Subunit vaccines– vaccines from purified macromolecules

In this article, I briefly describe the types of subunit vaccines.

Table of Contents

Purified macromolecules as vaccines

Attenuated or inactivated whole-organism vaccines carry certain risks, prompting the development of alternative options that utilize specific purified macromolecules from pathogens. Currently, three main types of such vaccines are used: capsular polysaccharides, toxoids, and recombinant microbial antigens.

Capsular polysaccharides

The polysaccharide capsule of a bacterial cell is a prominent structure that often forms the outermost layer of the cell. It plays a key role in facilitating direct interactions between the bacteria and its surrounding environment. This capsule is a tightly organized layer, resistant to being washed away, and can contribute to developing various diseases. The virulence of certain pathogenic bacteria largely relies on the antiphagocytic properties of their hydrophilic polysaccharide capsule. However, coating the capsule with antibodies and/or complement significantly enhances the ability of macrophages and neutrophils to phagocytose these pathogens.

Vaccine development against Streptococcus pneumoniae

Encapsulated bacteria, including Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae serogroup B (Hib), significantly contribute to the disease globally. Vaccine development against these pathogens has focused on their capsular polysaccharides (CPS), as antibodies targeting the capsule often protect against disease. Streptococcus pneumoniae was independently discovered by Louis Pasteur and George Sternberg in 1880. It was later identified as the primary causative agent of pneumonia, meningitis, and other infectious diseases. In many underdeveloped nations, pneumonia caused by S. pneumoniae remains a leading cause of death among children under five and adults over 50 years old.

S. pneumoniae are encapsulated, aerotolerant anaerobic, gram-positive bacteria that are non-motile, non-sporulating, and capable of utilizing a wide range of carbohydrates as carbon sources. In 1916, Dochez and Avery successfully isolated the capsular polysaccharides of S. pneumoniae. A few years later, capsular antigens were identified as the basis for pneumococcal serotyping. By the 1930s, the critical role of pneumococcal CPS as a virulence factor was established, highlighting its immunogenic properties. The virulence and invasiveness of pneumococcal strains vary by serotype and depend on the chemical composition and quantity of CPS produced, which influence bacterial survival in circulation and the likelihood of causing invasive disease.

The purification of CPS is essential for the development of next-generation S. pneumoniae vaccines, which are composed of CPS alone or conjugated to proteins. Such vaccines are both effective and safe, though they remain costly. The current vaccine for S. pneumoniae, responsible for pneumococcal pneumonia, contains 23 antigenically distinct capsular polysaccharides. It induces the formation of opsonizing antibodies and is now included in the list of vaccines recommended for all infants.

Limitations of polysaccharide vaccines

Though polysaccharide vaccines have potential within them, they have certain limitations too. These vaccines are unable to activate T_H cells. Generally, polysaccharide (PS) antigens elicit a T cell-independent immune response, characterized by a lack of memory, and poor immunogenicity at the extremes of life. The poor immunogenicity of CPS vaccines has led to the development of conjugate vaccines.

When CPS is conjugated to carrier proteins, a T cell-dependent immune response is induced against these antigens. Haemophilus influenzae type b (Hib), causes bacterial meningitis in children less than 5 years of age. The vaccine for Haemophilus influenzae type b (Hib) consists of type b capsular polysaccharide covalently linked to a protein carrier, tetanus toxoid. The polysaccharide-protein conjugate is considerably more immunogenic than the polysaccharide alone and it activates T_H cells. Although this type of vaccine can induce memory B cells, it can’t induce memory T cells specific to the pathogen.

The Vi capsular polysaccharide vaccine (or ViCPS) is a typhoid vaccine recommended by the World Health Organization for the prevention of typhoid (another is Ty21a). It is a subunit vaccine and was first licensed in the US in 1994. This is made from the purified Vi capsular polysaccharide from the Ty2 Salmonella Typhi strain. It is important to repeat the vaccine every three years to maintain immunity. The Vi polysaccharide, or Vi antigen, is part of the bacterial capsule found outside of the typhoid bacterium, Salmonella enterica subsp. enterica ser. Typhi. It is produced by the action of a single gene cluster in the cytoplasm and transported to the surface. This antigen plays a main role in typhoid virulence and is important for the infection of intestinal epithelial cells.

