key: cord-0715560-dt2xankb
authors: Takeshita, Masaru; Nishina, Naoshi; Moriyama, Saya; Takahashi, Yoshimasa; Ishii, Makoto; Saya, Hideyuki; Kondo, Yasushi; Kaneko, Yuko; Suzuki, Katsuya; Fukunaga, Koichi; Takeuchi, Tsutomu
title: Immune evasion and chronological decrease in titer of neutralizing antibody against SARS-CoV-2 and its variants of concerns in COVID-19 patients
date: 2022-04-07
journal: Clin Immunol
DOI: 10.1016/j.clim.2022.108999
sha: 335c81ae2eeae84a83392027307af909e6b32a0a
doc_id: 715560
cord_uid: dt2xankb

Many variants of SARS-CoV-2 have emerged, and decreased neutralizing antibodies after vaccination and breakthrough infections have become a problem. The importance of monitoring titers of neutralizing antibodies is getting higher. We enrolled 146 COVID-19 patients, who were thought to be infected with Wuhan-hu-1 or D614G strains, and examined the time course of neutralizing titers against six concerning strains (Wuhan-hu-1, Alpha, Beta, Gamma, Kappa, and Delta) using newly developed ELISA. The acquisition of neutralizing titer was positively associated with disease severity. Immune evasions were observed approximately 20 to 30% for Alpha, Kappa, and Delta variant, and 40 to 45% for Beta and Gamma variant. The titers against all strains decreased over time, and interestingly, while titers against Wuhan-hu-1 decreased by 23%, those to Delta variant decreased by 70%. Our simple, cost-effective, and non-hazardous system will be applicable to process numerous samples, such as monitoring titers against prevalent strains after infection or vaccination.

SARS-CoV-2 acquires mutations as the infection spreads. In particular, mutations in the receptor binding domain (RBD) region of the spike protein, which is used by the virus to enter cells, may cause changes in infectivity and in the reactivity of neutralizing antibodies [1] [2] [3] .

Even in the early stage of the pandemic, the D614G mutation increased and replaced the previous strain within a few months; however, since this mutation was outside the RBD, it did not have a serious impact on the titers of neutralizing antibodies, and increased transmissibility is thought to be the cause of its spread [3, 4] .

Three variants have been reported from 2020 and designated as variants of concerns (VOCs) by the World Health Organization (WHO): B.1.1.7 (Alpha strain), which first emerged in England; B.1.351 (Beta strain), which first emerged in South Africa; and P.1 (Gamma strain), which first emerged in Brazil. The Alpha strain is reported to have little effect on neutralizing titer [5] [6] [7] or only a mild decrease of 22% [8] . In contrast, strong immune evasion was reported for the Beta and Gamma variants. The Beta variant shows an 8-to 13-fold reduction in convalescent plasma, and a 7-to 9-fold or 65% reduction in vaccinated serum [6, 7, 9] . The Gamma variant shows a 3-to 5-fold reduction in convalescent and J o u r n a l P r e -p r o o f Journal Pre-proof vaccinated plasma/serum [10, 7, 11] . Even therapeutic monoclonal antibodies have been reported to have reduced or lost neutralizing ability against variants with the E484K mutation [7, 9, 12] .

In April and May 2021, there was a rapid increase in the number of patients in India, mainly due to the B.1.617 strain [13] . This lineage has three main subtypes: B.1.617.1 (Kappa strain) and B. 1.617.3, characterized by L452R and E484Q mutation in RBD, and B.1.617.2 (Delta strain), characterized by L452R and T478K in RBD. Due to their high transmissibility, these variants have spread to 96 countries as of the end of June, and the WHO has classified the Kappa variant and the Delta variant as a variant of interest (VOI) and a variant of concern (VOC), respectively [13] . These variants have also been reported to escape from humoral immunity, with the Kappa strain showing a 2-to 7-fold decrease in recovered sera and a 3-to 7-fold decrease in post-vaccination sera [14] [15] [16] , and the Delta strain showing a 6-fold In Japan, variants with the D614G mutation were initially the majority, and VOC and VOI strains were not detected until the end of 2020. Three VOC strains, Alpha, Beta, and Gamma strains, were first detected in airport quarantine on December 25, 2020 , December 28, 2020 , and January 6, 2021 , respectively. A sample taken on January 22 was the first time that the Alpha strain was detected in Japan from a person with no history of travel abroad [20] . Therefore, by the end of 2020, most infected patients were infected with variants having only the D614G mutation. The Alpha strain became dominant in early 2021 and the Delta strain has been dominant since June/July [21] . Therefore, it seems important to examine the titers of neutralizing antibodies against these VOCs, after infection with non-mutated strains or vaccination. It is also important to examine the time-dependent attenuation of the titers, as previously reported [22] .

