Introduction
Emerging data are now surfacing suggesting that recombinant protein vaccines indeed might offer an advantage or complement to the nucleic acid or viral vector vaccines that will likely reach the clinic faster. Here, we summarize the current public information on the nature and on the development status of recombinant subunit antigens and adjuvants targeting SARS-CoV-2 infections. WHO lists 42 candidate vaccines in clinical evaluation, among them 12 based on recombinant protein technology (Table 1).
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. In the S-protein trimer, three S1 subunits sit on top of a stem of three S2 subunits. SARS-CoV-2 shares extensive sequence homology, as well as structural and functional homologies with prior coronaviruses, namely SARS-CoV, but also MERS-CoV. Early on in the pandemic, it was shown that anti-SARS Sprotein 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 Receptor binding domain (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. In Antibody-dependent enhancement (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).
Full-length S-protein based vaccines
The NVX-CoV2372 trimeric nanoparticle produced by Novavax is made from the full-length S-protein. 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 the case of SARS-CoV-2 S-protein, the group has shown that the final antigen forms a homotrimer similar to the natural conformation of the spike protein, likewise able to assume both open and closed conformations. Clover has genetically fused the SARSCOV-2 S-protein (aa residues 1-1211) to human C-propeptide of alpha1(I) collagen.The fusion protein self-trimerizes and aids purification by affinity chromatography using a collagen-receptor-derived resin.
RBD-based vaccines
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 RBDencoding 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 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 1 clinical trials.
Multi-epitope vaccines
Many vaccine candidates in the literature employ neither the native viruses 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. 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 COVID19 vaccine to induce broad T-cell immunity. It will be interesting to see how the ongoing studies shift the focus between the full-length S-protein based and the RBD vaccines.
Protein production and delivery platforms
Escherichia coli
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. 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 while Bexsero® , developed by GSK, uses three immunogenic meningococcal antigens (fHbp, NadA, and NHBA) synthesized in E. coli. E. coli expression systems do not typically provide post-translational modifications (PTMs), which can affect the immune response and vaccine function.
Yeasts
Yeasts are another well-known microbial expression platform. Unlike E. coli, yeasts can secrete recombinant proteins extracellularly, which makes the downstream purification process simpler and less costly. 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. 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.
Mammalian cell culture expression
systems
Most current COVID-19 recombinant protein vaccine candidates are expressed in mammalian cell culture-based expression systems 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 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, uses Chinese Hamster Ovary (CHO) cells to produce recombinant glycoprotein E from the virus as its antigen.
Insect cells
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. Insect cells can reach higher densities in a shorter period when compared to mammalian cells. Insect cells do not cause hyperglycosylation, which may be an issue if sophisticated glycans are required to maintain the function of a recombinant protein. However, this system does not cause N-glycosysylation. Kentucky BioProcessing and other tobacco growers, for example, are employing tobacco plants to express SARS-CoV-2 vaccine antigens.
Vaccine Delivery
Parenteral Vaccination
COVID-19 subunit vaccine candidates currently at an advanced clinical stage of development are being administered either by intramuscular (i.m.) or subcutaneous (s.c.) injection, and while some novel vaccine platforms require specialized administration equipment, protein-based vaccines can be administered using conventional low-cost hypodermic needles. Intradermal (i.d.) immunizations could probably generate a stronger immune response because the dermis contains higher numbers of dendritic cells, which will facilitate the uptake of antigens. Local inflammation in the dermis induces the maturation of the dendritic cells and stimulates migration into draining lymph nodes. However, i.d. needle injections are technically complex and allow for only small volumes.
Mucosal Vaccination
Wang et al., (2020) have designed a strategy to produce an oral vaccine based on the SARSCoV-2 spike protein. Oral vaccines promise to be particularly suitable for low-and middle-income countries since they can be administered without trained personnel and can be transported and stored without requiring a cold chain. In addition, the vaccine designed in this study in the benign probiotic bacterium Lactobacillus plantarum is expected to specifically trigger an enhanced mucosal immune response, desirable for preventing viral respiratory infections such as COVID19. Intranasal vaccination for COVID-19 has also been investigated by many groups, mostly with live attenuated flu viruses that are genetically modified to express the spike protein.
Adjuvants
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.
Conclusions and outlook
DNA and mRNA vaccines inactivated viruses, as well as vector-based strategies, were able to attract more attention. Whether this first-generation vaccine will have the necessary efficacy to prevent infection in humans, and whether its, likely, new vaccine technology, will be received well by an increasingly vaccine-hesitant public remains to be seen. While recombinant protein vaccines may lag in development, they may offer the better solution in the long run, in particular with respect to transferring a proven vaccine technology to low and middle-income countries, where the infrastructure to distribute fastidious nucleic acid vaccines, e.g. storage required at – 94° F is out of reach. Moreover, the true efficacy of nucleic acid-based vaccines remains unproven in humans and vector-based vaccines carry the risk of immunity to the vector which would make booster vaccinations challenging.
Source: Advanced Drug Delivery Reviews, Volume 170, 2021, Pages 71-82, ISSN 0169-409X, https://doi.org/10.1016/j.addr.2021.01.001.