Glycoconjugate Vaccine: An Effective Way to Combat Infectious Diseases and Cancers

Abstract

Glycoconjugate is a molecule of carbohydrate covalently linked to another compound. In glycoconjugate vaccine, carbohydrate antigen is linked to another molecule, particularly a protein carrier. Vaccines targeting capsular polysaccharides can prevent bacterial infection. However, capsular polysaccharide alone is weak immunogenic as it produces a B cell immune response independent of T lymphocyte. To increase the immunogenicity, the capsular polysaccharide can be covalently linked to a protein carrier that converts carbohydrate antigen from T lymphocyte independent to T lymphocyte dependent antigen. Several carrier proteins such as tetanus toxoid (TT), diphtheria toxin (DT), the outer membrane protein complex (OMPC) of N. meningitides serogroup B, and Haemophilus protein D are currently used in licensed conjugate vaccines. The protein carrier in the glycoconjugate vaccine engages with T cell dependent immune response and the carbohydrate part engages with T cell independent immune response. The involvement of T cells in the immune response against the glycoconjugate vaccine helps in B cell proliferation and differentiation into memory B cell which is utmost important for long-term immunity. Carbohydrate structures decorated on the surface of pathogens and malignant cells can be considered as a key target in developing safe and effective vaccines to combat cancer, bacterial infections, viral infections.

1. Introduction

Vaccines since its first use played a crucial role in the prevention, control and eradication (such as smallpox in humans and rinderpest in animals) of diseases. The mass immunization program against the diseases for which vaccines are available save countless live and economics losses in both humans and animals. The recent example include mass immunization of humans against SARS-CoV-2 (COVID-19) helped to reduce the mortality and hospitalization numbers. As per WHO data, current mass vaccination in children saved 2–3 million lives of children every year as result of that mortality in children of less than 5 years of age reduced from 93 deaths/1000 live births in year 1990 to 39 deaths/1000 live births in 2018. Vaccine is defined as a biological product that induces specific immune response against specific antigen and protects the individual against that disease on subsequent exposure to similar antigen [1]. An antigen may be anything such as a whole pathogen, small component of a pathogen, toxins produce by pathogen, etc. In the beginning of vaccine development, whole pathogen in the form of either live inactivated or killed was being used. With the advancement of medical science and biotechnological techniques, instead of using whole pathogen a small component of pathogens (subunit vaccine) that have both antigenicity and immunogenicity are being used for vaccine development. Moreover, many other platforms are being used for vaccine development such as DNA vaccines, recombinant protein vaccines, mRNA vaccine etc. The basic principle behind the protection given by vaccine is that vaccine induces the immune response against specific whole pathogen (either live, inactivated or killed) or small component of pathogen and immune system of body create a memory of this exposure in form of memory plasma cell, so that if individual gets exposure to same pathogen, it respond quickly without any delay, and clear the infection as soon as possible [2]. Whereas the naïve immune system starts developing antibodies after 7–10 days of its first exposure to antigen, called as primary response. But this primary antibody response consists of antibodies of IgM isotype that have low affinity for antigen. However, in primary response antibody titer is not enough and consists of IgM that is not sufficient to eliminate the infection completely. So, it is possible that animal may die due to delay immune response. The immune system requires more time approximately several days to few weeks to produce antibodies of either IgG, IgA or IgE isotypes that have the same antigen specificity but have high affinity to antigen [3]. Thus, mass immunization has been proved to be a very effective and economical way to prevent and control the diseases.

The word immunity is derived from the Latin word “immunitas” which is a legal status of Roman city-states that grants immunity to individuals from paying tributes to Rome or immunity from prosecution. In the first century, the Roman poet Lucan described the Psylli of North Africa as immune to the bites of venomous snakes in the Roman poem De Bello Civile. Similarly, the term diplomatic immunity indicates immunity to foreign government officials in the jurisdiction of the host country. Diplomatic immunity was first time guaranteed in 1709 by the British Parliament under the Diplomatic Privileges Act to foreign ambassadors after Count Andrey Matveyev, a Russian resident in London, was harassed verbally and physically by British bailiffs. Immunity is referred to as the ability of the immune system to protect the body from harmful pathogens and other substances/antigens. The fundamental function of the immune system is to differentiate between self and non-self antigens. And then, the non-self antigen is neutralized or eliminated by the immune system. The immune system is divided into two subtypes i.e. innate immune system and adaptive (acquired) immune system. The innate immune system consists of physical barriers (such as skin and mucous membranes), physiological barriers (such as temperature and pH), inflammatory mediators (such as complement, cytokines, interferon, acute phase protein, leukotrienes, etc.) and cellular components (polymorphonuclear cells, neutrophils, eosinophils, basophils, mast cells, monocytes and macrophages, dendritic cells). Using pattern recognition receptors (PRRs), innate immune cells may identify pathogens and tissue injury. Toll-like receptors (TLRs), which are found on the cell surface and in endosomes, were the first to be identified and are the best characterized. There are also more PRRs, including C-type lectin receptors on the cell surface and Retinoic acid-inducible gene-I (RIG-I) and Nucleotide oligomerization domain (NOD)-like receptors in the cytoplasm. Innate immune system mediators are naturally present in a host since birth and are constitutive. However, innate immunity can also be induced, such as in the case of viral infection, virus-infected cells produce interferon that acts on non-infected cells and activates innate immunity against viral infection. The innate immune system is considered to be fast but rather nonspecific. The adaptive immune system consists of humoral and cell-mediated immune responses. The adaptive immune response is mediated by B lymphocytes and T lymphocytes. The humoral immune response is mediated by B lymphocytes and the cell-mediated response is mediated by T lymphocytes. In contrast to innate immunity, adaptive immunity has specificity in the recognition of foreign antigens by functional receptors residing on the cell surfaces of B lymphocyte (B cell receptor) and T lymphocyte (T cell receptor). An individual can acquire adaptive immunity either by direct contact of antigen with the immune system that leads to an immune response (either humoral or cell-mediated), called active immunity or by the acquisition of pre-formed antibodies and immune-reactive lymphocytes from another individual. Active adaptive immunity remains longer period (a few years) or sometimes gives lifelong immunity, whereas passive adaptive immunity remains for short period (a few weeks or months).

