People who have been infected with the COVID-19 virus have immune memory to protect against reinfection for at least eight months

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Australian researchers have revealed – for the first time – that people who have been infected with the COVID-19 virus have immune memory to protect against reinfection for at least eight months.

The research is the strongest evidence for the likelihood that vaccines against the virus, SARS-CoV-2, will work for long periods. Previously, many studies have shown that the first wave of antibodies to coronavirus wane after the first few months, raising concerns that people may lose immunity quickly.

This new work allays these concerns.

The study is the result of a multi-center collaboration led by Associate Professor Menno van Zelm, from the Monash University Department of Immunology and Pathology, with the Alfred Research Alliance between Monash University, The Alfred hospital and the Burnet Institute, and published today in the prestigious journal, Science Immunology.

The publication reveals the discovery that specific cells within the immune system called memory B cells, “remembers” infection by the virus, and if challenged again, through re-exposure to the virus, triggers a protective immune response through rapid production of protective antibodies.

The researchers recruited a cohort of 25 COVID-19 patients and took 36 blood samples from them from Day 4 post infection to Day 242 post infection.

As with other studies – looking only at the antibody response – the researchers found that antibodies against the virus started to drop off after 20 days post infection.

However – importantly – all patients continued to have memory B cells that recognized one of two components of the SARS-CoV-2 virus, the spike and nucleocapsid proteins. These virus-specific memory B cells were stably present as far as eight months after infection.

According to Associate Professor van Zelm, the results give hope to the efficacy of any vaccine against the virus and also explains why there have been so few examples of genuine reinfection across the millions of those who have tested positive for the virus globally.

“These results are important because they show, definitively, that patients infected with the COVID-19 virus do in fact retain immunity against the virus and the disease,” he said.

“This has been a black cloud hanging over the potential protection that could be provided by any COVID-19 vaccine and gives real hope that, once a vaccine or vaccines are developed, they will provide long-term protection.”


The rapidly spreading severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) betacoronavirus has infected millions of people and killed hundreds of thousands worldwide in 2020. Infection causes the coronavirus disease 2019 (COVID-19), which ranges in presentation from asymptomatic to fatal.

The vast majority of infected individuals experience mild symptoms that do not require hospitalization (Wu and McGoogan, 2020). It is critically important to understand if SARS-CoV-2-infected individuals who recover from mild disease develop functional immune memory cells capable of protection from subsequent SARS-CoV-2 infections, thereby reducing transmission and COVID-19 disease.

Immunological memory is primarily mediated by cells of the adaptive immune system. In response to most acute viral infections, B and T cells that can bind viral proteins through their antigen receptors and become activated, expand, differentiate, and begin secreting effector molecules to help control the infection.

Upon resolution of infection, approximately 90% of these virus-specific “effector cells” die, whereas 10% persist as long-lived “memory” cells (Ruterbusch et al., 2020). Immune memory cells can produce a continuous supply of effector molecules, as seen with long-lived antibody-secreting plasma cells (LLPCs).

In most cases, however, quiescent memory lymphocytes are strategically positioned to rapidly reactivate in response to re-infection and execute effector programs imprinted upon them during the primary response. Upon re-infection, pathogen-specific memory B cells (MBCs) that express receptors associated with antigen experience and the transcription factor T-bet rapidly proliferate and differentiate into protective immunoglobulin (Ig)G+ antibody-secreting plasmablasts (PBs) (Kim et al., 2019; Knox et al., 2019; Nellore et al., 2019).

Reactivated T-bet-expressing memory CD4+ T cells proliferate, “help” activate MBCs, and secrete cytokines (including interferon [IFN]-γ) to activate innate cells (Ruterbusch et al., 2020). Meanwhile, memory CD8+ T cells also secrete cytokines and kill virus-infected cells directly through the delivery of cytolytic molecules (Schmidt and Varga, 2018).

These quantitatively and qualitatively enhanced virus-specific memory populations coordinate to quickly clear the virus, thereby preventing disease and reducing the chance of transmission. It is therefore critical to assess the full cadre of SARS-CoV-2-specific immune memory responses to determine whether mild infection induces a lasting, multilayered defense.

To infect cells and propagate, SARS-CoV-2 relies on the interaction between the receptor-binding domain (RBD) of its spike (S) protein and angiotensin converting enzyme 2 (ACE2) on host cells (Hoffmann et al., 2020). Multiple studies have shown that the majority of SARS-CoV-2-infected individuals produce S- and RBD-specific antibodies during the first 2 weeks of the primary response and that RBD-specific monoclonal antibodies can neutralize the virus in vitro and in vivo (Long et al., 2020; Robbiani et al., 2020; Shi et al., 2020). Therefore, RBD-specific antibodies would likely contribute to protection in response to reinfection if maintained in the plasma by LLPCs or rapidly expressed by MBCs.

