Instituto Valenciano de Microbiología

Masía El Romeral
Ctra. de Bétera a San Antonio Km. 0.3
46117 Bétera (Valencia)
Phone. 96 169 17 02
Fax 96 169 16 37
CIF B-96337217


Haemophilus influenzae - Serum bactericidal antibodies (SBA) assay  

Haemophilus influenzae type b (Hib) is a human pathogenic gram-negative bacterium that mainly affects children under 5 years. It can cause severe invasive infections such as meningitis, sepsis and pneumonia. In addition, it can cause otitis media, epiglottitis and osteoarticular infections, among others.

According to the chemical structure of the capsular polysaccharide, H. influenzae has been divided into six serotypes (a, b, c, d, e and f). Furthermore, there are non-capsulated strains lacking cap gene and are referred to as non-typeable because they are not typable for any of the six serotypes mentioned. Of the 6 existing serotypes, type b is the most virulent globally, and in the early 1980s, before the vaccine was introduced against this serotype, was the leading cause of meningitis in children. The second most important serotype is serotype a. This serotype is rare in most of the world, but is prevalent in specific geographical regions such as northern and western Canada, Alaska and southern US, affecting almost exclusively to indigenous peoples.

The expression of the capsular polysaccharide, a virulence factor, protects the bacteria of defense mechanisms, such as complement-dependent bacteriolysis.

Haemophilus influenzae type b conjugated vaccines induce the production of antibodies to the capsular polysaccharide, providing protection against invasive disease caused by this serotype. Since the introduction of the Haemophilus influenzae type b conjugated vaccine there was a decrease of serious infections in which it was involved. In U.S.A. the vaccine was introduced in 1988 and more than a 99% reduction in the incidence of invasive disease in children under 5 years was achieved.

Vaccination against H. influenzae type b does not offer protection against other serotypes of H. influenzae. In the post-vaccination against H. influenzae type b era, strains of other serotypes have become important in the etiology of invasive disease by this bacterium. 

Protecting disease H. influenzae type b, it is correlated with the presence of antibodies to capsular polysaccharide antigen of this serotype corresponding to molecules polyribosylribitol phosphate (PRP). Some studies have shown that the use of bactericidal antibodies from different strains of H. influenzae type b, are nearly equal or it differs by one or two dilutions.

Nontypeable H. influenzae strains (NTHI) unlike other serotypes lack the polysaccharide capsule. These strains are commensal of human nasopharyngeal mucosa and can occasionally act as a pathogen initiating infection of the upper or lower airways, causing otitis media, chronic obstructive pulmonary disease, and invasive infection e. gr. meningitis and sepsis, particularly in infants and elderly people. A vaccine is needed for these strains, but the main problem is the high genetic diversity among them, so it needs to identify antigens having functional epitopes capable of inducing cross-reactive antibodies to the different strains. It has sought an outer membrane protein and lipooligosaccharides. Moreover, a protective antigen must express epitopes available for binding of antibodies on the surface of the intact bacterium. Immersed molecules at the outer membrane of the cell wall or blocked by steric joints of adjacent structures such as the sialic acid are not available for antibody binding and therefore unable to generate protective antibodies.

Both protein and polysaccharide vaccines have been evaluated using the test of complement-mediated death in the SBA assay (Serum bactericidal Antibody Assay). This test was developed in the early 1960s with the objective to find the presence of functional specific antibodies in Neisseria meningitidis infections. Specific functional antibodies are those which in addition to binding the bacteria activate a biological defense system such as phagocytosis (opsonophagocytosis), or activation of the complement system. When the specific antibodies bind to the bacteria changes in the structure of the antibody (Fc region) promotes the activation of the complement system starting fixing C1q subunit and subsequently the activation of the whole cascade of subunits and factors occurs (C1q, r, s, C4, C2, C3, C5, C6, C7, C8 and C9). Once have been activated all factors sequentially, the bacterial die by lysis. In various tests to determine the bactericidal activity mediated by such antibodies, with the participation of the complement system, proposed by various authors, all share three elements: bacteria, antibodies and complement. Methods of Serum bactericidal antibody (SBA) differ in the number of bacteria (CFU) used; in the buffer used during the test; growth of the strain used; the incubation time of the test; the source of complement, human or rabbit; and starting dilution of the serum. This traditional method (SBAm: Serum bactericidal microassay colony counting Assay) is considered very laborious and inappropriate when numerous samples should be studied, which involves seeding and bacterial counts. For these reasons, there have been proposed alternative methods using a system to facilitate the reading of the results, such as: triphenyltetrazolium use (TTCmSBA) to display the results by the change of color of this element rather than counting bacteria; or using a colorimetric method (CSBA) based on the ability to degrade a substrate, in the presence of a pH indicator to estimate the growth of the surviving bacteria.

There are children with high antibody titers by ELISA and serum bactericidal activity low, and viceversa (Townsend et al Evaluation and validation of a serum bactericidal antibody assay for Haemophilus influenzae type b and the threshold of protection Vaccine, 2014, 32: 5650-5656). This is because the ELISA does not distinguish between functional and non-functional antibodies. For this reason in order to determine the presence of functional antibodies it is required to perform the SBA (Serum bactericidal Antibody Assay).

Several studies defined a cutoff of protection an antibody titer ≥1:4. Subsequently, Borrow et al. (Serological basis for serogroup C meningococcal use of conjugate vaccines in the United Kingdom: reevaluation of correlates of protection Infect Immun 2001, 69: 1568-1573), established the correlation of test results using human serum or rabbit serum as complement source, and proposed for the case of using rabbit serum a cut protective antibody titer of ≥1: 8. Other authors, a more conservative approach using human serum as a complement source, the cutoff proposed protection titer ≥1: 8 . (Donelly, J. et al, Qualitative and quantitative assessment of the meningococcal antigens to evaluate-potential strain average of protein based vaccines PNAS, 2010, 107: 19490-19495).

In the method of serum bactericidal antibody (SBA) assay against Haemophilus influenzae type b, the titer of functional antibody is determined in the presence of human or rabbit complement, using serum dilutions that are faced to a suspension of capsulated Haemophilus influenzae, and after an incubation period, they are added complement, turning to incubate and detecting the death of the bacteria by seeding and subsequent counting of the surviving bacteria.

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