Senior Scientist Molecular Virology and vaccine development

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Dr. Vikram Srivastava

Senior Scientist NeoVaxSyn, Inc.

Mobile : +1-215-***-**** e-mail : **********.****@*****.*** Immigration Status: Lawful Permanent Resident

SKILLS & EXPERTISE IN BIOTECHNIQUES

1. Techniques of MAb: Preparation of Anti-Sera, Hybridoma Fusion, Antibody Gene Cloning from Hybridoma and single B-Cells, Expression and purification of Recombinant Antibody. 2. Protein Biochemistry: Protein expression, purification (FPLC, Unicorn 6, SoftMax Pro, GE AKTA) (size exclusion, and affinity chromatography) and characterization methods, SDS PAGE, western blot.

3. Immunochemical Techniques ELISA, Immuno-histochemistry, Fluorescent In-Situ Hybridization and Fluorescent Microscopy. Panel design and gating strategy for multiparametric FACS (FACS Aria III) and 8 color Flowcytometry LSRII for intracellular and surface staining to determine phenotype and cytokine profile of cell population. 4. Vaccine Design and Evaluation: Immunogen design and preclinical evaluation of immunogenicity and neutralizing and non-neutralizing antibody effector functions such as pseudovirus neutralization and ADCC assay. Analysis of humoral and T-cell immune responses in small animal model mouse and rabbits.

5. Molecular Biology: Cloning of genes in E. coli, Yeast, insect and mammalian vectors, site- directed-mutagenesis.

6. Cell Biology: Cell culture, transfection, and protein expression and purification from bacteria, yeast, insect (Sf9, Sf21), and mammalian (HEK293T, 293F expi293 and CHO, cell line) cells. 7. Virological Techniques: Influenza Virus culture in MDCK cells, HIV culture in H9 cells and preparation of virus from infectious molecular clones. Generation and purification of recombinant Vaccinia Virus. Virus quantification by enumeration of plaque forming unit. Pseudovirus preparation, TCID50 quantification and neutralization assay using luciferase/Beta-glo system and plaque reduction test. Virus and infectious manipulations in BSL-3 facility. 8. SOFTWARES: Delta Graph, SPSS, GraphPad Prism, Microsoft Office, EndNote, Flowjo, Snapgene for cloning, UCSF Chimera and Alphafold 2 for protein structure modelling and various other online software tools used in the field of molecular biology vaccine design and antibody research.

EDUCATION

1. Ph.D. in Medical Microbiology (September 2007) from the Department of Medical Microbiology, Faculty of Medical Sciences, University of Delhi, Delhi India. Thesis entitled “Role of Apoptosis in the Pathogenesis of Influenza A Virus, Correlation of Virological and Immunological Parameters: A study in Human & Murine Model”. Award Date February 2008. Status 2. Master of Science (M.Sc.) in Biotechnology (1997-1999) from the "Department of Biosciences & Biotechnology Indian Institute of Technology, Roorkee (Formerly University of Roorkee,), Roorkee INDIA”.

3. Bachelor of Science (B.Sc.) in Microbiology (1994-1997) from the "Department of Microbiology, D. Ram Manohar Lohia Awadh University, Faizabad (U.P.), INDIA". 2

SUMMARY OF PROFESSIONAL & RESEARCH EXPERIENCE

1. Senior Scientist NeoVaxSyn Inc. Ames IA August 2022 – Continued: Using a variety of molecular biology, immunology, and biochemical techniques, we study immune responses against vaccine candidates and adjuvants. My basic projects involve development of vaccine against HIV-1 using 10E8 based neutralizing antibody as a target epitope. I have cloned and expressed several recombinant antibody genes from the hybridoma generated, HIV-1Fusion peptide-based vaccine candidate immunized mice. These antibodies will be used for cryo-EM in complex with gp140 to analyze target epitopes. This will aid in better design of vaccine candidates. (Proprietary in nature). I am also responsible for the design, implementation, and execution of experiments. I also supervise the lab techs and scientific staff and review their goals and project deadlines. 2. Research Scientist II, Department of Biomedical Sciences, College of Veterinary Medicine, Iowa State University January 2020 – August 2022: This position was an extension of previous postdoc position at Iowa State University and my job responsibilities included review of SOPs, developing new methods, writing reports, manuscripts, and mentoring graduate students. Experimental and scientific responsibilities were structure-based immunogen design and preclinical evaluation of immunogenicity and neutralizing and non-neutralizing antibody effector functions such as pseudovirus neutralization and ADCC assay. Analysis of T-cell immune responses in small animal model mice and rabbits. I cloned and expressed two potent neutralizing recombinant antibody genes from the hybridoma generated, SARS-CoV-2 RBD-based vaccine candidate immunized mice. These antibodies are being used for cryo-EM in complex with spike to analyze target epitopes on spike. This will aid in better design of vaccine candidates. (Proprietary in nature) 3. Postdoctoral Research Associate, Department of Biomedical Sciences, College of Veterinary Medicine, Iowa State University October 2017- January 2020: I was associated with design, development, and preclinical evaluation of vaccine candidate. Cloning of SARS-CoV-2, HIV-1 and Influenza Virus vaccine candidates. Expression of recombinant fusion protein in mammalian and bacterial cells. Purification of protein using affinity and size exclusion chromatography. Characterization by ELISA for antigenicity evaluation. Assessment of immunogenicity and induction of neutralizing antibody in small animal model mice and rabbits. Analysis of neutralizing antibody response and generation of pseudovirus. Fusion generation and establishment of hybridoma and cloning of recombinant antibody gene from hybridoma and single B-cells. HIV and Influenza virus propagation and culture in H9 and MDCK. Generation and purification of recombinant vaccinia virus. Virus quantification by enumeration of plaque forming unit. Pseudo- virus preparation, TCID50 quantification and neutralization assay using luciferase/Beta-glo system, and plaque reduction test.