Toxoids

A toxoid is a deactivated toxin, usually an exotoxin, that has undergone chemical treatment (such as with formalin) or heat treatment to neutralize its toxicity while preserving its ability to trigger an immune response. Certain bacterial pathogens, like those responsible for diphtheria, tetanus, and botulism, produce exotoxins.

Diphtheria is an infectious disease that starts with a sore throat and fever. The bacterium Corynebacterium diphtheria is the causative organism of the disease. The infection generally spreads between people by direct contact or through the air. It may also be spread by contaminated objects. A diphtheria vaccine is effective for prevention and is available in several formulations. Three or four doses, given along with tetanus vaccine and pertussis vaccine, are recommended during childhood.

Tetanus and Botulism

Tetanus is a bacterial infection that leads to muscle spasms. Some additional symptoms such as fever, sweating, headache, difficulty swallowing, high blood pressure, and a rapid heart rate are also seen. It is caused by the bacterium Clostridium tetani, which is commonly found in soil, saliva, dust, and animal manure. The infection typically occurs when the bacteria enter the body through a break in the skin, such as a cut or puncture wound from a contaminated object. The bacteria produce toxins that disrupt normal muscle function. Tetanus can be prevented through vaccination with the tetanus vaccine. For individuals with significant wounds who have received fewer than three doses of the vaccine, both vaccination and tetanus immune globulin are advised.

Botulism is a rare but potentially life-threatening condition caused by a toxin produced by the bacterium Clostridium botulinum. The spores of this bacterium are commonly found in soil and water. Under low oxygen conditions and specific temperatures, the spores produce the botulinum toxin. Foodborne botulism occurs when food contaminated with the toxin is consumed. Whereas, Infant botulism arises when the bacteria grow in the intestines and release the toxin. Clostridium botulinum is a large, anaerobic, Gram-positive bacillus that forms subterminal endospores. Although vaccines are being developed, they have limitations, including concerns about the potential for reversion to harmful native activity. Treatment for botulism typically involves the administration of botulism antitoxin and supportive medical care.

Toxin and toxoid

Bacteria secrete toxins and toxoids are altered forms of toxins. As bacteria do not secrete toxoids, when used during vaccination, an immune response is mounted and immunological memory is formed against the molecular markers of the toxoid without resulting in toxin-induced illness.

The bacterial exotoxin is purified to prepare diphtheria and tetanus vaccines. The toxin is then inactivated with formaldehyde to form a toxoid. Vaccination with the toxoid induces anti-toxoid antibodies, which are also capable of binding to the toxin and neutralizing its effects (figure 1). The tetanus toxoid is derived from tetanospasmin, a toxin produced by Clostridium tetani, the bacterium responsible for tetanus. Immunization against tetanus is provided through the DTaP vaccine.

**Figure 1: Immune response to exotoxin**

Although some individuals may report side effects following vaccination, these are typically linked to the body’s immune response and the clearance of the toxoid, rather than the toxoid itself. The production of toxoid vaccines requires precise control to ensure detoxification while preserving the structure of the epitopes. To facilitate this process, large amounts of exotoxin can be produced by cloning the toxin genes and expressing them in easily cultured host cells. It is followed by purification and inactivation.

Recombinant antigen vaccine

A recombinant vaccine is a vaccine produced through recombinant DNA technology. Live recombinant bacteria or viral vectors effectively stimulate the immune system as in natural infections and have intrinsic adjuvant properties. There are a variety of expression systems with different advantages, allowing the production of large quantities of proteins depending on the required characteristics. Many genes encode surface antigens from viral, bacterial, yeast, insect, or mammalian expression systems and the expressed antigens used for vaccine development. The first such recombinant antigen vaccine approved for human use is the hepatitis B vaccine.

