Study Title: Developing Indian SARS-CoV-2 Pseudo Virus panel to evaluate the effectiveness of COVID-19

vaccines against SARS-CoV-2 variants in Indian population.

Rationale: Over one year after its inception, the coronavirus disease-2019 (COVID-19) pandemic caused by
severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) remains difficult to control despite the
availability of several excellent vaccines1. Progress in controlling the pandemic is hampered by the emergence of
variants that appear to be more transmissible and more resistant to antibodies 2–6. India had faced the world's
worst coronavirus outbreak earlier this year, witnessing lakhs of cases and thousands of fatalities daily. Daily
cases in India had peaked at 4,14,000 in May20217. A robust test to detect neutralizing antibodies of SARS-
CoV-2 is urgently needed to determine not only the infection rate, herd immunity and predicted humoral
protection, but also vaccine effectiveness after large-scale vaccination in India. Also, early identification of
evolving SARS-CoV-2 variants and understanding its underlying mechanisms of immune evasion from natural
and vaccine-induced immunity will be crucial for the development of next-generation vaccines that elicit broadly
neutralizing activity against current and potential future variants. With the onset of the vaccine being rolled-out
in the country [Covishield, Covaxin and Sputnik], and the uncertainty of how effective the vaccine elicited
adaptive immunity towards VoC of SARS-CoV-28 needs to be explored.
Specific aims:
The present study is aimed to investigate the following:
Objective 1: To develop and validate pseudo typed virus neutralisation test (pVNT) panel to measure SARS-CoV-2
specific neutralization responses among naturally infected and vaccinated individuals.
Objective 2: To identify the functional ability of neutralizing antibodies and to check if an individual has
developed protective immunity towards SARS-CoV-2 VoC from convalescent patients and volunteers inoculated
with candidate vaccine.

Novelty/Innovation: The increasing prevalence and diversity of SARS-CoV-2 spike variants in India raises
concerns for current vaccines or for an increased risk of reinfection. Hence, developing indigenous SARS-CoV-2
variant lineage-based pseudo typed VNT would be useful for evaluating antibody responses against SARS-CoV-2
and also to facilitate the timely design and testing of next generation vaccines and therapeutic mAbs.
Study Design: Cross-sectional.
The research proposal will be submitted to YRG CARE & SRM Institute (Deemed to be University)
Institutional Review Board (IRB) for ethical clearance before initiating the study. The study
participants will be enrolled from SRM Medical College Hospital and Research Centre (SRM
MCHRC), Chennai.
Full-length open reading frame of the S gene of SARS-COV2 (Genbank accession: YP_009724390.1) will be
synthetized by GenScript (Piscataway, NJ) and cloned into pCDNA3.1 followed by site directed mutagenesis using
the QuikChange Lightning SiteDirected Mutagenesis kit (Agilent Technologies) to create WHO defined VoC
pseudovirus panel as given in table 1. HEK293T/17 cells (ATCC cat. no. CRL-11268) and 293T/ACE2.MF (will be
obtained from Dr David C. Montefiori, Duke University Medical Center, Durham, NC, USA).
Group-1 COVID-19 Vaccinated individuals (n=40):
Inclusion criteria:
• Both male and female with age 18 – 60.
• Completed both the doses of COVID-19 vaccination.
• No history of past COVID-19 infection.
Exclusion criteria: Chronic viral infections (eg. HIV, HBV and HCV) and pregnant and lactating women.
Group-2 Convalescent Individuals (n=40):
Inclusion criteria:
• SARS-CoV-2 convalescent individuals, consisting of 20 asymptomatic, 10 mild and 10 moderate infections.
• No history of vaccination.

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 Exclusion criteria: Chronic viral infections (eg. HIV, HBV and HCV) and pregnant and lactating women.
Group-3 SARS-CoV-2 infected (n=40):
Inclusion Criteria:
• SARS-CoV-2 infected asymptomatic or symptomatic.
• SARS-CoV-2 RNA positive.
• Not vaccinated for COVID-19.
Exclusion criteria: Chronic viral infections (eg. HIV, HBV and HCV) and pregnant and lactating women.