We have previously measured the titer of neutralizing antibodies from convalescent patients using magnet beads [23] . In this study, we developed this system as a competitive enzyme-linked immunosorbent assay (ELISA), and validated it using commercially available ELISA kits and sera that had been previously evaluated by an authentic virus neutralization assay [23] . In addition to the ELISA using RBD of the original Wuhan-hu-1 strain-derived sequences, we also produced ELISA using RBD derived from five different variants (Alpha, J o u r n a l P r e -p r o o f Journal Pre-proof Beta, Gamma, Kappa, and Delta) . Here, we measured the titers of neutralizing antibodies against each variant in the sera of patients infected by 2020, who are assumed to have been affected by the original strain, and in the sera of vaccinated volunteers. The attenuation of the titers of neutralizing antibodies was also examined using serum samples collected over the course of time.

We recruited patients who had COVID-19, diagnosed by approved reverse transcriptase polymerase chain reaction (RT-PCR) tests for SARS-CoV-2 using swabs from the nose or saliva, and hospitalized at Keio University Hospital between April and December 2020.

Serum samples from patients were collected at outpatient visits over a period of 6 months to 1 year. We also collected the serum from healthy volunteers 1.5 to 2 months after their second Pfizer/BioNTech vaccination.

The following parameters were collected from medical charts: signs and symptoms; neutrophil and lymphocyte counts; serum parameters of lactate dehydrogenase (LD), J o u r n a l P r e -p r o o f Journal Pre-proof C-reactive protein (CRP), ferritin, D-dimer, sialylated carbohydrate antigen KL-6, estimated glomerular filtration rate (eGFR); and medication history. Pneumonia was diagnosed based on lung computed tomography (CT). The titers of neutralizing antibodies against the authentic virus for some sera were derived from the previous study [23] . This study was approved by the Ethics Committee of Keio University School of Medicine and conducted in compliance with the tenets of the Declaration of Helsinki. Informed consent was obtained from all participating individuals.

Recombinant soluble ACE2 (1-708 AA) and RBD (Spike 319-541AA) were produced as previously described with minor modifications [23] . Briefly, the extracellular domain of ACE2 was inserted into the pcDNA3.4 expression vector (Thermo Fisher Scientific, MA, USA) with a polyhistidine tag at the N-terminus. The RBD of the spike was inserted into pcDNA3.4 with an SBP tag at the C-terminus. The RBD vectors of Alpha, Beta, Gamma, Kappa, and Delta strains were produced by PCR using mutated primers. The recombinant RBD and ACE2

were produced using the Expi293 Expression System (Thermo Fisher Scientific) according to manufacturer's instruction. The supernatants of RBD were concentrated and buffer The inhibition rate was calculated as follows: Inhibition rate = 1 -(OD450 of sample) / (OD450 of blank). To show the results as comparable linear values, inhibition rates were converted to titers of neutralizing antibody based on a standard curve as follows: the sample with the highest titer of neutralizing antibody was defined as the standard serum, its titer against the Wuhan-hu-1 strain was set to 100 U/ml, and the standard curve was obtained from the dilution series of the standard serum. The conversion formula is as follows; The serum titers of neutralizing antibody were also measured by commercially available kit, a SARS-CoV-2 Neutralization Antibody Detection Kit (MBL, Tokyo, Japan) at 1:10 dilution, and the inhibition rate between RBD and ACE2 was calculated according to manufacturer's instructions.

