Development of an Opsonophagocytic Killing Assay Using HL-60 Cells for Detection of Functional Antibodies against Streptococcus pyogenes.

The clinical development of group A streptococcal (GAS) vaccines will require the implementation of a standardized, high-throughput assay to measure the activity of functional opsonic antibodies in vaccine recipients. In the present study, we adapted and modified the HL-60-based protocol that was developed for the detection of opsonic antibodies against Streptococcus pneumoniae for use with multiple M types of GAS. Modifications of the assay conditions permitted the evaluation of 21 different M types of GAS in the assay. The specificity of the antibody-mediated opsonization was demonstrated by inhibition with homologous, but not heterologous, M proteins. Maximum rates of opsonophagocytic killing (OPK) of 14 different M types promoted by rabbit antiserum against the 30-valent M protein-based vaccine were comparable in whole-blood and HL-60 assays. Data are also presented showing OPK serum titers (opsonic index) of naturally acquired human antibodies present in IVIG [intravenous immune globulin (human)]. Results of the HL-60 assay performed on different days using 21 different M types of GAS and IVIG as the antibody source were significantly concordant. This report indicates that the OPK assay conditions may be optimized for the measurement of opsonic antibodies against a number of epidemiologically important M types of GAS and, once standardized, should facilitate the clinical development of effective vaccines to prevent these infections.IMPORTANCE Measuring functional opsonic antibodies against group A streptococci is an important component of the clinical development path for effective vaccines. Prior studies have used an assay developed over 60 years ago that relied on whole human blood as the source of phagocytes and complement, both of which are critical components of antibody-mediated killing assays. In this study, we adapted an assay that uses the HL-60 human promyelocytic leukemia cell line as phagocytic cells and baby rabbit serum as a source of complement for detection of opsonic antibodies against group A streptococci. On the basis of some of the known biological characteristics of the bacteria, we modified the assay conditions to support the evaluation of 21 epidemiologically important M types and demonstrated the utility and reproducibility of the assay for measurement of functional opsonic antibody levels.

Trivalent M-related protein as a component of next generation group A streptococcal vaccines.

PURPOSE: There is a need to broaden protective coverage of M protein-based vaccines against group A streptococci (GAS) because coverage of the current 30-valent M protein vaccine does not extend to all emm types. An additional GAS antigen and virulence factor that could potentially extend vaccine coverage is M-related protein (Mrp). Previous work indicated that there are three structurally related families of Mrp (MrpI, MrpII, and MrpIII) and peptides of all three elicited bactericidal antibodies against multiple emm types. The purpose of this study was to determine if a recombinant form containing Mrp from the three families would evoke bactericidal antiserum and to determine if this antiserum could enhance the effectiveness of antisera to the 30-valent M protein vaccine. MATERIALS AND METHODS: A trivalent recombinant Mrp (trMrp) protein containing N-terminal fragments from the three families (trMrp) was constructed, purified and used to immunize rabbits. Anti-trMrp sera contained high titers of antibodies against the trMrp immunogen and recombinant forms representing MrpI, MrpII, and MrpIII. RESULTS: The antisera opsonized emm types of GAS representing each Mrp family and also opsonized emm types not covered by the 30-valent M protein-based vaccine. Importantly, a combination of trMrp and 30-valent M protein antiserum resulted in higher levels of opsonization of GAS than either antiserum alone. CONCLUSION: These findings suggest that trMrp may be an effective addition to future constructs of GAS vaccines.

Structure-based design of broadly protective group a streptococcal M protein-based vaccines.

BACKGROUND: A major obstacle to the development of broadly protective M protein-based group A streptococcal (GAS) vaccines is the variability within the N-terminal epitopes that evoke potent bactericidal antibodies. The concept of M type-specific protective immune responses has recently been challenged based on the observation that multivalent M protein vaccines elicited cross-reactive bactericidal antibodies against a number of non-vaccine M types of GAS. Additionally, a new "cluster-based" typing system of 175M proteins identified a limited number of clusters containing closely related M proteins. In the current study, we used the emm cluster typing system, in combination with computational structure-based peptide modeling, as a novel approach to the design of potentially broadly protective M protein-based vaccines. METHODS: M protein sequences (AA 16-50) from the E4 cluster containing 17 emm types of GAS were analyzed using de novo 3-D structure prediction tools and the resulting structures subjected to chemical diversity analysis to identify sequences that were the most representative of the 3-D physicochemical properties of the M peptides in the cluster. Five peptides that spanned the range of physicochemical attributes of all 17 peptides were used to formulate synthetic and recombinant vaccines. Rabbit antisera were assayed for antibodies that cross-reacted with E4 peptides and whole bacteria by ELISA and for bactericidal activity against all E4GAS. RESULTS: The synthetic vaccine rabbit antisera reacted with all 17 E4M peptides and demonstrated bactericidal activity against 15/17 E4GAS. A recombinant hybrid vaccine containing the same E4 peptides also elicited antibodies that cross-reacted with all E4M peptides. CONCLUSIONS: Comprehensive studies using structure-based design may result in a broadly protective M peptide vaccine that will elicit cluster-specific and emm type-specific antibody responses against the majority of clinically relevant emm types of GAS.

Protective immunogenicity of group A streptococcal M-related proteins.

Many previous studies have focused on the surface M proteins of group A streptococci (GAS) as virulence determinants and protective antigens. However, the majority of GAS isolates express M-related protein (Mrp) in addition to M protein, and both have been shown to be required for optimal virulence. In the current study, we evaluated the protective immunogenicity of Mrp to determine its potential as a vaccine component that may broaden the coverage of M protein-based vaccines. Sequence analyses of 33 mrp genes indicated that there are three families of structurally related Mrps (MrpI, MrpII, and MrpIII). N-terminal peptides of Mrps were cloned, expressed, and purified from M type 2 (M2) (MrpI), M4 (MrpII), and M49 (MrpIII) GAS. Rabbit antisera against the Mrps reacted at high titers with the homologous Mrp, as determined by enzyme-linked immunosorbent assay, and promoted bactericidal activity against GAS emm types expressing Mrps within the same family. Mice passively immunized with rabbit antisera against MrpII were protected against challenge infections with M28 GAS. Assays for Mrp antibodies in serum samples from 281 pediatric subjects aged 2 to 16 indicated that the Mrp immune response correlated with increasing age of the subjects. Affinity-purified human Mrp antibodies promoted bactericidal activity against a number of GAS representing different emm types that expressed an Mrp within the same family but showed no activity against emm types expressing an Mrp from a different family. Our results indicate that Mrps have semiconserved N-terminal sequences that contain bactericidal epitopes which are immunogenic in humans. These findings may have direct implications for the development of GAS vaccines.