key: cord-1050286-ar8muxwk
authors: Kurahashi, Yukiya; Sutandhio, Silvia; Furukawa, Koichi; Tjan, Lidya Handayani; Iwata, Sachiyo; Sano, Shigeru; Tohma, Yoshiki; Ohkita, Hiroyuki; Nakamura, Sachiko; Nishimura, Mitsuhiro; Arii, Jun; Kiriu, Tatsunori; Yamamoto, Masatsugu; Nagano, Tatsuya; Nishimura, Yoshihiro; Mori, Yasuko
title: Cross-Neutralizing Breadth and Longevity Against SARS-CoV-2 Variants After Infections
date: 2022-02-24
journal: Front Immunol
DOI: 10.3389/fimmu.2022.773652
sha: 6f94378bf32ecc1b6d0ab52acb084826b5b881f3
doc_id: 1050286
cord_uid: ar8muxwk

BACKGROUND: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the virus responsible for the Coronavirus Disease 2019 (COVID-19) pandemic. The emergence of variants of concern (VOCs) has become one of the most pressing issues in public health. To control VOCs, it is important to know which COVID-19 convalescent sera have cross-neutralizing activity against VOCs and how long the sera maintain this protective activity. METHODS: Sera of patients infected with SARS-CoV-2 from March 2020 to January 2021 and admitted to Hyogo Prefectural Kakogawa Medical Center were selected. Blood was drawn from patients at 1-3, 3-6, and 6-8 months post onset. Then, a virus neutralization assay against SARS-CoV-2 variants (D614G mutation as conventional strain; B.1.1.7, P.1, and B.1.351 as VOCs) was performed using authentic viruses. RESULTS: We assessed 97 sera from 42 patients. Sera from 28 patients showed neutralizing activity that was sustained for 3-8 months post onset. The neutralizing antibody titer against D614G significantly decreased in sera of 6-8 months post onset compared to those of 1-3 months post onset. However, the neutralizing antibody titers against the three VOCs were not significantly different among 1-3, 3-6, and 6-8 months post onset. DISCUSSION: Our results indicate that neutralizing antibodies that recognize the common epitope for several variants may be maintained for a long time, while neutralizing antibodies having specific epitopes for a variant, produced in large quantities immediately after infection, may decrease quite rapidly.

variants of concern (VOC) (13) . B.1.1.7, which was firstly detected in the United Kingdom, has an N501Y mutation in the receptor binding domain (RBD) of S protein. P.1, which was identified in Brazil, has three mutations (K417T, E484K, and N501Y) in the RBD. B.1.351, which was found in South Africa, has three mutations (K417N, E484K, and N501Y) in the RBD (14) . B.1.617.2, which was confirmed in India, has mutations (L452R and T478K) in the RBD (15) , leading to higher viral load in infected individuals, in addition to the P681R mutation which increases the virus transmissibility (16) . Finally, B.1.1.529, which was detected in Botswana on November 11, 2021 and South Africa on November 14, 2021, has 15 mutations (G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, and Y505H) in the RBD (17) . The N501Y mutation shared among B.1.1.7, B.1.351, P1 and B.1.1.529 have influence on the affinity between the angiotensinconverting enzyme 2 (ACE2) receptor and the RBD of the S protein, which causes high transmissibility of the virus. The variant B.1.1.7 has proven to be more transmissible than original strains (18) . The E484K mutation in the RBD of B.1.351 and P1 is involved in immune escape (19, 20) . The K417N mutation of B.1.351 and B.1.1.529 and K417T mutation of P.1 are suggested to change the conformation of S protein, allowing escape from Nabs (19, 20) . Nabs have reduced activity on the variants B.1.351 and P.1 (14) . Especially, the variant B.1.351 is much more resistant to neutralization by Nabs, possibly because of differences in the mutations of the N terminal domain (NTD), which are associated with escape from immunity (21) . Nabs against B.1.1.529 from sera of convalescent patients infected with the ancestral SARS-CoV-2 and sera of vaccinated people with two dose of ChAdOx1 nCoV-19 or BNT162b2 is also lower than that against the other variants (22, 23) .

