A multidisciplinary team of researchers is the first to show combining yeast-expression technology and a novel adjuvant formulation to produce a COVID-19 vaccine candidate is effective against SARS-COV-2 and promises to be easy to produce at large scale and cost-effective, important aspects for vaccinating people worldwide, especially in low- to middle-income countries.
Results from the study, which applied lessons learned from the hepatitis b vaccine platform technology, are published online today in Science Immunology.
Researchers from the Yerkes National Primate Research Center (NPRC) at Emory University, Infectious Disease Research Institute (IDRI), 3M and Texas Children’s Hospital’s Center for Vaccine Development at Baylor College of Medicine paired Baylor’s SARS-CoV-2 Receptor Binding Domain (RBD) recombinant protein formulation vaccine candidate with IDRI’s aluminum-based formulation of 3M’s Toll-like receptor 7 and 8 agonist 3M-052 (3M-052/Alum) to enhance immune response against SARS-CoV-2 and, thus, increase vaccine effectiveness against COVID-19.
“Working with rhesus macaques, we found 3M-052/Alum formulation produced a significant and superior overall immune response than alum alone, a licensed adjuvant,” says corresponding author Sudhir Kasturi, Ph.D. “The superior immune response from our RBD+ 3M-052/Alum vaccine resulted in a significant reduction of SARS-CoV-2 in upper and lower respiratory tracts and a markedly reduced severity of lung disease when compared with unvaccinated animals.
We also showed a substantial reduction in virus shedding from the upper airways, which suggests our vaccine may also slow or halt virus transmission,” Kasturi continues. Kasturi is an assistant professor in the Emory School of Medicine (SOM) Department of Pathology and Laboratory Medicine and a research assistant professor in Yerkes’ Microbiology and Immunology (M&I) division and the Emory Vaccine Center.
The researchers believe their vaccine comprising a recombinant RBD protein with its novel 3M-052 adjuvant formulation may be strongly effective against the emerging variants because the vaccine has the capability to induce both neutralizing antibodies and CD8+ T cells, which can kill the virus if it enters cells. They say this is critical for reducing disease transmission and the virus’ impact worldwide.
“Also critical is we showed the vaccine potently reduces the levels of SARS-CoV-2 and limits inflammation by blocking the expansion of pro-inflammatory monocytes, which provides a better understanding of how the vaccine works,” says Mirko Paiardini, Ph.D.
“Furthermore, in collaboration with our Emory colleagues Drs. Susan Pereira Ribeiro and Rafick P. Sekaly, we identified via our study a combination of blood markers that predict the virus burden in lungs.
Such a diagnostic could potentially help healthcare professionals monitor the disease and adjust treatments for increased effectiveness,” Paiardini continues. He is also a corresponding author of the study and an associate professor in the Emory SoM Department of Pathology and Laboratory Medicine as well as a researcher in Yerkes’ M&I division.
To the authors’ knowledge, their SARS-CoV-2 research is first to report use of a recombinant RBD immunogen and the 3M-052/Alum adjuvant to induce CD8+ T cell responses. The researchers say such T cell responses should be easily translatable from the rhesus monkey model into broad-based protection in humans, especially against emerging SARS-CoV-2 variants.
The researchers vaccinated two groups of five rhesus monkeys each with RBD+alum (Group 2) or RBD+ 3M-052/Alum (Group 3). All animals in these groups received three immunizations over 10 weeks. Based on previous HIV studies, the researchers reasoned a third vaccination could substantially improve the magnitude and quality of neutralizing activity and effectiveness. An additional five rhesus monkeys (Group 1) served as unvaccinated controls for evaluation purposes.
The researchers challenged all animals with SARS-CoV-2 (WA1Ä2020 isolate) via a combined intranasal and intratracheal route one month after the third vaccination. The Group 3 animals, which received RBD+ 3M-052/Alum, showed clear advantages in antibody response, neutralizing activity and effectiveness over the Group 2 animals, which received RBD+alum.
Adding to the appeal of the Baylor RBD vaccine candidate is the large-scale production capacity in low- to middle-income countries that use this established yeast-expression platform for producing the hepatitis B vaccine. Such production capacity addresses the ability for transferring this vaccine technology in an effort to improve global health.