2. History of vaccines and immunization

Edward Jenner, an English Physician who discovered the smallpox vaccine using cow pox virus in year 1796, is considered as father of vaccine. Jenner collected matter from a cowpox sore on the hand of a milkmaid and inoculated into 8-year-old boy named James Phipps. Initially, James Phipps suffered from local reaction and ailing for several days but recovered soon and got lifelong immunity from smallpox. However, there are several evidence which show that the immunization practice has been started much before the Edward Jenner. In fifteenth century, people in different parts of world used to expose themselves intentionally to smallpox (by inhaling the crushed scab) to prevent the disease. In seventeenth century the Buddhist monks in China used to follow the practice of drinking snake venom to get immunity against the snake bite. Lady Mary Wortley Montagu brought smallpox inoculation to Europe in 1721. It is Benjamin Jesty, who in year 1774 tested his hypothesis that a cowpox virus when inoculated to human gives protection against smallpox. In May 1796, English Physician Edward Jenner expands on this discovery and inoculates 8-year-old James Phipps. Two months later, in July 1796, Jenner inoculates Phipps with matter from human smallpox sore in order to test Phipps’ resistance. Phipps remains in perfect health and becomes the first human to be vaccinated against smallpox. The term ‘vaccine’ is later coined, taken from the Latin word for cow, vacca. It is very difficult to forget the achievements of Louis Pasteur in the development of vaccines. In 1872, despite enduring a stroke and the death of two of his daughters due to typhoid, Louis Pasteur creates the first laboratory-produced vaccine for fowl cholera in chickens. In 1885, Louis Pasteur was able to prevent the rabies successfully through 13 injections of post-exposure vaccination to 9 year old boy named Joseph Meister by using formalin inactivated rabies virus. Since then, many vaccines have been developed and many are in developing phase. On 30 January, 2020 the WHO Director General declares the outbreak of novel coronavirus 2019 (SARS-CoV-2) to be a Public Health Emergency of International Concern. On 11 March, WHO confirms that COVID-19 is a pandemic. Effective COVID-19 vaccines are developed, produced and distributed with unprecedented speed, some using new mRNA technology. In December 2020, just 1 year after the first case of COVID-19 was detected, the first COVID-19 vaccine doses are administered. The vaccine development usually take 10 to 15 years including clinical trials, however, it is the first time in history when COVID-19 vaccine was developed and administered to the patient in one year. The reason for this was that different phases of clinical trials overlap with each other.

3. Immunogenic vaccine antigen

Antigen is a substance (molecule) when introduced into the body induce a specific immune response (either humoral or cellular) or capable of binding with products of an immune response such as antibodies or lymphocytes [4]. Examples of antigens include proteins, carbohydrate, lipids, nucleic acid, toxins, any foreign particle (non-self-antigen) and sometimes bodies own tissue and cells (self-antigen). All immunogens are antigen but it is essential that all antigens are immunogens. For better understanding, few definitions are given below:

