Abstract
Infections caused by Gram-negative bacteria are leading causes of mortality worldwide. Due to the rise in antibiotic resistant strains, there is a desperate need for alternative strategies to control infections caused by these organisms. One such approach is the prevention of infection through vaccination. While live attenuated and heat-killed bacterial vaccines are effective, they can lead to adverse reactions. Newer vaccine technologies focus on utilizing polysaccharide or protein subunits for safer and more targeted vaccination approaches. One promising avenue in this regard is the use of proteins released by the Type 5 secretion system (T5SS). This system is the most prevalent secretion system in Gram-negative bacteria. These proteins are compelling vaccine candidates due to their demonstrated protective role in current licensed vaccines. Notably, Pertactin, FHA, and NadA are integral components of licensed vaccines designed to prevent infections caused by Bordetella pertussis or Neisseria meningitidis. In this review, we delve into the significance of incorporating T5SS proteins into licensed vaccines, their contributions to virulence, conserved structural motifs, and the protective immune responses elicited by these proteins.
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Introduction
Infectious diseases are a leading cause of mortality worldwide. However, the number of deaths are amplified by antimicrobial resistance (AMR), which results from the extensive and inappropriate use of antibiotics used to combat infections1. A recent study revealed AMR associated deaths reached 4.95 million in 2019, making it a global health threat2. Moreover, a comprehensive evaluation of peer-reviewed studies and healthcare data predicts a potentially dire scenario: by 2050, up to 10 million lives annually might succumb to AMR-related causes3. With the incidence of deaths due to bacterial infections increasing annually, the need for alternative approaches to treat bacterial infections is increasing4. A pivotal strategy in averting AMR infections lies in vaccination. Vaccination is a prophylactic measure to fight infectious diseases, thus reducing the need for antibiotics and the long-term consequences of infection. Vaccines induce protection against a pathogen by eliciting an immune response that decreases the severity of the diseases5. Remarkably, vaccines rank among the most successful medical interventions in human history; for instance, the smallpox vaccine alone elevated life expectancy from 58.5 to 70 years4. According to the WHO, due to increased vaccine availability and compliance across the globe, since 2017 there has been a 90% decrease of vaccine-preventable infectious diseases such as diphtheria, measles, tetanus, pertussis, polio, and rubella6.
Despite the success of vaccines in preventing bacterial infections, vaccines are still lacking against most major bacterial diseases. Numerous challenges impede vaccine success, including the prolonged duration for vaccine development and licensure, limited knowledge about immunogenic antigens, and an incomplete understanding of the immune responses linked to protection7. Furthermore, obstacles like inadequate health infrastructure, inadequate vaccine cold-chain management in low- and middle-income countries restrict vaccine accessibility in these regions. Additionally, the intricate diversity and complexity of bacterial pathogens further complicates the development of new vaccines8. To address these challenges, innovative technologies are needed to overcome the limitations of current vaccines and enhanced comprehension of protective immune responses against infections would propel the development of new vaccines against other emerging bacterial pathogens9. This review explores the potential use of T5SS proteins as vaccine candidates and examines the outcomes of licensed bacterial vaccines employing these proteins.
Antibodies as mediators of protection
Vaccines function primarily by eliciting immune protection through the production of antibodies and the promotion of humoral immunity. This humoral immune response is initiated when antigens interact with antigen presenting cells (APCs) like B-cells. T-helper cells or CD4+ T cells are required for the activation of B-cells and the production of antibodies. CD4 + T cells are further divided into Th (T helper) 1, Th2, Th9, Th17, Th22, Treg (regulatory T cells) and Tfh (follicular helper T cells)10,11. Th1 cells produce interferon-gamma (IFN-γ), interleukin (IL-2) and tumour necrosis factor (TNF), promote cell-mediated immunity and control infections by intracellular pathogens. Th2 cells produce IL-4, IL-5, IL-10 and IL-13, and promote antibody mediated immunity. Studies have shown that a balanced Th1/Th2 response is required to optimise the protective effect of vaccines, such as the Group A Streptococcus vaccine12,13. Th17 produce IL-17 which plays an important role in protective immunity against pathogens by recruitment of neutrophils to the site of infection. Tfh cells help B-cells to produce antibodies and are particularly important with respect to vaccination as they play a role in the development of a protective antibody response against pathogens14. Almost all vaccines, including those for smallpox, diphtheria, tetanus, pertussis, polio, and Haemophilus influenzae type b (Hib), operate by triggering the production of antibodies. In these diseases, antibody titres generated post immunisation have been shown to correlate with protection7.