Materials and Methods:

Specimen collection:
Blood samples: From each study participants, about 50 mL of whole blood specimens will be collected in different
anticoagulant tubes as follows (i) 10 mL EDTA (n=3), 6 mL EDTA (n=2), 4 mL SST (n=2) for routine, study
specific parameters and future study storage.

Nasopharyngeal and oropharyngeal swabs specimens: Nasopharyngeal and oropharyngeal swabs specimens will
be collected only from SARS Cov2 RNA positive individuals.

Routine parameters: Complete Blood Counts, CD4 Count and HbA1C testing will be processed on real time basis.

Study-specific parameters: The study-specific parameters will be processed with batch testing and until then the
processed samples will be stored in appropriate storage conditions (Serum & plasma at -75 ±5°C and PBMCs at < -
134°C in LN2 system).

Amplification, detection and Sequencing of SARS Cov2 spike region from clinical samples: Nasopharyngeal and
oropharyngeal swabs specimen will be used to amplify spike region from individuals with SARS Cov2 variants
including delta plus (B.1.617.2.1 or AY.1)
Construction and identification of S expressing plasmid: The SARS-CoV-2, Indian spike will be cloned into
pCDNA3.1 and then subjected to Site Directed Mutagenesis (Agilent Technologies) to include WHO defined VoC
mutations8 including delta plus lineages. All mutations will be confirmed by full-length spike gene sequencing by
Sanger Sequencing. As shown in the table 1, pseudo virus neutralisation testing panel would be developed to assess
the breadth and magnitude of SARS CoV2 neutralising antibody elicited through natural and artificial adaptive
immunity.
Table 1: WHO SARS CoV2 Variants of Concern updated 25June2021.
Lentiviral pseudovirus production
and SARS-CoV-2 Pseudo-virus
neutralization assay: Pseudo viruses
will be produced by co-transfection
with a lentiviral backbone (HIV-1
pNL4.luc encoding the firefly
luciferase gene as previous described
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. Pseudovirus and serially diluted test
plasma/sera will be incubated for 1 h
at 37 °C, 5% CO2. Cells will be added
at 1 × 104 cells per well after 72 hrs of incubation at 37 °C, 5% CO2, luminescence will be measured using
luminometer. Neutralization will be measured as described by a reduction in luciferase gene expression
after single-round infection of 293T/ACE2.MF cells with spike-pseudotyped viruses. Titres will be
calculated as the reciprocal plasma dilution (ID50) causing 50% reduction of relative light units. Equivalency

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will be established through participation in the SARS-CoV-2 Neutralizing Assay Concordance Survey 1 run
by EQAPOL and VQU, Duke Human Vaccine Institute.

Distribution of tasks between collaborator institution:

Study Activities YRG CARE SRM

Institutional Ethical committee review and approval X X
Participant enrolment X X
Complete Blood Counts X
Specimen processing (PBMC, Plasma and Serum) and Storage X
Amplification, detection, and Sequencing of SARS Cov2 spike
X
region from Nasopharyngeal and oropharyngeal swabs
Construction and identification of S expressing plasmid X
Viral gene and viral genome sequencing X
Lentiviral pseudovirus production and SARS-CoV-2 Pseudo-virus
X
neutralization assay
Data analysis X X
Final report preparation and submission. X X

FEASIBILITY: Obtaining 40 SARS-CoV-2 infected patients might not be a major problem, since SRIHER is well equipped
with IP to treat SARS-CoV-2 infection. Also, even enrolling SARS-CoV-2 recovered and uninfected vaccinated individuals is
also possible with SRIHER, which is a multi-speciality hospital with more than >200 patients visit/day.
Timelines: The total duration of this study will be 24 months. Enough start-up time of 4 months is required to
prepare for the project, order the reagents and planning the manpower. Patient enrolment, assay standardization,
training, assay performance and data analysis will take place for the next 20 months ‘time.

