Recent advances in the molecular design of synthetic vaccines

Abstract

Vaccines have typically been prepared using whole organisms. These are normally either attenuated bacteria or viruses that are live but have been altered to reduce their virulence, or pathogens that have been inactivated and effectively killed through exposure to heat or formaldehyde. However, using whole organisms to elicit an immune response introduces the potential for infections arising from a reversion to a virulent form in live pathogens, unproductive reactions to vaccine components or batch-to-batch variability. Synthetic vaccines, in which a molecular antigen is conjugated to a carrier protein, offer the opportunity to circumvent these problems. This Perspective will highlight the progress that has been achieved in developing synthetic vaccines using a variety of molecular antigens. In particular, the different approaches used to develop conjugate vaccines using peptide/proteins, carbohydrates and other small molecule haptens as antigens are compared.

Main

Vaccination has significantly improved human health and continues to prevent millions of deaths worldwide every year. Historically, successful vaccines have been prepared from live attenuated bacteria or viruses (mediated by multiple passages through non-human hosts) or inactivated viruses (often using heat or formaldehyde to effectively kill the pathogen, Fig. 1). Issues arising from these approaches include the potential for infections due to the use of whole organisms, reversion of the pathogen to a virulent form through mutations, and the potential for unproductive reactions to vaccine components. Large-scale culturing of pathogenic organisms can also be problematic, leading to variability between batches and the heterogeneity of vaccine mixtures often requires complicated analysis and characterization protocols to be developed.

Figure 1: Modular approach to rationally designed synthetic vaccines.

The empirical nature of traditional vaccine development, which relies on the use of live attenuated or whole inactivated viruses, is being replaced by a more sophisticated molecular approach that harnesses computational design and advanced synthetic techniques to create well-defined conjugate vaccines.

In recent years, significant progress has been made to enable a more mechanistic approach to vaccine research and development compared with the traditional empirical approaches¹.

One method to avoid the issues arising from using attenuated or inactivated forms of a pathogen is to develop well-defined synthetic vaccines. These can be designed to elicit an immune response to the desired target more reliably and with more precision than a vaccine based on a whole organism. The antigens to which the immune system responds in a traditional vaccine are generally relatively small sequences of amino acids or carbohydrates that are present either as contiguous regions or created through conformational alignment. However, small molecule antigens (called haptens) are poorly immunogenic on their own, and therefore need to be conjugated to carrier proteins to stimulate or boost an immune response (Fig. 1). Carrier proteins impart suitable immunogenicity to the conjugate as they are perceived as 'non-self' to activate helper T cells in the immunized animal. Keyhole limpet haemocyanin (KLH) is a massive glycoprotein that has often been used as a carrier since it elicits a strong T cell immune response in experimental animals and humans due to the evolutionary distance between giant keyhole limpets (from which KLH is isolated) and mammals. The surface of KLH is decorated with multiple copies of the hapten using an amide-bond-forming reaction between the lysine residues of the carrier and a carboxylic acid in the linker to the target antigen (Fig. 2a). The resulting multivalent presentation of haptens on the carrier also causes the B cell immune receptors to crosslink, which is important for initiating a specific antibody response to the hapten. Nowadays, inactive toxins (for example, CRM₁₉₇) and virus-like particles (VLPs) are often used in clinical vaccine design. This Perspective is divided into sections based on the nature of the molecular antigen to emphasize the high molecular diversity of targets to which a vaccine may be generated.

Figure 2: Modern peptide-protein vaccines are well-defined synthetic antigens that possess considerable molecular diversity.

a, Diagram of a typical conjugate vaccine, using KLH as the carrier protein (image created using a cryoEM structure of KLH showing the multiunit nature of the protein)³⁵. The carrier is decorated with multiple copies (n) of the peptide epitope (purple) that trigger immune receptor crosslinking. b, Crystal structure showing the extensive interactions between a chemically enhanced melanoma-associated epitope (gold) and an MHC protein (Protein Data Bank 4WJ5) indicating the potential for structure-based immunogen design (key interacting residues in green, α-helices in red and β-sheets in cyan). c, Trifunctional scaffold (based on lysine in red) using sequential and orthogonal triazole click chemistry to append two different peptide epitopes in a modular fashion to a carrier protein using maleimide chemistry. d, A crystal structure of TNFα (PDB 2TNF) highlighting the surface-located tyrosine residue Y86 in purple that was mutated to nitro-Tyr using amber suppression to create a homogeneous molecular vaccine that terminated self-tolerance.

