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Prior COVID-19 protects against reinfection, even in the absence of detectable antibodies

      Graphical Abstract

      Several studies, including from our own centres, have shown the strong protection from reinfection conferred by previous SARS-CoV-2 infection
      • Hanrath Aidan T.
      • Payne Brendan A.I.
      • Duncan Christopher J.A.
      Prior SARS-CoV-2 infection is associated with protection against symptomatic reinfection.
      • Breathnach Aodhán Seán
      • Riley Peter Andrew
      • Cotter Meaghan Patricia
      • Houston Angela Cara
      • Habibi Maximillian Shahin
      • Planche Timothy David
      Prior COVID-19 significantly reduces the risk of subsequent infection, but reinfections are seen after eight months.
      • Hall Victoria Jane
      • Foulkes Sarah
      • Charlett Andre
      • Atti Ana
      • Monk Edward J M
      • Simmons Ruth
      • Wellington Edgar
      • Cole Michelle J
      • Saei Ayoub
      • Oguti Blanche
      • Munro Katie
      • Wallace Sarah
      • Kirwan Peter D
      • Shrotri Madhumita
      • Vusirikala Amoolya
      • Rokadiya Sakib
      • Kall Meaghan
      • Zambon Maria
      • Ramsay Mary
      • Brooks Tim
      • Brown Colin S
      • Chand Meera A
      • Hopkins Susan
      SIREN Study Group. SARS-CoV-2 infection rates of antibody-positive compared with antibody-negative health-care workers in England: a large, multicentre, prospective cohort study (SIREN).
      • Holm Hansen Christian
      • Michlmayr Daniela
      • Gubbels Sophie Madeleine
      • Mølbak Kåre
      • Ethelberg Steen
      Assessment of protection against reinfection with SARS-CoV-2 among 4 million PCR-tested individuals in Denmark in 2020: a population-level observational study.
      • Lumley SF
      • O'Donnell D
      • Stoesser NE
      • Matthews PC
      • Howarth A
      • Hatch SB
      • Marsden BD
      • Cox S
      • James T
      • Warren F
      • Peck LJ
      • Ritter TG
      • de Toledo Z
      • Warren L
      • Axten D
      • Cornall RJ
      • Jones EY
      • Stuart DI
      • Screaton G
      • Ebner D
      • Hoosdally S
      • Chand M
      • Crook DW
      • O'Donnell AM
      • Conlon CP
      • Pouwels KB
      • Walker AS
      • Peto TEA
      • Hopkins S
      • Walker TM
      • Jeffery K
      • Eyre DW
      Oxford University Hospitals Staff Testing Group. antibody status and incidence of SARS-CoV-2 infection in health care workers.
      However these studies did not address whether prior infection is protective in the absence of a detectable humoral immune response. Patients with primary or secondary antibody deficiency syndrome and reduced or absent B cells can recover from COVID-19
      • Quinti I
      • Lougaris V
      • Milito C
      • Cinetto F
      • Pecoraro A
      • Mezzaroma I
      • Mastroianni CM
      • Turriziani O
      • Bondioni MP
      • Filippini M
      • Soresina A
      • Spadaro G
      • Agostini C
      • Carsetti R
      • Plebani A.
      A possible role for B cells in COVID-19? lesson from patients with agammaglobulinemia.
      ,
      • Montero-Escribano P
      • Matías-Guiu J
      • Gómez-Iglesias P
      • Porta-Etessam J
      • Pytel V
      • Matias-Guiu JA.
      Anti-CD20 and COVID-19 in multiple sclerosis and related disorders: a case series of 60 patients from Madrid, Spain.
      . Although there have been few mechanistic studies, preliminary data show that such individuals generate striking T-cell immune responses against SARS-CoV-2 peptide pools
      • Schwarzkopf S
      • Krawczyk A
      • Knop D
      • Klump H
      • Heinold A
      • Heinemann FM
      • Thümmler L
      • Temme C
      • Breyer M
      • Witzke O
      • Dittmer U
      • Lenz V
      • Horn PA
      • Lindemann M.
      Cellular immunity in COVID-19 Convalescents with PCR-confirmed infection but with undetectable SARS-CoV-2-specific IgG.
      . SARS-CoV-2 specific T cell immune responses but not neutralising antibodies are associated with reduced disease severity suggesting the immune system may have considerable redundancy or compensation following COVID-19
      • Rydyznski Moderbacher C
      • Ramirez SI
      • Dan JM
      • Grifoni A
      • Hastie KM
      • Weiskopf D
      • Belanger S
      • Abbott RK
      • Kim C
      • Choi J
      • Kato Y
      • Crotty EG
      • Kim C
      • Rawlings SA
      • Mateus J
      • Tse LPV
      • Frazier A
      • Baric R
      • Peters B
      • Greenbaum J
      • Ollmann Saphire E
      • Smith DM
      • Sette A
      • Crotty S.
      Antigen-specific adaptive immunity to SARS-CoV-2 in acute COVID-19 and associations with age and disease severity.
      . It is plausible that mucosal immunity, memory B-cells, or other classes of antibody may also play a significant role in protection, although direct evidence is lacking
      • Russell MW
      • Moldoveanu Z
      • Ogra PL
      • Mestecky J.
      Mucosal immunity in COVID-19: a neglected but critical aspect of SARS-CoV-2 infection.
      .
      We examined datasets from four UK laboratories and identified a subset of patients with proven SARS-CoV-2 infection, defined as laboratory detection of RNA, in the first wave of the pandemic between March and May 2020, but with negative serology results in June and July. SARS-CoV-2 RNA test results (PCR or other nucleic acid amplification technology) between August 2020 and January 2021 were reviewed to identify patients with likely reinfection in the second wave of the UK pandemic. Repeat positive results within 90 days were discounted. A comparator group of patients with no evidence of infection in the first wave – i.e. negative serology with either a negative or no RNA assay performed - was used to calculate the relative risk of infection in those with and without prior infection. A second comparator group was also examined, who were RNA-positive and antibody-positive in the first wave. A significant proportion of the patients were healthcare workers, who were offered serology as part of a national policy. We terminated the study at the end of January, as we judged that the national vaccination rollout might interfere with the reliability of results thereafter.
      The results are summarised in Table 1. We identified 224 RNA-positive antibody-negative patients in the first wave, with two laboratory-confirmed reinfections in the second wave (0.89%), compared to 2054 second-wave infections in the 47139 patients with previous negative serology and either no RNA result, or a negative RNA result (4.36%.) This implies a significantly reduced risk of reinfection (relative risk 0.20, 95% CI 0.05 to 0.81) in those with prior SARS-CoV-2 infection but without detectable antibodies, compared to those with no previous evidence of infection. We also found 2087 RNA-positive antibody positive patients in the first wave, with 18 reinfections (0.86%) – this was similar to the proportion in the RNA-positive antibody-negative patients (relative risk 1.04).
      Table 1Numbers of patients and SARS-CoV-2 (re)infections identified in the participating laboratory datasets.
      SWLPNWLPNCLEKCHTotalSignificance; 95% confidence intervals
      Test group: Confirmed infection, serology negative in first waveRNA-positive, Antibody negative in first wave98752823224
      Reinfected in second wave11002
      Proportion reinfected1.02%1.33%0%0%0.89%
      Comparator Group 1:

