de Pinho Favaro et al. Microbial Cell Factories https://doi.org/10.1186/s12934-022-01929-8 (2022) 21:203 Microbial Cell Factories REVIEW Open Access Recombinant vaccines in 2022: a perspective from the cell factory Marianna Teixeira de Pinho Favaro1,2†, Jan Atienza‑Garriga1,3,4†, Carlos Martínez‑Torró1,3,4†, Eloi Parladé1,3,4†, Esther Vázquez1,3,4*, José Luis Corchero3,1,4*, Neus Ferrer‑Miralles1,3,4* and Antonio Villaverde1,3,4* Abstract The last big outbreaks of Ebola fever in Africa, the thousands of avian influenza outbreaks across Europe, Asia, North America and Africa, the emergence of monkeypox virus in Europe and specially the COVID-19 pandemics have glob‑ ally stressed the need for efficient, cost-effective vaccines against infectious diseases. Ideally, they should be based on transversal technologies of wide applicability. In this context, and pushed by the above-mentioned epidemiologi‑ cal needs, new and highly sophisticated DNA-or RNA-based vaccination strategies have been recently developed and applied at large-scale. Being very promising and effective, they still need to be assessed regarding the level of conferred long-term protection. Despite these fast-developing approaches, subunit vaccines, based on recombinant proteins obtained by conventional genetic engineering, still show a wide spectrum of interesting potentialities and an important margin for further development. In the 80’s, the first vaccination attempts with recombinant vaccines consisted in single structural proteins from viral pathogens, administered as soluble plain versions. In contrast, more complex formulations of recombinant antigens with particular geometries are progressively generated and explored in an attempt to mimic the multifaceted set of stimuli offered to the immune system by replicating pathogens. The diversity of recombinant antimicrobial vaccines and vaccine prototypes is revised here considering the cell factory types, through relevant examples of prototypes under development as well as already approved products. Keywords: Recombinant proteins, Vaccines, Antigens, Nanovaccines, Nanoparticles, VLPs Introduction taken a dominant rule in the scientific literature but fea- Immune protection against infectious diseases is a main tures of specific immunization strategies and vaccine- goal in human and animal health [1, 2]. The current vac- induced immune responses are also finely dissected and cine narrative is flooded by COVID-19, for which an overtly discussed in the media. This situation has stressed extremely rapid vaccination response has been impera- the challenges posed by emerging viral pandemics and tive at global scale. Nowadays, vaccination has not only more generically, the transversal needs associated to vac- cine development, irrespective of the involved pathogen- host pair [2, 3]. The usual failing in incorporating the † Marianna Teixeira de Pinho Favaro, Jan Atienza-Garriga, Carlos-Martínez-Torró whole infectious agent in a safe and protective vaccine and Eloi Parladé contributed equally to this work formulation, either in inactivated or attenuated versions, *Correspondence: Esther.Vazquez@uab.cat; jlcorchero@ciber-bbn.es; Neus. pushes towards considering recombinant subunit vac- Ferrer@uab.cat; antonio.villaverde@uab.cat cines [4, 5]. This is also supported by the inherent bio- 1 Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de logical risks associated to bottlenecks in the large-scale Barcelona, Cerdanyola del Vallés, 08193 Barcelona, Spain 3 Centro de Investigación Biomédica en Red de Bioingeniería, chemical inactivation of pathogens, either bacterial cells Biomateriales y Nanomedicina, Instituto de Salud Carlos III, Cerdanyola or virus particles [6, 7], or to the potential of reverting to del Vallès, 08193 Barcelona, Spain virulence in the case of attenuated strains [7–9]. Also, the Full list of author information is available at the end of the article © The Author(s) 2022. 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The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. de Pinho Favaro et al. Microbial Cell Factories (2022) 21:203 Page 2 of 17 manipulation of subunit vaccines can dismiss the use of In the context of emerging vaccination technologies, P3 laboratories and high-biosafety facilities. such as those based on expressible DNA or mRNA, Being and old concept [10–12], subunit vaccines are recombinant antigens show remarkable interest. Recom- based on particularly immunodominant antigens or binant proteins have been used as drugs for decades cocktails of selected antigens purified from the pathogen. [31–34] and their intrinsic clinical safety and industrial The main challenges in designing usable subunit vaccines scalability in their production have been largely demon- are not only linked to their biological efficacy but also strated. In addition, the functional and structural ver- to the fastness, cost-effectiveness, biosafety issues and satility of polypeptides allow designing presentations as transversal nature of the process design and large-scale multimeric, nanoscale materials in which self-assem- production. Therefore, the industrial-scale production of bling is achieved through several alternative approaches the relevant antigens is desired over their extraction from [35–43]. This set of properties make them excellent can- natural sources, often costly or even unfeasible. Impor- didates for new generation approaches in contemporary tantly, the geometry of the selected antigen presentation vaccinology. In addition, novel natural or engineered cell might be critical [13–15], especially in anti-viral vaccines factories with appealing properties have been incorpo- [16]. Then, virus-like particles (VLPs) have been achieved rated in the last decades to the biofabrication of protein in different pathogenic and non-pathogenic viruses from drugs [44], beyond the more classical bacterial and yeast more than 35 families through the spontaneous self- species and mammalian and insect cells. In the present assembling of recombinant versions of capsid proteins, review, we discuss the contemporary approaches in the upon strong expression of the encoded genes [17]. VLPs biological fabrication of recombinant subunit vaccines have been developed as vaccines but also as carriers for from alternative cell factories and how these antigens drugs or imaging agents, proving the enormous potential are adapted to comply with the requirements for vaccine of such technology in different fields of precision nano- effectiveness regarding stability, formulation and multi- medicines [18]. In immunization, VLP-based vaccines or valent nanoscale presentation. vaccine prototypes have been generically successful prob- ably because of the virus-like oligomeric presentation of Nanovaccines the antigens [19]. However, so far, VLPs have been only The immunogenicity of plain subunit vaccines, that is, approved for a limited number of diseases (Table 1). In non-oligomeric antigens, is often moderate. This fact is a step further and following the concept of multiple and usually