As the world moves into a second wave of the coronavirus disease 2019 (COVID-19) pandemic, the need for an affordable and widely available vaccine continues to remain urgent. Researchers at the Texas Children's Hospital Center for Vaccine Development, USA, have published a report on the preprint server bioRxiv* that describes a yeast-expressed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor-binding domain (RBD) protein, a key target of current vaccine development efforts. The yeast RBD is shown to be immunogenic in mice and to elicit a high neutralizing antibody titer, making it a promising foundation for a human vaccine.
The major areas to focus on in terms of developing an attractive vaccine include its effectiveness, ease of production, scalability, and suitability for widespread delivery.
RBD-based aluminum-adjuvanted vaccine
The current study is based on the results of earlier coronavirus vaccine candidates that are built around recombinant viral RBD proteins formulated with the aluminum-containing adjuvant Alhydrogel to counteract SARS (severe acute respiratory syndrome) and MERS (Middle East respiratory syndrome) – the earlier lethal coronavirus outbreaks of the current century. These were shown to be effective at inducing high neutralizing antibody titers and protecting against these earlier viruses, in mouse studies. Moreover, they did not cause eosinophilic immune enhancement, unlike the wild-type spike protein (or S-protein).
A) Amino acid sequence alignment between SARS-CoV-2 RBD219-WT (S2-RBD) and RBD219-N1C1 (S2-RBD-N1C1). In the N1C1-mutant, the N-terminal glutamine residue (N331, green) is removed and a C538A mutation (yellow) was introduced. Neither mutation is inside the receptor-binding motif (RBM, purple). B) The structure model of RBD219-WT was extracted from the crystal structure of the SARS-CoV-2 spike protein (PDB ID 6VXX). The RBM (N436-Y508) is again shown in purple while the deleted asparagine (N331) and mutated cysteine (C538, mutated to alanine) in RBD219-N1C1 are highlighted in green and yellow, respectively
Affordable and immunogenic
The researchers decided to use the yeast Pichia pastoris to produce the recombinant RBD in large amounts. This is already in extensive use in many low and middle-income countries (LMICs) to produce hepatitis B vaccines. They selected and compared two variants of the RBD, the wild-type and the N1C1 variant, to test their potential to elicit high neutralizing antibody titers, as well as cellular immunity, and to assay their protective capability.
The N1 variant of the SARS-CoV RBD vaccine, when formulated with aluminum oxy-hydroxide (Alhydrogel), was already known to induce a high neutralizing antibody response, without any unwanted immune enhancement, as shown by the absence of eosinophilic lung infiltrates when challenged by the virus following immunization. In fact, it was more effective than the full-length spike at eliciting specific antibodies, and it protected the treated mice from SARS-CoV-2 infection completely.
Modified RBD enhances protein yield
The current study used the recombinant SARS-CoV-2 N1C2-RBD analog for immunization. The researchers reduced the excessive glycosylation of the RBD to allow for easier purification, while the introduction of a free cysteine residue by mutation enhanced protein production within the yeast system. At the same time, the changes did not reduce the functionality of the antigen, nor did they impact its immunogenicity when used as in the current experiment, in an Alhydrogel-containing formulation.
Both the wild-type and N1C1 RBD antigen-based vaccines formulated with Alhydrogel induced high IgG titers after two doses in mice. The SARS-CoV-2 pseudovirus also elicited high neutralizing titers in the range of 1:1,000 to 1:10,000 dilutions, for both the antigens. They induced a wide range of cytokines, including interferon-gamma, IL-6 and IL-10. The robust neutralizing antibody response, along with the T-cell activation profile, are reassuring findings.
No immune enhancement
Moreover, the researchers suggest that this formulation may reduce the risk of immune enhancement, which also throws new light on the mechanism of immune enhancement. Thus, Alhydrogel may be an important adjuvant for coronavirus vaccine research. At present, there is no agreement on how to select the best antigens, adjuvants, delivery vehicles or biotechnology tools based only on established principles of vaccine development or the preclinical information available so far.
Such recombinant vaccines may be useful both as independent vaccines or as part of a prime-boost regimen using different vaccines at various stages – as is already used with malaria. Aluminum is already used in seven COVID-19 trial vaccines at present, and often along with other immunostimulants so as to avoid unbalanced immune responses.
This yeast platform's advantages include its low cost of production and the simplicity of the manufacturing process. This allows for it to be used by LMICs on a large scale, thus providing successful vaccines that are produced cheaply and without the need for a sophisticated infrastructure. The platform has been validated already by earlier vaccines, such as the recombinant hepatitis B vaccine, which is produced in yeast by several countries that belong to the Development Country Vaccine Manufacturers Network (DCVMN). This means that the same process can be used for the production of a yeast-based COVID-19 vaccine without additional investment or training being necessary.
In fact, the process of production and the research cell lines for the recombinant RBD antigen used in the current study are already licensed to an Indian firm, and the cGMP production of this antigen is underway for the purpose of further clinical development. Simultaneously, the antigen in wild-type and N1C1 form is still being tested in preclinical models to optimize its physiological activity as well as to evaluate other newer formulations.
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.