Perspectives on passive antibody therapy and peptide-based vaccines against emerging pathogens like SARS-CoV-2

Abstract

The current epidemic of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is raising awareness of the need to act faster when dealing with new pathogens. Exposure to an emerging pathogen generates an antibody response that can be used for preventing and treating the infection. These antibodies might have a high specificity to a target, few side effects, and are useful in the absence of an effective vaccine for treating immunocompromised individuals. The approved antibodies against the receptor-binding domain (RBD) of the viral spike protein of SARS-CoV-2 (e.g., regdanvimab, bamlanivimab, etesevimab, and casirivimab/imdevimab) have been selected from the antibody repertoire of B cells from convalescent patients using flow cytometry, next-generation sequencing, and phage display. This encourages use of these techniques especially phage display, because it does not require expensive types of equipment and can be performed on the lab bench, thereby making it suitable for labs with limited resources. Also, the antibodies in blood samples from convalescent patients can be used to screen pre-made peptide libraries to identify epitopes for vaccine development. Different types of vaccines against SARS-CoV-2 have been developed, including inactivated virus vaccines, mRNA-based vaccines, non-replicating vector vaccines, and protein subunits. mRNA vaccines have numerous advantages over existing vaccines, such as efficacy, ease of manufacture, safety, and cost-effectiveness. Additionally, epitope vaccination may constitute an attractive strategy to induce high levels of antibodies against a pathogen and phages might be used as immunogenic carriers of such peptides. This is a point worth considering further, as phage-based vaccines have been shown to be safe in clinical trials and phages are easy to produce and tolerate high temperatures. In conclusion, identification of the antibody repertoire of recovering patients, and the epitopes they recognize, should be an attractive alternative option for developing therapeutic and prophylactic antibodies and vaccines against emerging pathogens.

Introduction

Despite the impact of improved sanitation and the availability of antibiotics and vaccines, infectious diseases are still the leading cause of death worldwide. Each year, many new infections threaten the health of the local and world populations. Several factors that may influence the appearance of emerging and re-emerging infectious diseases have been described by Morse, including microbial adaptation, ecological and demographic factors, movement of people and goods, industry, and worsening public health services.¹

Recently, the outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), known as coronavirus disease 2019 (COVID-19), began in Wuhan and spread rapidly around the world. This outbreak resulted in more than 144,099,374 confirmed cases, with more than 3,061,912 deaths worldwide by 23 April 2021.² Infection control focuses on quarantining infected persons and restricting the mobility of persons while vaccinating the world population until the threat disappears. Globalization and the movement of people are spreading new, emerging, and re-emerging pathogens worldwide. Therefore, the scientific community must be ready to put all its skills into the search for ways to act quickly to save lives. Advances in technology and knowledge in the life sciences make it possible to identify and isolate epitopes derived from a pathogen that has induced an immune response and the antibodies that have been generated during the infection. This article discusses the prospects for passive antibody therapy and active immunization, including peptide-based vaccines to combat pathogens, with a particular focus on emerging and re-emerging pathogens.

Methods

Literature search

An extensive literature research was conducted using keyword filters to select articles related to “therapeutic antibodies”, “neutralizing antibodies”, “peptide vaccine” in combination with “SARS-CoV-2”, “emerging” or “re-emerging pathogen”, and “infectious diseases”. This research was carried out on articles published in the PubMed and Scopus databases for the English language from 1 January 2020 to 12 December 2020. Also, articles on the SARS-CoV-2 vaccines were examined. Information about approved therapeutic antibodies was collected from the Antibody Society website,³ the incidence of COVID-19 cases was obtained from the World Health Organization (WHO) website,² and information of the approved COVID-19 vaccines was obtained from the Regulatory Affairs Professionals Society (RAPS) website.⁴ The review process is graphically shown in Figure 1.

Figure 1.

Flow diagram showing study inclusion and exclusion

Clinical trials search

The entire database at ClinicalTrials.gov was searched on 10 January 2021 using the following search terms: condition or disease = “infectious disease”; other terms = “antibody” OR “therapeutic antibody” OR “monoclonal antibodies” OR “therapeutic peptide” OR “peptide vaccine”. The number of clinical trials registered for therapeutic antibodies during the last ten years were counted for all diseases and infectious diseases. Tables were prepared that listed pathogen-specific antibodies, or therapeutic antibodies specific for human proteins, that were used for treatment of infectious diseases and were registered during 2020. Furthermore, a list was prepared of peptide vaccines that were used for the prevention of infectious diseases and that were registered in the last ten years.

