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Review
Serum bactericidal antibody assays – The role of complement in infection and immunity E.D.G. McIntosh a,∗ , M. Bröker b , J. Wassil c , J.A. Welsch c , R. Borrow d a
Sir Alexander Fleming Building, Imperial College, London SW7 2AZ, United Kingdom Novartis Vaccines GmbH, Marburg, Germany GSK Vaccines, Inc., 350 Massachusetts Avenue, Cambridge, MA, USA d Vaccine Evaluation Unit, Public Health England, Manchester Royal Infirmary, Manchester, UK b c
a r t i c l e
i n f o
Article history: Received 28 November 2014 Received in revised form 21 June 2015 Accepted 7 July 2015 Available online xxx Keywords: Complement Serum bactericidal antibody Meningococcal infection Meningococcal vaccine
a b s t r a c t Complement is an essential component of the immune system and human pathogenic organisms have developed various mechanisms for evading complement mediated serum killing. The “gold standard” for measuring the ability of vaccine-induced antibody to kill Neisseria meningitidis is the serum bactericidal antibody (SBA) assay which measures complement mediated killing via antibody. This assay requires active complement, either intrinsic from the serum being tested or the addition of exogenous complement, either from a human or from another species such as rabbit. For serogroup C, an SBA titre of ≥4 was established as the correlate of protection when using human complement and ≥8 as the threshold when using rabbit complement, based on comparative assay results. Licensure of meningococcal vaccines, including polysaccharide protein conjugate vaccines and serogroup B vaccines has been based on the immune responses measured with the SBA assay, thus on a surrogate of vaccine efficacy. This review examines the use of complement and the SBA assay to assess immunity to meningococcal infection, and provides examples of vaccine trials in different age groups where various assays have been used. © 2015 Elsevier Ltd. All rights reserved.
1. Subversion of host defenses The normal immune system is in a constant state of alertness. Circulating antibodies and memory B cells signify a state of responsiveness, and herald the elimination in vivo of invading organisms by targeting them, and alerting circulating immune cells to home in on the pathogen. The defence against capsulated bacteria relies mainly on naïve B-cells in the marginal zone of the spleen. These cells produce polyreactive antibodies that play a crucial role in this defence. The antibodies often perform this function with the aid of complement, especially if the invading organisms are capsulated bacteria. In patients with late complement component deficiencies, both capsulated and non-capsulated organisms, especially N. meningitidis are reported to cause disease such as sepsis and
Abbreviations: anti-B PS IgM Ab, anti-serogroup B capsular polysaccharide IgM antibodies; BCA, bactericidal activity test; CFU, colony forming unit; ELISA, enzymelinked immunosorbent assay; fH, factor H; fHbp, factor H-binding protein; GMT, geometric mean titre; IMD, invasive meningococcal disease; MCC, meningococcal serogroup C conjugate; OPA, opsonophagocytic assay; SBA, serum bactericidal antibody. ∗ Corresponding author. Tel.: +44 7990530294. E-mail address: [email protected] (E.D.G. McIntosh).
meningitis at a higher frequency than in the general population [1]. Haemophilus influenzae has developed various mechanisms, mostly involving outer surface structures such as lipooligosaccharide glycans and outer surface proteins, for evading the host’s immune system [2]. Similarly, Staphylococcus aureus has the staphylococcal superantigen-like protein 7 (SSL7) that binds both immunoglobulin A and complement C5, in order to inhibit complement mediated haemolytic and bactericidal activity [3]. A surface-exposed neuraminidase is used by the oral spirochaete Treponema denticola both as a nutrient and as a decorating molecule, in order to disguise it from complement mediated serum killing [4]. Borrelia spp. uses CspA for binding of host-derived proteins and/or direct interaction with the formation of the terminal complement complex, as a mechanism for evasion [5] and [6]. These and other examples, including mechanisms used by N. meningitidis, are shown in Table 1 [2–16]. The meningococcus has several important adaptations that allow it to survive in the human host: the presence of the capsule and the ability to bind factor H (fH) which is an inhibitor of complement activation. Normally, factor H is one of the alternative pathway complement regulatory proteins that prevents excessive complement activation from injuring the host. Persons with genetic fH deficiencies have uncontrolled complement activation due to an
http://dx.doi.org/10.1016/j.vaccine.2015.07.019 0264-410X/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: McIntosh EDG, et al. Serum bactericidal antibody assays – The role of complement in infection and immunity. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.07.019
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Table 1 Mechanisms by which selected organisms evade complement mediated killing. Organism
Molecule(s)
Mechanism of evasion
Reference
Neisseria meningitidis
Polysaccharides and outer membrane proteins
