key: cord-0866348-11zdc418
authors: Rieke, Gereon J; van Bremen, Kathrin; Bischoff, Jenny; To Vinh, Michael; Monin, Malte B; Schlabe, Stefan; Raabe, Jan; Kaiser, Kim M; Finnemann, Claudia; Odainic, Alexandru; Kudaliyanage, Anushka; Latz, Eicke; Strassburg, Christian P; Boesecke, Christoph; Schmidt, Susanne V; Krämer, Benjamin; Rockstroh, Jürgen K; Nattermann, Jacob
title: Induction of NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) against SARS-CoV-2 after natural infection is more potent than after vaccination
date: 2022-03-22
journal: J Infect Dis
DOI: 10.1093/infdis/jiac060
sha: 8143ea239429dca99f141aa50affb35b630d1826
doc_id: 866348
cord_uid: 11zdc418

We compared the ability of SARS-CoV2 Spike-specific antibodies to induce natural killer (NK) cell-mediated antibody dependent cellular cytotoxicity (ADCC) in patients with natural infection and vaccinated persons. Analyzing plasma samples from 39 COVID-19 patients and 11 vaccinated individuals, significant induction of ADCC could be observed over a period of more than three months in both vaccinated and recovered individuals. Although plasma antibody concentrations were lower in recovered patients, we found antibodies elicited by natural infection induced a significantly stronger ADCC response compared to those induced by vaccination, which may affect protection conferred by vaccination.

A c c e p t e d M a n u s c r i p t However, increasing data indicate that immunity declines over time after both natural infection [1] and vaccination [2] . In a recently published study, natural immunity was shown to confer longer-lasting and stronger protection against infection and symptomatic disease than immunity induced by two doses of the BioNTech/Pfizer mRNA BNT162b2 vaccine [3] .

The mechanisms underlying these differences remained unclear but it is tempting to speculate that a more pronounced immune response triggered by natural infection compared to vaccination-induced immune activation may play a role here.

In both post-infection patients and vaccinated individuals, SARS-CoV-2-specific antibodies are important in mediating immunity. In particular, neutralising antibodies that can directly block infection appear to be associated with protective immunity [4] . However, the development and persistence of neutralising antibodies seems to vary according to the degree of disease severity [5] . Beyond neutralisation, antibodies can also provide antiviral protection by recruiting complement and/or Fc receptors present on all immune cells. These extra-neutralising functions include the ability to activate antibody-dependent cellular cytotoxicity (ADCC) [6] . Natural killer (NK) cells, a heterogeneous lymphocyte subset, are a main mediator of ADCC. Here, binding of NK cell-expressed FcγRIII (CD16) to the Fc-part of an antibody bound to an antigen on a target cell induces NK cell activation and subsequent killing of the target cell. NK cell-mediated ADCC has been shown to contribute to both natural and vaccine-induced antiviral immunity [7] and recent studies suggest ADCC to also play a role in natural SARS-CoV-2 infection [8] .

A c c e p t e d M a n u s c r i p t 6 The extent to which mRNA-based COVID-19 vaccination can induce NK cell-mediated ADCC and whether this differs from natural infection is currently unclear and was therefore investigated in the present study.

A total of 39 patients with natural SARS-CoV2 infection (female gender 17/39 (44%); mean age 57 years (range: 25-83)) and 11 healthcare workers vaccinated with mRNA-1273 (ModernaTX, Inc) (female gender 5/11 (45%); mean age 39 years (27-60)) were enrolled into this study after written informed consent was obtained. All vaccinated donors had tested negative for SARS-CoV2 infection multiple times prior to study inclusion. In addition, all plasma samples from this group were tested negative for NP antibodies, further ruling out a history of SARS-CoV2 infection. COVID-19 patients were stratified according to the WHO Ordinal Scale for Clinical improvement with grade 2 to 4 being considered moderate disease and 5 to 8 severe disease.

In COVID-19 patients, blood samples were obtained between day 3 and 244 after onset of symptoms. 12 of these patients were sampled 2 times, once during active infection and once at the post COVID unit, making it a total of 51 samples. In the vaccine group blood samples were analyzed at four different timepoints: day 1-7, day 21-28, day 48-70, and day 105-127 after the first dose. All individuals received the second dose on day 28 after the first vaccination. As a control, historic samples collected before December 2019 were used. 

First, we analyzed plasma antibody levels in vaccinated persons at four different timepoints.

Significant anti-SARS-CoV-2 Spike IgG levels were detectable three to four weeks after the first vaccination, peaked around day 60, after the second dose was administered, and then decreased again ( Figure 1A In vaccinated individuals, robust induction of ADCC and IFN- production was observed three to four weeks after the first dose of vaccination and remained stable for more than 100 days ( Figure 1C ). Both plasma-induced NK cell cytotoxicity and IFN- production correlated positively with SARS-CoV-2 Spike-specific antibody concentrations ( Figure 1D ).

In COVID-19 patients highest plasma concentrations of anti-SARS-CoV-2 Spike levels were detected between weeks 2-4 after symptom onset. Over time, there was a gradual decrease in antibody levels, but significant antibody concentrations were still detectable after more than 100 days (Figure 2A ).

