The onset of the coronavirus disease 2019 (COVID-19) brought about intensive research on the biological characteristics of the pathogen responsible, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The immune response mediated by neutralizing antibodies, elicited by the virus or by vaccines, is of paramount importance in determining the type of intervention necessary to achieve population immunity, which is agreed to be the sole hope of arresting the pandemic and emerging from its shadow.
Now, a new study recently released as a preprint bioRxiv* server reports the essential nature of Fc-mediated functionalities in determining the efficacy of such antibodies.
Antibodies have two functional parts, the antigen-binding fragment (Fab) and the crystallizable fragment (Fc). The former determines the specificity of the antibody and allows it to bind its cognate antigen, but the Fc mediates its biological activity via interaction with the Fc receptor (FcR).
FcRs are of various types, including gamma receptors (FcγRs) which engage the antibodies to produce various biological reactions against the particle bearing the antigen in question. These include complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity 79 (ADCC) and antibody-dependent cellular phagocytosis (ADCP).
As is obvious from the names, these activities result in the activation of the coagulation and inflammatory cascade components called the complement system, which results in the tagging and destruction of the pathogen. Additionally, the pathogen is attacked by immune cells based on the attached antibody, and either destroyed or engulfed by phagocytes.
The Fc region thus attracts and triggers the activation of natural killer cells (NK cells), monocytes and neutrophils, in order to promote the clearance of the infected cells and prevent the propagation of the virus.
Moreover, the FcR-mediated response also determines the cytokine profile produced by these cells following activation, promoting adaptive immunity against the pathogen.
Fc-mediated responses are also associated with harmful effects on the host, such as antibody-dependent enhancement (ADE) of disease. This has been reported with SARS-CoV and the respiratory syncytial virus (RSV).
The current study aimed to understand the role played by NAbs via their Fc portions in COVID-19 protection or pathology, given that these are being widely employed in the treatment of this condition.
It describes using an imaging modality called bioluminescence imaging (BLI) to explore the actual effects of treatment with NAbs in mice, both before and after challenging them with SARS-CoV-2. The virus was tagged with the nanoluciferase enzyme to allow for BLI.
Most NAbs attach to the viral spike protein, at the receptor-binding domain (RBD) of the S1 subunit, to block the spike's attachment to the host cell receptor, the angiotensin-converting enzyme 2 (ACE2). The N-terminal domain (NTD) of the S1 subunit, and the S2 subunit, are also targeted by NAbs.
The use of BLI allowed the spread of the virus to be traced throughout the body, to a variety of anatomical sites, identifying the tissues involved and visualizing the effects of various NAb treatment protocols.
What were the results?
The researchers tracked the spread of the SARS-CoV-2 virus from the nose to the lungs, and then to various organs via the systemic circulation, including the central nervous system.
The administration of potent NAbs led to the effective resolution of infection when given within 72 hours of infection, and was also capable of preventing it. The NAbs came from a convalescent COVID-19 patient, whose plasma showed robust neutralizing activity with high NAb levels.
The breadth of the B cell response showed multiple NAbs, of which the two most potent were CV3-1 and CV3-25. These were characterized for specificity by a variety of assays, and showed very high affinity for the viral spike, at low nanomolar concentrations. The latter also showed cross-reactivity with the SARS-CoV-2 S2 subunit, and the SARS-CoV spike.
Both antibodies blocked SARS-CoV-2 spike-expressing pseudovirus as well as the authentic virus, and prevented syncytia formation driven by the spike protein following infection. CV3-1 showed ten-fold higher activity than CV3-25.
In three different prophylactic regimens, the NAbs reduced the viral load in the lungs and its spread. When CV3-1 was given either alone or in combination with CV3-25, in an equimolar cocktail, almost 100% protection against infection was achieved.
Moreover, both these regimens prevented the induction of the hyperinflammatory cytokine storm, though this was not seen with CV3-25 pretreatment.
