Microbial complement inhibitors as vaccines

Vaccine 26S (2008) I113–I117

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Microbial complement inhibitors as vaccines Seppo Meri ∗ , Markus Jördens, Hanna Jarva Haartman Institute, Department of Bacteriology and Immunology, University of Helsinki, Finland

a r t i c l e Keywords: Complement Factor H C4bp Meningococcus Borrelia Yersinia

i n f o

a b s t r a c t Complement inhibiting surface proteins of pathogenic bacteria provide candidates for vaccines because of two reasons. First, an immune response against them would recognize the microbes and secondly, it would neutralize the key bacterial virulence mechanism. Prerequisites for a vaccine protein include the following: (i) it should show limited variability, (ii) it should be immunogenic and the immune response against it should cover a sufficiently broad range of microbial strains, (iii) it should not be hidden beneath a capsule, long LPS O-polysaccharide side chains or a protein coat and (iv) it should not raise unwanted immune responses against host structures. Bacterial complement inhibitors often act by binding the soluble inhibitors factor H or C4 bp, by blocking C3 or C5 activation or by enzymatically cleaving key complement components. Inhibitors have been found from all major types of pathogens and may offer promise as rational vaccine candidates for preventing diseases such as meningococcal meningitis, systemic pneumococcal or group B streptococcal disease and Lyme borreliosis. © 2008 Elsevier Ltd. All rights reserved.

1. Introduction Current vaccines use primarily killed or live attenuated viruses or inactivated protein toxins or capsular polysaccharides from bacteria. Dating back over 100 years, the main underlying philosophy in their development was to induce sufficient immunity for neutralizing the pathogen or its toxin. With the existing new knowledge on microbial virulence mechanisms and genomes it is possible to develop new innovation- or knowledge-based vaccines [1]. An essential factor in microbial virulence is to escape the host’s microbicidal complement system. Although both complement and microbes have been known for over 100 years it is only during the last 20 years that essential information on the various complement evasion mechanisms of pathogens has emerged. A natural development in this line of research is to design ways to counteract the microbial evasion mechanisms and use this information for the development of new vaccines. The need for new vaccines exists, e.g. for group B meningococcus, pneumococcus, tuberculosis, Lyme borreliosis, malaria and various viral illnesses, notably HIV/AIDS. In this review we will discuss principles and provide some exam-

Abbreviations: CRP, C-reactive protein; MAC, membrane-attack complex; C4bp, C4b binding protein; GAS, group A streptococcus S. pyogenes; fHbp, factor H binding protein. ∗ Corresponding author at: Haartman Institute, Department of Bacteriology and Immunology, University of Helsinki, P.O. Box 21, FI-00014 Helsinki, Finland. Tel.: +358 9 1912 6758; fax: +358 9 1912 6382. E-mail address: seppo.meri@helsinki.fi (S. Meri). 0264-410X/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2008.11.058

ples on the potential use of microbial complement inhibitors as vaccines. 2. The complement system The complement system is an important part of innate immunity. The functions of complement include defence against invading microbes and, on the other hand, clean-up of the host body of cell and tissue debris, immune complexes and apoptotic cells. Complement consists of over 30 soluble or membrane-bound proteins. The activation proceeds via three different pathways, all leading to the deposition of C3b on target surfaces and activation of the terminal pathway. The classical pathway is initiated via contact of blood plasma with, e.g. surface deposited IgG, IgM, C-reactive protein (CRP) or lipid A of bacterial lipopolysaccharides. The alternative pathway is constantly in a semi-activated state via the tick-over hydrolysis of plasma C3, resulting in the deposition of C3b on nearby surfaces. The lectin pathway is activated through the binding of mannose-binding lectin or ficolins to microbial surface carbohydrates containing, e.g. N-acetyl-glucosamine or mannose. All three primary pathways lead to formation of C3 convertases and the deposition of C3b on surfaces. When complement activation continues via the terminal pathway, C5a is released and, ultimately, the membrane-attack complex (MAC) is formed. C5a is a strong anaphylatoxin and enhances the inflammatory reaction. MAC makes a pore on the target membrane and potentially leads to cell lysis. A particularly important point in complement activation is the alternative pathway amplification loop. Amplification occurs on targets that lack protective host cell structures. Even when

