Bactericidal Titers Research Articles

Release of Gentamicin and Vancomycin from Preformed Spacers in Infected Total Hip Arthroplasties: Measurement of Concentrations and Inhibitory Activity in Patients’ Drainage Fluids and Serum

Gentamicin (G) and vancomycin (V) concentrations in drainage fluids obtained from patients during the first 24 hours after implantation of antibiotic-loaded polymethylmethacrylate (PMMA) spacers in two-stage revision of infected total hip arthroplasty were studied. The inhibitory activity of drainage fluids against different multiresistant clinical isolates was investigated as well. Seven hips were treated by implantation of industrial G-loaded spacers. Vancomycin was added by manually mixing with PMMA bone cement. Serum and drainage fluid samples were collected 1, 4, and 24 hours after spacer implantation. Antibiotics concentrations and drains bactericidal titer of combination were determined against multiresistant staphylococcal strains. The release of G and V from PMMA cement at the site of infection was prompt and effective. Serum levels were below the limit of detection. The local release kinetics of G and V from PMMA cement was similar, exerting a pronounced, combined inhibitory effect in the implant site. The inhibitory activity of drainage fluids showed substantial intersubject variability related to antibiotic concentrations and differed according to the pathogens tested. Gentamicin and vancomycin were released from temporary hip spacers at bactericidal concentrations, and their use in combination exerted strong inhibition against methicillin-resistant S. aureus and Coagulase Negative Staphylococci strains.

Persistence of antibodies for 42 months following vaccination of adolescents with a meningococcal serogroups A, C, W-135, and Y tetanus toxoid conjugate vaccine (MenACWY-TT)

A primary dose of the European Union (EU)-licensed meningococcal A, C, W-135, and Y tetanus toxoid conjugate vaccine (MenACWY-TT) was immunogenic and well-tolerated in subjects aged 15-19 years. This study assessed antibody persistence at 3.5 years after vaccination with a MenACWY-TT or a tetravalent ACWY polysaccharide vaccine (MenPS, control). In the original study, participants were randomized to receive a single dose of MenACWY-TT or MenPS. Serum bactericidal activity using rabbit serum as exogenous complement source was evaluated up to 42 months post-vaccination. At 42 months post-vaccination with MenACWY-TT (n = 19) or MenPS (n = 17), all subjects in each group had serum bactericidal activity titers ≥1:8 against all serogroups, except for two subjects in the MenPS group against serogroup C. Geometric mean antibody titers were higher than pre-vaccination levels at all post-vaccination time-points in both groups, and were significantly higher in the MenACWY-TT group versus the MenPS group for serogroup W-135 at month 42 (exploratory analysis). These results indicate that seroprotection following primary vaccination with MenACWY-TT extends more than 3 years. Ongoing persistence data are required to understand the duration of protection following vaccination and to guide decisions on the need for future booster doses.

Safety and Immunogenicity of a Quadrivalent Meningococcal Polysaccharide Diphtheria Toxoid Conjugate Vaccine in Infants and Toddlers

Quadrivalent meningococcal conjugate vaccine (Menactra [MenACWY-D]), was licensed in the United States in 2005 to prevent meningococcal disease in adolescents and adults. The license was extended to children aged 2-10 years in 2007 and extended again in 2011 to infants aged 9 months and older based, in part, on results from 3 phase III studies presented herein. The safety and immunogenicity of 2 doses of MenACWY-D was assessed in study-eligible children: dose 1 was administered at 9 months of age and dose 2 was administered 3 months later with or without routine childhood vaccines. Thirty days after vaccination, protective serum bactericidal assay-human complement titers ≥1:8 for meningococcal serogroups A, C, Y and W-135 were achieved by 86-100% of children receiving 2 doses of MenACWY-D. When MenACWY-D was concomitantly administered with measles, mumps, rubella and varicella or heptavalent pneumococcal conjugate vaccine, 81-98% of participants achieved protective responses (serum bactericidal assay-human complement titers ≥1:8 for all serogroups). All seroprotection rates were >91% when the protective titer was defined as serum bactericidal assay-human complement ≥1:4. MenACWY-D did not interfere with measles, mumps, rubella or varicella vaccine responses (98-100% achieved protective titers). When heptavalent pneumococcal conjugate vaccine was given concomitantly with MenACWY-D, antipneumococcal antibody levels, although decreased, were protective for all serotypes by enzyme-linked immunosorbent assay (98-100% ≥ 0.35 μg/mL) and opsonophagocytic assay (99-100% ≥ 1:8). Adverse events were generally mild and similar across groups. Serious adverse events were uncommon. MenACWY-D was safe and immunogenic when given in 2 doses to infants and toddlers; this vaccine can be given with other common childhood immunizations.