Antibodies consist of two light and two heavy chains bound together by disulphide bonds (Fig. 1). Within the antibody structure, the fragment of antigen binding (Fab) portion engages in antigen recognition, while the fraction crystallisable (Fc) portion plays a pivotal role in interactions with host cells, significantly influencing antibody function15. Based on the heavy chain constant region the antibodies are classified into various isotypes that differ in function: IgM, IgG, IgA, IgD and IgE. IgD is an enigmatic antibody whose function remains unknown and IgE is involved in allergic reactions. IgD and IgE are not considered further here. IgM is the first antibody produced during an immune response following infection or immunisation and is found in the blood. IgM is more efficient at fixing complement than IgG. This is attributed to the pentameric structure of IgM; a single pentameric IgM molecule has five Fc regions as opposed to a single Fc region in IgG, thereby making it more potent than IgG at binding complement components16. Research using IgM knockout models has underscored the crucial role of IgM in reducing susceptibility to typhoid infection17. Moreover, immunisation of mice with Salmonella enterica Typhimurium porin OmpD (Outer membrane protein D), revealed that IgM was required for vaccine-mediated protection18. IgG appears a little later and is the most abundant isotype in human serum. IgG is divided into four subclasses in humans (IgG1, IgG2, IgG3 and IgG4), and in mice (IgG1, IgG2a, IgG2b and IgG3). The subclasses differ in the length of the hinge region. Studies have shown that human IgG3 and IgG1, and mouse IgG2a, IgG2b and IgG3 are more efficient at fixing complement. This reason is attributed to the length and flexibility of the hinge region, which results in less steric hindrance for complement binding when compared to the other IgG subclasses19,20. Evidence from multiple studies show that the human IgG3 followed by IgG1 is crucial for mediating phagocytosis of the pathogen21. Since IgG1 accounts for 70% of the total IgG and is the most abundant subtype of IgG, deficiency of IgG1 is associated with a decline in total IgG concentration22. Capsular polysaccharide antigens predominantly induce IgG2 antibodies and studies have shown that deficiency of this antibody subtype results in increased susceptibility to bacterial infections19,23. Protein conjugate vaccines such as Pneumococcus type-6B tetanus toxoid vaccine induce IgG1 and IgG3 antibodies24. IgG4 is the least abundant immunoglobulin subtype and, like IgE is associated with allergic reactions25. IgA is the most abundant antibody and exists in monomeric form in serum and dimeric form in mucosal secretions. Evidence shows that patients with IgA and IgG deficiency are more susceptible to severe respiratory infections26. Thus, the induction of specific antibody isotypes and subclasses holds paramount importance in vaccine-mediated protection. However, the effectiveness of these antibodies depends on their functionality and quantity27.
In humans, Ig classes are made up of two light chains (purple) and two heavy chains (orange) that are joined by disulphide bonds. The Fab (Fragment of antigen binding) region is involved in antigen binding. The variable region is made up of heavy and light chains VH and VL respectively and are involved in antigen binding. The constant region includes CH1, CH2 and CH3. The Fc portion of the Ig is involved in interaction with cells and effector molecules. a IgM exists as a pentamer or hexamer joined by disulphide bonds and a Joining (J) chain. b IgG exists as 4 subclasses in humans: IgG1, IgG2, IgG3 and IgG4 which differ in hinge length. c IgA exist as two subclasses IgA1 and IgA2. The two subclasses exist in monomeric from in serum and dimeric form in mucosal secretions. The figure was created using BioRender.com.
Antibodies play various roles in defending against pathogens, including neutralization, phagocytosis, complement-mediated killing, activation of mast cells and eosinophils, and antibody-dependent cellular toxicity (ADCC) (see Fig. 2). For instance, to safeguard against infection, the meningococcal vaccine (Bexsero) induces antibodies that function by mediating complement-dependent killing of Neisseria meningitidis28. The importance of functional antibodies is illustrated by numerous studies showing increased susceptibility to bacterial infections in individuals with primary immunodeficiencies. Furthermore, several studies have demonstrated that patients with myeloma and chronic renal failure have a higher risk of developing Hib disease when compared to healthy individuals. As circulating antibodies typically protect against invasive Hib disease by activating complement and phagocytosis, this phenomenon was attributed to a lack of functional antibodies29. Hence, comprehending the functionality of specific antibodies and their relationship with protection against infections can offer valuable insights into vaccine effectiveness and design. Specifically, antibodies targeting outer membrane proteins play a pivotal role in Salmonella eradication, emphasizing the significance of complement-mediated killing in controlling Salmonella infections. However, the O-Antigen (O-Ag) of the lipopolysaccharide (LPS) can hinder antibody access to the protein antigens30. Furthermore, excess antibodies to LPS can inhibit complement-mediated killing of Salmonella31, Uropathogenic Escherichia coli (UPEC) and Pseudomonas aeruginosa, and is correlated with poor outcomes27,32. The development of an LPS-based conjugate vaccine, Aerugen against P. aeruginosa was suspended during a randomized phase III trial due to poor outcomes. The failure of this vaccine is likely due to excess IgG2 antibodies to the O-Ag that blocks complement mediated killing of the pathogen27,33. Hence, understanding antibody function, the accessibility of antibodies to the antigen, antibody titres, and the target site are important for vaccine design.
The mechanism of antibody mediated protection includes a Neutralisation- Neutralising antibodies bind to a bacterial toxin and prevent it from binding to the toxin receptors on the host. b Opsonisation and phagocytosis- Antibodies bind to the antigen on the surface of the bacterial cell and are recognised by the Fc receptor on macrophages resulting in phagocytosis of the pathogen. c Complement mediated killing- Binding of complement (C1 complex) to the antibody activates the complement pathway and leads to the formation of the membrane attack complex (MAC) resulting in lysis of the bacterial cell. d Antibody dependent cellular toxicity (ADCC)- the Fc receptor on the effector cell such as Natural killer (NK) cell recognises and kills the antibody coated target cell. e Mast cell activation- IgE antibodies are involved in allergic reactions and bind to the receptors of mast cells resulting in activation and degranulation of mast cells. The figure was created using BioRender.com.
Licensed bacterial vaccines
Vaccines can be classified into live attenuated vaccines and non-live vaccines in which the antigenic component of the vaccine can be composed of whole cells, outer membrane vesicles (OMVs), polysaccharides, and/or proteins. A list of the currently licensed bacterial vaccines is presented in Table 1.
