DT India & South Asia No. 9, 2020

COVID-19 is a disease of human progress- Guest editorial by Dr Sanjiv Hyoju / ORF8 & ORF3b antibodies: Accurate serological markers of early & late SARS-CoV-2 infection / Cycle Threshold (Ct) value of RTPCR can tell us if a SARS-CoV-2 infected person can spread disease / Masks and PPE with hydrophilic surfaces could reduce infection risk – IIT Bombay research / Humans develop robust immune response & memory that may provide lasting protection against Covid-19

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DENTALTRIBUNE
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ORF8 & ORF3b

Cycle Threshold

Accurate
serological
markers of early
& late SARSCoV-2 infection

Cycle Threshold (Ct)
value of RT-PCR can
tell us if a SARSCoV-2 infected
person can spread
disease

” Page 03

09/20

Masks and PPE

Immune memory

Masks and PPE with
hydrophilic surfaces
could reduce infection
risk – IIT Bombay
research
” Page 04

” Page 05

Humans develop
robust immune
response & memory
that may provide
lasting protection
against Covid-19

” Page 06

COVID-19 is a disease of
human progress- Guest
editorial by Dr Sanjiv Hyoju
by Dental Tribune South Asia
This guest editorial by Dr
Sanjiv Hyoju talks about a
possible connection between a
diet-induced disturbance in the
gut microbiome equilibrium
and its impact on our immune
response to viral infections
like SARS-CoV-2. Dr Hyoju is
a surgeon and researcher from
University of Chicago, with his
research focused on Microbiome,
Sepsis, Obesity, Bowel leak,
Infections, Inflammatory bowel
disease (IBD). Dr Hyoju‘s
article on the same topic has
been published in Medical
Hypotheses (Nov, 2020).
We are in the middle of
COVID-19
pandemic.
The
latest data shows 14.5 million
infections reported globally, and
the disease growing at a rate of
1.4% per day. As of today, 607,266
people have died due to COVID19, and 8.7 million people have
recovered from the illness. The
overall Infection Fatality Rate
(IFR) of Severe Acute Respiratory
Syndrome Coronavirus 2 (SARCoV-2) remains as low as 0.6% for
the entire population. However,
the Case Fatality Rate (CFR) is
significantly high among older
people (aged 65 and above) and with
underlying comorbid conditions
like obesity, diabetes mellitus,
hypertension,
heart
disease,
cancer. After observing such
vulnerable populations infected
with the virus it has become
clear that SARS-CoV-2 induces
an overwhelming inflammation
leading to multiorgan damage.
With this observation, one
can speculate that “it‘s not the
virus that is lethal, it‘s the host
immune response to the virus
that causes the detrimental effects
and death“. Hence, it is utmost
important to understand the

Guest editorial by Dr Sanjiv Hyoju talks about a possible connection between a disturbed equilibrium in the gut microbiome and its impact on
our hyper inflammatory response to viral infections like SARS-CoV-2

reasons behind such erratic host
immune response toward virus
among vulnerable populations.
Ageing and other comorbid
conditions create complex disease
processes involving interactions
between the gut microbiome, the
nervous system, the endocrine
system and the immune cells.
Within these interactions, the
main driver is the gut microbiome
which is highly influenced by
dietary habits and host stress.
Lately, the rapid advancements
in civilization and agricultural
development have introduced
highly processed high-sugar,
high-fat diets in our society.
Subsequent
epidemiological
studies have demonstrated that
there has been a rapid increase
in the incidence of diseases such
as obesity, diabetes mellitus,

hypertension,
cardiovascular
disease, cancer, autoimmune
disease and stroke, thus the
name “Diseases of Civilization“.
Such observation has led to the
conclusion that western-diet
associated comorbidities harbour
a disrupted microbiome which is
in sharp contrast to our ancestors
who survived on high-fibre, highprotein and low-sugar diet.
Disrupted microbiome in the
gut has a profound effect on the
brain and immune cell functions
through the enteric nervous
system, leading to chronic
stress situations with exhausted
and senescent immune cells
making the host susceptible to
poor outcomes following a viral
infection. In an animal model,
high-fat diet feeding increased
the influenza A virus-associated

cardiovascular
damage.
Similarly, the stress-animal model
demonstrated
increased
the
susceptibility to coronavirus and
influenza virus. Stress animals are
found to have an altered immune
cell activity, making them more
susceptible to viral infections
and subsequent complications.
Not only chronic stress but also
psychological stress has been
associated with an increased risk
of acute coronavirus infection.
In one study, it has been found
that white blood cells harvested
from stressed patients‘ plasma
produced an excessive level of
pro-inflammatory
cytokines
upon stimulation.
Our brain has two autonomic
functions,
sympathetic
and
parasympathetic. For the body
homeostasis, sympathetic and

parasympathetic outflow must
equilibrate regularly. One of the
prime drivers for maintaining such
equilibrium is the gut microbiome
and its microbial metabolites via
the enteric nervous system, also
known as the Gut-Brain Axis.
Studies have shown that there is
a disrupted microbiome in the
western population compared
to an underdeveloped noncivilized tribe. Hence, it has
been speculated that this newly
developed disrupted-microbiome
led to a stage of disequilibrium in
the autonomic function causing
chronic sympathetic hyperactive
immune stress that manifests as
various diseases such as obesity,
diabetes, and hypertension.
Though
the
sympathetic
response is made to protect
the host from detrimental