Budget: An estimated cost of Rs. 80,89,630 is required (YRGCARE –Rs. 47,76,870 & SRM – 33,12,760). A
total of Rs. 53,05,000 will be utilized for Assay standardization, performance, reagents supplies, consumables, and
office supplies. A total of Rs. 27,84,630 will be utilized for personnel, travel
and administrative costs.

Expected outcome: Compared to a neutralizing assay with live virus, SARS-CoV-2 pseudotyped virus neutralization
assay is safer and can be easily carried out in BSL-2 conditions as the quantitative detection
of infected virus is realized by recording data objectively with a luminescence meter, which makes the assay
amenable to high-throughput detection and easy to standardize. Considering the evolving nature of SARS-CoV-2,
early identification and characterization of newly emerging variants requires robust genetic surveillance coupled with
rapid laboratory and clinical investigation of neutralizing antibodies would facilitate the timely design and testing of
next generation vaccines and therapeutic.

Dr. Shanmugam Saravanan, Associate Professor, YRG Centre for AIDS Research and Education (YRG CARE),
Chennai. I have been involved in HIV & Hepatotropic viral interaction research for more than 16 years and heading
state of the art Division of Molecular biology and Regional HIV-1 Genotyping laboratory. My key area involves
studying molecular characterization of HBV, HCV, HIV Drug resistance, sequence analysis and characterization of
different HIV subtypes. In the past several years I was awarded grants to study (i) HIV Drug Resistance in Different
HIV-1 Subtypes, (ii) HIV Drug Resistance Monitoring in Chennai, India, (iii) Validation of cost effective pooling
HBV DNA PCR for detecting occult HBV among HIV-1 infected drug naïve individuals in southern India (iv) the
prevalence of virological failure among HIV infected women on first-line ARV therapy, and to investigate inter-
compartmental difference in evolution of drug resistance and (v) Immune Activation in virologically Suppressed
Indian HIV- Infected Patients. For the first time in India, our lab has developed an ultra-sensitive allele specific PCR
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 to detect key drug resistance mutations in India. Recently I have received grant from DBT Welcome trust Team
Science Grant (TSG) for developing broadly neutralizing monoclonal antibody and exploring bNAbs mediated
prevention and treatment strategy by assessing their effectiveness in neutralizing HIV-1 subtype C circulating in
India across different regions and distinct risk groups (2019-2023) in collaboration with Dr. Jayanta Bhattacharya
(IAVI, Delhi) and Dr Vainav Patel, National Institute for Research in Reproductive Health (NIRRH), ICMR,
Mumbai. As a research convener, 4 students have been awarded PhD degree in the field of HIV drug resistance,
resistance in genital compartments, Inflammation, Immune Activation and HIV Reservoir with Affiliation of
University of Madras. For this proposal I will be responsible for all laboratory work in YRG CARE and will work
closely with Dr. Parani Madasamy, Professor & Head, Department of Genetic Engineering, SRM Institute of
Science and Technology (SRMIST), Chennai, according to the specific aim of the proposal.
List of 10 most recent publications
1. Munusamy Ponnan S, Thiruvengadam K, Kathirvel S, Shankar J, Rajaraman A, Mathaiyan M, Thongadi Ramesh
Dinesha, Selvamuthu Poongulali, Saravanan S et al. Elevated Numbers of HIV-Specific Poly-Functional CD8+ T
Cells With Stem Cell-Like and Follicular Homing Phenotypes in HIV-Exposed Seronegative Individuals. Front
Immunol. 2021;12:638144.
2. Rodgers MA, Gomathi S, Vallari A, Saravanan S, Lucas GM, Mehta S, et al. Diverse HCV Strains And HIV
URFS Identified Amongst People Who Inject Drugs In India. Sci Rep. 2020 Apr 29;10(1):7214.
3. Saravanan S, Gomathi S, Delong A, Kausalya B, Sivamalar S, Poongulali S, et al. High discordance in blood and
genital tract HIV-1 drug resistance in Indian women failing first-line therapy. J Antimicrob Chemother. 2018 Aug
1;73(8):2152–61.
4. Pallikkuth S, Pahwa R, Kausalya B, Saravanan S, Pan L, Vignesh R, et al. Cardiac morbidity in HIV infection is
associated with checkpoint inhibitor LAG-3 on CD4 T cells. PLoS One. 2018;13(10):e0206256.
5. Nandagopal P, Bhattacharya J, Srikrishnan AK, Goyal R, Ravichandran Swathirajan C, Patil S, et al. Broad
neutralization response in a subset of HIV-1 subtype C-infected viraemic non-progressors from southern India. J
Gen Virol. 2018 Mar;99(3):379–92.
6. Wallis CL, Viana RV, Saravanan S, Silva de Jesus C, Zeh C, Halvas EK, et al. Performance of Celera RUO
integrase resistance assay across multiple HIV-1 subtypes. J Virol Methods. 2017 Mar;241:41–5.
7. Sivamalar S, Dinesha TR, Gomathi S, Pradeep A, Boobalan J, Solomon SS, et al. Accumulation of HIV-1 Drug
Resistance Mutations After First-Line Immunological Failure to Evaluate the Options of Recycling NRTI Drugs
in Second-Line Treatment: A Study from South India. AIDS Res Hum Retroviruses. 2017 Mar;33(3):271–4.
8. Sharma S, Aralaguppe SG, Abrahams M-R, Williamson C, Gray C, Balakrishnan P, et al. The PTAP sequence
duplication in HIV-1 subtype C Gag p6 in drug-naive subjects of India and South Africa. BMC Infect Dis. 2017
Jan 24;17(1):95.
9. Saravanan S, Kausalya B, Gomathi S, Sivamalar S, Pachamuthu B, Selvamuthu P, et al. Etravirine and
Rilpivirine Drug Resistance Among HIV-1 Subtype C Infected Children Failing Non-Nucleoside Reverse
Transcriptase Inhibitor-Based Regimens in South India.AIDS Res Hum Retroviruses. 2017 Jun;33(6):567–74.
10. Duarte HA, Panpradist N, Beck IA, Lutz B, Lai J, Kanthula RM, et al. Current Status of Point-of-Care Testing for
HIV Drug Resistance.J Infect Dis. 2017 Dec 1;216(suppl_9):S824–8.