Live-attenuated vaccines
Live-attenuated bacterial vaccines, including the BCG, Typhoid (Ty21a) and Vibrio cholerae (CVD 103-HgR) vaccines, have been extensively used over years34,35,36. Live attenuated vaccines mimic a natural infection and deliver multiple antigens that have the potential to mediate both a humoral and cell-mediated immune response. The main disadvantages of this category of vaccines are the potential to cause infection in immunocompromised individuals, the risk of releasing live recombinant bacteria into the environment, reversion of the mutants to the wild type phenotype, and short-term efficacy37. For example, Ty21 is a live attenuated vaccine which protects against Salmonella Typhi infections38,39, however, the vaccine is limited by poor immunogenicity in children, is not suitable for immunocompromised individuals, and is only protective against S. Typhi, and no other Salmonella infections40.
Inactivated whole cell vaccines
Whole cell vaccines are obtained by inactivation of bacteria by heat, chemicals, or irradiation. Immunisation with whole cell vaccines induces the humoral immune response and the activation of CD4+ T cells that together mediate protection. Currently, licensed inactivated whole cell vaccines include those protecting against V. cholerae, Coxiella burnetii and Bordetella pertussis. The whole cell pertussis vaccine (wPV) contributes to bacterial clearance by inducing the production of opsonising antibodies and activation of the complement system41. The drawbacks associated with the wPV are high incidences of swelling, induration and fever following immunisation42. Unsubstantiated claims of a causal link to neurological disorders diminished parental confidence in this vaccine and as a result most industrialised countries have now switched to the acellular pertussis vaccine (aP) described later; however, wPV is still used in low- and middle-income countries and has a better efficacy than aP43,44.
Polysaccharide-based vaccines
The bacterial capsular polysaccharide (CPS) is attached to the outer membrane (OM) and is an important virulence factor preventing phagocytosis and complement-mediated killing45,46. Polysaccharide-based vaccines have been licensed against N. meningitidis, Haemophilus influenzae, Streptococcus pneumoniae and S. Typhi. Purified polysaccharides can overcome some of the safety concerns associated with live-attenuated and whole cell vaccines47. Immunisation with polysaccharide-based vaccines induces T-cell independent responses and anti-polysaccharide antibodies that protect against infection by mediating complement-dependent killing and opsonophagocytosis48. Limitations of polysaccharide-based vaccines include that polysaccharide antigens elicit poor antibody titres in immunocompromised individuals, immune responses are short-lived, and are not effective in children49. For example, the Vi vaccine has not been licensed in children due to its poor immunogenicity in this age group and does not protect against infections with nontyphoidal strains of S. enterica40. One way to address these constraints is by conjugating polysaccharides to a protein carrier, a strategy employed in the licensed typhoid conjugate vaccine and pneumococcal vaccines like Prevnar 13 and Pneumovax 23; these conjugate vaccines trigger long-lasting immune responses that are well-tolerated and significantly bolster protection50,51.
Outer membrane vesicle (OMV)-based vaccines
OMVs are naturally occurring nanoscale bi-layered membrane structures produced by vesiculation of the outer membrane and have a diameter of 20–250 nm52. They are composed of LPS, outer membrane proteins (OMPs) and phospholipids on the outside, and phospholipids and inner membrane proteins on the inside52. The lumen of the OMV contains periplasmic and cytoplasmic proteins, RNA, DNA and peptidoglycan53. OMVs possess pathogen associated molecular patterns (PAMPS) that are required for APC-mediated activation of T-cells. OMVs have emerged as an attractive vaccine platform which was first used to develop vaccines against N. meningitidis infections54. OMV vaccines were first used to address the meningococcal epidemics in Cuba (VA-MENGOC-BC)55, Brazil (VA-MENGOC-BC)56, Norway (MenBVac)57 and New Zealand (MeNZB)58. However, the immunogenicity of these vaccine largely depended on an immunodominant protein Porin A (PorA) that is highly variable across different strains. As a result, the protection offered by these vaccines was limited and was only effective against strains dominated by a single PorA59. To address this, the Bexsero vaccine was developed. It consists of three subunit proteins, identified by reverse vaccinology, combined with detergent extracted OMVs from the New Zealand strain NZ98/254, which contains PorA60. Studies have shown that the OMV vaccine platform can induce a long lasting humoral and cellular immune response. Immunisation with OMVs induce high levels of IgG antibodies, which correlate with complement-mediated killing and opsonophagocytic antibodies61,62.
Limitations to preparing OMVs by detergent extraction include LPS toxicity and the potential loss of protective antigens that are naturally associated with the OM. Lipid A is the endotoxic part of the LPS and is composed of 6 acyl chains. To reduce toxicity, genetic modification of the parent strain can be used to alter the structure of lipid A to a penta-acylated form63. Notably, genetically produced GMMA (generalised modules for membrane antigens) are OMVs generated through the deletion of genes such as tolR, msbB, and pagP. Deletion of tolR enhances OMV release from the outer membrane and deletion of msbB and pagP results in penta-acylated lipid A53. Studies have shown that penta-acylated lipid A had reduced endotoxicity64, thereby improving its use in vaccine studies. Currently, GMMA-based vaccines are in development against Shigella, Salmonella and Neisseria65,66,67. The OMV vaccine platform presents distinct advantages over alternative approaches. These vaccines are self-adjuvating, encompass numerous antigens from the outer membrane, maintain these antigens in their natural conformation, boost cost-effectiveness in manufacturing, and production can be readily replicated for various other Gram-negative bacteria68.
Protein-based vaccines
Protein-based vaccines are composed of one or more purified proteins obtained from an infectious agent. In some instances, these proteins are chemically rendered inactive. For instance, widely used vaccines for diphtheria and tetanus are produced by isolating toxins from bacterial growth medium and subsequently rendering them non-toxic using formaldehyde. In contrast, acellular protein-based vaccines designed to safeguard against infections caused by entire bacteria, like those targeting B. pertussis (aP) and N. meningitidis serogroup B (Bexsero), require multiple antigenic components. In the case of the aP and Bexsero vaccines, the antibodies induced by these vaccine components serve several crucial functions. They neutralize the function of the disease-causing agents, trigger complement-dependent killing mechanisms, and enhance opsonophagocytosis69. These protein antigens are typically outer membrane proteins or proteins transported to the bacterial cell surface through various secretion systems.
