Development of flow cytometric opsonophagocytosis and antibody-mediated complement deposition assays for non-typeable Haemophilus influenzae.

BACKGROUND: Haemophilus influenzae is found in the nasopharynx of 80% of the human population. While colonisation with non-typeable Haemophilus influenzae (NTHi) is usually asymptomatic, it is capable of causing acute and chronic otitis media (OM) in infants, invasive disease in susceptible groups and is the leading cause of exacerbations of patients with chronic obstructive pulmonary disease (COPD). Current methods for assessing functional antibody immunity to NTHi are limited and labour intensive. Flow cytometric assays could provide an attractive alternative to evaluate immune responses to candidate vaccines in clinical trials. RESULTS: We have developed a duplexed flow-cytometric uptake and oxidative burst opsonophagocytosis assay (fOPA). We have also developed a duplexed antibody-mediated complement C3b/iC3b and C5b-9 deposition assay (CDA). Antibody-mediated C3b/iC3b deposition correlated with opsonophagocytic uptake (r = 0.65) and with opsonophagocytic oxidative burst (r = 0.69). Both fOPA and CDA were reproducible, with the majority of samples giving a coefficient of variation (CV) of < 20% and overall assay CVs of 14% and 16% respectively. CONCLUSIONS: The high-throughput flow cytometric assays developed here were successfully optimised for use with NTHi. Assays proved to be sensitive and highly reproducible for the measurement of bacterial uptake and oxidative burst opsonophagocytosis and antibody-mediated deposition of C3b/iC3b and C5b-9. These assays are useful tools for use in large scale epidemiological studies and to assist in the assessment of functional antibody induced by NTHi candidate vaccines.

Pneumococcal Haemophilus influenzae protein D conjugate vaccine induces antibodies that inhibit glycerophosphodiester phosphodiesterase activity of protein D.

Haemophilus influenzae outer membrane protein D (PD) is a glycerophosphodiester phosphodiesterase (GlpQ) activity-possessing virulence factor and a promising vaccine antigen, providing 35.3% efficacy against acute otitis media caused by nontypeable H. influenzae (NTHI) when it was used as a carrier protein in a novel pneumococcal PD conjugate (Pnc-PD) vaccine. To study if PD-induced protection against NTHI could be due to antibodies that inhibit or neutralize its enzymatic activity, a GlpQ enzyme inhibition assay was developed, and serum samples collected from Finnish infants before and after Pnc-PD vaccination were analyzed for enzyme inhibition and anti-PD immunoglobulin G (IgG) antibody concentration. Before vaccination at age 2 months, the majority (84%) of infants (n = 69) had no detectable anti-PD IgG antibodies, and all were enzyme inhibition assay negative (inhibition index, <20). At age 13 to 16 months, all infants receiving three or four doses of Pnc-PD had detectable anti-PD IgG antibodies and 36% (8/22 infants) of the infants receiving three doses and 26% (6/23 infants) of the infants receiving four doses of Pnc-PD were inhibition assay positive (inhibition index, >/=20). No significant rise in anti-PD IgG antibodies or enzyme inhibition among control vaccinees (n = 24) receiving three doses of hepatitis B vaccine was detected. A modest correlation (r(s), approximately 0.66) between anti-PD IgG concentration and enzyme inhibition was detected; however, their kinetics were clearly different. These data suggest that measurement of antibody responses that inhibit PD's enzymatic activity could be a useful tool for assessing Pnc-PD vaccine-induced protective immunity against NTHI.

Safety and immunogenicity of an investigational 4-component Staphylococcus aureus vaccine with or without AS03B adjuvant: Results of a randomized phase I trial.