Immunogen: It is an antigen which is capable of inducing a specific immune response, called as immunogen. They can mobilize immune system and provoke immune response. Incomplete antigen: It is an antigen which can bind with specific antibody but unable to induce immune response by its own. These types of antigen need the help of other carrier molecule to behave as a complete antigen. They are also known as hapten. Autoantigens: There are some proteins such as lens proteins, sperm protein, myelin basic protein, thyroglobulin, kidney protein and some heart muscle protein that never participated in the process of immunogenic tolerance. Therefore, these proteins are recognized as foreign by T and B cells such that immune response is produced. Immunogenic tolerance to self-antigen is acquired by clonal deletion or inactivation of developing lymphocytes. Allo-antigens: These antigens are individual specific antigen present in one individuals but not in other. Examples of these antigens are blood group antigen and graft rejection. Heterophilic antigen: Antibodies produced by one antigen binds cross react with another antigen then such types of antigen are called as heterophilic or cross reacting antigen. For example, antibodies produced against Rickettsia bind with some Proteus species. Similarly, antibody produced against M protein of Streptococcus pyogens cross reacts with heart muscle protein of human. Super antigens: These types of antigens stimulate and cause proliferation of large fraction of T lymphocytes in non-specific manner. For example Staphylococcus enterotoxins, shock toxins, exfoliating toxin, pyrogenic exotoxins. Antigenicity: It is the ability of foreign molecule to combine specifically with products of immune response such as antibody or lymphocytes, is known as antigenicity. Immunogenicity: It is the ability of the foreign molecule (antigen) to induce immune response. Immunogenicity of an antigen depends upon four factors; how foreign an antigen compared to body, molecular size (antigen of large the molecular size is more immunogenic; >10,000 dalton), chemical composition (decreasing order of immunogenicity; Proteins > carbohydrate > Lipid > nucleic acids) generally do not act as antigen (immunogen) unless they are complexed with protein or carbohydrates) and their ability to be processed and presented on the surface of antigen presenting cells (APCs) such as dendritic cells, macrophage and B-cell. Some antigen needs the help of T cell for the production of antibodies. These types of antigens are called T cell dependent or thymus dependent antigen. Thymus dependent antigen induce both humoral as well as cell mediated immune response. On the other hand some antigen induces the antibody production without the help of T cell are called T cell independent or thymus independent antigen. Thymus independent antigen induces only humoral immune response. Antigens can be classified into exogenous antigen and endogenous antigen based on their origin. Exogenous antigens originate from outside and are foreign to host body. These antigens enter to the body either through inhalation, ingestion or injection. Whereas, endogenous antigens originate inside own body. These antigens are body’s own tissues or cells or sub fragments or compounds or the antigenic products that are produced as a result of normal cell metabolism, or because of viral or intracellular bacterial infection. In vaccine production, mainly four types of antigens are used viz.; live inactivated whole pathogen, killed whole pathogen, toxoids and small component of whole pathogen.

4. Immune cells involved in immunization

First, it is very important to understand the different components of immune system. The immune system can be divided into two main subsystems; the innate immune system and acquired/adaptive immune system. The effective immune system is the result of interaction and co-ordination between innate and acquired immune system. There are four main differences between innate and acquired immune system. The first difference is that innate immune system is effective by birth, whereas, effective adaptive immune system develop with time as body acquires natural infections. The second difference is that innate immune system act in non-specific manner, whereas, adaptive immune system act in specific manner for specific pathogen/antigen. In literal meaning, innate immune system uses same weapon to all pathogen, whereas acquired immune system uses different weapons to different pathogen. The third difference is that innate immune system keeps no memory of antigen exposure, whereas adaptive immune system keeps memory of antigen exposure. The fourth difference is that the innate immune system is very fast (takes minutes to hours to respond) compared to adaptive immune response (takes few days to weeks to respond). However, the adaptive immune system has memory which means that the adaptive immune system will respond more rapidly to that particular pathogen with each successive exposure. The innate immune system includes anatomic barriers such as intact skin and mucous membranes, physiologic barriers as the normal body temperature, fever, gastric acidity, lysozyme, interferon, and collectins. The complement pathways are also a part of the defensive measures of the innate immune system. The inflammatory response is another essential part of the innate immune response. The inflammatory response allows products of immune system into area of infection or damage. The adaptive immune response is composed of the B cells/antibodies and T cells. Natural killer cells are also from the lymphocyte lineage like B cells and T cells; however, natural killer cells are only involved in innate immune responses. For an immune response to be effective, both the innate and adaptive immune systems must function. B cells that make antibodies (humoral immunity) and T cells (cellular immunity) are the mediators of the adaptive immune response. Additionally, in order for immunization to be effective, effector cells and memory cells need to be produced in order to cause long-term stimulation of the adaptive system’s humoral and cell-mediated arms. The body must first recognize the threat, whether it be a pathogenic agent or an immunization, as with any challenge to the immune system. Although B cells may also carry out this initial detection, the innate immune system typically handles this task. When the immune system detects antigen epitopes, the detection process starts. Small areas on antigens called epitopes mimic immune recognition. The innate immune system’s various components will then react to this threat. These innate immune system elements will opsonize or bind to the pathogen, assisting antigen-presenting cells like macrophages or monocytes in engulfing it. The pathogenic agent’s antigens will then be processed by these antigen-presenting cell(s), and they will be added to the surface of the antigen-presenting cell along with the MHC protein. If the antigen is viral or endogenous, the MHC-I protein will bind to it and the antigen-presenting cell will present it to a CD8+ T cell, which is likely to result in cell-mediated immunity. If the antigen is bacterial or parasitic or other exogenous antigen, MHC-II protein will bind to it and the antigen will be presented by the antigen-presenting cell to a CD4+ T cell, likely inducing antibody-mediated immunity. The majority of current vaccines are thought to confer protection primarily through the induction of antibodies, with the exception of BCG (which is thought to induce T cell responses that prevent severe disease and innate immune responses that may inhibit infection). There is a number of evidence to support the idea that different types of functional antibodies play a crucial role in vaccine-induced immunity, and it primarily comes from three different places: studies of passive immunity, immunological data, and immunodeficiency conditions.

