key: cord-0979109-lahh2l34
authors: Inada, Makoto; Ishikane, Masahiro; Terada, Mari; Matsunaga, Akihiro; Maeda, Kenji; Iwamoto, Noriko; Ujiie, Mugen; Kutsuna, Satoshi; Morioka, Shinichiro; Ishizaka, Yukihito; Mitsuya, Hiroaki; Ohmagari, Norio
title: Antibody responses after two doses of SARS-CoV-2 mRNA-1273 vaccine in an individual with history of COVID-19 re-infection
date: 2022-03-16
journal: Int J Infect Dis
DOI: 10.1016/j.ijid.2022.03.017
sha: dd5e4171dc7f704f611c08733aa3d00a0dc491fa
doc_id: 979109
cord_uid: lahh2l34

We present a case of 58-year-old Japanese man with history of two previous COVID-19 infections who received two doses of mRNA-1273 vaccine. We are not aware of any previous study regarding antibody tendency following two infections and two vaccinations. We evaluated his IgG titer of anti-spike protein and neutralizing activity from the first infection before and after two doses of vaccine. Both anti-spike IgG titer and neutralizing activity showed a tendency to decline almost 1 year after initial infection, and they rapidly increased after the first vaccination, and they remained high after the second vaccination. Although this is a single case-report, it seems to have generalizability because the findings are consistent with previous reports regarding single infections or three doses of vaccination. Our findings suggest that a single booster shot may provide sufficient protection, and aid the understanding of immunologic responses of vaccination, in COVID-19 patients with history of re-infection.

Since December 2019, coronavirus disease 2019 caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide (Hayakawa et 5 al., 2020) . As of January 25, 2022, 544 re-infected COVID-19 cases have been reported worldwide (BNO news. COVID-19 reinfection tracker; Inada et al., 2021) .

Medical history of COVID-19 appears to have a protective effect against re-infection but especially among older people, protection against repeat infection is merely 47% (Hansen et al., 2021) . Since the end of 2020, two messenger RNA vaccines, mRNA-1273 [Moderna] and BNT162b2 [Pfizer] , which induce the anti-spike protein of SARS-CoV-2, have shown high efficacy in preventing COVID-19 onset and severe disease (Golob et al., 2021) . Many countries, including Japan and the USA, recommend that everyone should be vaccinated, regardless of past history of COVID-19 (Ministry of Health, Labour and Welfare; Centers for Disease Control and Prevention). Only a single dose of mRNA vaccine can elicit rapid immune responses in seropositive participants (Krammer et al., 2021) . However, to the best of our knowledge, there are no reports of antibody responses and implications of vaccination among individuals with a history of COVID-19 re-infection.

Here, we evaluate the trend in anti-spike protein antibody titers, including neutralizing antibodies, in a patient with COVID-19 re-infection after two doses of mRNA vaccine, and discuss the implications of vaccination in re-infected patients.

A 58-year-old Japanese man with a medical history of hyperlipidemia was diagnosed with COVID-19 re-infection four months after his initial COVID-19 infection (Inada et al., 2021) . Although phylogenic investigations were not done for both episodes, according to the epidemiology of SARS-CoV-2 in Japan, the causative variants were assumed to be PANGO lineage B.1.1.162 and B.1.1.284, which were domestic minor variants of SARS-CoV-2 in Japan (Sekizuka et al., 2021) . Fifteen months after the initial infection, he received two doses of mRNA-1273 vaccinations 4 weeks apart. After each vaccination, he developed a fever of 40℃ for one and two days, respectively, which improved with antifebrile medication. We evaluated the anti-spike protein antibody titer, including neutralizing antibody, approximately 1 year after the initial infection and after each vaccination.

The measurement of anti-SARS-CoV-2 spike IgGs was performed as per standard protocol. Briefly, recombinant spike protein-coated plates were incubated with 1/800-diluted patient sera at 37 °C for 1 h. After washing, the plate was incubated with horseradish peroxidase-conjugated anti-human IgG (GeneTex, Irvine, CA, USA) at 37 °C for 30 min and developed with 3,3,5,5-tetramethylbenzidine substrate (Nacalai Tesque, Kyoto, Japan). Samples from healthy volunteers without SARS-CoV-2 infection were 7 used as negative controls, whereas those from infected patients with high levels of anti-spike IgGs were used as positive controls. Each sample was assayed in triplicate. The cut-off value was the negative control mean +3 standard deviation.

