key: cord-0830203-fm9eitto authors: Wang, Qiao; Feng, Lili; Zhang, Haohai; Gao, Juehua; Mao, Changchuin; Landesman-Bollag, Esther; Mostoslavsky, Gustavo; Lunderberg, Justin M; Zheng, Weina; Hao, Shushun; Gao, Wenda title: Longitudinal waning of mRNA vaccine-induced neutralizing antibodies against SARS-CoV-2 detected by an LFIA rapid test date: 2022-01-15 journal: Antib Ther DOI: 10.1093/abt/tbac004 sha: 075ad8fa325d254e68f8dcf74d0532d5d3bd9964 doc_id: 830203 cord_uid: fm9eitto While mRNA vaccines against SARS-CoV-2 were highly efficacious against severe illness and hospitalization, they seem to be less effective in preventing infection months after vaccination, especially with the Delta variant. Breakthrough infections might be due to higher infectivity of the variants, relaxed protective measures by the general public in “COVID-19 fatigue”, and/or waning immunity post-vaccination. Determining the neutralizing antibody levels in a longitudinal manner may address this issue, but technical complexity of classic assays precludes easy detection and quick answers. We developed a lateral flow immunoassay NeutraXpress™ (commercial name of the test kit by Antagen Diagnostics, Inc.), and tested fingertip blood samples of subjects receiving either Moderna or Pfizer vaccines at various time points. With this device, we confirmed the reported clinical findings that mRNA vaccine-induced neutralizing antibodies quickly wane after 3–6 months. Thus, using rapid tests to monitor neutralizing antibody status could help identify individuals at risk, prevent breakthrough infections and guide social behavior to curtail the spread of COVID-19. Statement of Significance. Mounting evidence suggests that mRNA vaccine-induced neutralizing antibody titres against SARS-CoV-2 wane in 3–6 months. Quick identification of fully vaccinated persons with high risk of breakthrough infections is key to control the COVID-19 pandemic. The described LFIA device having a control/sample dual-lane design serves this purpose with successful field-test data. patients hospitalized with COVID- 19 in Massachusetts had been fully vaccinated [1] . In another study by Yale University, among the fully vaccinated breakthrough patients admitted to the New Heaven Hospital between March 23 and July 1, 2021, 20% had moderate disease, and 26% had severe or critical illness [2] . Furthermore, fully vaccinated individuals with breakthrough infections can have peak viral loads similar to unvaccinated subjects and are able to efficiently transmit infection in household settings, including to fully vaccinated contacts [3] . Even though the viral loads in breakthrough vaccinated individuals may be as high as in unvaccinated individuals, the former clear the virus faster and remain contagious for shorter periods than the latter [4] . Clearly, even in breakthrough infections, vaccination shortens the time window of high transmission potential, minimizes symptom severity and duration, and may restrict tissue dissemination of the virus [5] . Protective immunity induced by natural infection or vaccination includes both humoral immunity and cellular immunity, the latter of which involves cytotoxic lymphocytes to clear virus- [8] . In another study, Israeli researchers found that neutralizing antibody levels among 4,800 health care workers who had received two doses of Pfizer vaccine fell rapidly in the first 3 months, especially among men, among individuals 65 years of age or older, and among individuals with weak immune systems [10] . It should be noted that the initial vaccine efficacies were calculated from clinical trials conducted with healthy volunteers without underlying medical conditions. In the real world, however, some individuals might not respond to vaccinations. This includes cancer patients receiving chemotherapy, organ transplant recipients and autoimmune patients taking certain immunosuppressant medications. There are 6 million such people in the U.S. alone. Therefore, when it is said that a vaccine is 95% protective, one has to consider its dynamic effectiveness, weaving in factors such as time-since-vaccination [6, 8, 9, 11] , subjects' immune status [12] , and neutralization-resistant variants that escape from vaccine-elicited immunity [13] [14] [15] . Even under the best scenario, efforts are needed to identify the 5% unprotected vaccine recipients and provide them with special instruction and care. Five percent in the U.S. alone means 16 million people. With the possibility that this pandemic will transition into a potential recurrent seasonal disease [16] , it is crucial to monitor the waning antibody response over time, in order to be able to address when and to whom to give a booster or another extra dose of COVID-19 vaccine. IgG responses whereas much lower titers were observed to the S2 subunit, and the majority of the neutralizing activity (90%) against SARS-CoV-2 is mediated by RBD-specific Abs blocking virus binding to ACE2 [17] . The most classic method to measure NAbs is the plaque reduction neutralization test (PRNT) that is considered the gold standard for NAb measurement [18, 19] . However, the assay takes several days for the virus plaques to form and to be counted. Engineered SARS-CoV-2 virus with a fluorescence reporter has been used to avoid manual counting and to improve assay throughput [20] . But this assay is quite cumbersome and requires Biosafety Level-3 lab setting to work with live and infectious SARS-CoV-2 virus. Several virus neutralization tests using pseudovirus with GFP or luciferase reporters have been developed [21] [22] [23] , and can be performed at Biosafety Level-1 or -2 facility. Still, there are several common issues with virus neutralization tests regardless of adopting a pseudovirus or not. In these tests, the neutralization ability of the antibodies is highly dependent on the maturation state or titre of the virus as well as the cell type and cell condition used in the assay [24] . Poor reproducibility or even false results can be generated if the virus and host cells are not at optimal assay conditions [24] . All the subjects are fully vaccinated U.S. residents having received two doses of mRNA vaccines from either Moderna or Pfizer, and participated in this study with their full consent on the disclosed purpose and data usage. None of the subjects had a prior PCR-confirmed SARS-CoV-2 infection, nor was on active immunosuppressant medication. Subject demographics in terms of gender, age and vaccine brand are listed in Table 1 . The detailed procedure for constructing the NeutraXpress test cassette is provided in For testing the sensitivity and specificity of NeutraXpress, we used Chinese hamster ovary (CHO) cells to express positive and negative control reagents, with detailed procedures described in the Supplementary Methods. In brief, we expressed a neutralizing human IgG1 antibody REGN10933 as a positive standard for analytical control, and an RBD-binding but nonneutralizing human IgG1 antibody CR3022 as a negative control. The anti-SARS nucleocapsid (N) human IgG1 antibody (TJ21) was expressed as a non-relevant control antibody. Finally, we expressed by CHO cells the hACE2-hIgG1 Fc fusion protein as a specificity control. In response to the unmet needs to monitor the levels of neutralizing antibodies after We emphasize here that a test that detects NAbs against SARS-CoV-2 must demonstrate the principle of blocking the interaction between ACE2 and Spike protein or its RBD domain (Fig. 1A) . Hence, it has to be an inhibition assay. Unlike other popular single-lane designs, the NeutraXpress cassette has two identical lanes, one for the specimen and the other for the "diluent only" negative control. In other LFIA products for SARS-CoV-2 NAb detection, it is very difficult to judge how much reduction of the test line signal is real reduction, as there is no control provided. Some product instructions even erroneously direct the users to compare the intensity of T line with that of the irrelevant C line. Our design provides a real-time internal control and eliminates ambiguity when performing the test. The contrast in line patterns, i.e., disappearance of T1 and appearance of T2 (Fig. 1E) , is very obvious and easy to interpret. Our test is also sensitive and specific. Using REGN10933, a recombinant human IgG1 NAb developed by Regeneron Pharmaceuticals, we showed that NeutraXpress can detect neutralization activity brought by >2.5 µg/mL NAb in undiluted serum at T1 line (about 30% inhibition), and can also detect RBD-binding hIgG1 >2.5 µg/mL in undiluted serum at T2 line (Fig. 1B) . The strong correlation of logarithmic NAb concentrations in serum with the reduction of T1 line intensity (R 2 =0.994) suggests that NeutraXpress can be used to accurately monitor the changes of serum NAb levels over a wide range (Fig. 1C) . The disappearance of T1 is specific for NAb, as an RBD-binding but non-neutralizing hIgG1 CR3022 as well as a nonrelevant anti-N-hIgG1 failed to dissipate T1 line even at a concentration as high as 500 µg/mL (Fig. 1D ). The specificity of T1 line was also shown by the complete competition of interaction