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activation is initiated through the classical or lectin pathway, the alternative pathway is needed for efficient complement activation to continue. On the other hand, as exemplified by antimeningococcal immunity, activation of the alternative pathway requires efficient initiation through the classical pathway by specific antibodies. Since activation of the complement system may lead to considerable tissue damage it has to be tightly regulated. Currently, five membrane-bound and six soluble regulators are known. Complement receptor type 1 (CR1, CD35), membrane cofactor protein (MCP, CD46), decay-accelerating factor (DAF, CD55) and CRIg (complement receptor of the immunoglobulin superfamily) inhibit complement activation at the C3 convertase level [2–4]. Protectin (CD59) inhibits the formation of MAC. Of the soluble regulators, factor H and FHL-1, an alternatively spliced product of the factor H gene, regulate the amplification loop of the alternative pathway. C1-inhibitor and C4b binding protein (C4bp) inhibit the classical pathway, clusterin and vitronectin the terminal pathway. 3. Factor H Factor H is a fluid-phase regulator of the alternative pathway amplification loop. It is a soluble 150 kDa protein composed of 20 short consensus repeat (SCR) domains. Factor H regulates the alternative pathway by inhibiting the binding of factor B to C3b, acting as a cofactor for factor I-mediated cleavage of C3b (cofactor activity) and accelerating the decay of the alternative pathway convertase C3bBb (decay-accelerating activity). All these steps are essential in keeping the alternative pathway amplification loop under control. By controlling the key steps of the amplification loop factor H inhibits also activation that has been initiated via the classical or the lectin pathway. The cofactor and decay-accelerating activities of factor H have been located to the SCR domains 1–4 [5]. These N-terminal SCRs bind to C3b, whereas the most C-terminal SCRs 19–20 of factor H bind to the C3d-region or the thiolester-containing TED-domain of C3b [6]. SCR7 and SCR20 regions also have binding sites for polyanions, like glycosaminoglycans and sialic acids. In addition to controlling alternative pathway activation in the fluid phase, factor H has an important function in the discrimination between complement activating (“nonself”) and non-activating (“self”) surfaces. As C3 undergoes spontaneous low level hydrolysis to produce C3(H2 O) that can bind factor B (tick-over), the alternative pathway is in a continuous state of alertness to react with target structures [7]. Upon contact with a surface the default for the alternative pathway is to become activated, unless inhibited. As a consequence, C3b molecules get constantly deposited on nearby surfaces. Surfaces of intact human cells are abundant in terminal sialic acids and glycosaminoglycans. As factor H has a relatively high affinity for C3b (over that of factor B), when the surface around C3b is coated with these polyanions, the alternative pathway activation is kept under control [8]. However, if the surface is devoid of polyanions, the affinity of factor H for surface-associated C3b is reduced, factor B binds to C3b and complement activation proceeds upon formation of the alternative pathway C3 convertase C3bBb. 4. Complement and microbes Complement has several functions in microbial defence. The most important function is opsonization by C3 activation products C3b and iC3b. Phagocytes have receptors for C3b (CR1) and iC3b (CR3 and CR4), of which CR3 (CD11b/18) is the major opsonophagocytic receptor. Enhancement of the inflammatory reaction by chemotactic and anaphylatoxic complement cleavage products C5a

and C3a is another anti-microbial function. C5a has multiple functions in recruiting and activating phagocytes. Formation of MAC is important for protection against many Gram-negative bacteria, most importantly against neisseriae (Neisseria meningitidis and Neisseria gonorrhoeae). Complement is also involved in the activation and strengthening of B and T cell responses by its natural adjuvant-like activities [9,10].