counteracted by combining proteins to immu- repetitive antigen presentation, vaccination platforms, nostimulant molecules (adjuvants) and nanoparticles, namely versatile antigen presentation systems with mod- which can also act as immune potentiators [45]. In the ular or interchangeable elements are highly desired and last decades, proteins have been displayed in multi- pointed out as main goals for development [20, 21]. In ple copies on a plethora of nanoparticle types, includ- the search of such transversal vaccine platforms, the mul- ing metals, polymers, lipids, and others [13], aiming to timeric antigen display on nanostructured materials has increase half-life, promote a deposit effect, or to specifi- been repeatedly noted to favor, improve and enhance the cally activate certain immune cells [46]. However, the use protective response [22, 23]. Such nanoscale multimeric of protein-only nanovaccines resulting from recombinant presentation mimics natural structural features of viral protein self-assembly has been also in use for a long time particles, what has prompted the development of univer- and represents a way to suit the requirements of an ideal sal antigen presentation systems in form of nanoparticles vaccine formulation (Fig. 1). Also, it avoids the use of (nanovaccines) [24–27]. Because the specific and promis- non-protein nanoparticles as carriers, what might repre- ing features of nanoscale vaccines, this strategy is given, sent an additional source of concern because of potential as a global, a special attention in the next section. side toxicities in the case of xenobiotic, recalcitrant or This increase of the structural complexity in the formu- poorly biocompatible materials [47–50]. lation of subunit vaccines contrasts with the earlier single The dedicated study of nano-sized oligomeric pro- soluble antigen approach that initially arose linked to the tein vaccines emerged with the development of VLPs, in recombinant DNA technologies at the late 70’s. Associ- which the multimerization of antigens seems to be essen- ated to vaccine formulation, stability during storage and tial for eliciting proper immune responses. The side-by- transportation and the suitability of a vaccine product for side presentation of antigens is recognized by immune mass administration need special attention [28]. Novel cells as a pathogen-associated molecular pattern (PAMP) adjuvants and formulation strategies are being explored [13], which promotes the activation of B-cell receptors to allow the simplest manipulation of the vaccine doses in a more efficient way since their cross-linking is a key [25, 29], especially at large scale administration programs and early step to activate B cells [51]. While the dense in which thermal stability is particularly demanded [30]. arrangement of antigens interacts with B-cell receptors, de Pinho Favaro et al. Microbial Cell Factories (2022) 21:203 Page 3 of 17 Table 1 Main recombinant, subunit or oligomeric vaccines approved for human or veterinary use Pathogen Antigen Names Year of 1st Production system Formulation Approval (FDA/ EMA) Human Hepatitis B virus Hepatitis B Surface Engerix-B, Heplisav-B, 1986 Yeast (S. cerevisiae) VLPs antigen (HBsAg) Pediarix, HBVaxPRO, Yeast (H. Polymorpha) Recombivax, Twinrix, Vaxelis, Heplisav-B Papillomavirus L1 capsid protein Gardasil, Gardasil 9 2006 Yeast (S. cerevisiae) VLPs Adjuvanted Cervarix 2009 Insect cells-BVES Hepatitis E virus ORF2 protein Hecolin 2012* E. coli VLPs Influenza A & B virus Hemagglutinin (HA) Flublok, Flublok RIV4, 2013 Insect cells-BVES NPs Supemtek No adjuvant Neisseria meningitidis 2 fHbp variants Trumenba 2014 E. coli No adjuvant serogrup B Malaria HBsAg + RTS chimera Mosquirix 2015 Yeast (S. cerevisiae) VLPs Varicella Zoster virus Truncated gE Shingrix 2017 CHO cells Adjuvanted SARS-CoV-2 Spike (S) protein Nuvaxovid 2021 Insect cells-BVES NPs SARS-CoV-2 Spike protein Covifenz 2022** Plant (Nicothiana VLPs benthamiana) Swine A. pleuropneumoniae ApxII, TbpB, CysL, Pleurostar APP n.s n.s Om1A proteins Classical Swine Fever E2 protein Porcilis Pesti, 2000 Insect cells-BVES Subunit virus Bayovac CSF E2 Withdrawn Adjuvanted Porcine Circovirus ORF-2 protein Ingelvac CircoFLEX, 2008 Insect cells-BVES Fom VLPs Adjuvanted Type 2 Porcilis PCV, Circogard, Circumvent PCV Porcine parvovirus PPV 27a VP2 Reprocyc ParvoFLEX 2019 Insect cells-BVEs VLPs (PPV) Adjuvanted Canine Borrelia burgdorferi OspA & C chimeric Vanguard crLyme, n.s E. coli No adjuvant OspA Recombitek Lyme Leishmania A2 different species Leish-Tec 2004 n.s Adjuvanted Leishmania Chimeric Protein Q Letifend 2016 E. coli No adjuvant Poultry Newcastle disease virus Hemagglutinin-neu‑ Approved by USDA, 2006 Plants (tobacco sus‑ raminidase not commercialized pension cells) Feline Feline leukemia virus P45 env. antigen Leucogen 2009 E. coli Subunit Nobivac LeuFel Adjuvanted Equine Streptococcus equi CCE, mEq84, IdeE Strangvac 2021 E. coli Adjuvanted * Only approved in China; ** Only approved in Canada; VLP, virus-like particle; NP, nanoparticle; fHbp, lipidated factor H binding protein; gE, glycoprotein E; n.s., not specified; LiESP, L. infantum Excreted-Secreted protein, BVES, baculovirus expression system nanovaccines ranging from 20 to 200 nm in size are without the capacity to self-assemble, several tools can better drained to lymph nodes and thus allow a better be used to modify scaffold, irrelevant VLPs to incorpo- uptake by antigen-presenting cells (APCs), which exem- rate foreign proteins, such as genetic fusion of the anti- plifies how size and geometry of the protein assembly gen to coat proteins with the ability to self-assemble, or contribute to the immune responses [13]. The success of by conjugation of antigens to the scaffold through a vari- the VLP platform is evident by its presence in the mar- ety of both covalent and non-covalent strategies [56]. A ket, with two formulations already being commercialized very promising and widely used strategy, named SpyTag/ against Human Papilloma Virus (HPV) [52, 53] and two SpyCatcher, functionalizes a VLP expressing SpyCatcher others against Hepatitis B Virus (HBV) [54, 55] (Table 1). sequence to covalently bind to recombinant antigens VLPs intended as immunogens for vaccination can fused to a SpyTag sequence, thus allowing the decora- be attained by producing the capsid of viral pathogens tion of VLPs by simply mixing them with the modified as recombinant proteins that thus self-assemble as an antigen [57]. This platform was evaluated against differ- empty, viral-mimicking particle. In the case of proteins ent pathogens and was recently applied to SARS-CoV-2, de Pinho Favaro et al. Microbial Cell Factories (2022) 21:203 Page 4 of 17 Fig. 1 Principles of antigen engineering and formulation as nanovaccines. A The multimeric presentation of antigens, either as NPs or VLPs, enhances the protective immune response upon administration. B Non relevant NPs or VLPs can be used as scaffolds for the multiple presentation of antigens that are reluctant to oligomerization, either as genetic fusions