Phage display strategy against outbreaks of unknown pathogens: learning from the SARS-CoV-2 pandemic.

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Despite the impact of improvements in sanitation and the availability of antibiotics and vaccines, infectious diseases remain the leading cause of death worldwide (1). There are a number of emerging infections that threaten people’s health both locally and globally every year. Several factors can influence the appearance of emerging and re-emerging infectious diseases including ecological factors, globalization, microbial adaptation, and human behavior (2).

Recently, the outbreak of the severe acute respiratory syndrome coronavirus (SARS-CoV) -2, known as COVID-19, appeared in Wuhan and spread rapidly all over the world, reaching more than 13 millions people infected and 0.57 millions death worldwide until mid-July 2020 (3). Infection control focuses on quarantining infected people and restricting people’s mobility while a protective vaccine is ready or the threat disappears.

Globalization and the movement of people make new emerging pathogens spread throughout the world. Therefore, the scientific community needs to be prepared to put all its skills into finding ways to act quickly to minimize damage when the disaster happens. Technological advances and knowledge in life sciences allow us to have a straightforward approach to act in such situations. Such approaches could be organized and coordinated by administrations and health authorities that need to provide the scientific community in each country with the necessary resources to apply these approaches.

In this article, a direct approach was developed to apply when such situations arise. It consists of two simple materials used as a starting point that are later developed as key components against an infection. These are the immunogenic epitopes that induce the immune system and the antibodies against them produced by patients who have passed the infection.

Blood samples from infected individuals at different times post infection are a valuable resource of components of the immune system that have been produced in response to infection. Also, the immune system of convalescent patients has provided them with all the tools and components to fight a pathogen. One of these components is the antibodies, therefore, the plasma of the patients that have these antibodies and the B cells that produce them are essential to fight infectious diseases. In recent studies with serological samples from individuals infected with SARS-CoV-2, it was found that most patients had a detectable level of IgM antibodies two weeks after the onset of symptoms. Unlike IgM, IgG antibody levels peaked between 16-30 days and remained relatively stable during the convalescence phase (4, 5). These antibodies have valuable information on immunogen epitopes recognition and neutralizing and opsonizing ability, thus we should take advantage of the antibodies found in convalescent plasma.

One of the most important parts when studying an emerging pathogen like SARS-CoV-2 is to identify its immunogenic epitopes. This can let to determine the identity of an emerging pathogen and the sequences recognized by antibodies from convalescent plasma. This is excellent starting point information for developing diagnosis assays and designing therapeutic compounds. Furthermore, peptides having immunogenic epitopes are selected from peptide libraries using antibodies purified from convalescent plasma. Having such libraries in a displayed system, such as phage display, is crucial, but the libraries must be diverse enough to carry peptides that mimic immunogenic epitopes of a pathogen. However, even when a library is highly diverse, it is probably necessary to screen several libraries that display different types of peptides (e.g., linear or circular peptides). There are several peptide libraries available that could be used for such studies. There are three types of commercial libraries available: a linear heptapeptide, a linear dodecapeptide, and a cysteine-constrained heptapeptide library (New England Biolabs). In addition, there are several publications that describe how to construct phage display peptide libraries. Construction of libraries of 10- and 15-mer peptides expressed at the N-terminus of the phage pIII coat protein has been described. One of these libraries is based on the sequence ADGSGGX₁₀GAPGA (6), and the other on the sequence AEXXX₁₅ P₆AE (where XXX represents random amino acid residues) (7). All these libraries could be used to build a platform to identify immunogenic epitopes of unknown pathogens. In brief, the peptide libraries are incubated with antibodies purified from convalescent patients. Antibody – phage complexes are captured with protein A- or G conjugated beads. To remove the peptide phages that bind protein A or G, the protein A beads are alternated with protein G beads between panning steps. Finally, bound phages are eluted with glycine-HCl, pH 2.2, or triethylamine (8). The binding capacity of the isolated phages displaying the peptides must be confirmed before continuing and producing them as synthetic peptides.

Also, this approach was used to select SARS-CoV epitopes by screening phage-displayed peptide libraries with convalescent human serum IgG. Selected peptides contain a sequence homologous to regions of the SARS-CoV S protein, M protein, and SARS-CoV structural proteins (9, 10). This has shown that phage display peptide libraries are useful in identifying immunogenic epitopes.