Covid-19 plasma samples obtained during the first few days after onset of symptoms did not trigger ADCC. Beginning after week 1, however, a significant induction of NK cell cytotoxicity and IFN- production was found, which reached its peak at around 10-20 days after first symptoms occurred. Over time the capacity of COVID-19 plasma to induce NK cell-mediated ADCC waned but was still visible in the majority of patients beyond day 100 (Figure 2A ).

severe disease compared to those with moderate COVID-19 in the first weeks after symptom onset. However, this was not statistically significant. No such differences were found in the samples analyzed at later time points ( Figure 2B ).

Of particular interest, however, was the comparison between the two groups. Here, we observed that plasma from recovered patients had lower antibody levels on average but was more effective in inducing NK cell-mediated ADCC and IFN- production than plasma from vaccinated individuals ( Figure 2C ). This difference was particularly prominent in samples collected more than 100 days after onset of symptoms or first vaccination.

Taken together, our data indicate a stronger and longer lasting induction of NK cell mediated

A c c e p t e d M a n u s c r i p t 10 Discussion:

In the present study, we investigated the extent and duration of NK cell-mediated ADCC induced by mRNA-based COVID-19 vaccination compared to natural infection.

Our data show that antibodies capable of mediating NK cell-mediated ADCC are induced in recovered patients as well as after vaccination, and that this can still be detected more than three months after the onset of disease or vaccination. Of particular interest was the observation that despite having similar antibody levels these antibodies were less effective in inducing ADCC in vaccinated compared to recovered individuals.

The underlying mechanisms are still unclear at the moment. However, it seems plausible that specific characteristics of the induced antibodies play an important role.

The effector function of antibodies is modulated by N-linked glycosylation in the Fc region of the antibody. In particular, the absence of nuclear fucose on the Fc-N glycan increases the binding affinity of IgG1-Fc to the FcγRIIIa on NK cells and results in enhanced ADCC activity [9, 10] . Thus, differences in fucosylation of vaccine-versus infection-induced antibodies would be a possible explanation here. Accordingly, it has been shown that high titers of antibodies with low fucosylation can be found especially in severe COVID-19 [8, 11] . Further studies are warranted to address this issue.

In the present study, plasma samples obtained from patients with severe disease in the first weeks after symptom onset tended to induce a stronger ADCC response than those from individuals with moderate COVID-19. Although these differences did not reach statistical significance, they are similar to results from a previous study that also found increased ADCC activity in severe disease [12] . The authors, therefore, discussed that the ADCC response might contribute to immunopathogenesis in COVID-19. However, a significant difference between patients with moderate and severe COVID-19 was only found between d21-30 after symptom onset. In addition, this study also showed that patients who survived A c c e p t e d M a n u s c r i p t 11 severe disease had higher ADCC activity than those who died, which might indicate a beneficial effect of ADCC. Indeed, several studies have shown that ADCC is important for an effective antiviral immune response [7] . Therefore, our finding of reduced triggering of ADCC following vaccination may contribute to the recent observation that vaccine-induced immunity provides less effective protection against infection and symptomatic disease than immunity induced by natural infection. However, it is likely that multiple mechanisms are relevant here.

In contrast to vaccination, the immune response after infection is not limited to the Spike protein. For example, in recovered patients we also found ADCC induction by NP-binding antibodies, whereas this was not the case in vaccinated individuals (Supplemental Figure   S2 ).

In our study, we compared the ability of antibodies induced by vaccination or infection to trigger NK cell-mediated ADCC. Therefore, we used NK cells from a healthy, non-vaccinated donor as effector cells to exclude a possible effect of qualitative or quantitative differences of the NK cell pool.

Recent work, however, indicates that alterations in lymphocyte functions may persist in convalescent COVID-19 patients [13] . We recently demonstrated severe COVID-19 to be associated with marked impairment of NK cell activity, which was still detectable more than 3

weeks after symptom onset [14] . Whether such an NK cell dysfunction persists in patients that recovered from COVID-19 and how this may affect the ability of NK cells to mediate ADCC is currently unclear and warrants further research.

In our study, we used antibody binding to plate bound spike protein as a surrogate assay.

Therefore, the question may arise whether our findings reflect the in vivo situation, since 

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Neutralizing antibody levels are highly predictive of immune protection from symptomatic SARS-CoV-2 infection

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Oligosaccharide Improves Binding to Human FcγRIII and Antibody-dependent Cellular Toxicity

High titers and low fucosylation of early human anti-SARS-CoV-2 IgG promote inflammation by alveolar macrophages

Antibody-dependent cellular cytotoxicity response to SARS-CoV-2 in COVID-19 patients

Alterations in T and B cell function persist in convalescent COVID-19 patients

Early IFN-α signatures and persistent dysfunction are distinguishing features of NK cells in severe COVID-19

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M a n u s c r i p t A c c e p t e d M a n u s c r i p t 13 A c c e p t e d M a n u s c r i p t