Potential risks of the new coronavirus vaccine: How terrible is the ADE effect?

Key points:

1. Antibody-dependent enhancement (ADE) effect mainly occurs in immune cells with Fc receptors. Evidence of ADE effect has been found in many viruses (including coronaviruses), which mainly manifests as enhanced viral infection ability.

2. The discovery of ADE phenomenon in in vitro experiments does not necessarily mean that it will affect clinical results.

3. Improving antibody quality is the key to reducing the risk of vaccine ADE.

Written by | Gene

Recently, the COVID-19 vaccine research and development in various countries has entered the Phase III clinical stage, and the safety of the COVID-19 (SARS-CoV-2) vaccine has once again entered the public eye. Many articles have mentioned that the ADE effect may be a potential risk of the COVID-19 vaccine.

What is the ADE effect? ​​ADE stands for Antibody-dependent enhancement, which means antibody-dependent enhancement. A more popular explanation is that when a virus infects cells, for some reason, the relevant antibodies already in the body will enhance the virus's ability to infect. In other words, after natural immunity or vaccination, when you come into contact with the relevant virus again, the antibodies produced in the body may enhance its ability to infect, eventually leading to a worsening of the disease.

So, how is ADE explained scientifically? Does the new coronavirus also have the ADE effect? ​​How can we avoid it? This article will introduce the ADE effect of the virus in depth, hoping to help everyone correctly understand scientific phenomena and scientific conclusions.

Discovery of antibody-dependent enhancement Antibodies were first discovered by German scientist Emil Adolf Von Behring and Japanese scientist Kitasato Shibasaburo. They found that injecting rabbit serum infected with tetanus bacteria into mice could protect mice from tetanus bacteria and tetanus toxin [1]. Subsequently, Behring injected inactivated diphtheria bacteria and diphtheria toxin into guinea pigs and found that guinea pig serum also had protection against diphtheria bacteria and diphtheria toxin [2]. Therefore, Behring believed that a protective substance called "antitoxin" would be produced in the serum of immunized animals, which could react with foreign antigens and take effect.

“Antitoxin” is what we later call antibody. In 1891, German scientist Paul Ehrlich first used the term “antibody” (antikörper)[3]. Later, scientists discovered that antibodies are mainly divided into five subtypes: IgA, IgD, IgE, IgG and IgM.

Antigen refers to a molecule on a pathogen that can be specifically recognized by immune cells. Each antigen can have one or more epitopes. Epitopes are more detailed, and they are chemical groups in antigen molecules that determine antigen specificity. Immune cells (or antibodies) interact with antigens mainly by recognizing epitopes, thereby triggering an immune response. (See the figure below)

In 1964, Australian scientist Royle Hawkes accidentally discovered in an experiment that in the presence of highly diluted chicken antibody serum, the infectivity of various viruses of the Flaviviridae family to chicken embryo fibroblasts increased[4]. This finding contradicted the understanding that "serum has a protective effect", and Hawkes began to doubt his own findings.

Three years later, Hawkes finally confirmed that serum could indeed enhance the infectivity of viruses, and further discovered that this phenomenon was related to IgG antibodies in serum[5]. Antibodies are the body's shield against viral invasion, but viruses can "use their shields as their own spears" and rely on antibodies to invade cells. This was the first time that humans recognized the antibody-dependent enhancement effect of viruses, but Hawkes was unable to explain the specific mechanism of this phenomenon at the time.

Now, the generalized ADE states that:

Some suboptimal antibodies can enhance the virus's ability to infect, or even assist the virus in entering cells that it was previously unable to enter, leading to massive viral replication or abnormal immune cell responses, ultimately aggravating the condition of the infected person and causing tissue pathological damage.

It was not until 1977 that Scott Halstead, a pioneer in the field of dengue and a famous virologist, linked severe dengue fever caused by dengue virus (DENV) in clinical practice to ADE - some infected people gained immunity to the dengue virus after recovery, but after a period of time, when these patients were infected with the dengue virus for the second time, their condition was more serious than the first time.

Dengue virus is divided into different serotypes (i.e., subspecies of the virus). Experiments have found that monkeys that are immune to types I, III, and IV, after being infected with type II virus, not only did not clear the dengue virus in their bodies, but the virus level was significantly higher than that of other monkeys. Halstead further found that dengue virus replicates faster in the peripheral blood white blood cells of immune monkeys or humans. Based on various evidences, Halstead concluded that ADE is related to white blood cells: in the presence of antibodies, the virus can replicate in large numbers in white blood cells [6-8].