4. Postdoctoral Fellow, College of Pharmacy, Touro University, California May 2017- October 2017: I was responsible for DNA based vaccine development projects for Influenza virus. My job responsibilities were cloning of genes, expression, and purification of recombinant protein. Flow- cytometry based analysis of T-cell populations in splenocytes and lungs, Virus culture, and neutralization assays.

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5. Postdoctoral Fellow, Centre for Inflammation, Translational and Clinical Lung Research, Temple University, Philadelphia, June,2016-May 2017: The role and responsibilities at Temple University was with study involved in elucidation of defective mechanisms that influence the aggravated inflammatory and innate immune responses of airway epithelium to rhinovirus infection. And effects of quercetin, a plant polyphenol on airway epithelial regenerative mechanisms that are defective in COPD. Techniques specifically used in the study are ELISA, Immuno-histochemistry, Fluorescent In-Situ Hybridization, and Fluorescent Microscopy. Panel design and gating strategy for multiparametric FACS (FACS Aria III) and 8 colors Flowcytometry LSRII for intracellular and surface staining to determine phenotype and cytokine profile of cell population in blood and lungs. BALF collection, lung tissue fixation, and preparation for sectioning and staining. 6. Research Scientist, Department of Molecular Microbiology, Quantum Genetics, Lucknow, India March 2013-May 2016: My job responsibilities were to mentor and train technical staff and facilitate and execute research and PCR based molecular diagnostic services for the infectious diseases to the clinics and hospitals affiliated with the company. 7. Postdoctoral Scientist, AIDS Institute/ Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong January 2011-February 2013: I was, associated with development of a system to determine ADCC dominant epitopes on HA glycoprotein of Influenza virus. The project was having two components first part was to establish a flowcytometry based assay system to analyze and evaluate ADCC activity in plasma and purified antibodies in blood sample collected from influenza virus infected and vaccinated patients. This involved processing of blood samples received from the clinics of Queen Mary Hospital affiliated to University of Hong Kong. Plasma separation and purification of total IgG. Development of ADCC assay, using flowcytometry. And second component was generation of Yeast antigen display system to detect dominant epitopes associated with ADCC activity by comparing yeast populations probed with ADCC+ and ADCC- samples.

8. Associate Research Scientist, Department of Molecular Microbiology, Quantum Genetics, Lucknow, India January 2009-January 2011: My job responsibilities were to execute research and PCR based molecular diagnostic services for infectious diseases to the clinics and hospitals affiliated with the company.

9. Senior Scientist, Auroprobe Laboratories, (Molecular Diagnostic Facility), Delhi, India March 2008-January 2009: As a Senior Scientist I was responsible for delivering molecular diagnostics services on established platforms for HCV and HBV COBAS AMLICOR and HPV for the diagnosis of infectious diseases.

10. Scientist-II Division of Virology, National Institute of Cholera & Enteric Diseases, Kolkata, India September 2007–March 2008: in the project entitled “Multi Site Monitoring of Influenza Virus” sponsored by Indian Council of Medical Research India and Centre for Disease Control USA. My Job responsibility was to provide diagnostics support in the event of any outbreak of influenza virus both in human and avian populations.

11. Visiting Scholar, Kresge Eye Institute; Wayne State University, 4717 St. Antoine; Detroit, MI 48201 February 2007 – April 2007: Visiting scholar to study effect of oxidative stress in diabetic retinopathy.