of chemically coupled upon assembling of the scaffold protein presenting encouraging results in pre-clinical trials [58]. exciting opportunity to take subunit vaccines to the spot- In fact, the COVID-19 pandemics encouraged the expan- light in terms of efficiency and safety. sion of different platforms of nanovaccines being assessed in pre-clinical trials. In this scenario, a recombinant nan- Approved recombinant vaccines oparticle vaccine based on the Spike protein from SARS- Many of the vaccines approved for clinical use still focus on CoV-2 was developed by Novavax and it passed phase III using whole viruses or pathogen cells in conventional live clinical trials with 89.7% protection against infection [59], or inactivated forms, being this fact particularly true for also showing cross-protection against antigenic variants veterinary vaccines [71–73]. However, recombinant vac- [60]. Its approval represents an important step in the vac- cines have gained interest as the application of recombi- cinology against COVID-19 [61]. nant DNA technologies in vaccinology solved most of the In light of all the potential advantages observed in the use problems posed by the classic strategy, as discussed above. of nanostructured subunit vaccines, a great deal of effort Therefore, the trend in immunization is shifting towards has been placed in the identification and development of the exploration of this technology, as demonstrated by alternative non-VLP strategies that allow self-assembly of the significant number of approved recombinant vaccines antigens. Ferritin, for instance, is a popular platform which (Table 1, [74, 75]). Among them, nanoparticle (NP) and in different studies has been associated to an increased VLP versions abound in this list, following the multimeric immune response towards the antigen, and lumazine syn- presentation principles discussed in the previous section. thase is also extensively used, and often preferred to recruit Escherichia coli, a few yeast species, insect cells, mamma- more subunits in the assembly than ferritin [62]. Overall, lian CHO cells and plant cells are the most common cell different protein oligomerization tags and strategies have factories used for production, whose appealing properties been explored and developed [63], including the straight- and bottlenecks are highlighted and discussed in the next forward use of poly-histidine tails, common as purifica- sections. tion tags, as self-assembling tools [40, 64, 65]. Despite the Most of these data are extracted from references [74, 75]. diversity of approaches, antigen assembly into a multi- meric version consistently results in enhanced immune Conventional cell factories for recombinant responses [66–70]. Altogether, nanovaccines represent an vaccines emerging field in the frontier between protein engineering Bacteria and materials sciences. By combining the power and chal- Bacteria are a straightforward choice to approach heter- lenges of protein production in heterologous systems to the ologous protein expression through recombinant DNA ones associated with nanoparticles, these tools provide an technologies. Their high productivity paired with the de Pinho Favaro et al. Microbial Cell Factories (2022) 21:203 Page 5 of 17 significantly lower culture cost of this platform when crLyme, Recombitek Lyme), Leishmania (Letifend), compared to other expression systems are very appeal- Feline leukemia virus (Leucogen & Nobivac LeuFel) and ing features for large-scale protein production. Unfor- Streptococcus equi (Strangvac) (see Table 1). tunately, several issues inherent to the bacterial systems Although E. coli remains as the main expression sys- have hindered their adoption as preferred tools for tem for recombinant production in bacteria, there are recombinant vaccine production. These constraints many other bacterial species being adapted in parallel revolve around the immunogenicity of residual bacterial that offer unique traits and advantages as protein pro- components such as lipopolysaccharides (LPS) from the ducers. Among Gram negative bacteria, Pseudomonas cell wall, or the inability to adequately fold complex pro- fluorescens has been of particular interest for its non- teins or those that undergo post-translational modifica- acetogenic nature and moderate oxygen needs, enabling tions when produced in their natural sources. a lax control over oxygen and glucose concentrations Regarding the first issue, procedures for LPS removal while still achieving good production yields [90]. In fact, from protein preparations are under continuous devel- there is a wide toolbox and strains available for recombi- opment. In fact, these efforts have provided alternative nant protein production that feature antibiotic-free selec- methods for their generic and successful industrial appli- tion or periplasmic protein expression [90–92]. To this cation to any protein produced in bacteria and intended date, two recombinant proteins produced in P. fluores- for clinical applications, including vaccinology [76–78]. cens have already been successful in eliciting protection In contrast, insolubility of recombinant proteins and the against malaria and anthrax infections in mouse and rab- consequent precipitation as cytoplasmic or periplasmic bit models, respectively [93, 94]. Several other interesting inclusion bodies have represented a more persistent bot- gram-negative bacteria have been explored as potential tleneck in the biological fabrication of protein drugs in E. options for expression systems, including Pseudomonas coli [79], reluctant to the generic application of most of putida, Ralstonia eutropha, Burkholderia glumae and the preventive metabolic strategies [80, 81]. This fact has Acinetobacter sp., but broad host-range tools still need to limited the number of bacterial protein products trans- be developed in terms of plasmid maintenance, plasmid ferred to the market, which are in fact those that natu- transfer and secretion signaling [95]. rally, or with cost-effective post-production protocols, Moreover, lactic acid bacteria and Bifidobacterium reach a conformation compatible with solubility while species are of particular interest due to their Gener- retaining the expected biological activity. Alternatively, ally Recognized as Safe (GRAS) status, as they do not since inclusion bodies might contain high proportions of require pre-market approval when used in the food folded protein species [82, 83], strategies for the straight- industry. These bacteria have been massively employed forward use of these protein aggregates as a formula- by the industry and consequently, their engineering and tion for recombinant antigens or immunomodulators scalability have been thoroughly explored. Based on the are interesting and have offered intriguing data [84–87]. assumption of safety, the development of expression sys- However, they have been in general discontinued. In tems in several strains of such species has thrived as of addition, they face important regulatory issues because lately. Many and diverse microbial