The modular synthetic approach described above provides considerable opportunities for the rational design of novel and well-defined vaccines as each component of the vaccine (hapten, linker and carrier protein) can be optimized. In particular, it facilitates the systematic elucidation of structure–function relationships that can be applied more broadly within vaccine research and development to improve efficiency (as seen in small molecule drug discovery, Table 1).

Table 1 Chemical innovations in small molecule drug discovery can be applied to vaccine research and development.

There are significant overlaps in the chemistry-based technologies that have underpinned advances in small molecule therapeutics and vaccines research. These include the creation of targeted covalent protein inhibitors, where electrophilic warheads are designed to react with a specific amino acid residue in a binding pocket, and the development of bioorthogonal/biocompatible linkage chemistries that can be used to construct complex biomolecules with additional functionalities, such as conjugate vaccines. In this area there is also a connection to chemical biology approaches that capture the interacting proteins of a small molecule drug and often rely on the use of finely tuned protein-reactive probes. These techniques will no doubt be used in the future to help elucidate the modes of action of molecular adjuvants with unknown mechanisms that are used to boost the efficacy of a vaccine. Modulation of biological systems using synthetic biology is being harnessed to optimize small molecule drug manufacture and to create natural product libraries. In vaccine development these techniques are applied to expanding the genetic code to unnatural amino acid residues that facilitate site-specific protein chemistry and the creation of inactive synthetic viruses. Computational chemistry and biology has been used to predict the physicochemical properties of small molecule drugs and how they interact with biological systems, and in vaccine research, epitope sequence and conformation prediction are extremely important techniques that expedite target selection, design and synthesis. And finally, consideration should be given to the pharmaceutical properties of both small molecules and vaccines, to ensure that the cost of goods sold (COGS) is kept as low as possible, for instance, which feeds back into the earliest stages of design of the original molecules.

The parallels between small molecule drug discovery and chemical vaccinology highlighted in this Perspective (Table 1) emphasize the need for medicinal chemists to broaden their outlook and embrace the challenges of vaccine research.

Protein/peptide antigen design and synthesis

The following section describes the design and synthesis of a well-defined protein or peptide antigen-based vaccine. Special focus is given to the selection of the target peptide epitope sequence, carrier protein and linker chemistry.

Peptide epitope selection. One of the key challenges in developing an effective vaccine against a specific protein is knowing which peptide epitope (the antigenic determinant recognized by antibodies) to select from the target sequence for conjugation to the carrier. To ensure specificity, the epitope sequence chosen will be unique to that protein to limit or avoid unwanted cross-reactivity of the vaccine-generated antibodies with other proteins, and a search using the basic local alignment search tool (BLAST) can easily provide this information². A sequence should also be chosen that will be likely to trigger a strong and specific immune response. Various bioinformatics approaches have been developed to predict peptide immunogenicity (called immunoinformatics) to aid in the selection and synthesis of the target epitopes. Sequence-based methods for T cell epitope prediction include the use of algorithms based on similarity to motifs known to elicit an immune response. T cell receptors bind peptide fragments presented by the major histocompatibility complex (MHC) molecules displayed on the surface of antigen-presenting cells, and therefore peptide epitopes can be selected based on similarity to known MHC-binding motifs. More sophisticated immunoinformatics use artificial neural networks, support vector machine and quantitative matrix-driven methods, which have been reviewed elsewhere^3,4.

Bioinformatics are also used in the analysis of the sequenced genomes of pathogens, searching for target proteins either expressed on the cell surface, or secreted into the extracellular milieu, which can then be combined with the epitope prediction methods described above to select vaccine candidates. This approach, originally called reverse vaccinology, has been applied to the development of vaccines against many important pathogens, including Neisseria meningitidis, Streptococcus pneumonia, Chlamydia pneumonia and Leishmania major⁵. An important innovation in this process has been the development of next-generation sequencing techniques to facilitate the rapid identification of new targets from large numbers of bacterial isolates.