Hepatitis B virus (HBV) infection is a widespread chronic liver disease characterized by its strong affinity for human liver cells. This tropism is partially attributed to the presence of a specific receptor on the surface of infected cells. The HBV vaccine was created by cloning the gene encoding the major surface antigen of the virus (HBsAg) and expressing it in yeast cells. These recombinant yeast cells are cultivated in large fermenters (figure 2), where HbsAg accumulates within the cells. The cells are then collected and broken apart using high pressure. They release the recombinant HBsAg, which is subsequently purified through standard biochemical methods.

**Figure 2: Preparation of Hepatitis B vaccine**

Another example of a recombinant vaccine is the one developed against human papillomaviruses (HPVs), which are among the most common sexually transmitted infections. HPV is associated with various mucocutaneous conditions, including cervical, vulvar, and vaginal cancers, as well as genital warts. Two vaccines currently available for HPV prevention were developed using virus-like particles (VLPs) derived from HPV subtypes 6, 11, 16, and/or 18.

Advancements and Challenges in Recombinant Vaccine Development

Recombinant vaccines rely on the ability of one or more specific antigens to stimulate immunity against a pathogen. They often do it with the aid of adjuvants or through expression by plasmids or non-pathogenic bacterial or viral vectors. These vaccines offer significant advantages over traditional macromolecule-based vaccines, such as eliminating the risks associated with co-purification of unwanted contaminants or the reversion of toxoids to their toxic forms, as seen with diphtheria and tetanus vaccines. However, a key challenge in developing recombinant vaccines lies in designing formulations that induce the appropriate immune response, particularly for intracellular pathogens that cause chronic, often lifelong infections.

Recombinant protein-based vaccines provide benefits such as enhanced safety and reduced production costs. However, they often exhibit low immunogenicity when administered alone, necessitating the use of adjuvants to generate a strong and durable immune response. The successful deployment of recombinant protein vaccines, such as those for hepatitis B and HPV, has been made possible through the use of aluminum salts as adjuvants. This highlights the critical importance of ongoing research into novel adjuvants as a vital area of study in vaccinology.

Conclusion

The three main types of subunit vaccines can be categorized as capsular polysaccharides, toxoids, and recombinant microbial antigens. The polysaccharide capsule of a bacterial cell is a prominent structure that often forms the outermost layer of the cell. It plays a key role in facilitating direct interactions between the bacteria and its surrounding environment. Coating the capsule with antibodies and/or complement significantly enhances the ability of macrophages and neutrophils to phagocytose these pathogens. Polysaccharide vaccines are unable to activate T_H cells. The Vi capsular polysaccharide vaccine (or ViCPS) is a typhoid vaccine recommended by the World Health Organization for the prevention of typhoid (another is Ty21a). It is a subunit vaccine and was first licensed in the US in 1994.

Bacteria secrete toxins and toxoids are altered forms of toxins. The bacterial exotoxin is purified to prepare diphtheria and tetanus vaccines. The toxin is then inactivated with formaldehyde to form a toxoid. The tetanus toxoid is derived from tetanospasmin, a toxin produced by Clostridium tetani, the bacterium responsible for tetanus.

A recombinant vaccine is a vaccine produced through recombinant DNA technology. Many genes encode surface antigens from viral, bacterial, yeast, insect, or mammalian expression systems. The expressed antigens are used for vaccine development. The first such recombinant antigen vaccine approved for human use is the hepatitis B vaccine.

Recombinant vaccines offer significant advantages over traditional macromolecule-based vaccines. They eliminate the risks associated with co-purification of unwanted contaminants or the reversion of toxoids to their toxic forms, as seen with diphtheria and tetanus vaccines. However, a key challenge in developing recombinant vaccines lies in designing formulations that induce the appropriate immune response, particularly for intracellular pathogens that cause chronic, often lifelong infections.