[2] =>

2

News

effects during short term stress,
such protective nature is lost
during prolonged sympathetic
hyperactivity driven by disrupted
microbial community, making
them more vulnerable to erratic
stress such as viral infections.
A respiratory viral infection is a
stressful condition that enhances
the sympathetic outflow from
the brain as part of its protective
mechanism. However, in a patient
with a chronic sympathetic
hyperactive condition, who then
contracts the virus, the interaction
tremendously
increases
sympathetic pro-inflammatory
response. In simple words, it is
just like pushing somebody off the
edge of a cliff. Observations made
in animal studies demonstrate
that activation of sympathetic
pro-inflammatory response has
a detrimental effect; and also,
an attenuation of such response
by drugs such as Clonidine
and Propranolol can be highly
effective in preventing death from
Influenza virus infection.
Although vaccine and antiviral
therapy are the mainstays of
treatment, development of such
therapeutic modalities take time.
Older populations with comorbid
conditions are the group that
require vaccination because of
increased fatality rates. However,

09/20

the immune status of the
population is already impaired, as
explained above. This brings up
the question that “How effective
will the vaccine be against SARCoV-2 among such populations?“
Because evidence from U.S
Influenza Vaccine Effectiveness
Network
demonstrates
the
overall effectiveness of the
influenza vaccine to be only
45% and vaccination does not
confer 100% protection. Likewise,
recently published literature
shows that antibodies produced
against COVID-19 might not last
long among those recovered.
Hence, there is a possibility that
SAR-CoV-2 virus may remain
“as another flu-like syndromic
virus with a substantial threat
to the community for a longer
period than expected“. Given
the fact that this virus is highly
contagious and difficult to
contain and the development
of an effective vaccine will take
time, FDA approved commonly
available drugs like clonidine and
propranolol should be considered
to mitigate the impact of the
disease.
Conclusion:
With
the
advancement of civilization,
consumption
of
westerndiet leading to a disrupted
microbiome will push the

normal health toward chronic
sympathetic hyperactive status
bringing up disease condition like
obesity, hypertension, diabetes
mellitus, altering the immune
cell response towards a proinflammatory phenotype leading
to a worse outcome to SAR CoV-2
once infected. This virus has
been otherwise well tolerated by
a healthy population. Although
a vaccine against the virus is the
foremost armour to bring back life
normal, scepticism remains high
regarding the effectiveness of the
vaccine. Again, another risk with
this virus is a lack of long lasting
immunity making the general
population susceptible to reinfections.
Take home message: Here
is a famous quote by the Greek
physician Hippocrates “Let food
be thy medicine“. The World
Health Organization should
come up with strict legislation
and program for the entire world
to change the dietary habit to high
fibre, low sugar and low fat. This
will prevent disruption of normal
microbiome, make the immune
system healthy, fight against
virus and prevent death. This is
the most sustainable solution for
current COVID-19 pandemic and
also avoid similar viral pandemic
in the near future.

1. After observing vulnerable
populations
infected
with
the virus it has become clear
that SARS-CoV-2 induces an
overwhelming
inflammation
leading to multiorgan damage.
With this observation, one
can speculate that “it‘s not the
virus that is lethal, it‘s the host
immune response to the virus
that causes the detrimental effects
and death“. Hence, it is utmost
important to understand the
reasons behind such erratic host
immune response toward virus
among vulnerable populations.
2. Disrupted microbiome in
the gut has a profound effect
on the brain and immune cell
functions through the enteric
nervous system, leading to
chronic stress situations with
exhausted and senescent immune
cells making the host susceptible
to poor outcomes following a viral
infection.
3.
A
respiratory
viral
infection is a stressful condition
that enhances the sympathetic
outflow from the brain as part
of its protective mechanism.
However, in a patient with a
chronic sympathetic hyperactive
condition, who then contracts
the
virus,
the
interaction

tremendously
increases
sympathetic pro-inflammatory
response.

Author:

Dr Sanjiv K Hyoju

Dr Sanjiv K Hyoju MBBS, MS
Surgeon
and
Research
Professional,
University
of
Chicago.
Research focus: Microbiome,
Sepsis, Obesity, Bowel leak,
Infection, Inflammatory bowel
disease (IBD).

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[3] =>

News

09/20

ORF8 & ORF3b antibodies: Accurate
serological markers of early & late
SARS-CoV-2 infection
by Rajeev Chitguppi, Dental
Tribune South Asia
The currently available
SARS-CoV-2
antibody
tests with their traditional
serological approaches just
tell us - yes or no, which has
resulted in an underestimation
of asymptomatic and mild
infections. The next-generation
serologic tests with a broader
landscape of antibody responses
to a wide range of viral proteins
may help us establish the
timing of infections too. This
editorial describes a recent
study (Nature, 17th Aug 2020)
that gives an idea about the
future of antibody testing in
Covid-19.
Serologic tests or the blood
tests that look for antibodies in
the blood (SARS-CoV-2 antibody
tests) form a major component
for the diagnosis of recent
and past COVID-19 infection.
Serological testing is important
for the diagnosis and seroepidemiology of SARS-CoV-2
virus infection and vaccines.
Several serology tests are
currently in use that primarily
assess Spike (S) and on occasion,
nucleocapsid (N) antibodies.
Problem with the current
antibody tests:
Most SARS-CoV-2 positive
patients develop robust T and B
cellular responses, along with S1specific antibody responses that
are high- titre and neutralizing,
which has formed the basis
of serological diagnosis and
vaccine development. However,
several studies have shown that
a proportion of individuals with
RT-PCR confirmed COVID-19,
do not develop robust antibody
responses. This possibility of low
or no antibody responses creates
a limitation for the traditional
serological
approaches
leading to an underestimation
of asymptomatic and mild
infection. It may also jeopardize
the success of a potential vaccine
that targets S alone. Therefore,
what we need is a broader
landscape of antibody responses
to a wide range of viral proteins
in order to better detect the
immunogenicity
of
SARSCoV-2 infection and understand
pathogenesis and immunity.
Let‘s first understand the
SARS-CoV-2 Genome:

The next-generation serology tests with a broader landscape of antibody responses to a wide range of viral proteins may help us establish the timing of
infections too. (Photo: rawpixel)

SARS-CoV-2 is a singlestranded RNA virus with a
genome size of 29,903 nucleotides,
which makes it the secondlargest known RNA genome.
The virus genome consists of
two untranslated regions (UTRs)
at the 5′ and 3′ ends and 11 open
reading frames (ORFs) that
encode 27 proteins.
An open reading frame is a
portion of a DNA molecule that,
when translated into amino acids,
contains no stop codons. The
first ORF (ORF1/ab) constitutes
almost 2/3 of the virus genome
and encodes 16 non-structural
proteins (NSPs). The remaining
1/3 of the genome encodes 4
structural proteins, and about
seven accessory proteins.

4 structural proteins=
spike glycoprotein (S), matrix
protein (M), envelope protein (E),
nucleocapsid protein (N).

7 accessory proteins=
ORF3a, ORF3b, ORF6, ORF7a,
ORF7b, ORF8, and ORF10
Structural Proteins and the
antibodies against them:
1.
We need to study
antibodies to the four structural
proteins = S, N, M and E. Also,
antibodies to three S subunits of
S = S1, S2 and S2′.
2.
Seroconversion
to
structural proteins S and N
generally occurs in 2- 3 weeks
of illness and is not suitable for
diagnosis of acute disease and
these assays have suboptimal
sensitivity and specificity.

3.
The S protein has
multiple stages of maturation,
which may present unique
antigens. First, the S protein is
cleaved into S1 (contains RBD)
and S2 subunits. Next, the S2 is
further cleaved into S2′ to form
the viral fusion peptide.
4.
Most
vaccines
in
development against COVID19 target the S protein to elicit
neutralizing antibodies to block
infection, as the S protein contains
the receptor-binding domain,
which is critical for viral entry.
However, it is unclear whether
neutralizing antibodies to S
protein are the major contributor
to a protective immune response.
Open Reading Frames (ORF)
1.
ORF1ab = encodes
for a large polyprotein that is
proteolytically cleaved into 16
nonstructural proteins (NSP 1 to
16).
2.
Seven other ORFs that
are available = ORF3a, ORF3b,
ORF6, ORF7a, ORF7b, ORF8 and
ORF10. They may encode for
proteins but their functions are
yet to be determined.
A recent study tested
antibodies to the four structural
proteins (S, N, M and E), three S
subunits (S1, S2 and S2′), the seven
available ORFs (ORF3a, ORF3b,
ORF6, ORF7a, ORF7b, ORF8 and
ORF10) and one relevant NSP
within ORF1ab (NSP1).
This study reports the
detection of antibody responses
directed against an extensive
spectrum of 15 SARS-CoV-2

antigens to identify new and
unique antigenic targets of the
humoral immune response of
COVID-19 patients.
Significant findings of the
study:
1. Antibody responses to
structural proteins vs nonstructural proteins
The study showed that
patients produce antibodies to
structural and nonstructural
proteins beyond S. N showed
elevated antibody responses,
better performing as a diagnostic
tool than the other structural
proteins.
2. Non-structural Protein N
and ORF antibodies:
ORF8
antibodies
(in
addition to N protein) due to
their immunodominance and
specificity, form a major marker
of acute, convalescent and longterm antibody response to
SARS-CoV-2. The combined use
of ORF3b and ORF8 provides
a highly sensitive and specific
method for the detection of
patients with COVID-19, both in
early and late stages of infection.
Combinational use of ORF3b,
ORF8 and N can be a highperforming marker of infection
at early and late time points.
3. Avoiding the confusion
with pre-existing cross-reactive
antibodies against other human
coronaviruses:
Endemic
human
coronaviruses like the common
cold coronaviruses could result
in the detection of pre-existing

cross-reactive antibodies, which
may reduce the specificity of
serological assays. However,
this study showed an absence
of cross-reactivity with N, ORF8
and ORF3b antibodies, which
is crucial for their wide use for
diagnosis.
4. Accurate diagnosis with
ORF3b and ORF8:
ORF3b and ORF8 antibodies
are stable over time, highlighting
their importance for early rapid
diagnosis of patients with
COVID-19. The levels of these
antibodies were stable up to day
100 post-symptom onset. Highly
accurate diagnosis of COVID19 is possible if two antibody
responses- ORF3b and ORF8 are
considered together.
Such studies will help antigen
targets for vaccine development
as well as monoclonal antibody
reagents.
References:
[1] Hachim, A., Kavian, N.,
Cohen, C.A. et al. ORF8 and
ORF3b antibodies are accurate
serological markers of early and
late SARS-CoV-2 infection. Nat
Immunol (2020). https://doi.
org/10.1038/s41590-020-0773-7.
[2] Yoshimoto, F.K. The
Proteins of Severe Acute
Respiratory
Syndrome
Coronavirus-2 (SARS CoV-2 or
n-COV19), the Cause of COVID19. Protein J 39, 198–216 (2020).
https://doi.org/10.1007/s10930020-09901-4

[4] =>

4 News

09/20

Cycle Threshold (Ct) value of RTPCR can tell us if a SARS-CoV-2
infected person can spread disease