Immunotherapy has become a focus area for the development of cancer therapies, and vaccines are being developed to stimulate the immune system to destroy tumours specifically. A new approach in this field relies on the use of tumour antigen-derived peptides that bind to the MHC molecule, so triggering a cytotoxic T cell response, although efficacy has mostly been limited thus far. Prolonged recognition of the target antigen by MHC through increases in affinity is recognized as a key driver of improved immunogenicity. Medicinal chemistry optimization of peptide–MHC interactions can therefore be used to enhance cytotoxic T cell production and improve anticancer vaccine efficacy. Unnatural amino acids could also be incorporated to improve protease stability and increase bioavailability of the antigen. In a recent study, immunotherapeutic antigens were optimized following the preparation and MHC screening of more than 3,000 chemically altered peptide ligands created using 90 non-proteogenic synthetic amino acids in a range of epitopes⁶. A crystal structure of one of these ligands (an enhanced melanoma-associated epitope) bound to the MHC (Fig. 2b) showed key interactions driving the significantly improved potency (through π–π, cation–π and hydrogen bonding interactions with tyrosine residues in particular). These results clearly demonstrate a potential future use of structure-based molecular design in the vaccine and immunotherapeutic areas. One could also imagine developing a covalent chemical probe to react in the MHC binding site to report on the relation between antigen occupancy and T cell pharmacological effects to aid in dose projection to the clinic for these agents, as used in small molecule drug discovery (Table 1)⁷.

Carrier protein selection and virus-like particles. The glycoprotein KLH is often used experimentally as a carrier protein to generate an immune response against haptens that are poorly immunogenic. Since KLH is a biological product derived from giant keyhole limpets, its supply is limited for clinical vaccine manufacture, and therefore KLH is mostly used in preclinical research. Other commonly used multivalent carriers are formaldehyde-inactivated diphtheria toxoid (DT) and cross-reacting material 197 (CRM₁₉₇), which is an inactive form of the diphtheria toxin that contains a single amino acid mutation. CRM₁₉₇ has found significant use in clinical vaccines due to its lack of toxicity and large-scale production in the Corynebacterium diphtheria C7(β197)^tox− strain to provide high purity protein⁸. Tetanus toxoid (TT) is another formaldehyde-inactivated neurotoxin that is a widely used carrier in licensed vaccines due to its ready availability from Clostridium tetani cultures and proven safety. It is used as the carrier in haemophilus B, streptococcal and meningococcal conjugate vaccines.

Virus-like particles (VLPs) are self-assembling bionanoparticle antigens that more closely mimic native virions by presenting multiple epitopes on their surface in an even more highly organized manner. When antigenic determinants are linked to, or associated with, the VLP they can therefore produce a stronger response than traditional carrier proteins such as KLH. VLPs do not contain viral genetic material and are thus non-infectious and provide significant advantages over live or attenuated viruses. The first recombinant human vaccine (expressed in yeast), called RECOMBIVAX HB, contains the hepatitis B virus surface antigen (HBsAg), which assembles into 22 nm lipid-containing particles. Other VLP-based human vaccines include Cervarix (against the cancer-causing human papillomavirus, HPV), which is produced in a baculovirus-based insect cell expression system, and Hecolin (hepatitis E vaccine, HEV), which uses an E. coli system⁹. A variety of other VLPs are also being explored specifically as carriers for linked antigens, including Qβ, parvovirus and norovirus. VLP selection is based on factors such as conjugate vaccine efficacy in preclinical trials, high manufacturing yield and low productions costs, which have been reviewed elsewhere¹⁰.

There have been numerous efforts to develop antimalarial vaccines due to the emergence of multi-drug resistant strains of Plasmodium falciparum, particularly to the widely used drug artemisinin, and insecticide-resistant mosquitos. Unfortunately, short-lived protection, limited funding and lack of key technologies have all played a role in hampering the development of a successful candidate. Irradiated and chemically treated 'whole parasite' approaches, although efficacious, have faced COGS issues and concerns regarding reversion to virulence¹¹. The lead vaccine candidate in clinical trials is RTS,S, which contains two proteins: the circumsporozoite protein (CSP) target antigen of P. falciparum (previously identified as a promising target of immune responses) and HBsAg (ref. 12). Once expressed in yeast cells, the proteins spontaneously form multimeric VLPs with the potential for enhanced immunogenicity. The Phase III results of RTS,S were published in 2015, and the vaccine will be evaluated as an addition to, not a replacement for, existing preventative (long lasting insecticide nets) and treatment (artemisinin) measures (http://www.who.int/malaria/areas/vaccine/en). However, more efficacious second-generation vaccines are urgently required, although this is a significant challenge due to the antigenic diversity of the parasite. To address this, vaccines based on VLP fusions of multiple epitopes from different P. falciparum proteins are being developed^13,14.