5. Vaccine types

Vaccines are generally classified as live or non-live/ inactivated. Live vaccines contain those attenuated replicating strains of the relevant pathogenic organism. Non-live/inactivated vaccines contain killed whole organisms. Several other platforms, such as viral vectors, nucleic acid-based RNA and DNA vaccines, and virus-like particles, have been created over the past few decades in addition to the “traditional” live and non-live vaccines. It’s crucial to understand the difference between live and non-live vaccines. The live vaccines may have the potential to replicate uncontrollably in immune-compromised individuals resulting in some limitations on their use. Non-live vaccines, on the other hand, pose no risk to immune-compromised individuals. Example of live vaccines include measles, mumps, rubella and rotavirus vaccines, oral polio vaccine, BCG vaccine, and live attenuated influenza vaccine. Inactivated vaccines, on the other hand, do not always elicit as strong or long-lasting immune responses as live attenuated vaccines. Examples of inactivated vaccines include inactivated Polio vaccine, Hepatitis A vaccine, etc. In subunit vaccines, which contain no whole bacteria or viruses. Instead, these vaccines often include one or more particular pathogen-surface antigens. Subunit vaccinations provide an advantage over complete pathogen vaccines in that the immune response can concentrate on identifying a limited set of antigen targets. Subunit vaccinations frequently fail to elicit the same robust or durable immune response as live attenuated vaccines. Initially, they often call for repeated dosages, followed by booster dosage the following year. Subunit vaccinations frequently have adjuvants added. Adjuvants are the substances that support and prolong the immunological response to the vaccine. Aluminum salts (alum) have been extensively used as adjuvants for more than 80 years (HPV). The oil-in-water emulsion MF59, AS01 and AS04, are examples of novel adjuvants. As a result, with these kinds of immunizations, typical local reaction could be more obvious and frequent. In recombinant vaccines, a tiny fragment of DNA from the virus or bacterium that we want to protect ourselves from is obtained and introduced into the production cells. For instance, a portion of the DNA from the hepatitis B virus is introduced into the DNA of yeast cells to create the hepatitis B vaccine. Once one of the hepatitis B virus’s surface proteins is produced by these yeast cells, it is purified and employed as the vaccine’s active component. Recombinant vaccine examples include MenB vaccine, HPV vaccine, and hepatitis B vaccine. This has proteins from the outer layer of meningococcal bacteria. The recombinant method was used to create three of the proteins. Toxins generated by bacteria are inactivated in toxoid vaccinations using formalin or heat to lessen pathogenicity and are used as vaccine. These toxins can elicit an immune response despite being inactive and safe. Diphtheria, tetanus, and pertussis (whooping cough) vaccines are a few examples of toxoid vaccines. Toxoid and surface-derived proteins from the pertussis bacteria are both included in the pertussis vaccine. The vaccination is frequently described as “acellular.” In virus-like particles (VLPs), viral genetic material is absent which are entities that closely resemble viruses but are not infectious. They exist naturally or are created through individual expression of viral structural proteins, after which they self-assemble into forms that resemble viruses. In other circumstances, the viral structural proteins themselves serve as VLP vaccination antigens. As an alternative, VLPs can be created to display antigens from many diseases on their surface or to do so simultaneously. Due to the fact that each VLP includes numerous copies of an antigen on its surface, it can more efficiently elicit an immune response than a single copy. Outer member vesicles (OMV) vaccines are a more recent development in vaccine technology. Membrane vesicles (OMVs), which contain many of the antigens on the cell membrane, are naturally produced by bacteria. To create vaccines, these OMVs can be extracted from bacteria. The OMVs can also be altered to keep the antigens that are good at triggering an immune response while removing the harmful antigens. OMVs also function as adjuvants by nature. The MenB vaccine is authorized to use this technology. In contrast to conventional vaccines, nucleic acid vaccines do not provide the protein antigen, thus they operate differently. Instead, they impart the genetic code for the antigen to body cells, which then produce the antigen and trigger an immune response. Nucleic acid vaccines are quick and easy to develop and provide significant promise for the development of vaccines in the future. RNA vaccines and