Literature review

Emerging pathogens

In addition to SARS-CoV-2, several other emerging pathogens have been reported to infect humans during the last two decades,⁵ including human bocavirus,⁶ human coronavirus HKU1,⁷ human T-lymphotropic virus (HTLV) 3 and ^4,8,9 human coronavirus NL63,¹⁰ SARS coronavirus,¹¹ human metapneumovirus,¹² Middle East respiratory syndrome coronavirus (MERS-CoV),¹³ and severe fever with thrombocytopenia syndrome (SFTS) virus.¹⁴ For example, the human bocavirus was identified for the first time in humans in 2005 in children with respiratory tract infections in Sweden. The human coronavirus HKU1 was isolated in January 2004 from a 71-year-old Chinese man who had been hospitalized with pneumonia and bronchiolitis. HTLV-3 and -4 were isolated in 2005 from two hunters of monkeys from the southern forests of Cameroon. The human coronavirus NL63 was isolated in 2004 from a 7-month-old child with bronchiolitis from the Netherlands. Cases of SARS coronavirus were first reported in China in 2002. The human metapneumovirus was isolated in 2001 from young children in the Netherlands. MERS-CoV was first reported in Saudi Arabia in 2012. SFTS was first clinically identified in a patient with several symptoms including fever and thrombocytopenia in China in 2009.

Some examples from before 2000 include the Nipah virus that appeared in 1998, Ebola in 1976, human immunodeficiency virus (HIV) in 1959, Marburg virus in 1967, and Lassa fever in 1969. Outbreaks of unknown infectious diseases often become a challenge due to lack of treatment and diagnosis, malnutrition, and the emergence of drug-resistant pathogens.

Antibody response

Exposure to an emerging pathogen, such as SARS-CoV-2, generates an antibody response that changes over time and between individuals. IgM antibodies against SARS-CoV-2 become detectable during the week following infection and are present for a month before progressively diminishing, while IgG antibodies are detectable within ten to 21 days up to a minimum of three months.¹⁵ The survival of individuals with severe SARS-CoV-2 infection can be attributed in part to a rapid and potent IgG class switching with high-affinity FcR binding capacities.¹⁶ Consequently, antibodies from patients who recover from COVID-19 or other emerging infections can provide valuable information about their neutralization and opsonization capabilities and the epitopes they recognize. The identification of these epitopes and the production of antibodies against them should be one of the main objectives of emerging pathogen research and this is what this review suggests.

Passive and active immunization

Convalescent plasma and therapeutic antibodies might be useful treatments for emergent pathogens when no other treatment is available, while vaccines are prophylactic and are designed to induce a specific host immune response against pathogens.

Convalescent plasma

Convalescent plasma therapy may be the most straightforward option for reducing symptoms and mortality in infected patients when vaccines or drugs are unavailable, but with varying degrees of success. The WHO gave priority to assessing treatment with convalescent whole blood or plasma derived from patients who recovered from Ebola virus disease during the outbreak in West Africa in 2014-2015. However, the report of Griensven et al. indicated that the convalescent plasma therapy did not induce a significant reduction of mortality among Ebola patients.¹⁷

On 23 August 2020, the Food and Drug Administration (FDA) issued an emergency use authorization for convalescent plasma as a potentially promising treatment of critically ill COVID-19 patients.¹⁸ They concluded that the convalescent plasma may be effective in reducing the severity or length of COVID-19 illness in some hospitalized patients. The advantages of convalescent plasma outweigh the potential risks in the absence of appropriate, approved and available alternative therapies. Since then, numerous studies have been published about how to apply this type of therapy to severe and critically ill COVID-19 patients.^19-28 The FDA released a new evaluation that indicates that convalescent plasma no longer meets clear criteria of being “maybe effective”.²⁹ According to the new information available, it seems that high titer convalescent plasma should only be given at the beginning of the infection (within 3 days of symptom onset) and it is probably not beneficial after 8 days or more after the onset of symptoms. Therefore, convalescent plasma therapy should be considered with some discretion, but alternative approaches should also be explored.

Passive antibody therapy

Administration of purified antibodies may be an alternative to convalescent plasma transfusion. It has been speculated that patients with COVID-19 could be treated with intravenous immunoglobulin (IVIg) or specific human monoclonal antibodies (mAbs) that inhibit SARS-CoV-2 binding to angiotensin-converting enzyme 2 (ACE-2) receptors and its entry into human cells.^30,31 The receptor binding domain (RBD) of the spike glycoprotein (S) protein on the surface of SARS-CoV-2 is responsible for the attachment of the virus to ACE-2 to gain access to host cells.³² This interaction can be neutralized with antibodies that compete with ACE-2 for binding to RBD. Neutralizing antibody levels depends on the time after the onset of symptoms, age of the patient, and the severity of the disease. It is not clear what level of neutralizing antibodies is needed to ensure protection against infection and re-infection.

The number of new therapeutic antibodies entering clinical trials in all stages of development increased over time (a linear trend in phases 1 to 4) for all diseases including infectious diseases (Figure 2). Between January 2020 and December 2020, 429 trials were initiated for therapeutic antibodies against different diseases compared with 41 (9.6%) trials against infectious diseases. Among them, 23 antibodies targeted pathogens (Table 1), while the other 18 antibodies targeted human molecules (Table 2). Twenty-six clinical trials were evaluating antibodies against SARS-CoV-2, 6 focused on HIV, 2 on malaria, 1 on herpes simplex virus 1 (HSV-1), 1 on chikungunya virus, 1 on BK virus, 1 on rabies virus, 1 on dengue virus, 1 on Pseudomonas aeruginosa, and 1 on Clostridioides difficile. Moreover, a significant number of neutralizing antibodies have not been evaluated in clinical trials.