Bioconjugation and click chemistry. Hapten bioconjugation often uses amide-bond-forming reactions between the carrier protein surface lysine residues and a carboxylic acid in the linker to a peptide epitope. Alternatively, a bifunctional linker may be used that contains an acid for coupling to the carrier and a maleimide group to enable subsequent coupling to a cysteine residue in the epitope. However, complex mixtures can arise when there are multiple reactive amino acid functionalities in the peptide epitope, which can compete with the linker chemistry, resulting in suboptimal presentation of the epitope to the immune system. To circumvent these issues, bioorthogonal click chemistry (which is modular, fast, reliable, biocompatible and provides high yields)¹⁵ can be used to effect hapten–carrier protein coupling in a more reliable fashion. The copper(I)-catalysed azide–alkyne cycloaddition (CuAAC)^16,17, resulting in the formation of a 1,2,3-triazole, is an example of a click reaction that can be used to link an azido-functionalized peptide to an alkyne-functionalized carrier.

Another remarkably simple click reaction uses dialkyl squarate reagents to sequentially stitch two amino-functionalized biomolecules together^18,19. Further work is required to understand the immunogenic potential of the aromatic rings formed following these click reactions (triazole and squaramide motifs), as carrier-induced epitopic suppression resulting from immunodominant reactivity of aromatic linkers is a known phenomenon^20,21. This is particularly important as more vaccine candidates are beginning to emerge that have utilized these linkers^{22,23,24,25,26}. Other recently developed methods applied to vaccine bioconjugation include selective tyrosine alkynylation of CRM₁₉₇ using triazoline dione chemistry (followed by CuAAC)²⁷, and a photoinduced thiol-ene click coupling²⁸. The latter reaction could be useful to avoid the formation of potentially immunodominant aromatic-containing linkers.

Related to multivalency and the chemistries that can be used to present multiple copies of the same epitope to the immune system on a carrier protein or VLP, synthetic polyvalent peptide vaccines can represent various epitopes from different viral and bacterial strains to increase cross-reactivity. When multiple activated epitopes are coupled to a carrier protein simultaneously using the same amide-bond-forming chemistry, a highly complex mixture would result, with a distribution that represents the reactivity of the different peptides. Bioorthogonal click chemistry is ideally suited to the construction of polyvalent vaccines in a more defined and controllable manner. By way of illustration, a chemical scaffold was developed recently for multiple epitope presentation that used two sequential and orthogonal click reactions — a strain-promoted azide–alkyne conjugation (SPAAC) and a CuAAC reaction appended two different azide-containing peptides to a carrier protein in a simple one-pot process (Fig. 2c)²⁹. These synthetic antigens illustrate the ability of click chemistry to build highly complex architectures in a modular fashion to avoid the batch-to-batch variability and polydispersity seen with traditional polyvalent vaccine synthesis.

Homogeneous synthetic protein vaccines. Self-proteins are poorly immunogenic and do not make suitable vaccines in their native form. However, several protein post-translational modifications (PTMs) can break self-tolerance and elicit undesired T cell-mediated immune responses. PTMs such as sulfation, nitration, halogenation, citrullination (produced from arginine) and glycosylation (of Arg and Lys resulting in advanced glycation end-products) play important roles in autoantigenicity and disease³⁰. These biosynthetic modifications may therefore serve as inspiration for the creation of homogeneous protein antigens — in essence the vaccine becomes a single synthetic protein molecule that does not require carrier protein bioconjugation. Tyrosine nitration in particular has been a focus of research due to its presence in proteins linked to many inflammatory diseases characterized by high nitrative stress³¹. Motivated by these observations, recent studies have found that site-specific mutation of surface-exposed residues in TNF-α to 4-nitrophenylalanine and 3-nitrotyrosine (probably retaining the natural conformation of the protein) using an amber suppressor tRNA/aminoacyl-tRNA synthetase pair in E. coli resulted in highly immunogenic proteins, which generated antibodies against natural TNF-α³². Nitro-TNF-α derivatives (such as the Y86 to 3-nitrophenylalanine mutation, Fig. 2d) were used to vaccinate mice, which were protected from a lethal challenge of lipopolysaccharide by the elicitation of protective antibodies against the cytokine, so demonstrating the potential of this approach.

Manufacturing a vaccine on a large scale using the amber suppression technique may be problematic due to relatively low yields, and therefore synthetic chemistry approaches could be explored to modify protein residues in a selective manner. For example, proof-of-principle for a semi-synthetic approach was recently reported using solid-phase peptide synthesis to incorporate a nitro-Tyr site-specifically into a peptide fragment, which was then stitched into a full length protein (α-synuclein in this case) using native chemical ligation³³. Selective synthetic tyrosine nitration of native recombinant proteins, using chemical nitrating agents, could be used as a general technique to break self-tolerance, although doing so in a site-specific manner will be challenging³⁴.