Ct values of RT-PCR can tell us more than whether a person is SARS-CoV-2 positive or negative.
(Dean Calma / IAEA/Flickr)

by Rajeev Chitguppi, Dental
Tribune South Asia
Currently, all SARS-CoV-2
detection
measures
make
use of RT-PCR test, and its
results are generally reported
as either positive or negative,
which tells us if a person is
infected. However, to contain
the pandemic, what we need to
know is whether that person
is infectious or in other words,
‚can he spread the disease?‘
It‘s important to know that the
RT-PCR test does provide an
additional measure of the viral
load in the sample. This reading
is called the cycle threshold
(Ct) value. Evidence suggests
that reporting this Ct value
(calculated viral load) can assist
in a better interpretation of the
condition, and also in taking
clinical decisions.
Our main objective is to
prevent the spread of COVID19 pandemic, for which we must
identify and isolate infectious
people. Hence, it is critical that
those with a high viral load
are isolated and not allowed to
transmit the virus to others.
Quantitative vs Qualitative
data
Currently,
all
detection
measures
viz.
diagnosis,
screening, and surveillance
depend
on
a
reverse
t r a n s c r i p t a s e – q u a n t i t a t i ve
polymerase chain reaction (RTqPCR) test, and we report its
results as either positive or
negative.

However, it‘s essential to
know that the RT-PCR test
does provide a measure of the
viral load in the sample, and
the reading is called the cycle
threshold (Ct) value. Evidence
suggests that reporting this Ct
value (calculated viral load) can
assist in the better interpretation
of the condition and support the
clinical decisions.
Measuring infectivity by
viral culture
RT-PCR testing can tell us
whether there is a detectable
virus present in an individual.
Still, it does not accurately tell
us whether that individual
is infectious or is capable of
spreading the disease. Infectivity
in cell culture is the standard for
determining whether a patient is
contagious.
In the absence of viral
culture data, one can use viral
load or cycle threshold (Ct)
values derived from RT-PCR
as a proxy for the likelihood
of transmission. The Ct is the
number of replication cycles
required for a signal of RT-PCR
product to cross a determined
threshold.
Early studies on symptoms
and viral shedding
Early studies showed the
highest viral load in throat swabs
at the time of symptom onset
and inferred that infectiousness
peaked on or before symptom
onset.
Another early study showed
pharyngeal virus shedding to
be very high during the first

week of symptoms, with a peak
happening on day 4.
Correlation
between
symptoms and Ct values:
(JAMA Internal Medicine, 6
Aug 2020)
In this cohort study, both
asymptomatic and symptomatic
SARS-CoV-2 patients showed
similar Ct values. Viral molecular
shedding was prolonged. Since
asymptomatic/ presymptomatic
transmission of SARS-CoV-2
infection may be a critical
factor in community spread,
population-based surveillance
and isolation of asymptomatic
patients may be required.
Infectivity: Ct values & viral
culture
In a German study the
researchers could readily isolate
the virus during the first week
of symptoms from a significant
fraction of samples. Still, no
isolates were obtained from
samples taken after day eight
despite ongoing high viral loads.
The study recommended an
early discharge with ensuing
home isolation for patients who
are beyond day 10 of symptoms
with less than 100,000 viral RNA
copies per ml of sputum. Both
criteria predict that there is a
little residual risk of infectivity,
based on cell culture.
In a French study published
in April 2020, the researchers
at the Méditerranée Infection
University Hospital Institute in
Marseille, France collected 183
samples from 155 patients. Out
of the 183 samples obtained, 174

were nasopharyngeal swabs, and
9 were sputum samples. They
assessed the patient samples
for SARS-CoV-2 RNA positivity
using real-time RT-PCR targeting
the E gene. All 183 samples were
inoculated in cell cultures. The
objective was to correlate viral
load to cultivable viruses.
Among the 183 samples
inoculated in the studied period,
129 led to virus isolation. Of
these, 124 samples had detectable
cytopathic effect between 24 and
96 h. Blind subcultures allowed
obtaining five additional isolates
only.
The study found a significant
relationship between Ct value
and culture positivity rate.
a.
Samples with Ct values
of 13–17 all led to a positive
culture.
b.
Culture positivity rate
then decreased progressively
according to Ct values to reach
12% at 33 Ct.
c.
No culture was obtained
from samples with Ct > 34.
d.
The five additional
isolates obtained after blind
subcultures had Ct between 27
and 34 (low viable virus load).
Thus the study found a
strong correlation between Ct
value and sample infectivity in
a cell culture model. The authors
could deduce that with this
system, patients with Ct values
equal or above 34 do not excrete
infectious viral particles.
A study done in Manitoba
(May 2020) evaluated the
relationship
between
cycle
threshold (Ct) values (wrt E
gene SARS-CoV-2 RT-PCR from
respiratory samples), symptom
onset to test (STT) and infectivity
in cell culture. They took SARSCoV-2 RT-PCR confirmed positive
samples and determined their
ability to infect Vero cell lines.
The study observed no growth
in samples with a Ct > 24 or STT
> 8 days. The authors concluded
that the infectivity of patients
with Ct >24 and the duration of
symptoms >8 days might be low.
For every 1 unit increase in
Ct, the odds ratio for infectivity
decreased by 32%. The high
specificity of Ct and STT suggests
that Ct values greater than
24, along with the duration of
symptoms lasting longer than
eight days may be used in