“Our results showed producing the RBD recombinant protein using the yeast expression platform would meet the demand for vaccinating communities around the world,” says Maria Elena Bottazzi, Ph.D. We are very excited to see our vaccine candidate is also beneficial in inducing a balanced antibody and CD8+T cells response previously not seen with other protein-based vaccine approaches.” Bottazzi is another corresponding author and associate dean of the National School of Tropical Medicine at Baylor and co-director of Texas Children’s Center for Vaccine Development.
Study author Peter Hotez, MD, Ph.D., notes the widespread use and outstanding safety track record of yeast-expressed recombinant protein immunizations offer promise for using this approach to produce and deliver COVID-19 vaccines for global health. Hotez is dean of the National School of Tropical Medicine at Baylor and co-director of Texas Children’s Center for Vaccine Development.
The research team also includes Christopher Fox, Ph.D., IDRI, and Mark Tomai, Ph.D., 3M.
“Especially because of its cost effectiveness, we believe this vaccine could serve as a great option against emerging SARS-CoV-2 variants as well as an attractive boost to select advanced clinical candidates where repeated vaccination might be a challenge,” Kasturi says.
More than a year into the COVID-19 pandemic and in light of unprecedented worldwide efforts to develop countermeasures, the first generation of vaccines have now reached the clinic. Russia  and China  were the first to start mass vaccination campaigns, and are now followed by mRNA vaccines recently authorized for use in Europe  and the Americas . As of December 8, 2020, the WHO lists 52 candidates in clinical evaluation and 162 in pre-clinical testing .
With this never-before-seen acceleration of research efforts, some of the front-runner platform technologies in this vaccine race have not previously been in the clinic, such as DNA or mRNA-based vaccines. More traditionally produced vaccines such as those based on recombinantly produced subunit proteins are lagging; nonetheless, there are currently 16 vaccines based on recombinant protein antigens in the clinic (Table 1 ), and 56 in pre-clinical testing. Arguably, the fact that this type of vaccine is lagging may not necessarily be a reflection of their validity or promise, but has multiple reasons, including possibly the way the initial funding was directed.
Here we will provide a review of the status of the most advanced recombinant protein vaccines for COVID-19. While other candidates undoubtedly have high scientific merit, we intentionally limited the scope of this review to allow a focus on those vaccines that will likely make the strongest impact in the short term.
Table 1 – Select recombinant protein vaccine candidates in clinical trials for COVID-19 as of December 8, 2020 
|Antigen||Vaccine developer||Platform/technology||Adjuvants||Most advanced clinical stage||References|
|Full-length S-protein based vaccines|
|Trimer||Novavax||Insect cells||Matrix M||Phase 3||[, , ]|
|S-protein||Sanofi Pasteur/GSK||Insect cells||2 different adjuvants (likely variants of AS03)||Phase 1 (to be repeated)|||
|SCB-2019 trimer||Clover Biopharmaceuticals Inc./GSK/Dynavax||CHO cells||Alum+CpG 1018 or AS03||Phase 1||[10,11]|
|S-2P (MVC-COV1901)||Medigen Vaccine Biologics Corporation/NIAID/Dynavax||CHO cells||Alum+CpG1018||Phase 1||[12,13]|
|Covax-19||Vaxine Pty Ltd/Medytox||Insect cells||AdvaxCpG55.2||Phase 1||[14,15]|
|SARS-CoV-2-RBDN1C1||Biological E/BCM||Yeast||Alum+CpG||Phase 1-2||[, , ]|
|FINLAY-FR-1/2||Instituto Finlay de Vacunas, Cuba||Phase 1||[20,21]|
|KBP-201||Kentucky Bioprocessing, Inc||Plants||Phase 1-2|||
|RBD Dimer||Anhui Zhifei Longcom Biopharmaceutical/Institute of Microbiology, Chinese Academy of Sciences||CHO Cells||Aluminum preparation||Phase 3||[23,24]|
|RBD||West China Hospital, Sichuan University P||Insect Cells||Alum||Phase 2||[, , ]|
|Multitope Peptide-based Vaccine (MPV)||COVAXX||Peptides||CpG and alum (AdjuPhos®)||Phase 1||[28,29]|
|EpiVacCoron||Vektor Laboratories, Russia||Chemical synthesis||Alum||Phase 1|||
|CoVac-1||University Hospital Tübingen||Peptides||Montanide ISA51||Phase 1||[31,32]|
The spike protein as a vaccine antigen candidate
The ~29.8 kb SARS-CoV-2 genome contains 14 open-reading frames encoding 27 proteins, including the four major structural proteins, E, envelope protein, M, matrix protein, N, nucleocapsid protein, and S, the spike protein . Among these, the immunodominant trimeric S protein is the primary source of all major vaccine antigen targets to date. Other proteins have received considerably less attention as vaccine antigen candidates for various reasons.