DNA vaccines are the two categories into which nucleic acid vaccines fall. In an RNA vaccination, messenger RNA is enclosed in a lipid membrane. When the mRNA first enters the body, its fatty layer protects it. However, it also facilitates entry into cells by joining with the cell membrane. The mRNA is translated into the antigen protein by internal cell machinery once it has entered the cell. Although this mRNA only persists for a few days on average, enough antigen is produced during that period to elicit an immunological response. The body then naturally breaks it down and eliminates it. RNA vaccines are unable to interact with the genetic code of humans (DNA). Currently, the UK has approved the use of two RNA vaccines for emergency situations. The COVID-19 vaccines from Moderna and Pfizer BioNTech are both made of RNA. DNA vaccines do not need the same initial protection because DNA is more stable than mRNA. DNA vaccinations are frequently given combined with a process known as electroporation. This enables the body’s cells to absorb the DNA vaccination by using low-frequency electrical waves. Before DNA can be translated into protein antigens that trigger an immune response, it first needs to be transcribed into mRNA in the cell nucleus. Although there are several DNA vaccines being developed, there are currently no licensed DNA vaccines available for commercial use. A more recent development in vaccine development is the use of viruses to carry the genetic code of the target antigens of the vaccine to body cells, where the cells can then create protein antigens to elicit an immune response. Viral vectored vaccines can be produced rapidly and easily on a wide scale since they can be generated in cell lines. When compared to nucleic acid vaccines and many subunit vaccines, viral vectored vaccines are typically produced at a significant cost savings. There are two types of viral vectored vaccines, depending on whether a replicating or non-replicating vector was utilized. When utilized as a platform for vaccine delivery, replicating viral vector vaccines retain the capacity to produce new viral particles in addition to delivering the vaccine antigen. This replicating virus, like live attenuated whole pathogen vaccines, has the intrinsic benefit over non-replicating vaccines in that it may supply a continuous stream of vaccine antigen over an extended period of time, which is likely to result in a higher immune response. Protection may be provided by a single vaccination. Replicating viral vectors are often chosen so that the viruses are attenuated or harmless, preventing disease while they are infecting the host. Despite this, there is a higher likelihood of moderate adverse reactions with these vaccines since viral replication is still occurring. Recombinant vesicular stomatitis virus is used in the Ebola vaccine Ervebo (rVSV-ZEBOV). Over 90,000 people were protected by this vaccination during several Ebola outbreaks in Europe in 2019 after it received approval. The vaccine has mostly been employed in “ring vaccination,” which immunizes a person’s close contacts in order to stop the virus from spreading. When a vaccine uses a non-replicating viral vector, the vector nevertheless has the capacity to produce new virus particles while delivering the vaccine antigen to the cell. This is due to the deletion of essential viral genes required for viral replication. The vaccine cannot induce disease and adverse events linked to viral vector replication are also decreased. But only while the initial vaccine is still present in infected cells, vaccine antigen can be generated (a few days). Accordingly, booster doses are probably necessary because the immune response is typically less than with viral vectors that can replicate themselves. A non-replicating viral vector with the name of ChAdOx1 is also used in the Oxford-AstraZeneca COVID-19 vaccine, which was authorized for use in emergency situations. The conjugate vaccine type is another challenging area with a numbers of successful vaccines available commercially. In conjugate vaccine, a hapten (polysaccharide or other molecules) is coupled to a carrier molecule. The polysaccharide is joined to diphtheria or tetanus toxoid protein (carrier) in the majority of conjugate vaccines which are specifically termed as glycoconjugate vaccine. These carrier molecules merely make the hapten more visible to the immune system. These proteins are relatively simple for the immune system to recognize, which contributes to a greater immunological reaction to the polysaccharide. MenC vaccine, Pneumococcal (PCV) vaccine, MenACWY vaccine, and Typhoid Conjugate Vaccine (TCV) are a few examples of glycoconjugate vaccines. Glycoconjugate vaccines are discussed further in detail. List of various platforms for vaccine development included in the Table 1.