Enhanced Opc expression associated with vitronectin binding and reduced membrane attack complex deposition; lipopolysaccharide immunotype switch; pile allelic exchange associated with enhanced autoaggregation Binds to factor H and enhances meningococcal resistance to complement The organism becomes surrounded by factor H and is no longer identified as foreign Binding of Msf preferentially to activated vibronectin (in addition to Opc binding to vibronectin) Decrease cell surface binding of the complement activator IgM Inhibition of complement Binds immunoglobulin A and complement C5 Resistance to complement killing and suppression of biofilm formation Surface exposed protein binds Factor H, Factor H-like protein-1, complement Factor H-related protein, and plasminogen Nutrient and as a decorating molecule Binding of host-derived proteins and/or direct interaction with the formation of the terminal complement complex Protection from envelope stress whilst cell wall damage is repaired Recruitment of regulator of classical and lectin pathway (C4b-binding protein), and regulator of alternate pathway (factor H)
[7]
Neisserial surface protein A (NspA) Factor H binding protein (fHbp)
Haemophilus influenzae
Staphylococcus aureus Acinetobacter baumannii
Novel adhesin: meningococcal surface fibril (Msf) Lipooligosaccharide glycans and outer surface proteins Vitronectin Staphylococcal superantigen-like protein 7 Secreted serine protease PKF
Pseudomonas aeruginosa
57-kDa dihydrolipoamide dehydrogenase (Lpd)
Treponema denticola Borrelia species
Surface-exposed neuraminidase Orthologs of CspA
Extraintestinal Escherichia coli (ExPEC)
Exopolysaccharide colanic acid
Yersinia pseudotuberculosis
Outer membrane protein Ail
inability to regulate the alternative pathway. Mutations in the gene are associated with diseases such as atypical hemolytic uraemic syndrome (aHUS) [17]. The meningococcus takes advantage of its ability to bind fH in order to shield itself from the action of the innate immune response. The meningococcus harbours at least three distinct proteins on its surface to exploit fH: factor H-binding protein (fHbp), Neisserial surface protein A (NspA) [8,10,18] and PorB [19,20]. With these proteins, the organism can bind human fH specifically – the organism becomes surrounded by fH which keeps complement C3b in check, thereby down-regulating complement activation by the human immune system. Consequently, the meningococcus is less susceptible to complement mediated antibody dependent lysis. The meningococcal capsule is also known to play a major role in the evasion of complement-mediated lysis and in addition to outer membrane proteins such as fHBP binding to fH, the capsule itself down-regulates complement activation and enhances resistance to bactericidal activity [21,22]. Expression levels of the surface proteins which bind fH therefore affect the susceptibility of the organism: more expression of the protein on the bacterial surface equates with increased resistance to killing and vice versa: a decrease in expression of the proteins equates with increased susceptibility of the organism [23,24]. 2. The serum bactericidal antibody (SBA) assay and correlates of protection The origins of the SBA to measure meningococcal killing was in the 1960s when, during an outbreak of invasive meningococcal serogroup C disease at an army base, Goldschneider and colleagues measured functional antibody concentrations in newly-enlisted military recruits [25]. Goldschneider and colleagues demonstrated that recruits with a defined amount of circulating functional meningococcal antibodies were at lowest risk of disease, whilst recruits with antibody levels below this concentration were at highest risk. The group employed the SBA (sometimes referred to in the literature as the bactericidal activity test or BCA) assay to measure the concentration of these functional antibodies was the SBA. For
[8] [9,10] [11] [2] [12] [3] [13] [14]
[4] [5,6] [15] [16]
the purposes of the present article, we take “SBA” to mean “serum bactericidal antibody”. The SBA assay measures the ability of the antibodies to lyse bacteria in the presence of complement. In their analysis of the recruits, serum from both healthy recruits and those who succumbed to disease was used. The minimum titre that correlated with protection was the dilution 1:4. Thus the protective titre measured using human complement was defined as ≥1:4 although it should be noted that in this analysis it was an individual correlate based on natural immunity, not vaccine induced antibodies. A subsequent study of vaccinated individuals, the Norwegian protection trial with 172,000 adolescent subjects, came to a similar conclusion: that an hSBA titre of 4 is a good estimate of both short- and long-term protection [26]. To substantiate the conclusion that a titre of ≥4 was the correlate of protection against invasive meningococcal disease, Goldschneider also performed a population-based study, measuring SBA in individuals from infancy through to adulthood. An inverse relationship exists between the presence of SBA titres at 1:4 and the incidence of invasive meningococcal disease [25]. Of note, SBA was measured for serogroups A, B and –C, with all three inversely correlating with the incidence of disease, although disease at the time in the USA was primarily due to serogroups B and C only. The serum bactericidal activity as measured by SBA assay has become the “gold-standard” correlate of both infection and successful immunisation [27]. The SBA assay measures the ability of circulating antibodies to lyse meningococci in the presence of complement, otherwise known as complement mediated killing via the classical pathway of the immune response. The bactericidal titre is defined as the dilution of the test serum that results in at least a 50% decrease in colony forming units (CFUs) per mL of bacteria [28]. As such, it is a functional assay in that it measures the killing of live bacteria. The conditions of the assay have a considerable impact on the results, so defined and validated conditions need to be used, developed by a panel of experts convened by the WHO [29]. As meningococcal vaccines were under development, the guidelines for the assay included the use of baby rabbit complement, due to the availability of this reagent and the difficulty sourcing human complement [29].