Protein secretion mechanisms in Gram-negative bacteria
Bacteria possess a diverse range of secretion systems that enable the transfer of proteins, DNA, and small molecules to the external environment. In the case of Gram-negative bacteria, eleven protein secretion pathways have been identified to date, including Type 1-Type 6, Type 8-Type 11 and the chaperone usher pathway70. These protein secretion systems operate through either a one-step or two-step translocation process to deliver proteins across the bacterial cell envelope. In one-step translocation, also known as Sec-independent systems, substrates are transported directly from the cytoplasm to the extracellular milieu or the interior of the host cell. Notable examples of this one-step translocation system are found in double-membrane-spanning secretion systems such as Type 1, Type 3, Type 4, and Type 6. Conversely, the Type 2, Type 5, Type 8 through Type 11, and the chaperone usher pathways employ a two-step translocation mechanism. For the latter systems, substrates are first translocated to the periplasm with the help of inner membrane-spanning transporters such as SecYEG translocon or Tat (twin-arginine translocation system)71,72,73,74. Subsequently, these substrates are transported to the OM or released into the extracellular space. In the case of the aP vaccine, its components encompass the Type 4 secreted pertussis toxin, two fimbrial antigens secreted via the chaperone usher pathway, and two Type 5 secreted proteins, Pertactin and Filamentous Haemagglutinin (FHA)75. Similarly, the Bexsero vaccine combines four distinct elements: the OMVs from the New Zealand strain NZ98/254 containing PorA, the cell surface-associated proteins factor H binding protein (fHbp) and neisserial heparin binding antigen (NHBA), and the Type 5 secreted protein NadA76. The effectiveness of the Type 5 proteins in the Bexsero and aP vaccines suggest similar proteins may form the basis of vaccines against other pathogens. The type of immune response and protection generated by the Type 5 proteins in the licensed pertussis and meningitis vaccines is discussed in the later sections.
Type 5 secretion system
Among the various secretion systems, the Type 5 secretion system (T5SS) is the most prevalent protein secretion system in Gram-negative bacteria and frequently plays a crucial role as virulence factors during infections71,77. T5SS proteins contribute to a wide array of functions, including adhesion, aggregation, biofilm formation, proteolysis, serum resistance, pathogenesis, and serve as significant virulence factors78. Proteins secreted via this mechanism generally exhibit a tripartite structure: an N-terminal signal sequence, a passenger, and a β-barrel translocator ___domain79. Initially, these proteins traverse the inner membrane (IM) through the SecYEG translocon. In the periplasmic space, signal peptidases cleave the signal sequence, and chaperones, such as Skp, SurA, and DegP, facilitate the transfer of the remainder of the protein across the periplasmic space to the BAM complex within the OM. The BAM complex plays a crucial role by inserting the translocator ___domain into the OM as a β-barrel and facilitating the passage of the passenger to the cell surface through the β-barrel’s pore80,81. Once at the cell surface, the passenger may either remain attached to the cell’s surface or be released into the extracellular space82.
Subclasses of the T5SS
Depending on the ___domain architecture, the T5SS can be divided into five subclasses: Type 5a-e71,77 (Fig. 3). Within the different classes of the T5SS proteins, the β-barrel ___domain is highly conserved but the functional passengers display much more heterogeneity indicative of evolutionary and immunological pressures77. Proteins that are members of the Type 5a secretion system (T5aSS) are also known as classical or monomeric autotransporters. Classical autotransporters are comprised of a signal sequence at the N-terminus, a passenger, and a 12-stranded β-barrel ___domain at the C-terminus. Examples of this pathway include IgA protease from N. meningitidis, the first member of this family to be described, Tsh from Escherichia coli, and Pertactin from B. pertussis81. The Type 5b secretion system (T5bSS) is also known as the two-partner secretion system. Type 5b proteins also have a passenger and β-barrel ___domain, but these are encoded on separate genes77,78,81. In addition, the β-barrel ___domain has a 16-stranded β-barrel instead of 12 and contains two polypeptide transport associated (POTRA) domains and bears homology to BamA, the central component of the BAM complex77. The passenger polypeptide is referred to as the TpsA protein and the translocator as TpsB. The best characterized member of this subfamily is Filamentous haemagglutinin (FHA) of B. pertussis. The Type 5c secretion system (T5cSS) is made up of trimeric autotransporter adhesins (TAAs). TAAs have all domains encoded on one gene, but the C terminal β-barrel ___domain is made of three polypeptide chains, each contributing four β strands to the 12-stranded β-barrel in the OM. As a result, the nascent polypeptide chains must trimerize to form a complete barrel to enable translocation of the passenger. While the best characterized trimeric autotransporter is YadA from enteropathogenic Yersinia enterocolitica, which is involved in adhesion to host cells, other examples include NadA, a component of the Bexsero vaccine against N. meningitidis77,83. The Type 5d secretion system (T5dSS) is also known as the fused two partner section; thus far these proteins are lipases that have patatin like domains77,81. Like the classical autotransporters, the T5dSS proteins are transcribed as a single polypeptide, but possess POTRA domains like the T5bSS. Examples of the T5dSS proteins include PlpD from P. aeruginosa and FplA from Fusobacterium nucleatum78. In contrast to the classical autotransporters, the order of the passenger and β ___domain are reversed in the Type 5e secretion system (T5eSS) such that the C-terminus of the protein is presented on the surface of the bacterial cell and the N-terminus encodes the β-barrel ___domain. Intimin from E. coli and Invasin from Y. enterocolitica are well characterized T5eSS proteins81.
