1
Instituto Tecnológico y de Estudios Superiores de Monterrey
Campus Monterrey
School of Engineering and Sciences
Development of a Pseudotyped Lentivirus-Based Assay to Measure Neutralizing Antibody activity against SARS-CoV-2 in Mexico
A thesis presented by
José Antonio Cruz Cárdenas
Submitted to the
School of Engineering and Sciences
in partial fulfillment of the requirements for the degree of Master of Science
In Biotechnology
Monterrey Nuevo León, June 1st, 2021
All rights reserved Dedicatoria
A mis padres, Julián Fernando y Gricelda, quienes han sido estimulo para mi crecimiento personal y profesional, por su apoyo incondicional, por su ayuda en los momentos mas difíciles y promover los valores como base para el éxito y ser un ejemplo a seguir.
A mis hermanos, por ser piezas claves en todo el proceso, desde mis estudios básicos, hasta el día de hoy y lo seguirán siendo, dándoles el ejemplo que todo lo que se propongan lo pueden lograr siempre y cuando se esfuercen para materializar sus sueños.
A mi novia, Alejandra López, por ser mi motivación a ser mejor persona cada día y ayudarme a crecer en todo sentido, por su apoyo incondicional y amor.
A mis abuelos, Norberta, Martha y José Antonio, por que, sin duda, sin ellos este logro no hubiera sido posible.
5 Agradecimientos
A la Dra. Marion Brunck, por permitirme formar parte de su grupo de investigación y darme la confianza para desarrollar este Proyecto.
A mis compañeros del Brunck Lab, del Tecnológico de Monterrey: Raúl Piñeiro Salvador, por su plena disposición a ayudar a resolver los problemas experimentales que surgieron durante el Desarrollo de este Proyecto, por ser una pieza fundamental en las optimizaciones realizadas en las etapas mas criticas del proyecto. A Milena Ureña Herrera, por su apoyo y disponibilidad para apoyar en cultivo cellular cuando se necesitaba. A Alejandrá de Jesús López Arredondo, por su ayuda en las optimizaciones de las tecnicas de proteomica y biologia molecular.
A las Dras. Laura Alicia Palomares Aguilera y Michelle Gutiérrez Mayret, del Instituto de Biotecnología de la UNAM, por su colaboración y ayuda para el desarrollo de las herramientas implementadas en este proyecto.
Al Dr. Cuauhtémoc Licona Cassani, por su disposición y apoyo para enriquecer el proyecto.
A los Drs. José Antonio Enciso Moreno y Julio Enrique Castañeda Delgado, del Instituto de Investigación Biomédica, IMSS-Zacatecas, por su colaboración y contribución con las muestras de suero usadas para el desarrollo de este proyecto.
A los Drs. Augusto Rojas Martínez Gerardo García Rivas, Christian Silva Platas, Eduardo Vázquez Garza, de Tec Salud, por su colaboración permitiéndonos el uso de sus instalaciones, específicamente la ultracentrífuga y cell sorter para el Desarrollo de partes fundamentales para este Proyecto
Al M. Sc Héctor Manuel Castañeda Aponte, por su apoyo y enseñanzas durante toda mi estancia en el posgrado.
A Conacyt por mi beca de investigación #1007842
A AMEXCID, por el recurso otorgado para la realización de este proyecto.
7
Development of a Pseudotyped Lentivirus-Based Assay to Measure Neutralizing Antibody activity against SARS-CoV-2 in Mexico
by
José Antonio Cruz Cárdenas
Abstract
The SARS-CoV-2 virus is responsible for the COVID-19 pandemic declared by the WHO in March 2020, which has caused more than 172 million confirmed cases and 3.69 million deaths worldwide to date. Infection with SARS-CoV-2 leads to the development of antibodies in patients. The presence of neutralizing antibodies protects against SARS-CoV-2 infections and is an essential parameter that confirms the success of vaccination. The titration of neutralizing antibodies by classical methods is not trivial since it requires the use of replicative virus, which implies a high risk of infection and requires facilities certified with the BSL-3 biosafety level.
Mexico is one of the countries most affected by SARS-CoV-2 and there are not enough facilities to carry out effective immunity monitoring. Here, this thesis presents the results of a multi-institutional national collaboration in the design of a non- replicative pseudovirus that expresses the SARS-CoV-2 spike protein on its surface, which is applied in the development of a method for quantifying SARS-CoV neutralizing antibodies. The development of this assay will facilitate the characterization and monitoring of humoral immunity against SARS-CoV-2, and can be easily modified to monitor immunity against emerging variants in the country. To the best of our knowledge, this work presents the first report of measuring neutralizing antibody to SARS-CoV-2 in the Mexican population using a pseudovirus system.
List of Figures
1 Constructs developed as part of a third-generation lentiviral plasmid
system for the development of SARS-CoV-2 S pseudovirus………... 15
2 Validation of pLenti-Nanoluc……… 17
3 Gel electrophoresis of 2 restriction maps obtained for pCMV-Spike (57)-Rev……… 19
4 Transfection optimization of HEK-293T validated by fluorescence microscopy and flow cytometry……… 21
5 HEK-293T/ACE2 production……… 22
6 Representative graph of a recombinant p24 standard curve Lentivirus- associated p24 titration graphs……… 23
7 Correct assembly of the produced lentiviral particles……….. 29
8 Kinetics of EGFP expression over 7 days post-infection of HEK-293T cells with VSV-G-EGFP pseudovirus……….. 25
9 Kinetics of EGFP expression over 7 days post-infection of HEK-293T cells with VSV-G-EGFP pseudovirus……… 26
10 Optimization of target cell number during infection of HEK-293T with VSV-G-Nanoluc pseudovirus……….. 28
11 ACE2 expression on cell lines………. 30
12 Susceptibility of cell lines to pseudovirus infection……….. 31
13 Optimization of virus dose for neutralization………. 33
14 Measurement of neutralizing activity in pre-pandemic human samples... 35
15 Measurement of neutralizing activity in COVID-19 positive human samples……..………. 36
9 Table
1 Summary of the demographic, clinical parameters and serum EC50 of patients which sera were tested in this work……… 37
Contents
Abstract ... 7
List of Figures ... 8
Table ... 9
1. Introduction... 13
2. Theoretical Framework ... 15
2.1. Human coronavirus ... 15
2.2. SARS-CoV-2 ... 16
2.3. SARS-CoV-2 S immunogenicity... 16
2.4. Assay to measure neutralizing antibodies (nAbs) ... 17
2.5. SARS-CoV-2 S pseudovirus to measure neutralizing antibodies ... 18
3. Thesis outline ... 19
3.1. Justification ... 19
3.2. Hypothesis ... 19
3.3. General objective ... 19
3.4. Specific objectives ... 19
4. Materials and methods ... 20
4.1. Primer design and PCR... 20
4.2. Vectors Construction ... 20
4.3. Cell culture ... 21
4.4. Transfection optimization ... 22
4.5. SARS-CoV-2 S pseudovirus production ... 22
4.6. Pseudovirus quantification ... 23
4.7. Flow cytometry ... 24
11
4.8. Infection assays ... 24
4.9. Western blot analysis of viral particles ... 24
4.10. TCID50 ... 25
4.11. Human samples ... 26
4.12. Neutralizing antibodies assay ... 26
4.13. Generation of ACE2-HEK-293T stably expressing cell line ... 26
5. Results ... 28
5.1. Production of pseudotyped lentiviral particles ... 28
5.2. Transfection optimization of HEK-293T ... 32
5.3. Generation of stable expressing ACE2 cell line ... 33
5.4. Quantification of produced lentiviral particles ... 35
5.5. Validation of pseudovirus assembly... 37
5.6. Transduction of HEK-293T with VSV-G-EGFP pseudovirus ... 38
5.7. HEK-293T infection with VSV-G-Nanoluc pseudovirus ... 40
5.8. Cell line selection ... 41
5.8.1. ACE2 expression on target cell lines ... 41
5.8.2. Infection susceptibility ... 43
5.8.3. TCID50 titration ... 45
5.8.4. Neutralizing Antibodies Assay ... 47
6. Discussion ... 52
7. Conclusions ... 58
7.1. Conclusions ... 58
7.2. Future work ... 58
8. Appendix ... 59
8.1. Appendix A. Primers for Nanoluc and Spike(D57) cloning ... 59
8.2. Appendix B. Sanger sequencing results ... 59
8.2.1. Spike D57 ... 59
8.2.2. Rev gene ... 60
8.2.3. 5’ LTR ... 61
8.2.4. 3’ LTR ... 61
8.2.5. Nanoluc gene ... 61
8.2.6. RRE ... 61
8.2.7. cPPT ... 61
8.2.8. WPRE ... 62
9. Bibliography... 63
10. Curriculum Vitae ... 70
Chapter 1. Introduction 13
Chapter 1
1. Introduction
The new -coronavirus SARS-CoV-2 is the causative agent of the COVID-19 pandemic which started in March 2020 and has resulted in more than 120 million confirmed cases and >2.6 million deaths worldwide at the time of writing. SARS- CoV-2 contains a positive- sense RNA genome and is assembled with four structural proteins: spike (S), membrane (M), nucleocapsid (N) and envelope (E). The S protein of SARS-CoV-2 mediates viral entry into target cells through binding to the angiotensin-converting enzyme 2 (ACE2) receptor (Lan et al., 2020). The S protein is strongly immunogenic and induces the production of neutralizing antibodies (nAbs). The presence of nAbs in humans is a correlate of protection (Addetia et al.,
2020) and passive transfer of nAbs prevents and mitigates disease in animal models of SARS-CoV-2 infection (Hassan et al., 2020; Zost et al., 2020). SARS- CoV-2 S is therefore the target of all currently available vaccines (Krammer, 2020).