Glycoconjugate vaccines

Polysaccharides that encapsulate pathogenic bacteria have been the target of conjugate vaccines for many years. On their own, polysaccharides are poorly immunogenic (they are T cell-independent type 2 antigens where an immunoglobulin M (IgM) response dominates), which enables the bacteria to evade the host immune system (a mechanism used by many pathogens, often called glycan shielding). By conjugating the polysaccharide to a carrier protein bearing T cell epitopes, IgM to IgG isotype switching occurs to provide a long-lasting immune response following vaccination. This approach has led to the widespread clinical use of glycoconjugate vaccines for highly virulent pathogens such as Streptococcus pneumonia, Haemophilus influenzae type b and meningococcus¹. The huge diversity of capsular polysaccharides, even within a single species, has resulted in the categorization of serotypes based on the structures of the surface carbohydrates. Vaccines that target multiple bacterial serotypes are composed of carrier proteins conjugated to the polysaccharides present in these different serotypes. For instance, a pneumococcal vaccine (Prevnar 13) composed of polysaccharides from thirteen different serotypes conjugated to CRM₁₉₇ provides protection for children against pneumococcus infection³⁶.

Although antibacterial vaccines have found great success in improving human health, vaccines to hypervariable RNA viruses, such as those for HIV, have met with limited success in clinical trials. HIV is a particularly difficult target due to its considerable genetic diversity and high mutation rate, resulting in protein-based vaccines and inactivated viruses failing to elicit antibodies that can effectively neutralize the virus and provide persistent protection. As a result, there is now a significant effort to explore opportunities to target glycan-dependent epitopes in the HIV glycoprotein envelope. The isolation of carbohydrate-reactive broadly neutralizing antibodies from HIV-infected patients has provided confidence that this approach could be successful and characterization of the targeted epitopes has laid the basis for carbohydrate immunogen design^37,38.

By using the tools of organic chemistry, the synthesis of well-defined, less heterogeneous glycoconjugate vaccines is facilitated, and structure–function relationships can be delineated to enable rational vaccine design. Additionally, some glycans are low expressing in bacterial cultures and therefore chemical synthesis can provide larger quantities of homogeneous carbohydrate antigens to enable vaccine preparation. Although the facile, iterative syntheses of peptides and oligonucleotides have become automated and widespread, oligosaccharide synthesis is more complex, and therefore methodological advances are required in this field. To this end, an iterative glycal assembly approach was developed to create anticancer vaccines containing the aberrant and overexpressed carbohydrates on the surface of various tumours (including the rare Globo-H antigen, which is difficult to obtain in high quantities and sufficient purity from cultured bacteria, Fig. 3)³⁹. Cancers express many complex sugars, and second-generation vaccine candidates with improved efficacy have addressed this heterogeneity by using peptide chemistry to present multiple tumour-associated carbohydrate antigens in a single molecule³⁹.

Figure 3: Glycal assembly.

Complex polysaccharide glycoconjugate vaccines are synthesized in a well-defined manner using iterative glycosylations. Glycal activation at the anomeric carbon creates an oxonium donor that reacts with the hydroxyl group of a glycal acceptor. This coupling process can be repeated, allowing the iterative glycal assembly of complex carbohydrate architectures. The structure of the Globo-H anticancer vaccine, which used the glycal assembly procedure to prepare the rare hexasaccharide antigen, is shown below.

Other synthetic strategies have been developed to prepare complex polysaccharides that have enabled vaccine candidate preparation. Solid phase automated synthesis uses suitably functionalized glycal monomers appended in a sequential manner to a solid support (instruments are now capable of preparing 50-mer oligosaccharides⁴⁰). One-pot syntheses rely on the different reactivity of similar (chemo-selective) or chemically diverse (chemo-orthogonal) anomeric leaving groups⁴¹.

However, immunological studies have also shown that small oligosaccharide epitopes (di/trisaccharides) can be sufficient to elicit protective antibodies in vivo in certain instances⁴². Reverse engineering of a monoclonal antibody against Candida albicans β-mannan identified a trisaccharide epitope, that when conjugated to peptides present in cell wall proteins predicted to be effective T cell epitopes, produced a vaccine that protected mice from candidiasis infection⁴³. This work utilized a simple heterobifunctional non-immunogenic triethylene glycol linker bearing an N-hydroxysuccinimidyl ester for coupling to the amino sugar epitope, and an acrylate motif that reacted smoothly with the thiopeptides. This approach, and the simplicity of the chemistry, should find significant utility in the development of glycopeptide vaccines.