Immunogenicity and safety of a new meningococcal A conjugate vaccine in Indian children aged 2–10 years: A Phase II/III double-blind randomized controlled trial

This study compares the immunogenicity and safety of a single dose of a new meningococcal A conjugate vaccine (PsA-TT, MenAfriVac™, Serum Institute of India Ltd., Pune) against the meningococcal group A component of a licensed quadrivalent meningococcal polysaccharide vaccine (PsACWY, Mencevax ACWY®, GSK, Belgium) 28 days after vaccination in Indian children. This double-blind, randomized, controlled study included 340 Indian children aged 2–10 years enrolled from August to October 2007; 169 children received a dose of PsA-TT while 171 children received a dose of PsACWY. Intention-to-treat analysis showed that 95.2% of children in PsA-TT group had a ≥4-fold response in serum bactericidal titers (rSBA) 28 days post vaccination as compared to 78.2% in the PsACWY group. A significantly higher rSBA GMT (11,209, 95%CI 9708–12,942) was noted in the PsA-TT group when compared to PsACWY group (2838, 95%CI 2368–3401). Almost all children in both vaccine groups had a ≥4-fold response in group A-specific IgG concentration but the IgG GMC was significantly greater in the PsA-TT group (89.1μg/ml, 95%CI 75.5–105.0) when compared to the PsACWY group (15.3μg/ml, 95%CI 12.3–19.2). Local and systemic reactions during the 4 days after immunization were similar for both vaccine groups except for tenderness (30.2% in PsA-TT group vs 12.3% in PsACWY group). None of the adverse events or serious adverse events was related to the study vaccines. We conclude that MenAfriVac™ is well tolerated and significantly more immunogenic when compared to a licensed polysaccharide vaccine, in 2-to-10-year-old Indian children.

Immunogenicity of a Single Dose of Meningococcal Group C Conjugate Vaccine Given at 3 Months of Age to Healthy Infants in the United Kingdom

From 1999, in the United Kingdom, meningococcal C conjugate (MCC) vaccines from 3 manufacturers were introduced to the infant immunization schedule at 2, 3 and 4 months of age. In 2006, the schedule was refined to a 2-dose primary schedule at 3 and 4 months of age, with a combined MCC/Haemophilus influenzae type b (MCC/Hib-TT) booster at 12 months of age. Recent data have demonstrated that 2 of the 3 MCC vaccines showed potential for use as a single priming dose in infancy. A randomized trial was undertaken with 2 MCC vaccines; one using tetanus toxoid carrier protein (MCC-TT) and one using CRM197 carrier protein (MCC-CRM197). Infants were immunized with MCC at 3 months of age followed by an MCC/Hib-TT booster at 12 months of age. The serum bactericidal antibody geometric mean titers 1 month after a single dose of MCC-TT or MCC-CRM 197 were 223.3 (95% confidence interval [CI]: 162.9-306.1) and 95.8 (95% CI: 66.4-138.2) with 100% and 95.5% of infants having serum bactericidal antibody titers ≥ 8, respectively. Before boosting, antibody titers had declined, and 1 month after the MCC/Hib-TT booster, serum bactericidal antibody geometric mean titers rose to 2251.0 (95% CI: 1535.3-3300.3) and 355.9 (95% CI: 235.4-538.1) for children primed with MCC-TT and MCC-CRM 197, respectively. In conclusion, a single priming dose of either MCC-TT or MCC-CRM197 administered at 3 months of age can be used together with the Hib/MCC-TT booster in the second year of life.