Why does it happen in white blood cells? This starts with the steps of virus infection of cells. After entering the human body, the virus first binds to the surface receptors of human cells through its own membrane proteins, then enters the cell through membrane fusion or cell endocytosis, then releases genetic material, replicates and assembles, and finally releases viral "offspring" to continue infecting other cells.

The process of virus invading white blood cells is no exception. Halstead explained that ADE is mediated by Fc receptors (FcR) on the surface of white blood cells. After the Fab segment of the antibody recognizes and binds to the virus, the Fc segment of the antibody interacts with the Fc receptors on the surface of white blood cells (including monocytes, macrophages, B cells, neutrophils, etc.), causing the virus to adhere to the surface of white blood cells and promoting the endocytosis of the virus by white blood cells, which is equivalent to "letting the wolf into the house" and enhancing the virus's ability to infect. This is also the main mechanism of ADE at present.

What are the Fab and Fc segments of antibodies? A picture will show you how to do it.

Figure 1. Antibodies are immunoglobulin (Ig) molecules, and their basic structure is in the shape of a "Y". The two arms of the Y are the key to identifying foreign antigens, so they are also called fragment antigen binding, or Fab segments; the root of the Y is called the crystallizable fragment, or Fc segment, which is mainly responsible for regulating immune cell activity. In addition, the Fc segment is also related to ADE. | Author drawing

Subsequently, Malik Peiris, a famous virologist and former dean of the School of Public Health at the University of Hong Kong, clarified this mechanism through more detailed experimental evidence [9, 10]. Peiris found that in the process of West Nile virus (WNV, belonging to the Flaviviridae family) infecting macrophage cell lines, blocking the binding of specific Fc receptors on the surface of white blood cells with the Fc segment of antibodies can block the ADE effect of viral infection. Other researchers have also reached the same conclusion in experiments on dengue virus and yellow fever virus (YFV, belonging to the Flaviviridae family) [11, 12]. The Flaviviridae family became famous for ADE.

There is more than one ADE mechanism. In 1983, Malaysian virologist Jane Cardosa discovered another ADE mechanism in the Flaviviridae family. In the experiment, in the presence of IgM antibodies, the infectivity of West Nile virus to lymphoma cells was enhanced. However, as in the past, blocking the Fc receptor on the cell surface was no longer effective; however, if the binding of the antibody Fc segment to the type III complement receptor (CR3) on the cell surface was blocked, the virus's infectivity enhancement effect could be stopped [13].

Complement is a group of biologically active proteins in serum that can complement and assist specific antibodies and mainly mediate nonspecific immune and inflammatory responses. The complement system includes complement intrinsic components, complement regulatory components, and complement receptors (CRs).

This means that the ADE effect in the Cardosa experiment is mediated by complement receptors on the cell surface. After the Fab segment of the IgM antibody recognizes and binds to the virus, the conformation of the antibody changes, exposing the complement binding site of the Fc segment - originally, this is to activate the complement system and help fight the virus, but the flaw is exposed as soon as the complement system is activated - after the complement system is activated, the virus-antibody complex binds to the complement receptors on the target cell, sending the virus into the cell, further enhancing the infection.

This pathway is independent of Fc receptor-mediated ADE because Fc receptors are only expressed in immune cells, while complement receptors are expressed in a relatively wide range of cell types[14], which aggravates the virus's invasion of a wider range of cells.

At present, Fc receptor-mediated and complement receptor-mediated are the two most common mechanisms of ADE. In addition to Flaviviridae, scientists have also discovered ADE in many viruses of other virus families, and the mechanisms are not exactly the same.

ADE effect in coronaviruses The ADE effect in coronaviruses (CoV) was first discovered in 1980 [15]. The famous coronavirus expert Niels Petersen conducted an infection experiment with kittens using feline coronavirus to induce feline infectious peritonitis (FIP). In the experiment, he found that under natural conditions, kittens with positive antibodies to feline infectious peritonitis virus (FIPV)* developed the disease earlier and died faster than kittens with negative antibodies. In other words, kittens that were immune to FIPV had more severe disease after being infected.

*Note: FIPV is a type of feline coronavirus FCoV.

A year later, researchers confirmed that kittens that had been pre-injected with anti-FIPV serum or antibodies (called passive immunization in experiments) also developed disease and died earlier than kittens in the control group when infected with FIPV [16]. In 1990, researchers vaccinated kittens with FIPV (called active immunization in experiments), confirmed the detection of antibodies in the body, and then infected these kittens with FIPV, and obtained the same results [17]. At this point, the ADE phenomenon in the FIPV infection process was finally widely known.