5. Bacterial evasion of complement Virulent microbes have developed multiple mechanisms to evade complement attack [11,12]. They can, e.g. avoid being recognized as “nonself”, inhibit deposition of opsonic complement components, cleave activation products or hijack host complement inhibitors, factor H, C4bp or CD59. Often multiple mechanisms are employed. The ability to escape complement is one of the key discriminatory features between pathogens and nonpathogens. Polysaccharide capsules, peptidoglycan, protein coating and lipopolysaccharide (LPS) structures provide basic protection for bacteria against complement activation. Sialic acid-containing capsules and terminal sialylation of LPS may in some, but not all, cases further inhibit opsonophagocytosis or activation of the alternative pathway. The thick peptidoglycan layer of Gram-positive bacteria is in general protective against MAC formation and lysis. Therefore, activation of the terminal pathway has only a minor role in the defence against Gram-positive bacteria. In Gram-negative bacteria, the O-polysaccharide side chains of the lipopolysaccharides also sterically hinder C3b/iC3b deposition and MAC formation on the outer cell membranes. Many pathogenic microbes bind soluble complement regulators, e.g. factor H or C4bp. Microbes do not naturally produce glycosaminoglycans but they can have, e.g. hyaluronic acid or various types of sialic acid moieties in capsules or on surface polysaccharides. For example, serotype III group B streptococci (Streptococcus agalactiae), group B meningococci (N. meningitidis) and Escherichia coli K1 produce capsules that are composed of polysialic acid. Although polymeric sialic acid is inefficient in binding factor H, the capsules of these bacteria mediate resistance to opsonophagocytosis by preventing adherence and binding of opsonins. Specific resistance to the alternative pathway can also be mediated by surface proteins that bind factor H. Once factor H is bound to the surface through these molecules, complement activation is restricted. For example, the M-protein of group A streptococcus (GAS, Streptococcus pyogenes) and the OspE and Bba68/CRASP-1 proteins of serum-resistant strains of Borrelia burgdorferi and Borrelia afzelii bind factor H from serum [13–15]. The factor H binding capacity is not restricted to bacteria as the yeast Candida albicans and the parasitic worms Onchocerca volvulus (microfilariae) and Echinococcus have also been shown to acquire factor H to their surfaces [16–18]. Microbes such as group A streptococci, N. gonorrhoeae, Bordetella pertussis and Yersinia enterocolitica bind also the classical pathway regulator C4bp to their surfaces [19–22]. In addition to the examples above many other microbial complement evasion mechanisms exist. However, despite the rapidly accumulating knowledge, the complement evasion mechanisms of pathogenic microbes are still only partially known. Also, the fact that a mechanism to avoid complement activation in vitro has been recognized does not necessarily yet indicate that this mechanism would be important for the microbial survival in the human host. Nevertheless, the recognition and functional analyses of complement inhibitors are extremely important for attempts to prevent certain serious infections. As the microbial complement evasion

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molecules often represent virulence factors they are obvious vaccine candidates and potential therapeutic agents.

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plement. This approach has largely not been taken yet although there is evidence that for example cyclic peptides, such as compstatin could act as effective complement inhibitors [26]. To start with, bacterial homogenates could be tested for complement inhibiting activity and the potential inhibitors identified by size fractionating the solubilizates and testing the fractions for their capacity to inhibit the lytic activity of serum. Again, staphylococci have proven to be efficient producers of a wealth of small molecular weight proteins.

6. Bacterial proteins for therapy? Fungi have given us penicillin, which has saved lives of millions of people. What are prospects for using microbial complement inhibitors as therapeutic agents to prevent excessive complement activation during sepsis or other forms of acute inflammation? Recent studies have shown that staphylococci (Staphylococcus aureus) are masters in producing soluble complement inhibitors. As an example, the extracellular fibrinogen-binding protein (Efb) and its homologous protein Ehp bind C3b and inhibit opsonophagocytosis by granulocytes [23,24]. Both proteins induce conformational changes in C3b converting it into an inactive form [24]. A major problem in using bacterial proteins as potential therapeutics is their immunogenicity as foreign molecules. This could be bypassed by allowing only a single use of the product. This was the case with the fibrinolytic agent streptokinase, isolated from streptococci and used in the treatment of acute myocardial infarction [25]. Because of possible allergic reactions, the use of streptokinase was not recommended after 4 days of the initial use. Another option for using bacterial proteins as therapeutics would be to work out the active site of the complement inhibitor protein and produce it recombinantly with a host protein, e.g. IgG-molecule. To reduce immunogenicity, other attempts to “humanize” the protein could be pursued but this can be expected to be a rather challenging task. In general, it may prove difficult to find better complement inhibitors from bacteria than from man itself. In the case of regulator deficiencies or complement overactivation the current approaches using native or recombinantly expressed human complement inhibitors (like factor H or soluble CR1) may be more realistic and likely to succeed, although the cost of producing high molecular weight recombinant proteins is a somewhat restrictive factor. Nevertheless, efficient bacterial complement inhibitors may provide leads for products that are effective in limiting complement attack. An alternative to finding and modifying bacterial proteins for therapy would be to find bacterial small molecule inhibitors of com-