The neutralizing activity of the sera of the patients was determined as previously described (Maeda et al.) . In brief, each serum was serially diluted 4-fold in the culture medium. The diluted sera were incubated with 50% tissue culture infectious dose (TCID 50 ) of viruses at 37°C for 20 min, after which the sera-virus mixtures were inoculated into VeroE6 TMPRSS2 cells (1.0 x 10 4 /well) in 96-well plates. The SARS-CoV-2 strain, SARS-CoV-205-2N (PANGO lineage B) (Maeda et al., 2021) , was used in this assay. After culturing the cells for 3 days, the levels of cytopathic effect (CPE) observed in SARS-CoV-2-exposed cells were determined using the WST-8 assay using the Cell Counting Kit-8 (Dojindo, Kumamoto, Japan). The serum dilution that resulted in 50% inhibition of CPE was defined as a 50% neutralization titer (NT 50 ).

The anti-SARS-CoV-2 spike protein IgG antibody level and NT 50 , which remained high after re-infection with SARS-CoV-2, declined about 1 year after the initial infection (anti-SARS-CoV-2 spike protein IgG antibody level was 0.67, and NT 50 value was x140).

However, after the first mRNA-1273 vaccination, the anti-SARS-CoV-2 spike protein IgG antibody level and NT 50 increased rapidly and were higher than their original levels 8 at the time of infection (anti-SARS-CoV-2 spike protein IgG antibody level = 3.29, NT 50 >1,000). Furthermore, the titer remained high after the second vaccination (anti-SARS-CoV-2 spike protein IgG antibody level = 3.24, NT 50 >1,000) (Figure 1 ).

Here, we have shown the trend of anti-spike protein antibody titers and NT 50 against COVID-19 re-infection, about 1 year after initial infection and after two doses of mRNA-1273 vaccine. An individual with past COVID-19 infection is less likely to suffer re-infection (Hansen et al., 2021) , but it is not clear whether an individual with two previous infections of COVID-19 may suffer from a third infection. An anecdotal case series has shown the occurrence of a third infection (Hasanzadeh et al., 2021) . According to a study of vaccine breakthrough participants who had a breakthrough infection tended to have a lower IgG level and lower NT 50 (Bergwerk et al., 2021) , suggesting that an individual who has a lower antibody titer might be more easily re-infected.

Our report highlights two important considerations. First, even if an individual has a history of re-infection with SASR-CoV-2, the anti-spike protein IgG antibodies and NT 50 decrease approximately 1 year after initial infection. This suggests the possibility of a third infection. Second, after the first vaccination, the anti-SARS-CoV-2 spike protein 9 IgG antibody level and NT 50 increase rapidly and are higher than at the time of infection.the antibody titer after vaccination is higher than those who had been infected only once (Terada et al., 2021) . This may suggest that SARS-CoV-2 re-infection prior to a mRNA vaccination could induce robust antibody response, and sufficient immunity could be obtained without -second vaccination. Some experts suggest that a single mRNA vaccine dose may provide effective protection, even in previously infected persons (Krammer et al., 2021) . At present, however, evidence to support this idea is lacking in the real world. It requires further examination whether individuals with history of COVID-19 re-infection need less doses of vaccination.

Our study has several limitations. First, this study evaluated anti-spike antibodies and NT 50 in a single case of re-infection. However, this antibody response seems similar to the trend among individuals with past infection or two doses of vaccination. Namely, antibody titers decrease after a single infection event (Chen et al., 2021) or two doses of vaccination (Doria-Rose et al., 2021) , and increase rapidly and strongly in response to vaccination after a single infection (Krammer et al., 2021) , or booster after two doses of vaccination. Therefore, although this study describes the antibody trend of a single case, the tendency is consistent with previous reports and plausible. Second, we evaluated the tendency of the antibody titer and NT 50 in vitro, but the relationship between antibody 10 trend and disease prevention or severity is still unclear. Despite these limitations, to the best of our knowledge, this report is the first to evaluate the trend in anti-spike protein antibody titers and NT 50 , in a re-infected COVID-19 patient after two doses of mRNA vaccination.

In conclusion, the anti-SARS-CoV-2 spike protein IgG antibody level and NT 50 increase rapidly after the first mRNA vaccination, and this high antibody titer is The patient was diagnosed with COVID-19 infection twice, on April 17 and July 31, 2020, and was vaccinated with mRNA-1273 on July 5 and on August 2, 2021. Two NT 50 values after vaccination were above the upper limit (NT 50 :>1,000).

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We would like to thank Drs. These funding sources had no involvement in the content of this study.

This study was approved by the ethics committee of the National Center for Global Health and Medicine (NCGM) (approval no: NCGM-G-003536-03 and NCGM-G-004136-00) and was conducted in accordance with the Declaration of Helsinki.Written informed consent was obtained from the patient for publication of the paper.

All authors declare no conflicts of interest regarding this study.