It took another two years for researchers to discover the mechanism of the ADE effect of feline coronavirus. It turns out that certain anti-FIPV IgG antibodies can enhance the ability of FIPV to infect macrophages, and this process is related to Fc receptors[18]. Since then, more and more studies have been conducted on the ADE effect of FIPV.

Figure 2. Petersen and Tony, a kitten who recovered from FIPV infection[19].

In 2005, researchers discovered for the first time in experiments that antibodies against human SARS coronavirus (SARS-CoV) can enhance the infection of host cells by another SARS strain [20], and that in human B cells and macrophages, the ADE effect of SARS virus is associated with a specific type of Fc receptor (FcγRII), and blocking this receptor can block the occurrence of ADE [21, 22].

It is worth noting that the process of SARS-CoV infecting macrophages through ADE is not to aggravate the infection by simply replicating a large number of viruses (Figure 3A), but to interfere with various cytokine signals (Figure 3B), causing macrophages to be overburdened in the middle and late stages, abnormal activation, and increased secretion of inflammatory factors, ultimately causing acute inflammation and pathological damage to the body [23, 24].

Figure 3. Two ways in which suboptimal antibodies exacerbate coronavirus infection. Green represents antibodies, yellow represents cells, and the blue protrusions on the cell surface are Fc receptors. | Adapted from reference [25].

Another in vitro study on ADE in MERS coronavirus (MERS-CoV) infection found that some suboptimal antibodies can change the conformation of the spike protein after binding to the spike protein on the virus surface. As a result, not only can the virus still bind to the corresponding cell surface receptors, but the Fc segment of the antibody can also bind to the Fc receptor on the cell surface, making it easier for the virus to enter the cell[26]. This shows that if the antibodies induced during the initial infection are not ideal, it may also directly trigger the ADE effect.

Based on the evidence of SARS and MERS coronaviruses and clinical studies, some researchers have reasonably speculated that the novel coronavirus SARS-CoV-2 infection also has an ADE effect [27, 28]. A recent in vitro study (preprint) showed that the monoclonal antibody MW05 against SARS-CoV-2 may bind to a specific receptor (FcγRIIB) on the surface of target cells through the Fc segment, causing an ADE effect. The specific results still need to be further verified [29]. In addition, another preprint study showed that in critically ill patients infected with the new coronavirus, IgG antibodies may induce macrophages to produce a hyperinflammatory response, thereby damaging the integrity of the pulmonary endothelial cell barrier and inducing microvascular thrombosis [30].

What are “suboptimal” antibodies? The factors that determine whether an antibody will cause ADE mainly include: antibody specificity, titer, affinity, and antibody subtype[25].

There are different types of SARS vaccines. The vaccines targeting the spike protein (S protein) and the nucleocapsid protein (N protein) use different antigens and induce different specific antibodies. In a mouse experiment, the titers of specific antibodies induced by these two types of vaccines were similar after the mice were vaccinated. Subsequently, these mice were infected with SARS-CoV, and it was found that the vaccine encoding the N protein induced the mice to secrete more pro-inflammatory factors, and the lung infiltration of certain white blood cells in the mice was relatively increased, and the lung pathological changes were relatively more severe [31].

Similarly, in the monkey model, antibodies targeting different epitopes of the SARS-CoV spike protein induced different responses, some of which could provide good protection, while others were more likely to cause ADE effects[32].

Low antibody titers can also easily cause the ADE effect. For example, during SARS or MERS coronavirus infection, increasing the antibody titer can inhibit ADE and promote the occurrence of neutralization reactions [26, 33]. During the neutralization reaction, high-affinity antibodies can also provide better protection than low-affinity antibodies [34].

Antibodies with neutralizing effects are called neutralizing antibodies. Neutralization refers to the binding of the antibody Fab segment to the corresponding antigen epitope, blocking its receptor binding site or causing its conformational change, making it impossible for the antigen to enter the cell.

The affinity of an antibody, in layman's terms, refers to how firmly the antibody binds to the antigen.

In addition, different antibody subtypes have different functions in regulating immune cells through their Fc segments: IgM can more effectively activate the complement system and produce pro-inflammatory responses, while IgG regulates immune responses based on different Fc receptors on the cell surface. For example, during SARS-CoV infection, some types of Fc receptors (FcγRIIa and FcγRIIb) can mediate ADE, while others (FcγRI and FcγRIIIa) cannot[33]. Furthermore, different splicing forms (isoforms) of the same type of Fc receptor can also induce different ADE effects[35].

How to avoid ADE in vaccine development? In the development of the new crown vaccine, the key to reducing the risk of ADE lies in improving the quality of antibodies, mainly including the selection of antigen epitopes and adjuvants.