Please cite this article in press as: McIntosh EDG, et al. Serum bactericidal antibody assays – The role of complement in infection and immunity. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.07.019
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An SBA titre of ≥4 using human serum as the exogenous complement source (hSBA) is the established correlate of protection against invasive meningococcal disease [25,27]. Some experts have suggested that a level of ≥8 (hSBA) should be used as a more conservative threshold (as used in the development of one of the CRM-based monovalent serogroup C meningococcal conjugates [30]). On occasions titres between ≥4 and ≥8 have been used (as used in the development of the serogroup B vaccine 4CMenB) [31]. Furthermore, some have concluded that the sensitivity of the hSBA assay may be low, meaning that there is the possibility that individuals with titres 8 had declined from 38% (range 35–41%) to 15% (range 12–19%). There was a rapid waning of protective titres in early childhood, continuing into the second decade of life. These studies led directly to a decision to introduce an adolescent dose of MCC vaccine in the UK and elsewhere. In standardised assays using rabbit complement, immunogenicity values such as geometric mean titres (GMTs) and seroresponse are generally higher than those using human complement [28,31,77]. SBAs are prone to inter-laboratory differences due to the biological nature of the assay, for example, in variables such as bacterial growth [89]. However successful inter-laboratory standardisation can be performed if the assay operating procedure is strictly followed and the same source of bacteria and complement are used [90]. Therefore, SBA results should really only be directly compared if the serum is tested in the same laboratory at the same time using the same complement source and the same indicator strains. Investigators at the University of Massachusetts Medical School, Boston University School of Public Health, Novartis Vaccines and the Novartis Vaccines Institute for Global Health set out to answer three questions relating to the similarities and differences between SBAs performed using rabbit vs human complement [78]: (1) What is the strength of correlation between hSBA and rSBA results for each of the four vaccine serogroups (ACWY), and does it vary significantly between serogroup? (2) Does the strength of correlation vary as a function of whether the response is measured before (i.e. at baseline) or at time points after vaccination?
Please cite this article in press as: McIntosh EDG, et al. Serum bactericidal antibody assays – The role of complement in infection and immunity. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.07.019
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Table 2 Selected recent meningococcal vaccine studies where analysis was performed using either human or rabbit complement. Vaccine (Company)
Complement
Comment
Reference
Tetravalent meningococcal conjugate with tetanus toxoid carrier (GSK)
Rabbit
3.5 years after vaccination of 15–19 year-olds, all subjects still had SBA titres >1:8 indicating persistence 2–10 yr olds vaccinated: higher GMTs for serogroups A, W and Y at 3 years than in those vaccinated with plain polysaccharide Adolescents received 1 dose: one month post-vaccination >85.4–97.1% had titres >1:8 for each subgroup
[50]
MenC titres ≥1:8 three years after vaccination at 12–18 m in 64.2% and 53.2% in Hib-MenC and Hib plus MenC (separately) respectively MenC titres ≥1:8 sixty six months after priming with Hib-MenC-TT 82.6% and 94.1% for those primed with MenC-TT alone MenC titres ≥1:8 at 5 years of age in subjects primed in infancy: 59.3% (52.0–66.3%) vs 44.4% (31.7–58.5%) in controls
[53]
Infants vaccinated at 2, 4, 6 and 12–15 m: MenC titres ≥8 after 3 doses 98.8% and MenY titres 95.8%; after fourth dose titres 98.5% and 98.8% respectively Infants vaccinated at 2,3,6 m: hSBA titre ≥8 95.9% for MenC and 85.8% for MenY
[56]
5 years after the primary vaccination, 70% of subjects were seropositive for serogroups C, W135 and Y 11–18 years olds vaccinated: hSBA titres ≥1:8 for all serogroups non-inferior for both concomitant and sequential administration of Tdap and HPV Children 2–10 years of age vaccinated: hSBA titres ≥1:4 one month after one dose 82% (MenA), 83% (MenC), 95% (MenW), 91% (MenY) Adolescents 11–18 years of age received 1 dose of vaccine or 1 dose of tetravalent vaccine with diphtheria toxoid carrier (Sanofi). Percent with hSBA tits ≥1:8 for serogroups A, C, W, Y: 75/67, 84/84, 96/98, 88/69 respectively Infants receiving doses at 2,4,12 months: hSBA titre of ≥1:4 in at least 94% for serogroups C, W, Y, and 79% against serogroup A
[58]
Combined Hib-MenC conjugate with tetanus toxoid carrier (GSK)
Rabbit
Combined Hib-MenC/Y conjugate with tetanus toxoid carrier (GSK)
Human