YRG CARE Institutional support: YRG CARE is a non-profit medical and research institution, located at Kilpauk,
Chennai and is one of the premiere institutions to provide comprehensive medical care for persons with HIV disease in
India. Our institution have state of art immunology and molecular biology laboratory installed with facilities such as
Coulter NAVIOS (Beckman Coulter, USA), 2 Victor X3 Luminometer (Perkin Elmer, USA), NSF certified Class-II
Bio-safety cabinets (ThermoElectron Co., USA) & Thermo Forma Scientific CO2 incubator (ThermoElectron Co.,
USA) to carry out immunological assays and PCR set-up (separate Pre-Amplification, Amplification and Post
amplification areas) with 3 Thermocyclers (Perkin Elmer, USA), Real Time PCR - m2000rt and m2000sp (ABBOTT
Molecular Diagnostics, USA), ABI 3500 Genetic Analyzer (Applied Biosystems, USA, ABI RealTime PCR 7500 Fast
Block (Applied Biosystems,USA), MiSeq Next Generation Sequencing (NGS) (Illumina Inc.,US) to carry out
molecular assays. Hence, we have competent enough to carry out SARS-2 spike amplification and Sequencing, Pseudo
virus preparation and Neutralization assays in our laboratory. Apart from that YRG CARE Infectious diseases
laboratory is ICMR approved for the COVID-19 testing facility, accredited by NABL, New Delhi and recognized by

