Immunological, Viral and Genetic Basis of COVID-19 in

local Patients (RRG # 211)

Principle Investigator:
Dr. Shah Jahan (Associate Professor)
shahjahan@uhs.edu.pk
Head of Department,
Department of Immunology,
University of Health Sciences, Lahore Pakistan
 Project Team

Dr. Shah Jahan

Prof. Dr. Javeed Akram
Principle Investigator
Co-Principle Investigator
PhD (Molecular Biology)

Miss
Dr. Muhammad Bilal Dr. Sadia Omer Miss Almina Shafiq
Muhammad Roman Alam Khan Sidra_tu_muntaha
Sarwar (MBBS) M.Phil. PhD
Research Scholar Research Assistant Research Assistant Research Scholar PhD Research
Research Associate
Scholar
 Objectives

|Viral Genetics| |Immunological Response| |Cellular Response| |Host Genetics|

1. SARS-CoV2 2. Analysis of Specific 3. Gene expression 4. Sequencing analysis

complete genome antibodies and cytokines analysis of Receptors of Receptor ACE-2 and
analysis from different Levels (TNFα, IFNα, β, γ (ACE-2 and TLRs) TLR2 and TLR 4 and host
infection clusters in and IL-1β,2,4,6,) in active Cytokines (TNFα, , IFNα, genes (IL4, IL6, IFNs,
Punjab and Epitopes and recovered patients β, γ and IL1β,2,4,6) in CCL2) and HLA typing
mapping of E, M and S in comparison of healthy local patients by Real (HLA B4061) in corelation
proteins to evaluate population. time PCR. with immune patteren of
impact on immunoge- COVID-19 patients.
nicity.
 Patients Collection Criteria and Case Definations

*Inclusion Criteria
• ≥18, PCR Confirmed, symptomatic,
Hospitalized
• Dyspnea, blood oxygen saturation ≤
90%, respiratory rate >30 breaths
min-1; severe respiratory distress
• cough, sore throat, shortness of
breath, coryza (head cold), anosmia
(loss of sense of smell), with or
without fever *Mild_to_Moderate *Sever/Critical
Group Group

Admitted in
ICU Patients, PCR
isolation wards,
Positive, Clinical
meeting case
Sign of Pneumonia
definition

Exclusion Criteria PCR Positive, with >30 breaths min-1, or

• Active respiratory infection, and without oxygen saturation
evidence of (SpO2) < 90, severe
Hospital Acquired Covid pneumonia pneumonia respiratory distress
• < 18 , Immunocompromised, PCR
negative or CT >35, Not. Admitted
to Hospital
*“World Health Organization Guidelines for COVID-19 Pneumonia Diagnosis and Treatment”.
 Involved Hospitals (N= 852)

Jinnah Hospital Lahore

N= 166
Mayo Hospital Lahore
Isolation Hospital and Infectious Treatment Center N=202
N=356

Pakistan Institute of Medical Sciences, Islamabad

N=128
Demographic Features of local COVID-19 Patients

Mean age: 58 ± 14 years

Male to Female Ratio: 61.7% to 38.2%
 Demographic Features of local COVID-19 Patients
Mild/Moderate p value
Variables Total (n=452) Severe (n=213)
(n=239)
Age (Years)
Average Age 55.75 (15.1) 61.62 (13.0)  
58.52 (14.42)  
≤ 50 years n (%) 126 (27.88), 83(34.73) 43(20.19) -

> 50 years n(%), 326(72.12) 156(65.27) 170(79.81%)

• 27.3% of the patients
Gender
had an exposure history
Male 279 (61.7%) 137 (49.2%) 142 (50.8%)  
Female 173 (38.2%) 102 (58.9%) 71 (41.1%) N/A
• 11.9% travel history.
Family Type
Nuclear 158 (35%) 88 (55.7%) 70 (44.3%) • 60% of patients, the
0.429
Extended 294 (65%) 151 (51.4%) 143 (48.6%) source of infection was
Source of COVID-19 Infection
unknown.
Travel History 55 (12.2%) 33 (60%) 22 (40%) • 65% were belong to the
Contact History 124 (27.4%) 66 (53.2%) 58 (46.8%) extended family type.
0.496
No known source
273 (60.4%) 140 (51.3%) 133 (48.7%)
for COVID-19

