Recent outbreaks of infectious diseases, such as the middle east respiratory syndrome, Zika infection, Ebola hemorrhagic fever, and Coronavirus disease (COVID-19) pose significant challenges on the rapid development of efficacious vaccines. Virus-vectored vaccines, as an important new vaccine, can be administrated noninvasively through aerosol inhalation or oral administration, which could stimulate humoral, cellular, and mucosal immune responses without the need for adjuvants, showing good immunogenicity and safety in clinical trials or in emergency use. With the deeper understanding of the viral genome and structural proteins, synthetic biology has enabled the design and modification of viruses to produce recombinant viral vector-based vaccines with high titer, safety, and immunogenicity, and such research has significant implications for the vaccine development. This review highlights major strategies employed in the construction of virus-vectored vaccines, including the construction method of replication-competent or replication-defective viral vectors, and the development of viral vectors commonly used in producing the recombinant vaccines. Among these viral vectors, replication-deficient adenovirus-based vectors with gene deletion in the E1 and E3 regions are most mature for use. Currently, adenoviral vectors that have been used in the approved recombinant vaccines include Ad5, Ad26 and ChAdOx1. Vesicular stomatitis virus and flavivirus with small genomes are negative-sense and positive-sense single-stranded RNA viruses, respectively, which are easy to prepare and more suitable for being used in developing recombinant vaccines with small antigen proteins. Poxviruses and herpesviruses have large genomes for high packing capacity, but they are most difficult to be modified with synthetic biology methods. Different viral vectors need to be prepared using different strategies, and consequently vaccines developed with these vectors have different immune effects. The construction strategies of different viral vector vaccines introduced in this review will provide valuable theoretical reference for the research and development of novel viral vector vaccines. In the future, virus-vectored vaccines will be iteratively developed for higher safety, stronger protection, better compliance and lower production cost.

Human diseases, especially infectious diseases and cancers, pose unprecedented challenges to public health and the global economy, making the development of preventive and therapeutic vaccines a top priority for addressing these challenges. Among all vaccines, vector vaccines that activate T cell immune responses have significant advantages. This article reviews the immunological principles of vector vaccines, strategies for designing T cell vector vaccines, and their research advances. T cells, upon infection, can differentiate into various effector T cell subsets that play a crucial role in clearing pathogens. Research on the functions and mechanisms of effector T cells is essential for designing vaccines that can elicit T cell-mediated immunity. Currently, the development of vaccines for many viruses such as HIV and HCMV as well as cancers focuses on T cell-based vaccines. Various vectors, including viral vectors, bacterial vectors, and nucleic acid vectors, exhibit excellent performance on antigen delivery capability, immunogenicity, and protective efficacy. In addition, this article summarizes strategies for designing T-cell vector vaccines, including identifying appropriate antigen presentation pathways and vector delivery routes, ensuring biological safety, selecting suitable vaccine vectors, and evaluating the advantages and disadvantages of various vector vaccines. Notably, mRNA vaccines have played a crucial role in addressing the challenges posed by the COVID-19 pandemic. Technological advancements in vector vaccines are expected to accelerate the development of novel vaccines and enhance preparedness for emerging public health events. This review provides insights for the design of vector vaccines that are both safe and efficient. With advancements in vector vaccine technology and the progress of various interdisciplinary approaches, the next generation of vaccine development will continue to drive the evolution of vaccinology.

A Public Health Emergency of International Concern (PHEIC) is defined by the World Health Organization (WHO) as “an extraordinary event which is determined to constitute a public health risk to other states through the international spread of disease and potentially requires a coordinated international response”. To date, WHO has declared seven PHEIC events, including the H1N1 influenza, Ebola, poliomyelitis, Zika, COVID-19 and mpox. Vaccination remains as an effective method in preventing infectious diseases. The International Health Regulations (IHR) Emergency Committee's recommendations for preventing or reducing the international spread of disease and avoiding unnecessary interference with international traffic include an emphases on the development of diagnostics and therapeutics for diseases, as well as the vaccine development. The mRNA vaccine represents a platform technology for the development of next-generation vaccines, and possesses distinct advantages, such as a shortened development cycle, scalable and cost-effective production, as well as enhanced amplification capacity, highlighting its potential in rapid responding to emerging and re-emerging infectious diseases. In recent decades, the development of mRNA synthesis technology and nucleic acid delivery system has facilitated the rapid development of mRNA vaccines and their clinical applications. Here, we overview the development of mRNA vaccines in response to the past PHEICs, and discuss challenges and trends in this regard. Currently, COVID-19 mRNA vaccines have been authorized for human use, while multiple mRNA vaccines against influenza, Zika, mpox and Ebola have been evaluated in clinical or pre-clinical studies. Despite their proven efficacy, there is still room for further improvement of the mRNA vaccines. The mRNA design, optimization, delivery, formulation, manufacturing, storage, and transportation can be further improved by integrating synthetic biology, biochemistry, artificial intelligence, and other multidisciplinary technologies. Although the emergence of the next PHEIC cannot be predicted with certainty, we are optimistic that the mRNA vaccine technology will play a pivotal role in preventing pandemics in the future.