Figure 2.

Trends in clinical trials registration of therapeutic antibodies during the last ten years

The closed circles with a solid trend line represent the number of clinical trials with therapeutic antibodies for all types of diseases registered each year for the last ten years at ClinicalTrials.gov. The open circles with a dashed trend line represent the number of clinical trials with therapeutic antibodies for infectious diseases registered each year for the last ten years at ClinicalTrials.gov.

Table 1.

Pathogen-specific antibodies for treatment of infectious diseases registered during 2020 at ClinicalTrials.gov

Antibody	Target	Pathogen	Phases	NCT Number
3BNC117-LS	CD4 binding site on the HIV-1 envelope	HIV	I, II	NCT04250636, NCT04319367
10-1074-LS	V3 glycan supersite on the HIV-1 envelope protein	HIV		NCT04250636, NCT04319367
PGT121.414.LS, VRC07-523LS	CD4 binding site of the HIV-1 envelope	HIV	I	NCT04212091
Herpevizumab (HDIT101)	Epitope on HSV-1/2 glycoprotein B (gB)	HSV-1	II	NCT04539483
SAR440894	E2 envelope protein of chikungunya virus	Chikungunya virus	I	NCT04441905
MAU868	Viral capsid protein, VP1	BK virus	II	NCT04294472
SYN023 (CTB011 + CTB012)	Non-overlapping epitopes on the rabies virus (RABV) glycoprotein (G)	Rabies virus	III	NCT04644484
AV-1	West Nile virus E protein	Dengue	I	NCT04273217
Antibodies against Pseudomonas from patients' B lymphocytes	Pseudomonas	Pseudomonas aeruginosa		NCT04335383
Bezlotoxumab	Toxin B	Clostridioides difficile	II	NCT03829475
SCTA01	S protein	SARS-CoV-2	I	NCT04483375
REGN10933+REGN10987	S protein	SARS-CoV-2	I, II, III	NCT04519437, NCT04426695, NCT04425629, NCT04452318
TY027	S protein	SARS-CoV-2	I, III	NCT04429529, NCT04649515
JS016	S protein	SARS-CoV-2	I	NCT04441918
DZIF-10c (BI 767551)	From recovered COVID-19 patients	SARS-CoV-2	I, II	NCT04631705
DZIF-10c	From recovered COVID-19 patients	SARS-CoV-2	I, II	NCT04631666
VIR-7831	From patient recovered from SARS in 2003	SARS-CoV-2	II, III	NCT04545060
CIS43LS	Circumsporozoite protein (PfCSP)	Malaria	I	NCT04206332
TB31F	Pfs48/45	Malaria	I	NCT04238689

Table 2.

Therapeutic antibodies specific for human proteins for treatment of infectious diseases registered during 2020 at ClinicalTrials.gov.

Antibody	Target	Pathogen	Phases	NCT Number
UB-421	CD4	HIV	II	NCT03164447, NCT04404049, NCT03743376
Garadacimab (CSL312)	Factor XII/XIIa	SARS-CoV-2	II	NCT04409509
Mavrilimumab (KPL-301, CAM3001)	GM-CSF α	SARS-CoV-2	II, III	NCT04447469
Mavrilimumab	GM-CSF α	SARS-CoV-2	II	NCT04397497
Leronlimab	CCR5	SARS-CoV-2	II	NCT04343651, NCT04347239
TJ003234	Granulocyte-monocyte stimulating factor (GM-CSF)	SARS-CoV-2	II, III	NCT04341116
Lenzilumab	GM-CSF	SARS-CoV-2	III	NCT04351152
Ravulizumab	Complement component C5	SARS-CoV-2	III	NCT04369469
CPI-006	CD73 cell-surface ectonucleotidase	SARS-CoV-2	I	NCT04464395
Crizanlizumab	P-selectin	SARS-CoV-2	II	NCT04435184
REGN-COV2, Tocilizumab	IL-6	SARS-CoV-2	II, III	NCT04381936
Gimsilumab	Granulocyte-monocyte stimulating factor (GM-CSF)	SARS-CoV-2	II	NCT04351243
Pamrevlumab	Connective tissue growth factor (CTGF)	SARS-CoV-2	II	NCT04432298
Canakinumab	IL-1β	SARS-CoV-2		NCT04348448
Tocilizumab	IL6	SARS-CoV-2	IV	NCT04377750

Jin and Simmons reviewed the available literature on neutralizing antibodies that block chikungunya virus from binding to host receptors thereby preventing membrane fusion and virus entry into cells.³³ Chikungunya virus is a type of emerging mosquito-transmitted alphavirus that can cause acute or chronic polyarthritis and foot swelling, and is currently spreading rapidly from endemic regions of Africa and Asia to Europe and America.³⁴ Corti et al. isolated LCA60, a potent neutralizing antibody derived from the memory B cells of an individual who had been infected with MERS-CoV. This antibody recognizes an epitope in the viral S protein and blocks virus binding to the host receptor CD26, reducing the disease and virus titers in a relevant mouse model.^35,36