For instance, while the abundant SARS-CoV-2 N-protein is used in virus diagnostics [, , ], it is not included in most COVID-19 vaccine candidates because its SARS-CoV homolog was shown to increase the number of eosinophils within inflammatory infiltrates upon vaccination and subsequent challenge . The S-protein is made up of two subunits, S1 and S2 that fulfill multiple functions related to the initial binding of the virus to its angiotensin-converting enzyme 2 (ACE-2) cell surface receptor and the subsequent endosome mediated entry of the virus into the host cell . In the S-protein trimer, three S1 subunits sit on top of a stem of three S2 subunits. Within S1, a distinct receptor-binding domain (RBD, residues 331-524) and within it, a distinct receptor-binding motif (RBM), is responsible for the initial docking to ACE-2 .
Despite each S1 domain having its own functional RBD domain, it appears though that only one at a time is active, folded into the exposed confirmation, while the other two are hidden from the immune system within the trimer . Moreover, there does not appear to be any cooperativity between the three RBDs within the S1 trimer when it comes to ACE-2 binding. Upon RBD/ACE-2 binding and catalyzed by a host protease, transmembrane protease serine 2 (TMPRSS2), S is then cleaved, allowing the S2-fusion peptide to facilitate cell entry.
While this process, in general, is similar to what is observed in SARS-CoV, SARS-CoV-2 is distinguished by the presence of a unique furin cleavage site proximal to the S1/S2 junction that might facilitate cell entry and thus may be responsible for the increased virulence of SARS-CoV-2 over SARS-CoV .
SARS-CoV-2 shares extensive sequence homology, as well as structural and functional homologies with prior coronaviruses, namely SARS-CoV, but also MERS-CoV, the causative agent of Middle East Respiratory Syndrome. Early on in the pandemic, it was shown that anti-SARS S-protein antibodies were also capable of inhibiting the binding of SARS-CoV-2 to ACE-2. These observations concentrated vaccine development on antigens derived from the spike protein . While some groups focus on the whole S1 subunit as their primary vaccine antigen candidate, others are using the RBD as their main vaccine antigen.
A reason for the focus on the RBD lies in observations with the homologous SARS-CoV S-protein vaccine in mice, made by Drs. Jiang and Tseng , who observed lung pathology in mice with the full-length S-protein as their vaccine antigen, but not with the RBD. As a possible underlying cause for this observation, antibody-dependent enhancement (ADE) is considered as a possible contributing factor. In ADE, antibodies present in vaccinated individuals facilitate the entry of virus particles into the host cell through an additional mechanism using the Fc receptor II (Fig. 1 ).
In particular, non-neutralizing antibodies that do not interfere with the binding of the RBD to ACE-2 might thus increase the risk of ADE. Thus, reducing the size of the antigen to limit exposure to non-neutralizing epitopes might reduce the risk of undesired immunopathology. Notably, though, the majority of ADE data almost exclusively stems from experiments in mice and has not been unequivocally reproduced in, for example, Rhesus models for either SARS-CoV or SARS-CoV-2.
Full-length S-protein based vaccines
The COVID-19 vaccines currently in the clinic, including the recombinant protein vaccines, use various versions of the S-protein as their vaccine antigen component. The NVX-CoV2372 trimeric nanoparticle produced by Novavax is made from the full-length S-protein (GenBank accession number, MN908947; nucleotides 21563–25384). One mutation, 682-QQAQ-685, was introduced at the S1/S2 junction to increase protease resistance, and two other mutations, K986P and V987P, were added to increase the stability of the recombinantly produced vaccine antigen . [, , ]
In an approach to increase the stability of the prefusion S-protein antigen (residues 1-1208), Medigen, with support from the NIAID, mutated the furin recognition site at the S1/S2 junction (682-RRAR-685 to GSAS) and exchanged amino acid residues K986 and V987 near the top of the central S-2P helix with two proline residues. The same mutations had also been inserted into S-2 by Wrapp et al.  to allow the determination of the SARS-CoV-2 structure by cryo-EM and had previously been used with Medigen’s MERS-CoV vaccine antigen . In addition, a C-terminal T4 fibritin trimerization domain, an HRV3C cleavage site, an octa-histidine tag as well as a Twin-Strep96 tag were added to the wild-type sequence .