Tetravalent meningococcal conjugate with CRM carrier (Novartis)
Human
[51] [52]
[54] [55]
[57]
[59] [60] [61]
[62]
Tetravalent meningococcal conjugate with diphtheria toxoid carrier (Sanofi)
Human
MenC titres ≥1:8 thirty days after two doses – first at 9 m then 3 m later, 85–100% for all serogroups
[63]
Tetravalent meningococcal conjugate with tetanus toxoid carrier (GSK)
Rabbit Human
2- to 10-year olds vaccinated: rSBA titres ≥8 for all serogroups 99.5–100% Adolescents and adults 11–25 years of age given one dose: hSBA titres ≥8 in 81.9–96.1% one month post-vaccination
[64] [65]
OMV vaccines (Norwegian Institute of Public Health NIPH and Finlay Institute Cuba)
Human
Infants, young children and adults received 3 doses: hSBA 4-fold or greater rise in 31–35% of young children, 37–60% of adults. 10% response in infants to heterologous strains but 90%+ response to homologous strains
[43,66]
4CMenB (Novartis)
Human
95–100% had hSBA titres ≥5 for all antigens after the booster dose; good immunogenicity in infants Adolescents 11–17 yrs of age: after 2 or 3 doses 99–100% had hSBA titre ≥4; at 6 months 91–100% still had titres ≥4. After 18–24 months 77–94% and 86–97% still had titres ≥4 after 2 and 3 doses respectively Vaccination at 2, 3, 4 or 2, 4, 6 m: hSBA titres ≥5 of 81.7% or 79–86.1% respectively against the NZ98/254 strain and 99% against strains 44/76-SL and 5/99 Adults 18–40 years of age: immunogenicity against multiple heterologous MenB strains Adult laboratory workers 18–50 years of age vaccinated at 0, 2, 6 m: hSBA titre ≥4 one-month post-vaccination 92–100%
[67]
Immunogenicity not performed in infants due to fever rates Post-dose 3 seroconversion rates against target strains of 68.8–95.3%; good immunogenicity in healthy children and adolescents Toddlers vaccinated at 0, 1 and 6 m: seroconversion ≥4-fold rise: 61.1–88.9% for homologous strains and 11.1–44.4% for heterologous strains Adults aged 18–25 years vaccinated at 0, 1, 6 months. Titres >1:4 in 47–90%, 75–100% and 88–100% at doses 10, 60 and 200 g respectively, against 6 serogroup B strains
[73] [74]
Bivalent fHbp rLP2086 (Pfizer)
Human
(3) Is the strength of correlation influenced by the kind of vaccine used (in this case purified polysaccharide vs proteinpolysaccharide conjugate)? Serum samples previously obtained from study subjects vaccinated with either a meningococcal serogroup ACWY-CRM conjugate vaccine or a plain polysaccharide ACWY vaccine, were tested in parallel using rSBA (Vaccine Evaluation Unit, PHE) or hSBA (Novartis Vaccines serology laboratory at Marburg). For serogroups A, W and Y, paired hSBA and rSBA results generated from the same samples correlated poorly: rSBA did not reliably predict hSBA status or vice versa, regardless of whether the conjugate or the plain polysaccharide vaccine had been administered, and regardless of the timing relative to vaccination. Interestingly, samples
[68,69]
[70] [71] [72]
[75] [76]
with titres
ARTICLE IN PRESS
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Contents lists available at ScienceDirect
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Review
Serum bactericidal antibody assays – The role of complement in infection and immunity E.D.G. McIntosh a,∗ , M. Bröker b , J. Wassil c , J.A. Welsch c , R. Borrow d a
Sir Alexander Fleming Building, Imperial College, London SW7 2AZ, United Kingdom Novartis Vaccines GmbH, Marburg, Germany GSK Vaccines, Inc., 350 Massachusetts Avenue, Cambridge, MA, USA d Vaccine Evaluation Unit, Public Health England, Manchester Royal Infirmary, Manchester, UK b c
a r t i c l e
i n f o
Article history: Received 28 November 2014 Received in revised form 21 June 2015 Accepted 7 July 2015 Available online xxx Keywords: Complement Serum bactericidal antibody Meningococcal infection Meningococcal vaccine
a b s t r a c t Complement is an essential component of the immune system and human pathogenic organisms have developed various mechanisms for evading complement mediated serum killing. The “gold standard” for measuring the ability of vaccine-induced antibody to kill Neisseria meningitidis is the serum bactericidal antibody (SBA) assay which measures complement mediated killing via antibody. This assay requires active complement, either intrinsic from the serum being tested or the addition of exogenous complement, either from a human or from another species such as rabbit. For serogroup C, an SBA titre of ≥4 was established as the correlate of protection when using human complement and ≥8 as the threshold when using rabbit complement, based on comparative assay results. Licensure of meningococcal vaccines, including polysaccharide protein conjugate vaccines and serogroup B vaccines has been based on the immune responses measured with the SBA assay, thus on a surrogate of vaccine efficacy. This review examines the use of complement and the SBA assay to assess immunity to meningococcal infection, and provides examples of vaccine trials in different age groups where various assays have been used. © 2015 Elsevier Ltd. All rights reserved.