Right now, there is an increasing tendency to develop neutralizing antibodies as a result of COVID-19. Antibodies to treat COVID-19 are currently being tested in a number of clinical trials (See Tables 1 and 2). On 9 November 2020, FDA issued an emergency use authorization (EUA) to Eli Lilly and Company for the combination of antibodies bamlanivimab (LY-CoV555) and etesevimab (LY-CoV016, also known as CB6 or JS016).³⁷ These are antibodies produced by B cells from convalescent patients that target the SARS-CoV-2 S protein. Antigen-specific memory B cells were identified and isolated by flow cytometry and the V_H and V_L were amplified, cloned, and produced in an expression system. On 20 November 2020, Regeneron Pharmaceuticals obtained approval for the therapeutic application of the antibody cocktail casirivimab/imdevimab (REGN10987+REGN10933),³⁸ which contains both humanized mice antibodies and antibodies from blood samples from recovered COVID-19 patients.³ The antibody variable regions were determined by next-generation sequencing, and the repertoire for the heavy and light chain pairs was identified, cloned, and produced in an expression system. Recently, the European Medicines Agency (EMA) approved the use of the antibody regdanvimab (also known as CT-P59) for treating COVID-19.³⁹ It functions by targeting the RBD of the viral S protein. Regdanvimab is a fully human immunoglobulin reformatted from a single-chain variable fragment that was selected from a phage display antibody library created by B cells from a convalescent patient.⁴⁰ Approved therapeutic antibodies against SARS-CoV-2 are shown in Table 4.

Table 3.

Peptide vaccines for the prevention of infectious diseases registered in the last ten years at ClinicalTrials.gov

Vaccine	Target	Pathogen	Phases	NCT Number	Start year
CMVPepVax (CMVpp65-A*0201)	HLA A*0201 restricted pp65 CD8 T-cell peptide epitope fused with the P2 peptide epitope of tetanus toxin, and mixed with a Toll-like receptor (TLR) 9 agonist	Cytomegalovirus	I, II	NCT01588015	2012
			II	NCT02396134	2015
Multi-peptide CMV-Modified Vaccinia Ankara Vaccine	Modified Vaccinia Ankara (MVA) viral vector encoding three herpes virus cytomegalovirus (CMV) tumor-associated antigens (TAAs), including UL83 (pp65), UL123 (IE1) and UL122 (IE2), with potential immunostimulating activity	Cytomegalovirus	II	NCT02506933	2015
			I, II	NCT03354728	2018
			II	NCT04060277	2019
CENV3	Synthetic peptide vaccine derived from HCV E1 and HCV E2	Hepatitis C virus	I, II	NCT01718834	2011
Multimeric 001 (M-001)	9 conserved peptides from influenza A and B	Influenza H5N1	II	NCT02691130	2015
DC-HIV04	DC-HIV vaccine with HIV peptides	HIV infection	I	NCT03758625	2018
Multipeptide cocktail (pVAC)	CoVac-1 (SARS-CoV-2 HLA-DR peptide)	COVID-19 vaccine	I	NCT04546841	2020
StreptInCor	55 amino acid residues of the C-terminal portion of the M protein	Rheumatic fever, Streptococcus pyogenes	I	NCT03998592	2021

Table 4.

Therapeutic antibodies against SARS-CoV-2 approved before 19 April 2021

Drug code	Sponsors	Source	Techniques	Approval	Ref.
Regdanvimab (CT-P59)	Celltrion	B cells from convalescent patients	Phage display	EU (Mar 2021), South Korea (Feb 2021)	Kim et al., 202140
LY-Co555 + LY-CoV016	AbCellera / Eli Lilly and Company	B cells from convalescent patients	Flow cytometry	US (Feb 2021), EU (Mar 2021)	Shi et al., 202037
Casirivimab/imdevimab (REGN-COV2) (REGN10987+REGN10933)	Regeneron	From mice + B cells from convalescent patients	Next-generation sequencing	US (No. 2020), EU (Feb 2021)	Hansen et al., 202038

All data were collected from The Antibody Society (antibodysociety.org).³

Only few antibodies have been approved to treat infections in humans (e.g., ruxibacumab, obiltoxaximab, palivizumab, bezlotoxumab, ibalizumab, KamRAB, etc.) compared to the approved antibodies for other types of disease such as cancer. Raxibacumab and obiltoxaximab neutralize the binding of the anthrax exotoxins to host receptors and their application is for the treatment of Bacillus anthracis infections.⁴¹ Palivizumab is specific for the F-glycoprotein of the respiratory syncytial virus (RSV) and has been successfully used prophylactically for severe RSV disease especially in infants.⁴² Bezlotoxumab has been developed for the prevention of Clostridioides difficile infection recurrence, ibalizumab prescribed for HIV,⁴³ and KamRAB for rabies infection.⁴⁴ Approved therapeutic antibodies against different pathogens are shown in Table 5.