Titers of nAbs against SARS-CoV-2 are reported to culminate between 2 and 4 weeks post virus exposure and to slowly decline over 2 to 3 months (Legros et al., 2021; Seow et al., 2020), while a recent study reports remaining titers of anti-S antibodies up to 6 to 8 months after infection (Dan et al., 2021). Demographic parameters affect the development and persistence of nAbs. For instance older individual and patients with BMI>25 have significantly higher titers, whereas female patients exhibit a slower decline of nAbs (Grzelak et al., 2020). Paradoxically, higher titers of NAbs have been measured in patients suffering from a more severe form of COVID-19 (Legros et al., 2021). Due to the relative novelty of the virus within the human population, and the wide heterogeneity in clinical manifestations of COVID- 19, many questions remain to be answered regarding SARS-CoV-2 nAbs.
Emerging SARS-CoV-2 variants exhibit mutations in the S protein. The D614G variant shows increased infectivity and replication rates in infected cells, while the B.1.531 variant escapes neutralization by antibodies elicited against a distinct SARS-CoV-2 lineage (Plante et al., 2021; Wibmer, s. f.). Host genetics also likely impact the severity of infection and development of an appropriate immune
response (Ovsyannikova et al., 2020). It is therefore critical to assess the impact of emerging variants on host humoral immunity and vaccine efficacy locally. Classic assays of antibody-mediated virus neutralization involve the use of wild-type, infectious SARS-CoV2. These assays carry a risk of infection and as such must be performed in a Biosafety Level 3 (BSL-3) type facility. These facilities are scarce in Latin America and not readily available in Mexico. An alternative strategy is to produce nonreplicative viral particles that express the SARS-CoV-2 S protein on their surface and contain a reporter gene to be delivered to infected cells (Garcia- Beltran et al., 2021; Nie et al., 2020a).
Mexico has experienced one of the highest COVID-19 mortality rate worldwide, which may be explained in part by the prevalence of co-morbidities (Suárez et al., 2020). The immune response in Mexican patients has not been reported at the time of writing. The development of a SARS-CoV-2 nAb assay in Mexico will facilitate the characterization and monitoring of humoral immunity to SARS-CoV-2 infections and to emerging variants in the country. This safe and sensitive alternative system also permits the high-throughput characterization of vaccine efficacy locally, and guide the selection of convalescent plasma for clinical trials. Finally, though this development we present the first report of humoral immunity to SARS-CoV-2 in the Mexican population.
Chapter 2. Theoretical Framework 15
Chapter 2
2. Theoretical Framework 2.1. Human coronavirus
Coronaviruses are pathogens that originate respiratory and intestinal infections in animals and humans. The coronaviruses genome size ranges from about 26 to 32 kilobases, one of the largest among RNA viruses. Hundreds of coronaviruses currently infect animals such as bats, pigs, camels, and cats, all of them considered the main reservoirs of these viruses (Chakrabarti et al., 2020; Poon et al., 2005). Coronaviruses first arose in the mid-1960s, and there are seven distinct variants of the virus classified into four groups: alpha, beta, gamma, and delta. Only seven known forms of coronaviruses cause disease in humans, subdivided into two different strains, one of them causing mild symptoms such as the common flu (HCoV-OC43, HCoV-HKU1, HCoV-229E, and HCoV-NL63), and the other group causing severe respiratory symptoms (MERS-CoV, SARS-CoV, and SARS-CoV-2).
Historically, coronaviruses were not considered highly pathogenic in humans.
During the end of 2002, an outbreak of Severe Acute Respiratory Syndrome (SARS- CoV) associated with influenza symptoms was detected in China, sometimes culminating in severe respiratory failure. Up to this point, the international scientific community highlighted the importance of studying these viruses and their pandemic potential. The SARS-CoV outbreak spread to 30 countries, causing more than 8000 cases.
Then in 2012, another outbreak of coronavirus emerged in Saudi Arabia. The pathological agent was named Coronavirus-associated Middle East Respiratory Syndrome (MERS-CoV), and 27 countries reported more than 2,500 cases (Su et al., 2016). By the ending of 2019, a new disease outbreak of unknown etiology causing severe respiratory syndrome emerged. Some days later, a coronavirus was identified as the pathological entity and was named SARS-CoV-2.
2.2. SARS-CoV-2
The new -coronavirus SARS-CoV-2 is the causative agent of the COVID-19 pandemic which started in March 2020 and has resulted in more than 166 million confirmed cases and >3.4 million deaths worldwide at the time of writing. SARS- CoV-2 is a single strand RNA virus of positive polarity and is assembled with four structural proteins: Spike (S), membrane (M), nucleocapsid (N) and envelope (E).
The Spike (S) protein of coronaviruses is responsible for internalizing the virus into the cell. Two subunits compose the S protein. The S1 is the ectodomain that contains the Receptor Binding Domain (RBD) that binds with the cellular receptor Angiotensin Converting Enzyme (ACE2) and promotes the onset of infection. The S2 subunit facilitates the fusion of the virion membrane and the cell membrane (Lan et al., 2020; Yurina, 2020).
2.3. SARS-CoV-2 S immunogenicity
Antibodies against most components of the virus have been reported during a SARS-CoV-2 infection, but only those directed against Spike have neutralizing activity. S protein plays an essential role in the induction of both humoral and cellular immunity (Gaebler et al., 2021). The S protein is strongly immunogenic and induces the production of neutralizing antibodies (nAbs), specifically those antibodies directed against RBD. This domain of the protein S specifically comprises the binding site of the virus with ACE2 receptor on the cell surface, therefore, an antibody that binds to that specific region would have neutralizing characteristics (Lan et al., 2020).
The presence of neutralizing antibodies has been shown to correlate with protection against SARS-CoV-2 (Addetia et al., 2020). In addition, several studies have been carried out that correlate the severity of the disease with the production of neutralizing antibodies, in which patients who presented severe COVID-19 present a neutralizing antibody titer compared to those patients who presented the moderate form or were asymptomatic (Garcia-Beltran et al., 2021).
