneumonia, as well as organ transplantation patients
and others on immunosuppressants. Dickey concluded
that “there are many strategies that could be developed,
now that we know combinations of innate ligands
delivered by aerosol to the lungs are capable of inducing
a high level of broad host resistance against a variety of
pathogens.”
14 | GENengnews.com
MRNA VACCINES: THE BODY AS BIOREACTOR
Billions of people worldwide are at risk from endemic and newly
emerging tropical infectious diseases. Although traditional
vaccines have had an enormous impact on preventing
disease and saving lives, hurdles remain to more rapid vaccine
development and deployment.
Some believe that the introduction of mRNA vaccines could
usher in a new era in vaccinology. Although early reports of
successful use of in vitro transcribed mRNA in animals appeared
more than 30 years ago, only recently have major technological
innovations allowed mRNA to begin taking its place as a viable
therapeutic.
“Typical vaccines often utilize recombinant proteins, but
the need to produce and purify them are major hurdles,”
explained Jeroen Pollet, PhD, assistant professor of pediatrics
at the National School of Tropical Medicine at Baylor College
of Medicine in Houston. “Once a platform is developed, the
process can be streamlined. It is even conceivable to combine
mRNAs against different antigens to increase potency.”
According to Pollet, mRNA vaccines offer some significant
advantages: “There is no risk of genomic integration. The
cellular immune response can be regulated both by nucleoside
modifications and delivery methods, and mRNA vaccines can be
produced by rapid, inexpensive, and scalable means.”
Pollet and colleagues at Texas Children’s Hospital Center
 DECIPHERING THE IMMUNE RESPONSE TO VIRAL INFECTION
for Vaccine Development are studying Chagas disease, which
is caused by the protozoan parasite Trypanosoma cruzi. An
estimated eight million people in Latin America are afflicted
with the parasite, and the incidence is increasing. Pollet
explained his approach: “We created a vaccine that combined
six unique mRNAs encoding different parasite proteins and
administered that to mice. We’ve had exciting results thus far;
however, our in vivo experiments are complex because we aim
to affect the lengthy chronic phase of Chagas disease.”
Pollet reported that this same strategy is being adapted to
develop therapeutics for other infectious diseases such as Zika
and rabies. In addition, Pollet pointed out that there are other
lucrative uses of the technology: “Any therapeutic mAb could be
developed into an mRNA-encoded antibody therapy. This would
allow patients to make their own Abs in any of their transfected
cells. Certainly, progress in overcoming challenges related to
mRNA stability, immunogenicity, and delivery can now begin to
drive a large and expanding commercial application of mRNA
therapeutics.”
CONSORTIUM FOR STANDARDIZING MABS
Although a number of investigators have produced and
evaluated mAbs against Ebola virus (EBOV), making
comparisons is challenging without standardization of assays
and interpretations among the various groups. To help solve
15 | GENengnews.com
the problem, the Viral Hemorrhagic Fever Immunotherapeutic
Consortium (VIC) assembled with the goal of gathering a broad
pool of antibodies to EBOV and other viruses and analyzing
them using a systematic strategy employing identical assay
conditions. Gary P. Kobinger, PhD, professor and director of the
Infectious Disease Research Centre at the Université Laval is a
member of VIC. He gave the keynote address at the conference
titled, “The Ascent of mAb Therapies against Infectious
Diseases.”
Issues confronting mAb therapeutics include identifying
which in vitro tests best predict the in vivo efficacy of mAbs.
VIC recently published a report describing development of
a comprehensive dataset examining more than 170 mAbs
evaluated in each of 30 assays. The various mAbs included
chimeric Abs, human survivor mAbs, and those raised by
immunization. They concluded that no single neutralization
assay alone can always predict protection, and that the mAb
epitope is not the sole determinant of neutralization behavior.
Despite these findings, the group compiled other sets of key
information to serve as a framework for future studies of EBOV
and other human pathogens.
SINGLE HUMAN MAB QUELLS EBOLA
At the National Institute of Allergy and Infectious Diseases
(NIAID) Vaccine Research Center (VRC), Nancy J. Sullivan,
 DECIPHERING THE IMMUNE RESPONSE TO VIRAL INFECTION
NIAD
A healthy volunteer receives an intravenous infusion
of mAb114 — an experimental treatment for Ebola
virus disease — in a Phase 1 clinical trial held at the NIH
Clinical Center in Bethesda, Maryland.
16 | GENengnews.com
PhD, chief of the Biodefense Research
Section, and colleagues have developed
a therapeutic mAb (mAb114) derived
from blood drawn from an Ebola survivor
11 years after infection.
Sullivan and team first verified the
presence of circulating antibodies against
the EBOV’s surface glycoprotein (GP).
Then, they sorted the patient’s memory B
cells, immortalized individual clones, and
chose one with specific properties they
had determined from previous research
to increase mAb potency. After cloning
its gene, purifying mAb protein, and
testing in a rhesus macaque model, they
found mAb114 could protect against
infection even when given five days after
challenge.
Seeking to identify the structural and
molecular basis for mAb114’s striking
activity, Sullivan and colleagues at
Dartmouth College obtained crystals
and solved the ternary structure of the
mAb/GP complex. Sullivan explained
that the GP, found in abundance on
the virus surface, consists of a trimer of
monomers with two subunits, GP1 and
GP2. They found the mAb binds to a novel
site of vulnerability on GP1, attesting to
the value of identifying natural defenses
targeted by the host immune system.
Subsequently, Julie E. Ledgerwood, DO,
chief, clinical trials program at the VRC,
and her team, led by Martin Gaudinski,
MD, medical director at the VRC,
completed the first-in-human open label-
Phase I trial of mAb114. Ledgerwood
reported (Lancet 2019) that even after
a single 30-minute intravenous infusion
(and patient monitoring for 24 weeks),
the mAb was well tolerated, showed
linear pharmacokinetics, and was rapidly
infused, making it suitable for rapid
deployment as a treatment for outbreaks.
Thus, one of the striking features of
mAb114 is that a single infusion, rather
than several over multiple days, protects
the subject.
Ridgeback Biotherapeutics is leading
further clinical development of mAb114.
 DECIPHERING THE IMMUNE RESPONSE TO VIRAL INFECTION