Organic synthesis is also influencing vaccine research and development through the use of synthetic glycan microarrays, both as diagnostics and in the selection of novel targetable epitopes. Sera from Leishmania-infected humans were screened against one such microarray (composed of leishmanial capping oligosaccharides) that identified a tetrasaccharide epitope that was subsequently conjugated to the CRM₁₉₇ carrier to elicit antibodies in mice that could detect the parasite (Fig. 3)⁴⁴.

A comprehensive description of glycoconjugate vaccine chemistry, and the targeting of glycans in the design of new vaccines, is beyond the scope of this Perspective, but there have been several excellent recent synopses of the field^38,39,45,46.

Other small molecule haptens

Small molecules by themselves are unable to elicit an immune response, and therefore need to be conjugated to carrier proteins or peptides to be recognized by the immune system. Drugs of abuse are targeted in the development of a therapeutic vaccine that can blunt the reinforcing effects of the drug by preventing it from crossing the blood–brain barrier, thus breaking the reward–addiction cycle (and simultaneously ameliorating drug toxicity)⁴⁷. Several legal and illicit drugs have been targeted through this approach, which involves conjugation of the small molecule hapten (a derivative of the target drug) to a carrier protein. In this area there is ample opportunity for molecular design and synthesis to explore and optimize immunogenicity and specificity of the resulting response. A significant focus of research into cocaine vaccines has provided numerous innovations in the field, and one such vaccine reached clinical trials (succinyl norcocaine (SNC) conjugated to recombinant cholera toxin B, called TA-CD)⁴⁸. To enhance T cell receptor recognition of the cocaine hapten, fluorination was used recently to potentially improve binding without drastically changing the structure or conformation of the parent small molecule⁴⁹. The fluorinated derivative GNF was conjugated to KLH and the construct appeared to generate a superior titre and retained similar cocaine affinity relative to the unfluorinated hapten (SNC), suggesting that this approach could find further utility (Fig. 4a). Enhanced cocaine vaccine immunogenicity was also achieved by improving the hapten serum stability (reduced hydrolysis by replacing esters in the linker with amides to provide hapten GNE, Fig. 4a)⁵⁰.

Figure 4: Conversion of small molecule haptens into conjugate vaccines can be achieved using medicinal chemistry optimization and a variety of linker chemistries.

a, Original cocaine KLH conjugate vaccine SNC, an optimized fluorinated GNF vaccine with improve efficacy, and an amide-stabilized GNE vaccine with improved bioavailability. b, Antinicotine conjugate vaccine candidate illustrating the attachment point to the 5-position of the pyridine ring of the nicotine molecule, and a relatively short polar linker to the DT carrier. c, 3-oxo-C₁₂-HSL antibacterial vaccine showing conjugation of bovine serum albumin (BSA, carrier protein) to the lipid tail of the quorum sensor hapten, preserving the chemical features of the oxo-homoserine lactone in the vaccine to generate specific antibodies.

In a similar way, antinicotine vaccines have been the subject of considerable effort in this field, driven by the high relapse rate of smokers attempting to quit on their own⁵¹. By way of illustration, recent work in this area exemplified a systematic approach to explore immunogenicity structure–function relationships through modifications of various features, including: (1) linker attachment point to the nicotine hapten, (2) linker lipophilicity and rigidity, and (3) the chemical handle used to attach the hapten to the carrier protein²⁵. This optimization effort led to the development of a vaccine candidate that gave high antibody titres and affinity in mice, significantly reducing administered nicotine from entering the brain. Interestingly, the best results (antibody titre, affinity and function) were obtained with shorter, flexible linkers between the hapten and DT carrier, enhancing and focusing the response to the hapten, and by attaching the linker to the 5-position of the pyridine ring (possibly presenting the hapten in a manner that retains the natural conformation or electrostatic surface of the target nicotine, Fig. 4b). Synthetic complexity and COGS are important parameters to consider in the vaccine design process (Table 1), and this vaccine preparation utilized an efficient synthetic functionalization of the nicotine molecule itself using a regioselective iridium-mediated borylation technique that had been reported previously⁵². This work suggests that other haptens, and possibly more complex natural products, could be amenable to semisynthetic functionalization. Other drugs of abuse being targeted by the immunopharmacotherapeutic approach include methamphetamine⁵³, oxycodone⁵⁴ and heroin⁵⁵.