The peptides selected from the libraries are sequenced to find a similarity to the proteins registered in the protein databases (e.g., NCBI). The sequences should be compared with each other to see if the analysis reveals common motifs. Even when such motifs do not exactly match the epitopes on the emerging pathogen, they are recognized by convalescent antibodies. They are called mimotopes, also sequences that mimic epitopes without matching completely but with enough similarity that they function in the same way. The similarity could be at the structural level rather than the sequence level. To analyze these motifs, the peptides containing them are produced as synthetic peptides or as a fusion protein conjugated to bovine serum albumin or other carrier protein or as a peptibody. It is important to determine whether the peptides synthesized with the motif continue to be recognized by the patient’s antibodies. Synthetic peptides that remain recognized by convalescent antibodies are used to identify antibodies against immunogenic epitopes from phage display antibody libraries.

It is important to identify antibodies that have recognized immunogenic epitopes and isolate them as recombinant antibodies for further analysis. These antibodies are applied to develop diagnostic assays and design therapeutic agents against pathogens. This is accomplished using phage display antibody libraries constructed from B cells isolated from convalescent patients. Furthermore, antibody libraries created in other display systems can also be used, including bacterial display, ribosome display, or baculovirus display. The V_H and V_L gene repertoires of B cells are amplified by PCR and cloning in fusion with the N-terminal region of the phage pIII protein (11). Effective display formats for antibodies are single-chain variable fragment (scFv), antigen-binding fragment (Fab) or single-chain domain fragments. Also, this strategy was applied to isolate antibodies from convalescent patients with SARS (12). The antibody library was screened against SARS coronavirus virions as antigens. Using another technique, monoclonal antibodies were isolated from convalescent COVID-19 patients by flow cytometry sorting of IgG+ memory B cells against virus the S-protein as a probe¹³. Peptides having immunogenic epitopes can be used to screen phage display antibody libraries or as probes to sort B cells.

The ideal way to obtain antibodies against immunogenic epitopes is through libraries generated from B cells from convalescent patients. However, this is time-consuming, and time is critical in epidemic and pandemic outbreak situations. But in the meantime, the libraries generated from B cells are under construction, synthetic or naive antibody libraries can be used if available. They should be screened against the immunogenic epitopes. However, it is important to make sure that antibody fragments isolated from these libraries compete with antibodies isolated from convalescent plasma. There are commercially available premade phage libraries (e.g., Dyax and Morphosys) or constructed by the researchers (14, 15, 16, 17). There are several studies that use these types of libraries. For example, they have been applied successfully to identify monoclonal antibodies against SARS-CoV from both naïve (14), synthetic (15)- and semi-synthetic antibody libraries (16, 17).

Whatever the source of isolated antibodies against immunogenic epitopes, research should go further by developing diagnostic assays and designing therapeutic molecules.

Gram-negative enteric pathogens, including Salmonella, have been associated with the autoimmune disease reactive arthritis (ReA). ReA followed outbreaks of Salmonella food poisoning (50). The titer and profiles of autoantibodies in the sera of patients with acute salmonellosis due to Salmonella enterica serovar Typhimurium (S. Typhimurium) or Salmonella enterica serovar Enteritidis (S. Enteritidis) infection, as well as in convalescent patients, indicate that anti-smooth muscle antibodies (ASMA) were the most prevalent in all salmonellosis (51).

Recently was demonstrated that Curli, thin amyloid fibers expressed on the surface of enteric bacterial cells, can induce inflammatory responses by stimulating pattern recognition receptors. The study showed that curli is expressed by S. Typhimurium in the cecum and colon of mice after natural oral infection, in both acute and chronic infection models. The infected mice generated increased levels of anti-dsDNA autoantibodies and joint inflammation. They hypothesized that the study provided the relation between bacterial amyloids like curli and human amyloids and their association with diseases such as Alzheimer’s Disease (52).

The sequence of the immunogenic epitopes may give some clue which is the unknown agent that causes the infectious disease. If that is not enough to identify the emerging pathogen, there are other techniques that can be used. For example, mass spectrometry is a powerful tool for diagnosis emerging pathogens. The technique is based on trypsin digestion of proteins extracted from inactivated virus samples, and the resulting peptides are analyzed with mass spectrometry (18). Using this technique, a study proposed peptides of the key structural proteins N, S, and M of SARS‐CoV‐2 for the development of diagnostic assays (19).

Ethical approval

Not required.

Author contributions

M.P. was the sole author of this article.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Declaration of competing interest

M.P. is Editor-in-Chief of Current Bioscience and a member of Current Bioscience editorial board. This article was reviewed by Current Bioscience editors and reviewers. The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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