7. Complement evasion molecules in vaccine development In contrast to the previous examples of using bacterial inhibitors of complement in therapeutics, as described above, immunization is a naturally desired effect when these molecules are developed into vaccines. Bacterial membrane proteins are thus suitable candidates for vaccines. Examples of bacterial proteins affecting the complement system, their properties and estimated potential as vaccines are listed in Table 1. What is the rationale behind the use of microbial complement inhibitors, including the factor H and C4bp binding proteins, as vaccines? Complement is important during the early stages of infection, in preventing the invasion and spreading of the foreign organism. Complement components are present on mucosal surfaces, e.g. on bronchial and intestinal epithelial surfaces. Thus, if a microbe is incapable of circumventing complement, it is destroyed before infiltrating the tissues. Current “complement vaccine” research has mostly focused on recognition of the bacterial proteins that bind complement inhibitors. If binding of the complement regulators is prevented, complement activation will proceed on and near the microbial surface leading to opsonization, inflammatory reaction and eventually to either phagocytosis or lysis of the microbe. Thus, it is the early action, activity in tissues and potency that make complement a very important effector in innate immunity. The rationale for vaccines is to rescue these activities against pathogens by neutralizing microbial complement inhibitors. Depending on the microbial source some complement inhibitors could be planned for use as general vaccines (e.g., group B meningococcus) and some others could be visioned for use as more “personalized” vaccines for individuals or patient groups with

Table 1 Examples of microbial inhibitors of complement, their properties and potential as vaccine candidates. Microbe

Interacting protein

Key structural featurea

Mechanism of actionb

Variability

Potential as a vaccine

Reference

GAS GBS Pneumococcus S. aureus

M-protein ␤ PspC Sbi

␣ ␣ ␣ ␣

H, C4bp H H C3

+++ + +++

++ +++ ++

[29] [38,39] [40,41] [42]

B. burgdorferi

OspE Bba68

␣

H H

+++ +

+ +

[14] [43]

B. pertussis

FHA

␤

C4bp

+

++

[19,44]

Y. enterocolitica

YadA Ail

␣ ␤

H, C4bp H, C4bp

++ ++

+ −

[20,45–47]

Y. pestis

Pla

␤

E

+

+

[48]

S. typhimurium

Rck PgtE

␤ ␤

TCC E

+ +

± ±

[45] [49]

N. meningitidis

GNA1870 PorA

␤ ␤

H C4bp

++ ++

++ +

[36,50]

N. gonorrhoeae

Por1A Por1B

␤ ␤

H, C4bp C4bp

+ +

+ +

[21,51,52]

␣, alpha-helical/coiled-coil; ␤, beta-barrel. H, binding of factor H; C4bp, binding of C4bp; C3, binding to C3/C3b; E, enzymatic activity towards complement proteins; TCC, inhibition of the terminal complement pathway. a

b

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undue susceptibility for infections caused by particular microbes (e.g., Pseudomonas in cystic fibrosis patients). 8. Prerequisites for rational vaccines The research until now has mainly focused on bacterial proteins that bind soluble inhibitors of complement. The prerequisites for a bacterial protein to be a good vaccine candidate are that it (1) is immunogenic, (2) does not exhibit extensive variation, (3) has suitable physicochemical properties and (4) raises an immune response that neutralizes an important virulence determinant, i.e. complement inhibitory activity in this case, on the bacteria. Several bacterial proteins that bind complement inhibitors have been identified in recent years (Table 1) [11,27,28]. It is somewhat striking that, structurally, the complement inhibitors on bacteria in most cases seem to fall into two major categories. Known structures and structural predictions indicate that they are either alpha-helices, often bundled into coiled-coils, or beta-barreltype structures with antiparallel beta-strands. The coiled-coils, like the streptococcal M-proteins (GAS), beta-protein (GBS) and PspC (pneumococci) as well as the stalks of yersinial YadA extend far from the membrane and can therefore more easily reach soluble complement inhibitors through bacterial surface layers (peptidoglycan or LPS oligosaccharides). The beta-barrels, found e.g. in bacterial omptins or autotransporters, apparently form templates for proteins with different functions on the microbial surfaces. These proteins can act, e.g. as porins (neisseriae), enzymes (Pla and PgtE) or adhesive proteins (Ail and FHA), and often have multiple functions. In order to be useful in vaccine development, the candidate protein must be stably expressed, have a relatively conserved structure and be present on most virulent strains of the bacterial species. Also, and perhaps most importantly, the protein must be essential for the bacterial virulence. Many bacteria interfere with complement activation by multiple mechanisms. For example, group A streptococcus (S. pyogenes) binds both the alternative pathway inhibitor factor H and the classical and lectin pathway inhibitor C4bp via the alphahelical M-protein [22,29]. However, there are two other recognized receptors for factor H on GAS: Scl1 and SpeB [30,31]. Also B. burgdorferi, the causative agent of Lyme borreliosis, expresses two major types of factor H binding proteins [32]. Evasion of complement activation is evidently so crucial for bacterial survival that redundant mechanisms are used for escape. Once the relative contributions of the different factors have been worked out it is possible to select the best candidates for vaccine studies. 9. Potential bacterial vaccine candidates N. meningitidis is a frequent colonizer of the nasopharynx but can also cause meningitis and sepsis. There is an efficient tetravalent polysaccharide vaccine against meningococcal serogroups A, C, W135 and Y in clinical use. However, the capsular polysaccharide of serogroup B consists of the ␣(2→8) N-acetyl neuraminic acid homopolymers, structures also found in human fetal neural tissue [33,34]. Thus, because of the low immunogenicity of the serogroup B capsular polysaccharides and risk for potential cross-reactions with neural tissue vaccine research in this field is concentrating on developing a protein vaccine against group B meningococci. Currently, two meningococcal vaccines have been in clinical trials. Somewhat coincidentally, they contain the outer membrane proteins porin A (PorA) and GNA1870 (factor H binding protein, fHbp) that have been shown to bind the soluble complement inhibitors C4bp or factor H, respectively [35,36]. However, the PorA-containing outer membrane vesicle (OMV) vaccines do not