Table 5.

Therapeutic antibodies against different pathogens approved before 22 April 2021

Antibody	Pathogen	Target	Approval (year)
Ansuvimab	Ebola virus	Receptor-binding domain	US (2020)
Antibody cocktail: atoltivimab, maftivimab, and odesivimab-ebgn	Ebola virus	Glycoprotein on the surface of Ebola virus	US (2020)
Obiltoxaximab	Bacillus anthracis	B. anthracis exotoxin	EU (2020), US (2016)
RabiMabs (antibody cocktail)	Rabies virus	Site II and III on G protein of rabies virus envelope	India (2019)
Ibalizumab	HIV	CD4	EU (2019), US (2018)
HyperRAB (HRIG*)	Rabies virus	Rabies virus	US (2018)
Bezlotoxumab	Clostridioides difficile	C. difficile enterotoxin B	EU (2017), US (2016)
KamRAB/KedRAB (HRIG*)	Rabies virus	Rabies virus	US (2017)
Rmab	Rabies virus	Amino acids 336–342 of the GP (antigenic site III)	India (2016)
Raxibacumab	Bacillus anthracis	B. anthracis protective antigen (PA)	US (2012)
Palivizumab	Respiratory syncytial virus	Antigenic site II region of F protein	EU (1999), US (1998)
Imogam (HRIG)	Rabies virus	Rabies virus	US (1984)

All data were collected from The Antibody Society (antibodysociety.org).¹⁰⁶

HRIG – human rabies immune globulin.

The increasing tendency to develop therapeutic antibodies against infectious diseases over the past ten years indicates that they have been accepted as an effective evolving treatment modality that may have a major clinical impact in the future.

Immune phage display libraries

Monoclonal antibodies can be generated by animal immunization and hybridoma techniques or by recombinant protein production.⁴⁵ However, the identification and recovery of individual antigen-specific antibodies from the antibody repertoire from peripheral blood mononuclear cells (PBMCs) from a human blood donor can be done using flow cytometry,⁴⁶ next-generation sequencing,⁴⁷ or different in vitro display techniques (e.g., phage display,⁴⁸ bacterial display,⁴⁹ yeast display,⁵⁰ or mammalian cell surface display⁵¹). Flow cytometry is a common method used for single B cell analysis and isolation and the next-generation sequencing enables simultaneous reading of up to several million variants of a single gene. However, the main disadvantage of these techniques is the high cost of their production equipment which makes them inaccessible to many labs, especially in developing countries. In contrast, phage display is a technique that only requires the types of equipment available in the majority of microbiology labs and can be done on the lab bench.

The genetic information encoding the selected antibodies from phage display libraries is immediately available which makes it easy to re-clone the isolated antibodies for further manipulation and expression. In addition, it can be used against toxic antigens, with no need for animals, the selection process is fast, and with it human antibodies can be obtained. Some disadvantages of phage display are that it is a laborious process to construct a phage display library and to obtain a library with sufficient diversity to represent the antibody repertoire. Raxibacumab and regdanvimab are the only antibodies approved by the FDA and EMA against pathogens, that had been isolated from phage display libraries. Raxibacumab was selected from a naive library, also generated from healthy individuals. However, regdanvimab was isolated from a immune phage display library constructed from B cells from a convalescent COVID-19 patient. The immune libraries may be a better option for infectious diseases⁵² because they mimic a natural vaccination with a live pathogen in a human host. This is an attractive option to obtain antibodies against immunogenic epitopes since they can be obtained from the V-gene repertoire specific to an infection. Currently, the antibody foravirumab (CR4098), selected from a phage display library constructed from the B cell repertoire of rabies-vaccinated individuals,⁵³ is in a phase II clinical trial. This indicates that phage display could be further exploited in finding therapeutic antibodies to treat infectious diseases as it has been exploited in oncology⁵⁴ and autoimmune diseases.⁵⁵

Developing immune or disease-specific antibody libraries helps understanding antibody reactions against specific disease antigens.

Several studies have used phage display to isolate human antibodies specific for bacterial, viral, and parasitic antigens. Recently, a set of fully human neutralizing antibodies was selected from phage display libraries generated from peripheral B cells collected from convalescent or severe COVID-19 patients. Several of these antibodies bind to distinct epitopes on the viral spike RBD and possess neutralizing activity for RBD binding to the human ACE2 receptor.⁵⁶ In another study, panning a phage-displayed single-domain antibody library against RBD and S1 subunit identified neutralizing antibodies that could be promising candidates for therapy of COVID-19.⁵⁷

