perform therapeutically essential post-translational modifications and express properly folded, glycosylated and functionally active proteins. however, efficacy of mammalian cells may be limited by epigenetic gene silencing and aggresome formation (recombinant protein aggregation). for mammalian cells, synthesised proteins were reported to be secreted into chemically defined media, potentially simplifying protein extraction and purification. the most prominent example under this class is chinese hamster ovary (cho) cells utilised for the synthesis of recombinant varicella zoster virus surface glycoprotein (ge) antigen for shingrix. cho cells are recognised for rapid growth and their ability to offer process versatility. they can also be cultured in suspension-adapted culture in protein-free medium, hence reducing risk of prion-induced contamination. baculovirus (insect) cells schematic representation of baculovirus structure and infection cycle. the baculovirus-insect cell expression system has the ability to express a variety of recombinant proteins at high levels and provide significant eukaryotic protein processing capabilities, including phosphorylation, glycosylation, myristoylation and palmitoylation. similar to mammalian cells, proteins expressed are mostly soluble, accurately folded, and biologically active. however, it has slower growth rate and requires higher cost of growth medium than bacteria and yeast, and confers toxicological risks. a notable feature is the existence of elements of control that allow for the expression of secreted and membrane-bound proteins in baculovirus-insect cells. licensed recombinant subunit vaccines that utilises baculovirus-insect cells include cervarix (papillomavirus c-terminal truncated major capsid protein l1 types 16 and 18) and flublok quadrivalent (hemagglutinin (ha) proteins from four strains of influenza viruses). extraction and purification throughout history, extraction and purification methods have evolved from standard chromatographic methods to the utilisation of affinity tags. however, the final extraction and purification process undertaken highly depends on the chosen expression system. please refer to subunit expression and synthesis for more insights. addition of adjuvants adjuvants are materials added to improve immunogenicity of recombinant subunit vaccines. adjuvants increase the magnitude of adaptive response to the vaccine and guide the activation of the most effective forms of immunity for each specific pathogen (e.g. increasing generation of t cell memory). addition of adjuvants may confer benefits including dose sparing and stabilisation of final vaccine formulation. appropriate adjuvants are chosen based on safety, tolerance, compatibility of antigen and manufacturing considerations. commonly used adjuvants for recombinant subunit vaccines are alum adjuvants (e.g. aluminium hydroxide), emulsions (e.g. mf59) and liposomes combined with immunostimulatory molecules (e.g. as01b). formulation and delivery delivery systems are primarily divided into polymer-based delivery systems (microspheres and liposomes) and live delivery systems (gram-positive bacteria, gram-negative bacteria and viruses) polymer-based delivery systems vaccine antigens are often encapsulated within microspheres or liposomes. common microspheres made using poly-lactic acid (pla) and poly-lactic-co-glycolic acid (plga) allow for controlled antigen release by degrading in vivo while liposomes including multilamellar or unilamellar vesicles allow for prolonged release. polymer-based delivery systems confer advantages such as increased resistance to degradation in gi tract, controlled antigen release, raised particle uptake by immune cells and enhanced ability to induce cytotoxic t cell responses. an example of licensed recombinant vaccine utilising liposomal delivery is shringrix. live delivery systems live delivery systems, also known as vectors, are cells modified with ligands or antigens to improve the immunogenicity of recombinant subunits via altering antigen presentation, biodistribution and trafficking. subunits may either be inserted within the carrier or genetically engineered to be expressed on the surface of the vectors for efficient presentation to the mucosal immune system. advantages and disadvantages advantages cannot revert to virulence meaning they cannot cause the disease they aim to protect against safe for immunocompromised patients can withstand changes in conditions (e.g. temperature, light exposure, humidity) disadvantages reduced immunogenicity compared to attenuated vaccines require adjuvants to improve immunogenicity often require multiple doses ("booster" doses) to provide long-term immunity can be difficult to isolate the specific antigen(s) which will invoke the necessary immune response it is not easy to supervise conjugation chemistry which leads to noncontinuous variation adverse effects and contraindications recombinant subunit vaccines are safe for administration. however, mild local reactions, including induration and swelling of the injection site, along with fever, fatigue and headache may be encountered after vaccination. occurrence of severe hypersensitivity reactions and anaphylaxis is rare, but can possibly lead to deaths of individuals. adverse effects can vary among populations depending on their physical health condition, age, gender and genetic predisposition. recombinant subunit vaccines are contraindicated to people who have experienced allergic reactions and anaphylaxis to antigens or other components of the vaccines previously. furthermore, precautions should be taken when administering vaccines to people who are in diseased state and during pregnancy, in which their injections should be delayed until their conditions become stable and after childbirth respectively. licensed vaccines hepatitis b engerix b (hepatitis b) vaccine engerix-b (produced by gsk) and recombivax hb (produced by merck) are two recombinant subunit vaccines licensed for the protection against hepatitis b. both contain hbsag harvested and purified from saccharomyces cerevisiae and are formulated as a suspension of the antigen