combination to determine the
period of infectivity in patients.
Contrary evidence
One study, published in the
NEJM in May 2020, coming
from a skilled nursing facility
in Kings County, Washington,
found viral growth in a patient
sample with a cycle threshold
(Ct) value of 34, as well as viral
growth in asymptomatic and
presymptomatic
individuals.
However, a systematic review
from
Stanford
University
analysing this study said that
the findings from an elder care
facility might not reflect the
general population. Reasons are
two-fold.
a.
It is difficult to recognise
early signs and symptoms of
respiratory viral infections in
the elderly populations due to
impaired immune responses
associated with ageing and the
high prevalence of pre-existing
and underlying conditions, such
as chronic cough and cognitive
impairments.
b.
Elderly and infirm
patients
have
blunted
physiological responses that
may allow them to remain
asymptomatic during infection.
Latest systematic review (21
Aug 2020)
The latest systematic review
on this topic MedRxiv, 21 Aug
2020 included 17 studies that
suggested a correlation between
the time from collection of a
specimen to test, cycle threshold
(as a proxy for viral load) and
symptom severity. The quality
of the studies was moderate
with lack of standardised
reporting and lack of testing
of PCR against viral culture or
infectivity in animals limiting
our current ability to quantify
the relationship between the
variables and ultimately the
usefulness of PCR use for
assessing
infectiousness
of
patients. However, the authors
mention that the infectivity
appeared to decline after about
a week of viral shedding around
the cycle threshold value of 24.
Infectivity in cell culture
is the gold standard for
determining whether a patient
is contagious.

[5] =>

5

News

09/20

Masks and PPE with
hydrophilic surfaces could
reduce infection risk – IIT
Bombay research

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IIT Bombay researchers show that hydrophilic surfaces could reduce Covid-19 spread

by Rajeev Chitguppi & Uday
Shetty
NEW YORK, U.S.: According
to the latest research from IIT
Bombay, masks and PPE materials
with hydrophilic surfaces could
help the respiratory droplets dry
faster, thus minimising the risk
of infection spread.
Evidence shows that SARSCoV-2 spreads through respiratory
droplets expelled from the mouth
or nose by coughing, sneezing or
talking. However, these droplets
lose their infectivity once they
dry, thus minimising the chance
of further infection spread.
Prompted by these findings, a
team of Indian researchers from
IIT Bombay conducted a study to
explore how these wet droplets
dry once they settle on various
surfaces like face masks or the
other frequently touched surfaces,
e.g. door handles or smartphone
touch screens etc.
Rajneesh Bhardwaj and Amit
Agrawal, two professors from
IIT Bombay, have published their
study in Physics of Fluids. They
state that by reducing the drying
time of droplets, one could reduce
the risk of SARS-CoV-2 infection.

Their study aimed to quantify
the droplet drying time on various
surfaces, based on which they
could make a recommendation
for the ideal types of surfaces for
masks and personal protective
equipment (PPE). They studied
the drying time of a droplet for
different contact angles, and then
using a mathematical physics
model, calculated the expected
survival chances of SARS-CoV-2
on a surface.

Drying time of a 5-nanoliter
droplet as a function of the
contact angle on the surface.
Credit: R. Bhardwaj and A. Agrawal
(phys.org)

Their calculations of the
drying time as a function of
contact angle showed that the

droplet dries roughly four times
faster on hydrophilic surfaces (that
attract water) than hydrophobic
surfaces (that repel water). Thus,
hydrophilic surfaces drastically
reduce the chances of virus
survival.
Therefore, by modifying the
surface wettability and drying
time, one can reduce the chances
of infection. Making a surface
more hydrophilic minimises the
drying time, and we can apply
this finding to the surfaces of
masks and PPE. We can also use
this finding on other frequently
touched surfaces inside the
hospitals, where outbreaks are
more likely to occur.
The
authors
recommend
reducing the contact angle of
the surface of face masks and
frequently touched surfaces. In the
case of N95 respirators, surgical
masks and PPE body-wear, the
contact angle reduction of a
hydrophilic surface could reduce
the chances of Covid-19 infection
by 50%.
This research provides a better
understanding of SARS-CoV-2
survival within a drying droplet,
which might help us in predicting
the survival of other transmissible

viruses, e.g. flu virus that spread
through respiratory droplets.
On
their
personal
communication to Dr Uday Shetty,
implantologist & aesthetic dentist
from Mumbai, the authors stated
that while they have not tested
cotton (which absorbs water) in
their study, it is very likely that it
helps in faster evaporation. This
could have a practical implication
in that cotton gowns might prove
to be better than the impervious
PPE that are in use currently.
Though this sounds logical, we
may have to wait for confirmatory
data to emerge.
Till then one can use cotton
masks on top of N95 masks
Indian
Institutes
of
Technology (IIT) are worldfamous institutes because of their
top-notch education standards
and their illustrious alumni. IIT
have made their way into Dilbert,
the immensely popular comic
strip about the corporate world.
IIT graduates hold prominent
positions in the US economy. IIT
Bombay is ranked 34th among
the top 200 Universities in Asia
category by the QS Asia Ranking
2020.

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[6] =>

6

News

09/20

Humans develop robust immune
response & memory that may provide
lasting protection against Covid-19
by Rajeev Chitguppi, Dental
Tribune South Asia
Currently, all SARS-CoV-2
deOne of the most critical
questions the world is waiting
for an answer is if the human
body can mount a protective
response that provides a lasting
immunity against SARS-CoV-2.
This understanding is crucial
to the success of vaccines, the
successful return of recovered
patients to their pre-pandemic
lifestyle, and also, how readily
the world can overcome the fear
of pandemic spread.
Sporadic reports of Covid-19
recovered people falling ill and
testing positive for the disease
again — have stoked fears
of re-infection due to a short
immune memory. Matters have
only got worse because of the
discouraging predictions made
by the mainstream media.
However,
the
latest
communication from the Indian
Council of Medical Research
(ICMR) says
1.
There has been no
evidence so far of repeat COVID19 infections or relapses in
recovered patients.
2.
All
the
supposed
cases of repeat infection from
some states may be post-viral
symptoms of Covid-19 that still
need to be studied thoroughly.
3.
The SARS-CoV-2 virus
seems to be persisting in some
individuals, but after ten days,
it‘s unlikely to spread & infect
others.
4.
The
commonly
performed tests to diagnose
Covid-19 can identify persistent
viral particles, but can‘t say if
they are live or inactivated.
5.
To establish SARSCoV-2 re-infection, one needs to
show a positive live virus in a
BSL-3 level lab. Epidemiologists
world over share a similar view.
Let‘s analyse the scientific
evidence that has emerged
recently in this regard.
First question: Is there any
evidence that shows the antiSARS-CoV-2 antibodies are
neutralising enough to be
called protective?
A recent study (MedRxiv,
20 July 2020) showed that the
presence of antibodies against
the Receptor Binding Domain
(RBD) region of the spike (S)
protein was highly predictive of