As elsewhere in the world, Japan is facing COVID-19 pandemic. As of mid-December 2021, nearly 1,730,000 Japanese people had been infected with COVID-19 and about 18,000 people had died (1) . So far, the country has been affected by five waves of exponential increase in new cases. D614G_KR, which had D614G mutation of S protein and 203_204delinsKR mutation of nucleocapsid protein, and its lineage were predominant during the first to third waves, from March 2020 to February 2021 (24) . VOCs have also spread to Japan. B. maintained up to six to twelve months post onset, although follow-up studies for longer duration are still needed (28) (29) (30) . Furthermore, the breadth and longevity of cross-neutralizing activities against VOCs have been minimally tested (28, 31) . In this study, we analyzed the longevity and breadth of neutralizing activity of COVID-19 convalescent sera across the VOCs (B1.1.7, P.1, and B.1.351) and the D614G.

Polymerase chain reaction (PCR) detection of the SARS-CoV-2 genome in nasopharyngeal swab samples was used to confirm the diagnosis of COVD-19. We used the same definitions of severities as in our previous report (32) . Asymptomatic patients had neither clinical symptoms nor hypoxia. Patients with mild illness had symptoms without evidence of pneumonia or hypoxia. Those with moderate illness had clinical symptoms of pneumonia with oxygen saturation levels over 90% on room air. Those with severe illness suffered from pneumonia with an oxygen saturation level under 90% on room air. Patients who needed mechanical ventilation were classified as critical. 

The SARS-CoV-2 Biken-2 (B2) strain including the D614G mutation was used as the conventional virus (accession number: LC644163), and was received from BIKEN Innovative Vaccine Research Alliance Laboratories. The three SARS-CoV-2 variants: B. 

The live virus neutralization assay against SARS-CoV-2 variants (D614G, B.1.1.7, P.1, and B.1.351) was done as previously reported according to Biosafety Level 3 regulations (32, 33) . At 24 hours before the assay, 4 × 10 4 Vero E6 (TMPRSS2) cells per well were seeded in 96-well tissue culture microplates. Serum samples were heat-inactivated at 56°C for 30 minutes, and twofold serially diluted using Dulbecco's Modified Eagle's Medium as the diluent. Diluted serum samples were mixed with 100 tissue culture infectious dose (TCID) 50 of SARS-CoV-2 variants and incubated at 37°C for one hour. The mixture of sera and virus was added to confluent Vero E6 (TMPRSS2) cells in a 96-well plate. Cells were incubated at 37°C with 5% CO 2 supplementation for six days. Then, the neutralizing titer was determined as the dilution factor in which cells showed no cytopathic effect. The titer was shown on a log2 scale. The cutoff titer was set at one; titer under one was defined as ND (not detected).

Continuous variables are described using medians and interquartile ranges (IQRs), defined by the 25th and 75th percentiles. Categorical factors were reported as counts and percentages. Cochran's Q test and Benjamini-Hochberg correction were performed to compare the proportion of patients whose Nab titer was ND among four variants. Friedman's test and the Benjamini-Hochberg correction was performed to compare the Nab titers among variants or sampling times. The level of statistical significance was set at p < 0.05. Statistical analyses were performed using STATA (version 14.2). Sample size calculation was not performed.

Our study was approved by the ethics committee of Kobe University Graduate School of Medicine (ID: B200200) and Hyogo Prefectural Kakogawa Medical Center. Written consent or the opt-out consent for our observational study was obtained.

We assessed 97 sera from 42 individuals in total, and the data are shown in Table 1 and Supplementary Table 1 . The median age with IQR was 56 (49-62) years. Fifty percent of patients were female. Patients' blood was taken two or three times serially. In terms of disease severity, four patients were asymptomatic (P1 to P4), fifteen had mild disease (P5 to P19), five had moderate disease (P20 to P24), fifteen had severe disease (P25 to P39) and three were critical (P40 to P42). We divided the post-onset data according to the three time periods (1-3 months, 3-6 months, and 6-8 months). The median days with IQR for these assessments were 47 (43) (44) (45) (46) (47) (48) (49) (50) (51) (52) (53) (54) , 117 (110-132), and 209 (199-219). Common chronic conditions were hypertension (28.6%), diabetes (26.2%), and pulmonary diseases (19.0%) including asthma and chronic obstructive pulmonary disease (COPD).

All data of neutralization assays are shown in Figure 1 . Among 42 patients, sera from 28 patients showed long-lasting neutralizing activities on the three VOCs (five out of fifteen mild: P6, P7, P9, P10, and P13, and all moderate to critical), in addition to D614G. On the other hand, sera from two patients (P8 and P18) showed no cross-neutralizing activity for any VOCs, and sera from six patients (P2, P4, P5, P11, P14, and P17) did not neutralize B.1.351 at all. Sera from the other six patients (P1, P3, P12, P15, P16, and P19) neutralized B.1.351 at first, but later could not. Totally, all four asymptomatic and 10 out of 15 mild patients could not obtain or maintain cross-Nabs for the three variants.