The selection of antigenic epitopes is particularly important. In the development of SARS vaccines, some vaccines could induce ADE effects or eosinophil-mediated immunopathological changes in mice or monkeys to a certain extent [20, 23, 36]. The reason for this may be that the quality (mainly titer) of antibodies induced by the dominant antigenic epitopes in the vaccine is not ideal.

Adjuvants are substances that are injected in advance or simultaneously with antigens. Adjuvants can effectively enhance the body's immune response to antigens, and can also change the type of immune response. Studies have shown that in aged mice, inactivated SARS vaccines enhanced with aluminum adjuvants can induce high titers of antibodies, but the antibody subtypes are not ideal. In addition, inappropriate adjuvants can also change the type of immune response, thereby affecting the immune response process and causing lung pathological changes[36].

In addition, the route of vaccination can also affect its effect. For the same SARS vaccine, recipients who were vaccinated via the nasal or intramuscular route had fewer lung pathological changes after viral infection[37]. Other studies have shown that using biological means to coat the surface of vaccine particles with a layer of shell, such as coating the surface of dengue vaccine particles with a calcium phosphate mineralized shell, can effectively avoid the occurrence of ADE without affecting its protective effect[38].

Starting from the mechanism of ADE, it is also possible to avoid risks in vaccine development. Since most ADE effects are mediated by Fc receptors on the cell surface, blocking specific Fc receptors on the cell surface can prevent the virus-antibody complex from binding to the Fc receptors, thereby preventing the ADE effect[39].

To achieve this process, specific antibodies against Fc receptors or small molecule inhibitors that inhibit the binding process are good choices. The former can be used as an immunosuppressant [40,41]. For example, intravenous immunoglobulin can improve the symptoms of severe COVID-19 patients in clinical practice [42, 43], but whether it is safe and effective on a large scale requires further study.

In short, blocking the binding of virus-antibody complexes to Fc receptors is also a means to prevent ADE from occurring. However, in addition to Fc receptors, ADE can still occur through other pathways mentioned above, such as complement mediation.

Therefore, when developing vaccines, it is necessary not only to ensure the induction of high-quality neutralizing antibodies, but most importantly, to try to choose vaccines that can induce strong cellular immunity. In fact, the body's clearance of viruses also depends on cellular immunity, because neutralizing antibodies can only work on viruses outside the cells, and are often powerless against "slippery fish" that have entered the cells. The virus will express its protein information on the surface of the infected cell, and the killer T cells can recognize this information, thereby launching an attack and killing the virus and the cells it infects together.

It is also important that the primary immunization (i.e. vaccination) not only induces antibodies, but also produces memory cells. The stronger the cellular immunity induced by the vaccine, the more killer T cells are activated and the more memory T cells are converted. In this way, the immune cells will function faster during the next viral infection, thereby effectively reducing the occurrence of ADE. Therefore, the choice of vaccine type is also crucial.

Conclusion Since the development of the COVID-19 vaccine, no clear evidence of ADE has been found in the published animal and clinical trial results. However, based on the experience of SARS and MERS vaccines, I believe that it is only a matter of time before the ADE effect is confirmed in very few monoclonal antibodies against the COVID-19 virus.

Although it was mentioned in the previous article that some studies have preliminarily shown that some monoclonal antibodies against the new coronavirus may have an ADE effect in vitro, the evidence is still insufficient. It is also important to note that there is often a large gap between in vitro experiments and in vivo conditions, and it is even further away from clinical manifestations. After the body is immunized with antigens, polyclonal antibody responses to multiple epitopes will appear. Even if a single antibody has an ADE effect, it is difficult to affect the neutralization of the serum.

Monoclonal antibodies are antibodies produced by a single B cell clone that target only a specific antigen epitope. Correspondingly, polyclonal antibodies are different antibodies that target multiple antigen epitopes.

In addition to vaccines, developing monoclonal antibodies and preparing antibody drugs is also a good option. Monoclonal antibodies have molecular precision and are easy to edit through genetic engineering. For example, using only the Fab segment of the antibody or modifying the Fc segment of the antibody through engineering (such as introducing mutations) can significantly improve safety[44].

Currently, there are hundreds of COVID-19 vaccines being developed by scientific research teams around the world, of which at least 30 have entered clinical trials (10 in China), the fastest of which has entered Phase III clinical trials, and the rest are being tested on animal models[45]. At the same time, the competition to develop monoclonal antibodies is also in full swing. The author believes that ADE will not become an obstacle in the development of COVID-19 vaccines.

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