A recombinantly produced homotrimer of the full-length S-protein also serves as the antigen in Clover Biopharmaceuticals’ S-Trimer vaccine . Using the company’s Trimer-tag platform, originally developed for cancer therapeutics, Clover has genetically fused the SARS-COV-2 S-protein (aa residues 1-1211) to human C-propeptide of alpha1(I) collagen. The fusion protein self-trimerizes and, as an added advantage, aids purification via affinity chromatography using a collagen-receptor-derived resin .
For Vaxine’s COVAX-19 candidate [, , , , , , , ], there is a significant amount of information about the adjuvant component of the vaccine, but details of the S-protein derived antigen  have not been published yet.
What the public is also reminded of now that the first front-runner clinical trials are completed though is that a clinical trial does not guarantee a successful product. At least two of the leading candidates, the University of Queensland’s Sclamp project  as well as Sanofi/GSK’s baculovirus produced S-protein vaccine  have been marred by unexpected results in Phase 1, leading to a halt or a significant delay of the project. This further reiterates the need to continue all efforts to bring additional vaccine candidates forward even if the first products have already been authorized.
Among those entities that focus on the RBD of the S-protein, Anhui Zhifei Longcom Biologic Pharmacy Co., Ltd., is developing an RBD-dimer produced in mammalian cells as their vaccine antigen. In addition to expressing an RBD monomer (aa residues 319-541), two copies of the RBD-encoding gene fragment (aa residues 319-537) were cloned in tandem, leading to the expression of a 60 kDa homodimer. Based on published reports, this dimerization increased the stability of the vaccine antigen, not just for SARS-CoV-2, but also in similar SARS-CoV and MERS-CoV constructs .
A slightly longer RBD (aa residues 319-545) is used in the vaccine candidate from West China Hospital . After the alum-adjuvanted vaccine had shown protection in non-human primates, it is now in Phase 2 clinical trials . Baylor College of Medicine in collaboration with Biological E is using an antigen comprising residues 331-549 of the RBD with mutations introduced to reduce glycosylation and aggregation [18,19]. Adjuvanted with Alum and CpG, this vaccine candidate is currently in Phase 1-2 clinical trials in India .
For some of the vaccine development efforts, for various reasons, little public information about the nature of the vaccine antigen is available. For example, while it is known that Cuba’s Soberana 01 vaccine is based on the RBD antigen, additional details have not yet been widely published, although original news reports suggest that a combination with the proven outer membrane vesicle platform of the Cuban meningococcus B vaccine was planned . AdimrSC-2f is a vaccine candidate developed by Adimmune, with the RBD antigen expressed in insect cells. The vaccine is currently in a Phase 1 clinical trial with or without aluminum as the adjuvant .
Many vaccine candidates in the literature employ neither the native virus’s full-length S-protein or its RBD as their antigen but instead are engineered multi-epitope vaccines synthesized from peptides. Among the most advanced candidates are COVAXX’s COVID-19 vaccine, made from epitopes of the RBD, the S2 protein, as well as other SARS-CoV-2 proteins, such as membrane and nucleoprotein regions. In guinea pigs, the company reports seeing neutralizing antibody titers that exceed those in human convalescent serum by a factor of 400 .
Also using peptides, and based on studies with convalescent sera, Tübingen University is advancing a multi-peptide vaccine made from HLA class I and HLA-DR T-cell epitopes of the S-protein as a potential COVID-19 vaccine to induce broad T-cell immunity , and Vektor Labs’ (Russia) EpiVacCorona vaccine is also reportedly composed of chemically synthesized peptides of SARS-CoV-2 epitopes, conjugated to a recombinant carrier protein and adsorbed on aluminum hydroxide . It should have recently completed Phase 1 trials, with no results published yet.
