key: cord-0875329-5wh63vck
authors: Cruz-Cardenas, José Antonio; Gutierrez-Mayret, Michelle; López-Arredondo, Alejandra; Castañeda-Delgado, Julio Enrique; Rojas-Martinez, Augusto; García-Rivas, Gerardo; Enciso-Moreno, José Antonio; Palomares, Laura A.; Brunck, Marion E. G.
title: A pseudotyped lentivirus-based assay to titer SARS-CoV-2 neutralizing antibodies in Mexico
date: 2022-01-31
journal: bioRxiv
DOI: 10.1101/2022.01.27.478128
sha: 1b373c5f5bc5f840655c1ffa24d82fafe1995800
doc_id: 875329
cord_uid: 5wh63vck

Measuring the neutralizing potential of SARS-CoV-2 antigens-exposed sera informs on effective humoral immunity. This is relevant to 1-monitor levels of protection within an asymptomatic population, 2-evaluate the efficacy of existing and novel vaccines against emerging variants, 3-test prospective therapeutic monoclonal neutralizing antibodies (NAbs) and, overall, to contribute to understand SARS-CoV-2 immunity. However, the gold-standard method to titer NAbs is a functional assay of virus-mediated infection, which requires biosafety level 3 (BSL-3) facilities. As these facilities are insufficient in Latin American countries, including Mexico, scant information has been obtained about NAb in these countries during the COVID-19 pandemic. An alternative solution to acquire NAb information locally is to use non-replicative viral particles that display the SARS-CoV-2 Spike (S) protein on their surface, and deliver a reporter gene into target cells upon transduction. Here we present the development of a NAb-measuring assay based on Nanoluc-mediated luminescence measurements from SARS-CoV-2 S-pseudotyped lentiviral particle-infected cells. The successive steps of development are presented, including lentiviral particles production, target cell selection, and TCID50 determination. We applied the optimized assay in a BSL-2 facility to measure NAbs in 15 pre-pandemic, 18 COVID-19 convalescent and 32 BNT162b2 vaccinated serum samples, which evidenced the assay with 100% sensitivity, 86.6% specificity and 96% accuracy. The assay highlighted heterogeneity in neutralization curves which are relevant in discussing neutralization potency dynamics. Overall, this is the first report of a BSL-2 safe functional assay to measure SARS-CoV-2 in Mexico and a cornerstone methodology necessary to measure NAb with a functional assay in the context of limited resources settings. Importance Evaluating effective humoral immunity against SARS-CoV-2 requires a functional assay with infectious virus. Handling the authentic SARS-CoV-2 virus requires specialized facilities that are not readily available in Latin America, including Mexico. Here we produce non-replicative viral particles pseudotyped with the SARS-CoV-2 S protein that are used as safe surrogate viral particles in an optimized BSL-2 ready neutralization assay. The establishment of this assay is critical to allow the evaluation of effective humoral immunity to SARS-CoV-2 post-infection and to monitor the efficacy of existing or novel vaccines against emerging variants in the Mexican population.

Western blot analysis of produced VP. A: The structural protein p24 was detected on 160 the 3 types of VP. B: VSV-G was selectively detected on VSV-G VP but not on either SARS-161 CoV-2 S or no-envelope protein VP. C: The S protein was detected as a 110 kDa protein on 162 SARS-CoV-2 VP using a chimeric monoclonal antibody, but not on VSV-G VP. Once assay parameters were optimized, neutralization of transduction with human sera was 226 implemented. Sera collected prior to the start of the COVID-19 pandemic, sera from COVID-227 19 diagnosed patients, and sera from health professionals that had received the BNT162b2 228 vaccine were used (Tables 1 and 2). Pre-pandemic sera showed neutralization of the SARS-229

CoV-2 pseudotyped-VP ranging between 11.6% and 41% at the lowest (1:5) dilution tested, 230 and ranging between 20.2% and 4.6% at the highest tested dilution (1:9860, Fig. 5A ). These 231 (Table 2) . Two individuals showed slight decrease in 248 neutralization after the second dose (Table 2) , which has been reported before 16 . Importantly, 249 the potency of vaccinated sera was higher than COVID-19 sera, with >18% vaccinated sera 250 (3/16) having a 30% neutralization titer of 9860, versus only 5.5% COVID-19 sera (1/18). 251

Using these values the presented assay has a 100% sensitivity, 86.6% specificity and 95.9% with a sharp slope around EC50 (ID 41, ID 44) and these differences could be observed 260 within a same individual, between 2 samples (ID 43). Interestingly, the curve of convalescent 261 patient ID 21 exhibited constant, moderately high neutralization >75% between 1:5 and 1: 262 540 serum dilutions, followed by a sharp decrease in neutralization between serum dilutions 263 1:540 and 1:9860. Due to the shape of this curve, EC50 is extremely low suggesting very 264 potent neutralization serum, however the patient suffered severe symptoms and passed away 265 (Table 1) 

To compare SARS-CoV-2 NAb and total IgG titers, 14 sera from either vaccinated (Pfizer-301