Design of Meningococcal Factor H Binding Protein Mutant Vaccines That Do Not Bind Human Complement Factor H

Meningococcal factor H binding protein (fHbp) is a human species-specific ligand for the complement regulator, factor H (fH). In recent studies, fHbp vaccines in which arginine at position 41 was replaced by serine (R41S) had impaired fH binding. The mutant vaccines elicited bactericidal responses in human fH transgenic mice superior to those elicited by control fHbp vaccines that bound human fH. Based on sequence similarity, fHbp has been classified into three variant groups. Here we report that although R41 is present in fHbp from variant groups 1 and 2, the R41S substitution eliminated fH binding only in variant group 1 proteins. To identify mutants in variant group 2 with impaired fH binding, we generated fHbp structural models and predicted 63 residues influencing fH binding. From these, we created 11 mutants with one or two amino acid substitutions in a variant group 2 protein and identified six that decreased fH binding. Three of these six mutants retained conformational epitopes recognized by all six anti-fHbp monoclonal antibodies (MAbs) tested and elicited serum complement-mediated bactericidal antibody titers in wild-type mice that were not significantly different from those obtained with the control vaccine. Thus, fHbp amino acid residues that affect human fH binding differ across variant groups. This result suggests that fHbp sequence variation induced by immune selection also affects fH binding motifs via coevolution. The three new fHbp mutants from variant group 2, which do not bind human fH, retained important epitopes for eliciting bactericidal antibodies and may be promising vaccine candidates.

The Effect of Human Factor H on Immunogenicity of Meningococcal Native Outer Membrane Vesicle Vaccines with Over-Expressed Factor H Binding Protein

The binding of human complement inhibitors to vaccine antigens in vivo could diminish their immunogenicity. A meningococcal ligand for the complement down-regulator, factor H (fH), is fH-binding protein (fHbp), which is specific for human fH. Vaccines containing recombinant fHbp or native outer membrane vesicles (NOMV) from mutant strains with over-expressed fHbp are in clinical development. In a previous study in transgenic mice, the presence of human fH impaired the immunogenicity of a recombinant fHbp vaccine. In the present study, we prepared two NOMV vaccines from mutant group B strains with over-expressed wild-type fHbp or an R41S mutant fHbp with no detectable fH binding. In wild-type mice in which mouse fH did not bind to fHbp in either vaccine, the NOMV vaccine with wild-type fHbp elicited 2-fold higher serum IgG anti-fHbp titers (P = 0.001) and 4-fold higher complement-mediated bactericidal titers against a PorA-heterologous strain than the NOMV with the mutant fHbp (P = 0.003). By adsorption, the bactericidal antibodies were shown to be directed at fHbp. In transgenic mice in which human fH bound to the wild-type fHbp but not to the R41S fHbp, the NOMV vaccine with the mutant fHbp elicited 5-fold higher serum IgG anti-fHbp titers (P = 0.002), and 19-fold higher bactericidal titers than the NOMV vaccine with wild-type fHbp (P = 0.001). Thus, in mice that differed only by the presence of human fH, the respective results with the two vaccines were opposite. The enhanced bactericidal activity elicited by the mutant fHbp vaccine in the presence of human fH far outweighed the loss of immunogenicity of the mutant protein in wild-type animals. Engineering fHbp not to bind to its cognate complement inhibitor, therefore, may increase vaccine immunogenicity in humans.

MF59 oil-in-water emulsion in combination with a synthetic TLR4 agonist (E6020) is a potent adjuvant for a combination Meningococcus vaccine

The inclusion of a potent TLR4 immune potentiator to a recombinant antigen vaccine formulation enhances both the magnitude and the breadth of the engendered immune response. One such immune potentiator (TLR4 agonist E6020) was evaluated with recombinant Men B antigens delivered in MF59 sub-micron adjuvant emulsion. The ability of this formulation to enhance serum antibody and bactercidal titers was investigated. The co-delivery of E6020 within MF59 enhanced both the serum and bactericidal titers for Men B antigens and for Men B antigens combined with Men ACWY-CRM conjugate vaccine. The delivery of TLR4 agonist within MF59 emulsion oil droplets leads to a more potent response in comparison to the TLR4 when admixed with MF59 emulsion.

