The ongoing SARS-CoV-2 crisis has brought the development of novel vaccines into the spotlight. Processes that used to take place behind closed doors in academic and commercial research institutions are taking center stage. We are witnessing traditional vaccines being rolled out alongside modern vaccines in a race to slow the spread of SARS-CoV-2. The current crisis is also showcasing just how essential fast, reliable and flexible vaccine platforms are if we want to win this race. New approaches are needed to support faster development of more efficacious vaccines.
Empirical vaccine development: The traditional path to vaccines
In traditional vaccine development, the live pathogen is inactivated or attenuated empirically by repeated passaging through cultured cells. The weakened pathogen is administered, and the immune system responds by producing antibodies. This approach works well for simple pathogens with highly conserved structures. However, for difficult pathogens, such as those with high rates of mutations and large numbers of different strains, this approach is not feasible to develop an effective vaccine that provides lasting protection. Notorious examples of difficult pathogens include the influenza virus or human immunodeficiency virus (HIV). To create vaccines that provide efficient, long-lasting protection from the flu or HIV/AIDS, broadly neutralizing antibodies are required.
While the rate of mutation in SARS-CoV-2 is lower than that of influenza or HIV,1 the emergence of SARS-CoV-2 mutations that may be less vulnerable to certain vaccines is cause for concern.2 With traditional vaccine development, the response time to fight emerging variants of SARS-CoV-2 might be too slow, too labor intensive and ultimately too expensive. Rational vaccine design may offer a more flexible and faster alternative to traditional empirical approaches.
Where we go from here: Reverse engineering and rational vaccine design
Instead of administering live or attenuated pathogens and simply observing the results, new technology allows rationally designed vaccines that consist of antigens, a delivery system, and adjuvants that are uniquely tailored to induce a specific and predictable immune response. The principle of reverse-engineering is used to identify the target epitopes from the neutralizing antibodies. Knowledge about mechanisms of protection, effector mechanisms and signaling pathways involved in processing the pathogen and adjuvants is used to design peptides that act as specific immunogenic epitopes. Rational vaccine design incorporates multiple cellular and molecular approaches to gain broader understanding of the viral pathology to identify more opportunities for specific vaccine targets.
Antigens that will drive a strong and very specific immune response are the essential component of a rationally designed vaccine. Identifying appropriate antigens starts by isolating potent neutralizing antibodies. However, the traditional empirical approach of measuring antibody titers or cellular response from blood is not feasible because it requires large numbers of donors, large sample volumes and access to high-throughput technology.3
Zost et al. used a rational design strategy to develop a cocktail of two antibodies that yielded stronger protection from SARS-CoV-2 virus in their mouse model than either antibody on its own.4 Their strategy relied on RT-qPCR with TaqMan primers to test the efficacy of monoclonal antibody (mAb) candidates for the cocktail. In these studies, they isolated mAbs from B-cells of donors who were recovering from SARS-CoV-2 and then selected a subset of antibodies that exhibited the strongest SARS-CoV-2 S-protein neutralizing impact in binding assays. The S-protein is the main antigen component among all SARS-CoV-2 structural proteins.5 To locate the sites within the S-protein receptor binding domain (SRBD) that were involved in interaction, the team used mutagenesis to alter residues in the SRBD. With the ability to readily synthesize mutated peptides, the team was able to conduct experiments with a wide range of technologies to identify the two most potent neutralizing mAbs. RT-qPCR revealed that levels of viral RNA as well as gene expression of cytokines and chemokines (indicators of inflammation) were both significantly reduced in the mouse lungs. The team concluded that the two antibodies tested as a cocktail provided preventive protection against SARS-CoV-2 infection.
Ku et al. also used a rational-design mutagenesis approach to develop a two-antibody cocktail to prevent viral escape. To identify mAb candidates for the optimum cocktail, they constructed a comprehensive SARS-CoV-2 SRBD mutation library. To establish the library, they used Sanger sequencing to confirm each of the unique clones in virus constructs. The team postulated that their approach might be broadly used to quickly and efficiently determine other types of mAb cocktails to combat the virus.6
Rational design of vector-based vaccines
Vector-based vaccines are well established and induce a robust T- and B-cell response. However, the manufacturing of vector-based vaccines is complex and offers limited scalability.7 Pre-existing immunity, due to common cold viruses or prime-boost (two-dose) immunization regimes, is another obstacle of vector-based vaccines. Prime-boost vaccination regimes can also come with distribution challenges. Manufacturing operations must be able to produce a stable supply to ensure enough vaccine is available for second doses. It can also be very difficult to ensure people and populations that have limited access to care can return for a second dose at the required time. Rational vector vaccine development strategies are focusing on solving these drawbacks.