antigens produced in of their heterogeneous chemical composition [88]. or surface-displayed on lactic acid bacteria have been E. coli stands as the most immediate host for recom- able to elicit significant immune and often protective binant protein production among suitable bacterial spe- responses [96–101]. These bacteria do not only serve as cies due to its fast growth rate, a broad repertoire of microbial cell factories [102, 103] but also as vaccine vec- genetic tools and a wide array of different strains engi- tors [103, 104], for orally and mucosally-administered neered to solve most of the production challenges [89]. formulations, as they are able to induce a potent immune Accordingly, the three approved recombinant vaccines response by expressing foreign antigens, while drawing a for human use that employ bacteria as the expression sys- minimal response against themselves [105–107]. tem are produced in E. coli. These vaccines are Hecolin All in all, the use of bacteria as the expression system (Xiamen Innovax Biotech, approved in 2012 in China), for vaccine production is trending upward, with three Bexsero (Novartis, commercialized since 2013) and Cec- approved vaccines in the last ten years. Moreover, the olin (Xiamen Innovan Biotech, approved in 2019), which engineering and further tailoring of endotoxin-free E. offer protection against Hepatitis E, Neisseria menin- coli strains might resolve one of the major issues of this gitidis serogroup B and human papillomavirus 16 and 18, expression system [108]. On the other hand, the cur- respectively (see Table 1). Outside of human application, rently ongoing efforts to export the Campylobacter jejuni there are other approved recombinant vaccines produced N-glycosylation system to E. coli [109–111] could repre- in E. coli prescribed to protect against canine, feline and sent a breakthrough in the production of antigenic pro- equine pathogens, such as Borrelia burgdoferi (Vanguard teins for vaccination purposes in bacteria [110]. de Pinho Favaro et al. Microbial Cell Factories (2022) 21:203 Page 6 of 17 Yeast purified protein, surface display or as a whole recombi- As it happens with other types of functional proteins, nant yeast cell [118]. Different candidate vaccines based antigens intended for immunization might require natu- on antigens, toxins, or VLPs are currently under devel- ral post-translational modifications to follow a native opment. Representative prototype examples are a subunit folding and produce a 3D structure with proper epitope RBD vaccine against COVID-19 [131, 132], a CRM137 presentation, especially for those that are conformational subunit vaccine against Typhoid fever caused by Salmo- rather than sequence-dependent [112–114]. Being good nella enterica [133], a LipL32 subunit vaccine against alternatives to bacteria and superior to them in solving leptosipirosis caused by Leptospira interrogans [134], a some protein folding issues [115], yeasts are considered NS1-based VLP vaccine against Zika virus disease [135] a useful system for the development of recombinant and and an HEV-VLP against hepatitis E [136]. unconventional vaccines for human and veterinary medi- Strain engineering (e.g., glycosylation) and pro- cine [116–118]. cess engineering (e.g., continuous processing, alterna- Several yeast species are commonly used for recombi- tive induction systems) will not only result in a greatly nant protein production including Kluyveromyces lactis, improved productivity of the P. pastoris system, but it is Schizosaccharomyces pombe, Arxula adeninivorans, Yar- also expected that these changes will expand the range rowia lipolytica, Hansenula polymorpha, Komagataella of available immunogens, including those currently phaffii (also known as Pichia pastoris) and Saccharo- only accessible through other expression methods. This myces cerevisiae [119]. From a metabolic point of view, improvement strategy considers the endpoint of applica- these species can be categorized as methylotrophic and tion before deciding how heterologous proteins are pro- non-methylotrophic [120]. S. cerevisiae, the best-known duced. Therefore, we can expect P. pastoris to become member of the non-methylotrophic group, has provided one of the first platforms of choice in the coming years important services in the bread and brewing industries as the biopharmaceutical application of this host system for thousands of years. As a result of its nonpathogenic improves. nature and lack of toxins, S. cerevisiae is one of the yeast expression systems most exploited to obtain biopharma- Insect cells ceutical products [31, 121–124]. It is also considered gen- The insect cell-baculovirus expression system (BVES) is erally safe (GRAS) by the Food and Drug Administration a robust platform that supports the production of differ- (FDA) and an accepted food additive by the European ent protein formats [137]. The ability of insect cell lines to Food Safety Authority (EFSA) [125]. However, major grow in suspension in serum-free medium and to reach drawbacks include N-hyperglycosylation of proteins and high density conditions positions this system as one with low secretion efficiency. In fact, S. cerevisiae manno- interesting advantages for the production of immuni- sylated glycoproteins are presumed to exhibit enhanced zation agents [138]. In addition, this system allows the immunogenicity because they specifically interact with introduction of several genes in the baculovirus genome mannose-binding receptors found on professional anti- backbone under control of the strong viral promoters gen-presenting cells such as dendritic cells and mac- [139] and the use of recombinant baculovirus as trans- rophages [125]. duction vectors for mammalian cells in the BacMam In any case, genes involved in the hypermannosylation expression system [140]. However, even though insect process have been identified in P. pastoris, and mutations cells BVES reproduces most of the post translational in these genes can reduce the production of undesirable modifications of mammalian cells, the distinctive gly- glycoforms [126]. Therefore, glycoproteins derived from cosylation pattern results in the synthesis of asialylated P. pastoris can act as adjuvants. P. Pastoris is a methy- glycoproteins [141]. Another drawback when compar- lotrophic yeast that has proven to be an excellent host ing with mammalian cell-based expression systems is system for recombinant expression. In addition to being the need to control and maintain the viral stock since the inexpensive, this yeast system is fast in terms of expres- expression experiments are based on a batch procedure, sion times, as well as in co-translational and posttran- and each infection regime implies the control of the cell scriptional processing. The cultivation of this species culture but also the quantification of the viral suspension in industrial bioreactors allows the production of large which is time consuming and expensive. Although this amounts of recombinant protein from high-density cell platform is considered more