Data are n(%) . P values suggest the disparity between age groups, family type, and source of infection among mild/moderate and severe COVID-19 patients. The significance level for p value ≤ is 0.05.
 Clinical Characterstics of local COVID-19 Patients
Clinical Features Total (n=452) Mild/Moderate (n=239) Severe (n=213) (%) p
(%) (%) value
Fever or Chills 364 (80.5) 187(78.2) 177(83.1) 0.234
Shortness of Breathing 343 (75.9) 162(67.8) 181(84.9) 0.000
Cough 261 (57.7) 134(56.1) 127(59.6) 0.448
Fatigue 138 (30.5) 93 (38.9) 45(21.1) 0.000
Pneumonia 89 (19.6) 16(6.7) 73(34.3) 0.000
Muscle or body aches 75 (16.6) 35(14.6) 40(18.8) 0.256
Most prevalent signs and
symptoms in ICU patients were
Vomiting 61 (13.5) 34 (14.2) 27(12.7) 0.680
dry coughs, myalgias, shortness
Headache 36 (7.9) 19 (7.9) 17(7.9) 1.000
of breath, gastrointestinal
Abnormal chest X-Ray 36 (7.9) 5(2.1) 31(14.6) 0.000
discomfort, and abnormal Chest
Sore throat 33 (7.3) 14(5.9) 19(8.9) 0.277 X-ray (p<0.001).
Diarrhea 33 (7.3) 15(6.3) 18(8.5) 0.469
Sputum production 19 (4.2) 13(5.4) 6(2.8) 0.240
Nausea 13 (2.9) 7(2.9) 6(2.8) 1.000
Loss of taste 12 (2.7) 10(4.2) 2(0.9) 0.040
Runny Nose 12 (2.7) 9(3.8) 3(1.4) 0.149
Loss of smell 7 (1.5) 5(2.1) 2(0.9) 0.455
Congestion 5 (1.1) 2(0.8) 3(1.4) 0.670
Data are n(%). P values suggest the disparity between mild/moderate and severe COVID-19 patients—significance level for p-value ≤ 0.050.
 Comorbidities of local COVID-19 Patients

Co-Morbidities Total Mild/Moderate Severe P value

(n=452) (n=239) (n=213)
Hypertension 190 (42.0) 86 (35.9) 104 (48.8) 0.007

Type 2 Diabetes Mellitus 157 (34.7) 75 (31.4) 82 (38.5) 0.115

Ischemic Heart Disease 66 (14.6) 30 (12.5) 36 (16.9) 0.230

Chronic Kidney Disease 19 (4.2) 9 (3.7) 10 (4.7) 0.646

High percentage of
hypertension, Type 2
Asthma 11 (2.4) 5 (2.1) 6 (2.8) 0.763
Diabetes, Ischemic
Immunocompromised state 10 (2.21) 4 (1.6) 6 (2.8) 0.527 Heart Disease were
Chronic Obstructive 8 (1.8) 1 (0.4) 7 (3.3) 0.029 leading comorbidities.
Pulmonary Disease
Smoking 5 (1.1) 2 (0.8) 3 (1.4) 0.670
Liver disease 3 (0.7) 1(0.4) 2 (0.9) 0.604

Data are n (%). P values suggest the disparity between mild/moderate and severe COVID-19 patients. Significance level for p value
≤ 0.050.
 Treatment Given to local COVID-19 Patients

Treatment Total (n=452) Mild/Moderate Severe (n=213) P value

(n=239)

Dexamethasone 418 (92.5) 207 (86.6) 211(99) 0.000

Remdesivir 314 (69.4) 136 (56.9) 178(83.5) 0.000

Drug Dexamethasone was
Azithromycin 206 (45.6) 119 (49.8) 87(40.8) 0.059 the leading prescribed and
administrated medicine to
Meropenem 197 (43.6) 76 (31.8) 121(56.8) 0.000 the COVID-19 patients,
followed by Remdesivir,
Clexane 192 (42.5) 100 (41.8) 92(43.2) 0.776 Meropenem, Heparin, and
Tocilizumab, respectively.
Heparin 187 (41.4) 75 (31.4) 112 (52.6) 0.000

Fluoroquinolones 152 (33.6) 87 (36.4) 65 (30.5) 0.196

Tocilizumab 15 (3.3) 1 (0.42) 14 (6.6) 0.000

Data are n (%). P values suggest the disparity between mild/moderate and severe COVID-19 patients. Significance level for p value
≤ 0.050.
Publication 1: Under Review process
Journal:
Frontiers in Cellular and Infection Microbiology
(4.3 IF)
Sub Section : Clinical Microbiology