Chapter 2. Theoretical Framework 17
There are studies where they report that comorbidities such as hypertension, diabetes, obesity, play a fundamental role in the development of the severe form of COVID-19, relating the presence of these comorbidities with the development of the disease (Zost et al., 2020).
Among the proteins that make up the structure of SARS-CoV-2, the Spike protein is considered to have strong immunogenicity and most of the antibodies produced are directed against this protein. Given the immunogenicity of the SARS- CoV-2 S protein, it is being used as a target for the development of vaccines, to date, most of the vaccines that are currently being used are directed against the S protein (Krammer, 2020)
2.4. Assay to measure neutralizing antibodies (nAbs)
One of the challenges that the pandemic generated by SARS-CoV-2 brought is the need to develop tests that allow the study of long-term immunity, either generated by a natural infection or induced by vaccination. Measurement of neutralizing antibodies is necessary to study the long-term duration of immunity. One of the techniques used to measure neutralizing antibody titers is the plaque assay, traditionally performed with the SARS-CoV-2 wild type (Burton et al., 2021). This test represents a useful tool to measure neutralizing antibody titers, however, it has disadvantages that limit its applicability in Mexico and the latin America (LATAM) countries. The infrastructure that is needed to manipulate SARS-CoV-2 is not so common in LATAM countries, this test must be carried out with real SARS-CoV-2 and it must be handled in level 3 biosafety laboratories(Beltrán-Pavez et al., 2021).
Mexico and LATAM there are few BSL-3 laboratories to perform plaque assays for the measurement of neutralizing antibodies, so the study of neutralizing antibodies with this class of assays is difficult to apply in Mexico.
There are other types of assays that allow the measurement of neutralizing antibody titers, an example of them is the one developed by Tan et al., 2020, which is an assay based on an Elisa, where a 96 plate with the ACE2 receptor is used.
adhered to its surface and soluble RBD coupled to HRP (Tan et al., 2020). However,
due to there are international limitations that prevent the importation of these reagents and make it difficult to implement them.
2.5. SARS-CoV-2 S pseudovirus to measure neutralizing antibodies
Pseudoviruses are a very versatile class of biological tools for studying emerging viruses. These allow the study of mechanisms of entry of viruses to cells (Nie et al., 2020a). SARS-CoV-2 is an emerging virus that must be handled in BSL- 3 laboratories, limiting the study of this virus in countries lacking in this infrastructure.
Pseudoviruses have been widely used for the study of humoral immunity (Schmidt et al., 2020). The development of assays based on pseudovirus that pseudotype the SARS-CoV-2 S protein are tools that allow us to manage this class of assays in BSL-2 laboratories. This class of pseudoviruses are based on using non-replicative viral vectors that contain the SARS-CoV-2 S in their membrane. The use of different viral vectors has been reported to generate pseudovirus, using different types of virus backbones. For example, Nie et al(Nie et al., 2020a), developed a SARS-CoV-2 S pseudovirus using the vesicular stomatitis virus (VSV) backbone, which is a negative polarity RNA vector and does not integrate its genetic material into the host cell genome. This kind of system presents a limitation, the viral titers obtained are lower, compared to other viral vectors, such as lentiviruses.
Other type of viral vector widely used to produce pseudovirus are the lentivirus-based systems, these use a non-replicative backbone based of HIV-1, they are positive polarity RNA viral vectors that have the characteristic that they integrate their genetic material into the host cell genome and have the advantage that they can higher viral titers than those obtained with other class of viral vectors will be produced (Kutner et al., 2009; Miyoshi et al., 1998a).
Chapter 3. Thesis outline 19
Chapter 3
3. Thesis outline 3.1. Justification
Due to the need to study neutralizing antibody-based immunity against SARS- CoV-2 in Mexico, and the lack of BSL-3 laboratories in Mexico, we aimed to develop a pseudovirus-based assay that can be implemented in BSL-2 laboratories and allows the study of humoral immunity in Mexico. This test facilitates the study of neutralizing antibody-based immunity in the Mexican population.
3.2. Hypothesis
The generation of a SARS-CoV-2 pseudovirus that contains the Spike protein in its membrane with a 19aa deletion at the C-terminal and the coding sequence for the Nanoluc gene in its genome, allows it to infect cells that express the ACE2 receptor and identify the infection by using Furimazine.
3.3. General objective
To develop a neutralizing antibody measuring assay based on a non- replicative lentivirus pseudotyping SARS-CoV-2 Spike.
3.4. Specific objectives
1. Design in silico and produce in vitro the plasmids required to assemble the SARS-CoV-2 Spike pseudovirus and relevants controls.
2. Produce stocks of the lentiviral particles and characterize their molecular and infective properties using western blot and a cell infection assay.
3. Optimize relevant parameters of the neutralizing antibodies assay such as host cell type, seeding density and infection protocol.
4. Measure neutralizing antibodies in the sera of pre-pandemic and COVID-19 confirmed patients in Mexico.
Chapter 4
4. Materials and methods 4.1. Primer design and PCR
The primers used in this Project were designed using SnapGene software v.4.3 and evaluated on the Oligo Analyzer online platform (Integrated DNA Technologies). Appendix A details all primer sequences designed. All PCR reactions were performed using Phusion flash high fidelity master mix (Thermo Fisher Scientific, cat. F548S).
4.2. Vectors Construction
pCAG-HIVgp and pCMV-VSV-G-RSV-Rev plasmids were kindly gifted by Dr.
Yukio Nakamura from Riken instate, Japan (Miyoshi et al., 1998). The pCMV14-3X- Flag-SARS-CoV-2 S plasmid encodes the codon-optimized SARS-CoV2 Spike lacking the last 19 amino acids at the C-terminal, and was a kind gift from Dr. Zhaohui Qian (Ou et al., 2020a). The pCMV-SARS-CoV2 S–RSV-Rev plasmid was obtained by cloning the SARS-CoV-2 S sequence in place of the VSV-G gene, between the NheI and XbaI sites. pLenti-Nanoluc was produced by amplifying the Nanoluc gene from pCCI-SP6-ZIKV-Nanoluc (Kindly gifted by Dr. Laura Palomares Aguilera from Instituto de Biotecnología, UNAM), and cloning it between the XbaI and BamHI sites of the pLentiCRISPR v2 (Kindly gifted by Dr. Laureano de la Vega from University of Dundee, Scotland) and removing the CAS9 gene using XbaI and BamHI resctriction enzymes. From pCMV-VSV-G-RSV-Rev, we produced the plasmid pCMV-Rev, which lacks an envelope protein gene, by removing the VSV-G gene with NheI and XbaI enzymes. Briefly, 5 g of each gene was mixed with 5 l of 10x Cutsmart buffer, 1 l of each enzyme, and up to 50 l of nuclease-free water. The digestion reaction was incubated at 37C overnight. For all ligation reactions, 30 ng of gene insert were mixed with 150 ng of vector, 2 l of 10x ligase buffer and 1 l of T4 DNA ligase (Promega cat. M1801). The ligation reaction was carried out in 20 l of final volume and incubated overnight at 4C. Afterwards, each construction was electroporated in E. coli strain Stbl4 cells, suitable for unstable gene fragments and
Chapter 4. Materials and methods 21
plasmid containing LTRs (Al-Allaf et al., 2013, p. 4). For electroporation, 50 l of electrocompetent E. coli Stbl4 cells were mixed with 3 l of ligation reaction and electroporated using a Gene Pulser XCell electroporator (Bio-rad cat. 1652666) with the pre-set program for bacterial electroporation using 0.2 cm2 cuvettes.