We assessed the safety, reactogenicity and immunogenicity of a staphylococcal vaccine combining capsular polysaccharides types 5 and 8 (CPS5/8), conjugated to tetanus toxoid (TT), with mutated detoxified α-toxin (AT) and clumping factor A (ClfA). In this phase I, randomized, placebo-controlled, observer-blind trial (NCT01160172), 88 healthy 18- to 40-year-olds received CPS5-TT/CPS8-TT/AT/ClfA vaccine (5/5/10/10 µg or 10/10/30/30 µg dose, each with or without AS03B adjuvant) or saline, at months 0, 1, 6. Solicited and unsolicited adverse events (AEs) were recorded for 7 and 30 d post-vaccination, respectively; potential immune-mediated diseases (pIMDs) and serious AEs (SAEs) were recorded throughout the study. Humoral and antigen-specific CD4(+)/CD8(+) T-cell immunity were assessed from Day (D) 0 to D540 post-vaccination. The most frequently reported solicited local and general AEs were pain (78.6%-100% of subjects), fatigue (36.4%-93.3% of subjects post-dose 1-2) and headache (20%-44.4% of subjects post-dose 3). Overall, 4 SAEs and 2 potential immune-mediated diseases (pIMDs) (none fatal or vaccine-related) were reported. For each antigen, pre-vaccination seropositivity rates were high (85.7%-100%) and geometric mean concentrations (GMCs) in vaccine recipients sharply increased from D0 to D14, then plateaued to study end. Exploratory group comparisons suggested higher GMCs with higher dosage, without AS03B effect. Vaccine-induced antibodies were functional (CPS5 opsonophagocytic assays, and AT/ClfA inhibition assays). AT- and ClfA-specific CD4(+) T-cells with Th0/Th1 cytokine profile were induced at low levels (median <0.05%) by each formulation (intracellular cytokine staining). In conclusion, no safety concerns were identified and each vaccine formulation induced robust humoral immune responses after the first vaccine dose.

Brucella pathogenesis, genes identified from random large-scale screens.

Pathogenicity islands, specialized secretion systems, virulence plasmids, fimbriae, pili, adhesins, and toxins are all classical bacterial virulence factors. However, many of these factors, though widespread among bacterial pathogens, are not necessarily found among bacteria that colonize eukaryotic cells in a pathogenic/symbiotic relationship. Bacteria that form these relationships have developed other strategies to infect and grow in their hosts. This is particularly true for Brucella and other members of the class Proteobacteria. Thus far the identification of virulence factors for Brucella has been largely dependent on large-scale screens and testing in model systems. The genomes of the facultative intracellular pathogens Brucella melitensis and Brucella suis were sequenced recently. This has identified several more potential virulence factors for Brucella that were not found in large screens. Here, we present an overall view of Brucella virulence by compiling virulence data from the study of 184 attenuated mutants.

Safety, immunogenicity, and antibody persistence following an investigational Streptococcus pneumoniae and Haemophilus influenzae triple-protein vaccine in a phase 1 randomized controlled study in healthy adults.

We investigated a protein-based nontypeable Haemophilus influenzae (NTHi) and pneumococcal (HiP) vaccine containing pneumococcal histidine triad D (PhtD), detoxified pneumolysin (dPly), and NTHi protein D (PD) in adults. In a phase I study, 40 healthy 18- to 40-year-old subjects were randomized (2:2:1) to receive two HiP doses administered 60 days apart, with or without AS03 adjuvant (HiP-AS and HiP groups, respectively), or Engerix B (GlaxoSmithKline, Belgium) as a control. Safety, antibodies, and antigen-specific CD4(+) T-cell immune responses were assessed before and until 480 days after vaccination. No serious adverse events were reported, and no subject withdrew due to an adverse event. Local and systemic symptoms were reported more frequently in the HiP-AS group than in the other two groups. The frequency and intensity of local and systemic symptoms appeared to increase after the second dose of HiP-AS or HiP but not Engerix B. Antibody geometric mean concentrations (GMCs) for PhtD, dPly, and PD increased after each dose of HiP-AS or HiP, with higher GMCs being observed in the HiP-AS group (statistically significant for anti-PD after dose 1 and anti-Ply after dose 2). GMCs remained higher at day 420 than prior to vaccination in both the HiP-AS and HiP groups. Antigen-specific CD4(+) T cells increased after each dose but were unmeasurable by day 480. Two doses of an investigational PhtD-dPly-PD protein vaccine induced humoral immunity and antigen-specific CD4(+) T-cell responses after each dose, with generally higher responses when the vaccine was administered with AS03. HiP combined with AS03 appeared to be more reactogenic than the antigens alone. (This study has been registered at ClinicalTrials.gov under registration no. NCT00814489.).