Quorum sensing, the means by which bacteria sense and respond to their environment using signalling molecules, has also been a target of vaccine research. Quorum sensing can control a number of essential functions, such as biofilm formation, virulence and aggregation, and Gram negative bacteria are known to use a series of N-acyl homoserine lactones to control these behaviours. Vaccines that elicit antibodies to sequester these molecules may therefore offer protection against bacterial infections on several fronts. 3-oxo-C₁₂-homoserine lactone (3-oxo-C₁₂-HSL) is involved in the quorum sensing of Pseudomonas aeruginosa, a major pathogen in the cystic fibrosis lung, and a 3-oxo-C₁₂-HSL–BSA conjugate vaccine was found to protect mice from lethal P. aeruginosa lung infection (Fig. 4c)⁵⁶. Quorum quenching vaccines could also be targeted to autoinducing peptides (AIPs; small cyclic peptide virulence factors), which may provide new opportunities for the treatment of drug-resistant bacteria such as methicillin-resistant Staphylococcus aureus. This could be enabled by structural information describing the mode of binding of quenching antibodies of AIPs, as recently described for the S. aureus AIP-4 (ref. 57).

Future directions and conclusions

Although numerous examples of the impactful use of organic chemistry in vaccine research are highlighted above, the chemical toolbox for vaccine synthesis still needs enhancing. Most vaccine bioconjugation relies on random surface lysine ligation, which can be difficult to control leading to significant variability in the final product, but some advances have been made in this area. Genetic code expansion using amber codon suppression and other methods of codon reassignment are finding considerable utility in chemical biology (as described above for the site-specific incorporation of nitrated amino acid residues that induce the termination of self-tolerance). Amber suppression-mediated incorporation of 'clickable' amino acid residues into carrier proteins and VLPs could enable the preparation of homogeneous synthetic immunogens through selective bioorthogonal conjugation to the antigen. Over 150 unnatural amino acids have thus far been genetically encoded into proteins, with over 50 for the purpose of bioconjugation, illustrating a considerable opportunity for future exploration of specific labelling technologies⁵⁸. An alternative application of amber suppression recently enabled the incorporation of unnatural amino acids (4-acetyl, 4-azido or 4-iodophenylalanine derivatives) into HIV proteins resulting in the preparation of live viral mutants that were unable to replicate in the host⁵⁹. This approach therefore provides a new synthetic paradigm for the preparation of chemically modified live attenuated viruses as vaccine candidates.

Structure-based design. Structural information will continue to significantly influence immunogen design. The immunotherapeutic potential for its application in optimizing T cell epitope binding to MHCs was described above⁶, and there are likely to be many more opportunities where this approach could be applied. Several artificial scaffolds have been developed to enable protein fold mimicry of target epitopes (an approach often called structural vaccinology)⁶⁰. Approaches have been developed that rely on the discovery of broadly neutralizing antibodies (bNAbs) that target vulnerable sites in the surface envelope proteins of RNA viruses⁶¹. Structural elucidation of these mAb-recognized epitopes has led to rational structure-based design of new immunogens (often using crystallographic analyses and computational modelling), resulting in considerable advances in the development of vaccines to HIV and RSV in recent years.

Nucleic acid-based vaccines. Synthetic DNA- or RNA-based vaccines provide an alternative and attractive approach for the induction of antigen-specific immunity as the nucleic acids drive antigen expression in vivo by the host, so avoiding the use of complex live attenuated viruses (although a nucleic acid-based vaccine has yet to be approved in humans). The key issue facing this field has been the difficulty in delivering the oligonucleotide to the desired site of action, although various approaches are being applied to try and address this, including the use of adenoviral vectors. A non-viral vaccine platform was recently developed to deliver synthetic, self-amplifying mRNA in a lipid nanoparticle (as used for siRNA delivery) for effective preclinical immunizations against HIV, RSV and H7N9 influenza⁶². If this approach proves to be safe and effective in humans, it would appear to be a relatively simple means to deliver entirely synthetic, long-acting nucleic acid-based vaccines against a variety of pathogens.