It will be interesting to see how the ongoing studies shift the focus between the full-length S-protein based and the RBD vaccines. The main argument in favor of the S-trimer is certainly the ambition to maintain the nature of the vaccine antigen as close to the natural confirmation as possible, while the interest in the RBD alone likely stems from concerns over adverse immune reactions triggered by full-length spike protein in SARS-CoV and also in RSV .
Protein production technologies
Over the last decades, recombinant protein technology has become efficient, relatively inexpensive, and widely available, allowing for cost-effective production of recombinant proteins in microbial and other expression host systems [63,64]. Among other advantages, since recombinant protein vaccines are non-replicating and lack any of the infectious components of an, albeit attenuated, viral particle, the vaccines are considered a safer approach compared to vaccines derived from live viruses. The technology has been tested widely and in general, these vaccines produce only very mild side-effects [65,66]. Consequently, multiple recombinant protein vaccines are now in clinical use worldwide .
For the production of recombinant proteins, a variety of expression platforms are now available, including microbial systems, such as Escherichia coli and various yeasts, as well as insect cells, mammalian cells, and even plants. Certainly, for non-industrial research purposes, E. coli is the most widely used system for recombinant protein production due to its rapid growth and general cost-effectivity, as well as the availability of the widest range of molecular manipulation tools. Several vaccine antigens have been produced in E. coli, including, in 1998, an FDA approved Lyme disease vaccine, which contained the recombinantly-expressed outer surface lipoprotein, OspA, from Borrelia burgdorferi.
While this particular vaccine was withdrawn from the market in 2002 due to concerns over adverse side effects , an improved version, VLA15, likewise produced in E. coli, is now in a Phase 3 clinical trial [69,70]. Other examples of E. coli produced antigens include vaccines against meningococcal serogroup B infections; Trumenba®, developed by Pfizer, uses two variants of the meningococcal factor H-binding protein (fHBP) as antigens [71,72], while Bexsero®, developed by GSK, uses three immunogenic meningococcal antigens (fHbp, NadA, and NHBA) synthesized in E. coli . These two vaccines were approved by the FDA in 2014 and 2017, respectively.
However, E. coli expression systems do not typically provide post-translational modifications (PTMs), such as glycosylation, which can affect the nature of the immune response and consequently, the functionality of the vaccine. PTMs also affect protein characteristics such as solubility and stability, and therefore it is critical to confirm correct folding and disulfide bond formation. In the case of SARS-CoV-2, depending on the product, the length of the vaccine antigen component ranges from ~200 to ~1,300 amino acids with 4-12 potential disulfide bonds . Due to this complexity, it is difficult to produce these antigens properly folded in E. coli, and other production platforms are favored.
Yeasts are another well-known microbial expression platform. Similar to E. coli, they grow rapidly and are easy to manipulate genetically. Unlike E. coli, yeasts can secrete recombinant proteins extracellularly, which makes the downstream purification process simpler and less costly. The inclusion of certain PTMs in this eukaryotic expression system also often facilitates proper folding of the recombinant protein . Several currently licensed hepatitis B vaccines, such as Engerix-B®, Recombivax HB, and HEPLISAV-B, use recombinant hepatitis B surface antigens (HBsAg) synthesized in yeast .
Another licensed vaccine, Gardasil®, uses the major capsid protein, L1, from four human papillomaviruses (HPV L1) as its antigens . While N-linked glycosylation in yeast resembles that in higher eukaryotes, a more controlled and humanlike N-glycosylation can be achieved in specialized, genetically engineered strains of the fungus .
For the production of the SARS-CoV-2 spike protein, it was discovered that the epitopes which are likely to trigger a potent neutralizing antibody response, are located in the N-terminal domain (NTD, residues 1-290 of S protein) and in the RBD (residues 306-577) of the spike protein , where the most potent ones could block ACE-2 binding . With respect to the NTD, there are eight potential N-glycosylation sites within this region , making it likely that different glycosylation of a potential recombinant vaccine antigen will affect the ability to trigger neutralizing antibodies within this region.
However, no N-glycosylation sites are within or proximal to the ACE-2 binding site, making glycosylation much less of a concern when expressing the antigen in yeast. A yeast-expressed RBD antigen (Residues 332-549 of the spike protein) is currently being pursued by Texas Children’s Center for Vaccine Development at Baylor College of Medicine (TCH-CVD) in partnership with Biological E [18,19,79]. Formulated with CpG as an adjuvant, it entered a Phase I/II clinical trial in India in November 2020 . This follows the prior production of a recombinant SARS-CoV RBD antigen in the same system; formulated with alum, this antigen-induced high neutralizing antibody titers and 100% protection in mice after viral challenge [81,82].
