Cell Human-derived protective neutralizing antibodies isolated from survivors of Crimean-Congo hemorrhagic fever virus infection

Written by Wangye Editor | xi In 2018, the WHO listed 10 viral diseases including Ebola, Marburg, MERS, and SARS as priority infectious pathogens, including Crimea -Crimean-Congo haemorrhagic fever virus (CCHFV).

CCHFV is a type of virus transmitted by ticks that can cause severe hemorrhagic fever symptoms.
The fatality rate of infection is usually 5%-30%, but in some outbreaks, it can even be as high as 80%.
It needs to be in a biosafety level 4 laboratory.
In operation.

CCHFV belongs to the Nairoviridae (Nairoviridae) under the Bunyavirales and is a segmented negative-strand RNA virus.

CCHFV is usually transmitted by the bite of the virus-carrying hyalomma (Hyalomma) or by contact with livestock contaminated by the virus.
There have also been reports of nosocomial infections.

The virus has spread to many countries in Europe, Asia and Africa and formed a local epidemic, becoming the most widespread tick-borne virus.

There is currently no specific therapeutic drug.

The genetic component of CCHFV is divided into three segments (S, L, M), which encode nucleoprotein, glycoprotein precursor, and RNA-dependent RNA polymerase, respectively.

According to the phylogenetic relationship of the M-segment sequence, CCHFV is divided into seven branches (clade I-VII).

The glycoprotein precursor is transcribed and processed to produce two structural proteins Gn and Gc on the surface of the viral envelope, and three secreted proteins (GP38, GP85, GP160), among which Gn is responsible for the adhesion of viral particles to recipient cells , Gc mediates membrane fusion.

Similar to other Bunyaviruses, it is currently believed that Gc of the genus Neirovirus is also a type II viral envelope protein, that is, a single transmembrane protein with the C-terminus in the membrane.

Studies have found that the absence of antibodies in the serum of CCHFV-infected patients is closely related to the mortality of patients, suggesting that antibodies play a role in protecting infected persons from death.

But currently only mouse-derived antibodies have been published, targeting Gc, Gn and GP38 of CCHFV, respectively.

According to recent studies in other virulent viruses, such as Ebola virus, neutralizing antibodies with therapeutic effects can be isolated from the blood of recovered patients, and they are still effective even when they are used when symptoms appear after infection.

Continuing this strategy, on June 1, 2021, Professor Kartik Chandran's research group from the Albert Einstein College of Medicine, in collaboration with Adimab, Mapp, and the collaborators of the US Army Institute of Infectious Diseases, published a paper entitled Protective neutralizing antibodies from in Cell.
A research article on human survivors of Crimean-Congo hemorrhagic fever reported for the first time that 361 human monoclonal antibodies targeting Gc and GP38 were found in four survivors of CCHFV infection.

Experiments have confirmed that the highly effective neutralizing antibodies target 6 conservative epitopes on Gc, and antibodies with different target epitopes can exert a preventive effect through synergistic action.

The high-efficiency antibody was further transformed into a bispecific antibody, and an antibody that can effectively protect mice from a lethal dose of CCHFV challenge was obtained.

The author first constructed the recombinant protein rGn/Gc as a bait, and performed flow sorting of a single memory B cell in the blood samples of four CCHFV recovered patients.
After sequencing, the monoclonal antibody was expressed heterologously in yeast engineering strains, and finally obtained 361 strains of antibodies.

Then, the researchers used biomembrane interference to detect the binding ability of all antibodies to rGn/Gc and Gc, and found that more than 80% of the antibodies can bind to both antigens, and some antibodies specifically bind to GP38, but all antibodies do not.
Gn.

Furthermore, the cross-binding ability of these antibodies to rGn/Gc derived from the three branches (Clade III, I, V) was tested, and it was found that most of the Gc-specific antibodies can simultaneously bind to the antigens derived from the three branches.
Seven GP38-specific monoclonal antibodies also have cross-reactions.

Next, by comparing all antibodies with 7 Fab fragments targeting different epitopes of Gc, the author confirmed that most of the antibodies target one of the six epitopes.

In order to confirm the specific epitope sequence targeted by the antibody, the authors performed epitope mapping experiments on the above-mentioned antibodies that have undergone epitope classification: using a yeast library expressing Gc mutants on the surface, three rounds of fluorescence-activated flow sorting were performed.
Sequencing of the Gc mutant finally determined the specific sequence information of the 6 sets of epitopes, and located these epitope information on the Gc structure simulation map (Figure 1).

Figure 1 The six groups of antibody epitope sequences and their positioning on the Gc structural model To determine the neutralizing effect of the monoclonal antibodies selected, the author constructed a virus-like particle containing all structural proteins and Nano-Glo luciferase reporter gene tecVLP can simulate the invasion of real viruses for the evaluation of small molecules and monoclonal antibodies.

With a constant antibody concentration (35 nM), the neutralizing effect of all antibodies against the branch IV Oman strain tecVLP was tested, and it was found that the antibody targeting the Gc fusion peptide and domain II showed the strongest neutralizing activity, and all GP38 specificity None of the monoclonal antibodies have neutralizing activity.

The author further tested the broad-spectrum neutralization ability of the more active antibodies, and determined the three antibodies ADI-37801 (epitope 1, targeting fusion peptide), ADI-36121 (epitope 3, targeting domain II) and ADI -36145 (epitope 6, targeting domain III) was used in subsequent experiments (Figure 2).

Figure 2 Summary of the neutralization effects of high-efficiency antibodies In response to the previous application of monoclonal antibodies for treatments that easily lead to escape mutations in strains, the author tested three candidate antibodies as a combination of synergistic antibody pairs before conducting animal experiments on antibodies may.

By mixing the antibodies of the non-competitive epitopes in an equimolar ratio, they tested their neutralizing effect on tecVLP from Oman and several other strains, and found that ADI-37801 can synergize with the other two antibodies.

Therefore, the author tested three kinds of antibodies and two combinations of antibodies.
A single injection of 50 mg/kg has the effect of preventing and protecting the mice.
The results show that all antibodies and combinations can be effective when injected 24 hours in advance.
Protect, but it fails to protect mice effectively when used after challenge.

In order to obtain a protective effect, the author combined the variable regions of the three antibodies to construct 4 bispecific antibodies.
After heterologous expression and purification, the neutralizing effects of these bispecific antibodies on different tecVLPs were tested.
A single injection of DVD-121-801 after 24 hours of poisoning can effectively protect the mice; the results also show that the construction of the bispecific antibody affects its protective effect, and the DVD-145-801 with the V region of the two groups of antibodies exchanged does not protect the mice.
Effect (Figure 3).

Figure 3 Bispecific antibody construction strategy and animal experiment results.
In this study, the author isolated hundreds of human CCHFV monoclonal antibodies from recovered patients for the first time.
Most of these antibodies target 6 conserved epitopes on Gc, some The antibody has strong neutralizing activity and preventive effect.
Through antibody modification, a bispecific antibody with therapeutic effect can be obtained, which is expected to be used in subsequent clinical development.

Of course, there are some problems in this article.
For example, the reason why none of the nearly 400 antibodies target Gn has yet to be determined.
The exact mechanism of high-efficiency neutralizing antibodies remains to be studied in structural biology.
The antibodies found in this study are in non-human primates.
And the effect in the human body still needs to be confirmed.

However, this strategy of directly isolating a single B cell from the plasma of recovered patients and quickly identifying candidate neutralizing antibodies through in vitro experiments can be extended to other virus prevention and control for which there is no effective treatment.

Original link: https://doi.
org/10.
1016/j.
cell.
2021.
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