SARS-CoV-2 infection. The IgG
antibodies targeting SARS-CoV-2
RBD showed high specificity
(100%) and sensitivity (97%)
against the infection after 14 days
from onset of illness. However,
in
comparison,
different
antibodies showed varying
durations of persistence. The
IgG seropositivity was sustained
in patients up to 75 days (last
time point measured). Still, the
IgM and IgA responses were
short-lived (many individuals
seroreverted within two months
of the onset of illness).
The
authors
identified
neutralising IgG antibodies
as a possible correlate of
protective immunity. While the
association between RBD-IgG
with neutralising titers and the
persistence of these antibodies at
late time points is encouraging,
further work is needed to define
the optimal antibody-mediated
correlates of protective immunity.
The authors, mentioning the
limitations of the study, stated
that the study does not provide
information about whether the
immune response protected the
individuals from subsequent
infection.
Is there any direct human
evidence that the anti-SARSCoV-2 neutralising antibodies
are protective against SARSCoV-2 infection „in humans“?
Our understanding of lasting
immunity and successful vaccine
development against SARSCoV-2 would be significantly
facilitated by the identification
of immunological parameters
that correlate with protection in
humans. However, to date, most
studies available on protective
immunity had been performed
in animal models, and correlates
of protection had not been
established in humans. A recent
study provided the first direct
evidence that anti-SARS-CoV-2
neutralising antibodies protect
against SARS-CoV-2 re-infection
in humans.
This new study (MedRxiv, 13
Aug 2020) done by Greninger lab
at the University of Washington
School of Medicine in Seattle
describes a SARS-CoV-2 outbreak
reported on a fishing vessel that
was associated with a high attack
rate (>85%).
One hundred twenty-two
sailors went on a ship, out of
which 6 had anti-SARS-CoV-2

Humans develop robust immune response & memory that may provide
lasting protection against Covid-19

antibodies before starting the
trip. Only three crew-members
tested seropositive before the
boat‘s departure in initial
serological screening and also
had neutralising and spikereactive antibodies in follow-up
assays. Then 104 people out of
122 got infected on the ship.
Metagenomic sequencing of 39
viral genomes suggested the
outbreak originated mostly from
a single viral clade.
However, the three crew
members with prior neutralising
antibodies did not become PCR
positive. None of these three
crew-members with neutralising
antibody titers showed evidence
of any bona fide viral infection
or experienced any symptoms
during the viral outbreak.
Therefore, the authors conclude
that the presence of neutralising
antibodies from a prior infection
was significantly associated with
protection against re-infection.
The study- a non-peerreviewed preprint has several
limitations (can be read in full
article), but even with such a
high attack rate (>85%), the lack
of infection in those three crewmembers with
neutralising
antibodies
was
statistically
significant in comparison the rest
of the boat‘s crew.
Overall, the study (famously
known as the Seattle Boat
Study) provides the first direct
evidence
that
anti-SARSCoV-2 neutralising antibodies
are protective against SARSCoV-2 infection in humans. This
evidence is encouraging as we
wait for more studies.
Introduction
to
immunological
mechanisms
before we go further:
Before we go to the next study,
let‘s summarise what we know
about the protective immune

responses that the body mounts
to fend off a re-infection.
The human body makes
use of two arms of the immune
system to fend off viral invaders
that keep returning.
1.
B cells that produce
antibodies that bind to the virus.
It also saves some memory in
the form of long-lived memory B
cells that are ready to act if the
virus comes back.
2.
T cells, which guard the
body checking and destroying
the infected cells, disrupt the
virus‘s ability to replicate. Even
T cells can also endure for years.
Immunological memory:
In some studies, the antibody
levels reduced to practically
undetectable
levels
within
about three months and many
as misinterpreted it as loss of
immunity. We should know
that it‘s very natural for the
antibodies to wane once the virus
disappears. Also that even those
very low levels of antibodies are
enough to provide protective
immunity - this means that even
if antibody levels drastically
reduce and reach low levels,
the immune system often has
a backup plan. The memory B
cells, which linger in the bone
marrow until a virus returns,
attain a new identity and become
plasma cells to produce specific
antibodies.
Another pertinent concept
is long-lasting T cell immunity:
An article published in Nature,
2002 showed how memory T cells
induced by previous pathogens
could shape susceptibility to,
and the clinical severity of,
subsequent infections.
SARS-CoV of 2003 induced
T cells against the virus, so longlasting that they have been found
17 years after infection.

