Establishment of a modified opsonophagocytic killing assay for anti-pneumococcal surface protein A antibody.

Streptococcus pneumoniae (pneumococcus) is a pathogenic gram-positive bacterium that causes pneumonia, meningitis, and sepsis. Pneumococcal surface protein A (PspA) induces antibodies that protect against lethal infections by pneumococci. PspA is a choline-binding protein present on the cell surface of almost all pneumococcal strains and is a non-capsular polysaccharide vaccine candidate. For research and development of PspA-based vaccines, an in-vitro test system to measure the activity of functional antibodies capable of killing pneumococci is essential. The opsonophagocytic killing (OPK) assay is used to evaluate the opsonic activity of functional antibodies induced by capsular polysaccharide (CPS)-based vaccines (standard OPK assay). Despite the potential of anti-PspA antibodies to protect against lethal infections in mice, the standard OPK assay fails to evaluate anti-PspA antibodies. Using a pneumococcal surface protein C-deficient strain and extending the incubation time of opsonized bacteria, complement, and HL-60 cells reportedly results in enhanced bactericidal activity (modified OPK assay). We aimed to measure the bactericidal activity of anti-PspA antibodies in intact pneumococcal strains. We optimized the pneumococcal culture method used in the OPK assay to increase the efficiency of anti-PspA antibody-mediated phagocytosis of HL-60 cells. As thick capsules hinder phagocytosis, we attempted to obtain pneumococci with thin capsules through an improved culture method. As pneumococci attached to cells exhibit thin capsules, pneumococci cultured in Todd Hewitt yeast extract (THY) broth were spread on blood agar plates and incubated for 4 h. cpsA mRNA transcript levels in pneumococci cultured on blood agar were lower than those in pneumococci cultured in THY broth. OPK activity against pneumococci expressing PspA of clades 1-5 was reasonably well detected using pneumococci cultured on blood agar in the modified OPK assay. The modified OPK assay for anti-PspA antibody using pneumococci cultured on blood agar represents a useful assay to determine the killing activity of functional anti-PspA antibodies against pneumococci.

Functional assays to evaluate antibody-mediated responses against Shigella: a review.

Shigella is a major global pathogen and the etiological agent of shigellosis, a diarrheal disease that primarily affects low- and middle-income countries. Shigellosis is characterized by a complex, multistep pathogenesis during which bacteria use multiple invasion proteins to manipulate and invade the intestinal epithelium. Antibodies, especially against the O-antigen and some invasion proteins, play a protective role as titres against specific antigens inversely correlate with disease severity; however, the context of antibody action during pathogenesis remains to be elucidated, especially with Shigella being mostly an intracellular pathogen. In the absence of a correlate of protection, functional assays rebuilding salient moments of Shigella pathogenesis can improve our understanding of the role of protective antibodies in blocking infection and disease. In vitro assays are important tools to build correlates of protection. Only recently animal models to recapitulate human pathogenesis, often not in full, have been established. This review aims to discuss in vitro assays to evaluate the functionality of anti-Shigella antibodies in polyclonal sera in light of the multistep and multifaced Shigella infection process. Indeed, measurement of antibody level alone may limit the evaluation of full vaccine potential. Serum bactericidal assay (SBA), and other functional assays such as opsonophagocytic killing assays (OPKA), and adhesion/invasion inhibition assays (AIA), are instead physiologically relevant and may provide important information regarding the role played by these effector mechanisms in protective immunity. Ultimately, the review aims at providing scientists in the field with new points of view regarding the significance of functional assays of choice which may be more representative of immune-mediated protection mechanisms.

The Development of Immunological Assays to Evaluate the Level and Function of Antibodies Induced by Klebsiella pneumoniae O-Antigen Vaccines.