Constructions were validated by restriction maps: to validate the plasmid pLenti-Nanoluc, a triple digestion was performed with the enzymes BamHI, PuvI and NheI. The reaction was incubated overnight at 37C and the product was visualized on a 1% agarose gel ran 1h at 90v. To validate the plasmid pCMV-Spike (57)-Rev, 2 digestion reactions were performed with different sets of enzymes. Reaction 1 was carried out with the enzymes MfeI, NheI and XbaI and reaction 2 was carried out with NheI and XbaI. Both reactions were incubated at 37C overnight and the result was visualized in a 1% agarose ran 1 h at 90v. Additionally, Sanger sequencing was applied to relevant fragments: Spike and Rev genes from the pCMV-Spike(57)- Rev, and 5’ and 3’ LTRs, Nanoluc gene, RRE, cPPT and WPRE genes from pLenti- Nanoluc plasmid to confirm nucleotide sequences were unaffected by the various processing steps associated with cloning (Appendix A2).
4.3. Cell culture
HEK-293T (ATCC CRL-3216), Vero (ATCC CCL-81), Vero E6 (ATCC CRL- 1586) and Caco-2 (HTB-37) cells were maintained in high glucose DMEM (Caisson, cat. DML10) supplemented with 10% heath-inactivated FBS (Sigma, cat. F2442) at 37C in a 5% CO2 atmosphere. Cells were passaged according to manufacturer’s instructions, using either gentle scrapping or brief exposure to trypsin (Hyclone, cat.
C838R55). For trypsinization, cells were washed with 2 ml of 1X PBS and then 2.5 ml of 1x trypsin and incubated 5 min at 37C. Then, 3 ml of fresh medium was added to inactivate trypsin and the culture was centrifuged 5 min at 250 g. All cell lines were used before passage 25. For the cell count, trypan blue was used. Briefly, 10 l of cells in culture were mixed with 10 l of trypan blue 0.5% and 10 l of this mixture was added to the Neubauer chamber. The 5 quadrants were counted and the formula (Cell number/5) x Dilution factor x 10,000 was applied to the number
obtained from the count. The number obtained is expressed as the number of cells per ml.
4.4. Transfection optimization
To optimize transfection conditions in packaging cells HEK-293T to produce viral particles, a calcium phosphate protocol was followed using various concentrations of pLenti-EGFP DNA. Briefly, HEK-293T cells were seeded in a 6- well plate at a concentration of 2.5x105 cells /ml. After 24 h, an expected confluence of 80% was reached. Culture medium was replaced with 1.8 ml of DMEM without FBS or antibiotics 2 h before transfection. Two different quantities of DNA were tested, 8 g and 15 g of pLenti-EGFP. The DNA was mixed with 80 l of H20, 10 l of 2M CaCl2 and 80 l of 2X HBS pH 7.1 in a final volume of 160 l. The transfection mix was incubated 15 min in an orbital shaker at 120 RPM and then the transfection mix was added to the cell culture and incubated 24 h at 37C in humid atmosphere with 5% CO2. At 24 h post transfection, EGFP expression was analyzed by fluorescence microscopy on a Nikon Eclipse Ti, using the 480/30 filter and by flow cytometry on a BD FACS Celesta by analyzing fluorescence after stimulation at 495 nm and emission acquired in a 530/30 filter. Briefly, for flow cytometry analysis, 2x105 cells were washed twice, by adding 1 ml of PBS, centrifugating at 250 g, and resuspending washed cells in a final volume of 200 l of PBS. Cell were read immediately on the flow cytometer and .fcs data files were analyzed using FlowJo v10 (BD Biosciences).
4.5. SARS-CoV-2 S pseudovirus production
pLenti-NanoLuc, pCAG-HIVgp, and pCMV-SARS-CoV-2 S-RSV REV plasmids were co-transfected using 2M CaCl2 to a 3:2:1 DNA ratio to confluent HEK- 293T cells cultured in T75 flasks. Briefly, 22.5 g of pLenti-Nanoluc, 15 g of pCAG- HIVgp and 7.5 g of pCMV-SARS-CoV-2 S-RSV-REV were mixed with 143 l of CaCl2 2M and 250 l of HBS 2X in a total volume of 500 l. The mixture was incubated in an orbital shaker at 200 RPM for 15 min at room temperature and the
Chapter 4. Materials and methods 23
full transfection preparation was added to the cells. Cell were incubated at 37C with 5% CO2 for 24 h prior to swapping the medium to DMEM 10% FBS. Lentiviral particles-containing supernatant was collected at 72 h post-transfection and filtered in 0.45 m PES filter (GE cat. 6780-2504). The supernatant containing viral particles was immediately aliquoted and stored at -80C until use. One aliquot per batch was titrated after a single freeze-thaw cycle using the QuickTiter Lentivirus Titer Kit (Lentivirus-associated HIV p24) (Cell biolabs, cat. VPK-107) according to the manufacturer’s protocol.
4.6. Pseudovirus quantification
In order to quantify the viral particles contained in our supernatant, the QuickTiter Lentivirus Titer Kit (Lentivirus-associated HIV p24) (Cell biolabs, cat.
VPK-107) was used. This kit, based on an ELISA, quantifies the p24 structural protein, both soluble and associated with lentiviruses, in addition, it includes the recombinant p24 protein to perform a standard curve. With this kit it is possible to accurately quantify the amount of p24 protein associated with pseudovirus.
Following the protocol described by the manufacturer, the p24 standard curve was prepared, in total 8 concentrations were carried out, ranging from 0 ng/ml to 100 ng/ml. Subsequently, 1 ml of viral supernatant was mixed with the Virabind reagent and centrifuged for 5 min at 5000 g, the pellet was resuspended in 200 l of sample diluent (supplied by the kit), 100 l were added to the plate and all incubated overnight at 4 C. Once the incubation was done, the sample wells were washed 3 times with the washing buffer and 100 l of FITC-Conjugated Anti-p24 was added to each well and incubated for 1 h on an orbital shaker at room temperature. Once the incubation time had elapsed, the wells were washed 3 times and 100 l of HRP- Conjugated anti-FITC antibody was added to each well and incubated 1 h on an orbital shaker at room temperature. Subsequently, it was washed 3 times with the washing buffer and 100 l of substrate solution was added to each well and incubated at room temperature. Immediately after a color change was observed in the well, 100 l of stop solution was added and the absorbance results were read immediately after on the microplate reader at 450 nm. With the absorbance data obtained from the standard curve, the values were plotted and the straight-line
equation y=mx+b was calculated, subsequently, the equation was solved and the absorbance results of our samples were substituted to calculate the concentration of p24-associated with lentivirus.
4.7. Flow cytometry
ACE2 expression was measured on cell lines on a FACS Celesta flow cytometer fitted with 405nm, 488nm and 533nm lasers (BD Biosciences). Briefly 3 x105 cells were incubated on ice with 5 l of a mouse anti-human ACE2 monoclonal antibody conjugated to AF-647 (Santa Cruz Biotechnology, cat. SC-390851). After incubation, cells were washed twice with 1 ml of PBS at 250 g and resuspended in 100 l of PBS + 2% FBS. Five l of propidium iodide (BD Biosciences, cat. 51- 6621E) was added shortly before reading and used as viability marker. The cells were passed through the cytometer and 2 x104 single cells events were acquired.
The obtained data was analyzed in FlowJo software v.10 (BD Biosciences). The staining index was calculated and plotted, it was obtained from the division of the median fluorescence intensity of the stained cells divided by the unstained cells.
4.8. Infection assays
Infection of cells by the produced viral particles was assessed by measuring nanoluc-mediated furimazine reduction-induced luminescence compared to uninfected control wells, as described in (Nie et al., 2020b). were infected using 140 pg of viral particles per well in a 96-well plate format, and incubated 24 h at 37C in a 5% CO2 atmosphere. Twenty-four hours post-infection, the culture supernatant was removed and replaced by reconstituted furimazine from a commercial kit, following the manufacturer’s protocol (Promega, cat. N1110). Luminescence was measured within 15 min on a Synergy HTX microplate reader (Biotek instruments) at 460nm. Obtained data were analyzed in GraphPad Prism v6 software (GraphPad).