A recent study published in
Nature, 15 July 2020 showed
that infections with beta
coronaviruses induce multispecific and long-lasting T cell
immunity against the structural
N protein. In this study, the
researchers evaluated T cell
responses against the structural
(nucleocapsid (N) protein) and
non-structural (NSP7 and NSP13
of ORF1) regions of SARS-CoV-2
in 36 individuals convalescing
from COVID-19. All these
individuals had developed CD4
and CD8 T cells that recognised
multiple regions of the N protein.
Next, the authors showed
memory T cells that are reactive
to the N protein of SARS-CoV in
23 patients who had recovered
from SARS-CoV infection of
2003. These cells found 17 years
after the outbreak of SARS in
2003) were not only long-lasting
but also displayed robust crossreactivity to the N protein of
SARS-CoV-2.
Understanding
the
distribution, frequency and
protective capacity of preexisting structural or nonstructural
protein-associated
SARS-CoV-2 cross-reactive T
cells could be necessary for
the explanation of some of the
differences in infection rates or
pathology observed during this
pandemic.
Common Cold coronavirus
and SARS-CoV-2 fragments that
are genetically similar show
cross-reactivity via memory T
cells. In some individuals, preexisting T cell memory against
common cold coronaviruses can
cross-recognise
SARS-CoV-2,
down to the exact molecular
structures, which could help us
understand why some people
show milder symptoms of Covid19, while others get severely sick.
Read more here.
A
study
published in
Cell, May 2020 evaluated
T -cell responses to SARSCoV-2 coronavirus in humans
infected with COVID-19 and
the unexposed individuals.
The
authors
demonstrated
memory CD4 T cells and CD8
T cells detected in 100% and
70% of patients who recovered,
respectively. Also, they detected
SARS-CoV-2-reactive CD4 T
cells in ∼40%-60% of unexposed
individuals, which suggested
cross-reactive T cell recognition

[7] =>

7

News

between circulating „common
cold“ coronaviruses and SARSCoV-2.
For more details, one can refer
to this article on Researchgate
that explains how there has been
a paradigm shift from B- cell
responses to T- cell responses in
our approach to SARS-CoV-2.
Is there any evidence that
suggests the human body
produces robust memory to
counter re-infection?
A recent study published
by Karolinska University in
Cell, 11 Aug 2020 showed that
SARS-CoV-2-specific
memory
T cells would be critical for
long-term immune protection
against COVID-19. Researchers
mapped the functional and
phenotypic landscape of SARSCoV-2-specific T cell responses
in different groups of people unexposed individuals, exposed
family members, and individuals
with acute or convalescent
COVID-19.
1.
Acute phase SARSCoV-2-specific T cells displayed
a highly activated cytotoxic
phenotype that correlated with
various clinical markers of
disease severity.
2.
C o nva le s c e n t-ph a s e
SARS-CoV-2-specific T cells were
polyfunctional and displayed a
stem-like memory phenotype.
3.
Importantly, SARS-CoV2-specific T cells were detectable
in
antibody-seronegative
exposed family members and
convalescent individuals with
a history of asymptomatic and
mild COVID-19.
Collectively, the data showed
that SARS-CoV-2 elicits robust,

09/20

broad and highly functional
memory T cell responses,
suggesting that natural exposure
or infection may prevent
recurrent episodes of severe
COVID-19.
Does this memory get better
with time?
A study published in
MedRxiv,
15
Aug
2020
longitudinally
assessed
a
group of individuals, who
had recovered from mildly
symptomatic
COVID-19,
to
determine if they develop and
sustain immunological memory
against SARS-CoV-2. The study
found that recovered individuals
developed SARS-CoV-2-specific
IgG antibody and neutralising
plasma, as well as virus-specific
memory B and T cells that not
only persisted but in some cases
increased numerically over three
months following symptom
onset.
The next level of the study
was to check how these mildly
symptomatic but recovered
individuals would respond
when they re-encounter the
virus - whether they will display
antiviral protective immunity
or not.
The study showed
that the SARS-CoV-2-specific
memory lymphocytes exhibited
characteristics associated with
potent antiviral immunity:
•
memory T cells secreted
IFN-γ and expanded upon
antigen re-encounter,
•
memory
B
cells
expressed receptors capable
of neutralising virus when
expressed as antibodies.
These findings demonstrate
that even mild COVID-19 elicits

memory
lymphocytes
that
persist and display functional
hallmarks
associated
with
antiviral protective immunity.
Although the study found
memory
B
cells
capable
of
producing
neutralising
antibodies that recognise SARSCoV-2, we need more studies
because these cells are more
challenging to locate and count
than antibodies — but thus far,
the evidence suggests that they
proliferate.
Is the immunity durable?
An extensive serological
study published in MedRxiv, 16
Aug 2020 quantified populationlevel exposure and defined the
correlates of immunity against
SARS-CoV-2. The researchers
found that the severe Covid19 cases (compared to mild
COVID-19 ones) exhibited higher
virus-neutralising titers and
antibody levels against three
regions - nucleocapsid (N), the
receptor-binding domain (RBD)
and the S2 region of the spike
protein. All cases, including
the asymptomatic individuals,
seroconverted by two weeks
post-PCR confirmation. Two
neutralising antibody titers
- those against RBD- and S2specific remained elevated and
stable for at least 2-3 months
post-onset. In contrast, those
against N were more variable
with rapid declines in many
samples. In contrast to other
reports, this report concluded
that immunity is durable for at
least several months after SARSCoV-2 infection.