between membrane-striped ACE2 and gold nanoparticle (GNP)-labeled RBD by soluble ACE2-hIgG1 Fc (Fig. 1D) . Moreover, the appearance of T2 line is also highly specific for RBD-binding moieties, shown by the signals with REGN10933-hIgG1, CR3022-hIgG1 and ACE2-hIgG1 Fc but no signal with anti-N-hIgG1 (Fig. 1B, D) line around 3 weeks after the 2 nd doses for both brands of mRNA vaccines (Fig. 1E) , suggesting that the mRNA vaccines are highly effective in inducing NAbs in the circulation. If tested only 7-10 days after the 2 nd dose, the body may not have developed high enough NAb titres to dissipate the T1 line, although T2 line could be strongly positive, indicative of the induction of RBD-binding antibodies (Fig. 1F) . Throughout this project, we were quite surprised that mRNA vaccine-induced NAb activities quickly waned after 3-4 months post the 2 nd vaccination ( Fig. 2A) . That is, when tested between 15 and 60 days post the 2 nd doses, the group of subjects had on average 62% inhibition of ACE2:RBD interaction by NAbs present in their blood, whereas this inhibition reduced to 25% when tested between 2-3 months, and further down to 16.7% when tested between 3-4 months ( Fig. 2A) . In fact, most subjects did not even show T2 lines after 4-6 months (Fig. 1G, Fig. 2D and Supplementary Figure 2) . Thus, our NeutraXpress rapid test confirms the clinical findings that mRNA vaccine-induced antibody responses against SARS-CoV-2 infection quickly waned after 3-6 months [6, 8, 9, 11] , which formed the basis for the recent government campaign for booster shots. It should be noted that after antigen stimulation, there is a natural contraction phase for Ag-specific B cells and the majority of these B cells undergo apoptosis with only a small fraction of them develop into memory B cells. If those primed B/plasma cells did not die and maintained their secretion of antibodies at peak levels, all the antibodies from the past antigen exposure would have made our blood too viscous to flow. But still, such quick waning of mRNA vaccineinduced NAb titres is somewhat unpredicted. It might be due to: 1) the mRNA vaccination schemes may not be optimal, i.e., the 2 nd dose is given 3-4 weeks after the 1 st dose. This may not be ideal when compared to spacing doses more apart, like the practice in the U.K.; and 2) perhaps for coronavirus, three doses of vaccine should be considered full vaccination. Whether previously infected or vaccinated, it is informative for individuals to learn if they generated high levels of neutralizing antibodies and how long the NAbs can last so that they can resume normal activities without fear of re-infection and transmitting the virus. However, as the pandemic unfolded, such a concept of "immunity passport" based on having antibodies didn't pan out. As we showed here (Fig. 1F) reported that they did not isolate any RBD-binding antibodies from 1 of the 5 COVID-19 patients with high NAb titres [28] . Seven of the 13 non-RBD binders from this patient were neutralizing antibodies and 2 of them were potent NAbs targeting NTD [28] . Thus, our current format could generate false-negative result for this type of individuals. By using S trimer, we probably won't be able to increase the sensitivity to detect those non-RBD binding NAbs, as NAbs against NTD, such as 4A8 [29] , do not directly block S/RBD:ACE2 interaction, and will not dissipate T1 line in our assay. Nevertheless, using S trimer for GNP labeling will increase the sensitivity of T2 line. Undoubtedly, COVID-19 vaccines have been highly effective in preventing severe symptomatic disease and death [30, 31] . Even though protection against SARS-CoV-2 infection appeared to wane rapidly following its peak after the 2 nd dose, protection against hospitalization and death persisted at a robust level for six months after the 2 nd dose, thanks to the elicited 14 cellular immunity [9] . While fully vaccinated people are eager to resume their normal social activities, it is an urgent call for researchers to find out whether there is a strong correlation of breakthrough infections with low NAb levels detectable by a POCT like NeutraXpress. In other words, it will be crucial to investigate whether individuals with low NAb levels have NeutraXpress and is seeking its EUA approval by the FDA. No animal is used in the present study. Consent was obtained from all the participants in this study on the purpose and data usage. Ethics approval was received from the Institutional Review Board of Antagen Diagnostics, Inc. 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