      No laboratory evidence of infection in first wave
      RNA negative or not tested; Antibody negative in first wave23289638910138732347139
      Infected in second wave6395624434102054
      Proportion infected2.74%8.80%4.37%5.60%4.36%
      Relative risk0.370.15000.20p = 0.02; CI = 0.05 to 0.81
      Comparator Group 2:

      Confirmed infection, serology positive in first wave
      RNA positive, Antibody positive in first wave8523113805442087
      Reinfected in second wave580518
      Proportion reinfected0.59%2.57%0%0.92%0.86%
      Relative risk1.740.52*01.04p = 0.96; CI = 0.24 to 4.43
      SWLP: South West London Pathology
      NWLP: North West London Pathology
      NCLE: Newcastle-upon-Tyne Laboratories
      KCH: King's College Hospital Laboratory
      * Relative risk undefined (0/0)
      Our results indicate that antibodies (as detected by routine laboratory assays) are not essential for protection against reinfection. To our knowledge this is a novel observation, though it is supported by a recent report that immunity to SARS-CoV-2 in patients without antibodies can occur if there is a significant T cell immune response
      • Schwarzkopf S
      • Krawczyk A
      • Knop D
      • Klump H
      • Heinold A
      • Heinemann FM
      • Thümmler L
      • Temme C
      • Breyer M
      • Witzke O
      • Dittmer U
      • Lenz V
      • Horn PA
      • Lindemann M.
      Cellular immunity in COVID-19 Convalescents with PCR-confirmed infection but with undetectable SARS-CoV-2-specific IgG.
      . IgG memory B cells against SARS-CoV-2 increase and exhibit greater affinity maturation over time despite a decline in serum antibody titres
      • Dan JM
      • Mateus J
      • Kato Y
      • Hastie KM
      • Yu ED
      • Faliti CE
      • Grifoni A
      • Ramirez SI
      • Haupt S
      • Frazier A
      • Nakao C
      • Rayaprolu V
      • Rawlings SA
      • Peters B
      • Krammer F
      • Simon V
      • Saphire EO
      • Smith DM
      • Weiskopf D
      • Sette A
      • Crotty S.
      Immunological memory to SARS-CoV-2 assessed for up to 8 months after infection.
      ,