Klebsiella pneumoniae, a Gram-negative bacterium, has been listed as a critical pathogen for urgent intervention by the World Health Organization. With no licensed vaccine and increasing resistance to antibiotics, Klebsiella pneumoniae causes a high incidence of hospital- and community-acquired infections. Recently, there has been progress in anti-Klebsiella pneumoniae vaccine development, which has highlighted the lack of standardized assays to measure vaccine immunogenicity. We have developed and optimized methods to measure antibody level and function after vaccination with an in-development Klebsiella pneumoniae O-antigen vaccine. We describe the qualification of a Luminex-based multiplex antibody binding assay and both an opsonophagocytic killing assay and serum bactericidal assay to measure antibody function. Serum from immunized animals were immunogenic and capable of binding to and killing specific Klebsiella serotypes. Cross-reactivity was observed but limited among serotypes sharing antigenic epitopes. In summary, these results demonstrate the standardization of assays that can be used to test new anti-Klebsiella pneumoniae vaccine candidates, which is important for moving them into clinical trials. IMPORTANCE There is no licensed vaccine for the prevention of Klebsiella pneumoniae infections, and increasing levels of antibiotic resistance make this pathogen a high priority for vaccine and therapeutic development. Standardized assays for testing vaccine immunogenicity are paramount for the development of vaccines, and so in this study, we optimized and standardized both antibody-level and function assays for evaluating in-development K. pneumoniae bioconjugate vaccine response in rabbits.

Preclinical evaluation of an investigational 21-valent pneumococcal conjugate vaccine, V116, in adult-rhesus monkey, rabbit, and mouse models.

Despite the widespread effectiveness of pneumococcal conjugate vaccines on the overall incidence of invasive pneumococcal disease, the global epidemiological landscape continues to be transformed by residual disease from non-vaccine serotypes, thus highlighting the need for vaccines with expanded disease coverage. To address these needs, we have developed V116,an investigational 21-valent non-adjuvanted pneumococcal conjugate vaccine (PCV),containingpneumococcal polysaccharides (PnPs) 3, 6A, 7F, 8, 9N, 10A, 11A,12F, 15A, 16F, 17F, 19A, 20, 22F, 23A, 23B, 24F, 31, 33F, 35B, anda de-O-acetylated 15B(deOAc15B) individually conjugated to the nontoxic diphtheria toxoid CRM197 carrier protein. Preclinical studies evaluated the immunogenicity of V116 inadult monkeys, rabbits, and mice. Following one dose, V116 was found to be immunogenic in preclinical animal species and induced functional antibodies for all serotypes included in the vaccine, in addition to cross-reactive functional antibodies to serotypes 6C and 15B. In these preclinical animal studies, the increased valency of V116 did not result in serotype-specific antibody suppression when compared to lower valent vaccines V114 or PCV13. In addition, when compared with naïve controls, splenocytes from V116 to immunized animals demonstrated significant induction of CRM197-specific T cells in both IFN-γ and IL-4 ELISPOT assays, as well as Th1 and Th2 cytokine induction through in vitro stimulation assays, thus suggesting the ability of V116 to engage T cell dependent immune response pathways to aid in development of memory B cells. V116 also demonstrated significant protection in mice from intratracheal challenge with serotype 24F, a novel serotype not contained in any currently licensed vaccine.

Opsonophagocytic Killing Assay to Measure Anti-Group A Streptococcus Antibody Functionality in Human Serum.

The opsonophagocytic killing assay (OPKA) is designed to measure the functionality of strain-specific antibodies and, therefore, assess protective immunity or the immunogenicity of Group A Streptococcus (GAS) (type A Streptococcus pyogenes) vaccines. Opsonization of GAS for phagocytosis is an important mechanism by which antibodies protect against disease in vivo. The Opsonophagocytic Index or Opsonic Index (OI) is the estimated dilution of antisera that kills 50% of the target bacteria. Here, we describe the protocol of the standardized GAS OPKA developed by Jones et al., 2018.

Optimization and validation of a microcolony multiplexed opsonophagocytic killing assay for 15 pneumococcal serotypes.

Background: To streamline and improve throughput, the agar-based multiplexed opsonophagocytic killing assay (MOPA) was optimized and validated on a microcolony platform for use in the Phase III clinical trial program for V114, an MSD 15-valent pneumococcal conjugate vaccine candidate. Results & methodology: The precision, dilutional linearity and specificity of the microcolony MOPA (mMOPA) were assessed for each serotype in validation experiments. All prespecified acceptance criteria on assay performance were satisfied. Accuracy was assessed by testing 007sp and the US FDA reference panel and comparing to consensus values. The mMOPA produced comparable results to other opsonophagocytic killing assays/MOPAs. Conclusion: The mMOPA is suitable for measuring functional antibodies in adult and pediatric samples. Benefits include throughput, reduced analyst-to-analyst variability and automation potential.