biosafe than the expression cultures [126]. system based on mammalian cells, infectious agents with Various viral proteins from hepatitis viruses [127], pap- potential risk for human and animal health have been illomavirus [128], porcine circovirus [129] or influenza detected and need to be screened [142]. virus [130], among many others, have been produced As in the mammalian expression system, the protein in P. pastoris, either in the form of viruses like particles, product can be accumulated intracellularly or sent to the de Pinho Favaro et al. Microbial Cell Factories (2022) 21:203 Page 7 of 17 medium by the addition of optimized insect secretion virus [156]. In 2016, a quadrivalent version of Flublok® signals in the recombinant gene [143]. However, more was approved to include VLPs of the haemagglutinin of a sophisticated protein presentations are obtained such as second seasonal virus of Influenza B virus to the trivalent spike protein (S) of SARS-CoV-2 (Nuvaxovid®) has been VLPs. These multiprotein structures can be isolated from formulation. Finally, a recombinant vaccine based on the the soluble cell fraction or from the medium when the membranous VLPs are budded [144–146]. approved [157]. In this instance, the full-length S glyco- The BEVS is well positioned in the vaccine area for vet- protein self-assembles into trimeric complexes which erinary applications (Table 1). In fact, several products organize into higher-order structures at the nanoscale. from this expression system have been approved by the Several strategies are being developed to improve the EMA and FDA to fight against main viral pathogens of insect cells BVES including the humanization of the swine (African swine fever virus/ASFV, Porcine circovirus glycosylations pattern [158], the delay of the apopto- 2/PCV-2 and Ungulate protoparvovirus 1/PPV) [147]. sis process in infected cells [159], the optimization of The composition of the vaccine products for ASFV is the secretion pathway and the control of proteolysis lope of the virus (Procilis® Pesti and Bayovac CSF E2®) based on subunits of glycoprotein E2 of the outer enve- [160] among others. Another interesting strategy that brings this expression system, close to the one based in sid protein VP2 for PCV-2 (Ingelvac CircoFlex®, Porcilis® [148] while the ones for PCV-2 and PPV are VLPs of cap- mammalian cells, is the establishment of transient gene PCV, Circogard®, and Circumvent® PCV) [149–151], and expression procedures or stable transgenic cell lines with the corresponding VP2 for PPV (Reprocyc® ParvoFLEX) the use of strong insect cell promoters [161, 162]. Therefore, the insect cell-BVES, is a flexible expres- [152]. The initial approvals of recombinant vaccines for sion system platform which supports the production of veterinary use were based on subunits, moving to mul- complex heterologous proteins. The synthesized proteins tiprotein complexes as VLPs. In fact, the vaccines based adopt conformations compatible with the formation of on protein subunits for circovirus have been withdrawn higher-order complexes with potential for nanovaccine and only higher order protein complexes are keeping the development. Further improvements to overcome the authorization status after review (Table 1). limitations of this expression system are being explored In the case of human vaccines, formulations based on as of now, which could help to secure its positioning in approved. Cervarix® is a vaccine against two viruses of protein nanoparticles (NPs) and VLPs have been recently the vaccine market. the Papillomaviridae family which are related to cancer Mammalian cells and is formulated as VLPs formed by the spontaneous Before the development of cell culture technologies, the assembly of the L1 capsid protein of the corresponding few available viral vaccines, based on whole virus parti- viruses [153]. In the production of the bivalent vaccine, cles, were produced in animal systems such as calf skin two independent infection processes are performed, each (smallpox), rabbit spinal cord (rabies), mouse brain with a recombinant baculovirus with the corresponding (Japanese encephalitis), or embryonated eggs (influenza cloned L1 gene. The VLPs are independently purified and and yellow fever viruses) [163]. Currently, embryonated of vaccine has also been obtained from yeast (Gardasil®) mixed for the final formulation of the vaccine. This type eggs are still a main source of conventional, whole virus vaccines, especially for the seasonal flu [164]. However, to include VLPs from type 16 and type 18 human pap- the use of eggs for vaccine production poses a number illomaviruses, and is even formulated as a quadrivalent of concerns regarding the risks of insufficient supply vaccine when VLPs of type 6 and type 11 human papil- especially in case of epidemics and pandemics, time- laviruses are added, after their production in separate consuming procedures, increased manufacturing costs fermentations [154]. Even when data on the pathogenic- and the potential allergic responses to eggs components varix® has not been reformulated to increase the number ity of other papillomaviruses has become available, Cer- [165]. Cell culture technology appeared as an approach to overcome limitations of egg-based vaccine production tion of Gardasil® was approved including five more dis- of distinct VLPs. Menwhile, a new nonavalent formula- and it was progressively incorporated. The development tinct virus VLPs in the Gardasil® 9 formulation [155]. of the polio vaccine in 1954 by Jonas Salk is considered a milestone in the field of vaccination. Later, a vaccine for influenza infections, Flublok®, was approved. In this case, In 2013, an alternative to egg-based vaccine for seasonal rubella was obtained from cultured human cells by Stan- ley Plotkin at the Wistar Institute (Philadelphia). There, due to the antigenic drift of the seasonal flu viruses, it is Plotkin developed a cell line (named WI-38) from lung necessary to review vaccine formulation every epidemic cells of an aborted fetus, in which many viruses, including season for a trivalent vaccine of two seasonal virus of rubella virus, could be grown. Since their establishment Influenza A virus and one seasonal virus of Influenza B in the 1960s, cell lines WI-38 and MRC-5 (also initiated de Pinho Favaro et al. Microbial Cell Factories (2022) 21:203 Page 8 of 17 from fetal lung cells) have been used to produce several contains these viral proteins, is immunogenic in humans viral vaccines based on infection, whole virus recovery [176]. The cytomegalovirus (CMV) glycoprotein B anti- and further attenuation or inactivation, like those for gen has also been stably expressed in CHO cells, lead- hepatitis A (VAQTA, Merck and Havrix/GlaxoSmith- ing to the development of a recombinant vaccine that is Kline), rubella (MERUVAX II, Merck, and ProQuad/ immunogenic in humans [177, 178]. Recently, CHO cell Merck), chickenpox (Varivax, Merck and ProQuad/ line was used by Glaxo Smith Kline (formerly Novartis Merck), shingles (zoster) (Zostavax, Merck), oral vaccine