Type 5 secreted proteins consist of a signal sequence, passenger, and translocator ___domain. Type 5 secretion is further divided into Types 5a–5e. Proteins that are members of the Type 5a secretion system (T5aSS) have a signal sequence at the N terminal, a passenger and β-barrel ___domain at the C terminal. An example of this subclass is Pertactin (retrieved from AlphaFold database, UniProt accession Q8RSU0). Type 5b is also known as two-partner secretion (TPS) where the passenger and the translocator ___domain are encoded for on two separate genes, which are translated into separate polypeptides. The TPS β-barrel ___domain has two POTRA domains. An example of this subclass is FHA with crystal structures for the N-terminal region of the passenger and translocator ___domain (retrieved from PDB 1RWR and 4QKY, respectively). The Type 5c secretion system (T5cSS) consists of proteins that assemble to form a trimer and are also known as trimeric autotransporter adhesins (TAAs). An example of this subclass is NadA with crystal structure for the N-terminal region of the passenger (retrieved from PDB 6EUN) and the translocator ___domain (residues 341–405) was generated using ColabFold194. The Type 5d protein is also known as fused two partner secretion, where the β-barrel ___domain also contains a POTRA ___domain. An example of this subclass is PlpD (retrieved from AlphaFold database, UniProt accession A0A0A8RFV7). A recent study by Hanson et al. has proposed that the passenger of PlpD is facing the periplasm195. Type 5e has the passenger at the C terminus and extends from the outer membrane of the cell. An example of this subclass is the crystal structures for Intimin (retrieved from PDB 1F02 and 6TQD for the passenger and 4E1S for the translocator ___domain). The figure was created using components from Alphafold, PDB and BioRender.com.
Licensed vaccines containing T5SS proteins
Numerous research studies have concentrated on harnessing T5SS proteins as viable vaccine antigens. These proteins exhibit several characteristics that render them ideal candidates for vaccines, including their proven role in pathogenesis and their localisation of the functional passenger ___domain on the bacterial cell surface, making them targets for immune responses mediated by antibodies. As noted above, two of the antigens of the aP vaccine belong to the type 5a (Pertactin) and type 5b (FHA) subclass of T5SS proteins84,85. The Bexsero vaccine is a recombinant meningococcal vaccine containing NadA, a trimeric autotransporter (type 5c)83. The following section delves into the outcomes of licensed vaccines based on T5SS proteins, providing insight into why these proteins are included in licensed vaccines, as well as their roles in infection and their immunogenic properties.
Bordetella pertussis vaccines
Pertussis, also known as whooping cough, is a severe acute respiratory disease caused by the Gram-negative bacterial pathogen B. pertussis86. Whooping cough was a leading cause of death worldwide until the introduction of vaccines. The aP vaccine induced high antibodies and low adverse reactions and is composed of 5 B. pertussis antigens; Pertactin, Pertussis toxin, FHA, Fimbriae 2 (FIM 2) and Fimbriae 3 (FIM 3)87,88,89. The criteria used for selection of the antigens in the aP vaccine is based on pathogenesis. FHA, Pertactin and FIM 2 and 3 were selected due to their role in adherence to respiratory epithelial cells, whereas pertussis toxin is a systemic toxin that interferes with the function of G-protein coupled receptors in host cell membranes90,91,92. Additionally, in vivo experiments in animal models demonstrated that each antigen had some protective effect93.
Clinical trials conducted in Italy and Sweden demonstrated that the efficacy of the three-component (Pertactin, FHA, and pertussis toxin) and five-component aP vaccines is significantly higher when compared to the two-component vaccine (FHA and pertussis toxin)94. In a specific clinical trial in Sweden, involving 9829 infants aged 6–12 months, the efficacy of the five-component vaccine, containing FHA, FIM 2 and 3, pertussis toxin, and Pertactin, reached 85%. Conversely, the two-component vaccine in the same trial exhibited a lower efficacy of 59%. These trial results strongly suggest that Pertactin may play a crucial role in enhancing the effectiveness of licensed aP vaccines84. The inclusion of multiple antigens, known for their high immunogenicity and protective properties, could potentially mitigate the impact of antigenic variation, which can occur when relying on a single antigen alone95,96. Furthermore, vaccines containing multiple antigens provoke a broader and more comprehensive immune response compared to single-component vaccines. Investigations of the antibodies produced in response to aP vaccine antigens have revealed that they neutralized the activity of the toxin. Given that pertussis toxin is released into the extracellular milieu, it is thought that these antibodies are unlikely to facilitate complement-mediated killing and phagocytosis. Additionally, studies have shown that anti-Fim antibodies neutralize the function of FIM 2 and 3 by reducing the attachment of bacteria to host cells97,98.