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 the DSIR, New Delhi. So far more than 27,000 COVID-19 RT-PCR tests and 10,000 COVID-19 IgG antibody tests at
YRG CARE Infectious Diseases Laboratory.
SARS CoV-2 Pseudo-virus assay technical capabilities at YRG CARE Infectious diseases laboratory: YRG
CARE Infectious Diseases Laboratory is a BS2-Plus lab equipped with 3 BSL-2 Biosafety Cabinet (Thermo Scientific,
USA), Biosafety Centrifuge (Thermo Scientific, USA), CO2 Incubator (Thermo Scientific, USA), Victor-3
Luminometer (PerkinElmer, USA) to perform SARS-CoV-2 Pseudo-virus neutralization assay. We will get regular
technical inputs from Prof. David Montefiori laboratory (Duke University, USA), hence we did not foresee any issues
in implementing SARS CoV-2 Pseudo-virus assay in our laboratory.
DSIR Recognition: YRG CARE laboratory is recognized by Department of Scientific & Industrial Research (DSIR,
New Delhi) till 31.3.2023.

SRM (Deemed to be University) - Institutional support: The SRM Institute of Science and Technology (SRMIST)
Department of Genetic Engineering takes immense care to impart knowledge in a highly interactive mode, hands-on
training in advanced molecular biology techniques and cutting-edge research opportunities. They had vast experience
in molecular biology and genomics for analyzing the viral genome sequences and designing primers. They have well
equipped state of the art genetic engineering lab extensively expertise on RNA isolation, RT-PCR, RT-LAMP,
genomics and gene expression. List of the major Equipment includes Automated DNA Sequencer (SRM-DBT
Platform),Illumina Next Generation Sequencer (NextSeq500), Real-time PCR Machines, Gradient PCR machines,
PCR machines, Gene Pulser, Gene Gun, Chromosome Karyotyping System, Phase Contrast Microscope, Anaerobic
Glove Box, Anaerobic Fermentor, HPLC, FPLC, Gas Chromatography, Vaccum Concentrator, UV visible
Spectrophotometer, Inverted Fluorescence Microscope, Bioanalyzer, Microarray (SRM-DBT
Platform),Biophotometer,CO2 incubators, Thermomixer, Hybridization Oven,2-D Gel Electrophoresis System,
Incubator (Static and Shaking),Centrifuges, Nanodrop, Gel Documentation System, Laminar Air Flow Chambers,
Walk-in Tissue Culture and Transgenic Plant Facility, Ultra-Low Temperature Storage (-20°C and -80°C) Facility and
Biosafety cabinets (level II).
The SRM Medical College Hospital and Research Centre (SRMCHRC) was the very first human clinical/vaccine trial
site for the COVID-19 vaccine (Covaxin) in Tamilnadu. SRM has 200 bedded COVID-19 clinical care centre and their
lab has been authorised testing centre by the ICMR.

References:
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vaccines-development-phases.png (accessed 9 June 2021).
2. Wibmer CK, Ayres F, Hermanus T, et al. SARS-CoV-2 501Y.V2 escapes neutralization by South African COVID-
19 donor plasma. bioRxiv. Epub ahead of print 19 January 2021. DOI: 10.1101/2021.01.18.427166.
3. Xie X, Liu Y, Liu J, et al. Neutralization of SARS-CoV-2 spike 69/70 deletion, E484K and N501Y variants by
BNT162b2 vaccine-elicited sera. Nat Med 2021; 27: 620–621.
4. Shinde V, Bhikha S, Hoosain Z, et al. Efficacy of NVX-CoV2373 Covid-19 Vaccine against the B.1.351 Variant.
New England Journal of Medicine. Epub ahead of print 5 May 2021. DOI: 10.1056/NEJMoa2103055.
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6. Planas D, Veyer D, Baidaliuk A, et al. Reduced sensitivity of infectious SARS-CoV-2 variant B.1.617.2 to
monoclonal antibodies and sera from convalescent and vaccinated individuals. bioRxiv 2021; 2021.05.26.445838.
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Increases Infectivity of the COVID-19 Virus. Cell 2020; 182: 812-827.e19.