1. Subversion of host defenses The normal immune system is in a constant state of alertness. Circulating antibodies and memory B cells signify a state of responsiveness, and herald the elimination in vivo of invading organisms by targeting them, and alerting circulating immune cells to home in on the pathogen. The defence against capsulated bacteria relies mainly on naïve B-cells in the marginal zone of the spleen. These cells produce polyreactive antibodies that play a crucial role in this defence. The antibodies often perform this function with the aid of complement, especially if the invading organisms are capsulated bacteria. In patients with late complement component deficiencies, both capsulated and non-capsulated organisms, especially N. meningitidis are reported to cause disease such as sepsis and
Abbreviations: anti-B PS IgM Ab, anti-serogroup B capsular polysaccharide IgM antibodies; BCA, bactericidal activity test; CFU, colony forming unit; ELISA, enzymelinked immunosorbent assay; fH, factor H; fHbp, factor H-binding protein; GMT, geometric mean titre; IMD, invasive meningococcal disease; MCC, meningococcal serogroup C conjugate; OPA, opsonophagocytic assay; SBA, serum bactericidal antibody. ∗ Corresponding author. Tel.: +44 7990530294. E-mail address: [email protected] (E.D.G. McIntosh).
meningitis at a higher frequency than in the general population [1]. Haemophilus influenzae has developed various mechanisms, mostly involving outer surface structures such as lipooligosaccharide glycans and outer surface proteins, for evading the host’s immune system [2]. Similarly, Staphylococcus aureus has the staphylococcal superantigen-like protein 7 (SSL7) that binds both immunoglobulin A and complement C5, in order to inhibit complement mediated haemolytic and bactericidal activity [3]. A surface-exposed neuraminidase is used by the oral spirochaete Treponema denticola both as a nutrient and as a decorating molecule, in order to disguise it from complement mediated serum killing [4]. Borrelia spp. uses CspA for binding of host-derived proteins and/or direct interaction with the formation of the terminal complement complex, as a mechanism for evasion [5] and [6]. These and other examples, including mechanisms used by N. meningitidis, are shown in Table 1 [2–16]. The meningococcus has several important adaptations that allow it to survive in the human host: the presence of the capsule and the ability to bind factor H (fH) which is an inhibitor of complement activation. Normally, factor H is one of the alternative pathway complement regulatory proteins that prevents excessive complement activation from injuring the host. Persons with genetic fH deficiencies have uncontrolled complement activation due to an
http://dx.doi.org/10.1016/j.vaccine.2015.07.019 0264-410X/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: McIntosh EDG, et al. Serum bactericidal antibody assays – The role of complement in infection and immunity. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.07.019
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Table 1 Mechanisms by which selected organisms evade complement mediated killing. Organism
Molecule(s)
Mechanism of evasion
Reference
Neisseria meningitidis
Polysaccharides and outer membrane proteins
Enhanced Opc expression associated with vitronectin binding and reduced membrane attack complex deposition; lipopolysaccharide immunotype switch; pile allelic exchange associated with enhanced autoaggregation Binds to factor H and enhances meningococcal resistance to complement The organism becomes surrounded by factor H and is no longer identified as foreign Binding of Msf preferentially to activated vibronectin (in addition to Opc binding to vibronectin) Decrease cell surface binding of the complement activator IgM Inhibition of complement Binds immunoglobulin A and complement C5 Resistance to complement killing and suppression of biofilm formation Surface exposed protein binds Factor H, Factor H-like protein-1, complement Factor H-related protein, and plasminogen Nutrient and as a decorating molecule Binding of host-derived proteins and/or direct interaction with the formation of the terminal complement complex Protection from envelope stress whilst cell wall damage is repaired Recruitment of regulator of classical and lectin pathway (C4b-binding protein), and regulator of alternate pathway (factor H)
[7]
Neisserial surface protein A (NspA) Factor H binding protein (fHbp)
Haemophilus influenzae
Staphylococcus aureus Acinetobacter baumannii
Novel adhesin: meningococcal surface fibril (Msf) Lipooligosaccharide glycans and outer surface proteins Vitronectin Staphylococcal superantigen-like protein 7 Secreted serine protease PKF
Pseudomonas aeruginosa