Pertactin
Pertactin belongs to the Type 5a subclass of autotransporters79,82. After translocation of the passenger to the cell surface, the Pertactin precursor polypeptide is processed into a separate β-barrel ___domain and the passenger. The processed passenger, sometimes referred to as P.69, remains attached to the cell surface through a noncovalent interaction with the β-___domain but can be released by mild heating99. Pertactin exhibits remarkable conservation across various Bordetella species and serves a crucial role in adhering to respiratory epithelial cells85,96,100. In vivo studies have demonstrated that Pertactin significantly contributes to pathogenesis by impeding the clearance of bacteria by neutrophils in the lungs85. The presence of high levels of anti-Pertactin IgG antibodies has been correlated with protection against B. pertussis101. A Pepscan analysis utilizing serum from both human and mouse subjects immunized with a Pertactin-containing vaccine, has revealed the specific epitopes on P.69 Pertactin to which protective antibodies bind102; the epitopes are shown in Fig. 4. The function of the protective antibodies varies depending on the immunogenic epitope of P.69 Pertactin the antibodies bind to. For example, the RGD motif of Pertactin is involved in adherence and antibodies to the RGD motif are likely to be neutralising in function; thus, preventing adhesion to the host. The antibodies that bind to P.69 Pertactin closer to the β-barrel ___domain are likely to facilitate complement-mediated killing. Furthermore, in vitro assays have demonstrated that antibodies targeting Pertactin possess bactericidal properties103. These antibodies bind to Pertactin, initiating the complement pathway, leading to the formation of the membrane attack complex (MAC), a critical component for bacterial lysis. The mechanism of action of Pertactin and the other components of the aP vaccine are shown in Fig. 5. Another potential mechanism for bactericidal action involves the deposition of C3b onto the bacterial cell surface. C3b is recognized by complement receptors (FcR) on phagocytes, thereby activating phagocytosis. This process represents a key immune mechanism for clearing B. pertussis from the respiratory tract104,105,106.
The crystal structure of P.69 Pertactin was retrieved from PDB (1DAB). The epitopes recognised by the human sera are shown in blue, epitopes recognised by mouse monoclonal antibodies (MAbs) are shown in yellow, and epitopes recognised by both human sera and MAbs are shown in red.
a Pertussis toxin (PT) elicits neutralising antibodies b Antibodies to FIM 2 and FIM 3 decrease bacterial adherence. c Anti-Pertactin antibodies activate the complement complex C1q resulting in (1) phagocytosis of bacteria or (2) formation of a membrane attack complex (MAC) on the surface of the bacterial cell, leading to lysis of bacteria. d FHA is secreted into the supernatant and anti-FHA antibodies decrease bacterial adherence. The crystal structure for PT was retrieved from PDB 1PRT. The structure for Pertactin was retrieved from AlphaFold database, UniProt accession Q8RSU0. The crystal structures for the N-terminal region of the passenger and translocator ___domain of FHA were retrieved from PDB 1RWR and 4QKY, respectively. The figure was created using components from BioRender.com, Alphafold, and PDB.
Filamentous hemagglutinin
FHA, a 220-kDa protein, belongs to the Type 5b subclass of the T5SS, where fhaB encodes the TpsA protein, and fhaC encodes the TpsB protein77,107. This surface-localized protein plays a pivotal role in the pathogenesis of B. pertussis. In in vitro studies, FHA has been identified as an adhesin, facilitating the attachment of B. pertussis to host cells108. Furthermore, FHA is indispensable for the initial adherence and colonization of the trachea during in vivo infections109. Remarkably, mutants lacking FHA exhibit a notable inability to bind human neutrophils, and this deficiency cannot be compensated for by other B. pertussis adhesins110. In mouse models, it has been observed that Bordetella bronchiseptica lacking FHA triggers lung inflammation, whereas the wild-type strain does not. This suggests that FHA plays a pivotal role in modulating inflammation during infection, thereby contributing to bacterial persistence111. Additionally, FHA is actively involved in the up-regulation of two cytokines: IL-6 and the anti-inflammatory cytokine IL-10, while concurrently down-regulating IL-12, a pro-inflammatory cytokine that regulates the differentiation of Th1 cells112,113. Beyond its anti-inflammatory functions, FHA also fosters phagocytosis and promotes the induction of a Th2 immune response112,114. Given its surface localization and its role in virulence, various investigations have explored the potential of anti-FHA antibodies in safeguarding against infection. Mice that were immunized with FHA demonstrated significantly reduced bacterial counts in their lungs and trachea, with this protective effect being mediated by anti-FHA antibodies115. Studies in mice have additionally shown that while FHA antibodies alone offer relatively limited protection, combining FHA with other B. pertussis antigens enhances the immune response, resulting in more robust bacterial clearance in immunized mice116. Furthermore, immunization with B. pertussis proteins, including pertactin and FHA, has provided protection against other Bordetella strains such as B. bronchiseptica117. Consequently, FHA was incorporated into licensed vaccines.
Emerging resistance to aP
Despite the success of the aP in controlling infections, there is evidence of a resurgence in pertussis. This resurgence is primarily attributed to antigenic variations between the Pertactin types in the circulating strains and the strains used in the vaccine96. Such antigenic variation can potentially undermine the efficacy of the vaccine. Studies have indicated an increase in the isolation of Pertactin-deficient strains following aP vaccination, suggesting a bacterial evasion mechanism against the bactericidal antibodies induced by Pertactin in the vaccine103. Recently, clinical isolates lacking FHA have been identified, and this has been linked to the phenomenon of phase variation in FHA expression. Phase variation involves the reversible switching on and off of protein expression118. It has been proposed that phase variation plays a role in the pathogen’s adaptation to different environments, potentially diminishing the efficacy of the current aP vaccine119. Furthermore, existing licensed pertussis vaccines are effective in protecting against severe pertussis infections but provide limited defense against bacterial colonization. To address this limitation, there is a growing need to develop new vaccines that not only prevent colonization but also reduce disease severity. Research conducted in mice and baboon models has shown that the licensed aP vaccine primarily triggers a Th2 immune response and antibody production. In contrast, natural infection with B. pertussis and immunization with wPV elicits a Th1 and Th17 immune response114. As described previously, Th17 cells are important for vaccine mediated protection. One strategy to enhance the Th1/Th17 immune response is to combine the vaccine with adjuvants that stimulate the production of Th1/Th17 cells120. Some of the adjuvants known to elicit a Th1/Th17 immune response include squalene, CpG oligodeoxynucleotides (ODN), and outer membrane vesicles (OMVs)114,120,121. Studies have shown that immunisation of mice with bivalent OMVs isolated from S. Typhi and Salmonella Paratyphi A induced a Th1 and Th17 immune response suggesting the inclusion of OMVs into current aP vaccines may provoke an increased protective effect and decrease the ability of the bacterium to escape the protective effects of the vaccine122. Indeed, OMVs have been used in the licensed Bexsero vaccine and are known to elicit a robust antibody response along with Th1, Th2 and Th17 cells120.