192 Urinary antibacterial activity of nitroxolin 250 mg versus trimethoprim 200 mg against uropathogens after single oral administration

MATERIAL & METHODS: In this phase 1, monocenter, prospectively randomized study the urinary antibacterial activity of NTX 250mg versus TMP 200mg against uropathogens was investigated after single dose in 6 healthy volunteers in a cross-over design using three different urine pH (native urine, urine adjusted to acid (pH 5.5) and alkaline (pH 8.0) milieu). Urine was collected before and after oral administration of the drugs in intervals: 0,0-4, 4-8, 812, 12-24 h. The urinary inhibitory (UIT) and urinary bactericidal titer (UBT) against 5 uropathogenic bacterial strains (E. coli TMP susceptible, E. coli TMP resistant, K. pneumoniae, P. mirabilis and S. saprophyticus) and a test strain (E. coli ATCC 25922) were determined. In 3 additional volunteers, the urinary bactericidal kinetics (UBK) were studied on the 3rd/4th day after oral administration of NTX 250mg t.i.d.

Immunogenicity and tolerability of a multicomponent meningococcal serogroup B (4CMenB) vaccine in healthy adolescents in Chile: a phase 2b/3 randomised, observer-blind, placebo-controlled study

Effective glycoconjugate vaccines against Neisseria meningitidis serogroups A, C, W-135, and Y have been developed, but serogroup B remains a major cause of severe invasive disease in infants and adolescents worldwide. We assessed immunogenicity and tolerability of a four-component vaccine (4CMenB) in adolescents. We did a randomised, observer-blind, placebo-controlled, study at 12 sites in Santiago and Valparaíso, Chile. Adolescents aged 11-17 years received one, two, or three doses of 4CMenB at 1 month, 2 month, or 6 month intervals. Immunogenicity was assessed as serum bactericidal activity using human complement (hSBA) against three reference strains for individual vaccine antigens, and assessed by ELISA against the fourth strain. Local and systemic reactions were recorded 7 days after each vaccination, and adverse events were monitored throughout the study. Participants were initially randomised to five groups (3:3:3:3:1) during the primary phase to receive either one dose, two doses 1 or 2 months apart, or three doses of 4CMenB, or three doses of placebo, with an additional three groups generated for the booster phase. All subjects received at least one dose of 4CMenB. Geometric mean titres, proportions of participants with serum bactericidal antibody titres of 4 or more, and Clopper-Pearson 95% CIs were calculated. The study is registered with ClinicalTrials.gov, number NCT00661713. Overall, 1631 adolescents (mean age 13·8 [SD 1·9] years) received at least one dose of 4CMenB. After two or three doses, 99-100% of recipients had hSBA titres of 4 or more against test strains, compared with 92-97% after one dose (p<0·0145) and 29-50% after placebo. At 6 months 91-100% of participants still had titres of 4 or more for each strain after two or three doses, but only 73-76% after one dose; seroresponse rates reached 99-100% for each strain after second or third doses at 6 months. Local and systemic reaction rates were similar after each 4CMenB injection and did not increase with subsequent doses, but remained higher than placebo. No vaccine-related serious adverse events were reported and no significant safety signals were identified. On the basis of immunogenicity responses this study provides evidence for an adolescent 4CMenB vaccine schedule of two doses, 1-6 months apart, to provide protection against meningococcal B infection. The extent of this protection against meningococcus B variants circulating worldwide will be determined by national surveys. Novartis Vaccines and Diagnostics.