Molecular adjuvants. Adjuvants are substances added to vaccines that enhance the immune response to the target antigen. Synthetic antigens in particular require some form of adjuvantation to replace some of the danger signals naturally provided by whole organism-based vaccines. Adjuvants aluminium oxohydroxide and aluminium hydroxyphosphate (often referred to collectively as 'alum') have been in widespread use for decades. They form nanoparticles to which antigens can often be adsorbed in the vaccine formulation, and they exert their immunomodulatory effects through the stimulation of cytokine release in dendritic cells. Novel adjuvants are required to improve overall vaccine safety and efficacy and potentially reduce the amount of antigen required to elicit the desired response, so reducing COGS and extending vaccine supply. As a result, the National Institutes of Health is committing up to US$70 million to adjuvant research over the next 5 years⁶³. However, the complex mechanisms by which nanoparticle material adjuvancy are elicited (for example, NLRP3 inflammasome activation, autophagy and phagocytosis) will need further elucidation for more rational approaches to be realized⁶⁴.

In the future it may be possible to produce synthetic vaccines in which the adjuvant and target epitope are conjugated together to form a single molecular species that avoids the need for a carrier protein, and the associated issues with polydisperse coupling. Toll-like receptor 2 (TLR2) agonists (such as the Gram negative bacteria-derived synthetic palmitoylated peptides Pam₂Cys and Pam₃Cys) are often used as vaccine adjuvants due to their ability to activate dendritic cells. An early report described the conjugation of synthetic viral peptides to Pam₃Cys to create a molecule that efficiently primed an influenza virus-specific response in vivo⁶⁵. Building on this work, completely synthetic trifunctional vaccines were created using Pam₂Cys conjugated to a T cell epitope and target epitope that provided protection from bacterial and viral infections in animal models⁶⁶. Additionally, structure–function relationships generated using synthetic libraries have been used to optimize the immunological effects and TLR2 agonism of these lipopeptides⁶⁷. In a recent example, a self-adjuvanting vaccine was developed by conjugating an allergen-derived T cell epitope to the glycolipid adjuvant α-galactosylceramide (α-GC) that specifically triggered activation of type 1 natural killer-like T (NKT) cells without the need for a carrier protein⁶⁸. Additionally, a cleavable linker between the α-GC and peptide epitope released these molecules in their native forms through enzymatic release within antigen-presenting cells, so avoiding non-specific systemic effects, and focusing the stimulatory activity. The resulting conjugate vaccine successfully reduced inflammation in a mouse model of allergic airway inflammation and the approach could therefore be of general utility for allergic diseases.

An important consideration in the development of lipidated peptide vaccines is their amphiphilic physicochemistry, which can lead to complex purification procedures at scale due to the formation of aggregates or vesicles⁶⁹. These issues can be addressed by controlling the vaccine formulation to deliberately create a well-defined physical form. In one approach, three different TLR2 ligands (lipid core peptide, Pam₂Cys and Pam₃Cys) were conjugated to an engineered polytope targeting group A streptococcus that then self-assembled into nanoparticles⁷⁰. Immunization of mice with this vaccine yielded high-titre antigen-specific IgG antibodies. To recapitulate the multivalency of VLP-based vaccines, the multiple antigenic peptide (MAP) system utilizes a synthetic dendritic core of an oligomeric branching lysine that is conjugated to a number of antigens and a lipophilic membrane-anchoring adjuvant, enabling formulation into a liposome for efficient delivery⁷¹.

Summary

This Perspective has attempted to capture the potential of synthetic biology and synthetic chemistry technologies to meet the demand for novel vaccines to respond rapidly to viral pandemics and address the economic burden of untreated diseases. Multidisciplinary teams that foster collaboration between medicinal chemists, molecular biologists, immunologists and vaccinologists will be essential to drive future innovations in this area and accelerate the discovery of urgently needed vaccines.

The complexities of many diseases will also necessitate the combination of both small molecule and vaccine modalities where appropriate to achieve maximum therapeutic efficacy, especially where synergistic combinations can be imagined. By way of illustration, a recent promising study showed that mTOR inhibition using an analogue of rapamycin (RAD001) ameliorated immunosenescence in the elderly, enhancing their response to influenza vaccination⁷². Improved multimodal immunotherapeutic strategies can be envisaged that use the chemically enhanced tumour-associated antigens described above with molecular adjuvants and checkpoint inhibitors loaded into the patient's own dendritic cells to induce antitumour immune responses with improved clinical outcomes⁷³. Such cooperativity will no doubt be seen in various other areas of drug discovery, where the molecular aetiology of disease will define the most appropriate modes of therapeutic intervention.