Antibodies to other pathogens than SARS-CoV-2 were identified through the use of immune libraries. For example, immune libraries against pentavalent botulinum toxoid (BoNT/A) revealed that single-chain variable fragments (scFvs) recognized a limited number of immunodominant epitopes exposed on the binding domain of BoNT/A. One group of these antibody fragments exhibited neutralizing activity.⁵⁸ Another example is when an scFv phage library was generated from a patient infected with HIV-1 subtype B. Screening of this library with recombinant soluble gp140 envelope proteins led to the identification of scFv clones that specifically recognize a conserved pocket binding domain in gp41.⁵⁹ Ekiert et al. identified antibodies from phage display libraries created from B cells that were collected from Turkish patients who survived H5N1 avian flu infection. These antibodies recognize conserved elements of the receptor-binding site on the hemagglutinin surface glycoprotein, enabling the neutralization of strains of multiple subtypes of influenza A viruses, including H1, H2, and H3.⁶⁰ In a study of helminth parasite infections, scFv fragments against a specific filarial antigen (BmR1) were isolated from an immune library developed with the repertoire of antibodies from individuals who had been confirmed positive for filariasis infection.⁶¹

Active immunization with peptides

Active immunity is defined as the process of exposing an individual to an antigen to generate an adaptive immune response.⁶² Vaccines are generally considered the gold standard for infection control and, in normal situations, require many years to develop.

According to the WHO, as of 23 April 2021, 91 vaccine candidates were under clinical development to treat COVID-19, and 184 candidate vaccines were in pre-clinical development.[63 More than 973 million vaccine doses have been administered worldwide, while 223 million people have been fully vaccinated by 23 April 23 2021.⁶⁴

Currently, different types of vaccines against SARS-CoV-2 have been developed including inactivated virus vaccines (e.g., CoronaVac, BBIBP-CorV, etc.), mRNA-based vaccines (e.g., BNT162b2 and mRNA-1273), non-replicating vector vaccines (e.g., Convidicea, AZD1222, and Janssen), protein-based vaccines (e.g., ZF2001), and peptide vaccines (e.g., EpiVacCorona).^65-67 Except for the inactivated virus vaccines, most of them target the surface glycoprotein (S-protein) of SARS-CoV-2 and were showed to have a minimal number of side effects, be risk-free, and efficient in phase III clinical trials. Approved vaccines against SARS-CoV-2 are shown in Table 6.

Table 6.

Vaccines against SARS-CoV-2 approved before 19 April 2021

Vaccine type	Vaccine name (Sponsors)	Origin	Approval
Inactivated virus vaccines	CoronaVac (Sinovac)	China	China, Brazil
	BBIBP-CorV (Sinopharm)	China	China
	WIBP-CorV (Sinopharm)	China	China
	Covaxin (Bharat Biotech)	India	India, Mexico
	CoviVac	Russia	Russia
mRNA-based vaccines	BNT162b2 (Pfizer-BioNTech)	Multinational	US, EU, UK
	mRNA-1273 (Moderna)	US	US, EU, UK
Non-replicating vector vaccines	Convidicea (CanSino)	China	China, Hungary, Mexico, Chile
	AZD1222 (AstraZeneca/Oxford)	UK	EU, UK
	Janssen (Johnson & Johnson)	The Netherlands, US	US, EU, Canada
Protein-based vaccines	ZF2001 (Anhui Zhifei Longcom)	China, Uzbekistan	China, Uzbekistan
Peptide vaccine	EpiVacCorona (Federal Budgetary Research Institution)	Russia	Belarus, Russia, Turkmenistan

All data were collected from The Regulatory Affairs Professionals Society (raps.org).⁴

Since peptides can be selected using serum from infected patients, they should represent or mimic the immunogenic epitopes that triggered the immune response. Therefore, epitope vaccination may constitute an attractive strategy to induce high levels of antibodies against a pathogen.⁶⁸ EpiVacCorona is a Russian SARS-CoV-2 peptide vaccine approved in Belarus, Russia, and Turkmenistan. Two peptide vaccines are under development, UB-612 developed by the U.S.-based company Vaxxinity which is in phase 2/3 trial, and pVAC, developed by the University Hospital Tuebingen in Germany and is in phase 1 trial.⁴

Peptide vaccines have been applied to induce neutralizing antibodies in different mice models using epitopes of the varicella-zoster virus,⁶⁹ Helicobacter pylori,⁷⁰ H1N1 influenza virus,⁷¹ Ebola virus,⁷² Plasmodium falciparum,⁷³ Nipah virus,⁷⁴ and Tropheryma whipplei.⁷⁵ There are 36 clinical trials evaluating peptide vaccines against different pathogens, primarily in phases I and II, of which only ten have begun in the past ten years (Table 3). Interestingly, only one study began in 2020 which was for SARS-CoV-2, and another is expected to begin during 2021 for Streptococcus pyogenes. The SARS-CoV-2 study is in phase I trial for evaluating a multi-peptide vaccine (CoVac-1) (ClinicalTrials.gov, identifier NCT04546841) that was developed based on identified multiple immunodominant human leukocyte antigen-D related (DR) T-cell epitopes in COVID-19 convalescent individuals.⁷⁶ In addition, several other studies are underway to develop peptide-based vaccines for SARS-CoV-2.⁷⁷