•Pre Print link:

https://www.medrxiv.org/content/10.1101/
2021.09.25.21264111v1
 Viral Genetics
SARS-CoV-2 Whole
Genome Sequencing
 SARS-CoV-2 Sequencing Work Flow
#
SARS-CoV-2 Sample Collection • Mayo#cDNA
hospital, Lahore
Synthesis and qPCR
Analysis
• Jinnah Hospital Lahore
• Nishter ~Hospital
60 min Multan
• Sheikh Zaid hospital Lahore

*ARTIC Library Preparation

• Sheikh Zaid Hospital Rahimyar Khan
• Alkhaidmat Diagnostic Laboratories (Karachi, Lahore,
~ 3.5 hours
Multan and Peshawar)
• Khyber Medical University, Peshawar
Viral RNA Extraction and
# • University of Sequencing
*NextSeq550 veterinary and
and animal Sciences, Lahore
Purification • Pakistan#Data Analysis
Institute of Medical Sciences, Islamabad
~45 min

# University of Health Sciences, Lahore

* Quadram Institute of Biosciences, UK.
SARS-CoV-2 Sampling Statistics

Male: 401
Female: 315
PANGO lineages of GISAID (as of 2020-05-14) data where Pakistan is the country
of exposure, with collection dates of first and most recent samples
 •April 2021 – October 2021
Detected SARS- •hCoV-19/Pakistan/UHSPK1- 01 to 102 (APRIL)
CoV-2 Strains •hCoV-19/Pakistan/UHSPK2- 01 to 325 (MAY, JUNE)
•hCoV-19/Pakistan/UHSPK3 –00. (JULY, AUGUST, SEPTEMBER)

PROVIENCE CIty Alpha (B.1.1.7-like) Beta (B.1.351-like) Delta (B.1.617.2-like) Eta (B.1.525-like) QC failed total
KPK Peshawar 35 2 25 1 8 71
KPK Swat 4 1 2 0 5 12
KPK Mardan 5  0 3 0 2 10
Punjab Bhakkar 5 0 10 0 6 21
Punjab Lahore 121 9 77 0 18 225
Punjab Multan 112 4 88 1 20 225
Punjab Sialkot 28 4 3 0 1 36
Sindh Karachi 3 16 69 0 31 119
Total 313 36 277 2 91 719
Phylogeny of B.1.1.7 (Alpha)
. At the top, the maximum-likelihood
dated tree of lineage B.1.1.7 (Alpha),
showing only samples close to
sequences related to Pakistan.
Branches are coloured red when all
descendant tips have Pakistan as the
country of exposure. Samples
sequenced in the present study are
highlighted as red dots.
histogram of introductions into and exportations out of Pakistan over time, as estimated by
ancestral state reconstruction.
Maximum-likelihood dated tree of lineage B.1.351 (Beta) focused on samples sequenced in this study. Branches are coloured
according to country of exposure, with Pakistan highlighted with a red circle. The two samples from the current study, UHS-
PAK-S45 and UHS-PAK-S93, are labelled in blue.
•Publication 2: Accepted
Title: SARS-CoV-2 Variants of Concern
Dominate in Lahore Pakistan in April
2021.
•Journal: Microbial Genomics (5.75IF)

•Publication 3: in writeup Process

Title: SARS-CoV-2 variants of concern from
Alpha to Delta in Pakistan March to October
2021.
Epitope Mapping
Publication 4: Insilico Epitope mapping and Vaccine
Antibodies
and cytokines
Levels
Comparison of the cytokines levels
in different symptomatic group

(a) Interleukin 1 beta, (b) Interleukin 2, (3) interleukin 4, (4) Interleukin 6.

(b) Means values were compared at the * P <0.05
 •Comparison of the cytokines levels in
different symptomatic group

(a) IFN-α, (b) IFN-β (3) IFN-γ (4) TNF-α.

 Antibodies (IgM and IgG)
measurement from the symptomatic
and recovered patients
 Gene expression analysis of
Receptors in local patients by Real
time PCR.
 Relative fold expression of cytokines and Cellular

IL-1beta, IL-2, IL-6 and IL-8

IFNg, TNF-alpha, NKF and COX-2
 Relative fold expression of cytokines and Receptors

IL-4 and IL-10 ACE-2, TLR-2, TLR-4

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