Age-stratified analysis of serotype-specific baseline immunity against group B streptococcus.

Group B streptococcus (GBS) vaccines are currently under development. Data on the natural immunity in diverse age groups will aid establishing the GBS immunization policy. In this study, thirty serum samples were collected from three age groups (neonates/infants, pregnant women, and the elderly) between August 2016 and July 2017. Serotype-specific opsonophagocytic activity (OPA) was assessed using a GBS multiplex opsonophagocytic killing assay (MOPA) against serotypes Ia, III, and V. The mean OPA titers for serotype Ia of the three age groups were not significantly different (p = .156), but tended to be lower in neonates/infants (mean ± standard deviation, 137 ± 278). For serotype III and V, the mean OPA titer of neonates/infants (338 ± 623 and 161 ± 445, respectively) was significantly lower than that of pregnant women (1377 ± 1167 and 9414 ± 6394) and the elderly (1350 ± 1741 and 3669 ± 5597) (p = .002). In conclusion, the lower levels of OPA titers against all tested serotypes in neonates/infants, despite high maternal titers, indicates that intrapartum GBS vaccinations may be required for efficient placental transfer of serotype-specific GBS antibodies with high avidity.

Immunogenicity and mechanisms of action of PnuBioVax, a multi-antigen serotype-independent prophylactic vaccine against infection with Streptococcus pneumoniae.

Streptococcus pneumoniae has multiple protein antigens on the surface in addition to the serotype specific polysaccharide capsule antigen. Whilst the capsule antigen is the target of the polysaccharide vaccines, bacterial proteins can also act as targets for the immune system. PnuBioVax (PBV) is being developed as a multi-antigen, serotype-independent prophylactic vaccine against S. pneumoniae disease. In this study we have sought to elucidate the immune response to PBV in immunised rabbits. Sera from PBV immunised rabbits contained high levels of IgG antibodies to the PBV vaccine, and pneumococcal antigens PspA, Ply, PsaA and PiuA which are components of PBV, when compared with control sera. The PBV sera supported killing of the vaccine strain TIGR4 in an opsonophagocytic killing assay and heterologous strains 6B, 19F and 15B. In addition, incubation in PBV sera led to agglutination of several strains of pneumococci, inhibition of Ply-mediated lysis of erythrocytes and reduced bacterial invasion of lung epithelial cells in vitro. These data suggest that PBV vaccination generates sera that has multiple mechanisms of action that may provide effective protection against pneumococcal infection and give broader strain coverage than the current polysaccharide based vaccines.

Development of an opsonophagocytic killing assay for group a streptococcus.

Group A Streptococcus (GAS) or Streptococcus pyogenes is responsible for an estimated 500,000 deaths worldwide each year. Protection against GAS infection is thought to be mediated by phagocytosis, enhanced by bacteria-specific antibody. There are no licenced GAS vaccines, despite many promising candidates in preclinical and early stage clinical development, the most advanced of which are based on the GAS M-protein. Vaccine progress has been hindered, in part, by the lack of a standardised functional assay suitable for vaccine evaluation. Current assays, developed over 50 years ago, rely on non-immune human whole blood as a source of neutrophils and complement. Variations in complement and neutrophil activity between donors result in variable data that is difficult to interpret. We have developed an opsonophagocytic killing assay (OPKA) for GAS that utilises dimethylformamide (DMF)-differentiated human promyelocytic leukemia cells (HL-60) as a source of neutrophils and baby rabbit complement, thus removing the major sources of variation in current assays. We have standardised the OPKA for several clinically relevant GAS strain types (emm1, emm6 and emm12) and have shown antibody-specific killing for each emm-type using M-protein specific rabbit antisera. Specificity was demonstrated by pre-incubation of the antisera with homologous M-protein antigens that blocked antibody-specific killing. Additional qualifications of the GAS OPKA, including the assessment of the accuracy, precision, linearity and the lower limit of quantification, were also performed. This GAS OPKA assay has the potential to provide a robust and reproducible platform to accelerate GAS vaccine development.