One advantage of peptide-based vaccines is that they may eliminate many side effects of allergic and auto-immune reactions associated with classical whole-organism vaccines.⁷⁸ Recently, a melanoma peptide vaccine was tested for immunogenicity and safety in 47 participants by Slingluff and colleagues.⁷⁹ The peptides were synthesized and purified in GMP conditions and the participants were vaccinated with them in an emulsion with incomplete Freund's adjuvant or/and polyICLC. They were found to be well tolerated, had clinical activity, and could elicit CD4+ T lymphocyte responses in most participants. Li et al. reviewed the progress and challenges of peptide vaccines,⁸⁰ Kaumaya et al. reviewed the peptide vaccines in cancer therapy,⁸¹ and Di Natale et al. reviewed peptide-based vaccines for COVID-19.⁸² Synthetic peptides are easily synthesized, cost-effective, and relatively safe.^78,83,84 One disadvantage of peptide-based vaccine is that peptides or a single epitope alone are not enough to induce a sufficiently high immune response, so they must be conjugated with biopolymers, encapsulated in nanoparticles⁸⁵ or applied to carrier or adjuvants⁶⁸ to improve the immune response. Virus-like particles can be an alternative to the nanoparticle delivery system of peptides including bacteriophages.⁸⁶

The use of phages as immunogenic carriers of peptides could be an attractive vaccine approach. Roehnisch et al. used phage particles as immunological carriers in a clinical phase I/II trial to vaccinate with idiotype vaccine 15 patients with advanced multiple myeloma.⁸⁷ They concluded that this approach might be a simple, time- and cost-efficient phage idiotype vaccination strategy. Therefore, this is an approach worthy of further consideration. Greenwood et al. showed that a peptide corresponding to Plasmodium falciparum surface protein displayed on phages was highly immunogenic, not requiring an adjuvant to induce an antibody response.⁸⁸ Currently, a phase II clinical trial is evaluating phage vaccine SNS-301 in patients with high-risk myelodysplastic syndromes and chronic myelomonocytic leukemia (ClinicalTrials.gov, identifier NCT04217720). Nonetheless, the potential for phage vaccination against pathogens, including emerging and re-emerging pathogens, needs to be further explored, in particular the behavior of phages in the human body and how they interact with cellular components.⁸⁹ However, this is a promising way of vaccination because it can first select peptide displaying phages using convalescent plasma or convalescent antibodies, and then evaluate them as vaccine candidates.

Peptide libraries

Many phage display peptide libraries have been constructed and used extensively over the years to characterize epitopes.^90,91 They allow us to select peptides that may share common sequence patterns or have similarities to proteins recorded in protein databases (e.g., NCBI) and thereby identify potential antigens from a pathogen. Screening random peptide libraries with antibodies from convalescent human serum from patients with SARS-CoV infections led to the identification of phages that were specifically bound to these antibodies. Phage derived peptides also show extensive sequence homology with structural proteins of SARS-CoV such as S and M-protein.⁹² Zamecnik et al. screened a programmable phage display library (HuCoV VirScan), containing overlapping peptide antigens from several coronavirus types (e.g., SARS-CoV-2), with COVID-19 patient sera. Peptide phages encoding the S (residues 783-839 and 1,124-1,178) and N proteins (residues 209-265 and 142-208) were enriched during panning against human sera and used to develop serological tests for SARS-CoV-2 detection.⁹³

Discussion

When an unknown pathogen appears and causes an outbreak, the B cells of the recovering patients become a valuable resource of the antibody repertoire that patients express during infections. This repertoire can be used in convalescent plasma transfusion or to screen for therapeutic antibodies and to identify immunogenic epitopes.

Passive antibody therapy can be an alternative to convalescent plasma therapy because the latter therapy may cause side effects, such as autoimmune reactions and its results can be unpredictable due to variability between lots. In recent years, the percent of approvals for antibodies has been increasing. Even so, there are only a few successful therapeutic monoclonal antibodies against infectious diseases that have reached the market compared with antibodies in other therapeutic areas.

An important challenge that therapeutic antibodies face is the strong competition with well-established treatments with antibiotics, vaccines, and anti-viral compounds. Laura DeFrancesco published an interesting article in which a group of experts commented on challenges with antibody therapy.⁹⁴ There are only a few successful therapeutic antiviral antibodies compared to antibodies in other medical fields such as cancer. Expectations for antiviral antibody therapy seemed to be low because of the high cost of production in comparison to the profits. It is not a profitable market for pharmaceutical companies, as emerging infectious diseases are rare and are mainly affecting developing countries. However, the cost could be reduced by producing antibody fragments such as scFv, antigen-binding fragments (Fab), or nanobodies in prokaryotic systems.⁹⁵ Different expression systems (e.g., mammalian, yeast, and prokaryotic) are suitable for both soluble and functionally active protein expression. Escherichia coli was found to be a faster and more cost-effective expression system than Pichia pastoris and Chinese hamster ovary (CHO).⁹⁶ However, it has higher levels of impurities and breakdown products than the other two systems. Therefore, an additional downstream purification step would be required to remove these impurities to improve the quality of antibodies obtained by this system for therapeutic purposes.⁹⁶ There are Fab fragments successfully generated in E. coli and approved by the FDA, such as certolizumab pegol (for treatment of Crohn's disease, rheumatoid arthritis, psoriatic arthritis, and ankylosing spondylitis)⁹⁷ and ranibizumab (anti-vascular endothelial growth factor).⁹⁸ This encourages investment in prokaryotic production of Fabs or other forms of antibody therapies for the prevention and treatment of emerging or re-emerging infectious diseases.