The authentic virus neutralization assay using SARS-CoV-2 virus (hCoV19/Japan/TY-WK-521/2020) and the variant strains (Alpha; hCoV19/Japan/QHN002/2020, Beta; hCoV19/Japan/TY8-612-P1/2021, Gamma; hCoV19/Japan/TY7-503/2021, and Delta; hCoV-19/Japan/TY11-927-P1/2021) were performed as previously described [24] . Briefly, a mixture of 100 TCID50 virus and serially diluted antibodies (2-fold serial dilutions starting at 1:5 dilution) was incubated at 37°C for 1 J o u r n a l P r e -p r o o f Journal Pre-proof hour before being placed on VeroE6/TMPRSS2 cells (JCRB Cell Bank, Osaka, Japan) seeded in 96-well flat-bottom plates. After culturing for five days at 37°C, cells were fixed with 20% formalin (Fujifilm Wako), and stained with crystal violet solution (Merck). Each sample was assayed in two wells and the lower cut-off dilution index with > 50% cytopathic effect was presented as neutralization titer.

Continuous data are presented as the median and interquartile range (IQR) or as a number with the percentage value, as appropriate. The chi-squared test was used to examine the categorical variables. The Wilcoxon rank sum test and Wilcoxon signed rank test were used to examine the continuous variables with and without correspondence, respectively. The Steel's multiple comparison test was used for comparison between multiple groups.

Correlations between two continuous variables were analyzed using Spearman's rank correlation coefficient. A model for the authentic virus neutralizing titer was made using multiple linear regression analysis. p-values < 0.05 were considered to be statistically significant. The variables that were associated with the neutralizing titer were entered into the J o u r n a l P r e -p r o o f model using forward selection with a threshold p-value of 0.05. All statistical analyses were performed with JMP 15 (SAS Institute, NC, USA).

We collected 220 sera from 146 patients in the convalescent phase of COVID-19, of which, 42 samples had been previously evaluated by authentic virus neutralization assay [23] , and sera from 10 vaccinated volunteers. The characteristics of participants and vaccinated volunteers are shown in Table 1 and 2. First, in order to confirm the performance of our in-house ELISA, we examined whether the results of the in-house ELISA correlated with the results of the authentic virus neutralization assay and commercially available ELISA kit for SARS-CoV-2 neutralizing antibody. As shown in Figure 1 , the titers of neutralizing antibodies measured by in-house ELISA were well correlated with those measured by authentic virus neutralizing assays and inhibition rates by commercially available ELISA. The titers by authentic virus neutralizing assays were also correlated with the inhibition rates by commercially available ELISA. Therefore, in-house ELISA was confirmed to have performance equivalent to that of the commercial kit. individuals were similar to those in patients with severe or critical disease severity. Next, we performed a multivariate analysis using the clinical parameters that correlated with the titers of neutralizing antibodies. As shown in Table 3 , disease severity, highest CRP level, and highest LD level were identified as the independent variables correlating with the titers of neutralizing antibodies, consistent with the previous studies [22, 23, 25] .

J o u r n a l P r e -p r o o f Next, we measured the titers of neutralizing antibodies against variant strains using the in-house ELISA system. In order to compare the effect of mutations only, mutated RBD protein production and ELISA procedures were performed in exactly the same manner, except that mutated vectors were used. First, we examined whether these ELISAs using mutated RBD correlated with the results of the authentic virus neutralization assay. We selected 10 sera from representative patients and confirmed that in-house ELISA using RBD with mutation correlated with the authentic virus neutralization titer as well as with Wuhan-hu-1 ( Figure 3A ). In overlaying these correlations ( Figure 3B ), the strains other than Beta showed similar correlations to those of Wuhan, although some variation was observed in Beta strain, suggesting that the neutralizing titers measured by in-house ELISA were comparable among strains with a small variation.

Because variant strains such as Alpha strain have been reported in Japan since January 2021 [20] , and because this study used samples from patients who had been infected by COVID-19 before that time, all patients were thought to be infected with the Wuhan-hu-1 strain or the strain with D614G. Figure 4A shows that the serum titers of neutralizing antibodies against the Wuhan-hu-1 strain were generally correlated with those against each variant strain. The distribution of some of the plots is skewed to the lower right,

Journal Pre-proof indicating that the neutralizing titers against the variant strain are lower than those against Wuhan-hu-1 strain. In particular, the beta and gamma strains, which commonly have the E484K mutation, are shifted more than the red line in the center.