confer widespread immunity against serogroup B strains but tend to be strain-specific. The antibodies generated by OMV vaccines are mostly directed against PorA, but the marked strain-to-strain variation of PorA has hindered subsequent development of this vaccine. The recently identified factor H binding protein designated as genome-derived neisserial antigen 1870 (GNA1870), also known as lipoprotein 2086 or fHbp, is an attractive candidate for vaccine development [36]. GNA1870 is widely expressed on serogroup B meningococcal strains. In animal models, antibodies binding to GNA1870 inhibit binding of factor H and thus render the bacterium susceptible to the alternative pathway. Furthermore, the bound antibodies activate the classical pathway, thereby initiating and enhancing complement attack [36]. However, the use of GNA1870 as a vaccine has some limitations since it exists in three forms that differ in their antigenicity [37]. Also, some strains naturally express only low amounts of GNA1870 and for these strains blocking GNA1870 does not result in bacteriolysis. The solution might be a combination of recombinant protein antigens containing nonoverlapping epitopes of GNA1870 [37]. A dilemma in vaccine development is the fact that one would like to use conserved regions of proteins but the functional sites are often located in the variable parts. The use of functionally active complement inhibiting proteins as vaccines also carries the potential risk that – by inhibiting complement or by binding to a host molecule – they could be less immunogenic or partially masked by the host protein. Thus, the vaccine proteins would need to be slightly modified so that they lose their functional activity but maintain the immunogenic potential, yet raise antibodies that neutralize the functional activity of the proteins on bacterial surfaces. 10. Words of caution Bacterial infections are often followed by immunological postinfectious complications, like rheumatic heart disease (GAS), glomerulonephritis (GAS), reactive arthritis (Y. enterocolitica, Salmonella, Campylobacter) or Guillain–Barré syndrome (Campylobacter). The possibility that these diseases are induced by bacterial proteins or lipopolysaccharides has not been excluded. In addition to the previously described neuron-cross-reactive group B meningococcal polysialic acid another potential example is the OspA vaccine against B. burgdorferi. This vaccine, now withdrawn from the market, was claimed to be associated with the development of arthritis, a disease it was supposed to protect against by preventing the transmission of the Lyme disease spirochetes from ticks to man. Proteins with structures prevalent in nature, like coiled-coils, or their post-translational modifications may exhibit unexpected cross-reactions even with human proteins. Therefore, one has to keep in mind that the use of bacterial proteins as vaccines may carry the, hopefully small, risk of complications that the trials have to monitor for. References [1] Rappuoli R. Bridging the knowledge gaps in vaccine design. Nat Biotechnol 2007;25(12):1361–6. [2] He JQ, Wiesmann C, van Lookeren Campagne M. A role of macrophage complement receptor CRIg in immune clearance and inflammation. Mol Immunol 2008;45(16):4041–7. [3] Walport M. Complement: first of two parts. N Engl J Med 2001;344:1058–66. [4] Walport M. Complement: second of two parts. N Engl J Med 2001;344:1140–4. [5] Gordon DL, Kaufman RM, Blackmore TK, Kwong J, Lublin DM. Identification of complement regulatory domains in human factor H. J Immunol 1995;155:348–56. [6] Jokiranta TS, Hellwage J, Koistinen V, Zipfel PF, Meri S. Each of the three binding sites on complement factor H interacts with a distinct site on C3b. J Biol Chem 2000;275(36):27657–62.

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