Antibody-Dependent Enhancement (ADE) Phenomenon Could Be Caused By SARS-COV-2 Antibody- Anti-N-Protein IgG, Increasing COVID-19 Severity

Antibody-Dependent Enhancement (ADE): Researchers from the University of Miami, Miami VA Healthcare System and Express Gene in a news study have identified the anti-N Immunoglobulin G (IgG) antibodies against SARS-CoV-2 as possibly being the  cause of the Antibody-Dependent Enhancement (ADE) phenomenon in patients experiencing COVID-19 severity. Furthermore the same antibodies could also act as non-neutralizing antibodies to the virus so that immune cells are activated but without viral destruction. This leads to the cascading release of excessive amounts of pro-inflammatory cytokines as well as the blockade of anti-inflammatory chemicals.


 
The study findings are published in a preprint server and are pending peer review. https://www.medrxiv.org/content/10.1101/2020.09.23.20197251v1
 
The novel SARS-CoV-2 coronavirus is a single-stranded RNA virus belonging to the Betacoronavirus family that has four key structural proteins of which the nucleocapsid protein or N protein is one.
 
Although the host immune response involves immunoglobulin G (IgG) developed against the various structural proteins, the anti-spike protein IgG is thought to be primarily responsible for neutralizing immunity and is, therefore the focus of many vaccine trials for COVID-19.
 
However the N protein is currently known to be required for the structure and function of viral RNA, being involved in the transcription of viral RNA as well as for assembly of viral particles, in addition to its RNA chaperone function. It comprises three regions, one repeating sequence which determines the length of its amino acids, one sumoylation motif, and one kinase-dependent phosphorylation-determining serine residue.
 
Typically most neutralizing antibodies are known to inhibit SARS-CoV-2 infection by blocking the binding of the viral spike protein with the ACE2 receptor on the host cell.
 
Alarmingly, there are some modes of antibody action that are harmful and damaging such as the antibody-dependent enhancement (ADE) phenomenon.
 
Antibody-dependent enhancement or ADE phenomenon occurs when non-neutralizing antibody attached to immune cells called macrophages and cytotoxic T cells, hence mediating the binding of the SARS-CoV-2 coronavirus to these host cells.
 
As a result, these cells are destroyed, weakening the immune response rather than strengthening it. The outcome is increased amounts of virus in the body and bloodstream.
 
Also another harmful route of antibody action on the host cell involves the binding of non-neutralizing antibodies to the virus so that immune cells are activated but without viral destruction. This leads to the cascading release of excessive amounts of pro-inflammatory cytokines as well as the blockade of anti-inflammatory chemicals, thus leading to a destructive immunological imbalance called the cytokine storm.
 
The researchers suspect that such processes may be at work in COVID-19 due to infections by cross-reacting strains of coronavirus in the past. In fact, the N protein of SARS-CoV-2 has a high degree of homology with other virulent coronaviruses. This agrees with the concept of a high level of cross-reacting n on-neutralizing antibodies present early in the disease, leading to ADE and precipitating a ‘cytokine storm’ as well as higher levels of infection.
 
The University of Miami study sought to explore the possibility that anti-N IgG was acting in this manner, leading to ADE and increased viremia in COVID-19.
 
Hence this should then be linked to higher chances of ICU admission, longer duration of hospitalization and ICU stay, and a higher death rate due to this illness.
 
The study team carried out a prospective study on patients confirmed to have COVID-19 by reverse transcriptase-polymerase chain reaction (RT PCR). They aimed to identify the risk factors for a worse outcome in these patients as well as the linkage between the serum anti-N IgG titer and clinical outcomes, as well as with viremia.
 
The research included about 400 patients. The mean age was 63 years. About a third was admitted to the ICU. Just over a quarter died in hospital.
 
The study team calculated a Charlson Comorbidity Index (CCI) from the chronic illnesses and age of each patient. They also looked at numerous other parameters, including clinical features and laboratory results.
 
Upon adjusting for confounding factors, the study team found that the odds of ICU admission were higher if they presented with breathlessness. Diabetic patients were also over two times the odds of ICU admission. ICU admission was associated with almost eight-fold higher odds of dying while in hospital. A higher CCI and raised IL6 titers were independently associated with this outcome.
 
Detailed serologic tests for anti-N antibodies were carried out on a hundred of these patients, almost equally male and female. They also measured the single to cut off (S/Co) ratio for all samples. They showed the presence of anti-N IgG in 55% at admission.
 
Interestingly anti-N protein IgG was positive in 55 (55%) patients at the time of admission. Further analysis showed that in comparison to race, CCI, lymphocyte counts, and the S/Co ratio, only the latter was associated with ICU admission. Again, only S/Co ratios over 1.5 were associated with hospitalization for over 16 days.
 
The study team also found that negative, low, and high IgG titers were associated with the presence of N protein, S protein, and ORF 1ab nucleotide sequences, respectively.
 
The research also indicated that males of advanced age with a higher CCI who developed COVID-19 were more likely to be hospitalized. The only independent predictor of ICU admission was dyspnea. Again, in-hospital mortality was linked with both higher CCI and higher IL-6 titers.
 
Significantly, COVID-19 patients who present with breathlessness due to hypoxemia have higher odds of high anti-N IgG titers, which are also linked to higher ICU admission risk and extended hospital stay.
 
Strangely, viremia was not found to be present in hospitalized COVID-19 patients, suggesting the virus is retained in the lymphatic system.
 
The study team says this research is “the first to report an association of high concentration of IgG against the N protein with poor outcome in COVID19.”
Although past studies have shown that total IgGs are at detectable levels even in the first week of the disease, this research findings show that a threefold risk of ICU admission when the patient has high anti-N IgG titers.
 
It is known that anti-S IgG does not cause ADE, which makes both vaccination and passive immunity directed at eliciting this antibody a worthwhile and safe exercise.
 
But on the other hand, “the high levels of non-neutralizing IgG for N protein might play a role as a Trojan Horse for SARS-CoV2 and facilitate virus entry to immune cells with attachment to fragment crystallizable (Fc) regions of lymphocytes, dendritic cells, macrophages, natural killer cells, and even platelets. The infected immune cells respond with cytokine production and autophagy.”
 
Also this could also be the reason for a higher rate of pulmonary embolism and thrombosis in severe COVID-19, secondary to platelet infection by the virus with secondary self-lysis leading to activation of platelet aggregation and clotting.
 
However another suggested mechanism involves infected cells with N protein expression in the cell wall. The anti-N IgG interacts with these particles on immune host cells like natural killer cells, neutrophils, and macrophages, triggering antibody-mediated cellular cytotoxicity.
 
Yet another route to the cytokine storm is the formation of immune complexes targeting the N protein. This elicits IL6 secretion from immune cells, which is known to be a potent stimulator of local and systemic inflammation.
 
The study team acknowledges that the research is limited by the uncertainty regarding the total non-neutralizing IgG and the failure to separately assess other non-neutralizing antibodies. The sample was too small to demonstrate a link between anti-N IgG and COVID-19 mortality.
 
The team calls for validation of these findings, as well as to elucidate the mechanism of pathogenicity of anti-N IgG in severe COVID-19, particularly relative to mortality in this condition. This may help to understand how ADE is involved in hypercoagulability via platelet activation, and thus to evolve better therapies.
 
The study recommends that titers of IgG targeting N-protein of SARS-CoV2 at admission is a prognostic factor for the clinical course of disease and should be measured in all patients with SARS-CoV2 infection.
 
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