In recent years, bacterial infections have emerged as the second leading cause of death globally, posing a serious threat to public health and demanding prioritized intervention from the healthcare community worldwide. While antibiotics have conventionally been used as the primary strategy to combat bacterial infections, their efficacy is increasingly compromised due to the emergence of drug-resistant bacteria, especially multi-drug-resistant and even pan-drug-resistant superbacteria. Vaccines are thus considered as one of the most scientific, economical, safe, and effective means to prevent infectious diseases and improve public health, which are estimated to save 2 to 3 million lives annually, and can serve as a critical tool in the battle against antimicrobial resistance. However, the complexity of bacterial structure and pathogenic mechanism has hindered the development of vaccines. Challenges include screening and rationally design of effective antigens, ensuring compatibility of various antigen combinations, establishing animal models for preclinical evaluation, and defining reliable endpoints for clinical efficacy assessment. As a result, only a small number of bacteria vaccines have been successfully developed so far, and none of them has been licensed to combat the most prevalent drug-resistant infections, such as Staphylococcus aureus, Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae. Synthetic biology is a brand-new multidisciplinary focusing on repurposing natural biological systems and inventing innovative biological tools, technologies, devices, and systems for practical applications, and its concepts, principles and technologies have been extensively employed to facilitate vaccine development, including rational design, screening, and optimization of antigen, carrier, adjuvant and delivery system as well as the modulation of bacterial pathogenicity and immune responses. Herein, we outline the current status of the development of bacterial vaccines and the advancement of clinical trials for drug-resistant bacterial vaccines. Then, we summarize the application of synthetic biology technology in the development of major bacterial vaccines. Finally, we prospect the potential of synthetic biology in creating novel bacterial vaccines. Researchers have access to a greater variety of design possibilities for bacterial vaccines through synthetic biology. To maximize these benefits, we should employ synthetic biology and related technologies more efficiently in developing bacterial vaccines. Meanwhile, we should develop a scientific, reasonable, effective, and feasible management system, as well as regulatory measures, to expedite the development of efficient bacterial vaccines, therefore addressing the problem of antibiotic resistance to protect human health.

The precise design and synthesis of carbohydrates with important biological functions and more complex structures is a frontier in synthetic biology. Recently, a novel strategy named Protein Glycan Coupling Technology (PGCT) based on bacterial oligosaccharyltransferases has been developed and widely used in the biosynthesis of bacterial glycoconjugate vaccines, which are one of achievements in modern medicine due to their effectiveness in fighting against infectious diseases. Herein, progress in developing key components for manufacturing glycoconjugate vaccines, such as oligosaccharyltransferases (PglL, PglS, PglB, and TfmP), carrier proteins (CRM197, diphtheria toxoid, recombinant Pseudomonas aeruginosa exotoxin A, and nanoparticles), polysaccharide biosynthesis gene circuits, and glyco-engineered strains is reviewed. Meanwhile, producing glycoconjugate vaccines through fermentation presents advantages in good product quality control for safety and efficacy, low production cost, and environmental-friendly manufacturing. PGCT has potentials to overcome some limitations of chemical conjugation production processes, such as complex purification and high cost, for competitiveness with existing chemical conjugates. As an emerging technology, more technological innovations are needed for PGCT. In the future, the directed evolution of oligosaccharyltransferases, the application of protein nanoparticle carriers, the combination rearrangement of glycosyltransferases, and the optimization of engineered bacterial strains with better metabolic pathways are expected to further promote the biosynthesis of conjugate vaccines. The next few years will be an important and exciting time for PGCT, as recent technological advances are being applied to the development of novel glycoconjugates, and ongoing large-scale clinic trials on the efficacy of glycoconjugate vaccines will also demonstrate the feasibility of this technology, making the future of PGCT vaccinology promising.