Development of a multiplexed opsonophagocytic killing assay (MOPA) for group B Streptococcus.

Group B Streptococcus (GBS) is a leading cause of sepsis in infants as well as chorioamnionitis in pregnant women. Opsonophagocytic killing assays (OPAs) are an essential technique in vaccine studies of encapsulated bacteria for estimating serotype-specific functional antibody levels in vitro. Here, we developed a three-fold multiplexed OPA (MOPA) to enable practical, large-scale assessment of GBS vaccine immunogenicity, including against serotypes Ia, III, and V. First, three target bacteria strains resistant to streptomycin, spectinomycin, or kanamycin were generated by natural selection through exposure to increasing antibiotic concentrations. Since a high level of nonspecific killing (NSK) of serotype V was observed in a 12.5% baby rabbit complement (BRC) solution, the BRC concentration was optimized. The final GBS-MOPA BRC concentration was 9%, which resulted in less than 20% NSK. The specificity was measured by preabsorbing serum with inactivated GBS. The opsonic index (OI) of preabsorbed serum with the homologous serotype GBS was significantly reduced in all three serotypes tested. The accuracy of the MOPA was compared with that of a single OPA (SOPA) with 35 serum samples. The OIs of the MOPA correlated well with those of the SOPA, and the r2 values were higher than 0.950 for all three serotypes. The precision of the MOPA assay was assessed in five independent experiments with five serum samples. The inter-assay precision of the GBS-MOPA was 12.5% of the average coefficient of variation. This is the first report to develop and standardize a GBS-MOPA, which will be useful for GBS vaccine development and evaluation.

Development and Qualification of an Opsonophagocytic Killing Assay To Assess Immunogenicity of a Bioconjugated Escherichia coli Vaccine.

The global burden of disease caused by extraintestinal pathogenic Escherichia coli (ExPEC) is increasing as the prevalence of multidrug-resistant strains rises. A multivalent ExPEC O-antigen bioconjugate vaccine could have a substantial impact in preventing bacteremia and urinary tract infections. Development of an ExPEC vaccine requires a readout to assess the functionality of antibodies. We developed an opsonophagocytic killing assay (OPA) for four ExPEC serotypes (serotypes O1A, O2, O6A, and O25B) based on methods established for pneumococcal conjugate vaccines. The performance of the assay was assessed with human serum by computing the precision, linearity, trueness, total error, working range, and specificity. Serotypes O1A and O6A met the acceptance criteria for precision (coefficient of variation for repeatability and intermediate precision, ≤50%), linearity (90% confidence interval of the slope of each strain, 0.80, 1.25), trueness (relative bias range, -30% to 30%), and total error (total error range, -65% to 183%) at five serum concentrations and serotypes O2 and O25B met the acceptance criteria at four concentrations (the lowest concentration for serotypes O2 and O25B did not meet the system suitability test of maximum killing of ≥85% of E. coli cells). All serotypes met the acceptance criteria for specificity (opsonization index value reductions of ≤20% for heterologous serum preadsorption and ≥70% for homologous serum preadsorption). The assay working range was defined on the basis of the lowest and highest concentrations at which the assay jointly fulfilled the target acceptance criteria for linearity, precision, and accuracy. An OPA suitable for multiple E. coli serotypes has been developed, qualified, and used to assess the immunogenicity of a 4-valent E. coli bioconjugate vaccine (ExPEC4V) administered to humans.

IL-17A and complement contribute to killing of pneumococci following immunization with a pneumococcal whole cell vaccine.