Safety and Immunogenicity of Quadrivalent Meningococcal Conjugate Vaccine in 2- to 10-year-old Human Immunodeficiency Virus-infected Children

Human immunodeficiency virus (HIV)-infected children are at increased risk of meningococcal infection and poor response to quadrivalent meningococcal conjugate vaccine (MCV4), but MCV4 has not been studied in preadolescent HIV-infected children. The P1065 trial enrolled 2- to 10-year-old HIV-infected children with CD4 ≥ 25% to receive MCV4 at entry and at week 24. Rates of response (≥ 4-fold increase in rabbit serum bactericidal antibody) against each meningococcal serogroup (A, C, Y, W-135), geometric mean titers, and rates of seroprotection (rabbit serum bactericidal antibody titer ≥ 1:128) were determined from sera obtained at entry and weeks 4, 24, 28, and 72. Adverse events were assessed for 6 weeks after each MCV4 dose. At entry, 47% of the 59 participants were male, 56% black, 31% Latino, median age was 6 years, 88% were receiving antiretroviral therapy, and 75% had viral load <400 copies/mL. There were no serious adverse events within 6 weeks after MCV4 doses; all vaccination reactions were mild. Response after a single MCV4 dose was high to serogroup A (92%) and W-135 (98%); responses improved after a second dose for serogroup C (43%-80%) (P < 0.0001) and Y (76%-84%) (P = 0.38). By week 72, seroprotection rates were 93%, 91%, 78%, and 46% for serogroups W-135, Y, A, and C, respectively. Two doses of MCV4 were safe and immunogenic in 2- to 10-year-old HIV-infected children. The second dose increased the proportion of children who made a response to serogroup C. Seroprotection waned substantially for serogroups A and C within 1 year of last MCV4 dose.

Management of postoperative spinal infections

Postoperative surgical site infection (SSI) is a common complication after posterior lumbar spine surgery. This review details an approach to the prevention, diagnosis and treatment of SSIs. Factors contributing to the development of a SSI can be split into three categories: (1) microbiological factors; (2) factors related to the patient and their spinal pathology; and (3) factors relating to the surgical procedure. SSI is most commonly caused by Staphylococcus aureus. The virulence of the organism causing the SSI can affect its presentation. SSI can be prevented by careful adherence to aseptic technique, prophylactic antibiotics, avoiding myonecrosis by frequently releasing retractors and preoperatively optimizing modifiable patient factors. Increasing pain is commonly the only symptom of a SSI and can lead to a delay in diagnosis. C-reactive protein and magnetic resonance imaging can help establish the diagnosis. Treatment requires acquiring intra-operative cultures to guide future antibiotic therapy and surgical debridement of all necrotic tissue. A SSI can usually be adequately treated without removing spinal instrumentation. A multidisciplinary approach to SSIs is important. It is useful to involve an infectious disease specialist and use minimum serial bactericidal titers to enhance the effectiveness of antibiotic therapy. A plastic surgeon should also be involved in those cases of severe infection that require repeat debridement and delayed closure.

Postoperative infections of the lumbar spine: presentation and management.

Postoperative surgical site infections (SSI) are a frequent complication following posterior lumbar spinal surgery. In this manuscript we review strategies for prevention, diagnosis and treatment of SSI. The literature was reviewed using the Pubmed database. We identified fifty-nine relevant manuscripts almost exclusively composed of Level III and IV studies. Risk factors for SSI include: 1) factors related to the nature of the spinal pathology and the surgical procedure and 2) factors related to the systemic health of the patient. Staphylococcus aureus is the most common infectious organism in reported series. Proven methods to prevent SSI include prophylactic antibiotics, meticulous adherence to aseptic technique and frequent release of retractors to prevent myonecrosis. The presentation of SSI is varied depending on the virulence of the infectious organism. Frequently, increasing pain is the only presenting complaint and can lead to a delay in diagnosis. Magnetic resonance imaging and the use of C-reactive protein laboratory studies are useful to establish the diagnosis. Treatment of SSI is centered on surgical debridement of all necrotic tissue and obtaining intra-operative cultures to guide antibiotic therapy. We recommend the involvement of an infectious disease specialist and use of minimum serial bactericidal titers to monitor the efficacy of antibiotic treatment. In the most cases, SSI can be adequately treated while leaving spinal instrumentation in place. For severe SSI, repeat debridement, delayed closure and involvement of a plastic surgeon may be necessary.