Mammalian cell culture expression systems
Most current COVID-19 recombinant protein vaccine candidates are expressed in mammalian cell culture-based expression systems (Table 1) that have been used to produce various biopharmaceuticals in recent years, including enzymes, antibodies, and vaccine antigens. Though more costly, mammalian systems are appreciated for their ability to express glycoproteins with their native structures and PTMs, and thus constitute the majority of the recently approved recombinant biologics . A successful example for this class of vaccines is Shingrix®, the herpes zoster vaccine manufactured by GSK, which uses Chinese Hamster Ovary (CHO) cells to produce recombinant glycoprotein E from the virus as its antigen .
COVID-19 subunit vaccine candidates, like those from Novavax, Sanofi and Adimmune are produced in a system that uses a baculovirus vector and insect cells as hosts. This system was first developed in 1983  and has since been used for several recombinant proteins [86,87]. Currently, there are two licensed vaccines in the USA, utilizing insect cell-expressed antigens; Cervarix®, an HPV vaccine that uses the recombinant HPV L1 antigen , and Flublok®, an influenza vaccine using a recombinant trivalent hemagglutinin antigen .
When compared to E. coli or yeast, the required growth medium is more costly and the cell growth rate is slower, but insect cells can reach higher densities in a shorter period when compared to mammalian cells [88,89]. Additionally, like mammalian cells, insect-cell expressed recombinant proteins are usually well-folded, soluble, and often contain the desired PTMs. However, even though this system does not cause hyperglycosylation, N-glycosylation by baculovirus-infected insect cells is not equivalent to those of higher eukaryotes , and thus, if sophisticated glycans are required to maintain the function of a recombinant protein, this system may not be the optimal option.
In addition to traditional vaccine manufacturing platforms, alternative expression systems are also being used to produce vaccine antigens. Kentucky BioProcessing and other tobacco growers, for example, are employing tobacco plants to express SARS-CoV-2 vaccine antigens . While the manufacturing of recombinant proteins in tobacco is a proven technology [, , , ], controlling cost at the pandemic scale might reserve this expression system to those with access to the necessary capacity.
Generally speaking, for any expression system, production cost will vary depending on the production yield, but based on the general cost comparison analyzed by Owczark et al. , and the example retail pricing for a few biopharmaceuticals , E. coli is the least expensive choice for protein production, and while mammalian cells are the most expensive option, the production cost for insect cells and yeasts is generally somewhere in between.
Recombinant proteins by themselves generally elicit only a weak immune response, unless they are assembled into larger particles . To augment the immune response and allow for antigen dose sparing, most protein-based COVID-19 vaccines are formulated in combination with adjuvants (Table 2 ). The addition of these immunostimulants can trigger specific cell receptors and induce an innate immune response at the site of injection and in the draining lymph nodes.
The innate immune response to the adjuvants then further activates the adaptive immune system by mobilizing antigen-presenting cells (APCs), thus improving antigen presentation to CD4 T helper cells. Depending on the phenotype, the activated T helper cell will stimulate the proliferation of antigen-specific antibody-producing B cells or CD8+ T cells (Fig. 1).
List of adjuvants used in recombinant protein COVID-19 vaccine candidates currently tested in the clinic.
|Name||Components||Receptor/pathway||Disease target tested in the clinic|
|Aluma||Aluminum salts (aluminum hydroxide,|
|NLRP3 uric acid, DNA||Anthraxa, Diphtheriaa, Tetanusa, Pneumococcusa, hepatitis Aa Hepatitis Ba, Japanese Encephalitisa, Meningococcal Ba and Ca, human papillomavirusa, SARS, COVID-19|
|MF59a, AS03a||Oil-in-water emulsion squalene oil|
|MyD88, ASC, ATP||Influenzaa, COVID-19|
|CpG 1018a||Synthetic DNA alone or formulated with Alum||TLR9||Hepatitis Ba, Malaria, Influenza, Anthrax, Cancer, COVID-19|
|Matrix M/IscoMatrix||Saponin||Unknown||Hepatitis C, Influenza, HSV, human papillomavirus, Malaria, Cancer, COVID-19|
|Advax||polysaccharide particle made from delta inulin||Unknown||HIV, Influenza, Hepatitis B, COVID-19|
aAdjuvants in licensed vaccines in the USA.