The pneumococcal whole cell vaccine (PWCV) has been investigated as an alternative to polysaccharide-based vaccines currently in use. It is a non-encapsulated killed vaccine preparation that induces non-capsular antibodies protecting mice against invasive pneumococcal disease (IPD) and reducing nasopharyngeal (NP) carriage via IL-17A activation of mouse phagocytes. Here, we show that PWCV induces antibody and IL-17A production to protect mice against challenge in a fatal aspiration-sepsis model after only one dose. We observed protection even with a boiled preparation, attesting to the stability and robustness of the vaccine. PWCV antibodies were shown to bind to different encapsulated strains, but complement deposition on the pneumococcal surface was observed only on serotype 3 strains; using flow cytometer methodology, variations in PWCV quality, as in the boiled vaccine, were detected. Moreover, anti-PWCV induces phagocytosis of different pneumococcal serotypes by murine peritoneal cells in the presence of complement or IL-17A. These findings suggest that complement and IL-17A may participate in the process of phagocytosis induced by PWCV antibodies. IL-17A can stimulate phagocytic cells to kill pneumococcus and this is enhanced in the presence of PWCV antibodies bound to the bacterial cell surface. Our results provide further support for the PWCV as a broad-range vaccine against all existing serotypes, potentially providing protection for humans against NP colonization and IPD. Additionally, we suggest complement deposition assay as a tool to detect subtle differences between PWCV lots.

Functional anti-polysaccharide IgG titres induced by unadjuvanted pneumococcal-conjugate vaccine when delivered by microprojection-based skin patch.

Adequate access to effective and affordable vaccines is essential for the prevention of mortality due to infectious disease. Pneumonia--a consequence of Streptococcus pneumoniae infection--is the world's leading cause of death in children aged under 5 years. The development of a needle-free, thermostable pneumococcal-conjugate vaccine (PCV) could revolutionise the field by reducing cold-chain and delivery constraints. Skin patches have been used to deliver a range of vaccines, with some inducing significantly higher vaccine-specific immunogenicity than needle-injected controls in pre-clinical models, though they have yet to be used to deliver a PCV. We dry-coated a licensed PCV onto a microprojection-based patch (the Nanopatch) and delivered it to mouse skin. We analysed resulting anti-polysaccharide IgG responses. With and without adjuvant, anti-polysaccharide IgG titres induced by Nanopatch immunisation were significantly higher than dose-matched intramuscular controls. These improved responses were primarily obtained against pneumococcal serotypes 4 and 14. Importantly, capsule-specific IgG correlated with functionality in an opsonophagocytic killing assay. We demonstrate enhanced anti-PCV immunogenicity when delivered by Nanopatch over intramuscular injection. As the first study of a PCV delivered by a skin vaccination technology, this report indicates the potential for reduced costs and greater global distribution of such a vaccine.

Phenotypic and functional analysis of HL-60 cells used in opsonophagocytic-killing assay for Streptococcus pneumoniae.

Differentiated HL-60 is an effector cell widely used for the opsonophagocytic-killing assay (OPKA) to measure efficacy of pneumococcal vaccines. We investigated the correlation between phenotypic expression of immunoreceptors and phagocytic ability of HL-60 cells differentiated with N,N-dimethylformamide (DMF), all-trans retinoic acid (ATRA), or 1α, 25-dihydroxyvitamin D3 (VitD3) for 5 days. Phenotypic change was examined by flow cytometry with specific antibodies to CD11c, CD14, CD18, CD32, and CD64. Apoptosis was determined by flow cytometry using 7-aminoactinomycin D. Function was evaluated by a standard OPKA against serotype 19F and chemiluminescence-based respiratory burst assay. The expression of CD11c and CD14 gradually increased upon exposure to all three agents, while CD14 expression increased abruptly after VitD3. The expression of CD18, CD32, and CD64 increased during differentiation with all three agents. Apoptosis remained less than 10% until day 3 but increased after differentiation by DMF or ATRA. Differentiation with ATRA or VitD3 increased the respiratory burst after day 4. DMF differentiation showed a high OPKA titer at day 1 which sustained thereafter while ATRA or VitD3-differentiated cells gradually increased. Pearson analysis between the phenotypic changes and OPKA titers suggests that CD11c might be a useful differentiation marker for HL-60 cells for use in pneumococcal OPKA.