Next, we analyzed the Nab titers among D614G and three VOCs by two severity groups. One group was named 'patients without pneumonia'. This group included the patients who did not present with pneumonia (asymptomatic and mild patients). The other was named 'patients with pneumonia', including the patients who presented with pneumonia (moderate, severe and critical patients). The Nab titers against B.1.351 in both groups were significantly lower than those against the other variants at 1-3 months post onset and 3-6 months post onset. The Nab titers against all four variants in 'patients with pneumonia' were higher than those in 'patients without pneumonia' (Figure 2A) .

Then, we analyzed the trend of ND (not detected, that is, Nab titer under one) among D614G and three VOCs by two severity groups in Figure 2B . All in 'patients with pneumonia' acquired and maintained cross-Nabs for three VOCs. However, many patients in 'patients without pneumonia' could not acquire or maintain the Nab titers for D614G and three VOCs. The proportions of patients with ND for D614G, B.1.1.7, and P.1 at 1-3 months post onset were 0%, 11.8%, and 17.6%, respectively. The proportion of patients with ND for B.1.351 was 47.1% and significantly higher than the other variants. The proportion of patients with ND for P.1 was the second-highest but without significant difference from D614G and B. Then, we focused on 28 patients with positive cross-Nabs for three VOCs and compared the Nab titer among the four variants by the timing of sampling ( Figure 3A ). These raw data were shown in Supplementary Table 2 . The median Nab titer (log2) with IQR against D614G, B.1.1.7, P.1, and B.1.351 at 1-3 months post onset were 5 (5-6), 5 (4-5), 4 (4-5), and 3 (2-4), respectively. The Nab titer against B.1.351 was significantly lower than that against the other three variants, and the Nab titer against P.1 was significantly lower than that against D614G. The median Nab titer (log2) with IQR against D614G, B.1.1.7, P.1, and B.1.351 at 3-6 months post onset were 4 (4-5.5), 4 (3-5), 5 (4-6), and 3 (2-4), respectively. The Nab titer against B.1.351 was also significantly lower than that against the other three variants at 3-6 months post onset. The median Nab titer (log2) with IQR against D614G, B.1.1.7, P.1, and B.1.351 at 6-8 months post onset were 4 (2-5), 5 (3-6), 4 (3-6), and 3 (2-3), respectively. The Nab titer against B.1.351 was significantly lower than that against B.1.1.7.

Finally, we analyzed the retention of neutralizing activities over time against the four variants ( Figure 3B ) by rearranging the same data shown in Figure 3A . The Nab titer against D614G significantly decreased at 6-8 months post onset compared to 1-3 months post onset. Interestingly, each Nab titer against the three VOCs did not significantly change among three time points.