57-kDa dihydrolipoamide dehydrogenase (Lpd)
Treponema denticola Borrelia species
Surface-exposed neuraminidase Orthologs of CspA
Extraintestinal Escherichia coli (ExPEC)
Exopolysaccharide colanic acid
Yersinia pseudotuberculosis
Outer membrane protein Ail
inability to regulate the alternative pathway. Mutations in the gene are associated with diseases such as atypical hemolytic uraemic syndrome (aHUS) [17]. The meningococcus takes advantage of its ability to bind fH in order to shield itself from the action of the innate immune response. The meningococcus harbours at least three distinct proteins on its surface to exploit fH: factor H-binding protein (fHbp), Neisserial surface protein A (NspA) [8,10,18] and PorB [19,20]. With these proteins, the organism can bind human fH specifically – the organism becomes surrounded by fH which keeps complement C3b in check, thereby down-regulating complement activation by the human immune system. Consequently, the meningococcus is less susceptible to complement mediated antibody dependent lysis. The meningococcal capsule is also known to play a major role in the evasion of complement-mediated lysis and in addition to outer membrane proteins such as fHBP binding to fH, the capsule itself down-regulates complement activation and enhances resistance to bactericidal activity [21,22]. Expression levels of the surface proteins which bind fH therefore affect the susceptibility of the organism: more expression of the protein on the bacterial surface equates with increased resistance to killing and vice versa: a decrease in expression of the proteins equates with increased susceptibility of the organism [23,24]. 2. The serum bactericidal antibody (SBA) assay and correlates of protection The origins of the SBA to measure meningococcal killing was in the 1960s when, during an outbreak of invasive meningococcal serogroup C disease at an army base, Goldschneider and colleagues measured functional antibody concentrations in newly-enlisted military recruits [25]. Goldschneider and colleagues demonstrated that recruits with a defined amount of circulating functional meningococcal antibodies were at lowest risk of disease, whilst recruits with antibody levels below this concentration were at highest risk. The group employed the SBA (sometimes referred to in the literature as the bactericidal activity test or BCA) assay to measure the concentration of these functional antibodies was the SBA. For
[8] [9,10] [11] [2] [12] [3] [13] [14]
[4] [5,6] [15] [16]
the purposes of the present article, we take “SBA” to mean “serum bactericidal antibody”. The SBA assay measures the ability of the antibodies to lyse bacteria in the presence of complement. In their analysis of the recruits, serum from both healthy recruits and those who succumbed to disease was used. The minimum titre that correlated with protection was the dilution 1:4. Thus the protective titre measured using human complement was defined as ≥1:4 although it should be noted that in this analysis it was an individual correlate based on natural immunity, not vaccine induced antibodies. A subsequent study of vaccinated individuals, the Norwegian protection trial with 172,000 adolescent subjects, came to a similar conclusion: that an hSBA titre of 4 is a good estimate of both short- and long-term protection [26]. To substantiate the conclusion that a titre of ≥4 was the correlate of protection against invasive meningococcal disease, Goldschneider also performed a population-based study, measuring SBA in individuals from infancy through to adulthood. An inverse relationship exists between the presence of SBA titres at 1:4 and the incidence of invasive meningococcal disease [25]. Of note, SBA was measured for serogroups A, B and –C, with all three inversely correlating with the incidence of disease, although disease at the time in the USA was primarily due to serogroups B and C only. The serum bactericidal activity as measured by SBA assay has become the “gold-standard” correlate of both infection and successful immunisation [27]. The SBA assay measures the ability of circulating antibodies to lyse meningococci in the presence of complement, otherwise known as complement mediated killing via the classical pathway of the immune response. The bactericidal titre is defined as the dilution of the test serum that results in at least a 50% decrease in colony forming units (CFUs) per mL of bacteria [28]. As such, it is a functional assay in that it measures the killing of live bacteria. The conditions of the assay have a considerable impact on the results, so defined and validated conditions need to be used, developed by a panel of experts convened by the WHO [29]. As meningococcal vaccines were under development, the guidelines for the assay included the use of baby rabbit complement, due to the availability of this reagent and the difficulty sourcing human complement [29].