One or two doses of quadrivalent meningococcal serogroups A, C, W-135 and Y tetanus toxoid conjugate vaccine is immunogenic in 9- to 12-month-old children.

BACKGROUND: The incidence of invasive meningococcal disease is highest in infants. A quadrivalent meningococcal serogroups A, C, W-135 and Y tetanus toxoid conjugate vaccine (MenACWY-TT) was evaluated in children 9-12 months of age. METHODS: We randomized infants (1:1) to receive 1 dose of MenACWY-TT at 12 months of age (ACWY-1 group) or 2 doses at 9 and 12 months (ACWY-2). We measured immunogenicity after each dose and 1 year after completing vaccination using human serum bactericidal antibody (hSBA) assays according to prespecified criteria of ≥ 1:8. Local and general symptoms were solicited for 8 days after vaccination. Adverse events were recorded for 6 months after the last dose. RESULTS: We enrolled and vaccinated 349 subjects, of whom 248 reenrolled at Year 1 for evaluation of antibody persistence. Percentages of subjects with postvaccination hSBA ≥ 1:8 in the ACWY-1 group were 79.5%, 94.6%, 50.8% and 56.1% and in the 2-dose group (ACWY-2) were 88.4%, 100%, 99.3% and 99.3% postdose 2 for serogroups A, C, W-135 and Y, respectively. At Year 1, 80.0-99.1% in each group had hSBA ≥ 1:8, except for serogroup A, for which 20.6% (ACWY-1) and 25.9% (ACWY-2) retained hSBA ≥1:8. Both schedules were well-tolerated, with no observed increase in reactogenicity after the second dose. CONCLUSIONS: MenACWY-TT was immunogenic when administered as a single dose at 12 months of age, or as 2 doses at 9 and 12 months, and had a clinically acceptable safety profile. Good antibody persistence was observed through 12 months postvaccination after both treatment schedules for serogroups C, W-135, Y.

Measurement of functional anti-meningococcal serogroup a activity using strain 3125 as the target strain for serum bactericidal assay.

Functional anti-N. meningitidis serogroup A (MenA) activity in human serum is detected by serum bactericidal assay (SBA), using either rabbit (rSBA) or human (hSBA) complement, with F8238 as the recommended MenA SBA target strain. However, the F8238 strain may not be optimal for this purpose because, as we show here, it expresses the L11 immunotype, whereas most MenA invasive strains express the L(3,7)9 or L10 immunotype. Moreover, SBA results may be strain dependent, because immunotypes differ in their sensitivity to complement, emphasizing the need to choose the most appropriate strain. Sera from random subsets of infants, toddlers, children, and adolescents in clinical trials of MenA conjugate vaccines were tested by rSBA using strains 3125 (L10) and F8238 (L11). In unvaccinated subjects from all age groups, the percentages of seropositive samples (rSBA-MenA titer, ≥1:8) was lower using strain 3125 than using strain F8238. However, in toddlers and adolescents immunized with a conjugate MenA vaccine, the percentages of seropositive samples generally were similar using either strain in the rSBA. In two studies, sera also were tested with hSBA. Using hSBA, the differences in the percentages of seroprotective samples (hSBA-MenA titer, ≥1:4) between strains 3125 and F8238 was less apparent, and in contrast with rSBA, the percentage of seroprotective samples from unvaccinated subjects was slightly higher using strain 3125 than using strain F8238. In adults vaccinated with plain MenA polysaccharide, the percentage of seroprotective samples was higher using strain 3125 than with strain F8238, and the vaccine response rates using strain 3125 were better aligned with the demonstrated efficacy of MenA vaccination. In conclusion, SBA results obtained using the MenA L10 3125 strain better reflected vaccine-induced immunity.

Immunogenicity and safety of H influenzae type b-N meningitidis C/Y conjugate vaccine in infants.