Sullivan also reported that due to a recent

African outbreak of EBOV, Jean Jacques
Muyembe, director general of the Institut
National de Recherche Biomédicale in
Kinshasa, DRC, is treating patients under
a WHO compassionate use protocol.

DESIGNER DNA IMMUNOTHERAPIES

Another form of immunotherapy, DNA
vaccines, sparked interest in the scientific
community in the 1990s with the
technology’s theoretical ability to generate
broad immune responses without the need
for live attenuated virus. However, early
clinical trials were disappointing, despite
a good safety record. Recently, many
of those hurdles have been overcome,
propelling “designer DNA vaccines” once
again into the limelight.
“Some of the earlier challenges
The Wistar Institute laboratory of David B. Weiner, PhD, has developed a workflow for generating DNA-encoding
were low response rates in humans, monoclonal antibodies (DMAbs) against infectious disease targets. (1) An antibody of interest is selected. (2)
poor reproducibility, and lack of an Sequence optimizations are performed. (3) The synthetic nucleotide is cloned into a plasmid backbone. (4) An
optimized buffer is formulated to improve shelf-life. (5) The plasmid is injected intramuscularly using facilitated in
immunogenic response,” reported David
vivo delivery at the same site to enhance plasmid uptake. (6) The plasmid is transcribed in vivo; the mRNA transcript
B. Weiner, PhD, executive vice president, is translated; and the encoded product is assembled and targeted to the cellular secretory pathway. (7) Sustained,
director of the Vaccine & Immunotherapy robust, and systematic expression of the antibody is achieved. [Ziyang Xu, Megan Wise, Ami Patel, James Hayden]