S. N.	Platform	Example
1	Live attenuated whole pathogen	Measles, mumps, rubella, yellow fever, influenza, oral polio, typhoid, Japanese encephalitis, rotavirus, BCG, varicella zoster
2	Killed whole pathogen	Whole-cell pertussis, polio, influenza, Japanese encephalitis, hepatitis A, rabies
3	Toxoid	Tetanus, diphtheria
4	Subunit vaccine	Pertussis, influenza, hepatitis B, meningococcal, pneumococcal, typhoid, hepatitis A, SARS-CoV-2
5	Virus like particle (VLP)	Human papillomavirus, hepatitis B virus
6	Protein-polysaccharide conjugate	Haemophilus influenzae type B, pneumococcal, meningococcal, typhoid
7	Viral vectored	Ebola, SARS-CoV-2
8	Outer membrane vessicle	Group B meningococcal
9	RNA vaccine (mRNA)	SARS-CoV-2
10	DNA vaccine	SARS-CoV-2, West Nile Virus in horses, Melanoma vaccine for dogs
11	Bacterial vectored	Experimental
12	Antigen presenting cells	Experimental

Table 1.

Different platforms used for vaccine development.

6. Glycoconjugate vaccine

Glycoconjugate vaccines are formed when a polysaccharide (mostly bacterial) is covalently linked with a protein providing epitopes for T lymphocyte which are required in the germinal centers for the affinity maturation of polysaccharide-specific B lymphocytes. Research has been shown that the bacterial conjugate vaccines can be used in all the age grouped including infants, adolescents, and the elderly, and are found to be among the safest and most successful vaccines developed during the last 40 years [5]. The theory of conjugate vaccine (glycans covalently linked to immunogenic proteins) was studied for the first time by Avery in the year 1931, and was introduced in the area of Antibacterial Vaccine in 90s [6]. There is a positive benefit of the glycoconjugate vaccines which when taken up by antigen-presenting cells, the conjugate molecule gets digested and the covalently-linked fragments of both polysaccharides and proteins are able to bind with the major histocompatibility complex II (MHC-II). This is then presented to T lymphocytes which in turn results to isotype switching from IgM to IgG and also induce B cell differentiation into memory cells, together act as a good vaccine. Tumor markers such as gangliosides, sialic acid-containing glycosphingolipids with extracellular polysaccharide head groups are expressed at high levels on the surface of cancer cells which are readily available to interact with the immune cells in turn acts as an antigen for cancer cells and becoming a potential cancer vaccine target. Though they have been proved to be a poor antigen but when synthetic antigen of gangliosides linked to polyamidoamine scaffolds to induce responses in γδ T lymphocyte receptor and CD8+ phenotypes was found to be therapeutic. For a glycoconjugate vaccine to be produced, each parts are prepared individually followed by conjugation of both the components into one molecule where only the polysaccharide part remains unique for each given vaccine. Traditionally a small group of proteins is used after inactivation of toxins that is toxoids. Toxins produced by some pathogenic cells or bacteria (can be used as bio-factories), are inactivated with formaldehyde or any other inactivating agents shown strong antigenicity and safe, for example tetanus toxoid (TT) has been used since 1924 till today. There is also possibility to use relatively nontoxic mutants including Cross Reactive Material 197 (CRM197) than the diphtheria toxoid (DT) [7]. The mechanism of how glycoconjugate vaccine works are briefed in the Figure 1.

Figure 1.

Proposed mechanism of action of glycoconjugate vaccine to induce immune response.

7. Glycoconjugate vaccine production

There are three pathways for glycoconjugate vaccine production including:

1. Coupling of monofunctional oligosaccharides (after depolymerisation of the parent polysaccharide) or bifunctional oligosaccharides at low coupling efficiency, and either through direct attachment to the carrier protein or indirect attachment through a linker.

2. Activation of higher molecular weight polysaccharides without depolymerisation and conjugation through non-specific chemistry to multiple carrier proteins to give a very high molecular weight complex of >1 MDa size.

3. Reduced mass polysaccharides with multiple activations coupled to LPS-depleted outer membrane protein (OMP) vesicles.

Though the most of the protein carriers are related to toxins, there are also a various known proteins which can be used as carrier including keyhole limpet hemocyanin is being used as a carrier for glycoconjugate vaccines against Candida albicans [8]. Bovine serum albumin (BSA) which is a well characterized protein is also used as carrier for glycoconjugate vaccine against Aspergillus fumigates [9]. The polysaccharide part on the other hand is produced enzymatically rendering polydispersity which again depends on the kinetics, thermodynamics, and relative proportions of subunits and enzymes during the polysaccharide production. It is also possible to synthesize the small fragments in vitro. Although advances have been made for synthesis and fractionation where polysaccharides are near-monodisperse. After both the components are ready, then the step includes is the covalent linking. As the surface of each molecule possesses a number of functional group with a potential to generate a covalent link under the right conditions. One way to reduce the polydispersity is to target the formation of specific bonds which has been described by Adamo et al. [10]. As a vaccine to be effective, the first and one of the important trait is the stability of the each components and their covalent linker, although the most important component which needs to be stable is the polysaccharide so that it is able to mimic accurately the structure of the target antigen, whereas the protein only needs to be recognized as non-self by the antigen presenting cell (APC). To learn about the integrity of the conjugated vaccines, there are physicochemical methods including high field nuclear magnetic resonance (NMR) spectroscopy [11], analytical ultracentrifugation (AUC) [12], which have been proven to be important in determining their molecular integrity.