To protect against COVID-19, high levels of neutralizing antibodies to the spike protein of SARS-CoV-2 are essential. However, similarly to antibody levels in patients that have recovered from SARS-CoV, SARS-CoV-2 antibody responses seem to wane rapidly within months after infection. In addition, while less severe cases of SARS were associated with accelerated induction of a Th1-type immune response, Th2 cell responses have been associated with enhancement of lung disease following infection in mice parenterally vaccinated with inactivated SARS-CoV viral vaccines. Therefore, the FDA specifically stated in their guidelines to the industry from earlier this year that COVID-19 vaccine candidates should preferably elicit a strong Th1-skewed CD4 T cell response, in addition to the induction of high levels of neutralizing antibodies .
We here provide an overview of the vaccine adjuvants that have been formulated in reported COVID-19 protein vaccine candidates.
Aluminum hydroxide (alum)
Semi-crystalline suspensions of aluminum are the most commonly used adjuvants in vaccine development worldwide . The aluminum salts have a high binding capacity and typically will adsorb the antigens on their surface. Although hundreds of millions of people have been vaccinated with aluminum-based vaccines, there is still discussion on the exact mechanism of action. The most widely accepted explanations include a possible depot effect, enhancement of phagocytosis of the antigen, and activation of the pro-inflammatory NLRP3 pathway .
Aluminum-based formulations generally induce a strong humoral immune response in combination with the secretion of Th2-biased cytokines (e.g., IL-4, IL-6, IL-10). While some studies found that candidate SARS-CoV vaccines formulated with aluminum induced specific Th2-biased responses and possibly induced lung eosinophilic immunopathology in mice , other studies found no direct evidence linking aluminum to enhanced eosinophilia .
The true cause of the undesired immune response remains under discussion [62,102]. Nonetheless, since the FDA guided the industry toward a Th1 immune response, most COVID-19 recombinant protein vaccine formulations that are formulated with aluminum hydroxide (alum) include a second adjuvant, such as CpG, in order to balance the immune response and also stimulate proliferation of Th1 type CD4(+) cells. Alum has an excellent safety record and can be produced at a relatively low cost, making it an ideal COVID-19 vaccine adjuvant for global health [102,103].
MF59® is an oil-in-water emulsion developed by Novartis. The adjuvant contains squalene oil and two surfactants, Tween-80 and Span-85, emulsified in a citric acid buffer . MF59 has been deemed safe and is well-tolerated in humans and MF59-adjuvanted vaccines have been approved for pandemic and seasonal influenza in over 38 countries worldwide . For example, Fluad®, an MF59-adjuvanted seasonal influenza vaccine, has been licensed since 1997. However, in the United States, FLUAD and FLUAD Quadrivalent are licensed only for persons over the age of 65 years . MF59 was also added to vaccines against the pandemic flu strain H1N1 (Focetria® and Celtura®). Within oil-in-water formulations, the antigen remains typically in the water phase and does not interact with the oil droplets. It provides neither direct transport nor depot effect for the antigen.
Antigens and MF59 are taken up by neutrophils and monocytes, and later followed by dendritic cells (DCs) and B cells, and moved to draining lymph nodes . MF59 effects the apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and stimulates IL-4 and Stat-6 signaling, while being independent of any type-1 interferon or inflammasome pathways. The emulsion has further been shown to significantly increase the IL-5 and IL-6 levels . MF59 has been selected as a COVID-19 vaccine adjuvant because it was proven to induce fast priming of antigen-specific CD4(+) T-cell responses, and to induce strong and long-lasting memory T- and B-cell responses, and to overall broaden the immune response against the vaccine antigens .
CpG adjuvants are synthetic DNA sequences containing unmethylated CpG sequences. These oligonucleotides are potent stimulators of the innate immune system through activation of Toll-like receptor-9. TLR9 agonists directly induce the activation and maturation of plasmacytoid dendritic cells and enhance differentiation of B cells into antibody-secreting plasma cells . As a vaccine adjuvant, CpG augments the induction of vaccine-specific cellular and humoral responses. Dynavax Technologies has developed a short CpG-containing oligonucleotide sequence named CpG 1018 and progressed it through clinical testing as an adjuvant for immunization against hepatitis B virus (HEPLISAV-B) . The immunostimulatory effects of CpG are optimized by keeping the oligonucleotide and the vaccine antigen in close proximity. Driven by electrostatic interaction, CpG 1018 binds well to aluminum hydroxide and is therefore well-suited for co-formulation with aluminum-based subunit vaccines .