BACKGROUND: Meningococcal disease incidence is highest in children younger than 2 years of age, yet there is no US-licensed vaccine for this age group. A phase III study evaluated the immunogenicity and safety of an investigational Haemophilus influenzae type b (Hib)-Neisseria meningitidis serogroups C and Y-tetanus toxoid conjugate vaccine (HibMenCY). MATERIALS AND METHODS: A total of 4180 infants were randomly assigned to receive the HibMenCY at the ages of 2, 4, 6, and 12 to 15 months or the licensed Hib tetanus toxoid conjugate vaccine (ActHIB) at 2, 4, and 6 months and Hib conjugated to N meningitidis outer membrane protein (PedvaxHIB) at 12 to 15 months. Routinely scheduled vaccines were coadministered. Serum bactericidal activity using human complement and anti-polyribosylribitol phosphate antibodies were assessed in 991 subjects. Local and systemic adverse reactions were recorded for 4 days after each dose. RESULTS: The percentage of HibMenCY recipients with serum bactericidal assay using human complement titers of 1:8 or higher after dose 3 was 98.8% for N meningitidis serogroup C (MenC) and 95.8% for N meningitidis serogroup Y (MenY). After dose 4, the percentages were 98.5% and 98.8%, respectively. The percentage of HibMenCY recipients with postdose 3 anti-polyribosylribitol phosphate antibody levels of ≥ 1.0 µg/mL was noninferior to that of control (96.3% vs 91.2%). After dose 4, MenC and MenY serum bactericidal assay using human complement antibody titers increased 12-fold over pre-dose 4 levels. Incidence of pain, redness, and swelling at the HibMenCY injection sites tended to be lower than with Hib type b after the first 3 doses and after the fourth dose. Rates of systemic symptoms were similar across groups. CONCLUSIONS: The HibMenCY was immunogenic against MenC and MenY and induced anti-polyribosylribitol phosphate antibody levels noninferior to those of licensed Hib conjugate vaccine. The safety profile of the HibMenCY was clinically acceptable and comparable to Hib conjugate vaccine.

Evaluation of pneumococcal polysaccharide immunoassays using a 22F adsorption step with serum samples from infants vaccinated with conjugate vaccines.

The history of the pneumococcal polysaccharide enzyme-linked immunosorbent assay (ELISA) is characterized by a continuous search for increased specificity. A third-generation ELISA that uses 22F polysaccharide inhibition has increased the specificity of the assay, particularly at low antibody concentrations. The present work compared various 22F ELISAs and non-22F ELISAs. The comparisons involved three different laboratories, including a WHO reference laboratory, and included sera from subjects from different geographic areas immunized with different pneumococcal conjugate vaccines, including the licensed 7-valent Prevenar vaccine and the 10-valent Synflorix vaccine. All comparisons led to the same conclusion that the threshold defined as 0.35 microg/ml for the WHO non-22F ELISA is lower when any 22F ELISA is used. The use of highly purified polysaccharides for coating further improved the specificity of the assay. In conclusion, we confirm that the 22F ELISA can be recommended as a reference method for the determination of antibodies against pneumococcal polysaccharides.

Systematic targeted mutagenesis of Brucella melitensis 16M reveals a major role for GntR regulators in the control of virulence.

In order to identify transcriptional regulators involved in virulence gene control in Brucella melitensis, we generated a collection of 88 mutants in the AraC, ArsR, Crp, DeoR, GntR, IclR, LysR, MerR, RpiR, and TetR families of regulators. This collection was named LiMuR (library of mutants for regulators). We developed a method to test several mutants simultaneously in one animal in order to identify those unable to survive. This method, called the plasmid-tagged mutagenesis method, was used to test the residual virulence of mutants after 1 week in a mouse model of infection. Ten attenuated mutants, of which six and three belong to the GntR and LysR families, respectively, were identified and individually confirmed to replicate at lower rates in mice. Among these 10 mutants, only gntR10 and arsR6 are attenuated in cellular models. The LiMuR also allows simple screenings to identify regulators of a particular gene or operon. As a first example, we analyzed the expression of the virB operon in the LiMuR mutants. We carried out Western blottings of whole-cell extracts to analyze the production of VirB proteins using polyclonal antisera against VirB proteins. Four mutants produced small amounts of VirB proteins, and one mutant overexpressed VirB proteins compared to the wild-type strain. In these five mutants, reporter analysis using the virB promoter fused to lacZ showed that three mutants control virB at the transcriptional level. The LiMuR is a resource that will provide straightforward identification of regulators involved in the control of genes of interest.