Vaccines) to produce a pentameric molecule consisting against adenovirus type 4 and type 7 (Barr Labs), and of the human CMV surface proteins. The pentamer could rabies vaccine (IMOVAX, Sanofi Pasteur). Compared to be recognized by monoclonal antibodies, and induced egg-based vaccine production, mammalian cell cultures neutralizing antibodies in mice, suggesting its suitability provide shorter production times in more controlled pro- as a vaccine in humans [179]. cesses that takes advantage of closed-system bioreactors, The third generation of the hepatitis B vaccine, Sci- and the opportunity to cultivate viral stocks without sig- B-Vac, contains three HBV antigens, including the S, nificant egg passage-dependent antigenic changes [166]. Pre-S1, and Pre-S2 antigens, and is also expressed in Apart from its role in whole virus production, immor- mammalian CHO cells [180]. CHO cells have also been talized mammalian cell lines are efficient factories for used to produce Hantavirus VLPs, which increase ­CD8+ recombinant protein production, also with the ability to T cell activity and induce antibody responses compara- make complex and precise post-translational modifica- ble to those seen with inactivated vaccines [181]. There tions, essential for correct folding and most likely needed is a human vaccine approved for human use (see Table 1) to mimic the antigenic structural and glycosylation pat- that is produced in CHO cells. Shingrix (GSK, Londres, terns the host encounters during natural infection [167]. UK) is a herpes zoster vaccine based on varicella zoster However, the possibility for cell lines to carry mammalian virus glycoprotein E [182]. pathogens or their potential tumorigenicity are consid- Another widely used mammalian cell line, the HEK293 ered key disadvantages for mammalian cells as produc- cell line, was created by transfection of a human primary ers of therapeutic molecules [168, 169]. In this regard, embryonic kidney cell culture taken from an aborted the Vero cell line was established in the early 1960s from embryo with sheared DNA of adenovirus type 5 (AD5) kidney cells of an African green monkey in 1962, and was [183]. The advantages of HEK293 cells are their ability the first cell line approved by the WHO to produce viral to grow in suspension in serum-free medium, suitability vaccines for human use under specified regulatory guide- for large scale transient gene expression, high transfect- lines [170]. Vero cells are considered non-tumorigenic ability and stable expression. Two genetic variants have below a certain passage number and safe to use as a sub- been described for the HEK293 cell line: the 293E line strate for vaccines, including vaccines against Japanese and the 293 T line, expressing the EBNA-1 antigen and encephalitis, rotavirus, polio, influenza or smallpox [170, the Simian Virus 40 large T Ag, respectively. These cell 171]. More recently, in a recent study to develop a can- lines sustain episomal replication of plasmids contain- didate VLP vaccine for COVID-19, a stable SARS-CoV-2 ing the EBV and SV40 origins, respectively [184]. As VLP has been produced using the Vero E6 cell line [172]. with the EBNA1-expressing CHO cell line, the fact that Apart from Vero cells, other cell lines such as Chi- these HEK293 genetic variants constitutively express nese hamster ovary (CHO), baby hamster kidney (BHK), viral antigens could present challenges for health author- human embryo kidney (HEK), CAP‐T cell line derived ity approval. In addition, the tumorigenicity of this cell from human amniocytes, and east lansing line-0 (ELL-0) line is still an issue [185]. An Ebola virus (EBOV) VLP are extensively utilized for the production of recombi- vaccine candidate has been produced by expressing the nant VLPs. CHO cells, the most frequently used cell line, EBOV VP40 and the virus envelop glycoprotein in HEK have an additional advantage over other cell lines due to 293 T cells. These VLPs were morphologically similar to its non-human origin, which prevents the risk of con- wild-type virus particles, highly immunogenic in in vitro tamination with human pathogens [146]. Additionally, and in vivo studies, and they effectively induced the CHO-based systems can be considered safer and cheaper maturation, activation, and secretion of cytokines and than, for example, those based on recombinant lentivi- chemokines. Mice vaccinated with EBOV VLPs showed rus, which require a higher biosafety capacity [173, 174]. B cell activation and produced high levels of EBOV-spe- CHO cells growing in suspension in serum-free medium cific antibodies. The VLPs also activated ­CD4+ and ­CD8+ can be used to produce recombinant viral proteins, such T cells and protected mice from deadly challenges [186]. as the S and PreS2 proteins of the hepatitis B virus (HBV) Nipah virus VLPs can also be formed in HEK293T cells particles [175]. Indeed, the GenHevac® B vaccine, which surface antigen, which are then assembled into HBV-like expressing the virus attachment glycoprotein (G), fusion (F) glycoprotein and matrix (M) protein [187]. Mice de Pinho Favaro et al. Microbial Cell Factories (2022) 21:203 Page 9 of 17 vaccinated with such VLPs produce specific antibodies affinity and efficacy [204]. Finally, another general draw- against Nipah virus and a strong CD8 + T cell response. back of human cell lines (like HEK) is that they are vul- Neutralizing antibodies have also been observed in nerable to human viral infections. Thus, viral inactivation pigs vaccinated with NiP VLPs, but in these animals no on human cell lines is essential [205]. CD8 + T cell responses were detected [188]. VLPs gen- In conclusion, despite these limitations associated to erated using proteins from other paramyxoviruses have low yield anf high cost, mammalian cell cultures provide also been developed, and have shown promising results a flexible and scalable platform that can benefit from in initial pre-clinical studies [189]. The COVID-19 pan- well-established biopharmaceutical bioreactor cell cul- demic drove the rapid development of adenoviral vec- ture infrastructures for vaccine production. The combi- tored vaccines and their eventual emergency use. Among nation of advances in cell culture as the use of serum-free the adenoviral vectored vaccines approved for emergency medium, suspension culture, microcarriers to increase use by the WHO, Ad5-nCOV [190] and ChAdOX1-nCoV cell densities and improvements in bioreactor design [191] are produced in HEK293 cells, while Ad26.COV2-S result in a greatly improved strategy to produce new and is produced in PER. C6 cells [192]. more effective vaccines for human and animal health. Regarding the expression system itself, recombinant proteins can be expressed transiently or stably. Mam- Alternative factories for recombinant vaccine malian cells constitutively producing recombinant pro- production teins are established by inserting the recombinant gene Despite the success of the aforementioned systems to into the host genome, a costly and time-consuming pro- produce recombinant proteins, other