Neisseria meningitidis serogroup B vaccine
N. meningitidis a Gram-negative bacterial pathogen, is responsible for causing meningitis and sepsis, particularly affecting children and young adults123. This bacterium is categorized into five pathogenic serogroups based on the composition of its bacterial capsule: A, B, C, Y, and W135. Polysaccharide conjugate vaccines have been effective against serogroups A, C, Y, and W135. However, due to concerns about potential autoimmune responses caused by cross-reactivity with human antigens, polysaccharide vaccines were not suitable for protection against serogroup B pathogens124. The Bexsero vaccine, represents a ground-breaking approach. It is a multicomponent vaccine that has received approval for use in Europe, Australia, and Canada. Remarkably, it is the first vaccine developed using the reverse vaccinology approach. This method involved analysing the complete genome sequence of N. meningitidis serogroup B strains to identify antigens secreted to the outer membrane. Antigens were selected based on their ability to induce serum bactericidal activity, a key indicator of protection, and their potential to offer cross-protection against a wide range of strains125,126. As noted above, the Bexsero vaccine includes four protein subunit antigens, one of which is the T5cSS adhesin NadA76 (Fig. 6). OMVs were added to the final formulation of the Bexsero vaccine to improve immunogenicity and strain coverage126.
The licensed Bexsero vaccine is made up 3 Neisseria meningitidis antigens that were identified by reverse vaccinology and outer membrane vesicles (OMVs). The 3 antigens include NadA which belongs to the T5cSS, NHBA and fHbp which are lipoproteins. The crystal structure for the N-terminal region of the passenger of NadA was retrieved from PDB 6EUN and the translocator ___domain (residues 341–405) was generated using ColabFold194. The crystal structures for NHBA and fHbp were retrieved from PDB 2LFU and 3KVD, respectively. The figure was created using components from BioRender.com, ColabFold, and PDB.
Each antigen in the Bexsero vaccine triggers the production of antibodies that work synergistically to boost bactericidal activity. The inclusion of multiple antigens in the licensed vaccine was driven by the stronger bactericidal response observed when compared to using a single antigen alone. Clinical development data for the Bexsero vaccine suggest that antibodies against these antigens can confer protection by 1) activating the classical complement pathway and 2) preventing binding to the host cell surface83,127,128. A study by Biolchi et al. assessed the potential of the Bexsero vaccine to offer cross protection against non-MenB serogroups. This study involved testing non-MenB isolates from various countries using sera from infants vaccinated with Bexsero. The results showed that immunization with Bexsero induced complement-mediated killing and offered cross-protection against non-MenB strains129,130. Additionally, studies in mice and humans have shown that immunisation with Bexsero could provide cross-protection against Neisseria gonorrhoeae, by inducing complement-mediated killing131,132.
Neisseria adhesin A (NadA)
NadA is a trimeric autotransporter and is involved in adhesion and invasion of N. meningitidis into host tissues133. NadA is present in ~50% of all the diseases associated serogroups of N. meningitidis83. A study by Commanducci et al. has shown that NadA elicited high titres of bactericidal antibodies that cross react with all variants and elicited protection in laboratory animals83. Based on the peptide sequence homology of NadA, four variants have been identified which include: NadA-1, NadA-2/3, NadA-4/5 and NadA-6. The Bexsero vaccine includes the variant NadA-2/3, which is highly immunogenic and provides cross-protection across strains displaying NadA-1 and NadA-2/3134. In vivo studies have shown that during meningitis infection, NadA interacts with monocytes, macrophages and epithelial cells and stimulates the activation of pro-inflammatory cytokines such as TNF-α and IL-8135. NadA is a trimer and is made up of an N-terminal head ___domain involved in cell adhesion, a stalk ___domain and a C-terminal membrane anchor region required for translocation to the bacterial surface. A study by Giulani et al. showed that anti-NadA antibodies against the head region prevent adhesion to cells. This study suggested that NadA was a crucial component in the Bexsero vaccine and anti-NadA antibodies were likely to prevent initial colonisation by neutralising the function of NadA136,137.
T5ss proteins tested as vaccines
Resurgence of diseases, as seen in the case of pertussis, is a common phenomenon and is associated with antigenic variation and reduction in vaccine efficacy overtime. Hence, new vaccine antigens need to be identified. The B. pertussis T5SS proteins BrkA and Vag8 belong to the T5aSS subclass and help the bacterium evade complement-mediated killing. Since complement-mediated killing is responsible for protection against B. pertussis infection, inclusion of BrkA and Vag8 might help improved vaccine efficacy against pertussis. Two separate studies have shown that immunisation of mice with BrkA and Vag8 resulted in reduction in bacterial numbers, which was attributed to complement-mediated killing138,139. Furthermore, immunisation with BrkA alone was not sufficient for protection and had to be combined with other B. pertussis antigens FHA and PT139. Hence, identifying new antigens and the mechanisms involved in evasion might help develop better vaccines.