Nanoparticle vaccines have been established firmly as a cornerstone of modern immunization strategies, with a compelling history that trace their pioneering use in human being back to 1981. Within the past four decades, these vaccines have not only demonstrated their efficacy, but have also been developed as powerful tools in fighting against a range of infectious diseases, most notably hepatitis B virus (HBV) and human papillomavirus (HPV). Their success can be attributed to their exceptional immunogenicity and impeccable safety as well, making them invaluable in curbing the spread of viruses and safeguarding the health and well-being of human being. The global outbreaks of the COVID-19 pandemic, driven by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has made vaccination into the forefront of public health priorities. This unprecedent challenge has accelerated the progress of various vaccine technologies, with nanoparticle vaccines attracting considerable attention. However, due to their relatively empirical design approaches and complicated manufacturing processes, progress in the clinical trials of SARS-CoV-2 nanoparticle vaccines has not been highlighted particularly. Therefore, the imperative for developing nanoparticle vaccines is to figure out their rational design, requiring groundbreaking advancement in novel technologies and theories. In this endeavor, synthetic biotechnology has emerged as an indispensable tool, driving the technological innovations of the production of nanoparticle vaccines. This article begins with an overview of technological advancements in the development of nanoparticle vaccines, encompassing progress from self-assembled nanoparticles to assist-assembled nanoparticles, and ultimately to antigen-display on formed nanoparticles. Furthermore, discoveries in understanding the unique roles of nanoparticle vaccines in enhancing antigen immunogenicity are updated, particularly in the function of nanoparticles with novel antigen presentation pathways. Finally, a comprehensive summary of the clinical trials of nanoparticle vaccines on fighting the COVID-19 pandemic is presented. In conclusion, we firmly believe that nanoparticle vaccines, bolstered by the scaffolding of synthetic biotechnology, are poised to emerge as steadfast guardians in the global battle against emerging and highly infectious diseases, and ongoing progress in this regard not only holds great promise, but also has potentials to revolutionize contagious disease prevention and control on a global scale.

Since the outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the end of 2019, it has evolved into different lineages, including Alpha, Beta, Delta, and Omicron. The development of broad-spectrum vaccines has become a necessity for preventing the highly mutated respiratory viruses. Traditional vaccine antigens, originating from prototype strains, cannot cover rapid mutations with these viruses, leading to breakthrough infections. With the development of synthetic biology, new technologies such as multivalent coupling of antigens, reconstructed dominate antigen modules, engineering design of conserved epitopes, epitope display, and computation-guided reconstruction have enabled redesigning antigens to achieve stronger immunogenicity with broader spectrum. The technology of synthetic biology is also applicable in the vaccine production process, such as antigen expression in nanoparticles, viral vectors, nucleic acids, and subunits. This article reviews the applications of synthetic biology technology in developing broad-spectrum vaccines in recent years, particularly for the broad-spectrum SARS-CoV-2 vaccines, and summarizes how to display common antigens and cross-antigenic sites by the reverse vaccinology for the activation of broad-spectrum immune responses against different mutant strains, achieving broad-spectrum vaccine protection effects through “remaining constant in response to ever-changing”. The article also provides a comprehensive comparison of the strengths and limitations of different broad-spectrum vaccine design strategies and discusses challenges to applying synthetic biology in the development of vaccines, offering valuable insights for universal against highly mutation viruses.