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12. Research Intern, Senior Research Fellow and PhD student, Department of Respiratory Virology V P Chest Institute University of Delhi, Delhi, India January 2001 – September 2007: Role of Apoptosis in the Pathogenesis of Influenza A Virus, Correlation of Virological and Immunological Parameters: A study in Human & Murine Model. (Ph.D. Thesis) The PhD research work was carried out at the Influenza Monitoring Center, Department of Respiratory Virology, Vallabhbhai Patel Chest Institute, University of Delhi. My research work focused on identification of influenza virus in clinical specimens, study of virus induced apoptosis and its role in viral pathogenesis in experimental murine model. I have developed murine model of influenza and have gone through study of influenza virus pathogenesis at cellular level, role of proinflammatory and anti- inflammatory cytokines by ELISA and flowcytometry, iNOS expression by RT-PCR and PKR activation by Western Blotting, histopathological examination of mouse lung samples to observe the changes associated with virus pathogenesis during influenza virus infection. AWARDS & FELLOWSHIPS RECEIVED

1. Graduate Aptitude Test in Engineering (GATE), Conducted by Indian Institute of Technology, Mumbai, India Qualified -1999.

2. Independent “Senior Research Fellowship” (SRF) from Indian Council of Medical Research

(ICMR), Delhi.

3. Foreign Travel Grant Awarded by Council for Scientific and Industrial Research, Government of India to Attend 44

th

Inter-science Conference on Antimicrobial Agents and Chemotherapy

(ICAAC), Washington, DC, USA, October 30 – November 2, 2004. 4. Foreign Travel Grant Awarded by Department of Science & Technology, Government of India to Attend XIII

th

International Congress of Virology 2005, Organized by 3 Divisions of the International Union of Microbiological Societies Based on the Theme “Microbes in Changing World” San Francisco, CA USA, July 23 - July 28, 2005. TRAINING & WORKSHOP

1. 3rd Winter School in Immunology, Goa, India, August 4th to August 10th, 2006) I have successfully completed a course of Basic and Applied Immunology organized National Cancer Research Institute, USA which comprised lectures on development of the immune system, cellular mechanisms involved in immune - regulation, effector and memory responses, innate immunity, complement system, role of Treg in lymphocyte activation, cell signaling pathways of lymphocyte activation.

2. Immunocyte Activation: In Vitro Protocols 3rd November to 14th November 2003) Organized by International Centre for Genetic Engineering & Biotechnology (ICGEB), New Delhi, India. The course consisted of 11 hours of lectures on various topics including development of the immune system, cellular mechanisms involved in immune - regulation, effector and memory responses, innate immunity, mucosal immunity, cell signaling pathways modulating lymphocyte activation and mass spectrometry. The course also consisted of 80 hours of laboratory experiments that covered topics such as maturation of dendritic cells and evaluation of their antigen - presenting abilities, measurement of cytokines, analysis of calcium responses in lymphocytes, nitric oxide responses in 5

activated macrophages, analysis of intracellular phosphorylation events, resolution of cellular proteins by 2-dimensional gel electrophoresis and protein identification by tryptic digestion followed by mass spectrometry.

PUBLICATIONS

1. Srivastava V, Niu L, Phadke KS, Bellaire BH and Cho MW (2021) Induction of Potent and Durable Neutralizing Antibodies Against SARS-CoV-2 Using a Receptor Binding Domain-Based Immunogen. Front. Immunol. 12:637982. doi: 10.3389/fimmu.2021.637982 2. Niu L, Wittrock KN, Clabaugh GC, Srivastava V and Cho MW (2021) A Structural Landscape of Neutralizing Antibodies Against SARS-CoV-2 Receptor Binding Domain. Front. Immunol. 12:647934. doi: 10.3389/fimmu.2021.647934 3. Gimenes JA Jr, Srivastava V, ReddyVari H, Kotnala S, Mishra R, Farazuddin M, Li W, Sajjan US. Rhinovirus-induces progression of lung disease in a mouse model of COPD via IL33/ST2 signaling axis. Clin Sci (Lond). 2019 Apr 29; 133 (8): 983-96. PMID: 30952808. doi: 10.1042/CS20181088 4. Farazuddin M, Mishra R, Jing Y, Srivastava V, Comstock AT Sajjan US (2018), Quercetin prevents rhinovirus-induced progression of lung disease in mice with COPD phenotype. PLoS ONE, 13, e0199612.

5. Srivastava V, Zheng Y, Hung I, Xu J, Zheng B Zhang MY (2013), Identification of dominant ADCC epitopes on hemagglutinin antigen of pandemic H1N1 influenza virus. J. Virol. 87, 10, 5831- 840.

6. Srivastava V, Khanna M, Sharma S and Kumar B, (2012) Resolution of immune response by recombinant transforming growth factor-beta (rTGF-β) during influenza A virus infection. IJMR:136,641-648.