      Hartley GE, Edwards ESJ, Aui PM, Varese N, Stojanovic S, McMahon J, Peleg AY, Boo I, Drummer HE, Hogarth PM, O'Hehir RE, van Zelm MC. Rapid generation of durable B cell memory to SARS-CoV-2 spike and nucleocapsid proteins in COVID-19 and convalescence. Sci Immunol. 2020 Dec 22;5(54):eabf8891. doi: 10.1126/sciimmunol.abf8891

      . This is consistent with the known development of the immune response: the loss of antibody may reflect not so much waning immunity but rather standard contraction of immune responses following SARS-CoV-2 infection, with development of antigen specific memory B cells. In addition, mucosal IgA or IgG may explain some of the protective effect we have observed. Furthermore, given the long incubation and slow onset of severe disease in SARS-CoV-2 infection, it is biologically plausible that the 2-3 day response time of antigen specific memory T- or B-cells is sufficient to protect against reinfection independently of circulating antibody, as is seen with Hepatitis B vaccination
      • Rosado MM
      • Scarsella M
      • Pandolfi E
      • Cascioli S
      • Giorda E
      • Chionne P
      • Madonne E
      • Gesualdo F
      • Romano M
      • Ausiello CM
      • Rapicetta M
      • Zanetti AR
      • Tozzi A
      • Carsetti R.
      Switched memory B cells maintain specific memory independently of serum antibodies: the hepatitis B example.
      The principal limitation of this study is that it is a retrospective pragmatic review of pooled clinical laboratory datasets. As such, SARS-CoV-2 RNA testing in many individuals will have been event-driven, rather than routine screening, and some cases of SARS-CoV-2 infection may have been missed, for example if asymptomatic. Furthermore, the criteria for seropositivity were set by the assay manufacturers; it is possible that some patients had specific antibody below the limit of assay detection, that nonetheless contributed to protection. Due to the deployment of different assays across laboratories, we were unable to examine the relationship between antibody index and risk of reinfection. The comparator group, of necessity, may have included some patients who also had antibody-negative infection in the first wave but who did not have an RNA assay performed. However, any such missed cases would have tended to reduce the apparent difference between the two groups, which increases confidence in our findings. We cannot exclude the possibility that positive test results may have influenced individual behavior, potentially increasing the risk of (re)infection or making seropositive individuals reluctant to come forward for further testing if they developed COVID-19 symptoms. However a recent Danish study showed no difference in protection from reinfection in health care workers tested regularly for SARS-CoV-2 infection, compared to other population groups
      • Holm Hansen Christian
      • Michlmayr Daniela
      • Gubbels Sophie Madeleine
      • Mølbak Kåre
      • Ethelberg Steen
      Assessment of protection against reinfection with SARS-CoV-2 among 4 million PCR-tested individuals in Denmark in 2020: a population-level observational study.
      . Finally, given the evolving epidemiology of the SARS-CoV-2 Pandemic, and the continual emergence of new strains, we can only say with confidence that our results apply to the situation in the UK up to the end of January 2021.
      In conclusion, our results add to the emerging evidence that detectable serum antibody may be an incomplete marker of protection against reinfection. This could have implications for public health and policy-making, for example if using seroprevalence data to assess population immunity, or if serum antibody levels were to be taken as official evidence of immunity – a minority of truly immune patients have no detectable antibody and could be disadvantaged as a result. Our findings highlight the need for further studies of immune correlates of protection from infection with SARS-CoV-2, which may in turn enhance development of effective vaccines and treatments. Serum antibody, whilst convenient to measure, is but a small window on the complex world of the human immune system.

      Acknowledgements

      We are grateful to the many clinical scientists and laboratory staff in our centres who evaluated and implemented the laboratory assays that enabled this study to be carried out.
      This study did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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        • Chionne P
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        • Romano M
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        • Carsetti R.
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