We therefore assessed SARS-CoV-2-specific immune responses at 1 and 3 months post-symptom onset in individuals that had experienced mild COVID-19. We found that a multipotent SARS-CoV-2-specific immune memory response forms and is maintained in recovered individuals for the duration of our study.

Furthermore, persistent memory lymphocytes display hallmarks of protective antiviral immunity, including a numerically increased population of virus-specific memory B cells capable of expressing SARS-CoV-2 neutralizing antibodies.

Discussion
Although a vaccine is needed to safely reach herd immunity against SARS-CoV-2, understanding if natural infection induces viral-specific immunological memory that could influence transmission and disease severity is critical to controlling this pandemic. We therefore investigated whether individuals that experienced mild COVID-19 developed and sustained multilayered, functional immune memory.

We found that 3 months after mildly symptomatic COVID-19, recovered individuals had formed an expanded arsenal of SARS-CoV-2-specific immune memory cells that exhibited protective antiviral functions. Recovered individuals had increased neutralizing antibodies, IgG+ classical MBCs with BCRs that formed neutralizing antibodies, Th1 cytokine-producing CXCR5+ circulating Tfh and CXCR5− non-Tfh cells, proliferating CXCR3+ CD4+ memory cells, and IFN-γ-producing CD8+ T cells.

These components of immune memory have all been associated with protection from other viruses in humans (Ahmed and Gray, 1996; Amanna et al., 2007; Morita et al., 2011). Together, these data demonstrate that all of the recovered individuals in our cohort formed a multifaceted defense, which suggests attenuated virus vaccines are likely to be similarly successful in eliciting a functional immune memory response.

Sustained production of neutralizing IgG+ virus-specific antibodies is a frequent correlate of protection from viral infection (Amanna et al., 2007). Some studies examining the longevity of the antibody response to coronaviruses have suggested that antibodies wane rapidly (Seow et al., 2020; Tang et al., 2011; Wu et al., 2007).

Our study, as well as other recent studies, has examined memory time points when only LLPCs, and not short-lived PBs, are thought to be producing circulating antibodies. Together, we demonstrate elevated IgG+ RBD-specific plasma antibodies and neutralizing plasma are generated and maintained at elevated levels for at least 3 months post-SARS-CoV-2 infection (Isho et al., 2020; Perreault et al., 2020; Ripperger et al., 2020; Wajnberg et al., 2020).

What level of antibody is needed to contribute to protection and whether that level will be maintained in the long term will require studies of later time points. Although we detected IgA+ RBD-specific antibodies early, the levels had dropped significantly by 3 months, suggesting that the early IgA was derived from short-lived PBs. IgA-producing LLPCs either do not form or are sequestered in a tissue such that antibodies are not secreted into the blood.

Functional virus-specific memory B and T cells are key mediators of protective immune memory (Plotkin, 2010) and, unlike LLPCs, can be directly measured. Although previous studies have described the emergence of SARS-CoV-2-specific MBCs within a month of infection (Grifoni et al., 2020; Juno et al., 2020), (Wilson et al., 2020) we characterized SARS-CoV-2-specific MBCs at 1 and 3 months from symptom onset in the same individuals to determine whether this population is maintained.

Our study also analyzed additional attributes of MBCs that have been associated with anti-viral protection. Our study revealed a prominent population of RBD-specific IgG+CD27+CD21+ MBCs, which, in other infections, has been associated with GC derivation, rapid differentiation into antibody-secreting PBs upon re-exposure (Nellore et al., 2019), and effective antiviral responses (Rubtsova et al., 2013).

Not only was this population maintained from 1 to 3 months, the numbers increased significantly. We also found that these cells express BCRs capable of neutralizing SARS-CoV-2 when expressed as monoclonal antibodies. Approximately half of the antibodies derived from the IgG+ MBCs analyzed were able to neutralize the virus in vitro.

The BCRs all exhibited SHM, and the number of mutations from Visit 1 BCRs was similar to those previously reported from samples obtained at a similar time point (Robbiani et al., 2020). SHM modestly increased in both heavy and light chains from Visit 1 to Visit 2, which could reflect additional affinity maturation in the GC, but further analysis of a larger numbers of samples is needed. Taken together, these data suggest that upon re-exposure with SARS-CoV-2, these individuals will have MBCs that can rapidly generate neutralizing SARS-CoV-2 antibody titers and help control the infection.

Memory CD4+ T cells can help reactivate MBCs through their expression of key molecules associated with T-B interactions including CXCR5, ICOS, CD40L, and a variety of cytokines (Vinuesa et al., 2016). SARS-CoV-2-specific CD4+ memory T cells in recovered individuals in our cohort exhibited the capacity to express all of these molecules and to undergo robust proliferation upon re-exposure to spike protein. Notably, spike-specific CD4+ memory T cells from CoV2+ individuals rapidly displayed increased levels of ICOS and CD40L on CXCR5+ and CXCR5– cells after stimulation as well as expression of Th1- and Th17-associated cytokines.