Glycoproteins with enveloped viruses, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza virus, and human immunodeficiency virus (HIV), display a trimeric conformation. Different from the monomeric form, the trimeric proteins exhibit superior immunogenicity. Several trimerization motifs, such as Foldon derived from phage T4 fibritin, have been used to promote the formation of trimeric proteins with natural conformations. Although the Foldon-induced trimeric proteins are stable, their high immunogenicity limits applications in the development of vaccine antigens. In a previous study, we developed a recombinant human collagen type Ⅲ protein and determined its crystal structure, revealing a triple-helix conformation. However, the potential of this recombinant protein as a trimerization motif remained unknown. In this study, we demonstrated that the recombinant humanized type Ⅲ collagen (Rh3C) was able to act as a trimerization motif, facilitating the spontaneous trimer formation of the Rh3C-conjugated receptor-binding domain (RBD) within the spike (S) protein of SARS-CoV-2. This trimeric protein could induce a stronger SARS-CoV-2 RBD-specific IgG, IgG1, and IgG2a immune response, when compared with the monomeric RBD protein in the immunized mice. Notably, the Rh3C-RBD protein, when adjuvanted with the novel STING agonist CF501, also elicited significantly higher neutralizing antibody responses against both the pseudotyped SARS-CoV-2 (D614G) and its variant Omicron (BA.2.2) in the immunized mice. To showcase the broad applications of the Rh3C trimerization motif, we further demonstrated that the Rh3C-conjugated HA1 of the influenza virus could also elicit a stronger antibody response than free HA1. Considering the wide distribution of the Rh3C protein in human bodies, its use as a trimerization motif would not induce an immune response due to immune tolerance, thereby allowing the immune response to concentrate on targeted viral proteins. Therefore, this Rh3C-based trimerization motif holds great potential for the design and optimization of vaccines consisting of trimeric protein antigens.

Prime Editor (PE) is an innovative gene editing tool based on the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein (CRISPR/Cas) system, which has revolutionized multiple fields, including genetics, medicine, and agriculture. Emerging as a successor to Base Editor (BE), PE has gained worldwide attention due to its ability to introduce base substitutions, insertions, and deletions without causing double-strand DNA breaks, which significantly reduces the risk of off-target effect and unwanted genetic change. Notwithstanding its immense potential, researchers need to address PE's long encoding sequence and low editing efficiency for its maximal applications. Researchers have been working relentlessly to explore and enhance the editing efficiency and safety of PE by modifying its protein scaffold, optimizing the guide RNA design, and identifying cellular factors that influence its activity. Improved PE variants have been developed with enhanced accuracy and efficiency as well as decreased off-target effect when compared with their initial versions, demonstrating their potential in gene editing-related applications. Several strategies have been investigated to enhance PE performance, including: ① Modifying the structure of PE proteins to increase their efficiency, specificity, and binding affinity, thereby significantly improving their editing activity. ② Optimizing the design of pegRNAs, such as modifying the length, composition, or structure, that can boost PE's editing efficiency. ③ Identifying and manipulating cellular factors, such as proteins and RNAs, that bear functional relationships with the PE system, thus greatly enhancing its gene editing capabilities. ④ Developing automated design tools to facilitate the customization of the PE system for specific applications, vastly improving its practicality in research and clinical settings. Finally, this article summarizes the applications of PE in engineering animals and plants and developing gene therapy. Despite much room for further improvement in PE, significant advances have been made in improving its editing efficiency and safety. The rapid development of Cas9 and BE for treating genetic diseases stands as compelling testimony to the potential of PE in advancing gene editing technologies and applications. With continued research and development, PE holds great promise for improving human health and well-being.

Over the past years, synthetic biology has seen significant development, establishing a typical "Design-Build-Test-Learn (DBTL)" cycle for engineering cell factories. This cycle has been becoming an enabling methodology for smart breeding to accelerate the development of biomanufacturing. In the DBTL cycle, the testing step primarily aims to evaluate the phenotypes of constructed cell factories, which can provide a large amount of data for further learning and iterative optimization. Due to the complexity of cellular metabolic networks and regulatory mechanisms, as well as the complicated associations between genotypes and phenotypes, the design and construction of cell factories have traditionally involved long-term and labor-intensive iterative experiments. In synthetic biology, the construction of cell factories with designed synthetic pathways is often combined with random mutation and evolution to build up a large screening library, which always requires a high throughput and efficient technology and equipment in the testing step. The testing step is the rate-limiting process in the entire DBTL cycle, and its efficiency is largely dependent on chassis cells themselves, as well as the throughput of bioreactors and instruments needed for their phenotype testing. Here, this review article focuses on an overview of bioreactors and instruments with different throughput scales used for the phenotype testing in synthetic biology. We introduce their characteristics and application scenarios, including single-cell detecting and screening technology as well as pico-, nano-, micro-, milli-, and liter-scale bioreactors. Moreover, this article also points out the application potential of existing phenotype testing bioreactors and instruments, and illustrates how they can be selected for specific research purposes. Finally, the challenges and perspectives for phenotype testing bioreactors and instruments are summarized, which hopefully provides a reference for a wide range of synthetic biology researchers to properly select and use phenotype testing instruments.