17 | GENengnews.com
 DECIPHERING THE IMMUNE RESPONSE TO VIRAL INFECTION
Center, and W.W. Smith Charitable Trust Professor in Cancer
Research at the Wistar Institute. Weiner, a pioneer in the field,
said several key innovations are driving the new resurgence of
interest: “First, we can now engineer inserts into DNA plasmids
that will result in 25–50 times more protein expression per cell.
Second, we’ve learned to increase DNA formulations that also
have reduced volume. Third, we’ve dramatically increased our
delivery with advanced electroporation-assisted devices that
provide for cellular update of plasmid more than 1000-fold as
compared to plasmid delivery alone.”
A search on clinicaltrials.gov revealed 610 current clinical
trials using DNA vaccines for the treatment of cancers, influenza,
and infectious diseases. Weiner feels that DNA vaccines provide
a valuable immunotherapy especially for rapidly emerging
infectious diseases. He gave the example of Zika virus. “During
the 2015 outbreak, there were no drugs and no vaccine
available to treat Zika,” he recalled. “Our vaccine, a synthetic
DNA cassette featuring a Zika-specific antigen, was the first
18 | GENengnews.com
into the clinic and took only about 6.5 months to develop from
bench to bedside.”
However, Weiner and colleagues have also taken another
approach to fighting Zika. They have engineered designer
synthetic plasmids with a DNA-encoding mAb, ZK190, that
can produce a full-length functional antibody known to
potently neutralize Zika in animal studies. The team found
that when delivered in vivo, the DMAb-ZK190 was produced
in the living animal and proved protective to Zika challenge in
both mice and rhesus macaques. Weiner explained, “Unlike
viral vector platforms, this platform is nonlive, nonintegrating,
and noninfectious while promoting rapid and transient highly
targeted DMAb generation.”
Weiner sees a broad horizon for DNA platforms, from
fighting emerging infectious diseases to attacking cancer. “As
improvements continue to broaden our scope and accelerate
the pace of success,” he says, “it is possible we are entering the
‘Designer DNA Vaccine and Immunotherapy Era.’” n
 DECIPHERING THE IMMUNE RESPONSE TO VIRAL INFECTION

Blood Clotting Patterns in Lungs of COVID-19 Patients

May Help Explain Apparent Differences in Mortality

A
study in Ireland has found that some Caucasian
patients hospitalized with severe COVID-19 develop a
form of abnormal blood clotting that can contribute to
death. The study, carried out by clinician scientists at the Irish
Centre for Vascular Biology, RCSI, and St. James’s Hospital,
Dublin, found that the abnormal blood clotting caused micro-
clots within the lungs, and that patients with higher levels of
blood clotting activity had a significantly worse prognosis
and were more likely to require admission to an intensive care
unit (ICU). The investigators say their results may also help to
explain emerging evidence of differences in racial susceptible to
COVID-19 mortality.

The study is published in the British Journal of Haematology.
“Our novel findings demonstrate that COVID-19 is associated
with a unique type of blood clotting disorder that is primarily
focused within the lungs and which undoubtedly contributes to
NIAID-RML
This scanning electron microscope image shows SARS-CoV-2 (yellow) also known
as 2019-nCoV, the virus that causes COVID-19 isolated from a patient in the United
States, emerging from the surface of cells (blue/pink) cultured in the lab.

the high levels of mortality being seen in patients with COVID-

ADDITIONAL CONTENT
19,” said James O’Donnell, MD, director of the Irish Centre for Blog Article: In our fight against viral pneumonia,
Vascular Biology, RCSI, and consultant hematologist at the the “Mighty Macrophage” packs a punch.

National Coagulation Centre in St James’s Hospital, Dublin.