Phenotypic and Functional Analysis of HL-60 Cells Used in Opsonophagocytic-Killing Assay for Streptococcus pneumoniae

Differentiated HL-60 is an effector cell widely used for the opsonophagocytic-killing assay (OPKA) to measure efficacy of pneumococcal vaccines. We investigated the correlation between phenotypic expression of immunoreceptors and phagocytic ability of HL-60 cells differentiated with N,N-dimethylformamide (DMF), all-trans retinoic acid (ATRA), or 1alpha, 25-dihydroxyvitamin D3 (VitD3) for 5 days. Phenotypic change was examined by flow cytometry with specific antibodies to CD11c, CD14, CD18, CD32, and CD64. Apoptosis was determined by flow cytometry using 7-aminoactinomycin D. Function was evaluated by a standard OPKA against serotype 19F and chemiluminescence-based respiratory burst assay. The expression of CD11c and CD14 gradually increased upon exposure to all three agents, while CD14 expression increased abruptly after VitD3. The expression of CD18, CD32, and CD64 increased during differentiation with all three agents. Apoptosis remained less than 10% until day 3 but increased after differentiation by DMF or ATRA. Differentiation with ATRA or VitD3 increased the respiratory burst after day 4. DMF differentiation showed a high OPKA titer at day 1 which sustained thereafter while ATRA or VitD3-differentiated cells gradually increased. Pearson analysis between the phenotypic changes and OPKA titers suggests that CD11c might be a useful differentiation marker for HL-60 cells for use in pneumococcal OPKA.

Phenotypic and Functional Analysis of HL-60 Cells Used in Opsonophagocytic-Killing Assay for Streptococcus pneumoniae

Differentiated HL-60 is an effector cell widely used for the opsonophagocytic-killing assay (OPKA) to measure efficacy of pneumococcal vaccines. We investigated the correlation between phenotypic expression of immunoreceptors and phagocytic ability of HL-60 cells differentiated with N,N-dimethylformamide (DMF), all-trans retinoic acid (ATRA), or 1alpha, 25-dihydroxyvitamin D3 (VitD3) for 5 days. Phenotypic change was examined by flow cytometry with specific antibodies to CD11c, CD14, CD18, CD32, and CD64. Apoptosis was determined by flow cytometry using 7-aminoactinomycin D. Function was evaluated by a standard OPKA against serotype 19F and chemiluminescence-based respiratory burst assay. The expression of CD11c and CD14 gradually increased upon exposure to all three agents, while CD14 expression increased abruptly after VitD3. The expression of CD18, CD32, and CD64 increased during differentiation with all three agents. Apoptosis remained less than 10% until day 3 but increased after differentiation by DMF or ATRA. Differentiation with ATRA or VitD3 increased the respiratory burst after day 4. DMF differentiation showed a high OPKA titer at day 1 which sustained thereafter while ATRA or VitD3-differentiated cells gradually increased. Pearson analysis between the phenotypic changes and OPKA titers suggests that CD11c might be a useful differentiation marker for HL-60 cells for use in pneumococcal OPKA.

IgG levels against 13-valent pneumococcal conjugate vaccine serotypes in non pneumococcal conjugate vaccine immunized healthy Japanese and intravenous immunoglobulin preparations.

No studies showed specific antibody levels against all serotypes covered by 13-valent pneumococcal conjugate vaccine (PCV13) among polyclonal intravenous immunoglobulin (IVIG) products. Our study aimed to assess whether we could expect the efficacy of IVIG therapy for invasive pneumococcal disease (IPD) and to clarify the age group which should be recommended for IVIG therapy in case of IPD. Serotype-specific immunoglobulin G (IgG) levels against PCV13 serotypes were measured in four IVIGs which were produced from Japanese donors who were not immunized with any pneumococcal conjugate vaccines (PCVs), and in the serum of 160 non-PCV immunized Japanese subjects, by enzyme-linked immunosorbent assay. The functional opsonic activities of the IVIGs against serotypes 6B and 19A were assessed by a multiplexed opsonophagocytic killing assay. Japanese infants aged <2 years had a geometric mean IgG concentration of <0.35 µg/ml against several serotypes. Serotype-specific IgG concentrations varied among IVIGs. In general, IgG antibodies against serotypes 6A, 14 and 19A were higher in each IVIG. Although opsonization indices also varied among preparations, each IVIG had the ability to opsonize both serotypes 6B and 19A. This study suggests that routine immunization with PCV is important for prevention of IPD, especially for children <2 years old and IVIGs might be effective for IPD patients.