4.9. Western blot analysis of viral particles
Chapter 4. Materials and methods 25
Incorporation of SARS-CoV-2 S, VSV-G, and p24 proteins in produced lentiviral particles were investigated by western blot. Nine ml of pseudotyped lentiviral particles were pelleted by centrifugation at 100,000 g for 2 h at 4C through a 25% sucrose cushion. The supernatant was removed and the viral pellet was resuspended in 100 l of PBS. Thirty l of concentrated samples were mixed with 5
l of 6x SDS-PAGE buffer, and the mixture was heated 15 minutes at 100C. The full volume was then loaded on an 8% acrylamide gel for SDS-PAGE, and ran for 1.5 h at 90v. The resultant gel was transferred onto a 0.45 m PVDF membrane (GE cat.
1060023) and blocked for 1 h with PBS + 5% skim milk. All types of viral particle tested were prepared following the same procedure. For membrane staining, a mouse anti-VSV-G in a dilution (1:500) (Santa Cruz, cat. SC-365019-HRP), mouse anti-HIV-1-P24 in a dilution (1:500) (Santa Cruz, cat. 69728-HRP), SARS-CoV-2 convalescent sera was used as primary antibody for Spike glycoprotein incorporated in pseudovirus in a dilution of 1:100 and a rabbit anti-human IgG (Abcam, cat.
Ab6759) was used as secondary antibody in a dilution (1:5000). Stained membrane was reveled using 1-Step TMB blotting solution (ThermoFisher, cat. 37574) following the manufacturer’s protocol.
4.10. TCID50
As a biological dose-response curve follows a sigmoid function, it is necessary to set-up our infection assay so that half of the cells in the assay (per well) are infected. This is known as the Tissue Culture Infectious Dose 50% (TCID50). We investigated the amount of viral particles necessary to infect 50% of cells in culture as described in the literature (Nie et al., 2020b), which is based on the original Reed- Muench protocol (Reed & Muench, 1938). A total of 9 2-fold dilutions of the viral particles stock suspension were tested, alongside a non-diluted sample. Each dilution was applied to 6 replicate wells. Briefly, 2.5 x104 Vero cells were seeded in a 96-well plate and incubated at 37C in a 5% CO2 atmosphere. Twenty-four h post- seeding, viral dilutions were added to cells and incubated for 24 h before measuring luminescence as described above.
4.11. Human samples
Human sera were kindly provided by Drs. José Antonio Enciso Moreno and Julio Enrique Castañeda Delgado from Unidad de Investigación Biomedica del IMSS in Zacatecas, Mexico. The study was approved by an Institutional Review Board, with protocol number R-2020-785-068. Peripheral blood of COVID-19 patients, diagnosed as positive for SARS-CoV-2 by qRT-PCR, were collected upon informed consent, aliquoted and stored at -20C until use. Pre-pandemic sera from earlier studies were used as negative control. Demographic and clinical characteristics of COVID-19 samples are provided in Table 1. A total of 25 COVID-19 patients were sampled to provide 39 COVID-19 sera (Table 1). Various hospitalized patients provided multiple samples. A total of 15 pre-pandemic sera was analyzed.
4.12. Neutralizing antibodies assay
Human sera were inactivated for 30 min at 56C and used immediately. Sera underwent a total of 7 serial dilutions in PBS, spanning 1:5 to 1:9680. Sera were co- incubated with 15 pg of viral particles at 37C for 1 h in a 96 well plate. At the end of this incubation, 2.5 x104 fresh trypsinized Vero cells were added to each well and the plate was further incubated for 24 h at 37C. Then, the culture medium was removed and replaced by furimazine as per the manufacturer’ s protocol.
Luminescence was measured at 460 nm using a microplate reader (Biotek Instruments). All samples were tested in duplicate of each dilution.
4.13. Generation of ACE2-HEK-293T stably expressing cell line
HEK-293T were genetically modified using viral transduction to incorporate the human ACE2 gene to its genome controlled by a constitutive CMV promotor also controlling EGFP expression. The ACE2 gene was cloned in the lentiviral vector pLenti. Briefly, the ACE2 gene was amplified from plasmid pCDNA3-ACE2 (Addgene cat. 1786). The pLenti-EGFP was produced in our laboratory according to methods similar to earlier described. The amplified insert and pLenti-EGFP were digested with XbaI and XhoI and ligated with T4 DNA ligase following the protocol described in vectors construction section above. The validation of the correct cloning of the gene in the vector was carried out by PCR and enzymatic digestion with XbaI and XhoI.
E. coli strain STBL4 cells were transformed with the validated recombinant vector.
Chapter 4. Materials and methods 27
Lentiviral particles that express VSV-G in the membrane and contain the ACE2 gene in their genetic material were produced following the procedure described in pseudovirus production section. Half a million HEK-293T cells were transduced with 500 l of the viral supernatant in 2 ml of total DMEM + 10% FBS.
The infected cells were incubated 48 h at 37C with a humid atmosphere and 5%
CO2. Supernatant was replaced by DMEM + 10% FBS and cells remained in culture for a further 72 h. Subsequently, 2.5 x105 cells were analyzed by flow cytometry to investigate the expression of ACE2 by transduced cells.
Chapter 5
5. Results
5.1. Production of pseudotyped lentiviral particles
We designed constructs for a third-generation system to allow assembly of lentiviral particles pseudotyping the SARS-CoV-2 Spike (Figure 1a), the vesicular stomatitis virus glycoprotein (Figure 1b), or a negative control for cell entry with lentiviral particles expressing no envelope protein in viral membranes (Figure 1c).
We chose a SARS-CoV-2 Spike (57) sequence that lacks the last 19 amino acids at the C-terminal. This sequence acts as a retention signal in the endoplasmic reticulum. Therefore, the absence of these last 19 amino acids improve viral packaging in the cell membrane (Ou et al., 2020a). To design the lentiviral transfer plasmid (Figure 1d), Nanoluc was selected as the reporter gene because it emits a signal 10x more intense than Renin Luciferase, having a higher sensitivity and allowing have a brigther signal. In addition, the Nanoluc is the gene commonly used in other published assays for measuring neutralizing antibodies (Nie et al., 2020a;
Schmidt et al., 2020)6/15/2021 4:13:00 PM. The plasmid codifying HIV-1 gag/pol proteins used to produce our pseudoviral system is described visually in Figure 1e.
Figure 1. Constructs developed as part of a third-generation lentiviral plasmid system for the development of SARS-CoV-2 S pseudovirus. (a) Plasmid
Chapter 5. Results 29
containing the gene encoding the SARS-CoV-2 Spike protein with a 19 amino acid deletion at the C-terminal. (b) Envelope plasmid containing Vesicular Stomatitis Virus Glycoprotein. (c) plasmid encoding Rev gene without envelope glycoprotein.
(d) Transfer plasmid containing the Nanoluc reporter gene. (e) Packaging plasmid containing the structural genes of HIV-1.
The plasmid system shown in figure 1 is a third-generation lentiviral system.
This class of systems improves safety by producing infective but replication-deficient viral particles. In addition, the plasmid pLenti-Nanoluc contains the U3 mutation in the 3’ LTR, it has been reported that this mutation minimizes transcriptional activity, reducing the possibility of the virus replicating itself and producing new infectious particles (Miyoshi et al., 1998b).
To clone plasmids, we selected the E. coli strain Stbl4 for transformation because of their relevance in the cloning of plasmids with unstable inserts, such as lentiviral genes and CDS included between LTR (Al-Allaf et al., 2013). To investigate clones, various colonies from each transformation experiment were analyzed by PCR using gene specific primers for Nanoluc and Spike genes (as detailed in primer appendix A) Figure 2a shows a gel electrophoresis of the PCR product of Nanoluc using transformed pLenti-Nanoluc as template. The Nanoluc amplicon was expected to have a size of 516 bp. Lane 1 (pLenti) contained the PCR product of a reaction using the pLenti backbone without insertion, used here as a negative control. Lane 2 contained the PCR product of the reaction using pLenti-Nanoluc as template. A dense band is observed between 500 bp and 750 bp which is consistent with the expected size for the insert. Lane 1 shows a dim band at the top of the gel, which most likely corresponds to supercoiled plasmid. This result confirms the insertion of the Nanoluc gene in the pLenti plasmid.