19 | GENengnews.com
 DECIPHERING THE IMMUNE RESPONSE TO VIRAL INFECTION

O’Donnell and colleagues reported their findings in a paper

titled, “COVID-19 Coagulopathy in Caucasian Patients.”
Severe COVID-19 infection is associated with marked lung
alveolar inflammatory cell infiltrate, together with a systemic

U.S. Navy / Chief Mass Communication Specialist Barry Rile

cytokine storm response, the authors wrote. Several studies
have also reported evidence of COVID-19-associated clotting
disorders, and post-mortem studies have indicated pathological
changes to the lung microvasculature, including microthrombi
and hemorrhagic necrosis. “Although the pathophysiology
underlying severe COVID-19 remains poorly understood,
accumulating data suggest that a lung-centric coagulopathy
may plan an important role,” the team noted. “Moreover,
emerging data suggest that severe COVID-19 is also associated
Processing blood samples from patients recovering from COVID-19 in a mobile
with a significant risk for developing deep vein thrombosis and
laboratory at the Javits New York Medical Station.
pulmonary embolism.”
Most published data on COVID-19-related coagulopathy are differences in coagulopathic features in COVID-10-infected
have been derived from studies on Chinese patients, but Caucasian compared to Chinese patients.”
race and ethnicity have major effects on thrombotic risk, the The investigators’ study included 83 consecutive COVID-19
investigators continued. Epidemiological studies have found patients admitted to the hospital between March 13, 2020, and
that the incidence of venous thromboembolism (VTE) is 3–4 April 10, 2020. There were 55 male and 28 female patients,
fold lower in Chinese patients than it is in Caucasians, and is with an age range of 26–92 years. Sixty-seven patients (81%)
significantly higher in African-Americans than it is in Caucasians. were Caucasian, 10 (12%) were Asian, 5 (6%) were African,
The lower incidence of VTE in Chinese patients means that and 1 (1%) was of Latino/Hispanic ethnicity. Underlying
thromboprophylaxis is used less in Chinese hospitals. “Given comorbidities were identified in 67 (80.7%) patients. All
these data, it is clearly important to determine whether there patients tested positive for SARS-CoV-2 by PCR, and all received