Vaccines are still lacking for many bacterial pathogens that infect humans and animals. Some of these pathogens have become resistant to multiple antibiotics and have been listed by the WHO as critical or high priority pathogens1. Hence, there is a need for the development of new therapeutics such as vaccines. The success of the T5SS proteins in the licensed acellular pertussis and Bexsero vaccines, and their wide distribution in Gram-negative bacteria, makes such proteins a rich hunting ground for vaccine antigens. Multiple studies have tested the potential of using T5SS proteins from different bacteria as vaccine antigens with varying success. The T5SS proteins tested (Table 2) in preclinical models showed varying degrees of protection against the relevant pathogen by blocking the functions of the pathogen. Some of the T5SS proteins such as DsrA against Haemophilus ducreyi, UspA1 and UspA2 against Haemophilus parasuis, SadA against S. Typhimurium, Pta against Proteus mirabilis showed only partial protection against infection140,141,142,143. Immunisation with Pta elicits antibodies that are neutralising in function. However, neutralising antibodies alone may not be involved in prevention or clearance of infection and other antibody mechanisms may be required. Therefore, the partial protection following immunisation with Pta might be attributed to neutralising antibodies alone. Inclusion of other antigens may be required to enhance protection and vaccine efficacy143. One study has shown that immunisation with a combination of T5SS proteins SigA, Pic and Sap showed more robust protection against Shigella flexneri than when used alone144. The licensed acellular pertussis and Bexsero vaccines include other antigens that are not T5SS proteins as well as OMVs. Since OMV production is a common phenomenon in Gram-negative bacteria and OMVs contain several surface antigens, the OMV vaccine platform can be used to enhance vaccine efficacy. Immunisation of chinchillas with a T5SS protein Hia in combination with OMVs showed protection against Nontypeable Haemophilus influenzae (NTHi)145. Hence, multiple antigen vaccines might work better than a single T5SS protein-based vaccine alone.
Future perspectives
The T5SS proteins are targets for antibodies and their potential as vaccines is currently being tested against Gram-negative pathogens. Nevertheless, there is no single surface protein vaccine licensed against any bacterium. To date, studies that have tested the use of a single protein as a vaccine have shown varying success and only provide partial protection when used alone. Currently, the only single subunit vaccines that have proved successful are based on toxins and capsular polysaccharides. One reason for the lack of protection following immunisation with some antigens could be due to the way antibodies to that antigen interact with the bacterial surface. Gram-negative bacteria have LPS on the outer leaflet that is made up of lipid A, core oligosaccharide and O-Antigen. Studies have shown that that the O-Ag can provide steric hindrance and block the antibodies from accessing some antigens30. Indeed, immunisation with OMPs such as OmpA from S. Typhimurium and Salmonella Enteritidis does not confer protection30,146. Nevertheless, immunisation with OmpD from S. Typhimurium provides partial protection against infection with S. Typhimurium. In contrast, immunisation with S. Typhimurium OmpD confers minimal cross protection against S. Enteritidis. The lack of protection against S. Enteritidis is due to a single amino acid difference between OmpD of the two serovars30. The difference in protection between OmpA and OmpD has been postulated to be due to the footprint created by the antigens in the LPS layer. OmpD creates a larger footprint in the O-Ag layer, which results in less steric hindrance and allows the antibody access to the membrane proximal OmpD where it can initiate serum killing. In contrast, the OmpA generated footprint is too narrow to enable access of the OmpA antibody to its target30. Thus, multiple antigens are required to ensure protection against infection, as seen in the licensed Bexsero and acellular pertussis vaccines84,94.
While LPS provides steric hindrance preventing access of antibodies to membrane proximal epitopes, the passenger ___domain of T5SS proteins are largely predicted to extend beyond the LPS layer and to be accessible to antibody, including the S. Typhimurium T5cSS SadA protein142. Although multiple studies have tested the potential of using T5SS proteins as vaccine antigens; FHA, Pertactin and NadA are the only T5SS proteins used in licensed vaccines. The reason only a few T5SS proteins have been tested to date may be attributed to the fact that only some of these proteins have been functionally characterized and thus their role in virulence and as vaccine targets is still unknown. Moreover, research on the immune mechanism and function that correlates with protection is important for designing and licensing vaccines against emerging and multi-drug resistant bacterial pathogens. The success of all T5SS proteins (Table 1) at providing at least partial protection against infection, suggests that the combination of T5SS proteins with other surface antigens, as seen for the aP and Bexsero vaccines, is a useful strategy for creating vaccines for many Gram-negative bacterial infections, and, as is the case for Bexsero, the incorporation of OMVs is likely to enhance the protective effect of these antigens.
Finally, the T5SS has been utilised for the surface display of several recombinant proteins and antigens on the cell surface and has been termed as autodisplay. In this approach, the passenger ___domain of the T5SS is replaced with a heterologous protein that is transported and displayed on the cell surface. The autodisplay system is a promising tool for a range of biotechnological and biomedical applications including the development of vaccines147. The surface display of an epitope from Plasmodium falciparum on the surface of an attenuated S. enterica strain using the T5SS protein MisL was shown to induce a strong antibody response to the epitope148. Thus, the display of multiple immunogenic epitopes on the surface of an attenuated bacterial vector may be more cost-effective than formulations containing a mixture of single antigens. Additionally, OMVs could be engineered to express multiple antigens of choice, such as T5SS proteins. This would effectively reduce the cost for manufacturing and makes them ideal platforms for vaccine development53.
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This work was supported by The University of Queensland Research Training Scholarship.
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Da Costa, R.M., Rooke, J.L., Wells, T.J. et al. Type 5 secretion system antigens as vaccines against Gram-negative bacterial infections. npj Vaccines 9, 159 (2024). https://doi.org/10.1038/s41541-024-00953-6
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DOI: https://doi.org/10.1038/s41541-024-00953-6