7. Srivastava V, Rawall S, Vijayan V.K. and Khanna M. (2009) Influenza A virus induced apoptosis: Inhibition of DNA laddering and caspase 3 activity by zinc supplementation in culture Hela cells. IJMR:129, 579-586.

8. Khanna M, Akther N, Srivastava V, Kumar P & Vijayan VK. (2006) Biological and epidemiological aspects of influenza virus H5N1 in context of India Ind J. Exp. Biol.;44(4):265-78. 9. Kumar P, Khanna M, Srivastava V, Tyagi Y, Ravi K, Raj HG, (2005) Effect of Quercetin Supplementation on Alterations in Antioxidant Defenses in Lung After Experimental Influenza Virus Infection. Experimental Lung Research, 31: 1-11. 10. Khanna M, Srivastava V, Kumar P (2002) Virological Studies in Clinical Practice: Utility & Indications: Medicine Update, Annual Ed. 7/2, 74-78. ISSN:0971-7684. CHAPTERS IN BOOKS

1. Khanna M, Srivastava V, Structure and Classification of Influenza Virus. P 11 – 23. In: Tripthi M. Mathew & Thankam Mathew, eds. Influenza and Its’ Global Public Health Significance Thajema Publishers 31 Glen view Dir. West Orange NJ 07052 USA: (6). ISBN:09727597-5-1 2. Khanna M, Srivastava V, Clinical and Laboratory Diagnosis of Influenza Virus. P 138 – 151. In: Tripthi M. Mathew & Thankam Mathew, eds. Influenza and Its’ Global Public Health Significance Thajema Publishers 31 Glen view Dir. West Orange NJ 07052 USA: (6). ISBN:09727597-5-1 3. Khanna M, Nazir R, Srivastava V, Prophylaxis Prevention and Treatment for Influenza A, B, C. p 169 – 191. In: Tripthi M. Mathew & Thankam Mathew, eds. Influenza and Its’ Global Public Health Significance Thajema Publishers 31 Glen view Dir. West Orange NJ 07052 USA: (6). ISBN:09727597-5-1

4. Khanna M, Srivastava V, Influenza Pandemic Preparedness. P 192 – 201. In: Tripthi M. Mathew

& Thankam Mathew, eds. Influenza and Its’ Global Public Health Significance Thajema Publishers 31 Glen view Dir. West Orange NJ 07052 USA: (6). ISBN:09727597-5-1 6

COFERENCES & SYMPOSIA

1. Srivastava V., Khanna M and Vijayan V.K. Immunomodulatory effect of rTGF-β1 on Nitric Oxide Synthase (iNOS) and dsRNA Dependent Protein Kinase (PKR) mediated inflammatory response During Experimental Influenza Infection in Mice presented in Third European Influenza Conference organized by European Scientific Working group on Influenza, B-2000 Antwerp, Belgium, at Vilamoura, Portugal, 14-17 September 2008.

2. Srivastava V., Khanna M and Vijayan V.K. Biphasic effect of rTGF-β1 on Nitric Oxide Synthase

(iNOS) and dsRNA Dependent Protein Kinase (PKR) During Experimental Influenza Infection in Mice presented in Options for the Control of Influenza – VI organized by Centre for Disease Control Foundation, Atlanta GA USA at Toronto, Canada, June 17 – June 23, 2007. 3. Srivastava V., Khanna M and Vijayan V.K Role of TGF- β on Nitric Oxide Synthase Dependent Inflammation During Experimental Influenza Infection presented in Mice presented in XIII th

International Congress of Virology 2005, Organized by 3 Divisions of the International Union of Microbiological Societies Based on the Theme “Microbes in Changing World” San Francisco, CA USA, July 23 - July 28, 2005.

4. Srivastava V., Khanna M, Rawall S and Vijayan V K, (2005) Effect of Zinc on Influenza A virus Induced Programmed Cell Death Virology, presented in Berlin International Influenza Conference 2005, Organized by Free University Berlin and German Society of Virology, 26 th

May 2005 – 29

th

May 2005.

5. Srivastava V., Khanna M and Vijayan V.K. Effect of TGF-β on the resolution of immune response after influenza A virus infection presented in 44th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, USA, October 30 – November 2, 2004. PROTOCOL AND ASSAY DEVELOPED

ADCC Assay Protocol Authors: Vikram Srivastava, Zheng Yang, Ivan Fan Ngai Hung, Jianqing Xu, Bojian Zheng and Mei-Yun Zhang, date: 1/20/2014, Protocol for ADCC assay using human peripheral blood mononuclear cells (PBMCs) as effector cells. Using this protocol, we determined the ADCC activity of convalescent plasma IgGs from six H1N1-infected patients. ADCC was determined using the flow-cytometry", http://www.bioprotocol.org/e1029

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