Virus comes from the Latin term "poison", the latter term having been elaborated from the works of Paracelsus and Ambroise Paré, during the sixteenth century.
In 1957, André Lwoff from Pasteur Institute was the first to state the fundamental characteristics that make viruses original entities (1):
- viruses contain only one type of small size nucleic acid (DNA or RNA, from 2 kb to 200kb) which constitutes the viral genome,
- viruses reproduction occurs by genetic replication and not by binary fission, allowing them to rapidly evolve,
- viruses are absolute intracellular parasites since they cannot create energy such as ATP on their own, due to the lack of a Lipmann system. They can only reproduce themselves within living cells, hijacking the cellular machinery to their own benefits.
A virus protects its genetic material from host nucleases with a shell - thanks to the assembly of multiple copies of a few proteins associated with the viral genome - named capsid. Beside protection, capsids can also be involved in viral transport within the host, escape immune system thanks to their mutations or serve as primer for replication.
Depending on the virus type, certain viruses - orthomyxoviruses, paramyxoviruses, rhabdoviruses, filoviruses, coronaviruses, bunyaviruses, arenaviruses, and retroviruses - can embed their capsids into a biological membrane containing lipids and proteins: the envelope.
Glycoproteins of viral envelopes: targets for innovative antiviral therapies
Most viral proteins located in the envelope are glycoproteins assembled as dimers or trimers, and are inserted into the lipid membrane in the same way as cellular transmembrane proteins.
The infectious particle, or virion, is the sum of viral capsid, genome and the envelope. No envelope, no infection. In fact, enveloped viruses enter cells by inducing fusion of viral and cellular membranes, process controlled by specialized membrane-fusion protein complexes.
Thanks to the advances in electron microscopy, we identify these envelope glycoproteins as “spikes” on the surface of purified virion. X-ray crystallography studies have now proven that three distinct classes of viral fusogenic membrane glycoproteins (FMG) - or fusogens - are represented within the viral kingdom:
- Class I fusogens are characterized by being synthesized as inactive precursors that require proteolytic processing to become fusion-competent:
- gp41 from HIV (Retroviridae),
- spike protein from COVID-19 (Coronaviridae),
- haemaglutinin from Influenza virus (Orthomyxoviridae)
- GP from Ebola virus (Filoviridae)
- F from Sendai virus (Paramyxoviridae)
- Class II fusion glycoproteins are derived from longer polyprotein precursors that are proteolytically processed during biosynthesis. The class II fusion proteins form icosahedral scaffolds of protein dimers at the viral surface:
- E1 from Semliki Forest virus (Alphaviridae)
- E from Dengue virus (Flaviviridae)
- Class III fusion proteins are not proteolytically processed:
- G from Vesicular stomatitis virus (Rhabdoviridae)
- gp64 from Baculovirus (Baculoviridae)
- gB from Herpes simplex virus (Herpesviridae)
Blocking attachment to the host cell and preventing penetration of the host cell membrane have then rapidly become the challenges of the scientific community in order to develop antiviral therapies.
Among all possible treatments, vaccination (active immunotherapy) and monoclonal antibodies injection (passive immunotherapy) are potential solutions to tackle virus threat. Both strategies use the potential of therapeutic neutralizing antibodies (NAbs) recognizing glycoproteins on the virion surface.
The risks of in-vivo neutralizing antibody generation
If the generation of in-vivo neutralizing antibody through vaccination seems a pragmatic and natural solution, the approach nevertheless presents several risks.
The antibody-dependant enhancement (ADE) effect
Certain viruses can utilize pre-existing antibodies, which potentially neutralize their capability of infecting through their natural receptor-ligand route, and bind to the FcR on phagocytes, to facilitate infection of their target cells. These viruses usually can replicate in the macrophages or monocytes, and may use them as reservoirs in order to reach other body tissues. The generation of non-neutralizing, sub-neutralizing antibodies or even neutralizing antibodies but at low circulating concentrations, may cause increased viral infection of monocytes or macrophages via FcγRIIa-mediated endocytosis, resulting in more severe disease (2).
Some examples have already been described such as yellow fever virus (3), dengue virus (4, 5, 6), human immunodeficiency virus type 1 (HIV-1) (7), respiratory syncytial virus (RSV) (8) or Ebola virus (9). Previous infections to coronavirus strains may increase COVID-19 severity.
The decline of circulating neutralizing antibodies
In the context of SARS-CoV-2 for instance, neutralizing activity appears to be transient with a decline, or even a loss, of the NAb titers associated with a decrease in systemic antibody levels, and may suggest that vaccine boosters are required to provide long-lasting protection (10, 11).
The technical challenges of Neutralizing antibody generation
Compared to the vaccine approach, the injection of neutralizing antibodies requires larger quantities of biological material but above all to overcome several technical difficulties to obtain the rare antibody of interest. An enormous workload to find the needle in the haystack.
Isolating rare B cell populations for effector mAbs identification
Whether starting from antiserum patients recovering from infection, de novo monoclonal antibody generation with non-immune phage display, or animal immunization anti-serum, a crucial problem remains in all cases.
How to isolate the rare clone of interest from the large population of B-secreting clones? Even if we stumble upon many antigen binders, how many monoclonals are actually effectors?
The following is the comparison of three different approaches for the generation of neutralizing antibodies for SARS-CoV.
Monoclonal antibodies generation from human naive phage display library
Starting from two human non-immune single-chain variable region fragment (scFv) libraries (2.7*10^10) constructed from B cells of 57 unimmunized donors, the team of Jianhua Sui faced the following workload. After three rounds of selection, a total of 288 clones were screened for specific binding by ELISA. Eight unique anti-S1 scFvs were identified by sequencing each one of the individual clones. Further, the eight ScFv tagged with His-6 were expressed in E. coli and purified by immobilized metal affinity chromatography. Amongst the eight scFvs, only one clone showed neutralization activity (12).