approaches are also cess. Even though stable cell lines based on CHO cells available, probably being plants the most relevant alter- are widely used to produce recombinant proteins [193], native platform as vaccine producers. The production there are inherent limitations in the synthesis and secre- of therapeutic proteins in plants is often referred to as tion of many complex polypeptides, such as low produc- molecular farming, a method first proposed in 1986 by tivity, growth restriction and expression instability, low introducing a human growth hormone gene in tobacco resistance to culture-related stresses and high costs of and sunflower plants [206]. Since then, different plants production. Random insertion of the foreign gene into were modified to express recombinant proteins including the host genome can result in clonal genotypic variation but not limited to maize, tobacco, potato and rice. After and phenotypic instability (which jeopardizes cell line some therapeutic proteins were successfully produced, stability and process reproducibility and consistency), the first plant-produced vaccine was approved by the US and in genomic instability over time, causing a drop in department of agriculture for veterinary use in 2006. It protein production [194, 195]. All these facts complicate consists of a subunit vaccine against Newcastle disease the procedure and increase the production costs [196]. virus (NDV) produced in cultured tobacco cells that suc- Thus, improvement of stable cell lines has become a clear cessfully protected poultry from a challenge with NDV need, using strategies involving genetic modification, [207, 208]. optimization of expression vector and process engineer- A hallmark for plant-made vaccines was the produc- ing [197, 198]. With the advancement of CHO cell line tion of 10 million doses of a candidate H1N1 influenza development and process optimization, yields of some virus vaccine composed of a plant-produced VLP within recombinant proteins (such as monoclonal antibodies) a month of receiving the sequence information, pre- have achieved as high as 5 g/L, or even more than 10 g/L senting a serious advantage in relation to the traditional [199, 200]. Faster and cheaper approaches for protein egg-derived influenza vaccines that are limited by time- production are preferred when many proteins (or sev- consuming and hard-to-scale manufacture [209]. The eral variants of a single protein) must be rapidly obtained plant-produced VLPs were successful in preclinical tri- and evaluated. In this context, transient gene expression als and were able to induce both humoral and cellular (TGE) is the strategy of choice. TGE has a relatively short immune responses in phases I and II trial in humans, period for protein harvesting, but usually results in low presenting a satisfactory safety profile and modest results yields, as the foreign gene is not integrated into the host in phase III trials [210–212]. In the last years, several genome and therefore is lost through time [201]. The plant-based vaccine strategies are being assessed in clini- TGE efficiency using HEK cells (the leading human cell cal trials, developed against hepatitis B, cholera, Ebola, line platform used in this approach) is restricted by low influenza, and other infectious diseases [213]. More transgene expression levels [202]. Therefore, transient recently, the COVID-19 pandemics pushed plant-made expression systems are only of short-term usage [203]. vaccines to the spotlight, with 3 phase 3 trials taking ing results [214]. Among them, Medicago® employed the Besides, proteins transiently expressed can show hetero- place targeting SARS-CoV-2 and presenting encourag- geneity in glycan content, resulting in inconsistency in de Pinho Favaro et al. Microbial Cell Factories (2022) 21:203 Page 10 of 17 same VLP technology initially developed for influenza Environmental risks such as gene transfer or exposure to and managed not only to reach a high rate of protection proteins used as selectable markers, as well as inadvert- against different SARS-CoV-2 strains but also to become ent exposure to the engineered antigens, are factors to the first plant-produced vaccine approved for human use be considered to establish the proper crop management [215, 216]. [222]. Concerns related to pollen improper dispersion are Even though plants can be stably modified, most being addressed by using tissue-specific promoters that approaches focus on transient expression, frequently drive the transgene expression to specific tissues. Beyond using Agrobacterium tumefaciens or to a lesser extent this, the downstream processing for protein purification other viruses such as Tobacco mosaic virus (TMV), a can become expensive as copious volumes of impuri- process that is less time-consuming and renders higher ties and cell debris must be removed, which reduces the and more consistent yields [213]. Lately, chloroplast financial advantage obtained by a cheaper production transformation has emerged as an alternative since it [219]. Overall, plant-based vaccines can particularly allows proteins to fold and accumulate in the subcellular thrive whenever an orally-administered formulation is compartment, even though this strategy still faces draw- viable, such as for veterinary use, or when the speed of backs regarding low efficiency and impaired glycosylation production is an important feature, such as for emerging [217]. In fact, glycosylation of plant-produced proteins is diseases, since transient expression is easier to scale-up a relevant aspect of this system since while this process in this system [219]. does occur, plant glycans differ slightly from human ones. Another alternative system is the use of algae to pro- This may render an increased immunogenicity. While duce recombinant proteins, and while different species this feature may be deleterious for therapeutic proteins can be used, the most advanced microalgal platform is for reducing their efficacy and likely eliciting side effects, Chlamydomonas reinhardtii, which has already been it may actually boost the effect of subunit vaccines. The used to produce several proteins. In this case, the gene configuration of plant polysaccharides may act as a nat- insertion is often performed in the chloroplast, which ural adjuvant, as they bind to receptors expressed by further allows the accumulation of the protein of inter- APCs and are recognized as PAMPs, which enhances est and thus facilitates the downstream purification antigen presentation by APCs [218, 219]. In fact, several steps [223]. In 2003 the first algae-produced vaccine was plant polysaccharides are currently being explored as reported [224]. It was followed by reports of antigen- adjuvants, with encouraging results that allowed many specific antibodies being elicited against the structural of them to advance to clinical trials [218]. In response protein E2 of classical swine fever virus (CSFV) after to concerns about allergies and immune responses elic- mice were vaccinated with antigens produced in C. rein- ited