Please cite this article in press as: McIntosh EDG, et al. Serum bactericidal antibody assays – The role of complement in infection and immunity. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.07.019
G Model JVAC-16672; No. of Pages 8
ARTICLE IN PRESS E.D.G. McIntosh et al. / Vaccine xxx (2015) xxx–xxx
An SBA titre of ≥4 using human serum as the exogenous complement source (hSBA) is the established correlate of protection against invasive meningococcal disease [25,27]. Some experts have suggested that a level of ≥8 (hSBA) should be used as a more conservative threshold (as used in the development of one of the CRM-based monovalent serogroup C meningococcal conjugates [30]). On occasions titres between ≥4 and ≥8 have been used (as used in the development of the serogroup B vaccine 4CMenB) [31]. Furthermore, some have concluded that the sensitivity of the hSBA assay may be low, meaning that there is the possibility that individuals with titres 8 had declined from 38% (range 35–41%) to 15% (range 12–19%). There was a rapid waning of protective titres in early childhood, continuing into the second decade of life. These studies led directly to a decision to introduce an adolescent dose of MCC vaccine in the UK and elsewhere. In standardised assays using rabbit complement, immunogenicity values such as geometric mean titres (GMTs) and seroresponse are generally higher than those using human complement [28,31,77]. SBAs are prone to inter-laboratory differences due to the biological nature of the assay, for example, in variables such as bacterial growth [89]. However successful inter-laboratory standardisation can be performed if the assay operating procedure is strictly followed and the same source of bacteria and complement are used [90]. Therefore, SBA results should really only be directly compared if the serum is tested in the same laboratory at the same time using the same complement source and the same indicator strains. Investigators at the University of Massachusetts Medical School, Boston University School of Public Health, Novartis Vaccines and the Novartis Vaccines Institute for Global Health set out to answer three questions relating to the similarities and differences between SBAs performed using rabbit vs human complement [78]: (1) What is the strength of correlation between hSBA and rSBA results for each of the four vaccine serogroups (ACWY), and does it vary significantly between serogroup? (2) Does the strength of correlation vary as a function of whether the response is measured before (i.e. at baseline) or at time points after vaccination?
Please cite this article in press as: McIntosh EDG, et al. Serum bactericidal antibody assays – The role of complement in infection and immunity. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.07.019
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Table 2 Selected recent meningococcal vaccine studies where analysis was performed using either human or rabbit complement. Vaccine (Company)
Complement
Comment
Reference
Tetravalent meningococcal conjugate with tetanus toxoid carrier (GSK)
Rabbit
3.5 years after vaccination of 15–19 year-olds, all subjects still had SBA titres >1:8 indicating persistence 2–10 yr olds vaccinated: higher GMTs for serogroups A, W and Y at 3 years than in those vaccinated with plain polysaccharide Adolescents received 1 dose: one month post-vaccination >85.4–97.1% had titres >1:8 for each subgroup
[50]
MenC titres ≥1:8 three years after vaccination at 12–18 m in 64.2% and 53.2% in Hib-MenC and Hib plus MenC (separately) respectively MenC titres ≥1:8 sixty six months after priming with Hib-MenC-TT 82.6% and 94.1% for those primed with MenC-TT alone MenC titres ≥1:8 at 5 years of age in subjects primed in infancy: 59.3% (52.0–66.3%) vs 44.4% (31.7–58.5%) in controls
[53]
Infants vaccinated at 2, 4, 6 and 12–15 m: MenC titres ≥8 after 3 doses 98.8% and MenY titres 95.8%; after fourth dose titres 98.5% and 98.8% respectively Infants vaccinated at 2,3,6 m: hSBA titre ≥8 95.9% for MenC and 85.8% for MenY