Monoclonal antibodies generation from microfluidic-based technique with patients starting material
Regarding SARS-CoV-2, and starting from patient’s material this time, the team of Yunlong Cao identified over 8,550 antigen-binding B cell clonotypes expressing immunoglobulin G1 (IgG1) antibodies from 60 convalescent patients. Cells are assigned to the same clonotype if they have identical heavy and light chain CDR3 DNA sequences. A total of 169 ideal candidates were selected from enriched clonotypes and were expressed in HEK293 cells through transfection. Yunlong Cao et al. finally identified 14 potent neutralizing mAbs, among which the most potent mAb, BD-368-2, exhibited an IC50 of 1.2. (13).
Monoclonal antibodies generation from transgenic immunized rodents
Starting with the immunization of transgenic Xenomouse® with ectodomain of S protein, from a total of 11,520 wells supernatants, 666 hybridomas capable of producing human antibodies specific to S protein were selected by Coughlin et al (14). A second screening identified 200 Abs that were specific to the S1 subdomain of the S protein. All of them were tested in microneutralization assay and 27 were selected. After this step, twenty-four out of 27 were put in limiting dilution, produced and purified by affinity columns. Nineteen of these were subsequently confirmed as monoclonal by Ig gene sequencing, and further divided into 8 categories based on their binding specificities (deduced from the usage of different V, J and D gene sequences). A cocktail of mAbs was then suggested as therapeutic that would simultaneously target several neutralizing epitopes and prevent emergence of escape mutants.
Generating antibody against native conformation transmembrane proteins
Like GPCR, ion channels, transporters or tyrosine kinase receptors, glycoproteins of viral envelopes are transmembrane proteins, requiring a strategy allowing the generation of monoclonal antibodies against conformational epitopes. DNA and cell immunizations strategies are potential solutions, and the screening step needs to differentiate prefusion from postfusion binders during the first round of selection, since the viral spike changes its conformation states before and after infecting cells.
While viral envelope proteins are promising targets for the development of new drugs, the production of therapeutic neutralizing antibodies (NAbs) is not so trivial. It still requires an enormous workload, expensive equipment and a great deal of expertise in isolating the potential, drug-active and promising effector antibody.
After all, didn't Alberich (15) say that the Lord of the Ring would become its slave?
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(2) Hawkes, R., 1964. Enhancement of the infectivity of arboviruses by specific antisera produced in domestic fowls. Aust J Exp Biol Med Sci 42, 465–82
(3) Schlesinger, J.J., and M.W. Brandriss. 1991. Growth of 17D yellow fever virus in a macrophage-like cell line, U937: role of Fc and viral receptors in antibody-mediated infection. J Immunol 127:659–665.
(4) Halstead, S.B., and E.J. O’Rourke. 1977. Antibody-enhanced dengue virus infection in primate leukocytes. Nature 265:739–741.
(5) Halstead, S.B., and E.J. O’Rourke. 1977. Dengue viruses and mononuclear phagocytes. I. Infection enhancement by non-neutralizing antibody. J Exp Med 146:201–217.
(6) Halstead, S.B., E.J. O’Rourke, and A.C. Allison. 1977. Dengue viruses and mononuclear phagocytes. II. Identity of blood and tissue leukocytes supporting in vitro infection. J Exp Med 146:218–229.
(7) Homsy, J., M. Meyer, M. Tateno, et al. 1989. The Fc and not CD4 receptor mediates antibody enhancement of HIV infection in human cells. Science 244:1357–1360.
(8) Gimenez, H.B., H.M. Keir, and P. Cash. 1989. In vitro enhancement of respiratory syncytial virus infection of U937 cells by human sera. J Gen Virol 70:89–96.
(9) Takada, A., S. Watanabe, K. Okazaki, et al. 2001. Infectivity-enhancing antibodies to Ebola virus glycoprotein. J Virol 75:2324–2330
(10) Marot, S., Malet, I., Leducq, V. et al. Rapid decline of neutralizing antibodies against SARS-CoV-2 among infected healthcare workers. Nat Commun 12, 844 (2021). https://doi.org/10.1038/s41467-021-21111-9
(11) Seow, J., Graham, C., Merrick, B. et al. Longitudinal observation and decline of neutralizing antibody responses in the three months following SARS-CoV-2 infection in humans. Nat Microbiol 5, 1598–1607 (2020). https://doi.org/10.1038/s41564-020-00813-8
(12) Sui J, Li W, Murakami A, Tamin A, Matthews LJ, Wong SK, Moore MJ, Tallarico AS, Olurinde M, Choe H, Anderson LJ, Bellini WJ, Farzan M, Marasco WA. Potent neutralization of severe acute respiratory syndrome (SARS) coronavirus by a human mAb to S1 protein that blocks receptor association. Proc Natl Acad Sci U S A. 2004 Feb 24;101(8):2536-41. doi: 10.1073/pnas.0307140101. PMID: 14983044; PMCID: PMC356985.
(13) Yunlong Cao, Bin Su, Xianghua Guo. Potent Neutralizing Antibodies against SARS-CoV-2 Identified by High-Throughput Single-Cell Sequencing of Convalescent Patients’ B Cells
(14) Coughlin M, Lou G, Martinez O, Masterman SK, Olsen OA, Moksa AA, et al. Generation and characterization of human monoclonal neutralizing antibodies with distinct binding and sequence features against SARS coronavirus using XenoMouse. Virology. 2007; 361: 93-102.
(15) Der Ring des Nibelungen, WWV 86, Richard Wagner.