20 | GENengnews.com
 DECIPHERING THE IMMUNE RESPONSE TO VIRAL INFECTION
Maxwell Photography
James O’Donnell, MD, director of the Irish Centre for
Vascular Biology, RCSI University of Medicine and
Health Sciences, and consultant hematologist in the
National Coagulation Centre in St James’s Hospital,
Dublin.
21 | GENengnews.com
supportive care, including the use of
supplemental oxygen where indicated,
and low molecular weight heparin
(LMWH) for thromboprophylaxis, unless
contraindicated.
The patients underwent a range
of tests at admission and throughout
hospitalization, including measures of
coagulation, D-dimer level, platelet
counts, and fibrinogen testing. The cohort
was also split into two groups based on
whether they had to be admitted to the
ICU for ventilator support or died due to
COVID-19 infection, versus those who were
discharged without requiring ICU support.
The researchers observed elevated
D-dimer levels at admission, which
remained higher in those patients who
eventually needed ICU admission.
“Similarly, fibrinogen and CRP levels were
also both significantly elevated in the
poor prognosis group,” the authors wrote.
“The marked increase in D-dimer levels
is consistent with progressive activation,
along with concurrent activation of
fibrinolysis within the lungs.” However,
they noted that despite increased D-dimer
levels, platelet counts were normal. “Thus,
despite the fact that thrombotic risk is
much higher in Caucasian patients and
the significant elevated levels of D-dimers
observed, overt DIC as defined according
to the ISTH SSC DIC score was present in
none of our COVID-19 patients at time of
admission.”
Reporting on their findings, the
investigators said that the observation
that abnormal coagulation parameters
on admission to hospital were linked with
poorer prognosis in Caucasian patients
with COVID-19 infection was in keeping
with previous Chinese data.
“In addition to pneumonia affecting
the small air sacs within the lungs, we are
also finding hundreds of small blood clots
throughout the lungs,” O’Donnell stated.
“This scenario is not seen with other types
of lung infection, and explains why blood
oxygen levels fall dramatically in severe
COVID-19 infection.”
 DECIPHERING THE IMMUNE RESPONSE TO VIRAL INFECTION
“Cumulatively, these data support the hypothesis that
COVID-19 associated coagulopathy probably contributes to
the underlying pulmonary pathogenesis.” But critically, the
investigators added, “despite the evidence of progressive
COVID-19 coagulopathy over time, none of our cohort
maintained on prophylactic LMWH developed systemic DIC.” In
the rare cases that DIC did develop, it tended to be restricted to
late-stage disease.
The investigators concluded that diffuse bilateral pulmonary
inflammation observed in COVID-19 is associated with a
novel pulmonary-specific vasculopathy, which is distinct from
DIC, and which they’ve termed “pulmonary intravascular
coagulopathy (PIC).”
“Understanding how these micro-clots are being formed
within the lung is critical so that we can develop more
effective treatments for our patients, particularly those in
high-risk groups,” O’Donnell commented. “Further studies will
be required to investigate whether different blood thinning
treatments may have a role in selected high-risk patients in
22 | GENengnews.com
order to reduce the risk of clot formation.”
The authors suggest that larger, controlled studies will be
needed to determine whether more intensive anticoagulation
and/or targeted anti-inflammatory therapies might help
reduce PIC in patients with severe COVID-19. The findings may
also be relevant to growing evidence that some ethnicities
are more likely to develop serious COVID-19 than others.
“Given that thrombotic risk is significantly impacted by race,
coupled with the accumulating evidence that coagulopathy
is important in COVID-19 pathogenesis, our findings raise
the intriguing possibility that pulmonary vasculopathy may
contribute to the unexplained differences that are beginning
to emerge highlighting racial susceptibility to COVID-19
mortality,” they concluded.
O’Donnell led the cross-disciplinary study, with joint first
authors Helen Fogarty, MD, and Liam Townsend, MD, together
with consultants from multiple specialties within St. James’s
Hospital and researchers at RCSI University of Medicine and
Health Sciences and Trinity College Dublin. n
 DECIPHERING THE IMMUNE RESPONSE TO VIRAL INFECTION

Dengue Shock Investigation

Clears CD4 T Cells of Wrongdoing

Laguna Design / Getty Images

Yuan Tian, La Jolla Institute for Immunology

I
n many episodes of Law & Order, prosecutors would uncover new
information that forced them to rethink their theory of the case.
Sometimes, they’d even drop all charges against their prime
suspect. Such a dramatic turn has occurred in an investigation
of dengue shock, a severe form of dengue fever. Dengue
shock has been thought to be incited by overactive CD4
T cells, but according to a review of transcriptional
signatures, CD4 T cells have been innocent all along.
“We found no evidence to support the common
dogma that these T cells are responsible for turning a
mild infection to a severe one,” said Yuan Tian, PhD, an
immunology fellow and bioinformatics student at La Jolla
Institute for Immunology (LJI). “This will help us narrow the
search for the true culprit.”
Tian was on an investigative team led by Alessandro
Sette, Dr. Biol. Sci., a professor of vaccine discovery at LJI, and
Daniela Weiskopf, PhD, an instructor at LJI. The team’s goal was ADDITIONAL CONTENT
Application Note: A Kinase Inhibitor Phenotypic
to define the molecular pattern of dengue-specific CD4 T cells
Screen Using A Multiplex T Cell Activation Assay
and to investigate whether there is a difference in the T-cell response