The role of fluoroquinolones in the management of urinary tract infections in areas with high rates of fluoroquinolone-resistant uropathogens

Fluoroquinolones have been recommended as the drugs of choice for the empirical treatment of uncomplicated and complicated urinary tract infections (UTIs) caused by trimethoprim-sulfamethoxazole-resistant uropathogens. However, because of the increased use of both oral and parenteral fluoroquinolones for other kinds of infections, increasing rates of resistance to fluoroquinolones among the most common uropathogens have challenged this recommendation, particularly in the Asia-Pacific region. The current interpretative criteria for the in vitro susceptibility of uropathogens to some fluoroquinolones, such as levofloxacin and ciprofloxacin, are set according to their therapeutic efficacy for bloodstream infections, and are not specific to UTIs. Fluoroquinolones exhibit concentration-dependent antibacterial activity, high renal excretion, and relatively early and prolonged urinary bactericidal titers. Whether or not current interpretative criteria for the in vitro susceptibility of uropathogens to fluoroquinolones predict clinical failure in treating UTIs is still controversial. The Clinical and Laboratory Standards Institute (CLSI) has established UTI-specific breakpoints for resistance to a few fluoroquinolones. However, the application of high-dose fluoroquinolone therapy for the treatment of mild to moderate UTIs caused by isolates with higher minimum inhibitory concentrations (MICs) of several fluoroquinolones needs to be re-validated based on more relevant clinical studies, prudent pharmacokinetic/pharmacodynamic (PK/PD) considerations, and thorough study of the mutant prevention concentration of fluoroquinolones in the treatment of UTI.

Persistence of Serum Bactericidal Antibody One Year After a Booster Dose of Either a Glycoconjugate or a Plain Polysaccharide Vaccine Against Serogroup C Neisseria meningitidis Given to Adolescents Previously Immunized With a Glycoconjugate Vaccine

Bactericidal antibody induced by immunization of infants with serogroup C Neisseria meningitidis (MenC) vaccines wanes rapidly during childhood. Adolescents are at particular risk from meningococcal disease, therefore they might benefit from a booster dose of vaccine. The duration of serologic response to such a booster in adolescents is unknown. In a previous study, English schoolchildren, aged 9 to 12 years, who had received a monovalent MenC glycoconjugate vaccine in 1999-2000, were given either a plain polysaccharide vaccine (MenC-PS group, n = 150) or a glycoconjugate vaccine (MenC-CRM group, n = 95) at 13 to 15 years of age. In this follow-up study, serum bactericidal antibody titers and specific immunoglobulin G concentrations were assessed 1 year later. Results were compared with unboosted controls of similar age (control group, n = 298). Compliance with study protocol was achieved for 146 of the MenC-PS group, 92 of the MenC-CRM group, and 293 of the control group. Compared with the control group, both the MenC-PS and MenC-CRM groups had a significantly higher (P < 0.0001) geometric mean serum bactericidal antibody titers 1 year after the booster dose (geometric mean titers for MenC-PS group 3388 [95% confidence interval {CI}: 2460-4665]; MenC-CRM group 4417 [95% CI: 2951-6609]; control group 316 [95% CI: 252-396]). Specific immunoglobulin G concentration also rose and remained elevated 1 year after the booster. A booster dose of MenC vaccine given to adolescents produced a marked rise in bactericidal antibody, which remained elevated 1 year later. Introduction of an adolescent booster of MenC vaccine might provide enhanced long-term population control of the disease.