Transformants with correct insert were further investigated with restriction maps of plasmids. Figure 2b shows the in silico digestion expected pattern for pLenti- Nanoluc. Three fragments are generated by digestion pLenti-Nanoluc with BamHI, PvuI and NheI enzymes of an expected size of 5033 bp, 3529 bp and 790 bp. Figure 2B shows a gel electrophoresis with the pattern obtained from the digestion of
pLenti-Nanoluc. Lane 1 corresponds to the molecular weight marker. Lane 2 shows gel electrophoresis of pLenti-Nanoluc plasmid digested with the enzymes BamHI, PuvI and NheI. Bands consistent with the size expected for the digestion of the plasmid (Figure 2c). This result confirms the correct cloning of the Nanoluc gene in the plasmid pLenti.
Figure 2. Validation of pLenti-Nanoluc. (a) PCR product consistent with the expected size for the Nanoluc gene. (b) In silico digestion of pLenti-Nanoluc plasmid with BamHI, PvuI and NheI restriction enzymes showing the expected size of 5033 bp, 3529 bp and 790 bp. (c) Gel electrophoresis of pLenti-Nanoluc digested with BamHI, PuvI and NheI.
Figure 3a shows an electrophoresis gel with the result of the PCR product for Spike (57), lacking the last 57 nucleotides at the 3’ end which consist to the last 19 aminoacids at the C-terminal, using as template recombinant DNA obtained from bacteria transformed with the recombinant vector. Lane 2 shows a dense band consistent with the expected size for the Spike gene (3762 bp) with the deletion of
Chapter 5. Results 31
19 amino acids at the C-terminal. This result confirms the correct insertion of the Spike gene (57) in the plasmid pCMV-Rev.
To obtain a suitable restriction standard to validate the plasmid pCMV-Spike (57) -Rev, an in silico restriction was performed. Figure 3b shows the restriction pattern obtained with the MfeI, NheI and XbaI enzymes, in which 4 bands of 3774 bp, 2295 bp, 1482 bp and 734 bp, are obtained and are consistent with the mapping of the complete plasmid. Figure 3c shows the digestion of the plasmid with the enzymes NheI and XbaI, generating two bands of 4508 bp and 3777 bp, which are consistent with the empty vector and the Spike gene, respectively. Figure 3d shows the restriction map obtained by digesting the pCMV-Spike (57) -Rev using two different sets of enzymes. In lane 1 the digestion pattern obtained with the MfeI, NheI and XbaI enzymes is observed, where the 4 bands are consistent with the expected sizes of 3774 bp, 2295 bp, 1482 bp and 734 bp (Figure 3a). Lane 2 contains the pattern of digestion obtained with the enzymes NheI and XbaI generating two bands of 4508 bp and 3777 bp, consistent with the expected pattern for the digestion of the plasmid pCMV-Spike (57)-Rev (Figure 3b). Additionally, the regions of interest were sequenced for the 3 plasmids to validate that there are no mutations in the coding genes and in the regulatory regions (Appendix B shows the sequenced regions). In appendix B are the sequences resulting from the sanger sequencing, in the results 100% homology was obtained, which suggests that our plasmids necessary for the development of the pseudovirus do not present mutations in their sequences.
Figure 3. Gel electrophoresis of 2 restriction patterns obtained for pCMV-Spike (57)-Rev. The construct was validated by restriction using two different combinations of enzymes. (a) Shows the 3,762 bp Spike (57) PCR product. (b) In silico restriction of the plasmid with the enzymes MfeI, NheI and XbaI, where four bands are seen consistent with the expected mapping for the plasmid. (c) In silico restriction with the enzymes NheI and XbaI where two bands are seen, consistent with the empty vector and the Spike gene. (d) Agarose gel of the enzymatic digestions, where we can see in lane 1 and 2 the expected patterns for the digestions with the two sets of enzymes mentioned above.
5.2. Transfection optimization of HEK-293T
To optimize the yield of produced lentiviral particles, we investigated optimal conditions to transfect HEK-293T cells in order to yield the highest possible percentages of transfected cells. To do so, we tested 2 doses, of 8 g and 15 g of pLenti-EGFP plasmid. The transfection mixes were prepared as described above with either of the concentrations to be tested, added to cells, and cells were incubated for 48 h at 37C and 5% CO2. We then investigated cell viability by manual counting in a Neubauer chamber using 0.5% trypan blue. In cells transfected with 8
g of plasmid, 94% viability was obtained, while in cells transfected with 15 g, viability was 83% (data not shown). Both conditions show a viability greater than
Chapter 5. Results 33
80%, however, at a higher concentration of DNA, cell viability is reduced, suggesting DNA toxicity (Maurisse et al., 2010). Transfected cells were observed by fluorescence microscopy. Figure 4a shows the image obtained in the bright field microscope of the cells transfected with 8 g of pLenti-EGFP, observing an approximate cell confluence of 60%. Figure 4b shows a representative image obtained using fluorescence microscopy after transfection with 8 g of pLenti-EGFP.
A large percentage of the total cells in the optical field, as shown on the bright-field image on Figure 4a express GFP suggesting the success of the transfection.
Transfection efficiency was further quantified by flow cytometry. Figure 4c show data analysis of transfected cells using 8 g of pLenti-EGFP yielded 74.2% positive cells at 2 days post-infection
On the other hand, using 15 g of pLenti-EGFP, similarly yielded the majority of cultured cells emitting EGFP. Figure 4d show a representative bright field of the cells and figure 4e shows its corresponding image obtained using fluorescence microscopy where we can see a majority of cells expressing EGFP. This suggests a good transfection efficiency. Figure 4f shows flow cytometry analysis of cells transfected with 15 g of pLenti-EGFP, where transfection efficiency reached 81.4%, which is the highest percentage achieved in our optimizations. On the basis of these results, we chose to perform HEK transfections with 8 g of DNA due to it keeps the cell viability above 90%.
5.3. Generation of stable expressing ACE2 cell line
To generate the VSV-G-ACE2 pseudoviruses, HEK-293T were transfected following the procedure described in the methodology section. Briefly, 5x105 HEK- 293T cells were infected with 500 l of the viral supernatant and the cells were analyzed by flow cytometry at day 2 post infection. The analysis by flow cytometry showed an increase in the positivity of the cell line, from 2.3% in control untreated cells, to 29.9% in cells infected with 500 l of VSV-G-ACE2 pseudovirus supernatant (Figure 5a). In the case of Median Fluorescense Intensity (MFI), we achieved an increase in cells infected with VSV-G-ACE2 pseudovirus of 2.5x compared to the uninfected control (Figure 5b), which
Figure 4. Transfection optimization of HEK-293T validated by fluorescence microscopy and flow cytometry. (a) HEK-293T cells transfected with 8 g observed in bright field. (b) HEK-293T transfected with 8 mg of pLenti-EGFP observed in the fluorescence microscope. (c) EGFP histograms of transfected cells reaching a maximum expression level of 74.2%. (d) HEK-293T transfected with 15
g of pLent-EGFP observed in bright field. (e) Cells transfected observed in the fluorescence microscope. (f) Histograms of EGFP in cells transfected with using 15 mg of pLenti-EGFP, reaching a maximum of 81.4% of EGFP expressing cells.
Indicates that cells infected with our lentiviral system correctly express the ACE2 receptor at least 2.5 times more than wild type cells, increasing the positivity from 2.3% to 29.9% (Figure 5c). Single cells were isolated using a FACS Melody cell sorter (BD Biosciences) on the basis of high EGFP fluorescence, as single cells per wells of 1 96-well plate and incubated for 3 weeks at 37C in humid atmosphere with 5% CO2 to promote clonal expansion.