23 | GENengnews.com
 DECIPHERING THE IMMUNE RESPONSE TO VIRAL INFECTION
Yuan Tian, La Jolla Institute for Immunology
Dengue-specific CD4 cells have both a pro-inflammatory function and an anti-
inflammatory function, which is typically not seen in acute viral infections. Each dot is
a double-positive cell with the color of each dot representing the expression level of a
protein named TIGIT, which is an inhibitory immunoreceptor, in each cell.
24 | GENengnews.com
between patients with mild dengue fever or with severe dengue
hemorrhagic fever.
Detailed findings appeared December 24 in the journal Cell
Reports, in an article titled, “Molecular Signatures of Dengue
Virus-Specific IL-10/IFN- γ Co-producing CD4 T Cells and Their
Association with Dengue Disease.”
“We identified antigen-specific IL-10+IFN- γ+ double-positive
CD4 T cells during acute dengue virus infection,” wrote the
article’s authors. “While the transcriptomic signatures of double-
positive cells partially overlapped with those of cytotoxic
and type 1 regulatory CD4 T cells, the majority of them were
noncytotoxic/Tr1 and included IL21, IL22, CD109, and CCR1.”
When analyzing dengue-specific CD4 T cells, the researchers
realized that the responding CD4 T cells, have both a
pro-inflammatory function (regulated by the cytokine interferon
gamma, or IFN- γ) and an anti-inflammatory function (regulated
by the cytokine IL-10) which is typically not seen in acute viral
infections. To comprehensively define these dengue-virus-
specific T cells in hospitalized patients, researchers used whole
transcriptome analysis to determine if there was a difference in
the quality of the increased response.
“Although we observed a higher frequency of double-positive
cells in dengue hemorrhagic fever, the transcriptomic profile of
double-positive cells was similar in dengue fever and dengue
hemorrhagic fever, suggesting that dengue hemorrhagic fever
 DECIPHERING THE IMMUNE RESPONSE TO VIRAL INFECTION
is not associated with the altered phenotypic or functional
attributes of double-positive cells,” the article’s authors
continued. “Overall, this study revealed a dengue-virus-specific
double-positive cell subset in patients with acute dengue
disease and argues against altered double-positive cells as a
determinant of dengue hemorrhagic fever.”
These findings are important because they improve our
understanding of dengue fever — the world’s most common
mosquito-borne illness. Also, they promise to narrow the hunt
for an effective vaccine for dengue.
The findings also demonstrate the power of whole
transcriptome analysis. In the current study, this approach was
used to evaluate dengue-specific CD4 T cells obtained from
patients who were being treated for either mild or for severe
dengue infection. (These patients were hospitalized in Sri Lanka,
where dengue fever is endemic.)
“This is a very powerful approach to detect gene expression
activity because all genes upregulated in response to the
virus can be identified,” noted Weiskopf, the current study’s
senior author. “It is completely unbiased and does not rely on
preselected genes.”
The research team, to their surprise, detected no difference
25 | GENengnews.com
in the genomic profile of dengue-virus specific CD4 T cells
regardless if they isolated them from patients with mild or
severe dengue infection.
“The CD4 T cell response in the severe disease does not look
different so that cannot be the switch we are all looking for,”
Tian elaborated. “In fact, based on some intriguing preliminary
findings, we speculate that to counteract the severe immune
response occurring in acute cases, these dengue-specific CD4
cells may have gradually acquired the ability to produce more
IL-10 by converting IFN- γ. It is as if they are trying to calm
themselves, calm the inflammation. The double-positive CD4 T
cells could actually be helping, rather than hurting.”
Tian added that he hopes these findings will serve to “help
guide efforts to develop effective dengue vaccines by improving
our understanding of this novel T-cell response.”
Such an outcome would be most welcome. Dengue fever is
spreading. Infected mosquitos have expanded beyond their
established tropical and subtropical territories in South East Asia
and Latin America to new continents, including Europe and the
United States. More than half of the world’s population is now at
risk; already, 390 million infections occur annually, according to
public health experts. n
 DECIPHERING THE IMMUNE RESPONSE TO VIRAL INFECTION