We further stratified the samples by disease severity, and compared the titers. As shown in Figure 4B , there was little difference in asymptomatic and mild cases with low titer but, especially in the group of severe and critical cases, there was an approximately 20 to 30% decrease in the Alpha, Kappa, and Delta strains, and an approximately 40 to 50% decrease in the Beta and Gamma strains. This attenuation of the titers of the neutralizing antibodies was also observed in post-vaccinated individuals to the same extent as in the severe and critical group.

Finally, we examine the changes in the titers of neutralizing antibodies over time. Because it is difficult to calculate the rate of decline for samples with a low neutralizing titer from the start, we selected patients with moderate to critical disease severity, and calculated the decline rate of the titers using the results of the first and second test (median, 47.5 and 112 days after first positive PCR, respectively). As shown in Figure 5A , the serum neutralization J o u r n a l P r e -p r o o f Journal Pre-proof titer against Wuhan-hu-1 strain decreased by about 23% in two months, and interestingly, the titer reduction against Beta, Gamma, and especially Delta strains was significantly faster than that against Wuhan-hu-1 strain. Figure 5b shows the titers of cases that could be measured more than three times. Although the number of patients was limited, the patients with higher neutralization titers tended to have a faster decrease in their titers up to 3 to 4 months after infection than later.

In this study, we established a highly versatile ELISA system for measuring the titers of neutralizing antibodies that correlates well with the results of authentic virus neutralization assay. In addition, the titers of neutralizing antibodies against the major variants were also examined in a comparable manner using the same protocol and mutation-introduced RBDs, and confirmed the correlation with the authentic virus neutralization assay using variant strains. The results showed that the titers of neutralizing antibodies correlated well with disease severity in COVID-19 patients. In addition, the vaccination-induced antibody titers were comparable to the neutralizing titers in severe and critical COVID-19 patients. Immune escape was observed in VOCs and the Kappa variant, and the titer decreased over time.

As for the immune evasion of variant strains, it is reported that the neutralizing titers decreased 6.3-fold in the Beta strain, 4.3-fold in the Kappa strain, and 5.1-fold in the Delta strain using a pseudovirus assay system [16] . The immune evasion of Kappa and Delta appears to be weaker than that of Beta and Gamma, according to other reports [14] , [17] .

Some of the neutralizing antibodies bind to N-terminal domain (NTD) of Spike protein, and the neutralizing ability of these antibodies is reduced by mutation of NTD such as in Alpha strain [26] . Even though only antibodies against RBD were examined in our assay, the results correlated with the authentic virus neutralization assay. This is probably due to the major epitope of the neutralizing antibody existing in the RBD [27] . The ranges of the reduction of the neutralizing titers were approximately 20 to 30% for the Alpha, Kappa, and Delta strains, and 40 to 50% for the Beta and Gamma strains in severe and critical cases and vaccinated samples in this study. Although there were some differences in the reduction rate depending on the assay system, the results of this method were correlated with those of the authentic virus neutralization assay, and the trend for each strain was similar.

The titers of neutralizing antibodies from COVID-19 patients decreased by about 23% in two months. The serum half-life of the neutralizing titers after vaccination was reported to be 14.7 weeks [22] , and the results of both studies were similar. In Figure 5B , the J o u r n a l P r e -p r o o f Journal Pre-proof results of the third sample indicate that the decrease in the neutralizing titers appears to be moderate, although the sample size is small. It was reported that the titers of neutralizing antibodies were maintained throughout a 13-month observation [28] , and the long-term trend of the titers is thus an issue to be addressed. In Figure 5A , the decrease in the neutralization titers was faster against Beta, Gamma, and Delta strain than against Wuhan-hu-1. In particular, the titer against Delta strain was reduced by a median of 70%. This could have contributed to the spread of infection and the replacement of existing strains. Therefore, it is important to establish a cutoff value of the neutralization titers against each strain that can protect against re-infection or breakthrough infection. Recently, breakthrough infections after vaccination have been reported to be linked to a lower antibody titer [29] , and the cutoff value will become important as an indicator for the additional vaccination of healthcare workers.

