The ongoing COVID-19 pandemic has been spreading rapidly, causing almost a million deaths from nearly 33 million reported infections. Scientists are eager to identify risk factors for increased mortality in this condition. A recent study by researchers at the University of Miami, Miami VA Healthcare System and Express Gene and published on the preprint server medRxiv* in September 2020 reports on the prognostic significance of the anti-N Immunoglobulin G (IgG) antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
The SARS-CoV-2 virus is a single-stranded RNA virus belonging to the Betacoronavirus family. It has four key structural proteins of which the nucleocapsid protein or N protein is one. 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.
What Role Does the N Protein Play?
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.
Neutralizing vs. Non-Neutralizing Antibody
Most neutralizing antibodies known to inhibit infection by SARS-CoV-2 block the binding of the viral spike protein with the ACE2 receptor on the host cell. There are, however, other modes of antibody action, some of which are deleterious, like the notorious antibody-dependent enhancement (ADE) phenomenon.
ADE occurs when non-neutralizing antibody attached to immune cells called macrophages and cytotoxic T cells mediates the binding of the virus 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.
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.
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 non-neutralizing antibodies present early in the disease, leading to ADE and precipitating a ‘cytokine storm’ as well as higher levels of infection.
Are Anti-SARS-CoV-2-N IgGs Related to ADE?
The current study sought to explore the possibility that anti-N IgG was acting in this manner, leading to ADE and increased viremia in COVID-19. 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 researchers 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 study included ~250 patients. The mean age was 63 years. About a third were admitted to the ICU. Just over a quarter died in hospital.
The researchers 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.
After adjusting for confounding factors, they found that the odds of ICU admission were higher if they presented with breathlessness. Diabetic patients 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.
Serum IgG analysis
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.
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.
RT PCR Analysis
The researchers 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.
Risk Factors for ICU Admission
The study, therefore, indicates 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.
Association with Anti-N IgG
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. However, viremia was not found to be present in hospitalized COVID-19 patients, suggesting the virus is retained in the lymphatic system.
The researchers say this study is “the first to report an association of high concentration of IgG against the N protein with poor outcome in COVID19.” Earlier studies have shown that total IgGs are at detectable levels even in the first week of the disease. However, the current paper reports a threefold risk of ICU admission when the patient has high anti-N IgG titers.
Mechanisms of Deleterious Anti-N IgG Activity
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. 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.”
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.
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.
Still, 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.
Implications and Future Directions
The current study 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 researchers call 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 concludes that subject to validation, “During the initial assessment of patients with COVID19, IgG targeting N-protein of SARS-CoV2 should be included among the measures.”
medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.