against the glycosylation pattern, several strategies hardtii [225]. Alternatively, these algae can be used for have been developed to modify plant glycosylation pat- the administration of edible vaccines for farmed ani- terns to be more human-like, mainly focusing on modi- mals such as fish and poultry, being dried and formu- fications of N-glycosilation or syalation pathways [209, lated along with the feed. For instance, a malaria vaccine 217]. Glycoengineering is evolving up to a point where composed of freeze-dried algae that produced a Plas- even entire human glycosylation biosynthetic pathways modium falciparum surface protein was used to orally can be transferred into plants, combined to the elimina- immunize mice and was able to elicit both IgG and tion of unwanted native glycosylation enzymes, aiming to IgA immune responses [226]. Despite its potential, it is increase product quality and safety [217, 220]. noteworthy that this system lacks the post-translational Overall, plant-produced vaccines present several modifications. advantages, as the production in this system tends to be Transgenic animals have also been investigated as an cheaper than in mammalian cell cultures but it allows alternative for recombinant protein production, mainly relatively similar protein folding, assembly and glyco- due to the possibility of producing large amounts of sylation, and unlike bacterial systems it renders endo- complex proteins in the milk [227]. In the context of vac- toxin-free products [221, 222]. Scale-up of production is cine production, two rotavirus proteins were produced easier as it consists of simply growing more plants rather in the milk of transgenic rabbits. The yields were up to than relying on bioreactor production optimizations, 200 μg/ml and they were able to induce antigen-specific and plant-based vaccines frequently dismiss the use of immune responses that conferred a high level of protec- cold-chain for transport [222]. This system requires less tion against virus challenge, either when partially purified sophisticated infrastructure than its counterparts and milk was intrarectally administered or when whole-milk could help overcome vaccine distribution issues around was orally administered to mice [228, 229]. In another the globe. On the other hand, some concerns have been study, mice were modified to produce an enterovirus 71 raised regarding the use of genetically modified plants. protein in the milk, and pups receiving transgenic milk de Pinho Favaro et al. Microbial Cell Factories (2022) 21:203 Page 11 of 17 orally developed antigen-specific antibodies and dem- room for engineering and improvement, and they show onstrated relatively better health conditions after the potential to overcome some of the limitations posed by challenge [230]. However, in the last years, there are less genetic vaccines. studies published that use transgenic animals as expres- Acknowledgements sion systems. This research was supported by CIBER-Consorcio Centro de Investigación Biomédica en Red-(CB06/01/0014), Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación. We also acknowledge the ICTS “NANBIOSIS”, more Recombinant vaccines for COVID‑19 specifically the support from the Protein Production Platform of CIBER-BBN/ Beyond the approved anti-COVID-19 front-line vaccines IBB, at the UAB SepBioES scientific-technical service (http://​www.​nanbi​osis.​es/​ based on expressible embedded mRNA and replication- unit/​u1-​prote​in-​produ​ction-​platf​orm-​ppp/). A.V. received an ICREA ACADEMIA award. incompetent adenovirus vectors [118, 231, 232], a recom- binant vaccine has come to market (Table 1, [157]) and Author contributions other many alternative recombinant vaccines are also All authors have collected bibliographic information and prepared respective sections as organized by AV. AV has combined such respective sections and approached through under fast developing strategies. generically organized the whole manuscript. All authors read and approved Because of the broad clinical experience in protein-based the final manuscript. vaccination, such protein-based vaccines are expected to Funding enter the prophylaxis landscape and complement or even The authors appreciate the financial support received for the develop‑ substitute, in the longer term, the nucleic acid versions ment of multimeric recombinant drugs, from AEI (PID2020-116174RB-I00 [233–236]. Due to the enormous investment in testing to A.V., PID2019-105416RB-I00/AEI/10.13039/501100011033 to E.V. and PID2019-107298RB-C22 to N.F.M.), AGAUR (2020PANDE00003 to A.V.), immunization strategies against SARS-CoV-2 infection, Instituto de Salud Carlos III (PI20/00770 to J.L.C.) and UAB-FEDER (NAUTILIS a comprehensive and updated overview of the spectrum Proof-of-Concept to A.V.). The authors also appreciate the support from of vaccine prototypes is just not feasible and it is beyond FAPESP (2021/08528-0 to M.T.P.F.) and from the Ministerio de Universidades (FPU20/02260) to J.A.G. C.M.T was supported by a postdoctoral fellowship the scope of this review. However, it can be stressed that from Ministerio de Universidades (Margarita Salas 2021). most of the taken approaches are based on the full-length spike protein (S) or its receptor binding domain (RBD), Availability of data and materials Not applicable. responsible for virus attachment to the host cell. Other SARS-CoV-2 proteins are also under consideration as Declarations epitope-full, including E, N and M [237, 238], with are explored in essentially all the protein production systems Ethics approval and consent to participate discussed above. Not applicable. Consent for publication Conclusions Not applicable. Through the recruitment of technologies and expertise Competing interests from more than 40 years of recombinant drug produc- The authors declare no competing interests. tion [31, 34], vaccinology has moved from the conven- Author details tional use of attenuated or inactivated live vaccines to 1 Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barce‑ recombinant antigens in different formulations. In par- lona, Cerdanyola del Vallés, 08193 Barcelona, Spain. 2 Laboratory of Vaccine ticular, the presentation of vaccine candidates in form of Development, Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil. 3 Centro de Investigación Biomédica oligomeric NPs or VLPs, mainly pushed by the develop- en Red de Bioingeniería, Biomateriales y Nanomedicina, Instituto de Salud ment of insect-based protein production platforms, has Carlos III, Cerdanyola del Vallès, 08193 Barcelona, Spain. 4 Departament de allowed to overcome the moderate immunogenicity of Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Cerdanyola del Vallés, 08193 Barcelona, Spain. plain subunit vaccines. The multiple and regular anti- gen presentation at the nanoscale boosts the immune Received: 10 June 2022 Accepted: 30 August 2022 response and increases protection, as well as proper anti- gen folding promoted by a native-like glycosylation pat- tern. 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