The efficacy of the NAbs was very high, with 50% protection being achieved with 0.75 mg/kg of CV3-1 against lethal infection. The therapeutic window within which maximal efficacy was present against lethal outcomes extended up to and including three days from infection with the virus.
At four days from the onset of infection, the administration of CV3-1 was unable to halt viral spread or neuroinvasion, with 75% of infected mice dying. These animals showed severe weight loss, high inflammatory cytokine levels, and viral loads in the tissues.
Absence of Fc effector function reduces efficacy
The researchers examined the role of FcR-mediated functions in the efficacy of these NAbs. They first created Leucine to Alanine (L234A/L235A, LALA) mutant versions of both NAbs.
These modifications would reduce interactions with the Fc receptors. The outcome was a decrease in ADCC and ADCP, but with intact antibody binding and neutralizing capacity for either NAb.
The mice treated with LALA-mutant NAbs showed changes such as loss of weight and non-survival following infection, with a high viral load in the tissues, indicating that “LALA mutations had indeed compromised the protective efficacy of both antibodies.”
The virus was able to replicate at a higher level, cause neuroinvasion and weight loss even after treatment with these NAbs, relative to the wildtype NAbs. Both mutants were found to be attached to neuronal surfaces.
LALA-NAbs fail to prevent infection
Following CV3-1 pretreatment, viral loads were undetectable at six days post-infection. However, with the LALA-CV3-1 mutant, pretreatment resulted in higher viral loads, indicating a failure of protection and rapidly extending infection of the lungs. All infected animals died by this time point despite pretreatment.
The same phenomenon was observed with CV3-25, where the viral loads in the tissues went down by a log, compared to those treated with the corresponding LALA mutant. In the latter, the viral loads were similar to those in untreated controls.
CV3-25 treatment led to a delay in the mice's death following infection, with only 25% of the mice being protected against a lethal outcome, unlike the 100% efficacy of CV3-1. However, this was reduced with the LALA mutations.
In fact, LALA-CV3-1 provided only less than 63% protection, and simultaneously, mice pretreated with either of the LALA mutants showed a significant increase in the inflammatory cytokine levels in the nose, lung and brain.
LALA-NAbs fail to treat infection
Similar findings were found following the treatment of established infection with the mutant NAbs. In fact, the viral loads in the lungs in the group of infected mice treated with CV3-1 LALA Nab were comparable to that of controls, but the cytokine levels were even higher, indicating the crucial need for Fc-effector functions to regulate cytokine release.
Neutrophils, monocytes and NK cells contributed to the antibody-dependent cure of mice from lethal SARS-CoV-2 infection and are critical for the success of SARS-CoV-2 NAb-directed therapies.”
Not only do these antibodies bring about direct neutralization, but they activate FcRs, along with monocytes, neutrophils and natural killer cells, to reduce the inflammatory cytokine response and thus mitigate systemic inflammatory damage.
What are the implications?
These findings imply that in the absence of Fc-effector functions, some of the virus particles failed to be cleared by the NAbs, and thus successfully established infection despite pretreatment. Immune cellular activity, involving monocytes, neutrophils and NK cells, is thus essential to CV3-1-mediated viral elimination.
The current study also shows that Fc effector functions are vital during the use of NAbs for both prevention and treatment of SARS-CoV-2 infection, unlike earlier hypotheses.
The S2-targeting CV3-25 NAb could become more important, even though less potent, as new variants of the virus show more resistance-conferring mutations in the S1 subunit, sparing the S2 domain. This antibody does, in fact, neutralize the South African variant, in contrast to other NAbs that target the NTD or RBD.
Our data add to the growing body of evidence that suggest the absence of an antibody-dependent enhancement (ADE) mechanism with a protective rather than a pathogenic role for Fc effects during SARS-CoV-2 infection.”
Further investigations should be carried out to confirm this and to identify the FcRs targeted by NAbs in order to design more potent therapeutic and prophylactic molecules.
bioRxiv 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.