8. Carriers of glycoconjugate vaccine

The most widely used carriers are protein based. The commonly used carrier proteins are TT (tetanus toxoid), DT (diphtheria toxoid), OMPC (outer membrane protein complex of Neisseria meningitides serogroup B), Haemophilus protein D, and CRM197 (Cross Reactive Material 197, mutant of DT). Studies showed that the stability of these carriers have negative effect when stored at −20°C, while they remain stable at 2–8°C storage temperature [7, 13]. A study performed by Togashi et al. [14] to compare the TT and CRM197 carriers for PRP (Polyribosyl Ribitol Phosphate) showed no significant differences, but it was observed that the CRM197 conjugate has higher local reaction. In another study by Akeda et al. [15] showed that the CRM197 has higher bactericidal action in comparison with TT. When the tri-component synapse of processed antigen; MHC- II; and T cell receptor is formed, the T helper cell provides stimulatory and cytokine-mediated signals to B-cells to release high affinity immunoglobulin G (IgG) and also memory B cells which gives a long-term immune response [6]. The other type of carriers which is also used for glycoconjugate vaccines is oligodeoxynucleotide and lipid carriers which targets TLRs (Toll-like receptors). Research is going on to find out the effectiveness of this carrier. The CpG-ODN (cytosine-phosphate-guanosine-oligodeoxynucleotides) has been used as an external adjuvant in a polysaccharide-protein conjugated vaccine to increase antibacterial immune response against S. pneumoniae polysaccharide types 19F and 6B [16]. The co-administration of CpG with H. influenzae type b (Hib) polysaccharide conjugate vaccine on mice model showed to increase the neutralizing antibody titer against both the polysaccharide and the Hib [17]. The N. meningitidis monophosphorylated lipid A (MPLA) conjugated with CPS of N. meningitides serotype C was evaluated in a mice model to learn the immunogenicity of MPLA conjugate. The MPLA glycoconjugates which were inoculated as liposomal formulations showed greater immunity as compared to the traditional protein glycoconjugates including adjuvant [18]. On the other side, when the tetrasaccharide of Mycobacterial LAM (lipoarabinomannan) was conjugated to the primary position of glucosamine residue of MPLA showed a robust IgG response in mice, indicating the structure of the linker and the conjugation site of the carbohydrate antigen epitope on MLPA has a key role to play in the immune response [6, 19]. The third type of carrier is nanoglycoconjugate where glycol-liposomes has been considered as a good alternative of covalent conjugation of protein and bacterial saccharide antigen [20]. The work of Hassane et al. [21] is a good example of use of nanoglycoconjugate carrier for Shigella flexneri vaccine formulation. In their studies, they have used synthetic liposomes with two sets of S. flexneri 2a synthetic pentasaccharides which are B cell epitopes mimicking the O antigen of S. flexneri and universal T_H epitope from hemagglutinin (HA) 307–319 of Influenza virus. It showed effective antibody response against the native lipopolysaccharide in vivo.

9. Advances in glycoconjugate vaccine

Recently there are glycoconjugate vaccines which are successfully licensed worldwide against H. influenzae; meningococcus serogroups A, C, and ACWY; pneumococcus serotypes 10 to 13; and Salmonella typhi. The list of licensed glycoconjugate vaccines are mentioned in the Table 2. Altogether these vaccines has made a history by reducing the global infant mortality and morbidity by eliminating some of these diseases including, meningococcus C eliminated from the United Kingdom after a huge vaccination campaign in 1999, and outbreaks of meningococcus A has also been eliminated from the African meningitis belt, resulting in great reduction in the global occurrence of bacterial meningitis and pneumonia [5]. Borja-Tabora et al. [22] and Holme et al. [23] experimented using a meningococcal vaccine (Men) with the serotypes A, C, W and Y polysaccharides which were either linked with TT (tetanus toxoid) as a glycoconjugate or as a polysaccharide only vaccine. In both the experiments, they have found higher and persistent antibody response where the patients were vaccinated with conjugate rather than polysaccharide only vaccine. Ramasamy et al. [24] in their experiment using conjugated and polysaccharide vaccines, observed differences in the antibody titer. They have observed that the polysaccharide vaccine has higher efficacy against meningococcus serotype C strains, whereas the conjugated vaccine showed higher efficacy against serotype W. They have concluded that these differences may be because of the lower titer of polysaccharide C which was conjugated with the CRM197 protein. Rothstein et al. [25] performed experiment using various combinations of conjugated and unconjugated vaccines against Haemophilus influenza type b on 7–15 month old infants with three time inoculations. They have observed that there was similar mean antibody titer when used all the three inoculations with conjugated vaccine, and when only the last inoculation was replaced with pure polysaccharides. On the other hand, there was decreased antibody titer when two inoculations were replaced with polysaccharide. These results tell us that the glycoconjugate vaccines show good antibody response as compared to polysaccharide vaccines. The human immunodeficiency virus (HIV) has been a curse in the humankind since decades. After years of research it is becoming more challenging to develop an effective vaccine against HIV. Question is whether this glycoconjugate vaccine can be a solution for this challenging disease. It was found that a good percentage of the broad neutralizing antibodies (bNAbs) in HIV-1 infected patients are against a dense high mannose region on envelope glycoprotein gp120 termed as high mannose patch (HMP) [26]. There have been many attempts to find out a suitable glycoconjugate vaccine with the principle of epitope-focusing targeting the HMP region of the HIV. McLellan et al. [27] identified that the PG9 antibodies made contacts with two glycans rich in mannose at Asn160 and Asn156 and a contiguous V1V2 (first and second variable loop of gp120) peptide b-strand. The other bNAbs including PGT121–123, 125–128 all target the V3 (third variable loop) region of gp120 [28, 29]. The V3 region of HIV-1 possesses three important N-linked glycosylation sites at the position N295, N332, N301 which are recognized by bNAbs. The synthetic V3 glycopeptide with high-mannose N-glycan at Asn332 was able to induce glycan dependent Ab responses in immunization studies in animals. In the follow-up experiment with a synthetic self adjuvating three-component immunogen made up of a 33-mer V3 glycopeptide epitope, a universal T-helper epitope P30, and a lipopeptide-based TLR-2 ligand showed glycan-dependent antibodies with a broader recognition of HIV-1 gp120 in comparison to the nonglycosylated V3 peptide. These observations indicating that the self adjuvating synthetic glycopeptide can be used as an important component to induce glycan-specific antibody response in HIV vaccine design [30]. Cancer is an another great havoc for the humankind. The tumor-associated carbohydrate antigens (TACAs) are considered as an important anticancer epitopes and have been targeted for anticancer glycoconjugate vaccines. Few of the TACA-based conjugate vaccines have reached randomized Phase III trials for melanoma, breast cancer, and nonsmall-cell lung cancer (including theratope, OPT822, GM2-KLH, racotumomab, and GD2-directed monoclonal Ab). On the other hand, the fully synthetic glycosphingolipid Globo-H epitope conjugated to CRM197 carrier was found to be more efficient in inducing IgG Ab production compared with KLH conjugates and also showed cross-reaction with Globo-H and Globo-H-related epitopes like SSEA3 and SSEA4 [31]. There is a high expression (around 100 times more than the normal) of mucin (MUC1) on the tumor cells, indicating MUC1 glycopeptides as an attractive target for cancer immunotherapy. Yin et al. [32] evaluated MUC1 peptides conjugated with bacteriophage Qb showed significant immune response against glycopeptides. In another study by Wu et al. [33], they have used a short synthetic Tn-nonapeptide of MUC1 (SAPDT*RPAP, * denotes glycosylation) conjugated with the bacteriophage Qb carrier showed higher anti-MUC1 IgG antibodies in immune-tolerant human MUC1 transgenic mice. These antibodies also showed high tumor binding and killing activities, good selectivity in glycopeptide recognition, and excellent recognition of human breast cancer over normal mammary tissues.