Chapter 5. Results 35
Figure 5. HEK-293T/ACE2 production. (a) Histograms of HEK-293T cells infected with the VSV-G-ACE2 pseudovirus. (b) MFI of HEK-293T cell lines. (c) Percentage of positivity for ACE2 in HEK-293T cell lines at day 7.
5.4. Quantification of produced lentiviral particles
Viral particles in the supernatant of packaging cells were quantified using the QuickTiter Lentivirus Titer Kit (Lentivirus-associated HIV p24) (Cell biolabs, cat.
VPK-107) following the protocol described by the manufacturer. This kit quantifies the concentration of the lentivirus-associated structural protein p24. The standard curve (Figure 6) was obtained using recombinant p24 resulting in a linear function with an R2 = 0.9933 and an equation y = 0.0171x + 0.0309. Subsequently, the equation was solved for x and normalized to input volume to obtain the concentration of viral particles in viral particle-containing supernatant samples. Briefly, the absorbance value obtained was substituted the resultant formula and the concentration of p24 was calculated with the formula obtained earlier.
Figure 6. Representative graph of a recombinant p24 standard curve. The obtained linear regression was used to estimate the concentration of each sample.
Using this standard curve, we calculated the concentrations in pg/ml of viral particles contained in the supernatant of VSV-G-EGFP, VSV-G-Nanoluc and Spike- Nanoluc pseudovirus (Figure 7). Figure 7a shows free and viral membrane-bound p24 proteins of Spike-Nanoluc lentiviral particles. Of the total 12 ng measured in 1 ml of viral supernatant, 8.02 ng correspond to p24-associated lentivirus and 7.66 ng to free p24. The graph observed in figure 7b corresponds to VSV-G-EGFP titration, showing a concentration of 3.83 ng of p24-associated pseudovirus and 7.66 ng of free p24 in 1 ml of supernatant. Finally, in figure 7c, the graph of VSV-G-Nanoluc is shown, a concentration of 3.55 ng of p24-associated lentivirus and 7.11 ng of free p24 were obtained in 1 ml of supernatant. There was a consistent trend of having more free p24 protein compared to membrane-bound p24. Yields of membrane- bound p24 were in a similar range for each type of engineered lentiviral particles produced. These results show that the protocol optimized for lentiviral particle production in this thesis is consistent for yield and reproducible across platforms.
Chapter 5. Results 37
Figure 7. Lentivirus-associated p24 titration graphs. (a) Graph of p24-associated Spike-Nanoluc pseudovirus, dark green corresponds to the concentration of lentivirus-associated p24 protein. (b) Graph of p24-associated VSV-G-EGFP pseudovirus, strong blue corresponds to the concentration of lentivirus-associated p24. (c) Graph of p24-associated VSV-G-Nanoluc, intense orange corresponds to the concentration of lentivirus-associated p24. Only one batch of VSV-G Nanoluc pseudovirus was tittered.
From the measurements of membrane-bound p24, it is possible to approximate the amount of viral particles in collected supernatant. This is because 1 ng of virus-associated p24 its about 1.25x107 (Data provided by the kit). Although for more accuracy in the rest of the thesis, we mention the actual amount of membrane-bound p24 added to wells, instead of a possibly less accurate number of added particles, or multiplicity of infection (MOI) per well.
5.5. Validation of pseudovirus assembly
The correct assembly of the pseudovirus was validated by western blot by detecting the structural protein p24, the VSV-G in the positive control pseudovirus and the SARS-CoV-2 S protein in our study pseudovirus. The p24 protein was detected in the negative control virus, envelope protein deficient virus and in the positive control pseudovirus. Figure 8 shows the result of the detection by WB of the p24 protein in the two viruses mentioned above. The structural protein p24 was detected both in the virus lacking the envelope glycoprotein and the pseudovirus
VSV-G-Nanoluc. Optimizations of antibody concentrations are currently ongoing to validate the presence of VSV-G and SARS-CoV-2 S on the surface of pseudovirus.
Figure 8. Correct assembly of the produced lentiviral particles. In lane 2, the p24 protein present in the envelope protein deficient virus (negative control) is observed. Lane 3 shows the p24 protein present in the VSV-G-Nanoluc virus (positive control).
5.6. Transduction of HEK-293T with VSV-G-EGFP pseudovirus
In this work, we set to produce a target cell line for the SARS-CoV-2. The strategy chosen, which has been previously published, was to generate a HEK-293T cell line which constitutively expresses the human ACE2 receptor in cell membranes.
While others have chosen to transfect HEK-293T cells with episomal plasmids, causing time-limited ACE2 expression (Inal, 2020, p. 2), we decided to engineer the genome of HEK-293T cells by inserting the ACE2 gene under a constitutive promoter (Beltrán-Pavez et al., 2021). We used our lentiviral system to apply this strategy, with the broad-spectrum VSV-G as entry protein to our cells. To optimize this protocol, we chose the EGFP reporter gene with is easily measured in our laboratory by flow cytometry.
The transduction protocol was developed trialing two loads of VSV-G-EGFP lentiviral particles and EGFP expression by infected cells was monitored over 7 days
Chapter 5. Results 39
by flow cytometry (Figure 9). In figure 9a the result of the kinetics of infection with 135 pg of p24-associated is observed. It can be seen that 2 days post infection, the expression of EGFP is detected in 5.46% of cells compared to untransduced control cells, validating our protocol. On day 3 post-infection, EGFP expression increased to 21.7%, which is 3 times more than the obtained in the previous day. On day 4, the percentage of EGFP positive cells was 23.5% and on day 5 a 24.1% EGFP was reached. On day 6 and day 7 of infection, we see that the expression level remained constant, and close to the maximum level obtained on day 5. In figure 9b the results of infecting HEK-293T cells with 270 pg of p24-associated VSV-G-EGFP is shown.
On day 2 post-infection, 8.88% of cells were obtained correctly expressing EGFP, while on day 3 an increase to 32.7% was obtained, approximately 4 times more EGFP than the percentage obtained on day 2. On day 4, 42.4 % EGFP-positive cells were measured and on day 5 the maximum level obtained in this kinetic was achieved, reaching 44.9% of HEK-293T expressing EGFP. On day 6 and 7, the expression remained close to the levels obtained on day 5, with 41.4% and 37.7%, for days 6 and 7, respectively. Therefore, regardless of the amount of viral particles added, the kinetics of expression of EGFP was similar, with maximum expression obtained at day 5. This result suggest almost half of transduced cells express the recombinant protein by day 5 post-infection using 270 pg of p24-associated to viral particles, and likely remains at this level thereafter, although in this experiments we only investigated EFGP expression over 7 days, others have investigated a longer time frame, more than 8 weeks and the expression persist consistent (Bai et al., 2003).
Figure 9. Kinetics of EGFP expression over 7 days post-infection of HEK-293T cells with VSV-G-EGFP pseudovirus. Flow cytometry of HEK-293T cells transduced with either (a) 135 pg (top row) or (b) 270 pg (bottom row) of p24- membrane associated lentiviral particles. Figure panels show overlap histograms of untransduced HEK-293T cells and transduced HEK-293T cells. Positive/negative gates were set according to the limit of autofluorescence of untransduced cells. Y axis represent the normalized count of cells and X axis represents EGFP expression.