Yersinia enterocolitica as a vehicle for a naked DNA vaccine encoding Brucella abortus bacterioferritin or P39 antigen.

Brucella is a facultative intracellular parasite that causes brucellosis in animals and humans. The protective immune response against Brucella involves both humoral and cell-mediated immunity. In previous studies, we demonstrated that the T-dominant Brucella antigens bacterioferritin (BFR) and P39 administered either as CpG adjuvant recombinant proteins or as naked-DNA plasmids induced a specific Th1-biased immune response in mice. In order to improve the protection conferred by the BFR and P39 vaccines and to evaluate the additive role of antilipopolysaccharide (anti-LPS) antibodies, we used live attenuated Yersinia enterocolitica serotypes O:3 and O:9 as delivery vectors for naked-DNA plasmids encoding these BFR and P39 antigens. Following two intragastric immunizations in BALB/c mice, the Yersinia vectors harboring a DNA vaccine encoding BFR or P39 induced antigen-specific serum immunoglobulin and Th1-type responses (both lymphocyte proliferation and gamma interferon production) among splenocytes. Moreover, as expected, antibodies recognizing Brucella abortus 544 lipopolysaccharide were detected in O:9-immunized mice but not in O:3-treated animals. Animals immunized with O:9 organisms carrying pCI or with O:9 organisms alone were found to be significantly resistant to infection by B. abortus 544. Our data demonstrated that pCI plasmids encoding BFR or P39 and delivered with live attenuated strains of Yersinia O:3 or O:9 can trigger Th1-type responses. The fact than only O:9 vectors induced a highly significant protective immunity against B. abortus 544 infection pointed out the crucial role of anti-LPS antibodies in protection. The best protection was conferred by a serotype O:9 strain carrying pCIP39, confirming the importance of the P39 T-cell antigen in this mechanism.

Identification of a quorum-sensing signal molecule in the facultative intracellular pathogen Brucella melitensis.

Brucella melitensis is a gram-negative alpha2-proteobacterium responsible for abortion in goats and for Malta fever in humans. This facultative intracellular pathogen invades and survives within both professional and nonprofessional phagocytes. A dichloromethane extract of spent culture supernatant from B. melitensis induces bioluminescence in an Escherichia coli acyl-homoserine lactone (acyl-HSL) biosensor strain based upon the activity of the LasR protein of Pseudomonas aeruginosa. HPLC fractionation of the extract, followed by mass spectrometry, identified the major active molecule as N-dodecanoylhomoserine lactone (C12-HSL). This is the first report of the production of an acyl-HSL by an intracellular pathogen. The addition of synthetic C12-HSL to an early log phase culture of either B. melitensis or Brucella suis 1330 reduces the transcription of the virB operon, which contains virulence genes known to be required for intracellular survival. This mimics events seen during the stationary phase of growth and suggests that quorum sensing may play a role in the control of virulence in Brucella.

The Ton system, an ABC transporter, and a universally conserved GTPase are involved in iron utilization by Brucella melitensis 16M.

Brucella spp. are gram-negative intracellular facultative pathogens that are known to produce 2,3-dihydroxybenzoic acid (DHBA), a catechol siderophore that is essential for full virulence in the natural host. The mechanism of DHBA entry into Brucella and other gram-negative bacteria is poorly understood. Using mini-Tn5Kmcat mutagenesis, we created a transposon library of Brucella melitensis 16M and isolated 32 mutants with a defect in iron acquisition or assimilation. Three of these transposon mutants are deficient in utilization of DHBA. Analysis of these three mutants indicated that the ExbB, DstC, and DugA proteins are required for optimal assimilation of DHBA and/or citrate. ExbB is part of the Ton complex, and DstC is a permease homologue of an iron(III) ABC transporter; in gram-negative bacteria these two complexes are involved in the uptake of iron through the outer and inner membranes, respectively. DugA is a new partner in iron utilization that exhibits homology with the bacterial conserved GTPase YchF. Based on this homology, DugA could have a putative regulatory function in iron assimilation in Brucella. None of the three mutants was attenuated in cellular models or in the mouse model of infection, which is consistent with the previous suggestion that DHBA utilization is not required in these models.