Vaccine (manufacturer)	Licensed in (year)	Target organism	Carrier protein
Pedvax-Hib (Merck Sharp & Dohme Corp, USA)	1990	Haemophilus influenzae	OMP
ActHib (Sanofi Pasteur SA, France)	1993	Haemophilus influenzae	TT
Menactra (Sanofi Pasteur Inc., USA)	2005	Neisseria meningitidis	DT
Hiberix (GSK, Belgium)	2009	Haemophilus influenzae	TT
Menveo (GSK, Italy)	2010	Neisseria meningitidis	CRM197
Prevnar 13 (Pfizer, USA)	2010	Streptococcus pneumoniae	CRM197
Typbar-TVC (Bharat Biotech Ltd., India)	2019	Salmonella typhi	TT
MenQuadfi (Sanofi Pasteur Inc., USA)	2020	Neisseria meningitidis	TT

Table 2.

Licensed glycoconjugate vaccines.

10. Conclusion

In the development of glycoconjugate vaccine, many factors play a crucial role such as saccharide size, carrier protein, conjugation chemistry and formulation. The methods are needed for faster identification of desired saccharide molecule on surface of pathogen, their characterization and their production in laboratory. The depth knowledge and extensive research of these factors will help in development of more effective glycoconjugate vaccines in short period of time. Moreover, we can expand their uses for cancer therapy. As we already have some licensed glycoconjugate vaccines for few infectious diseases, and for cancer therapy, there is ongoing phase lll trial; altogether we can see that this glycoconjugate vaccine has a great potential for some incurable disease conditions. In future to come up with effective glycoconjugate vaccines, we need more qualitative research and a good source of funding to solve the present disease scenario.

Conflict of interest

The authors declare no conflict of interest.

Abbreviations

Ab	antibody
APC	antigen presenting cell
AS	aluminum salt (Alum)
AUC	analytical ultracentrifugation
BCG	Bacillus Calmette Guérin
bNAbs	broad neutralizing antibodies
BSA	bovine serum albumin
CD	cluster of differentiation
CpG-ODN	cytosine phosphate guanosine oligodeoxynucleotides
CPS	capsular polysaccharide
CRM	cross reactive material
DNA	deoxyribonucleic acid
DT	diphtheria toxin
HA	hemagglutinin
Hib	H. influenzae type b
HIV	human immunodeficiency virus
HMP	high mannose patch
HPV	human papillomavirus
IgM	immunoglobulin M
LAM	lipoarabinomannan
LPS	lipopolysaccharide
MenB	meningococcus B
MHC	major histocompatibility complex
MPLA	monophosphorylated lipid A
mRNA	messenger ribonucleic acid
NMR	nuclear magnetic resonance
NOD	nucleotide oligomerization domain
OMPC	outer membrane protein complex
OMV	outer member vesicle
PCV	pneumococcal vaccine
PRP	polyribosyl ribitol phosphate
PRR	pattern recognition receptor
RIG-I	retinoic acid-inducible gene-I
ROS	reactive oxygen species
TACAs	tumor-associated carbohydrate antigens
TCV	typhoid conjugate vaccine
TT	tetanus toxoid
TLR	Toll-like receptor
VLP	virus like particle
WHO	World Health Organization