Global management of the SARS-CoV-2 crisis depends not only on the development of safe and protective vaccine candidates but also on the rapid production and deployment of billions of doses. Single-dose vaccine strategies could be critical to worldwide immunization.
Tostanofski et al. designed an adenovirus vector-based vaccine that requires just a single dose to protect against severe pathogenesis in hamsters.8 In the model used, severe pathogenesis can progress as an inflammatory response even after viral replication has diminished, so protection against viral replication does not ensure protection against severe infection. In developing their vaccine, this team focused on reducing the hallmark symptoms of severe infection in addition to neutralizing viral expression and boosting antibody response. To select candidate variants for the vaccine, they designed multiple adenovirus vectors containing various mutations in the SARS-CoV-2 S-protein. As for the antibody-based approaches above, Sanger sequencing confirmed the intended mutations in the vectors. A custom TaqMan assay was designed to measure subgenomic viral RNA. RT-PCR expression tests revealed reduced infectious virus in animals vaccinated with either construct. A model such as this one that enables testing of vaccine candidates against severe infection may ultimately contribute to the development of vaccines and therapeutics that reduce the incidence of severe disease and even mortality.8
Single-dose vaccines have also been developed using nanoparticle constructs of spike protein multimers expressed on ferritin, yielding three antigenic elements and enhanced antibody response.9
The more the merrier: Multi-epitope vaccines
An antigenic epitope is the segment of an antigen recognized by the immune system. A multi-epitope vaccine therefore uses a series of epitopes to offer protection from viral infections. Ideally, multi-epitope vaccines induce a broad immune response, targeting cytotoxic T-cells, Th and B-cells, and compensate for a lack of pattern-associated molecular patterns (PAMPs).10
In a crisis, rapid development of vaccines is vital to stemming the spread of disease.Chiuppesi et al. constructed a multi-epitope vaccine platform that efficiently generated recombinant vectors built entirely from chemically synthesized DNA.11 The platform was based on a unique three-plasmid system that enabled multiple antigen sequences to coexist in a single vector. They used the platform to rapidly produce fully synthetic vectors that simultaneously expressed SARS-CoV-2 S and N (nucleocapsid) antigens and induced a strong immune response in mice upon immunization. Sanger sequencing was used to confirm vector construction and design. The team speculated that a vaccine platform based on synthetic DNA will streamline development of virus vaccine vectors to rapidly respond to spreading of the virus.
While the efforts to vaccinate the global population against SARS-CoV-2 progress, the efforts to develop efficient, cost-effective vaccines that offer lasting protection are progressing simultaneously. It is too soon to give a definitive answer about whether SARS-CoV-2 will become less prevalent, but it is safe to assume that this virus is here to stay.12
Future vaccine design strategies are likely to continue to leverage an expanding range of cellular and molecular technologies to develop more powerful vaccines and new vaccine platforms. However, in order to develop completely new approaches, it will be essential to incorporate a wider range of methodologies, which may include new antigen forms and sequences; synthetic DNA variants; antibody, antigen and viral gene expression; and vaccine stability and immunogenicity. A plethora of genomic analysis technologies is becoming more important in vaccine design: next-generation sequencing to identify gene variants that may be relevant to viral pathology, PCR and fragment analysis to amplify and isolate intended vector variants, Sanger sequencing to confirm variants for mutagenesis, and RT-qPCR to assess the presence of viral nucleic acid and expression of cellular indicators. Population genomics and pharmacogenomics studies may reveal key genetic contributors to pathogen infection, vaccination and therapeutic responses in different populations, bringing precision medicine to vaccine development.
Rational vaccine design is an essential part of the puzzle to not only slow the spread of SARS-CoV-2, but to also be prepared for emerging viruses we don’t even know about yet.13 These strategies may contribute to creating vaccines that induce broadly neutralizing antibodies, offering protection even from mutant strains. Ultimately, the more people we protect from an infection, the less room we give the virus to evolve.14,15 It’s a win-win. When new variants and new coronaviruses emerge, rational vaccine design may enable the rapid development of new vaccines or updates to existing vaccines, helping to speed up licensing, production and distribution to stay ahead in the race against the spread of the virus.
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