BioNTech, 2 nd dose) or COVID-19 diagnosed individuals were selected to measure total 302 SARS-CoV-2 S IgG from samples that together exhibited a spectrum of neutralization 303 ranging between 58% and 97.5%. Total IgG against SARS-CoV-2 S1+S2 were measured in 304 the aforementioned samples, and in 7 randomly selected pre-pandemic serum samples, using 305 a quantitative ELISA. Vaccinated and COVID-19 samples contained similar average titers 306 of anti-SARS-CoV-2 S-specific IgG antibodies (Fig. 7A, 0 .095 and 0.085 mg/L, 307 respectively). These concentrations were on average 30% higher compared to titers measured 308 in pre-pandemic sera (Fig. 7A) . A similar increase over naïve serum concentration has been 309 previously observed using an ELISA specific for SARS-CoV-2 S1 40 . A significant difference 310 in total IgG concentration was evidenced between the vaccinated group and pre-pandemic 311 group only, as the COVID-19 group presented a larger distribution of concentrations. Larger 312 differences have been described in anti-SARS-CoV-2 S total IgG between pre-pandemic sera 313 and sera from individuals exposed to SARS-CoV-2 antigens 41 . To investigate neutralization 314 in these samples, we used the pseudotyped VP-based neutralization assay developed here. 315

As evidenced earlier, an overall significantly higher neutralization was observed for both 316 COVID-19 and vaccinated groups compared to the pre-pandemic group (p = 0.0006 for both 317 comparisons). In addition, there was significantly more neutralization from vaccinated 318 samples compared to individuals exposed to the virus through infection (Fig. 7B , p = 0.0006), 319 in contrast with total IgG concentrations between these groups. Higher titers of anti-SARS-320

CoV-2 NAb in individuals vaccinated with BNT162b2 compared to COVID-19 patients have 321 been extensively described 16,42 . In summary, after exposure to the antigen, a wide range of 322 concentrations of anti-SARS-CoV-2 S total IgG could be measured while neutralization was 323 restricted between 58% and 97.5% (Fig. 7C) . Others have similarly evidenced a range of 324 concentrations for total IgG between 10-100 mg/ml for COVID-19 patients while 325 neutralization was constrained to >95% 42 . components of the immune system strongly contribute to recovery 59 . As evaluating T cell-408 mediated immunity to SARS-CoV-2 in vitro remains a challenge, monitoring NAb is 409 mandatory to provide clues needed to elucidate immune requirements for protection against, 410 and recovery from, SARS-CoV-2 infections. Vaccine development also requires an easily 411 adapted and safe-to-use platform to measure the induction of immune response, in particular, 412 the detection of NAbs generated in response to the inoculated antigen. The assay developed 413 in this work could be easily adapted to emerging variants by either applying directed 414 mutagenesis to the S sequence, or replacing it with a synthetic gene 53 . As vaccines may need 415 to be adapted to target emerging variants of concern, effective immunity brought by novel 416 vaccines and variant-mediated infections can be monitored locally using this assay. We with SARS-CoV-2 was also recorded (Table 2) . Briefly, sera were enriched from coagulated 501 blood by centrifugation, inactivated for 30 min at 56 °C, and aliquoted and stored at -80 °C 502 until use. On the day of the assay, sera were serially diluted 7-folds, spanning 1:5 to 1:9860, 503 and 100 μL of each dilution was incubated for 1 h at 37 °C and 5% CO2, together with 15 pg 504 of SARS-CoV-2 VP in duplicate in a 96-well plate. Post-incubation, 25,000 Vero cells were 505 added to each well and the plate was incubated for 24 h at 37 °C and 5% CO2. As a positive 506 control for transduction, SARS-CoV-2 VP were incubated with Vero cells. Vero cells seeded 507 in triplicate were used for basal luminescence background assessment as described earlier. 508

After 24 h, Nluc levels were measured as described above. Neutralization is described as % 509 inhibition of transduction, calculated as: Inhibition (%)=(mean RLUs of infected control 510 wells -mean RLUs (duplicate) of sera-treated well) x100. 511 512

Statistical analysis 513 All statistical analyzes were performed using GraphPad Prism v.9 software and p-values < 514 0.05 were considered statistically significant. In the case of categorical values for calculation 515 of Sn and Sp, a contingency 2x2 table was used with a fisher exact test. For flow cytometry 516 analyzes, medians of fluorescence intensity and percentage of positive cells were compared 517 using Mann-Whitney and P values are reported. To estimate EC50, neutralization curves 518 were log transformed, normalized, and fitted to the most appropriate model between 519 log(inhibitor) vs. response (three parameters) and log(inhibitor) vs. response, variable slope 520 (four parameters). For calculating sensitivity and specificity of the assay, we determined sera 521 positivity and negativity at a final 1:20 dilution and using a 30% neutralization threshold, as 522 previously reported to evidence true/false positives and true/false negatives 43 . 523 524 Acknowledgements 525

We warmly thank Dr. Zhaohui Qian (Institute of Pathogen Biology, Beijing, China) for 526 sharing pCMV14-3X-Flag-SARS-CoV-2 S with us. We acknowledge the financial support 527 received by CONACYT (scholarships 1007842 and 657487 Sup. Fig. 2 : Determination of the TCID50 of SARS-CoV-2 S pseudovirus using 25,000 Vero 834 cells as target in 96 well plate with infection assessed at 24 h. The dotted line represents 50% 835

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1: Sequences of relevant plasmid portions obtained through Sanger sequencing

D19 RLU equivalent to 15 pg VP per well. The average of 2 independent experiments, 836 ran in triplicate, is shown. Error bars indicate SD 837