The CDC controversy:
Quoting the evidence, The
Centres of Disease Control and
Prevention (CDC), stated in their
recent (16 Aug 2020) guideline
that very low levels of SARSCoV-2 virus could stick around in
people‘s systems for up to three
months, so there is no need to get
tested again in that timeframe
unless new COVID-19 symptoms
develop. However, if a person
who has recovered from COVID19 develops symptoms again
within three months, he should
get evaluated for re-infection and
isolate himself.
But
mainstream
media
misinterpreted this statement
as “People who have been
infected with COVID-19 and
recovered are protected for up
to three months and can safely
interact with others, according
to new CDC guidance.“
A backlash came from Carl T.
Bergstrom Professor in Biology,
the University of Washington,
“CDC guidelines say that people
are protected for *at least*
three months, not *up to* three
months.“ in his twitter thread
that offers a good explanation on
the CDC guidelines.
His thread says „There
is nothing at all in the CDC
statement about evidence that
immunity wanes after three
months. Rather, they are making
a recommendation based on
positive evidence that immunity
doesn‘t wane before three
months. The 3- month window
is driven by the availability of
evidence. This is a new virus. We
haven‘t had enough cases here
in the US that are four or five

months old to have had time to
determine whether you get reinfection after that duration.“
Comparison of SARS-CoV-2
with other viruses and relevance
to vaccine development:
HIV: We all, including the
vaccine developers, want a longterm immunity & that too, a
sterilising immunity (antibodymediated) that can rapidly
prevent a returning virus from
infecting the body. However, not
all vaccines or infections elicit the
neutralising antibodies needed
to induce a sterilising immunity.
HIV infection rarely produces
neutralising antibodies; that‘s
why we don‘t have vaccines
against HIV. Read more here
Influenza Virus: SARSCoV-2 does not seem to mutate as
rapidly as the influenza viruses,
which is an encouraging finding
for the success of vaccines.
Conclusion:
The evidence is very clear that
people are not getting reinfected
at least in the short term. CDC
says there are no confirmed
reports to date of a person
being reinfected with COVID19 within three months of initial
infection. Since it is something
that we need to follow long
term, it is too early to give any
definitive statement. Although
we cannot predict how long
this protective immunity will
last, researchers are optimistic
about the emerging data that
the human immune system will
most likely fend off SARS-CoV-2
if exposed to the virus again.

First direct evidence: SARS-CoV-2
neutralizing antibodies protect against
re-infection in humans
by Rajeev Chitguppi, Dental
Tribune South Asia
Currently, all SARS-CoV-2
deVaccine development against
SARS-CoV-2 would be greatly
facilitated by the identification
of immunological parameters
that correlate with protection in
humans. However, to date, most
studies on protective immunity
have only been performed in
animal models and correlates
of protection have not been
established in humans. This
study provides the first direct
evidence that SARS-CoV-2
neutralizing antibodies protect
against
SARS-CoV-2
reinfection in humans.

This new study (MedRxiv, 13
Aug 2020) done by Greninger lab
at the University of Washington
School of Medicine in Seattle
describes a SARS-CoV-2 outbreak
reported on a fishing vessel that
was associated with a high attack
rate (>85%).
122 sailors went on a ship, out
of which 6 had anti-SARS-CoV-2
antibodies prior to starting the
trip. Only three crew-members
tested seropositive prior to
the boat‘s departure in initial
serological screening and also
had neutralizing and spikereactive antibodies in follow-up
assays. Then 104 people out of
122 got infected on the ship.
Metagenomic sequencing of 39

viral genomes suggested the
outbreak originated largely from
a single viral clade.
However,
the
3
crew
members with prior neutralising
antibodies did not become PCR
positive. None of these 3 crewmembers with neutralizing
antibody titers showed evidence
of any bona fide viral infection
or experienced any symptoms
during the viral outbreak.
Therefore, the authors conclude
that the presence of neutralizing
antibodies from a prior infection
was significantly associated with
protection against re-infection.
The study, a non-peerreviewed preprint has several
limitations, but even with such a

The Seattle boat study provides the first direct evidence that SARS-CoV-2
neutralizing antibodies protect against SARS-CoV-2 re-infection in humans.

high attack rate (>85%), the lack
of infection in those three crewmembers with neutralizing
antibodies
was
statistically
significant in comparison the rest
of the boat’s crew.

Overall, the study provides
the first direct evidence that
anti-SARS-CoV-2
neutralizing
antibodies are protective against
SARS-CoV-2 infection in humans.
This evidence is encouraging as
we wait for more studies.

[8] =>

7 News

7/19

and

Strictly physiologic!

Duo Quattro Centrifuge

‘’

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Position 1: A-PRF + : 1300 rpm / 14 min
Position 2 : i-PRF : 700 rpm / 3 min
Position 3 : i-PRF M : 700 rpm / 4 min
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Position 5 : A-PRF Liquid : 1300 rpm / 5 min
Position 6 : Custom : 1300 rpm / 3 min
Position 7 : Manual : Free settings

+ tubes S« PRF Box » allow you to get the membranes always hydrated and of
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tubes

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) [page_count] => 8 [pdf_ping_data] => Array ( [page_count] => 8 [format] => PDF [width] => 794 [height] => 1134 [colorspace] => COLORSPACE_UNDEFINED ) [linked_companies] => Array ( [ids] => Array ( ) ) [cover_url] => [cover_three] =>

[cover] => DT India & South Asia No. 9, 2020

[toc] => Array ( [0] => Array ( [title] => COVID-19 is a disease of human progress- Guest editorial by Dr Sanjiv Hyoju [page] => 01 ) [1] => Array ( [title] => ORF8 & ORF3b antibodies: Accurate serological markers of early & late SARS-CoV-2 infection [page] => 03 ) [2] => Array ( [title] => Cycle Threshold (Ct) value of RTPCR can tell us if a SARS-CoV-2 infected person can spread disease [page] => 04 ) [3] => Array ( [title] => Masks and PPE with hydrophilic surfaces could reduce infection risk – IIT Bombay research [page] => 05 ) [4] => Array ( [title] => Humans develop robust immune response & memory that may provide lasting protection against Covid-19 [page] => 06 ) ) [toc_html] =>

Table of contents

[toc_titles] =>

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