Even with a vaccine, antibodies could still be needed to treat people who do not respond significantly to vaccination (e.g., infants, elderly, and immunocompromised individuals). From a technical point of view, by Fc region engineering, the half-life of therapeutic antibodies can be increased and their functions such as antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis can be controlled by modulating their recognition by effector cells of the immune system.⁹⁹ It is interesting to note that smaller-sized antibodies such as Fabs, scFvs, and nanobodies that can be obtained by phage display can penetrate cells and target intracellular viral proteins.¹⁰⁰ This means that passive antibody therapy with small antibodies could have valuable potential for targeting intracellular pathogens that escape the host immune response.

An alternative promising therapy for infectious diseases is gene therapy. The pathogen can be neutralized by supplying genes coding recombinant antibodies against the pathogen in a gene therapy vector, such as adeno-associated viral vectors (AVAs) to be expressed by the host's own cells (e.g., muscle cells).¹⁰¹ Currently, an antibody gene therapy is being evaluated in a phase I clinical trial (VRC 603), which is testing the safety of adeno-associated virus vector expressing an HIV-1 neutralizing antibody that was originally isolated from the blood of a person with HIV infection (VRC 603 trial is available on ClinicalTrials.gov using identifier NCT03374202).

Human B cell cloning might be the most common approach to identify neutralizing antibodies, and phage display is one of the techniques used to isolate the antibody repertoire from the convalescent B cells. Regdanvimab, an anti-SARS-CoV-2 antibody, is an example of phage display's effectiveness since it is a technique that can be used in any lab without requiring costly equipment, making it accessible to developing countries where the majority of emerging diseases occur and samples are easy to obtain. However, there are some drawbacks, such as the fact that it is a time-consuming process and there is a need to create a new library for each pathogen or disease. Another issue is the formation of artifacts during V_H and V_L combinations or the fact that certain bacterial clones grow faster than others. However, this can be avoided by continuously monitoring each step during library creation, for example by sequencing multiple clones and ensuring that the bacteria culture grows only for a short period of time.

Peptide vaccine is a promising alternative or complement to the already existing therapeutic approaches. However, more work is required to develop peptide vaccines before they can be used in clinical settings. The most difficult aspect of developing an epitope-based vaccine is determining the epitopes of interest with proper accuracy. Another challenge with peptide vaccine is the low immunogenicity which could be improved with nontoxic adjuvants. Another limitation is that multiple peptides may be required because a single peptide may not provide a sufficient immune response. Also, peptides that are susceptible to enzymatic digestion may need to be encapsulated and delivered with nanoparticles such as copolymer poly(lactic-co-glycolic acid) (PLGA).^102,103 The safety of the delivery approach is of utmost concern when selecting strategies for peptide delivery. However, PLGA is an FDA-approved polymer for use in drug delivery, diagnostics, and other applications that have been in the market for many years.

mRNA vaccines represent a new type of vaccine and they have several advantages over other vaccines including safety, efficacy, speed of production, and cost-effectiveness.¹⁰⁴ The mRNA enters the cells and is used to make viral antigen proteins from inside the cell, which involves natural, post-translational modifications. mRNA vaccine production does not require cell cultures and/or fermentation-based manufacturing processes. They can be naturally degraded with no metabolic toxicity and mRNA vaccines do not enter the nucleus. Some of their disadvantages include the fact that they are not as stable as DNA and proteins and are susceptible to degradation. Also, there are few studies on mRNA vaccines for bacteria and parasites. A major disadvantage of mRNA vaccines is their instability at high temperatures, which makes them unavailable in remote locations. Therefore, phage-based vaccines are a strategy worth further consideration, as phages are stable at high temperatures.

Henry et al. reviewed studies of the filamentous bacteriophages' properties, as well as their various applications in research.¹⁰⁵ Filamentous phage can be used to display polypeptides as fusion proteins with the major coat protein pVIII. In addition, each phage can display more than 2500 copies of pVIII, making it a good way to present peptides to the immune system. Phage-based vaccines are easy to develop and do not require expensive equipment, so they may be a useful tool for studying emerging and re-emerging pathogens in developing countries.

Conclusions

In conclusion, the antibody repertoire of recovering patients and the epitopes they recognized should be considered as a scientific priority for the development of therapeutic and prophylactic antibodies and vaccines against emerging pathogens. Several tools can be taken advantage of that can help us improve their efficacy and reduce their production costs, such as phage display, antibody engineering, and prokaryotic protein expression.