The purpose of this study was to examine the longevity of Nab activity of COVID-19 convalescent sera against D614G, and their neutralizing breadth against B.1.1.7, P.1, and B.1.351. We performed live virus neutralization assays against D614G and (Figures 1,  2A, B) . This reason might be that weak immune-response against SARS-CoV-2 lead to the low Nab titers of 'patients without pneumonia' against D614G compared to those of 'patients with pneumonia' (21, (37) (38) (39) (40) . The Nab titer against B.1.351 was lower than that against the other variants ( Figure 3A) , as similarly reported by other studies (14, 28, 41) . The neutralizing titer against D614G significantly decreased in sera of 6-8 months post onset compared to those of 1-3 months post onset ( Figure 3B ). Several studies have reported that the peak neutralizing antibody titer is three to five weeks post onset, and decreases rapidly, then is sustained at low level for several months (42) (43) (44) . The rapid early decay was shown to be caused by the short half-life of serum antibodies and by the short life of antibody-secreting cells; the maintenance of neutralizing antibody titers was supported by long-lived plasma cells to produce long-term antibodies (45) . Further follow-up will be needed to confirm whether the specific Nab titer against D614G is maintained or not. Interestingly and surprisingly, Nab titers against the three VOCs did not decrease until 6-8 months post onset ( Figure 3B) , possibly indicating that Nabs that recognize common epitopes were produced after infection, were selected for survival and sustained for a long time, while Nabs that recognized specific epitopes for a variant were produced in greater numbers after infection and decreased rapidly. Our results may reflect the increasing neutralizing breadth of antibodies which recognize common epitopes among VOCs (46) (47) (48) . Importantly, SARS-CoV-2 RNA and proteins were detected in intestinal enterocytes several months post onset (46) . These persisting viral antigens may stimulate memory B cells continuously. Then, B cells that produce neutralizing antibodies targeting the common epitope among variants can undergo further maturation in germinal center and as a result, produce high-affinity antibodies several months later. B cells that produce neutralizing antibodies targeting the common epitope among variants can undergo further maturation in germinal center and as a result, produce high-affinity antibodies several months later. Simultaneously, low-affinity antibodies disappear over time (48) . Similar to our study, other study also showed that sera from convalescent patients infected with the ancestral SARS-CoV-2 maintained the cross-neutralizing antibody titers against B. (2) . Neutralizing antibodies that recognize a common epitope for variants may be kept for a long time. SARS-CoV-2 specific functional CD 4 + T cell has also an important role to help the long lived S-specific B cell to produce high-affinity antibodies (50, 51) . Recent study showed that mild COVID-19 patients induced fewer but functionally superior B cell than critical patients with mechanical ventilation (52) . The diversity of B cell might be brought by CD4 + T cell. Further follow-up would be required to clarify the mechanism to get the long-lived immunity and cross-neutralizing activity against VOCs. We should be careful in interpreting the meaning of ND. It remains unclear how much Nab titer determined by our method is required to protect reinfection. In this study, we did not examine whether fragment crystallizable (Fc) portions worked to recruit immune cells or serum complement as effectors. Some studies have shown that the Fc-mediated effector function of neutralizing antibodies against SARS-CoV-2 was essential for optimal therapy (53, 54) . Therefore, the recoverees with low titer or ND in our study may still be protected against reinfection or severe disease after infection. Furthermore, we need to evaluate not only humoral immunity but also cell-mediated immunity. Cell-mediated immunity might be obtained because few mutations in the T-cell epitope of VOCs are known (41, 55) , although L452R mutation, which is present in some variants (B.1.167 and B.1.427/429), escapes from HLA-24 cell-mediated immunity (56) . Therefore, even if the neutralization activity falls below the detection limit in the long term, convalescent COVID-19 patients might be protected from VOCs.

The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and approved by The ethics committee of Kobe University Graduate School of Medicine (ID: B200200) and Hyogo Prefectural Kakogawa Medical Center. The patients/participants provided their written informed consent to participate in this study. 

Tackling COVID-19 With Neutralizing Monoclonal Antibodies

Efficacy and Safety of COVID-19 Vaccines: A Systematic Review and Meta-Analysis of Randomized Clinical Trials. Vaccines (Basel) (2021)

The Effectiveness of Convalescent Plasma and Hyperimmune Immunoglobulin for the Treatment of Severe Acute Respiratory Infections of Viral Etiology: A Systematic Review and Exploratory Meta-Analysis

The Use of Ebola Convalescent Plasma to Treat Ebola Virus Disease in Resource-Constrained Settings: A Perspective From the Field

Convalescent Plasma or Hyperimmune Immunoglobulin for People With COVID-19: A Living Systematic Review

Mortality Benefit of Convalescent Plasma in COVID-19: A Systematic Review and Meta-Analysis

Development of Therapeutic Antibodies for the Treatment of Diseases

FDA Approves Antibody Cocktail for Ebola Virus

The Antibody Cocktail, RONAPREVE for Intravenous Infusion Set Receives the World's First Regulatory Approval From MHLW for COVID-19

The Coronavirus is Mutating -Does it Matter?

Antibody Evasion by the P.1 Strain of SARS-CoV-2

The Impact of Mutations in SARS-CoV-2 Spike on Viral Infectivity and Antigenicity

Furin Cleavage Site Is Key to SARS-CoV-2 Pathogenesis

Omicron and Delta Variant of SARS-CoV-2: A Comparative Computational Study of Spike Protein

Estimated Transmissibility and Impact of SARS-CoV-2 Lineage B.1.1.7 in England

Complete Mapping of Mutations to the SARS-CoV-2 Spike Receptor-Binding Domain That Escape Antibody Recognition

Molecular Dynamic Simulation Reveals E484K Mutation Enhances Spike RBD-ACE2 Affinity and the Combination of E484K, K417N and N501Y Mutations (501Y.V2 Variant) Induces Conformational Change Greater Than N501Y Mutant Alone, Potentially Resulting in an Escape Mutant

Emerging SARS-CoV-2 Variants of Concern Evade Humoral Immune Responses From Infection and Vaccination