[56]
5 years after the primary vaccination, 70% of subjects were seropositive for serogroups C, W135 and Y 11–18 years olds vaccinated: hSBA titres ≥1:8 for all serogroups non-inferior for both concomitant and sequential administration of Tdap and HPV Children 2–10 years of age vaccinated: hSBA titres ≥1:4 one month after one dose 82% (MenA), 83% (MenC), 95% (MenW), 91% (MenY) Adolescents 11–18 years of age received 1 dose of vaccine or 1 dose of tetravalent vaccine with diphtheria toxoid carrier (Sanofi). Percent with hSBA tits ≥1:8 for serogroups A, C, W, Y: 75/67, 84/84, 96/98, 88/69 respectively Infants receiving doses at 2,4,12 months: hSBA titre of ≥1:4 in at least 94% for serogroups C, W, Y, and 79% against serogroup A
[58]
Combined Hib-MenC conjugate with tetanus toxoid carrier (GSK)
Rabbit
Combined Hib-MenC/Y conjugate with tetanus toxoid carrier (GSK)
Human
Tetravalent meningococcal conjugate with CRM carrier (Novartis)
Human
[51] [52]
[54] [55]
[57]
[59] [60] [61]
[62]
Tetravalent meningococcal conjugate with diphtheria toxoid carrier (Sanofi)
Human
MenC titres ≥1:8 thirty days after two doses – first at 9 m then 3 m later, 85–100% for all serogroups
[63]
Tetravalent meningococcal conjugate with tetanus toxoid carrier (GSK)
Rabbit Human
2- to 10-year olds vaccinated: rSBA titres ≥8 for all serogroups 99.5–100% Adolescents and adults 11–25 years of age given one dose: hSBA titres ≥8 in 81.9–96.1% one month post-vaccination
[64] [65]
OMV vaccines (Norwegian Institute of Public Health NIPH and Finlay Institute Cuba)
Human
Infants, young children and adults received 3 doses: hSBA 4-fold or greater rise in 31–35% of young children, 37–60% of adults. 10% response in infants to heterologous strains but 90%+ response to homologous strains
[43,66]
4CMenB (Novartis)
Human
95–100% had hSBA titres ≥5 for all antigens after the booster dose; good immunogenicity in infants Adolescents 11–17 yrs of age: after 2 or 3 doses 99–100% had hSBA titre ≥4; at 6 months 91–100% still had titres ≥4. After 18–24 months 77–94% and 86–97% still had titres ≥4 after 2 and 3 doses respectively Vaccination at 2, 3, 4 or 2, 4, 6 m: hSBA titres ≥5 of 81.7% or 79–86.1% respectively against the NZ98/254 strain and 99% against strains 44/76-SL and 5/99 Adults 18–40 years of age: immunogenicity against multiple heterologous MenB strains Adult laboratory workers 18–50 years of age vaccinated at 0, 2, 6 m: hSBA titre ≥4 one-month post-vaccination 92–100%
[67]
Immunogenicity not performed in infants due to fever rates Post-dose 3 seroconversion rates against target strains of 68.8–95.3%; good immunogenicity in healthy children and adolescents Toddlers vaccinated at 0, 1 and 6 m: seroconversion ≥4-fold rise: 61.1–88.9% for homologous strains and 11.1–44.4% for heterologous strains Adults aged 18–25 years vaccinated at 0, 1, 6 months. Titres >1:4 in 47–90%, 75–100% and 88–100% at doses 10, 60 and 200 g respectively, against 6 serogroup B strains
[73] [74]
Bivalent fHbp rLP2086 (Pfizer)
Human
(3) Is the strength of correlation influenced by the kind of vaccine used (in this case purified polysaccharide vs proteinpolysaccharide conjugate)? Serum samples previously obtained from study subjects vaccinated with either a meningococcal serogroup ACWY-CRM conjugate vaccine or a plain polysaccharide ACWY vaccine, were tested in parallel using rSBA (Vaccine Evaluation Unit, PHE) or hSBA (Novartis Vaccines serology laboratory at Marburg). For serogroups A, W and Y, paired hSBA and rSBA results generated from the same samples correlated poorly: rSBA did not reliably predict hSBA status or vice versa, regardless of whether the conjugate or the plain polysaccharide vaccine had been administered, and regardless of the timing relative to vaccination. Interestingly, samples
[68,69]
[70] [71] [72]
[75] [76]
with titres