Maintenance of Immune Response throughout Childhood following Serogroup C Meningococcal Conjugate Vaccination in Early Childhood

The objectives of this study were to evaluate the kinetics of antibody decline through childhood in a longitudinal study of a single cohort following serogroup C meningococcal (MenC) vaccine immunization in early childhood and to calculate the proportion of 11 to 13 year olds with protective levels of bactericidal antibody 10 years after immunization. United Kingdom children aged 11 to 13 years in 2010 who had previously taken part in a longitudinal study at the Oxford Vaccine Group had blood samples drawn between 2001 and 2010. Sera from each time point were analyzed for the MenC serum bactericidal antibody titer using a baby rabbit complement (rSBA) assay. The median age at MenC immunization was 21 months (range, 1 year 3 months to 3 years 9 months). The MenC rSBA geometric mean titer (GMT) at age 3.5 to 5 years was 8.0 (95% confidence interval, 6.5 to 9.9; n = 287). By age 11.5 to 13.5 years, the rSBA GMT had declined to 3.3 (2.5 to 4.4; n = 98). The percentage of children with rSBA titers of ≥1:8 (the threshold for protection) also declined from 38% (35% to 41%) to 15% (12% to 19%). We concluded that MenC rSBA titers wane rapidly following vaccination in early childhood and continue to decline into the second decade of life. Since nasopharyngeal colonization in adolescents probably provides the major reservoir for MenC in the population, declining immunity in this cohort is of concern. Sustaining high levels of antibody through booster vaccination in this cohort is likely necessary to avoid a resurgence of disease in the decade ahead.

Study of a Localized Meningococcal Meningitis Epidemic in Burkina Faso: Incidence, Carriage, and Immunity

To better understand localized meningococcal meningitis epidemics, we evaluated a serogroup A (NmA) epidemic in Burkina Faso by surveillance, carriage, and seroprevalence studies. During March-April 2006, cerebrospinal fluid samples from patients suspected to have meningitis in 3 epidemic villages were analyzed by culture or polymerase chain reaction. We assessed meningococcal carriage and serogroup-specific serum bactericidal antibody titers with baby rabbit complement (rSBA) in a representative population sample (N = 624; age range, 1-39 years). A serogroup A/C polysaccharide vaccine campaign occurred in parallel. Cumulative incidence of Nm meningitis was 0.45% and varied among villages (0.08%-0.91%). NmA carriage prevalence was 16% without variation by vaccination status. NmA carriage and anti-NmA seroprevalence varied by village and incidence. In the 2 villages with highest incidence and seroprevalence, presence of rSBA titers ≥8 was associated with NmA carriage (odds ratio [OR], 9.33 [95% confidence interval {CI}, 1.90-45.91]) and vaccination ≤4 days earlier (OR, 0.10 [95% CI, .03-.32]). Visibly purulent or Nm meningitis was significantly associated with recent flulike symptoms and exposure to kitchen smoke (risk ratios >15). A surge of NmA carriage may be involved in the development of meningococcal epidemics and rapidly increase anti-NmA seroprevalence. Flulike infection and kitchen smoke may contribute to the strength of epidemics.

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb (BCG &lt;em&gt;lux&lt;/em&gt;/M.Tb &lt;em&gt;lux&lt;/em&gt;)

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal titers. Studies of serum bactericidal titers in response to childhood vaccines have enabled us to develop and validate cut-off levels for protective immune responses and such cut-offs are in routine use. No such assays have been taken forward into the routine assessment of vaccines that induce primarily cell-mediated immunity in the form of effector T cell responses, such as TB vaccines. In the animal model, the performance of a given vaccine candidate is routinely evaluated in standardized bactericidal assays, and all current novel TB-vaccine candidates have been subjected to this step in their evaluation prior to phase 1 human trials. The assessment of immunogenicity and therefore likelihood of protective efficacy of novel anti-TB vaccines should ideally undergo a similar step-wise evaluation in the human models now, including measurements in bactericidal assays. Bactericidal assays in the context of tuberculosis vaccine research are already well established in the animal models, where they are applied to screen potentially promising vaccine candidates. Reduction of bacterial load in various organs functions as the main read-out of immunogenicity. However, no such assays have been incorporated into clinical trials for novel anti-TB vaccines to date. Although there is still uncertainty about the exact mechanisms that lead to killing of mycobacteria inside human macrophages, the interaction of macrophages and T cells with mycobacteria is clearly required. The assay described in this paper represents a novel generation of bactericidal assays that enables studies of such key cellular components with all other cellular and humoral factors present in whole blood without making assumptions about their relative individual contribution. The assay described by our group uses small volumes of whole blood and has already been employed in studies of adults and children in TB-endemic settings. We have shown immunogenicity of the BCG vaccine, increased growth of mycobacteria in HIV-positive patients, as well as the effect of anti-retroviral therapy and Vitamin D on mycobacterial survival in vitro. Here we summarise the methodology, and present our reproducibility data using this relatively simple, low-cost and field-friendly model. Note: Definitions/Abbreviations BCG lux = M. bovis BCG, Montreal strain, transformed with shuttle plasmid pSMT1 carrying the luxAB genes from Vibrio harveyi, under the control of the mycobacterial GroEL (hsp60) promoter. CFU = Colony Forming Unit (a measure of mycobacterial viability).