AS03 is a squalene-based oil-in-water emulsion produced by GSK. It has been tested extensively in the clinic and is used for the H1N1 pandemic flu vaccine Pandemrix . It is also in Arepanrix and the new Q-pan for H5N1 influenza . Similar to MF59, AS03 can induce proinflammatory cytokines and chemokines, including CXCL10, but independently of type-1 interferon. This pro-inflammatory response is associated with improved recruitment, activation, and maturation of antigen-presenting cells at the injection site .
Novavax’s proprietary Matrix-M adjuvant consists of two individually nanosized particles, made with a different saponin fraction (Fraction-A and Fraction-C). The saponin particles are stabilized with cholesterol and phospholipid . As a part of different vaccine formulations, Matrix-M has been proven to augment both Th1 and Th2 type responses, inducing high levels of neutralizing antibodies, and enhancing immune cell trafficking [116,117]. Based on clinical data from over a dozen studies Matrix-M is considered safe and potent [, , , , ], however, it has not yet been part of a commercially available vaccine.
Advax made by Vaxine (Australia) is a microcrystalline polysaccharide particle composed of delta inulin . In published studies covering many years of research, delta inulin has been shown to provide a robust humoral and cellular immune response when formulated with recombinant vaccine antigens. Advax adjuvant has recently also successfully been tested in several human trials including vaccine studies to prevent seasonal and pandemic influenza, hepatitis B, and hyperallergic reactions to insect venom . Compared to the controls, the Advax adjuvant [56,123] seems to improve antibody and T-cell responses, while being safe and well-tolerated . It should however be noted that Advax is still a relatively new adjuvant, which has only been tested on small groups of patients and has not yet been part of a marketed vaccine.
Assessment of the adjuvant landscape for COVID-19 vaccines and outlook for novel adjuvant systems
Potential vaccines against the coronavirus should induce a strong humoral response with high neutralizing antibody titers, in combination with a Th1-type cellular response . There has also been discussion on the role of Th17 induced by an IL-6 inflammatory response, as this seems to contribute to severe lung pathology and mortality .
Except for alum, all adjuvants listed above have the potential to induce an antigen-specific, safe, and adequate Th1-type immune response when added to a SARS-CoV-2 recombinant vaccine formulation. Aluminum hydroxide still has value as part of a vaccine formulation, as it is known to promote a stronger humoral response; it will, however, require an additional immunostimulant to skew the cellular response towards Th1.
In addition to the adjuvant combinations currently evaluated for COVID-19, other experimental adjuvants might be beneficial for a vaccine against SARS-CoV-2 based on earlier coronavirus research studies. For a detailed overview of adjuvants for coronaviruses, we refer to recent reviews by Liang  and Gupta .
Aside from the functional aspect of the adjuvants, production capacity and costs are of key importance to guarantee vaccine availability to citizens of all nations, independent of their status or wealth. From that perspective, protein-based adjuvants may offer a significant advantage. They can be made using the same technologies used to produce the vaccine antigen (Section 3) and can easily be scaled up at a low cost. One example is rOv-ASP-1, a helminth-derived molecule that has been shown to induce potent Th1-type immune responses when added to influenza, helminth, and rabies vaccines [, , , ].
Another example is dmLT or LT(R192G/L211A), an ADP-ribosylating enterotoxin from Escherichia coli. What distinguishes dmLT is that it enhances antigen-specific IgA antibodies and long-lasting memory to co-administered antigens, in addition to an increase in Th1, Th2, Th17, cytotoxic T lymphocytes (CTLs), and antigen-specific antibodies [130,131]. Secretory IgA can act as an immune barrier and neutralize SARS-CoV-2 before it reaches and binds to epithelial cells . Therefore, dmLT may be an excellent choice to add to any mucosal or parental vaccine targeting SARS-CoV-2 RBD.
reference link : https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7788321/
reference link : Science Immunology (2021). DOI: 10.1126/sciimmunol.abh3634