These results are consistent with other recent reports of SARS-CoV-2-specific cTfh cells (Juno et al., 2020), although they detected a high frequency of Th17-like cTfh cells, which could be due to the earlier time point they were examining as Th17 cells can develop into Th1 cells late in an immune response (Lee et al., 2009). The expression of IFN-γ and IL-17 by cTfh cells is notable as these cytokines are associated with class switching to IgG and IgA isotypes, respectively (Hirota et al., 2013; Peng et al., 2002).

We also likely found cross-reactive memory B and T cells in healthy controls. In response to a viral infection, B cells that could recognize a viral antigen, but did not enter a GC, predominantly form IgM+ and IgD+ MBCs which tend to be low affinity and do not rapidly form PBs upon re-exposure, but might be able to recognize a variant of the viral protein (Weisel and Shlomchik, 2017).

Because we detected RBD-specific IgM+ and IgD+ MBCs in HCs, we hypothesize that some of these could be cross-reactive MBCs generated in response to a previous human coronavirus infection as recent work suggests (Song et al., 2020). We also found a small number of spike-responsive CD4+ memory T cells in HCs, which other groups have similarly attributed to cross-reactive memory T cells potentially associated with a previous human coronavirus infection (Braun et al., 2020; Grifoni et al., 2020; Sekine et al., 2020; Sette and Crotty, 2020). We also found SARS-CoV-2-specific CD8+ memory T cells in equal numbers in HCs and CoV2+ individuals.

This finding suggests cross-reactivity within a population of IFN-γ producing CD8+ memory cells in our HC samples and raises the possibility that our inability to interrogate CD8+ resident memory cells in the lungs could mask the true expansion of this compartment in CoV2+ individuals. How these cross-reactive cells contribute to the SARS-CoV-2 memory response in recovered individuals is difficult to discern without knowledge of each individual’s SARS-CoV-2-specific BCR and TCR repertoires prior to infection. However, we can conclude that mild COVID-19 induces an expanded population of MBCs and CD4+ memory T cells with markers of increased functionality in comparison with the cross-reactive pool present in our controls.

It is currently impossible to perform controlled SARS-CoV-2 reinfection studies to test the protective capacity of the SARS-CoV-2-specific memory lymphocytes we have described in humans. However, previous studies of human coronaviruses have shown protection from homologous rechallenge that correlated with antibody titers (Callow et al., 1990).

Although studies of SARS-CoV-2 have confirmed rare second exposures months after the first, they suggest prior exposure can be protective (Abu-Raddad, 2020; To et al., 2020). Additional studies have supported this finding, including evidence from a fishing vessel where 85% of the crew became infected, yet 3 previously exposed individuals with neutralizing antibodies did not get sick (Addetia et al., 2020).

More recently, during an outbreak at an overnight camp, none of the attendees that were seropositive (16%) prior to attending the camp tested positive for infection, whereas 91% of the remaining susceptible population tested positive for infection (Pray, 2020).

Animal studies provide additional support, as macaques infected with SARS-CoV-2 were protected from rechallenge (Chandrashekar, 2020). Although these studies support the role of immune memory contributing to protection from SARS-CoV-2 re-exposure, future studies will require data on the SARS-CoV-2-specific immune memory compartment prior to re-exposure to assess a correlation to protection.

It is also important to note that different levels of severity of COVID-19 could be associated with different levels of immune memory and subsequent immune protection. We focused on the immune memory response to mild COVID-19, but whether similar memory populations form after severe COVID-19 is still unclear.

In one largely histological study of post-mortem tissues from patients that succumbed to severe COVID-19, the lack of GC formation or the generation of CD4+ Tfh lymphocytes required for an optimal immune memory response suggested that forming immune memory could be difficult (Kaneko et al., 2020).

However, as these patients died of acute disease, it is impossible to determine if germinal centers were transiently disrupted due to acute inflammation as has been seen in other highly inflammatory diseases like malaria (Keitany, 2016). Although additional studies are needed to determine how long memory to SARS-CoV-2 infection lasts, our work suggests that mild COVID-19 induces persistent, multifaceted immune memory.

These functional antiviral memory lymphocytes are poised for a coordinated response to SARS-CoV-2 re-exposure that could contribute to immunity and help to curtail the pandemic.

reference link: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7682481/


More information: Rapid generation of durable B cell memory to SARS-CoV-2 spike and nucleocapsid proteins in COVID-19 and convalescence, Science Immunology  22 Dec 2020: Vol. 5, Issue 54, eabf8891, DOI: 10.1126/sciimmunol.abf8891 , immunology.sciencemag.org/content/5/54/eabf8891

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