The Significant Immune Escape of Pseudotyped SARS-CoV-2 Variant Omicron

Reduced Neutralisation of SARS-CoV-2 Omicron B.1.1.529 Variant by Post-Immunisation Serum

Introductions and Evolutions of SARS-CoV-2 Strains in Japan. medRxiv (2021)

Sera Neutralizing Activities Against SARS-CoV-2 and Multiple Variants Six Month After Hospitalization for COVID-19

Antibody Kinetics Eight Months From COVID-19 Onset: Persistence of Spike Antibodies But Loss of Neutralizing Antibodies in 24% of Convalescent Plasma Donors

Maintenance of Neutralizing Antibodies Over Ten Months in Convalescent SARS-CoV-2 Afflicted Patients

Neutralizing Antibodies Against SARS-CoV-2 Variants After Infection and Vaccination

Early Differences in Cytokine Production by Severity of Coronavirus Disease

Seroepidemiological Survey of the Antibody for Severe Acute Respiratory Syndrome Coronavirus 2 With Neutralizing Activity at Hospitals: A Cross-Sectional Study in Hyogo Prefecture

Japan Mutation Report: Outbreak.Info (2021)

SARS-CoV-2 B.1.1.7 Lineage Rapidly Spreads and Replaces R.1 Lineage in Japan: Serial and Stationary Observation in a Community

The Neutralizing Antibody Response Against Severe Acute Respiratory Syndrome Coronavirus 2 and the Cytokine/Chemokine Release in Patients With Different Levels of Coronavirus Diseases 2019 Severity: Cytokine Storm Still Persists Despite Viral Disappearance in Critical Patients

Prevalence of Neutralising Antibodies Against SARS-CoV-2 in Acute Infection and Convalescence: A Systematic Review and Meta-Analysis

Frontiers in Immunology | www.frontiersin.org

Stable Neutralizing Antibody Levels 6 Months After Mild and Severe COVID-19 Episodes

Sensitivity of SARS-CoV-2 Variants to Neutralization by Convalescent Sera and a VH3-30

SARS-CoV-2 Variants of Concern Partially Escape Humoral But Not T-Cell Responses in COVID-19 Convalescent Donors and Vaccinees

Defining the Features and Duration of Antibody Responses to SARS-CoV-2 Infection Associated With Disease Severity and Outcome

Rapid Decay of Anti-SARS-CoV-2 Antibodies in Persons With Mild Covid-19

SARS-CoV-2 Infection Induces Sustained Humoral Immune Responses in Convalescent Patients Following Symptomatic COVID-19

Plasma Cell Survival in the Absence of B Cell Memory

Evolution of Antibody Immunity to SARS-CoV-2

Naturally Enhanced Neutralizing Breadth Against SARS-CoV-2 One Year After Infection

Temporal Maturation of Neutralizing Antibodies in COVID-19 Convalescent Individuals Improves Potency and Breadth to Circulating SARS-CoV-2 Variants

Neutralizing Antibody Activity in Convalescent Sera From Infection in Humans With SARS-CoV-2 and Variants of Concern

SARS-CoV-2-Specific T Cell Immunity in Cases of COVID-19 and SARS, and Uninfected Controls

Targets of T Cell Responses to SARS-CoV-2 Coronavirus in Humans With COVID-19 Disease and Unexposed Individuals

Memory B Cells Targeting SARS-CoV-2 Spike Protein and Their Dependence on CD4(+) T Cell Help

Human Neutralizing Antibodies Against SARS-CoV-2 Require Intact Fc Effector Functions for Optimal Therapeutic Protection

The Fc-Mediated Effector Functions of a Potent SARS-CoV-2 Neutralizing Antibody, SC31, Isolated From an Early Convalescent COVID-19 Patient, are Essential for the Optimal Therapeutic Efficacy of the Antibody

SARS-CoV-2 Human T Cell Epitopes: Adaptive Immune Response Against COVID-19

SARS-CoV-2 Spike L452R Variant Evades Cellular Immunity and Increases Infectivity

We thank Kazuro Sugimura MD, PhD (Superintendent, Hyogo Prefectural Hospital Agency and Professor, Kobe University) for his full support to promote this study. We express our sincere gratitude for cooperation and participation of staffs of Hyogo Prefectural Kakogawa Medical Center. We thank for BIKEN Innovative Vaccine Research Alliance Laboratories providing SARS-CoV-2 B2 strain. We thank the National Institute of Infectious Disease Japan for providing SARS-CoV-2 B. 

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.