There are several limitations in this study: first, because most of the participants were infected by the Wuhan-hu-1 strain with or without D614G mutation, we cannot assess Figure 3B ). Only the Beta strain differed slightly from the other strains, which may have been due to impurities in the purified RBD (Supplementary Figure 1A) , and this may lead to the variation in the neutralization titer of Beta strain.

The authentic virus and pseudo-virus neutralization assay may reflect a more native infection process than the competitive ELISA used in this study, however, our cell-and virus-free method, as well as the previously reported one [30] , are simple, cost-effective, and non-hazardous. In situations where the measurement of a large number of samples is necessary, such as for monitoring the titers of medical personnel, the competitive ELISA, which is applicable to prevalent strains and has been confirmed to correlate with virus neutralization assay, will be widely applicable. J o u r n a l P r e -p r o o f J o u r n a l P r e -p r o o f Wilcoxon's signed rank test. 

Deep Mutational Scanning of SARS-CoV-2 Receptor Binding Domain Reveals Constraints on Folding and ACE2 Binding

Establishment and lineage dynamics of the SARS-CoV-2 epidemic in the UK

Tracking Changes in SARS-CoV-2 Spike: Evidence that D614G Increases Infectivity of the COVID-19 Virus

SARS-CoV-2 D614G spike mutation increases entry efficiency with enhanced ACE2-binding affinity

mRNA-1273 vaccine induces neutralizing antibodies against spike mutants from global SARS-CoV-2 variants

Neutralizing Activity of BNT162b2-Elicited Serum

SARS-CoV-2 variants B.1.351 and P.1 escape from neutralizing antibodies

Neutralization of SARS-CoV-2 lineage B.1.1.7 pseudovirus by BNT162b2 vaccine-elicited human sera

Evidence of escape of SARS-CoV-2 variant B.1.351 from natural and vaccine-induced sera

Increased Resistance of SARS-CoV-2 Variant P.1 to Antibody Neutralization

Age-Dependent Neutralization of SARS-CoV-2 and P.1 Variant by Vaccine Immune Serum Samples

Reduced sensitivity of SARS-CoV-2 variant Delta to antibody neutralization

World Health Organization. COVID-19 Weekly epidemiological update

SARS-CoV-2 variant B.1.617 is resistant to bamlanivimab and evades antibodies induced by infection and vaccination

Infection and vaccine-induced neutralizing antibody responses to the SARS-CoV-2 B.1.617.1 variant

Reduced neutralisation of the Delta (B.1.617.2) SARS-CoV-2 variant of concern following vaccination

.617.2 Delta variant emergence, replication and sensitivity to neutralising antibodies

Effectiveness of the BNT162b2 Covid-19 Vaccine against the B.1.1.7 and B.1.351 Variants

New variant of a novel coronavirus (SARS-CoV-2) with concerns of increased transmissibility and altered antigenicity

New variant of a novel coronavirus (SARS-CoV-2) with concerns of increased transmissibility and altered antigenicity

Kinetics and correlates of the neutralizing antibody response to SARS-CoV-2 infection in humans

Incomplete humoral response including neutralizing antibodies in asymptomatic to mild COVID-19 patients in Japan

Temporal maturation of neutralizing antibodies in COVID-19 convalescent individuals improves potency and breadth to circulating SARS-CoV-2 variants

Persistence assessment of SARS-CoV-2-specific IgG antibody in recovered COVID-19 individuals and its association with clinical symptoms and disease severity: A prospective longitudinal cohort study

Antibody resistance of SARS-CoV-2 variants B.1.351 and B.1.1.7

Mapping Neutralizing and Immunodominant Sites on the SARS-CoV-2 Spike Receptor-Binding Domain by Structure-Guided High-Resolution Serology

Dynamic Characteristic Analysis of Antibodies in Patients With COVID-19: A 13-Month Study

Covid-19 Breakthrough Infections in Vaccinated Health Care Workers

A high-throughput cell-and virus-free assay shows reduced neutralization of SARS-CoV-2 variants by COVID-19 convalescent plasma

Highest CRP level (mg/dl)

Immune evasion was observed all variant strains, including Kappa and Delta strain

The neutralizing titer against the infected Wuhan-hu-1 decreased 23% in two months. 5. The neutralizing titers against Beta, Gamma, and Delta strains decreased faster

We