Importance of Antibodies to Lipopolysaccharide in Natural and Vaccine-Induced Serum Bactericidal Activity against Neisseria meningitidis Group B

Analysis of the specificity of bactericidal antibodies in normal, convalescent, and postvaccination human sera is important in understanding human immunity to meningococcal infections and can aid in the design of an effective group B vaccine. A collection of human sera, including group C and group B convalescent-phase sera, normal sera with naturally occurring cross-reactive bactericidal activity, and some postvaccination sera, was analyzed to determine the specificity of cross-reactive bactericidal antibodies. Analysis of human sera using a bactericidal antibody depletion assay demonstrated that a significant portion of the bactericidal activity could be removed by purified lipopolysaccharide (LPS). LPS homologous to that expressed on the bactericidal test strain was most effective, but partial depletion by heterologous LPS suggested the presence of antibodies with various degrees of cross-reactivity. Binding of anti-L3,7 LPS bactericidal antibodies was affected by modification of the core structure, suggesting that these functional antibodies recognized epitopes consisting of both core structures and lacto-N-neotetraose (LNnT). When the target strain was grown with 5'-cytidinemonophospho-N-acetylneuraminic acid (CMP-NANA) to increase LPS sialylation, convalescent-phase serum bactericidal titers were decreased by only 2- to 4-fold, and most remaining bactericidal activity was still depleted by LPS. Highly sialylated LPS was ineffective in depleting bactericidal antibodies. We conclude that natural infections caused by strains expressing L3,7 LPS induce persistent, protective bactericidal antibodies and appear to be directed against nonsialylated bacterial epitopes. Additionally, subsets of these bactericidal antibodies are cross-reactive, binding to several different LPS immunotypes, which is a useful characteristic for an effective group B meningococcal vaccine antigen.

Antibodies in Action: Role of Human Opsonins in Killing Salmonella enterica Serovar Typhi

Although vaccines have been available for over a century, a correlate of protection for typhoid fever has yet to be identified. Antibodies are produced in response to typhoid infection and vaccination and are generally used as the gold standard for determining vaccine immunogenicity, even though their role in clearance of Salmonella enterica serovar Typhi infections is poorly defined. Here, we describe the first functional characterization of S. Typhi-specific antibodies following vaccination with a new vaccine, M01ZH09 (Ty2 ΔaroC ΔssaV). We determined that postvaccination sera increased the uptake of wild-type S. Typhi by human macrophages up to 2.3-fold relative to prevaccination (day 0) or placebo samples. These results were recapitulated using immunoglobulins purified from postvaccination serum, demonstrating that antibodies were largely responsible for increases in uptake. Imaging verified that macrophages internalized 2- to 9.5-fold more S. Typhi when the bacteria were opsonized with postvaccination sera than when the bacteria were opsonized with day 0 or placebo sera. Once inside macrophages, the survival of S. Typhi was reduced as much as 50% when opsonized with postvaccination sera relative to day 0 or placebo serum samples. Lastly, bactericidal assays indicated that antibodies generated postvaccination were recognized by complement factors and assisted in killing S. Typhi: mean postvaccination bactericidal antibody titers were higher at all time points than placebo and day 0 titers. These data clearly demonstrate that there are at least two mechanisms by which antibodies facilitate killing of S. Typhi. Future work could lead to improved immunogenicity tests associated with vaccine efficacy and the identification of correlates of protection against typhoid fever.