5.7. HEK-293T infection with VSV-G-Nanoluc pseudovirus
To initially evaluate our lentiviral system using the Nanoluc transgene instead of EGFP as shown earlier, VSV-G-Nanoluc pseudoviruses were produced and various infection conditions were evaluated. Two concentrations of viral particles and 2 numbers of cells, creating a matrix of 4 parameters. Figure 10 shows the result of infecting 1.25x104 and 2.5x104 cells with 70 pg and 140 pg of p24-associated viral particles. Results show that uninfected cells produce background signal corresponding to 3200 RLU. The lowest RLU in infected cells was obtained by infecting 1.25x104 cells infected with 70 pg, 4.7x105 RLU. This is 12 times more than the background luminescence. Others have suggested a cuttoff should be a luminescence of at least 3x the control or even 10x more than the control (Beltrán- Pavez et al., 2021; Nie et al., 2020a) When 1.25x104 cells were infected with 140 pg a maximum expression of 1.7x106 RLU was reached, approximately 3-fold more than the result obtained with 70 pg. In the results obtained from the infection of 2.5x104 cells we can see that with 70 pg we obtained 1.5x106 RLUs while with 140 pg we obtained 3.6x106 RLUs. The results obtained suggest that Nanoluc is an
Chapter 5. Results 41
appropriate reporter protein in this system as expected, and that the level of luminescence is proportional to the number of cells infected and the concentration of viral particles. Therefore, in our assay we decided to work with 2.5x104 cells with 140pg of p24-associated VSV-G-Nanoluc pseudovirus per well in order to maximize luminescence signal.
Figure 10. Optimization of target cell number during infection of HEK-293T with VSV-G-Nanoluc pseudovirus. Results obtained from the infection of 1.25x104 and 2.4x104 HEK-293T cells with different concentrations of VSV-G-Nanoluc pseudovirus. Uninfected control was included for the two different number of cells tested. The graphs shown here are an average of three technical replicates.
5.8. Cell line selection
5.8.1. ACE2 expression on target cell lines
To investigate what target cell would be most appropriate to use in the antibody-mediated SARS-CoV-2 spike lentiviral particles neutralizing assay, we measured ACE2 expression by flow cytometry in various cell lines known to express the ACE2 receptor.
Flow cytometric analyzes were performed in triplicate. Representative histograms of these analyzes are shown in Figure 11. HEK-293T expressed 2.3% of the ACE2 receptor on their membrane surface (Figure 11a), consistent with that reported in the literature, HEK-293T cells are ACE2 receptor deficient (Schrom et al., 2017). Subsequently, the expression of ACE2 on Caco2, Vero and Vero E6 cells was analyzed. Figure 11b shows the percentage of positivity of the ACE2 receptor in the Caco2 cell membrane, with 16.1% of positive cells. In figure 11c we observe the level of expression of ACE2 in Vero cells, the histogram shows an expression percentage of 93%, indicating a high level of expression of the ACE2 receptor in the Vero membrane. In figure 11d we have the histogram corresponding to the Vero E6 cells, obtaining 82% of cells that express the ACE2 receptor in their membrane.
These results show various cell lines reported to express ACE2 do so at different levels. Additionally, using the median intensity fluorescence (MFI) of ACE2 for each cell type, we calculated the staining index (SI), which is an indication of the density of ACE2 expression on cell surfaces. Figure 11e shows the graph obtained from the levels of expression of the ACE2 receptor on the membrane of the different cell lines analyzed, as in the percentage of positivity for ACE2 cells, the SI of these cells is 0.4, indicating that HEK-293T cells express the ACE2 receptor on their membrane in very low levels. The graph corresponding to the Caco2 cells shows an SI of 6.34, showing low levels of receptor in their membrane. For Vero cells, we have an SI of 339, this value indicates a high level of expression of the ACE2 receptor in the membrane of Vero cells, which is consistent with the percentage of expression shown in figure 11c, confirming the high abundance of the receptor on the cell membrane. In the case of Vero E6 cells, an SI of 172, showing a relatively high levels of the receptor in their membrane, but at least 50% lower than those obtained in Vero cells. The results obtained show that of the cell lines analyzed in this assay, Vero cells are those with most positivity for ACE2 and the highest levels of the ACE2 receptor, while HEK-293T cells have the lowest levels as expected. Therefore in future infection assays with SARS-CoV2-spike pseudotyped lentiviral particles, we decided to use Vero cells as target.
Chapter 5. Results 43
Figure 11. ACE2 expression on cell lines. The expression of the ACE2 receptor was evaluated in the membrane of three cell lines. Representative histograms of (a) HEK-293T, (b) Caco2, (c) Vero, and (d) Vero E6 are shown. (e) Staining index (SI) for each cell lines, which was calculated for each of the four cell lines analyzed as the ratio of ACE2-stained cells MFI over unstained control cells.
5.8.2. Infection susceptibility
We then investigated the susceptibility of each cell line to be infected by SARS-CoV-2 S lentiviral particles, which in theory should correlate to results of the flow cytometry experiment, as infection is mediated through the ACE2 receptor on the surface of target cells (Inal, 2020). For this assay, we use 25,000 cells and 140 pg of p24-associated lentiviral particles.
Figure 12a shows the results obtained from the infection tests where it can be seen that Caco2 cells are the least susceptible to being infected with SARS-CoV-2
S pseudovirus, reaching average RLUs of 6 x105. In the case of Vero E6 cells, they show a greater susceptibility than Caco2 cells but less than Vero cells, obtaining about 8 x105 RLUs. The Vero was the cell line in which we reached higher levels of luminescence emission, suggesting a greater susceptibility to infection by SARS- CoV-2 S. The average RLUs levels for the Vero cells were 1 x106, the cell line being with best levels obtained. This result is correlated with the level of expression of the ACE2 receptor, since of the 4 lines analyzed, the Vero cells are the ones that present the highest level of expression of ACE2. Based on the results obtained, Vero cells are the most suitable for the optimization of the neutralizing antibody f.
Figure 12. Susceptibility of cell lines to pseudovirus infection. Vero, Vero-E6 and Caco2 cells were tested for their susceptibility to the SARS-CoV-2 S
Chapter 5. Results 45
pseudovirion (a), VSV-G lentiviral particles (b), and no membrane protein lentiviral particles (c).
The VSV-G-Nanoluc pseudovirus was used as positive control and a lentivirus lacking envelope protein was used as negative control. Figure 12b show the positive control with the VSV-G-Nanoluc and figure 12c show the negative control, a viral vector lacking envelope protein. In the case of cells infected with VSV- G-Nanoluc, Vero cells have a greater susceptibility to being infected with this virus reaching 1x106 RLUs. This may be due to the fact that in their membrane they express a greater amount of LDL receptor, necessary for the internalization of VSV -G lentivirus. Additionally, it is noted that the susceptibility of Vero cells to be infected by VSV-G pseudovirus is greater than that of cells to be infected with SARS-CoV-2 S pseudovirus. Interestingly, in Vero E6 and Caco2 cells the susceptibility to being infected by VSV-G pseudovirus is lower than that of being infected with SARS-CoV- 2 S pseudovirus, contrary to what is observed in Vero cells. While in the cells infected with the lentiviruses lacking the envelope protein, we observed a very low expression in the 4 cell lines analyzed.
5.8.3. TCID50 titration
Working with an inhibition response in a biological assay, it was necessary to define the amount of viral particles necessary to infect 50% of the cells in culture.
This is because biological dose-response curves follow a sigmoid function.
Therefore, we investigated the Tissue Culture Infectious Dose 50% (TCID50). We tested a range of viral particle concentrations ranging from 0.15 pg to 140 pg per well, according to the range of dilutions available to us, defined by the original concentration of supernatants. Figure 13 shows the graph obtained from the TCID50. At concentrations from 0.15 pg to 2.5 pg of p24-associated SARS-CoV-2 S pseudovirus, the RLUs obtained were less than 2.5x103, being a weak signal for our tests as reported in published papers (Nie et al., 2020a). Using 5 pg p24-associated pseudovirus, 2.3 x105 RLUs were obtained, which is a 2 order of magnitude increase compared to the RLUs obtained with 2.5 pg. With 10 and 15 pg, the RLUs were 3.2 x105 and 4.3 x105, obtaining a 2-fold increase with respect to the RLUs obtained with