Zika Biomarkers Could Lead to Prenatal Diagnostic

I
nvestigators at USC believe that after immunoprofiling a
number of symptomatic Zika virus positive (ZIKV+) pregnant
patients and extensive multiplexing analysis of their cytokine
levels that they have identified a panel of biomarkers that
are “specifically associated with symptomatic ZIKV+ infection
during pregnancy.” This is an important discovery that could
lead to screening tests and a better understanding about
how the infection leads to fetal abnormalities. Findings from
the new study were published today in the Journal of Clinical
Investigation Insight through an article titled “Biomarkers and
immunoprofiles associated with fetal abnormalities of ZIKV-
positive pregnancies.”
“The highest risk of birth defects is from Zika virus infection
during the first and second trimester. A prenatal test has the
potential to relieve the concerns of many expectant mothers,”
explained lead study investigator Suan-Sin Foo, Ph.D., a
research associate in the department of molecular microbiology
and immunology at the Keck School of Medicine of USC. “We
ADDITIONAL CONTENT
still have a lot to learn about how the Zika virus affects the Application Note: An Optimized, Multiplexed Assay
immune responses in the mother, and how infection can for Screening Ex Vivo Conditions which Increase
Memory T Cell Frequency
negatively impact her baby.”

26 | GENengnews.com
 DECIPHERING THE IMMUNE RESPONSE TO VIRAL INFECTION
Zika virus, which is spread by Aedes aegypti mosquitoes,
typically does not affect most of those it infects — low-grade fever
being the most noticeable symptom. However, fetuses exposed to
Zika in the womb are at risk for devastating neurological defects.
One of those defects, microcephaly — a smaller-than-usual head
size — gained prominence in 2015 with Brazil reporting an unusual
number of cases in babies born to mothers infected with the virus.
Sadly, as those infants have become toddlers, many can’t see,
walk, chew, or talk and will require a lifetime of care, according
to the U.S. Centers for Disease Control and Prevention. In the
U.S., there have been approximately 2,483 pregnant women
infected with Zika and 116 infants born with Zika-associated
birth defects since 2015.
In the current study, the researchers examined the immune
systems of pregnant women through blood samples taken
during the first, second, and third trimesters of pregnancy. They
compared blood samples from 30 Zika-infected, pregnant
women in Brazil with 30 healthy pregnant women in Brazil and
14 in Los Angeles.
Specifically, the researchers were looking at cytokines, which
are messenger chemicals released by the body in response
to an infection. Across a panel of 69 cytokines screened, they
identified 16 cytokines that appeared to be associated with
Zika-induced abnormal births.
27 | GENengnews.com
“Extensive multiplexing analysis of 69 cytokines revealed
that CXCL10, CCL2, and CCL8 chemokines were specifically
associated with symptomatic ZIKV+ infection during
pregnancy, and distinct immunoprofiles were detected at
different trimesters in ZIKV-infected pregnant women,” the
authors wrote. “Intriguingly, the high CCL2 level and its
inverse correlation with CD163, TNFRSF1A, and CCL22
levels were apparently associated with ZIKV-induced
abnormal birth.”
At the moment, the research team isn’t sure whether the
messenger chemicals cause the birth defects or are secreted in
response to something else.
“Ultrasound is routinely used during pregnancy to check
a baby’s condition, but there’s a limit to what can be seen.
Magnetic resonance imaging can give clear, high-resolution
‘snapshots’ of the fetus, but there are safety concerns for the
baby, and it is recommended for second- and third-trimester
pregnancy,” concluded co-lead study investigator Weiqiang
Chen, Ph.D., a research associate in the department of
molecular microbiology and immunology at the Keck School of
Medicine. “Our findings identified a panel of biomarkers which
may potentially be useful in predicting Zika-associated fetal
outcomes regardless of pregnancy stages, simply by evaluating
the mothers’ blood.” n
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