adjuvanted with alum. antibody concentration ≥10miu/ml against hbsag are recognized as conferring protection against hepatitis b infection. it has been shown that primary 3-dose vaccination of healthy individuals is associated with ≥90% seroprotection rates for engerix-b, despite decreasing with older age. lower seroprotection rates are also associated with presence of underlying chronic diseases and immunodeficiency. yet, gsk hepb still has a clinically acceptable safety profile in all studied populations. human papillomavirus (hpv) gardasil vaccine and box cervarix, gardasil and gardasil9 are three recombinant subunit vaccines licensed for the protection against hpv infection. they differ in the strains which they protect the patients from as cervarix confers protection against type 16 and 18, gardasil confers protection against type 6, 11, 16 and 18, and gardasil9 confers protection against type 6, 11, 16, 18, 31, 33, 45, 52, 58 respectively. the vaccines contain purified vlp of the major capsid l1 protein produced by recombinant saccharomyces cerevisiae. it has been shown in a 2014 systematic quantitative review that the bivalent hpv vaccine (cervarix) is associated with pain (or 3.29; 95% ci: 3.00–3.60), swelling (or 3.14; 95% ci: 2.79–3.53) and redness (or 2.41; 95% ci: 2.17–2.68) being the most frequently reported adverse effects. for gardasil, the most frequently reported events were pain (or 2.88; 95% ci: 2.42–3.43) and swelling (or 2.65; 95% ci: 2.0–3.44). gardasil was discontinued in the u.s. on may 8, 2017, after the introduction of gardasil9 and cervarix was also voluntarily withdrawn in the u.s. on august 8, 2016. influenza flublok quadrivalent is a licensed recombinant subunit vaccine for active immunisation against influenza. it contains ha proteins of four strains of influenza virus purified and extracted using the baculovirus-insect expression system. the four viral strains are standardised annually according to united states public health services (usphs) requirements. flublok quadrivalent has a comparable safety profile to traditional trivalent and quadrivalent vaccine equivalents. flublok is also associated with less local reactions (rr = 0.94, 95% ci 0.90–0.98, three rcts, fem, i2 = 0%, low‐ certainty evidence) and higher risk of chills (rr = 1.33, 95% ci 1.03–1.72, three rcts, fem, i2 = 14%, low‐certainty evidence). herpes zoster shingrix is a licensed recombinant subunit vaccine for protection against herpes zoster, whose risk of developing increases with decline of varicella zoster virus (vzv) specific immunity. the vaccine contains vzv ge antigen component extracted from cho cells, which is to be reconstituted with adjuvant suspension as01b. systematic reviews and meta-analyses have been conducted on the efficacy, effectiveness and safety of shingrix in immunocompromised 18-49 year old patients and healthy adults aged 50 and over. these studies reported humoral and cell-mediated immunity rate ranged between 65.4 and 96.2% and 50.0%-93.0% while efficacy in patients (18-49 yo) with haematological malignancies was estimated at 87.2% (95%ci, 44.3–98.6%) up to 13 months post-vaccination with an acceptable safety profile. covid-19 nuvaxovid is a recombinant subunit vaccine licensed for the prevention of sars-cov-2 infection. market authorization was issued on 20 december 2021. the vaccine contains the sars-cov-2 spike protein produced using the baculovirus expression system, which is eventually adjuvanted with the matrix m adjuvant. history while the practice of immunisation can be traced back to the 12th century, in which ancient chinese at that time employed the technique of variolation to confer immunity to smallpox infection, the modern era of vaccination has a short history of around 200 years. it began with the invention of a vaccine by edward jenner in 1798 to eradicate smallpox by injecting relatively weaker cowpox virus into the human body. the middle of the 20th century marked the golden age of vaccine science. rapid technological advancements during this period of time enabled scientists to cultivate cell culture under controlled environments in laboratories, subsequently giving rise to the production of vaccines against poliomyelitis, measles and various communicable diseases. conjugated vaccines were also developed using immunologic markers including capsular polysaccharide and proteins. creation of products targeting common illnesses successfully lowered infection-related mortality and reduced public healthcare burden. emergence of genetic engineering techniques revolutionised the creation of vaccines. by the end of the 20th century, researchers had the ability to create recombinant vaccines apart from traditional whole cell vaccines, for instance hepatitis b vaccine, which uses the viral antigens to initiate immune responses. as the manufacturing methods continue to evolve, vaccines with more complex constitutions will inevitably be generated in the future to extend their therapeutic applications to both infectious and non-infectious diseases, in order to safeguard the health of more people. future directions currently, recombinant subunit vaccines are used in development for tuberculosis, dengue, soil-transmitted helminths, feline leukaemia and covid-19. along with technology development, investigators are now able to reach a great control and supervision over parameters of multiple variations to decrease toxicity, to improve immunogenicity and stability of antigen. furthermore, subunit vaccines are not only considered effective for sars-cov-2, also candidates for evolving immunizations against malaria, tetanus, salmonella enterica and many other diseases in the future. covid-19 research has been conducted to explore the possibility of developing a heterologous sars-cov receptor-binding domain (rbd) recombinant protein as a human vaccine against covid-19. the theory is supported by evidence that convalescent serum from sars-cov patients have the ability to neutralise sars-cov-2 (corresponding virus for covid-19) and that amino acid similarity between sars-cov and sars-cov-2 spike and rbd protein is high (82%). category:subunit vaccines